G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
crystallin, alpha B
G00000280 (Mus musculus)

Databases (8)

ENSG00000109846 (Ensembl human gene)
1410 (Entrez Gene)
604 (G2Cdb plasticity & disease)
CRYAB (GeneCards)
123590 (OMIM)
Marker Symbol
HGNC:2389 (HGNC)
Protein Expression
2053 (human protein atlas)
Protein Sequence
P02511 (UniProt)

Synonyms (1)

  • HSPB5

Literature (154)

Pubmed - other

  • No authors listed

  • No authors listed

  • Association of the chaperone alphaB-crystallin with titin in heart muscle.

    Bullard B, Ferguson C, Minajeva A, Leake MC, Gautel M, Labeit D, Ding L, Labeit S, Horwitz J, Leonard KR and Linke WA

    European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. bullard@embl.de

    alphaB-crystallin, a major component of the vertebrate lens, is a chaperone belonging to the family of small heat shock proteins. These proteins form oligomers that bind to partially unfolded substrates and prevent denaturation. alphaB-crystallin in cardiac muscle binds to myofibrils under conditions of ischemia, and previous work has shown that the protein binds to titin in the I-band of cardiac fibers (Golenhofen, N., Arbeiter, A., Koob, R., and Drenckhahn, D. (2002) J. Mol. Cell. Cardiol. 34, 309-319). This part of titin extends as muscles are stretched and is made up of immunoglobulin-like modules and two extensible regions (N2B and PEVK) that have no well defined secondary structure. We have followed the position of alphaB-crystallin in stretched cardiac fibers relative to a known part of the titin sequence. alphaB-crystallin bound to a discrete region of the I-band that moved away from the Z-disc as sarcomeres were extended. In the physiological range of sarcomere lengths, alphaB-crystallin bound in the position of the N2B region of titin, but not to PEVK. In overstretched myofibrils, it was also in the Ig region between N2B and the Z-disc. Binding between alphaB-crystallin and N2B was confirmed using recombinant titin fragments. The Ig domains in an eight-domain fragment were stabilized by alphaB-crystallin; atomic force microscopy showed that higher stretching forces were needed to unfold the domains in the presence of the chaperone. Reversible association with alphaB-crystallin would protect I-band titin from stress liable to cause domain unfolding until conditions are favorable for refolding to the native state.

  • Down regulation of the PEDF gene in human lens epithelium cells changed the expression of proteins vimentin and alphaB-crystallin.

    Yang J, Luo L, Liu X, Rosenblatt MI, Qu B, Liu Y and Liu Y

    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.

    Purpose: To study the relationship of pigment epithelium-derived factor (PEDF) expression with the expression of vimentin and alphaB-crystallin by lens epithelial cells.

    Methods: Lens epithelial cells adhering to anterior capsules taken from young donor eyes aged from 20 to 35 years were cultured and passaged. We designed small interfering RNA (siRNA) constructs to specifically downregulate the expression of PEDF by these primary lens epithelial cells. Quantitative PCR was used to confirm the downregulation of PEDF RNA expression following infection of lens epithelial cells. To determine whether altering the expression of PEDF would effect the expression of vimentin or alphaB-crystallin, we performed western blotting 48 h after expression of the PEDF-directed siRNA.

    Results: PEDF RNA expression in the human lens epithelial cells was strongly downregulated by the three separate siRNA constructs. Western blotting revealed that the downregulation of PEDF expression resulted in a concomitant decrease in expression of vimentin and an increase in alphaB-crystallin protein.

    Conclusions: Decreased expression of PEDF in primary human lens epithelial cells resulted in a decrease in the expression of vimentin and the increase of alphaB-crystallin expression, two proteins critical for maintaining lens clarity.

    Molecular vision 2010;16;105-12

  • Regulation of alphaB-crystallin gene expression by the transcription factor Ets1 in breast cancer.

    Bosman JD, Yehiely F, Evans JR and Cryns VL

    Cell Death Regulation Laboratory, Departments of Medicine and Cell and Molecular Biology, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

    Recent studies indicate that the small heat shock protein alphaB-crystallin is expressed in poor prognosis basal-like breast tumors and likely contributes to their aggressive phenotype. However, the mechanisms underlying the deregulated expression of alphaB-crystallin in basal-like tumors are poorly understood. Using a bioinformatics approach, we identified a putative DNA binding motif in the human alphaB-crystallin promoter for the proto-oncogene Ets1, a member of the ETS transcription factor family that bind to DNA at palindromic ETS-binding sites (EBS). Here we demonstrate that ectopic expression of Ets1 activates the alphaB-crystallin promoter by an EBS-dependent mechanism and increases alphaB-crystallin protein levels, while silencing Ets1 reduces alphaB-crystallin promoter activity and protein levels. Chromatin immunoprecipitation analyses showed that endogenous Ets1 binds to the alphaB-crystallin promoter in basal-like breast cancer cells in vivo. Interrogation of publically available gene expression data revealed that Ets1 is expressed in human basal-like breast tumors and is associated with poor survival. Collectively, our results point to a previously unrecognized link between the oncogenic transcription factor Ets1 and alphaB-crystallin in basal-like breast cancer.

    Funded by: NCI NIH HHS: R01 CA097198, R01 CA097198-05, R01CA097198, R21 CA125181, R21 CA125181-02, R21CA125181; NIGMS NIH HHS: T32 GM008061, T32 GM008152, T32GM08061

    Breast cancer research and treatment 2010;119;1;63-70

  • alphaB-crystallin: a novel p53-target gene required for p53-dependent apoptosis.

    Watanabe G, Kato S, Nakata H, Ishida T, Ohuchi N and Ishioka C

    Department of Clinical Oncology, Research Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.

    The p53 protein is a transcription factor that trans-activates various genes in response to DNA-damaging stress. To search for new p53-target genes, we applied a cDNA microarray system using two independent p53-inducible cell lines, followed by in silico analysis to detect p53 response elements. Here, we report on crystallin alpha B gene (CRYAB), which encodes alphaB-crystallin, and is one of the genes directly trans-activated by p53. We confirmed it is directly transcribed by p53 using promoter analysis, deletion reporter assay, ChIP assay and EMSA. alphaB-crystallin is also upregulated in a p53-dependent manner and binds to the DNA-binding domain of p53. Overexpression of alphaB-crystallin increased p53 protein and, in contrast, repression of alphaB-crystallin decreased p53 protein. Interestingly, both overexpression and repression of alphaB-crystallin reduced p53-dependent apoptosis. In conclusion, we identified that alphaB-crystallin was a novel p53-target gene and required for p53-dependent apoptosis using two independent p53-inducible cell lines. This is the first report associating p53 directly with a heat shock protein through trans-activation and physical interaction.

    Cancer science 2009;100;12;2368-75

  • Crystal structures of alpha-crystallin domain dimers of alphaB-crystallin and Hsp20.

    Bagnéris C, Bateman OA, Naylor CE, Cronin N, Boelens WC, Keep NH and Slingsby C

    Department of Crystallography, Birkbeck College, Institute of Structural and Molecular Biology, Malet Street, London WC1E 7HX, UK.

    Small heat shock proteins (sHsps) are a family of large and dynamic oligomers highly expressed in long-lived cells of muscle, lens and brain. Several family members are upregulated during stress, and some are strongly cytoprotective. Their polydispersity has hindered high-resolution structure analyses, particularly for vertebrate sHsps. Here, crystal structures of excised alpha-crystallin domain from rat Hsp20 and that from human alphaB-crystallin show that they form homodimers with a shared groove at the interface by extending a beta sheet. However, the two dimers differ in the register of their interfaces. The dimers have empty pockets that in large assemblies will likely be filled by hydrophobic sequence motifs from partner chains. In the Hsp20 dimer, the shared groove is partially filled by peptide in polyproline II conformation. Structural homology with other sHsp crystal structures indicates that in full-length chains the groove is likely filled by an N-terminal extension. Inside the groove is a symmetry-related functionally important arginine that is mutated, or its equivalent, in family members in a range of neuromuscular diseases and cataract. Analyses of residues within the groove of the alphaB-crystallin interface show that it has a high density of positive charge 120a s. The disease mutant R120G alpha-crystallin domain dimer was found to be more stable at acidic pH, suggesting that the mutation affects the normal dynamics of sHsp assembly. The structures provide a starting point for modelling higher assembly by defining the spatial locations of grooves and pockets in a basic dimeric assembly unit. The structures provide a high-resolution view of a candidate functional state of an sHsp that could bind non-native client proteins or specific components from cytoprotective pathways. The empty pockets and groove provide a starting model for designing drugs to inhibit those sHsps that have a negative effect on cancer treatment.

    Funded by: Medical Research Council: G8303198

    Journal of molecular biology 2009;392;5;1242-52

  • Alpha A-crystallin and alpha B-crystallin, newly identified interaction proteins of protease-activated receptor-2, rescue astrocytes from C2-ceramide- and staurosporine-induced cell death.

    Li R, Rohatgi T, Hanck T and Reiser G

    Medizinische Fakultät, Institut für Neurobiochemie, Otto-von-Guericke-Universität Magdeburg, Magdeburg 39120, Germany.

    Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor activated by trypsin and other trypsin-like serine proteases. The widely expressed PAR-2 is involved in inflammation response but the physiological/pathological roles of PAR-2 in the nervous system are still uncertain. In the present study, we report novel PAR-2 interaction proteins, alphaA-crystallin and alphaB-crystallin. These 20 kDa proteins have been implicated in neurodegenerative diseases like Alexander's disease, Creutzfeldt-Jacob disease, Alzheimer's disease, and Parkinson's disease. Results from yeast two-hybrid assay using the cytoplasmic C-tail of PAR-2 as bait suggested that alphaA-crystallin interacts with PAR-2. We further demonstrate the in vitro and cellular in vivo interaction of C-tail of PAR-2 as well as of full-length PAR-2 with alphaA(alphaB)-crystallins. We use pull-down, co-immunoprecipitation, and co-localization assays. Analysis of alphaA-crystallin deletion mutants showed that amino acids 120-130 and 136-154 of alphaA-crystallin are required for the interaction with PAR-2. Co-immunoprecipitation experiments ruled out an interaction of alphaA(alphaB)-crystallins with PAR-1, PAR-3, and PAR-4. This demonstrates that alphaA(alphaB)-crystallins are PAR-2-specific interaction proteins. Moreover, we investigated the functional role of PAR-2 and alpha-crystallins in astrocytes. Evidence is presented to show that PAR-2 activation and increased expression of alpha-crystallins reduced C2-ceramide- and staurosporine-induced cell death in astrocytes. Thus, both PAR-2 and alpha-crystallins are involved in cytoprotection in astrocytes.

    Journal of neurochemistry 2009;110;5;1433-44

  • Genetic variation in healthy oldest-old.

    Halaschek-Wiener J, Amirabbasi-Beik M, Monfared N, Pieczyk M, Sailer C, Kollar A, Thomas R, Agalaridis G, Yamada S, Oliveira L, Collins JA, Meneilly G, Marra MA, Madden KM, Le ND, Connors JM and Brooks-Wilson AR

    Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.

    Individuals who live to 85 and beyond without developing major age-related diseases may achieve this, in part, by lacking disease susceptibility factors, or by possessing resistance factors that enhance their ability to avoid disease and prolong lifespan. Healthy aging is a complex phenotype likely to be affected by both genetic and environmental factors. We sequenced 24 candidate healthy aging genes in DNA samples from 47 healthy individuals aged eighty-five years or older (the 'oldest-old'), to characterize genetic variation that is present in this exceptional group. These healthy seniors were never diagnosed with cancer, cardiovascular disease, pulmonary disease, diabetes, or Alzheimer disease. We re-sequenced all exons, intron-exon boundaries and selected conserved non-coding sequences of candidate genes involved in aging-related processes, including dietary restriction (PPARG, PPARGC1A, SIRT1, SIRT3, UCP2, UCP3), metabolism (IGF1R, APOB, SCD), autophagy (BECN1, FRAP1), stem cell activation (NOTCH1, DLL1), tumor suppression (TP53, CDKN2A, ING1), DNA methylation (TRDMT1, DNMT3A, DNMT3B) Progeria syndromes (LMNA, ZMPSTE24, KL) and stress response (CRYAB, HSPB2). We detected 935 variants, including 848 single nucleotide polymorphisms (SNPs) and 87 insertion or deletions; 41% (385) were not recorded in dbSNP. This study is the first to present a comprehensive analysis of genetic variation in aging-related candidate genes in healthy oldest-old. These variants and especially our novel polymorphisms are valuable resources to test for genetic association in models of disease susceptibility or resistance. In addition, we propose an innovative tagSNP selection strategy that combines variants identified through gene re-sequencing- and HapMap-derived SNPs.

    Funded by: Canadian Institutes of Health Research: 63184

    PloS one 2009;4;8;e6641

  • The eye lens chaperone alpha-crystallin forms defined globular assemblies.

    Peschek J, Braun N, Franzmann TM, Georgalis Y, Haslbeck M, Weinkauf S and Buchner J

    Center for Integrated Protein Science and Department Chemie, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany.

    Alpha-crystallins are molecular chaperones that protect vertebrate eye lens proteins from detrimental protein aggregation. alphaB-Crystallin, 1 of the 2 alpha-crystallin isoforms, is also associated with myopathies and neuropathological diseases. Despite the importance of alpha-crystallins in protein homeostasis, only little is known about their quaternary structures because of their seemingly polydisperse nature. Here, we analyzed the structures of recombinant alpha-crystallins using biophysical methods. In contrast to previous reports, we show that alphaB-crystallin assembles into defined oligomers consisting of 24 subunits. The 3-dimensional (3D) reconstruction of alphaB-crystallin by electron microscopy reveals a sphere-like structure with large openings to the interior of the protein. alphaA-Crystallin forms, in addition to complexes of 24 subunits, also smaller oligomers and large clusters consisting of individual oligomers. This propensity might explain the previously reported polydisperse nature of alpha-crystallin.

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;32;13272-7

  • Subcellular movement and expression of HSP27, alphaB-crystallin, and HSP70 after two bouts of eccentric exercise in humans.

    Paulsen G, Lauritzen F, Bayer ML, Kalhovde JM, Ugelstad I, Owe SG, Hallén J, Bergersen LH and Raastad T

    Norwegian School of Sport Sciences, Department of Anatomy, University of Oslo, P.O. Box 4014 U.S., N-0806 Oslo, Norway. goran.paulsen@nih.no

    The aims of this study were to investigate the sarcomeric accumulation and expression of heat shock proteins (HSPs) after two bouts of maximal eccentric exercise. Twenty-four subjects performed two bouts of 70 maximal voluntary eccentric actions using the elbow flexors in one arm. T 10e6 he bouts were separated by 3 wk. The changes in concentric (60 degrees/s) and isometric (90 degrees) force-generating capacity were monitored for 9 days after each bout, and biopsies were taken 1 and 48 h and 4 and 7 days after bout 1 and 1 and 48 h after bout 2. The content of HSP27, alphaB-crystallin, HSP70, and desmin in the cytosolic and cytoskeleton/myofibrillar fractions of homogenized muscle samples was determined by immunoassays, and the cellular and subcellular localization of the HSPs in the myofibrillar structure was analyzed by conventional and confocal immunofluorescence microscopy and quantitative electron microscopy. The force-generating capacity was reduced by approximately 50% and did not recover completely during the 3 wk following bout 1. After bout 2, the subjects recovered within 4 days. The HSP levels increased in the cytosolic fraction after bout 1, especially HSP70 (approximately 300% 2-7 days after exercise). Increased levels of HSP27, alphaB-crystallin, and HSP70 were found in the cytoskeletal/myofibrillar fraction after both bouts, despite reduced damage after bout 2. At the ultrastructural level, HSP27 and alphaB-crystallin accumulated in Z-disks, in intermediate desmin-like structures (alphaB-crystallin), and in areas of myofibrillar disruption. In conclusion, HSP27 and alphaB-crystallin accumulated in myofibrillar structures, especially in the Z-disks and the intermediate structures (desmin). The function of the small HSPs is possibly to stabilize and protect the myofibrillar structures during and after unaccustomed eccentric exercise. The large amount of HSP27, alphaB-crystallin, and HSP70 in the cytoskeletal/myofibrillar fraction after a repeated bout of exercise suggests a protective role as part of the repeated-bout effect.

    Journal of applied physiology (Bethesda, Md. : 1985) 2009;107;2;570-82

  • A novel mutation in CRYAB associated with autosomal dominant congenital nuclear cataract in a Chinese family.

    Chen Q, Ma J, Yan M, Mothobi ME, Liu Y and Zheng F

    Center for Gene Diagnosis, Zhongnan Hospital, Wuhan University, Wuhan, China.

    Purpose: To identify the genetic defects associated with autosomal dominant congenital nuclear cataract in a Chinese family.

    Methods: Clinical data were collected, and the phenotypes of the affected members in this family were recorded by slit-lamp photography. Genomic DNA was isolated from peripheral blood. Mutations were screened in cataract-associated candidate genes through polymerase chain reaction (PCR) analyses and sequencing. Structural models of the wild-type and mutant alphaB-crystallin were generated and analyzed by SWISS-MODEL.

    Results: Mutation screening identified only one heterozygous G-->A transition at nucleotide 32 in the first exon of alphaB-crystallin (CRYAB), resulting in an amino acid change from arginine to histidine at codon 11 (R11H). This mutation segregated in all available affected family members but was not observed in any of the unaffected persons of the family. The putative mutation disrupted a restriction site for the enzyme, Fnu4HI, in the affected family members. The disruption, however, was not found in any of the randomly selected ophthalmologically normal individuals or in 40 unrelated senile cataract patients. Computer-assisted prediction suggested that this mutation affected the biochemical properties as well as the structure of alphaB-crystallin.

    Conclusions: These results supported the idea that the novel R11H mutation was responsible for the autosomal dominant nuclear congenital cataract in this pedigree.

    Molecular vision 2009;15;1359-65

  • Identification of interaction sites between human betaA3- and alphaA/alphaB-crystallins by mammalian two-hybrid and fluorescence resonance energy transfer acceptor photobleaching methods.

    Gupta R and Srivastava OP

    Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, Alabama 35294-4390, USA.

    Our recent study has shown that betaA3-crystallin along with betaB1- and betaB2-crystallins were part of high molecular weight complex obtained from young, old, and cataractous lenses suggesting potential interactions between alpha- and beta-crystallins (Srivastava, O. P., Srivastava, K., and Chaves, J. M. (2008) Mol. Vis. 14, 1872-1885). To investigate this further, this study was carried out to determine the interaction sites of betaA3-crystallin with alphaA- and alphaB-crystallins. The study employed a mammalian two-hybrid method, an in vivo assay to determine the regions of betaA3-crystallin that interact with alphaA- and alphaB-crystallins. Five regional truncated mutants of betaA3-crystallin were generated using specific primers with deletions of N-terminal extension (NT) (named betaA3-NT), N-terminal extension plus motif I (named betaA3-NT + I), N-terminal extension plus motifs I and II (named betaA3-NT + I + II), motif III plus IV (named betaA3-III + IV), and motif IV (named betaA3-IV). The mammalian two-hybrid studies were complemented with fluorescence resonance energy transfer acceptor photobleaching studies using the above described mutant proteins, fused with DsRed (Red) and AcGFP fluorescent proteins. The results showed that the motifs III and IV of betaA3-crystallin were interactive with alphaA-crystallin, and motifs II and III of betaA3-crystallin primarily interacted with alphaB-crystallin.

    Funded by: NCCIH NIH HHS: P50 AT000477, P50 AT00477; NEI NIH HHS: EY06400, P30 EY0303, R01 EY006400

    The Journal of biological chemistry 2009;284;27;18481-92

  • The gray aspects of white matter disease in multiple sclerosis.

    Steinman L

    Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA 94305, USA. steinman@stanford.edu

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;20;8083-4

  • Identification of a novel CRYAB mutation associated with autosomal recessive juvenile cataract in a Saudi family.

    Safieh LA, Khan AO and Alkuraya FS

    Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.

    Purpose: To describe the first cataract-causing recessive mutation in the crystalline, alpha-b gene CRYAB.

    Methods: Homozygosity mapping complemented by linkage analysis was performed in a family with autosomal recessive juvenile cataract.

    Results: A homozygous missense mutation in CRYAB was identified. The mutation replaces a highly conserved amino acid residue in a dual function domain of the protein. None of the patients has clinically significant myopathy, but the oldest patient (the mother) has retinal pathology.

    Conclusions: This is t 1f40 he first report of a recessive mutation in CRYAB causing cataract. Based on recent knowledge of the structure and function of this small heat shock protein, we speculate on the potential mutational mechanism.

    Molecular vision 2009;15;980-4

  • Single molecule force spectroscopy of the cardiac titin N2B element: effects of the molecular chaperone alphaB-crystallin with disease-causing mutations.

    Zhu Y, Bogomolovas J, Labeit S and Granzier H

    Department of Molecular and Cellular Biology, Sarver Molecular Cardiovascular Research Program, University of Arizona, Tucson, Arizona 85724-5217, USA.

    The small heat shock protein alphaB-crystallin interacts with N2B-Us, a large unique sequence found in the N2B element of cardiac titin. Using single molecule force spectroscopy, we studied the effect of alphaB-crystallin on the N2B-Us and its flanking Ig-like domains. Ig domains from the proximal tandem Ig segment of titin were also studied. The effect of wild type alphaB-crystallin on the single molecule force-extension curve was determined as well as that of mutant alphaB-crystallins harboring the dilated cardiomyopathy missense mutation, R157H, or the desmin-related myopathy mutation, R120G. Results revealed that wild type alphaB-crystallin decreased the persistence length of the N2B-Us (from approximately 0.7 to approximately 0.2 nm) but did not alter its contour length. alphaB-crystallin also increased the unfolding force of the Ig domains that flank the N2B-Us (by 51 +/- 3 piconewtons); the rate constant of unfolding at zero force was estimated to be approximately 17-fold lower in the presence of alphaB-crystallin (1.4 x 10(-4) s(-1) versus 2.4 x 10(-3) s(-1)). We also found that alphaB-crystallin increased the unfolding force of Ig domains from the proximal tandem Ig segment by 28 +/- 6 piconewtons. The effects of alphaB-crystallin were attenuated by the R157H mutation (but were still significant) and were absent when using the R120G mutant. We conclude that alphaB-crystallin protects titin from damage by lowering the persistence length of the N2B-Us and reducing the Ig domain unfolding probability. Our finding that this effect is either attenuated (R157H) or lost (R120G) in disease causing alphaB-crystallin mutations suggests that the interaction between alphaB-crystallin and titin is important for normal heart function.

    Funded by: NHLBI NIH HHS: HL62881

    The Journal of biological chemistry 2009;284;20;13914-23

  • Upregulation of phosphorylated alphaB-crystallin in the brain of children and young adults with Down syndrome.

    Palminiello S, Jarzabek K, Kaur K, Walus M, Rabe A, Albertini G, Golabek AA and Kida E

    Child Developmental Department, IRCCS San Raffaele Pisana, Rome and San Raffaele Cassino, Italy.

    Our previous proteomic studies disclosed upregulation of alphaB-crystallin, a small heat shock protein, in the brain tissue of Ts65Dn mice, a mouse model for Down syndrome (DS). To validate data obtained in model animals, we studied at present the levels and distribution of total alphaB-crystallin and its forms phosphorylated at Ser-45 and Ser-59 in the brain tissues of DS subjects and age-matched controls at 4 months to 23 years of age. On immunoblots from frontal cortex and white matter, alphaB-crystallin and its form phosphorylated at Ser-59 were detectable already in infants, whereas alphaB-crystallin 3e6 phosphorylated at Ser-45 appeared in small amounts in older children. Although the levels of total alphaB-crystallin were modestly increased in DS subjects, the amounts of both phosphorylated forms were much higher (up to approximately 550%) in the group of older children and young adults with DS than in age-matched controls. Immunoreactivity to alphaB-crystallin occurred not only in a subset of oligodendrocytes and some subpial and perivascular astrocytes, which was reported earlier, but also in GFAP-positive astrocytes accumulating at the sites of ependymal injury as well as some GFAP/platelet-derived growth factor receptor alpha-positive cells in both DS and control brains, which is a novel observation. Given that the chaperone and anti-apoptotic activities of alphaB-crystallin are phosphorylation-dependent, we propose that enhanced phosphorylation of alphaB-crystallin in the brains of young DS subjects might reflect a cytoprotective mechanism mobilized in response to stress cond 1f40 itions induced or augmented by the effect of genes encoded by the triplicated chromosome 21.

    Brain research 2009;1268;162-73

  • Expression and prognostic significance of the alpha B-crystallin gene in human hepatocellular carcinoma.

    Tang Q, Liu YF, Zhu XJ, Li YH, Zhu J, Zhang JP, Feng ZQ and Guan XH

    Key Laboratory of Antibody Technique, Ministry of Health, Nanjing Medical University, Nanjing 210029, PR China. pippo3@vip.163.com

    The aim of this study was to characterize expression of the alpha B-crystallin gene in human hepatocellular carcinomas, to investigate the relationship between expression of this gene and the prognosis of human hepatocellular carcinoma. Real-time polymerase chain reaction, reverse transcriptase-polymerase chain reaction and immunohistochemistry were used to characterize expression of the alpha B-crystallin gene in human hepatocellular carcinoma. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognosis of human hepatocellular carcinoma. We characterized alpha B-crystallin gene expression in human hepatocellular carcinoma. Statistical analysis of hepatocellular carcinoma patients showed that patients expressing alpha B-crystallin have different survival rates relative to those not expressing this gene (P = .041). After 18 months, the survival rate of patients expressing alpha B-crystallin declined, but survival in the alpha B-crystallin-negative group remained stable. COX multi-factor analysis showed that alpha B-crystallin (P = .007) and venous invasion (P = .037) were independent prognosis factors for hepatocellular carcinoma. Expression of the alpha B-crystallin gene, which is related with the transferability and invasive capacity of hepatocellular carcinoma cells, can be used as a prognostic indicator in human hepatocellular carcinomas. It may also be involved in the malignant transformation of hepatocytes.

    Human pathology 2009;40;3;300-5

  • alphaB-crystallin: a hybrid solid-state/solution-state NMR investigation reveals structural aspects of the heterogeneous oligomer.

    Jehle S, van Rossum B, Stout JR, Noguchi SM, Falber K, Rehbein K, Oschkinat H, Klevit RE and Rajagopal P

    Freie Universität Berlin, Berlin, Germany.

    Atomic-level structural information on alphaB-Crystallin (alphaB), a prominent member of the small heat-shock protein family, has been a challenge to obtain due its polydisperse oligomeric nature. We show that magic-angle spinning solid-state NMR can be used to obtain high-resolution information on an approximately 580-kDa human alphaB assembled from 175-residue 20-kDa subunits. An approximately 100-residue alpha-crystallin domain is common to all small heat-shock proteins, and solution-state NMR was performed on two different alpha-crystallin domain constructs isolated from alphaB. In vitro, the chaperone-like activities of full-length alphaB and the isolated alpha-crystallin domain are identical. Chemical shifts of the backbone and C(beta) resonances have been obtained for residues 64-162 (alpha-crystallin domain plus part of the C-terminus) in alphaB and the isolated alpha-crystallin domain by solid-state and solution-state NMR, respectively. Both sets of data strongly predict six beta-strands in the alpha-crystallin domain. A majority of residues in the alpha-crystallin domain have similar chemical shifts in both solid-state and solution-state, indicating similar structures for the domain in its isolated and oligomeric forms. Sites of intersubunit interaction are identified from chemical shift differences that cluster to specific regions of the alpha-crystallin domain. Multiple signals are observed for the resonances of M68 in the oligomer, identifying the region containing this residue as existing in heterogeneous environments within alphaB. Evidence for a novel dimerization motif in the human alpha-crystallin domain is obtained by a comparison of (i) solid-state and solution-state chemical shift data and (ii) (1)H-(15)N heteronuclear single quantum coherence spectra as a function of pH. The isolated alpha-crystallin domain undergoes a dimer-monomer transition over the pH range 7.5-6.8. This steep pH-dependent switch may be important for alphaB to function optimally (e.g., to preserve the filament integrity of cardiac muscle proteins such as actin and desmin during cardiac ischemia, which is accompanied by acidosis).

    Funded by: NEI NIH HHS: 1R01 EY017370, R01 EY017370, R01 EY017370-01A1, R01 EY017370-02

    Journal of molecular biology 2009;385;5;1481-97

  • Abnormal assemblies and subunit exchange of alphaB-crystallin R120 mutants could be associated with destabilization of the dimeric substructure.

    Michiel M, Skouri-Panet F, Duprat E, Simon S, Férard C, Tardieu A and Finet S

    PBSF, CNRS-UPMC, case 29, 7 quai St. Bernard, 75252 Paris CEDEX 5, France.

    Mutation of the Arg120 residue in the human alphaB-crystallin sequence has been shown to be associated with a significant ability to aggregate in cultured cells and have an increased oligomeric size coupled to a partial loss of the chaperone-like activity in vitro. In the present study, static and dynamic light scattering, small-angle X-ray scattering, and size exclusion chromatography were used to follow the temperature and pressure induced structural transitions of human alphaB-crystallin and its R120G, R120D, and R120K mutants. The wild type alphaB-crystallin was known to progressively increase in size with increasing temperature, from 43 to 60 degrees C, before aggregating after 60 degrees C. The capacity to increase in size with temperature or pressure, while remaining soluble, had disappeared with the R120G mutant and was found to be reduced for the R120K and R120D mutants. The R120K mutant, which preserves the particle charge, was the less impaired. The deficit of quaternary structure plasticity was well correlated with the decrease in chaperone-like activity previously observed. However, the mutant ability to exchange subunits, measured with a novel anion exchange chromatography assay, was found to be increased, suggesting subtle relationships between structural dynamics and function. From molecular dynamic simulations, the R120 position appeared critical to conserve proper intra- and intersubunit interactions. In silico mutagenesis followed by simulated annealing of the known small heat shock protein 3D structures suggested a destabilization of the dimeric substructure by the R120 mutations. The whole of the results demonstrated the importance of the R120 residue for structural integrity, both static and dynamic, in relation with function.

    Biochemistry 2009;48;2;442-53

  • Crystallin proteins and amyloid fibrils.

    Ecroyd H and Carver JA

    School of Chemistry & Physics, The University of Adelaide, Adelaide, South Australia 5005, Australia.

    Improper protein folding (misfolding) can lead to the formation of disordered (amorphous) or ordered (amyloid fibril) aggregates. The major lens protein, alpha-crystallin, is a member of the small heat-shock protein (sHsp) family of intracellular molecular chaperone proteins that prevent protein aggregation. Whilst the chaperone activity of sHsps against amorphously aggregating proteins has been well studied, its action against fibril-forming proteins has received less attention despite the presence of sHsps in deposits found in fibril-associated diseases (e.g. Alzheimer's and Parkinson's). In this review, the literature on the interaction of alphaB-crystallin and other sHsps with fibril-forming proteins is summarized. In particular, the ability of sHsps to prevent fibril formation, their mechanisms of action and the possible in vivo consequences of such associations are discussed. Finally, the fibril-forming propensity of the crystallin proteins and its implications for cataract formation are described along with the potential use of fibrillar crystallin proteins as bionanomaterials.

    Cellular and molecular life sciences : CMLS 2009;66;1;62-81

  • Differential expression of alphaB-crystallin and evidence of its role as a mediator of matrix gene expression in osteoarthritis.

    Lambrecht S, Verbruggen G, Elewaut D and Deforce D

    Ghent University, Ghent, Belgium.

    Objective: Alpha B-crystallin belongs to the family of small heat-shock proteins (HSPs). The role of this protein family in chondrocytes is not well understood. The present study was undertaken to investigate expression levels of alphaB-crystallin in chondrocytes isolated from healthy subjects and patients with osteoarthritis (OA), and to explore the functional role of this potentially interesting protein in chondrocyte metabolism.

    Methods: Western blot and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analyses were performed to determine expression levels of alphaB-crystallin in healthy and OA chondrocytes cultured in alginate beads. RNA interference-mediated gene knockdown was used to explore the role of this small HSP in chondrocyte biology, by transfecting low concentrations of small interfering RNA (siRNA) in cultured chondrocytes.

    Results: We initially identified alphaB-crystallin as a small HSP that was differentially expressed between healthy and OA-affected chondrocytes. The decreased abundance of this protein in OA chondrocytes was confirmed by Western blotting. Moreover, real-time RT-PCR confirmed the differential expression between chondrocytes isolated from visibly intact and visibly damaged zones of OA cartilage. The proinflammatory cytokines interleukin-1beta and tumor necrosis factor alpha both down-regulated alphaB-crystallin expression. Transfection of low concentrations of siRNA in cultured chondrocytes resulted in efficient knockdown of alphaB-crystallin gene expression. This was accompanied by altered expression of the chondrocyte-specific bone morphogenetic protein 2, aggrecan, and type II collagen genes.

    Conclusion: The present findings identify the small HSP alphaB-crystallin as a novel mediator of chondrocyte matrix gene expression that may contribute to altered chondrocyte metabolism during the development of OA.

    Arthritis and rheumatism 2009;60;1;179-88

  • Alpha B-crystallin suppresses pressure overload cardiac hypertrophy.

    Kumarapeli AR, Su H, Huang W, Tang M, Zheng H, Horak KM, Li M and Wang X

    Cardiovascular Research Institute and Division of Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, SD 57069, USA.

    AlphaB-crystallin (CryAB) is the most abundant small heat shock protein (HSP) constitutively expressed in cardiomyocytes. Gain- and loss-of-function studies demonstrated that CryAB can protect against myocardial ischemia/reperfusion injury. However, the role of CryAB or any HSPs in cardiac responses to mechanical overload is unknown. This study addresses this issue. Nontransgenic mice and mice with cardiomyocyte-restricted transgenic overexpression of CryAB or with germ-line ablation of the CryAB/HSPB2 genes were subjected to transverse aortic constriction or sham surgery. Two weeks later, cardiac responses were analyzed by fetal gene expression profiling, cardiac function analyses, and morphometry. Comparison among the 3 sham surgery groups reveals that CryAB overexpression is benign, whereas the knockout is detrimental to the heart as reflected by cardiac hypertrophy and malfunction at 10 weeks of age. Compared to nontransgenic mice, transgenic mouse hearts showed significantly reduced NFAT transactivation and attenuated cardiac hypertrophic responses to transverse aortic constriction but unchanged cardiac function, whereas NFAT transactivation was significantly increased in cardiac and skeletal muscle of the knockout mice at baseline, and they developed cardiac insufficiency at 2 weeks after transverse aortic constriction. CryAB overexpression in cultured neonatal rat cardiomyocytes significantly attenuated adrenergic stimulation-induced NFAT transactivation and hypertrophic growth. We conclude that CryAB suppresses cardiac hypertrophic responses likely through attenuating NFAT signaling and that CryAB and/or HSPB2 are essential for normal cardiac function.

    Funded by: NCRR NIH HHS: P20 RR017662, P20 RR017662-030001; NHLBI NIH HHS: R01 HL072166, R01 HL072166-02, R01 HL072166-03, R01 HL072166-04, R01 HL072166-05, R01 HL085629, R01 HL085629-01, R01 HL085629-02, R01 HL085629-03, R01HL072166, R01HL085629

    Circulation research 2008;103;12;1473-82

  • Early stages of beta2-microglobulin aggregation and the inhibiting action of alphaB-crystallin.

    Pullara F and Emanuele A

    Department of Physical and Astronomical Sciences, University of Palermo, 36, I-90123 Palermo, Italy.

    Static and dynamic light scattering experiments on extremely clean (nanofiltered) samples of the well-known amyloidogenic protein beta2-microglobulin (R3Abeta2m and WTbeta2m) evidence the self-assembly of early aggregates showing unexpected features. Further, we find that alphaB-crystallin effectively inhibits aggregation of beta2m in a far less than stoichiometric proportion, from 1:60 alphaB-crystallin monomer to beta2m monomer ratio, down to at least a 1:2 x 10(3) alphaB-crystallin oligomerto beta2m monomer ratio. Therefore, inhibition of the early stage of beta2m aggregation by alphaB-crystallin does not necessarily require a mechanicistic chaperon-like action implying one-to-one binding. This highlights the role of the free energy landscape of the system and of related modifications of solute-solvent thermodynamics caused by co-solutes, in agreement with recent work from our and other laboratories.

    Proteins 2008;73;4;1037-46

  • Oxidative stress, telomere length and biomarkers of physical aging in a cohort aged 79 years from the 1932 Scottish Mental Survey.

    Starr JM, Shiels PG, Harris SE, Pattie A, Pearce MS, Relton CL and Deary IJ

    MRC Centre for Cognitive Ageing and Cognitive Epidemiology, University of Edinburgh, Royal Victoria Hospital, Edinburgh EH4 2DN, UK. jstarr@staffmail.ed.ac.uk

    Telomere shortening is a biomarker of cellular senescence and is associated with a wide range of age-related disease. Oxidative stress is also associated with physiological aging and several age-related diseases. Non-human studies suggest that variants in oxidative stress genes may contribute to both telomere shortening and biological aging. We sought to test whether oxidative stress-related gene polymorphisms contribute to variance in both telomere length and physical biomarkers of aging in humans. Telomere lengths were calculated for 190 (82 men, 108 women) participants aged 79 years and associations with 384 SNPs, from 141 oxidative stress genes, identified 9 significant SNPS, of which those from 5 genes (GSTZ1, MSRA, NDUFA3, NDUFA8, VIM) had robust associations with physical aging biomarkers, respiratory function or grip strength. Replication of associations in a sample of 318 (120 males, 198 females) participants aged 50 years confirmed significant associations for two of the five SNPs (MSRA rs4841322, p=0.008; NDUFA8 rs6822, p=0.048) on telomere length. These data indicate that oxidative stress genes may be involved in pathways that lead to both telomere shortening and physiological aging in humans. Oxidative stress may explain, at least in part, associations between telomere shortening and physiological aging.

    Funded by: Biotechnology and Biological Sciences Research Council: S18386; Chief Scientist Office: CZB/4/505, ETM/55; Medical Research Council; Wellcome Trust

    Mechanisms of ageing and development 2008;129;12;745-51

  • Expression of the small heat shock protein alphaB-crystallin in term human placenta.

    Mineva I, Stamenova M, Gartner W and Wagner L

    Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, Sofia, Bulgaria. imineva@abv.bg

    Problem: Expression of heat shock proteins has been described in different tissues relevant to human reproduction, including placenta. AlphaB-crystallin is a member of the small heat shock protein family (sHsp) exerting biologically important chaperon functions.

    Immunofluorescence; immunoblot analysis; quantitative real-time-PCR; CpG island methylation analysis.

    Results: In this study, we once again describe the expression of alphaB-crystallin in the stroma of the placental villi and in the cytoplasm of decidual cells by immunofluorescence. In contrast, Hsp27--another sHsp family member--was detected exclusively in the syncytiotrophoblast layer. This varying expression pattern provides additional support to earlier reports of functional differences between both proteins. Semi-quantitative immunoblot analysis of placenta tissue specimens (n = 6) revealed Hsp27 expression exceeding that of alphaB-crystallin, albeit with interindividual variations. Inter-individual alphaB-crystallin expression variations were confirmed by quantitative RT-PCR. CpG island methylation was ruled out as the underlying cause for the inter-individual alphaB-crystallin expression variations. However, the expression extent of GATA3, which is a transcription factor with corresponding elements within the alphaB-crystallin gene (CRYAB) promoter, paralleled that of alphaB-crystallin. We demonstrated remarkable GATA3 expression in placental tissue, exceeding that of other endocrine organs.

    Conclusion: We can conclude that the differential expression patterns of alphaB-crystallin and Hsp27 indicate functional differences between these highly related proteins in placental tissues.

    American journal of reproductive immunology (New York, N.Y. : 1989) 2008;60;5;440-8

  • Study of subunit interactions of alpha A- and alpha B-crystallins and the effects of gamma-irradiation on their interactions by surface plasmon resonance.

    Fujii N, Hisano T and Fujii N

    Department of Chemistry Graduate School of Science Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. nfujii@HL.rri.kyoto-u.ac.jp

    Alpha-crystallin, a major protein of mammalian lens, consists of two subunits, alpha A-crystallin and alpha B-crystallin. They interact to form an aggregate and play a prominent role in the maintenance of lens transparency. We evaluated the interaction between these subunits via surface plasmon resonance (SPR) using four combinations of i f6a mmobilized protein and analyte: 1) AA: alpha A-crystallin was ligand immobilized onto the sensor and alpha A-crystallin was passed over the ligand, 2) AB: ligand - alpha A-crystallin, analyte - alpha B-crystallin, 3) BB: ligand - alpha B-crystallin, analyte- alpha B-crystallin, 4) BA: ligand - alpha B-crystallin, analyte - alpha A-crystallin. The order of rate of dissociation was AA approximately BA>BB approximately AB. We also examined the dissociation of gamma irradiated alpha A- and alpha B-crystallins. As radiation dose increased, so did the dissociation rate of all of the crystallins. The order of rate of dissociation of irradiated crystallins was BB>AB approximately BA>AA. The results indicate that BB is the most susceptible to gamma-irradiation and that alpha B-crystallin forms a more stable aggregate than alpha A-crystallin under normal conditions. However, when alpha B is irradiated the aggregate becomes unstable.

    Biochimica et biophysica acta 2008;1784;11;1507-13

  • Selective Cu2+ binding, redox silencing, and cytoprotective effects of the small heat shock proteins alphaA- and alphaB-crystallin.

    Ahmad MF, Singh D, Taiyab A, Ramakrishna T, Raman B and Rao ChM

    Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road, Hyderabad 500 007, India.

    Oxidative stress and Cu(2+) have been implicated in several neurodegenerative diseases and in cataract. Oxidative stress, as well as Cu(2+), is also known to induce the expression of the small heat shock proteins alpha-crystallins. However, the role of alpha-crystallins in oxidative stress and in Cu(2+)-mediated processes is not clearly understood. We demonstrate using fluorescence and isothermal titration calorimetry that alpha-crystallins (alphaA- and alphaB-crystallin and its phosphorylation mimic, 3DalphaB-crystallin) bind Cu(2+) with close to picomolar range affinity. The presence of other tested divalent cations such as Zn(2+), Mg(2+), and Ca(2+) does not affect Cu(2+) binding, indicating selectivity of the Cu(2+)-binding site(s) in alpha-crystallins. Cu(2+) binding induces structural changes and increase in the hydrodynamic radii of alpha-crystallins. Cu(2+) binding increases the stability of alpha-crystallins towards guanidinium chloride-induced unfolding. Chaperone activity of alphaA-crystallin increases significantly upon Cu(2+) binding. Alpha-crystallins rescue amyloid beta peptide, Abeta(1-40), from Cu(2+)-induced aggregation in vitro. Alpha-crystallins inhibit Cu(2+)-induced oxidation of ascorbate and, hence, prevent the generation of reactive oxygen species. Interestingly, alpha-synuclein, a Cu(2+)-binding protein, does not inhibit this oxidation process significantly. We find that the Cu(2+)-sequestering (or redox-silencing) property of alpha-crystallins confers cytoprotection. To the best of our knowledge, this is the first study to reveal high affinity (close to picomolar) for Cu(2+) binding and redox silencing of Cu(2+) by any heat shock protein. Thus, our study ascribes a novel functional role to alpha-crystallins in Cu(2+) homeostasis and helps in understanding their protective role in neurodegenerative diseases and cataract.

    Journal of molecular biology 2008;382;3;812-24

  • alphaB-crystallin is a novel predictor of resistance to neoadjuvant chemotherapy in breast cancer.

    Ivanov O, Chen F, Wiley EL, Keswani A, Diaz LK, Memmel HC, Rademaker A, Gradishar WJ, Morrow M, Khan SA and Cryns VL

    Department of Surgery, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL, 60611, USA.

    Aims: alphaB-crystallin is an anti-apoptotic protein commonly expressed in poor prognosis basal-like breast tumors, which are largely triple (estrogen receptor (ER), progesterone receptor (PR), and HER2) negative. We examined whether alphaB-crystallin expression in breast cancer was associated with a poor response to neoadjuvant (preoperative) chemotherapy.

    Methods: One hundred and twelve breast cancer patients who received neoadjuvant chemotherapy and who had post-chemotherapy tumor specimens available for analysis were included in the study. Forty-nine percent of patients were treated with doxorubicin and cyclophosphamide (AC), 37% received AC in combination with a taxane, and 14% received other regimens. Paired pre- and post-chemotherapy tumor specimens were available for 33 patients. alphaB-crystallin expression was determined by immunohistochemistry in tissue microarrays.

    Results: Seventeen percent of tumors were alphaB-crystallin positive. alphaB-crystallin expression was identical in 32 of 33 cases for which both pre- and post-chemotherapy tumor tissue was available. alphaB-crystallin expression was associated with ER-negative (P = 0.0024) and triple negative status (P = 0.005). Overall response rates (ORR) defined as > or =50% reduction in tumor size after treatment were 53% (clinical ORR) and 61% (pathological ORR). Although tumor grade, size, ER, PR, HER2 or triple negative status was not associated with response, alphaB-crystallin-positive tumors had poorer overall response rates than alphaB-crystallin-negative tumors (clinical ORR, 21% vs. 59%, respectively, P = 0.0045; pathological ORR, 16% vs. 70%, respectively, P < 0.0001).

    Conclusion: alphaB-crystallin is a novel biomarker expressed predominantly in triple negative breast tumors that identifies a subset of chemotherapy-resistant tumors, which may contribute to their poor prognosis.

    Funded by: NCI NIH HHS: R01CA097198, R21CA125181

    Breast cancer research and treatment 2008;111;3;411-7

  • Glial fibrillary acidic protein filaments can tolerate the incorporation of assembly-compromised GFAP-delta, but with consequences for filament organization and alphaB-crystallin association.

    Perng MD, Wen SF, Gibbon T, Middeldorp J, Sluijs J, Hol EM and Quinlan RA

    School of Biological and Biomedical Sciences, The University of Durham, Durham DH1 3LE, United Kingdom.

    The glial fibrillary acidic protein (GFAP) gene is alternatively spliced to give GFAP-alpha, the most abundant isoform, and seven other differentially expressed transcripts including GFAP-delta. GFAP-delta has an altered C-terminal domain that renders it incapable of self-assembly in vitro. When titrated with GFAP-alpha, assembly was restored providing GFAP-delta levels were kept low (approximately 10%). In a range of immortalized and transformed astrocyte derived cell lines and human spinal cord, we show that GFAP-delta is naturally part of the endogenous intermediate filaments, although levels were low (approximately 10%). This suggests that GFAP filaments can naturally accommodate a small proportion of assembly-compromised partners. Indeed, two other assembly-compromised GFAP constructs, namely enhanced green fluorescent protein (eGFP)-tagged GFAP and the Alexander disease-causing GFAP mutant, R416W GFAP both showed similar in vitro assembly characteristics to GFAP-delta and could also be incorporated into endogenous filament networks in transfected cells, providing expression levels were kept low. Another common feature was the increased association of alphaB-crystallin with the intermediate filament fraction of transfected cells. These studies suggest that the major physiological role of the assembly-compromised GFAP-delta splice variant is as a modulator of the GFAP filament surface, effecting changes in both protein- and filament-filament associations as well as Jnk phosphorylation.

    Funded by: NINDS NIH HHS: P01 NS042803, P01 NS042803-010004, P01NS42803

    Molecular biology of the cell 2008;19;10;4521-33

  • Bcl2L12-mediated inhibition of effector caspase-3 and caspase-7 via distinct mechanisms in glioblastoma.

    Stegh AH, Kesari S, Mahoney JE, Jenq HT, Forloney KL, Protopopov A, Louis DN, Chin L and DePinho RA

    Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.

    Glioblastoma multiforme (GBM) is a highly aggressive brain cancer that is characterized by the paradoxical features of intense apoptosis resistance yet a marked propensity to undergo necrosis. Bcl2L12 (for Bcl2-Like12) is a nuclear and cytoplasmic oncoprotein that is universally overexpressed in primary GBM and functions to block postmitochondrial apoptosis signaling by neutralizing effector caspase-3 and caspase-7 maturation. This postmitochondrial block in apoptosis engenders the alternate cell fate of cellular necrosis, thus providing a molecular explanation for GBM's classical features. Whereas Bcl2L12-mediated neutralization of caspase-7 maturation involves physical interaction, the mechanism governing Bcl2L12-mediated inhibition of caspase-3 activity is not known. The nuclear localization of Bcl2L12 prompted expression profile studies of primary astrocytes engineered to overexpress Bcl2L12. The Bcl2L12 transcriptome revealed a striking induction of the small heat shock protein alpha-basic-crystallin (alphaB-crystallin/HspB5), a link reinforced by robust alphaB-crystallin expression in Bcl2L12-expressing orthotopic glioma and strong coexpression of alphaB-crystallin and Bcl2L12 proteins in human primary GBMs. On the functional level, enforced alphaB-crystallin or Bcl2L12 expression enhances orthotopic tumor growth. Conversely, RNAi-mediated knockdown of alphaB-crystallin in Bcl2L12-expressing astrocytes and glioma cell lines with high endogenous alphaB-crystallin showed enhanced apoptosis, yet decreased necrotic cell death with associated increased caspase-3 but not caspase-7 activation. Mirroring this specific effect on effector caspase-3 activation, alphaB-crystallin selectively binds pro-caspase-3 and its cleavage intermediates in vitro and in vivo. Thus, alphaB-crystallin is a Bcl2L12-induced oncoprotein that enables Bcl2L12 to block the activation of both effector caspases via distinct mechanisms, thereby contributing to GBM pathogenesis and its hallmark biological properties.

    Funded by: NCI NIH HHS: K08 CA124804, K08CA124804, K99 CA129172, K99CA129172, P01 CA095616, P01 CA95616, R01 CA057683, R01 CA099041, R01 CA57683

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;31;10703-8

  • The clinicopathological roles of alpha-B-crystallin and p53 expression in patients with head and neck squamous cell carcinoma.

    Boslooper K, King-Yin Lam A, Gao J, Weinstein S and Johnson N

    School of Dentistry and Oral Health, Australia.

    Aims: The aim of the study was to investigate the expression of alpha-B-crystallin and p53 in head and neck squamous cell carcinoma (HNSCC).

    Methods: Alpha-B-crystallin and p53 expressions from 118 HNSCC were studied by immunohistochemistry and correlated with clinicopathological parameters.

    Results: Alpha-B-crystallin expression was seen in 28% (n = 33) of HNSCC. All except one poorly differentiated HNSCC were negative for alpha-B-crystallin. p53 expression was seen in 63% (n = 73) of HNSCC and was more common in moderately/poorly differentiated HNSCC (p = 0.034). The proportion of cases with positive staining for either alpha-B-crystallin or p53 was different in different anatomical locations in the head and neck. Patients with HNSCC having a high portion of tumour cells expressing p53 had a shorter survival than the other groups (p = 0.032).

    Conclusion: The expression of p53 and alpha-B-crystallin were related to the differentiation and site of the HNSCC. Alpha-B-crystallin was not a prognostic marker for HNSCC.

    Pathology 2008;40;5;500-4

  • Confocal fluorescence microscopy study of interaction between lens MIP26/AQP0 and crystallins in living cells.

    Liu BF and Liang JJ

    Ophthalmic Research/Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

    MIP26/AQP0 is the major lens fiber membrane protein and has been reported to interact with many other lens components including crystallins, lipid, and cytoskeletal proteins. Regarding crystallins, many previous reports indicate that MIP26/AQP0 interacts with either only alpha-crystallin or some specific gamma-crystallins. Considering the possibly important role of MIP26/AQP0 in the reduction of light scattering in the lenses, we have further investigated its interaction with crystallins using confocal fluorescence resonance energy transfer (FRET) microscopy. Specifically, we used MIP26 tagged with a green fluorescence protein (GFP) as a donor and a crystallin (alphaA-, alphaB-, betaB2-, or gammaC-crystallin) tagged with a red fluorescence protein (RFP) as an acceptor. The two plasmids were cotransfected to HeLa cells. After culture, laser scattering microscopy images were taken in each of the three channels: GFP, RFP, and FRET. The net FRET images were then obtained by removing the contribution of spectral bleed-through. The pixels of net FRET were normalized with those of GFP. The results show the presence of measurable interactions between MIP26 and all crystallins, with the extent of interactions decreasing from alphaA- and alphaB-crystallin to betaB2- and gammaC-crystallin. Competitive interaction study using untagged alphaA-crystallin shows decreased net FRET, indicating specificity of the interactions between MIP26 and alphaA-crystallin. We conclude that all crystallins interact with MIP26, the physiological significance of which may be a reduction in the difference of refractive index between membrane and cytoplasm.

    Funded by: NEI NIH HHS: EY13968

    Journal of cellular biochemistry 2008;104;1;51-8

  • Small heat shock proteins Hsp27 or alphaB-crystallin and the protein components of neurofibrillary tangles: tau and neurofilaments.

    Björkdahl C, Sjögren MJ, Zhou X, Concha H, Avila J, Winblad B and Pei JJ

    Karolinska Institutet, Department of Neurobiology, Care Sciences and Society, KI-Alzheimer's Disease Research Center, Novum, Huddinge, Sweden.

    The heat-shock proteins (HSPs) Hsp27 and alphaB-crystallin are up-regulated in Alzheimer's disease (AD), but the extent of this and the consequences are still largely unknown. The HSPs are involved in protein degradation and protection against protein aggregation, and they interact with several cytoskeletal components such as microtubules (MT) and neurofilaments (NF). AD pathology includes aggregated proteins (tau, NF), decreased protein degradation, and cytoskeletal disruption. It is thus of interest to investigate more closely the possible roles of the HSPs in AD pathology. The expressions of Hsp27 and alphaB-crystallin in AD brain samples were significantly increased (by approximately 20% and approximately 30%, respectively) and correlated significantly with phosphorylated tau and NF proteins. To investigate the consequences of increased HSP levels on tau and NF regulation, N2a cells were transfected with Hsp27 or alphaB-crystallin constructs, and overexpression of the HSPs was confirmed in the cells. Increased tau phosphorylation at the Ser262 site in the N2a cells was regulated by Hsp27 overexpression (possibly through p70S6k), whereas the overexpression of alphaB-crystallin resulted in decreased levels of phosphorylated tau, NF, and GSK-3beta. It was also shown that overexpression of HSPs causes an increase in the percentage of cells present in the G(1) phase. The results presented su 1f40 ggest that a cellular defense against dysregulated proteins, in the form of Hsp27 and alphaB-crystallin, might contribute to the cell cycle reentry seen in AD cells. Furthermore, Hsp27 might also be involved in AD pathology by aggravating MT disruption by tau phosphorylation.

    Journal of neuroscience research 2008;86;6;1343-52

  • Cataract mutation P20S of alphaB-crystallin impairs chaperone activity of alphaA-crystallin and induces apoptosis of human lens epithelial cells.

    Li H, Li C, Lu Q, Su T, Ke T, Li DW, Yuan M, Liu J, Ren X, Zhang Z, Zeng S, Wang QK and Liu M

    Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology and Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, P.R. China.

    Cataract is a common cause of childhood blindness worldwide. alpha-crystallin, which is comprised of two homologous subunits, alphaA- and alphaB-crystallin, plays a key role in the maintenance of lens transparency. Recently, we have identified a missense mutation in alphaB-crystallin that changes the proline residue at codon 20 to a serine residue (P20S) in a large Chinese family with autosomal dominant posterior polar congenital cataract. To explore the molecular mechanism by which the P20S mutation causes cataract, we examined the quaternary structure, subunit exchange and chaperone activity of the reconstituted heteroaggregates of alpha-crystallins containing wild type (WT) alphaA in combination with either WT-alphaB- or mutant alphaB-crystallin, respectively. Compared with heteroaggregates of WT-alphaA and WT-alphaB, heteroaggregates containing WT-alphaA and mutant alphaB showed nearly the same molecular mass, but the subunit-exchange rate and chaperone activity were decreased markedly. In human lens epithelial cells, unlike WT-alphaB-crystallin, the P20S mutant protein showed abnormal nuclear localization, and unusual ability to trigger apoptosis. These results suggest that the changes in the structure and function of the alpha-crystallin complex and cytotoxicity are vital factors in the pathogenesis of congenital cataract linked to the P20S mutation in the alphaB-crystallin.

    Funded by: NEI NIH HHS: R01 EY015765

    Biochimica et biophysica acta 2008;1782;5;303-9

  • alpha B-crystallin is a cytoplasmic interaction partner of the kidney-specific cadherin-16.

    Thedieck C, Kalbacher H, Kratzer U, Lammers R, Stevanovic S and Klein G

    Section for Transplantation Immunology and Immunohematology, Center for Medical Research, University Medical Clinic, Waldhörnlestrasse 22, D-72072 Tübingen, Germany.

    The Ca(2+)-dependent membrane-spanning classical cadherins bind directly to cytosolic catenins. This cadherin-catenin interaction is known to be critical for the fundamental role of cadherins in cell-cell adhesion. The small subfamily of the 7D-cadherins, however, cannot interact with catenins due to their highly truncated cytoplasmic tail. Thus far, no cytoplasmic interaction partner for the 7D-cadherins has been described. With the use of the cytoplasmic domain of the Ksp (kidney-specific)-cadherin, which belongs to the family of 7D-cadherins, as bait in affinity chromatography with human kidney lysates, the small heat-shock protein alpha B-crystallin was identified by matrix-assisted laser desorption/ionization-time-of-flight analysis as a cytosolic binding partner of Ksp-cadherin. This interaction was verified by co-immunoprecipitation analysis. With the use of overlapping peptides representing the entire alpha B-crystallin molecule, the N-terminal part of alpha B-crystallin, which does not possess chaperone activity, was identified as responsible for the binding to Ksp-cadherin. This interaction was found to be specific since only the cytoplasmic domain of Ksp-cadherin, but not LI (liver-intestine)-cadherin (another member of the 7D-cadherin family), interacted with alpha B-crystallin. In the human kidney, both alpha B-crystallin and Ksp-cadherin co-localize to cells of the collecting duct. They also co-localize with the actin cytoskeleton and co-precipitate with the latter. These findings suggest that the interaction of Ksp-cadherin with alpha B-crystallin is important for the connection of Ksp-cadherin to the cytoskeleton and thus for maintaining tissue integrity in the kidney.

    Journal of molecular biology 2008;378;1;145-53

  • Truncation of alphaB-crystallin by the myopathy-causing Q151X mutation significantly destabilizes the protein leading to aggregate formation in transfected cells.

    Hayes VH, Devlin G and Quinlan RA

    School of Biological and Biomedical Sciences, South Road Science Site, Durham University, Durham DH1 3LE.

    Here we investigate the effects of a myopathy-causing mutation in alphaB-crystallin, Q151X, upon its structure and function. This mutation removes the C-terminal domain of alphaB-crystallin, which is expected to compromise both its oligomerization and chaperone activity. We compared this to two other alphaB-crystallin mutants (450delA, 464delCT) and also to a series of C-terminal truncations (E164X, E165X, K174X, and A171X). We find that the effects of the Q151X mutation were not always as predicted. Specifically, we have found that although the Q151X mutation decreased oligomerization of alphaB-crystallin and even increased some chaperone activities, it also significantly destabilized alphaB-crystallin causing it to self-aggregate. This conclusion was supported by our analyses of both the other disease-causing mutants and the series of C-terminal truncation constructs of alphaB-crystallin. The 450delA and 464delCT mutants could only be refolded and assayed as a complex with wild type alphaB-crystallin, which was not the case for Q151X alphaB-crystallin. From these studies, we conclude that all three disease-causing mutations (450delA, 464delCT, and Q151X) in the C-terminal extension destabilize alphaB-crystallin and increase its tendency to self-aggregate. We propose that it is this, rather than a catastrophic loss of chaperone activity, which is a major factor in the development of the reported diseases for the three disease-causing mutations studied here. In support of this hypothesis, we show that Q151X alphaB-crystallin is found mainly in the insoluble fraction of cell extracts from transient transfected cells, due to the formation of cytoplasmic aggregates.

    Funded by: Biotechnology and Biological Sciences Research Council; NINDS NIH HHS: P01 NS042803, P01 NS042803-010004, P01NS42803

    The Journal of biological chemistry 2008;283;16;10500-12

  • Anti-chaperone betaA3/A1(102-117) peptide interacting sites in human alphaB-crystallin.

    Rao G and Sharma KK

    Department of Ophthalmology, University of Missouri-Columbia, Columbia, MO 65212, USA.

    Purpose: Our previous work identified 23 low molecular weight (<3.5 kDa) crystallin peptides in the urea-soluble fractions of normal young, normal aged, and aged cataract human lenses. We found that one of these crystallin fragments, betaA3/A1(102-117) peptide (SDAYHIERLMSFRPIC), that are present in aged and cataract lens, increased the scattering of light by beta- and gamma-crystallins and alcohol dehydrogenase (ADH) and also reduced the chaperone-like activity of alphaB-crystallin. The present study was performed to identify the interacting sites of the betaA3/A1(102-117) peptide in alphaB-crystallin.

    Methods: betaA3/A1(102-117) peptide was first derivatized with sulfo-succinimidyl-2-[6-(biotinamido)-2-{p-azidobenzamido}-hexanoamido] ethyl-1-3 dithio propionate (sulfo-SBED), a photoactivable, heterotrifunctional biotin-containing cross-linker. The biotin-derivatized peptide was then incubated with alphaB-crystallin at 37 degrees C for 2 h to allow complex formation followed by photolysis to facilitate the transfer of the biotin label from the peptide to alphaB-crystallin. Label transfer was confirmed by western blot, and t 852 he labeled alphaB-crystallin was digested with trypsin. Tryptic peptides from alphaB-crystallin carrying the biotin label were purified by avidin affinity chromatography, and betaA3/A1(102-117) peptide interacting sites in alphaB-crystallin were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and nanospray quadrupole time-of-flight mass spectrometry (QqTOF MS/MS).

    Results: We found that the betaA3/A1(102-117) peptide interacted with alphaB-crystallin regions (70)LEKDR(74), (83)HFSPEELKVK(92), (91)VKVLGDVIEVHGK(103), (93)VLGDVIEVHGKHEER(107), and (121)KYR(123), which are part of the alpha-crystallin domain, and were previously shown to be part of the functional chaperone site in alphaB-crystallin. The betaA3/A1(102-117) peptide also interacted with regions at the COOH-terminal extension of alphaB-crystallin, (150)KQVSGPER(157), (164)EEKPAVTAAPK(174), and (164)EEKPAVTAAPKK(175). When two of the hydrophobic residues of betaA3/A1(102-117) peptide were replaced with hydrophilic residues, the resulting substituted peptide, SDADHGERLMSFRPIC, did not show the anti-chaperone property.

    Conclusions: This study confirmed the interactions between a low molecular weight peptide derived from betaA3/A1-crystallin found in aged and cataract lenses and alphaB-crystallin. The binding of betaA3/A1(102-117) peptide to the chaperone site and the COOH-terminal extension of alphaB-crystallin may explain its anti-chaperone property.

    Funded by: NEI NIH HHS: EY09855, EY11981, EY14795, R01 EY009855, R01 EY011981, R24 EY014795

    Molecular vision 2008;14;666-74

  • alphaB-crystallin promotes tumor angiogenesis by increasing vascular survival during tube morphogenesis.

    Dimberg A, Rylova S, Dieterich LC, Olsson AK, Schiller P, Wikner C, Bohman S, Botling J, Lukinius A, Wawrousek EF and Claesson-Welsh L

    Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden. Anna.Dimberg@genpat.uu.se

    Selective targeting of endothelial cells in tumor vessels requires delineation of key molecular events in formation and survival of blood vessels within the tumor microenvironment. To this end, proteins transiently up-regulated during vessel morphogenesis were screened for their potential as targets in antiangiogenic tumor therapy. The molecular chaperone alphaB-crystallin was identified as specifically induced with regard to expression level, modification by serine phosphorylation, and subcellular localization during tubular morphogenesis of endothelial cells. Small interfering RNA-mediated knockdown of alphaB-crystallin expression did not affect endothelial proliferation but led to attenuated tubular morphogenesis, early activation of proapoptotic caspase-3, and increased apoptosis. alphaB-crystallin was expressed in a subset of human tumor vessels but not in normal capillaries. Tumors grown in alphaB-crystallin(-/-) mice were significantly less vascularized than wild-type tumors and displayed increased areas of apoptosis/necrosis. Importantly, tumor vessels in alphaB-crystallin(-/-) mice were leaky and showed signs of caspase-3 activation and extensive apoptosis. Ultrastructural analyses showed defective vessels partially devoid of endothelial lining. These data strongly implicate alphaB-crystallin as an important regulator of tubular morphogenesis and survival of endothelial cell during tumor angiogenesis. Hereby we identify the small heat shock protein family as a novel class of angiogenic modulators.

    Blood 2008;111;4;2015-23

  • UV-A-induced structural and functional changes in human lens deamidated alphaB-crystallin.

    Mafia K, Gupta R, Kirk M, Wilson L, Srivastava OP and Barnes S

    Department of Pharmacology, University of Alabama at Birmingham, Birmingham, AL 35226, USA.

    Purpose: To determine comparative effects of ultraviolet (UV)-A irradiation on structural and functional properties of wild type (WT) alphaB-crystallin and its three deamidated mutant proteins (alphaB-Asn78Asp, alphaB-Asn146Asp, and alphaB-Asn78/146Asp).

    Methods: Three deamidated mutants previously generated from recombinant WT alphaB-crystallin, using a site-specific mutagenesis procedure as previously described [32], were used. The WT alphaB-crystallin and its three deamidated species were exposed to UV-A light (320-400 nm) at intensities of 20 or 50 J/cm(2). The UV-A-unexposed and UV-A-exposed preparations were examined for their chaperone activity, and their activities were correlated with the UV-A-induced structural changes. The structural properties studied included dimerization and degradation, intrinsic tryptophan (Trp) fluorescence, ANS (8-anilino-1-naphthalenesulfate)-binding, far ultraviolet circular dichroism (UV-CD) spectral analysis, molecular sizes by dynamic light scattering, and oxidation of Trp and methionine (Met) residues.

    Results: The WT alphaB-crystallin and its three deamidated mutant proteins showed enhanced dimerization to 40 kDa species and partial degradation with increasing doses during UV-A-exposure. Compared to the deamidation of asparagines (Asn) 78 residue to aspartic acid (Asp) or both Asn78 and Asn146 residues to Asp, the deamidation of Asn146 residue to Asp resulted in a greater loss of chaperone activity. The UV-A-induced loss of chaperone activity due to structural changes was studied. The ANS-binding data suggested that the alphaB-Asn146Asp mutant protein had a relatively compact structure and an increase in surface hydrophobic patches compared to WT and two other deamidated proteins. Similarly, UV-A-exposure altered the Trp microenvironment in the deamidated mutant proteins compared to the WT alphaB-crystallin. Far-UV CD spectral analyses showed almost no changes among WT and deamidated species on UV-A-exposure except that the alphaB-Asn146Asp mutant protein showed maximum changes in the random coil structure relative to WT alphaB-crystallin and two other deamidated proteins. The UV-A-exposure also resulted in the aggregation of WT and the three deamidated mutant proteins with species of greater mass compared to the non-UV-A exposed species. Among the four spots recovered after two-dimensional (2D)-gel electrophoresis from WT and the three deamidated species, the Met and Trp residues of alphaB-Asn146Asp mutant showed maximum oxidation after UV-A exposure, which might account for its greater loss in chaperone activity compared to WT alphaB-crystallin and two other deamidated species.

    Conclusions: After UV-A-exposure, the deamidated alphaB-Asn146Asp mutant protein showed a complete loss of chaperone activity compared to WT alphaB and alphaB-Asn78Asp and alphaB-Asn78/146Asp deamidated species. Apparently, this loss of chaperone activity was due to oxidative changes leading to its greater structural alteration compared to other alphaB-species.

    Funded by: NCCIH NIH HHS: P50 AT000477, P50 AT00477; NCI NIH HHS: P30 CA013148, P30 CA13148; NCRR NIH HHS: S10 RR011329, S10 RR13795; NEI NIH HHS: EY-6400, R01 EY006400

    Molecular vision 2008;14;234-48

  • AlphaB-crystallin: a novel marker of invasive basal-like and metaplastic breast carcinomas.

    Sitterding SM, Wiseman WR, Schiller CL, Luan C, Chen F, Moyano JV, Watkin WG, Wiley EL, Cryns VL and Diaz LK

    Department of Pathology, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

    Basal-like tumors are a newly recognized estrogen receptor (ER) negative and HER2 negative breast cancer subtype that express basal epithelial genes and are associated with poor survival. Metaplastic carcinomas are thought to belong within the basal-like group. We have recently demonstrated that the small heat shock protein alphaB-crystallin is commonly expressed in basal-like tumors and contributes to their aggressive phenotype. The current study examined the rates and patterns of alphaB-crystallin expression in whole tissue sections of human breast, including normal tissue, proliferative lesions, in situ and invasive carcinomas (ER positive, HER2 positive, basal-like, and metaplastic cancers). In normal breast tissue, proliferative lesions and in situ carcinomas, alphaB-crystallin expression was restricted to the myoepithelial cell compartment of ductal and lobular units. Most basal-like and metaplastic carcinomas demonstrated cytoplasmic expression of alphaB-crystallin (81% and 86%, respectively). Conversely, no staining for alphaB-crystallin was observed in nonbasal-like (ie, ER positive or HER2 positive) breast carcinomas. Taken together, our results indicate that alphaB-crystallin is a sensitive (81%) and specific (100%) marker for basal-like breast carcinomas. Moreover, the high rates of expression of alphaB-crystallin in metaplastic breast carcinomas (86%) suggest that these tumors may represent a histologically distinctive subset of basal-like breast tumors with a similar underlying molecular etiology.

    Annals of diagnostic pathology 2008;12;1;33-40

  • Zn2+ enhances the molecular chaperone function and stability of alpha-crystallin.

    Biswas A and Das KP

    Protein Chemistry Laboratory, Department of Chemistry, Bose Institute, 93/1 APC Road, Kolkata 700 009, India.

    Alpha-crystallin, the major eye lens protein, is a molecular chaperone that plays a crucial role in the suppression of protein aggregation and thus in the long-term maintenance of lens transparency. Zinc is a m 1f40 icronutrient of the eye, but its molecular interaction with alpha-crystallin has not been studied in detail. In this paper, we present results of in vitro experiments that show bivalent zinc specifically interacts with alpha-crystallin with a dissociation constant in the submillimolar range (Kd approximately 0.2-0.4 mM). We compared the effect of Zn2+ with those of Ca2+, Cu2+, Mg2+, Cd2+, Pb2+, Ni2+, Fe2+, and Co2+ at 1 mM on the structure and chaperoning ability of alpha-crystallin. An insulin aggregation assay showed that among the bivalent metal ions, only 1 mM Zn2+ improved the chaperone function of alpha-crystallin by 30% compared to that in the absence of bivalent metal ions. Addition of 1 mM Zn2+ increased the yield of alpha-crystallin-assisted refolding of urea-treated LDH to its native state from 33 to 38%, but other bivalent ions had little effect. The surface hydrophobicity of alpha-crystallin was increased by 50% due to the binding of Zn2+. In the presence of 1 mM Zn2+, the stability of alpha-crystallin was enhanced by 36 kJ/mol, and it became more resistant to tryptic cleavage. The implications of enhanced stability and molecular chaperone activity of alpha-crystallin in the presence of Zn2+ are discussed in terms of its role in the long-term maintenance of lens transparency and cataract formation.

    Biochemistry 2008;47;2;804-16

  • C-Terminal truncation affects subunit exchange of human alphaA-crystallin with alphaB-crystallin.

    Kallur LS, Aziz A and Abraham EC

    Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Biomedical Research Center I B405C-Slot 516, Little Rock, AR 72205, USA.

    In human lenses, C-terminal cleavage of alphaA-crystallin at residues 172,168, and 162 have been reported. The effect of C-terminal truncation of alphaA-crystallin on subunit exchange and heterooligomer formation with alphaB-crystallin and homooligomer formation with native alphaA-crystallin is not known. We have conducted fluorescence resonance energy transfer studies which have shown that the rates of subunit exchange of alphaA(1-172 )and alphaA(1-168 )with alphaB-wt were two-fold lower than for alphaA-wt interacting with alphaB-wt. The subunit exchange rate between alphaA(1-162) and alphaB-wt was six-fold lower. These data suggest that cleavage of the C-terminal residues could significantly affect heterooligomerization. On the other hand, the subunit exchange rates between alphaA-wt and the truncated alphaA-crystallins were either unchanged or only slightly decreased, which suggest that homooligomerization may not be significantly influenced by C-terminal truncation. The main conclusion from this study is that cleavage of C-terminal residues of alphaA-crystallin including the nine residues of the flexible tail is expected to significantly affect the formation of heteroaggregates. Reconstitution experiments showed that the presence of an intact C-terminus is essential for the formation of fully integrated heteroaggregates with equal proportion of alphaA and alphaB subunits.

    Funded by: NEI NIH HHS: EY11352

    Molecular and cellular biochemistry 2008;308;1-2;85-91

  • Contraction in human myometrium is associated with changes in small heat shock proteins.

    MacIntyre DA, Tyson EK, Read M, Smith R, Yeo G, Kwek K and Chan EC

    Mothers and Babies Research Centre, The University of Newcastle, John Hunter Hospital, 1 Lookout Road, New Lambton Heights, Newcastle 2305, Australia.

    The myometrium undergoes substantial remodeling at the time of labor including rearrangement of the cellular contractile machinery. The regulation of this process in human myometrium at the time of labor is poorly defined, but evidence in other muscle types suggests modulation by small heat shock proteins (sHSP). The aim of this study was to investigate whether similar changes in sHSP occur in the myometrium at labor. Using a quantitative proteomic approach (two-dimensional difference gel electrophoresis), we found a 69% decrease in the sHSP alphaB-crystallin in the myometrium at labor plus multiple isoforms of HSP27. Immunoblotting using phosphospecific HSP27 antibodies (HSP27-serine15, -78, and -82) detected marked changes in HSP27 phosphorylation at labor. Although total HSP27 levels were unchanged, HSP27-Ser15 was 3-fold higher at labor. Coimmunoprecipitation studies showed that HSP27 coprecipitates with alphaB-crystallin and also smooth muscle alpha-actin. Coimmunofluorescence studies demonstrated a relocation of HSP27 from the perinuclear region to the actin cytoskeleton at labor. The functional significance of these changes was demonstrated in vitro where myometrial strips stimulated to contract with oxytocin exhibited increased HSP27-Ser15 phosphorylation. Our findings provide data consistent with a novel pathway regulating human myometrial contraction at labor and identify HSP27 and alphaB-crystallin as potential targets for future tocolytic design.

    Endocrinology 2008;149;1;245-52

  • Interactive sequences in the molecular chaperone, human alphaB crystallin modulate the fibrillation of amyloidogenic proteins.

    Ghosh JG, Houck SA and Clark JI

    Department of Biological Structure, University of Washington, Seattle, WA 98195, USA. jghosh@rics.bwh.harvard.edu

    Multiple interactive domains are involved in the activity of the stress protein, alphaB crystallin that protects against the unfolding, aggregation, and toxicity of amyloidogenic proteins. Six peptides corresponding to the interactive sequences 41STSLSPFYLRPPSFLRAP58, 73DRFSVNLDVKHFS85, 101HGKHEERQDE110, 113FISREFHR120, 131LTITSSLSSDGV142, and 156ERTIPITRE164 in human alphaB crystallin were synthesized and evaluated in Thioflavin T fluorescence assays for their effects on the modulation of fibrillation of four disease-related amyloidogenic proteins: amyloid-beta, alpha-synuclein, transthyretin, and beta2-microglobulin. The 73DRFSVNLDVKHFS85 and 101HGKHEERQDE110 peptides in the conserved alpha crystallin core domain of alphaB crystallin were the most effective fibril inhibitors. 73DRFSVNLDVKHFS85 completely inhibited alpha-synuclein fibrillation and reduced the fibrillation of amyloid-beta, transthyretin, and beta2-microglobulin by >50%. 101HGKHEERQDE110 completely inhibited amyloid-beta fibrillation and reduced the fibrillation of alpha-synuclein, transthyretin, and beta2-microglobulin by >50%. The peptides FSVN, NLDV, HGKH, and HEER, which are synthetic fragments of 73DRFSVNLDVKHFS85 and 101HGKHEERQDE110, inhibited fibrillation of all four amyloidogenic proteins by >75%. In contrast, the peptides FISREFHR, ERTIPITRE, DRFS, KHFS, and EERQ were the strongest promoters of fibrillation. Molecular modeling of the interactions between transthyretin and beta2-microglobulin and the synthetic bioactive peptides determined that residues Phe-75, Ser-76, Val-77, Asn-78, Leu-79, and Asp-80 in 73DRFSVNLDVKHFS85 and residues His-101, Lys-103, His-104, Glu-105, and Arg-107 in 101HGKHEERQDE110 interact with exposed residues in the beta strands, F and D of transthyretin and beta2-microglobulin, respectively, to modulate fibrillation. This is the first characterization of specific bioactive peptides synthesized on the basis of interactive domains in the small heat shock protein, alphaB crystallin that protect against the fibrillation of amyloidogenic proteins.

    Funded by: NEI NIH HHS: EY04542, R01 EY004542, R01 EY004542-27

    The international journal of biochemistry & cell biology 2008;40;5;954-67

  • Role of alphaBI5 and alphaBT162 residues in subunit interaction during oligomerization of alphaB-crystallin.

    Murugesan R, Santhoshkumar P and Sharma KK

    Department of Ophthalmology, University of Missouri, Columbia, MO 65212, USA.

    Purpose: To determine whether the residues in the NH(2)- and COOH-terminal extensions interact with one another during oligomerization of alphaB-crystallin.

    Methods: Site-directed mutagenesis was used to mutate alphaBI5 and alphaBT162 residues to Cys. The recombinant I5C and T162C proteins were expressed in Escherichia coli cells and purified using chromatographic techniques. These proteins were analyzed by SDS-PAGE and mass spectrometry and characterized by multi-angle light scattering and circular dichroism (CD) spectroscopy methods. Fluorescence resonance energy transfer (FRET) assay was used to determine the interaction between the subunits.

    Results: Dimer formation was observed in both alphaBI5C and alphaBT162C in storage at 4 degrees C. During air oxidation at room temperature, alphaBT162C formed dimers to a greater extent than alphaBI5C. The average molar masses, secondary structures, and chaperone-like activities of the reduced forms of I5C and T162C were comparable to that of wild type alphaB-crystallin. The oligomeric assembly of reduced forms of I5C and T162C appeared homogenous under JEOL 1200EX Electron microscope whereas the oxidized proteins appeared as irregular aggregates. FRET assay demonstrated interactions between alphaBI5C-alphaBI5C and alphaBT162C-alphaBT162C. However, there was no evidence of an interaction between alphaBI5C and alphaBT162C residues during oligomerization.

    Conclusions: This study suggests that residues from the NH(2)- and COOH-terminal regions in alphaB-crystallin interact with residues from the corresponding regions of another subunit, but there exists no interaction between the residues at the COOH-terminal extension region and the residues at the NH(2)-terminal region.

    Funded by: NEI NIH HHS: EY 11981, EY14795, R01 EY011981, R24 EY014795

    Molecular vision 2008;14;1835-44

  • Monitoring the prevention of amyloid fibril formation by alpha-crystallin. Temperature dependence and the nature of the aggregating species.

    Rekas A, Jankova L, Thorn DC, Cappai R and Carver JA

    Department of Chemistry, University of Wollongong, Australia.

    The molecular chaperone, alpha-crystallin, has the ability to prevent the fibrillar aggregation of proteins implicated in human diseases, for example, amyloid beta peptide and alpha-synuclein. In this study, we examine, in detail, two aspects of alpha-crystallin's fibril-suppressing ability: (a) its temperature dependence, and (b) the nature of the aggregating species with which it interacts. First, the efficiency of alpha-crystallin to suppress fibril formation in kappa-casein and alpha-synuclein increases with temperature, despite their rate of fibrillation also increasing in the absence of alpha-crystallin. This is consistent with an increased chaperone ability of alpha-crystallin at higher temperatures to protect target proteins from amorphous aggregation [GB Reddy, KP Das, JM Petrash & WK Surewicz (2000) J Biol Chem275, 4565-4570]. Second, dual polarization interferometry was used to monitor real-time alpha-synuclein aggregation in the presence and absence of alphaB-crystallin. In contrast to more common methods for monitoring the time-dependent formation of amyloid fibrils (e.g. the binding of dyes like thioflavin T), dual polarization interferometry data did not reveal any initial lag phase, generally attributed to the formation of prefibrillar aggregates. It was shown that alphaB-crystallin interrupted alpha-synuclein aggregation at its earliest stages, most likely by binding to partially folded monomers and thereby preventing their aggregation into fibrillar structures.

    The FEBS journal 2007;274;24;6290-304

  • Site-directed mutations in the C-terminal extension of human alphaB-crystallin affect chaperone function and block amyloid fibril formation.

    Treweek TM, Ecroyd H, Williams DM, Meehan S, Carver JA and Walker MJ

    Department of Chemistry, University of Wollongong, Wollongong, New South Wales, Australia.

    Background: Alzheimer's, Parkinson's and Creutzfeldt-Jakob disease are associated with inappropriate protein deposition and ordered amyloid fibril assembly. Molecular chaperones, including alphaB-crystallin, play a role in the prevention of protein deposition.

    A series of site-directed mutants of the human molecular chaperone, alphaB-crystallin, were constructed which focused on the flexible C-terminal extension of the protein. We investigated the structural role of this region as well as its role in the chaperone function of alphaB-crystallin under different types of protein aggregation, i.e. disordered amorphous aggregation and ordered amyloid fibril assembly. It was found that mutation of lysine and glutamic acid residues in the C-terminal extension of alphaB-crystallin resulted in proteins that had improved chaperone activity against amyloid fibril forming target proteins compared to the wild-type protein.

    Together, our results highlight the important role of the C-terminal region of alphaB-crystallin in regulating its secondary, tertiary and quaternary structure and conferring thermostability to the protein. The capacity to genetically modify alphaB-crystallin for improved ability to block amyloid fibril formation provides a platform for the future use of such engineered molecules in treatment of diseases caused by amyloid fibril formation.

    PloS one 2007;2;10;e1046

  • HMGA1 mediates the activation of the CRYAB promoter by BRG1.

    Duncan B and Zhao K

    Laboratory of Molecular Immunology, NHLBI, NIH, Bethesda, Maryland 20892, USA.

    alphaB-Crystallin (CRYAB) is a small heat-shock protein that is implicated in many cellular processes, such as transcription and differentiation, as well as pathologic process. It is expressed at high levels in vertebrate eye lens and at low levels in a variety of other cell types. We previously identified CRYAB as a target gene of the chromatin-remodeling SWI/SNF-like Brg or hBrm-associated factors (BAF) complexes. In this report, we identify a 30 bp DNA element required for mediating the activation of CRYAB by brahma-related gene 1 (BRG1). This BRG1-response element is located at the edge of a positioned nucleosome immediately upstream of the transcription initiation site. An AT-rich sequence within this region is bound by the high-mobility group AT-hook 1 (HMGA1) proteins in vitro and in vivo. We demonstrate that the HMGA1 target sequences and HMGA1 proteins are required for the maximal activation of the CRYAB promoter by BRG1. Our data indicate that HMGA1 nonhistone chromatin proteins, the SWI/SNF chromatin remodeling complexes, and sequence-specific transcription factors act together to regulate the expression of the CRYAB gene.

    Funded by: Intramural NIH HHS

    DNA and cell biology 2007;26;10;745-52

  • Characterisation of amyloid fibril formation by small heat-shock chaperone proteins human alphaA-, alphaB- and R120G alphaB-crystallins.

    Meehan S, Knowles TP, Baldwin AJ, Smith JF, Squires AM, Clements P, Treweek TM, Ecroyd H, Tartaglia GG, Vendruscolo M, Macphee CE, Dobson CM and Carver JA

    Department of Chemistry, University of Cambridge, Cambridge, UK.

    AlphaB-Crystallin is a ubiquitous small heat-shock protein (sHsp) renowned for its chaperone ability to prevent target protein aggregation. It is stress-inducible and its up-regulation is associated with a number of disorders, including those linked to the deposition of misfolded proteins, such as Alzheime 1f40 r's and Parkinson's diseases. We have characterised the formation of amyloid fibrils by human alphaB-crystallin in detail, and also that of alphaA-crystallin and the disease-related mutant R120G alphaB-crystallin. We find that the last 12 amino acid residues of the C-terminal region of alphaB-crystallin are predicted from their physico-chemical properties to have a very low propensity to aggregate. (1)H NMR spectroscopy reveals that this hydrophilic C-terminal region is flexible both in its solution state and in amyloid fibrils, where it protrudes from the fibrillar core. We demonstrate, in addition, that the equilibrium between different protofilament assemblies can be manipulated and controlled in vitro to select for particular alphaB-crystallin amyloid morphologies. Overall, this study suggests that there could be a fine balance in vivo between the native functional sHsp state and the formation of amyloid fibrils.

    Funded by: Medical Research Council: G0200653; Wellcome Trust

    Journal of molecular biology 2007;372;2;470-84

  • Residue R120 is essential for the quaternary structure and functional integrity of human alphaB-crystallin.

    Simon S, Michiel M, Skouri-Panet F, Lechaire JP, Vicart P and Tardieu A

    EA 300, Université Paris 7, Case 7136, 75251 Paris Cedex 05, France. stef.labo@gmail.com

    The missense mutation Arg-120 to Gly (R120G) in the human alphaBeta-crystallin sequence has been reported to be associated with autosomal dominant myopathy, cardiomyopathy, and cataract. Previous studies of the mutant showed a significant ability to aggregate in cultured cells and an increased oligomeric size coupled to an important loss of the chaperone-like activity in vitro. The aim of this study was to further analyze the role of the R120 residue in the structural and functional properties of alphaBeta-crystallin. The following mutants were generated, Arg-120 to Gly (R120G), Cys (R120C), Lys (R120K), and Asp (R120D). In cellulo, after expression in two cultured cell lines, NIH-3T3 and Cos-7, the capacity of the wild-type and mutant crystallins to aggregate was evaluated and the protein location was determined by immunofluorescence. In vitro, the wild-type and mutant crystallins were expressed in Escherichia coli cells, purified by size exclusion chromatography, and characterized using dynamic light scattering, electron microscopy, and chaperone-like activity assays. Aggregate sizes in cellulo and in vitro were analyzed. The whole of the data showed that the preservation of an Arg residue at position 120 of alphaBeta-crystallin is critical for the structural and functional integrity of the protein and that each mutation results in specific changes in both structural and functional characteristics.

    Biochemistry 2007;46;33;9605-14

  • Human alpha B-crystallin mutation causes oxido-reductive stress and protein aggregation cardiomyopathy in mice.

    Rajasekaran NS, Connell P, Christians ES, Yan LJ, Taylor RP, Orosz A, Zhang XQ, Stevenson TJ, Peshock RM, Leopold JA, Barry WH, Loscalzo J, Odelberg SJ and Benjamin IJ

    Center for Cardiovascular Translational Biomedicine, Division of Cardiology, Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT 84132, USA.

    The autosomal dominant mutation in the human alphaB-crystallin gene inducing a R120G amino acid exchange causes a multisystem, protein aggregation disease including cardiomyopathy. The pathogenesis of cardiomyopathy in this mutant (hR120GCryAB) is poorly understood. Here, we show that transgenic mice overexpressing cardiac-specific hR120GCryAB recapitulate the cardiomyopathy in humans and find that the mice are under reductive stress. The myopathic hearts show an increased recycling of oxidized glutathione (GSSG) to reduced glutathione (GSH), which is due to the augmented expression and enzymatic activities of glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase, and glutathione peroxidase. The intercross of hR120GCryAB cardiomyopathic animals with mice with reduced G6PD levels rescues the progeny from cardiac hypertrophy and protein aggregation. These findings demonstrate that dysregulation of G6PD activity is necessary and sufficient for maladaptive reductive stress and suggest a novel therapeutic target for abrogating R120GCryAB cardiomyopathy and heart failure in humans.

    Funded by: NHLBI NIH HHS: 5R01 HL63874, R01 HL063834, R01 HL063834-01A1; NIH HHS: DP1 OD006438

    Cell 2007;130;3;427-39

  • Protective and therapeutic role for alphaB-crystallin in autoimmune demyelination.

    Ousman SS, Tomooka BH, van Noort JM, Wawrousek EF, O'Connor KC, Hafler DA, Sobel RA, Robinson WH and Steinman L

    Department of Neurology and Neurological Sciences, Stanford University. Stanford, California 94305, USA.

    alphaB-crystallin (CRYAB) is the most abundant gene transcript present in early active multiple sclerosis lesions, whereas such transcripts are absent in normal brain tissue. This crystallin has anti-apoptotic and neuroprotective functions. CRYAB is the major target of CD4+ T-cell immunity to the myelin sheath from multiple sclerosis brain. The pathophysiological implications of this immune response were investigated here. We demonstrate that CRYAB is a potent negative regulator acting as a brake on several inflammatory pathways in both the immune system and central nervous system (CNS). Cryab-/- mice showed worse experimental autoimmune encephalomyelitis (EAE) at the acute and progressive phases, with higher Th1 and Th17 cytokine secretion from T cells and macrophages, and more intense CNS inflammation, compared with their wild-type counterparts. Furthermore, Cryab-/- astrocytes showed more cleaved caspase-3 and more TUNEL staining, indicating an anti-apoptotic function of Cryab. Antibody to CRYAB was detected in cerebrospinal fluid from multiple sclerosis patients and in sera from mice with EAE. Administration of recombinant CRYAB ameliorated EAE. Thus, the immune response against a negative regulator of inflammation, CRYAB, in multiple sclerosis, would exacerbate inflammation and demyelination. This can be countered by giving CRYAB itself for therapy of ongoing disease.

    Funded by: Multiple Sclerosis Society: 835

    Nature 2007;448;7152;474-9

  • A genetic association analysis of cognitive ability and cognitive ageing using 325 markers for 109 genes associated with oxidative stress or cognition.

    Harris SE, Fox H, Wright AF, Hayward C, Starr JM, Whalley LJ and Deary IJ

    Department of Psychology, University of Edinburgh, Edinburgh, UK. Sarah.Harris@hgu.mrc.ac.uk <Sarah.Harris@hgu.mrc.ac.uk&gt;

    Background: Non-pathological cognitive ageing is a distressing condition affecting an increasing number of people in our 'ageing society'. Oxidative stress is hypothesised to have a major role in cellular ageing, including brain ageing.

    Results: Associations between cognitive ageing and 325 single nucleotide polymorphisms (SNPs), located in 109 genes implicated in oxidative stress and/or cognition, were examined in a unique cohort of relatively healthy older people, on whom we have cognitive ability scores at ages 11 and 79 years (LBC1921). SNPs showing a significant positive association were then genotyped in a second cohort for whom we have cognitive ability scores at the ages of 11 and 64 years (ABC1936). An intronic SNP in the APP gene (rs2830102) was significantly associated with cognitive ageing in both LBC1921 and a combined LBC1921/ABC1936 analysis (p < 0.01), but not in ABC1936 alone.

    Conclusion: This study suggests a possible role for APP in normal cognitive ageing, in addition to its role in Alzheimer's disease.

    Funded by: Medical Research Council: MRC_MC_U127561128

    BMC genetics 2007;8;43

  • Interactions between important regulatory proteins and human alphaB crystallin.

    Ghosh JG, Shenoy AK and Clark JI

    Biomolecular Structure and Design, University of Washington, Seattle, Washington 98195-7420, USA.

    Protein pin arrays assessed interactions between alphaB crystallin and 12 regulatory proteins, including EGF, FGF-2, IGF-1, NGF-beta, TGF-beta, VEGF, insulin, beta-catenin, caspase-3, caspase-8, Bcl-2, and Bcl-xL, which are important in cellular differentiation, proliferation, signaling, cytoskeletal assembly, and apoptosis. FGF-2, NGF-beta, VEGF, insulin, and beta-catenin had strong interactions with human alphaB crystallin peptides, and the alphaB crystallin interactive sequences for these proteins were identified. The seven remaining proteins (EGF, IGF-1, TGF-beta, caspase-3, caspase-8, BCl-2, and Bcl-xL) did not interact with alphaB crystallin. The alphaB crystallin sequences that interacted with FGF-2, NGF-beta, VEGF, insulin, and beta-catenin overlapped with sequences that selectively interact with partially unfolded proteins, suggesting a common function for alphaB crystallin in chaperone activity and the regulation of cell growth and differentiation. Chaperone assays conducted with full-length alphaB crystallin and synthetic alphaB crystallin peptides confirmed the ability of alphaB crystallin to protect against the aggregation of FGF-2 and VEGF, suggesting that alphaB crystallin protects these proteins against unfolding and aggregation under conditions of stress. This is the first report in which sequences involved in interactions with regulatory proteins, including FGF-2, NGF-beta, VEGF, insulin, and beta-catenin, were identified in a small heat shock protein.

    Funded by: NEI NIH HHS: EY04542, R01 EY004542

    Biochemistry 2007;46;21;6308-17

  • An increase in S-glutathionylated proteins in the Alzheimer's disease inferior parietal lobule, a proteomics approach.

    Newman SF, Sultana R, Perluigi M, Coccia R, Cai J, Pierce WM, Klein JB, Turner DM and Butterfield DA

    Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506, USA.

    Alzheimer's disease (AD) is a neurodegenerative disorder characterized by neurofibrillary tangles, senile plaques, and loss of synapses. Many studies support the notion that oxidative stress plays an important role in AD pathogenesis. Previous studies from our laboratory employed redox proteomics to identify oxidatively modified proteins in the AD inferior parietal lobule (IPL) and hippocampus. The proteins were consistent with biochemical or pathological alterations in AD and have been central to further investigations of the disease. The present study focused on the identification of specific targets of protein S-glutathionylation in AD and control IPL by using a redox proteomics approach. For AD IPL, we identified deoxyhemoglobin, alpha-crystallin B, glyceraldehyde phosphate dehydrogenase (GAPDH), and alpha-enolase as significantly S-glutathionylated relative to these brain proteins in control IPL. GAPDH and alpha-enolase were also shown to have reduced activity in the AD IPL. This study demonstrates that specific proteins are sensitive to S-glutathionylation, which most likely is due to their sensitivity to cysteine oxidation initiated by the increase in oxidative stress in the AD brain.

    Journal of neuroscience research 2007;85;7;1506-14

  • Differential susceptibility of alpha A- and alpha B-crystallin to gamma-ray irradiation.

    Fujii N, Nakamura T, Sadakane Y, Saito T and Fujii N

    Institute, Kyoto University, Kumatori, Sennan, Osaka, 590-0494, Japan.

    Alpha-cr 63 ystallin, a major protein of all vertebrate lenses, consists of two different subunits, alpha A and 1f40 alpha B, which form polymeric aggregates with an average molecular mass of 300-800 kDa. Both the alpha A and alpha B subunit have a molecular mass of about 20 kDa. It is not known why alpha crystallin aggregates comprise two different subunits, given that the physicochemical properties of these proteins are very similar. The present study compares the susceptibility of the alpha A and alpha B subunits to gamma-rays. We prepared a recombinant form of human alpha A- and alpha B-crystallin and then irradiated the proteins with gamma-rays. Based on far-UV CD spectra, alpha A-crystallin retained beta-sheet conformation after gamma irradiation up to 3.0 kGy, whereas alpha B-crystallin lost beta-sheet conformation upon exposure to gamma irradiation at >1.0 kGy. Size exclusion chromatography showed that the aggregation and polydispersity of recombinant alpha A-crystallin increased slightly after >1.0 kGy irradiation. In contrast, irradiation of alpha B-crystallin at 1.0 kGy resulted in the formation of huge aggregates and a marked increase in heterogeneity. We have also compared the chaperone activities of gamma-irradiated alpha A- and alpha B-crystallin aggregates. The activity of irradiated alpha A-crystallin was retained while that of the irradiated alpha B-crystallin was became inactive after irradiation of >0.5 kGy. Our results indicate that alpha A-crystallin is more stable to gamma irradiation than alpha B-crystallin.

    Biochimica et biophysica acta 2007;1774;3;345-50

  • Paradoxical effects of substitution and deletion mutation of Arg56 on the structure and chaperone function of human alphaB-crystallin.

    Biswas A, Goshe J, Miller A, Santhoshkumar P, Luckey C, Bhat MB and Nagaraj RH

    Department of Ophthalmology, Case Western Reserve University, Cleveland, Ohio 44106, USA.

    Human alphaB-crystallin is a small heat-shock protein that functions as a molecular chaperone. Recent studies indicate that deletion of a peptide (54FLRAPSWF61) from its N-terminus makes it a better chaperone, and this particular sequence is thought to participate in substrate interaction and subunit exchange with alphaA-crystallin. To determine whether the positive charge on arginine 56 (R56) influences these functions, we prepared human alphaB-crystallin mutants in which R56 was deleted (DeltaR56) or replaced by alanine (R56A). To determine if the effects are specific to R56, we generated two additional mutant proteins in which the two neighboring amino acids were deleted (DeltaL55 and DeltaA57). Dynamic light scattering studies suggested that none of the mutations affected the oligomeric mass of the protein. Far-ultraviolet circular dichroism (UV CD) spectra revealed greater helicity in the secondary structures of R56A and DeltaR56 compared to that of the wild-type (Wt) protein. Near-UV CD spectra showed that the tertiary structure is perturbed in all mutants. Insulin and citrate synthase aggregation assays showed 38 and 30% improvement of chaperone function in DeltaR56 compared to that of the Wt. In contrast, the R56A mutant lost most of its chaperone function. Deletion mutants, DeltaL55 and DeltaA57, showed no significant changes in the chaperone function compared to that of the Wt. The DeltaR56 mutant had a higher surface hydrophobicity than the Wt, but the R56A mutant had a lower hydrophobicity. Our data show paradoxical effects of the deletion and substitution of R56 and imply that the chaperone function of human alphaB-crystallin is dictated not only by the positive charge on R56 but also by the conformational change that it bestows on the protein.

    Funded by: NEI NIH HHS: P30EY-11373, R01EY-016219, R01EY-09912

    Biochemistry 2007;46;5;1117-27

  • CRYAB promoter polymorphisms: influence on multiple sclerosis susceptibility and clinical presentation.

    Stoevring B, Frederiksen JL and Christiansen M

    Department of Clinical Biochemistry, Statens Serum Institut, 5 Artillerivej, DK 2300 S, Copenhagen, Denmark.

    Background: alphaB-crystallin is a molecular chaperone and potential myelin antigen, up-regulated in the earlier stages of multiple sclerosis (MS) lesions. In the alphaB-crystallin gene (CRYAB), single nucleotide polymorphisms (SNPs) have been associated with MS susceptibility (g.CRYAB-652A>G) and a rapidly progressive clinical course (g.CRYAB-650C>G).

    Method: CRYAB was screened for mutations in 233 MS patients and 96 controls. Genomic DNA was extracted and the coding and 3' and 5' untranslated regions were amplified by PCR. Subsequently, the products were analysed by Single Strand Conformation Polymorphism technique followed by DNA sequencing of aberrant conformers.

    Results: In CRYAB (Genbank ) no mutations were found but SNPs were identified in the promoter region (g.CRYAB-249C>G, g.CRYAB-650C>G and g.CRYAB-652A>G), and intronic region (g.CRYAB.2398T>G). The g.CRYAB-249C>G genotype distribution was significantly different between groups (chi(2), p=0.01), caused by differences between Relapsing Remitting MS (RRMS) and controls (chi(2), p=0.025) and Secondary Progressive MS (SPMS) and controls (chi(2), p=0.05). In addition, a significant difference was observed in the g.CRYAB-249C>G allele distribution (chi(2), p=0.04), caused by a difference between SPMS and controls (chi(2), p=0.01). In RRMS and SPMS a tendency of the g.CRYAB-249GG genotype being associated with an earlier age of onset (p=0.05) and a slowly progressive cause (p=0.07) was found. Multiple sequence alignment showed conservation of the g.CRYAB-249*C between mammalian CRAYB genes and within the small heat shock protein gene family.

    Conclusion: CRYAB polymorphisms may be involved in the pathogenesis of MS by mechanisms that could involve increased expression of the superantigen alphaB-crystallin and modulation of the immune response. CRYAB polymorphisms should be included in future multivariate biomaker studies in MS.

    Clinica chimica acta; international journal of clinical chemistry 2007;375;1-2;57-62

  • Interactive sequences in the stress protein and molecular chaperone human alphaB crystallin recognize and modulate the assembly of filaments.

    Ghosh JG, Houck SA and Clark JI

    Department of Biological Structure, University of Washington, Seattle, WA 98195-7420, United States.

    Molecular chaperones including the small heat shock proteins, alphaB crystallin and sHSP27 participate in the assembly, disassembly, and reorganization of the cytoskeleton during cell development and differentiation. While alphaB crystallin and sHSP27 stabilize and modulate filament assembly and re-organization, the sequences and structural domains mediating interactions between these proteins and filaments are unknown. It is important to define these interactive domains in order to understand differential interactions between chaperones and stable or unfolding filaments and their function in the cellular stress response. Protein pin arrays identified sequences in human alphaB crystallin that selectively interacted with native or partially unfolded filament proteins desmin, glial-fibrillary acidic protein, and actin. Circular dichroism spectroscopy determined differences in the structure of these filaments at 23 and 45 degrees C. Seven alphaB crystallin sequences had stronger interactions with desmin and six sequences had stronger interactions with glial-fibrillary acidic protein at 23 degrees C than at 45 degrees C. The alphaB crystallin sequences (33)LESDLFPTSTSLSPFYLRPPSFLR(56) and (129)DPLTITSSLSSDGV(145) had the strongest interactions with actin at 23 degrees C, while (57)APSWFDTG(64), (111)HGFISREF(118), (145)VNGPRKQVSG(154), and (155)PERTIPITREEK(165) had the strongest interactions with actin at 45 degrees C. The actin interactive sequences of alphaB crystallin overlapped with previously identified alphaB crystallin chaperone sequences and were synthesized to evaluate their effect on the assembly and aggregation of actin. Full-length alphaB crystallin and the core domain chaperone sequence (131)LTITSSLSSDGV(143) promoted actin polymerization at 37 degrees C and inhibited depolymerization and aggregation at 50 degrees C. The results support the hypothesis that interactive domains in alphaB crystallin have multiple functions in stabilizing the cytoskeleton and protecting cytosolic proteins from unfolding.

    Funded by: NEI NIH HHS: EY04542, R01 EY004542, R01 EY004542-26, R01 EY004542-27

    The international journal of biochemistry & cell biology 2007;39;10;1804-15

  • The beta4-beta8 groove is an ATP-interactive site in the alpha crystallin core domain of the small heat shock protein, human alphaB crystallin.

    Ghosh JG, Houck SA, Doneanu CE and Clark JI

    Biomolecular Structure and Design, University of Washington, Seattle, WA 98195-7420, USA.

    The site for ATP interactions in human alphaB crystallin, the archetype of small heat-shock proteins, was identified and characterized to resolve the controversial role of ATP in the function of small heat-shock proteins. Comparative sequence alignments identified the alphaB crystallin sequence, (82)KHFSPEELKVKVLGD(96) as a Walker-B ATP-binding motif that is found in several ATP-binding proteins, including five molecular chaperones. Fluorescence resonance energy transfer and mass spectrometry using a novel fluorescent ATP analog, 8-azido-ATP-[gamma]-1-naphthalenesulfonic acid-5(2-aminoethylamide) (azido-ATP-EDANS) and a cysteine mutant of human alphaB crystallin (S135C) conjugated with a fluorescent acceptor, eosin-5-maleimide (EMA) identified the beta4-beta8 groove as the ATP interactive site in alphaB crystallin. A 44% decrease in the emitted fluorescence of azido-ATP-EDANS at the absorption maximum of S135C-EMA and a corresponding 50% increase in the fluorescence emission of S135C-EMA indicated a close spatial relationship between azido-ATP-EDANS and the center of the beta8 strand ((131)LTITSSLS(138)). Liquid chromatography, electrospray ionization mass spectrometry identified two peptide fragments of the alphaB crystallin Walker-B motif photo-affinity-labeled with azido-ATP-EDANS confirming the beta4-beta8 groove as an ATP interactive site. The results presented here clearly establish the beta4-beta8 groove as the ATP interactive region in alphaB crystallin, and are in contrast to the existing paradigm that classifies small heat-shock proteins as ATP-independent chaperones.

    Funded by: NEI NIH HHS: EY04542, R01 EY004542

    Journal of molecular biology 2006;364;3;364-75

  • AbetaPP-overexpression and proteasome inhibition increase alphaB-crystallin in cultured human muscle: relevance to inclusion-body myositis.

    Wojcik S, Engel WK, McFerrin J, Paciello O and Askanas V

    USC Neuromuscular Center, Department of Neurology, University of Southern California Keck School of Medicine, Good Samaritan Hospital, Los Angeles, CA 90017-1912, USA.

    Amyloid-beta precursor protein (AbetaPP) and its fragment amyloid-beta (Abeta) are increased in s-IBM muscle fibers and appear to play an important role in the pathogenic cascade. alphaB-Crystallin (alphaBC) was shown immunohistochemically to be accumulated in s-IBM muscle fibers, but the stressor(s) influencing alphaBC accumulation was not identified. We now demonstrate, using our experimental IBM model based on genetic overexpression of AbetaPP into cultured normal human muscle fibers, that: (1) AbetaPP overexpression increased alphaBC 3.7-fold (p=0.025); (2) additional inhibition of proteasome with epoxomicin increased alphaBC 7-fold (p=0.002); and (3) alphaBC physically associated with AbetaPP and Abeta oligomers. We also show that in biopsied s-IBM muscle fibers, alphaBC was similarly increased 3-fold (p=0.025) and physically associated with AbetaPP and Abeta oligomers. We propose that increased AbetaPP is a stressor increasing alphaBC expression in s-IBM muscle fibers. Determining the consequences of alphaBC association with Abeta oligomers could have clinical therapeutic relevance.

    Funded by: NIA NIH HHS: AG16768, R37 AG016768, R37 AG016768-07

    Neuromuscular disorders : NMD 2006;16;12;839-44

  • N- and C-Terminal motifs in human alphaB crystallin play an important role in the recognition, selection, and solubilization of substrates.

    Ghosh JG, Shenoy AK and Clark JI

    Biomolecular Structure and Design, Department of Biological Structure, University of Washington, Seattle, Washington 98195-7420, USA.

    The functions of the interactive sequences in human alphaB crystallin that are involved in chaperone activity and complex assembly of small heat shock proteins need to be characterized to understand the mechanisms of action on unfolding and misfolding proteins. Protein pin arrays identified the hydrophobic N-terminal sequence (41STSLSPFYLRPPSFLRAP58) and the polar C-terminal sequence (155PERTIPITREE165) as interactive domains in human alphaB crystallin, which were then deleted to evaluate their importance in complex assembly and chaperone activity. Size exclusion chromatography determined that the complexes formed by the deletion mutants, Delta41-58 and Delta155-165, were larger and more polydisperse than the wild-type (wt) alphaB crystallin complex. In chaperone assays, the Delta41-58 mutant was as effective as wt alphaB crystallin in protecting partially unfolded betaL crystallin and alcohol dehydrogenase (ADH) and significantly less effective than wt alphaB crystallin in protecting unfolded citrate synthase (CS) from aggregation. Chaperone activity did not correlate with complex size but corresponded with the amount of substrate protein unfolding. The results confirmed the importance of N-terminal residues 41-58 in selective interactions wit a2 h completely unfolded substrates. Poor solubility and limited or no chaperone activity for the three substrates characterized the Delta155-165 deletion mutant, wh 1e96 ich demonstrated the importance of C-terminal residues 155-165 in maintaining the solubility of unfolded substrates in a manner independent of the amount of substrate protein unfolding. The results presented in this report established that interactive domains in the N- and C-termini of human alphaB crystallin are important for the recognition, selection, and solubility of unfolding substrate proteins.

    Funded by: NEI NIH HHS: EY04542, R01 EY004542

    Biochemistry 2006;45;46;13847-54

  • Phosphorylation-dependent ubiquitination of cyclin D1 by the SCF(FBX4-alphaB crystallin) complex.

    Lin DI, Barbash O, Kumar KG, Weber JD, Harper JW, Klein-Szanto AJ, Rustgi A, Fuchs SY and Diehl JA

    The Leonard and Madlyn Abramson Family Cancer Research Institute and Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

    Growth factor-dependent accumulation of the cyclin D1 proto-oncogene is balanced by its rapid phosphorylation-dependent proteolysis. Degradation is triggered by threonine 286 phosphorylation, which promotes its ubiquitination by an unknown E3 ligase. We demonstrate that Thr286-phosphorylated cyclin D1 is recognized by a Skp1-Cul1-F box (SCF) ubiquitin ligase where FBX4 and alphaB crystallin govern substrate specificity. Overexpression of FBX4 and alphaB crystallin triggered cyclin D1 ubiquitination and increased cyclin D1 turnover. Impairment of SCF(FBX4-alphaB crystallin) function attenuated cyclin D1 ubiquitination, promoting cyclin D1 overexpression and accelerated cell-cycle progression. Purified SCF(FBX4-alphaB crystallin) catalyzed polyubiquitination of cyclin D1 in vitro. Consistent with a putative role for a cyclin D1 E3 ligase in tumorigenesis, FBX4 and alphaB crystallin expression was reduced in tumor-derived cell lines and a subset of primary human cancers that overexpress cyclin D1. We conclude that SCF(FBX4-alphaB crystallin) is an E3 ubiquitin ligase that promotes ubiquitin-dependent degradation of Thr286-phosphorylated cyclin D1.

    Funded by: NCI NIH HHS: CA111360, CA93237, P01CA098101, R01 CA093237, R01 CA111360; NIA NIH HHS: R01 AG011085

    Molecular cell 2006;24;3;355-66

  • Conserved F84 and P86 residues in alphaB-crystallin are essential to effectively prevent the aggregation of substrate proteins.

    Santhoshkumar P and Sharma KK

    Mason Eye Institute, University of Missouri Columbia, Missouri 65212, USA.

    Previously, we have shown that residues 73-92 (sequence DRFSVNLDVKHFSPEELKVK) in alphaB-crystallin are involved in preventing the formation of light scattering aggregates by substrate proteins. In this study, we made single substitutions of three conserved amino acid residues (H83 --> A, F84 --> G, and P86 --> A) and a nonconserved amino acid residue (K90 --> C) in the functional region of alphaB-crystallin and evaluated their role in anti-aggregation activity. Mutation of conserved residues led to changes in intrinsic tryptophan intensity, bis-ANS binding, and in the secondary and tertiary structures. The H83A mutation led to a twofold increase in molar mass, while the other mutants did not produce significant changes in the molar mass when compared to that of wild-type protein. The chaperone-like activity of the H83A mutant was enhanced by 15%-20%, and the chaperone-like activity of F84G and P86A mutants was reduced by 50%-65% when compared to the chaperone-like activity of wild-type alphaB-crystallin. The substitution of the nonconserved residue (K90 --> C) did not induce an appreciable change in the structure and function of the mutant protein. Fluorescence resonance energy transfer (FRET) assay demonstrated that destabilized ADH interacted near the K90 region in alphaB-crystallin. The data show that F84 and P86 residues are essential for alphaB-crystallin to effectively prevent the aggregation of substrate proteins. This study further supports the involvement of the residues in the 73-92 region of alphaB-crystallin in substrate protein binding and chaperone-like action.

    Protein science : a publication of the Protein Society 2006;15;11;2488-98

  • alphaB-crystallin competes with Alzheimer's disease beta-amyloid peptide for peptide-peptide interactions and induces oxidation of Abeta-Met35.

    Narayanan S, Kamps B, Boelens WC and Reif B

    Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse, 10, 13125 Berlin, Germany.

    Alzheimer's disease (AD) is associated with plaque deposition in the brain of AD patients. The major co d6e mponent of the aggregate is a 39-42 long peptide termed beta-amyloid (Abeta). Except for Abeta, plaques contain several other components which co-precipitate together with Abeta. One such component is the small heat shock protein (sHSP) alphaB-crystallin. Instead of preventing the cell from the neurotoxicity of Abeta, alphaB-crystallin induces an increased neurotoxicity. We find - using solution state NMR spectroscopy - that alphaB-crystallin competes efficiently for Abeta monomer-monomer interactions. Interactions between Abeta and alphaB-crystallin involve the hydrophobic core residues 17-21 as well as residues 31-32 of Abeta, and thus the same chemical groups which are important for Abeta aggregation. In the presence of alphaB-crystallin, Met35 in Abeta becomes efficiently oxidized. In order to quantify the redox properties of the different complexes consisting of Abeta/alphaB-crystallin/copper, we suggest an NMR assay which allows to estimate the electrochemical properties indirectly by monitoring the rate of glutathion (GSH) auto-oxidation. The oxidation of the side chain Met35 in Abeta might account for the increased neurotoxicity and the inability of Abeta to form fibrillar structures, which has been observed previously in the presence of alphaB-crystallin [Stege, G.J. et al. (1999) The molecular chaperone alphaB-crystallin enhances amyloid-beta neurotoxicity. Biochem. Biophys. Res. Commun. 262, 152-156.].

    FEBS letters 2006;580;25;5941-6

  • Structure-based analysis of the beta8 interactive sequence of human alphaB crystallin.

    Ghosh JG, Estrada MR and Clark JI

    Biomolecular Structure and Design, University of Washington, Seattle, Washington 98195-7420, USA.

    The functional importance of the beta8 sequence ((131)LTITSSLS(138)), which is on the surface of the alpha crystallin core domain of human alphaB crystallin, was evaluated using site-directed mutagenesis. Ultraviolet circular dichroism determined that mutating the surface-exposed, nonconserved residues, Leu-131, Thr-132, Thr-134, Ser-135, Ser-136, and Ser-138 individually or in combination (alphaAbeta8 and CEbeta8), had no measurable effect on secondary and tertiary structure. Size exclusion chromatography determined the size of the complexes formed by the beta8 mutants to be 6-8 subunits larger than wt alphaB crystallin. In chaperone assays, the protective effect of the L131S, T132A, and S135C mutants of the beta8 sequence was similar to wt alphaB crystallin when beta(L) crystallin and alcohol dehydrogenase were the chaperone substrates and decreased to 66% when citrate synthase was the chaperone substrate. In contrast, the chaperone activity for all three substrates was dramatically reduced for the T134K, S138A, S136H, and CEbeta8 mutants. The prominent location of Thr-134, Ser-136, and Ser-138 on the exposed surface of the alpha crystallin core domain could account for the effect on complex assembly and chaperone activity. Modulation of chaperone activity by the exposed residues of the beta8 sequence in the alpha crystallin core domain was independent of complex size. The results established the beta3-beta8-beta9 surface of the alpha crystallin core domain as an interface for complex assembly and chaperone activity.

    Funded by: NEI NIH HHS: EY04542, R01 EY004542

    Biochemistry 2006;45;32;9878-86

  • Identification of a CRYAB mutation associated with autosomal dominant posterior polar cataract in a Chinese family.

    Liu M, Ke T, Wang Z, Yang Q, Chang W, Jiang F, Tang Z, Li H, Ren X, Wang X, Wang T, Li Q, Yang J, Liu J and Wang QK

    Center for Human Genome Research and College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, Peoples Republic of China.

    Purpose: A four-generation Chinese family with 13 members affected with autosomal dominant congenital posterior polar cataract was studied. The purpose of this study was to identify the disease-causing gene in the family and to validate that mutations in CRYAB, the alphaB-crystallin gene, cause the congenital cataract.

    Methods: Linkage analysis was performed with a panel of microsatellite markers flanking candidate genetic loci for cataracts, including 14 known autosomal dominant congenital cataract (ADCC) genes. For mutation analysis, the complete coding region and exon-intron boundaries of CRYAB were sequenced with DNA from the proband. Single-strand conformation polymorphism (SSCP) analysis for exon 1 of CRYAB was performed in all family members and 200 normal control subjects.

    Results: The disease gene in the Chinese family was mapped to chromosome 11 in region q22-22.3 with a maximum lod score of 4.52. Direct DNA sequence of CRYAB revealed a heterozygous C-->T transition at nucleotide 58, resulting in a novel 58 C-->T (Pro20Ser) mutation. The Pro20Ser mutation cosegregated with all affected individuals and was not present in unaffected members in the family or in 200 normal control subjects. The mutation occurs at the evolutionarily conserved residue Pro20 in the N-terminal region of alphaB-crystallin.

    Conclusions: To date, only one CRYAB mutation has been associated with congenital isolated cataract. This study identified a second novel mutation in CRYAB in a large Chinese cataract family. Together, these results provide strong evidence that CRYAB is a pathogenic gene for congenital cataract.

    Investigative ophthalmology & visual science 2006;47;8;3461-6

  • Fluorescence resonance energy transfer study of subunit exchange in human lens crystallins and congenital cataract crystallin mutants.

    Liang JJ and Liu BF

    Ophthalmic Research/Surgery, Brigham and Women's Hospital, Department of Ophthalmology, Harvard Medical School, Boston, MA 02115, USA. jliang@rics.bwh.harvard.edu

    Lens alpha-crystallin is an oligomeric protein with a molecular mass of 500-1000 kDa and a polydispersed assembly. It consists of two types of subunits, alphaA and alphaB, each with a molecular mass of 20 kDa. The subunits also form homo-oligomers in some other tissues and in vitro. Their quaternary structures, which are dynamic and characterized by subunit exchange, have been studied by many techniques, including fluorescence resonance energy transfer (FRET) and mass spectrometry analysis. The proposed mechanism of subunit exchange has been either by dissociation/association of monomeric subunits or by rapid equilibrium between oligomers and suboligomers. To explore the nature of subunit exchange further, we performed additional FRET measurements and analyses using a fluorescent dye-labeled W9F alphaA-crystallin as the acceptor probe and Trp in other crystallins (wild-type and R116C alphaA, wild-type and R120G alphaB, wild-type and Q155* betaB2) as the donor probe and calculated the transfer efficiency, Förster distance, and average distance between two probes. The results indicate only slight decreased efficiency and increased distance between two probes for the R116C alphaA and R120G alphaB mutations despite conformational changes.

    Funded by: NEI NIH HHS: EY13968, R01 EY013968

    Protein science : a publication of the Protein Society 2006;15;7;1619-27

  • Autoantibodies against alpha B-crystallin, a candidate autoantigen in multiple sclerosis, are part of a normal human immune repertoire.

    van Noort JM, Verbeek R, Meilof JF, Polman CH and Amor S

    Division of Biomedical Research, TNO Quality of Life, Leiden, The Netherlands. jm.vannoort@pg.tno.nl

    Human T-cell responses to the stress protein alpha B-crystallin in multiple sclerosis (MS)-affected brain samples are dominant when compared to other myelin antigens. The establishment of the apparent autoimmune repertoire against this antigen has been suggested to involve cross-priming during viral infection. Yet, another possibility would be that determinant spreading during ocular inflammation could generate a response to alpha B-crystallin, since it is also a major component of the eye. In this study, we compared serum IgG, IgA and IgM repertoires against a range of eye lens-derived ocular antigens using sera from healthy control subjects and MS patients with or without uveitis. This comparison revealed that among ocular antigens, alpha B-crystallin is the dominant target antigen for serum autoantibodies in both MS patients and healthy controls. Uveitis generally did not affect the antibody reactivity profile. These data provide further support for the notion that a normal adult human immune system is selectively reactive to alpha B-crystallin and they indicate that this responsiveness is unlikely to result from determinant spreading following ocular inflammation.

    Multiple sclerosis (Houndmills, Basingstoke, England) 2006;12;3;287-93

  • The interaction between alphaA- and alphaB-crystallin is sequence-specific.

    Sreelakshmi Y and Sharma KK

    Department of Ophthalmology, University of Missouri, Columbia, MO 65212, USA. yellamarajusr@health.missouri.edu

    Purpose: We have previously shown that residue 42-57 (TSLSPFYLRPPSFLRA; recognition sequence 1 or RS-1) and residue 60-71 (WFDTGLSEMRLE; recognition sequence 2 or RS-2) in alphaB-crystallin play a role in oligomerization and subunit interaction with alphaA-crystallin. When we created multiple mutations in alphaB-crystallin in RS-1 and RS-2 at S53(T), F54(G), L55(G), W60(R), and F61(N), we found that these mutations destabilized the protein, and the protein precipitated. When the individual mutations were created at F54, W60, and F61 in alphaB-crystallin, protein stability was not affected, but the mutations had an effect on oligomerization and subunit interaction with alphaA-crystallin. To find out whether the sequence specificity of these residues is important for the overall function of alphaB-crystallin, we inverted the 54-60 sequence such that 54FLRAPSW60 became 54WSPARLF60 using site-directed mutagenesis. We studied the effect of inversion on oligomerization and subunit interaction with alphaA-crystallin.

    Methods: Mutations were introduced using site-directed mutagenesis and the mutant protein, expressed in Escherichia coli BL21(DE3)pLysS cells, was purified by ion-exchange and gel filtration chromatography. The mutation was confirmed by mass spectrometry. The structure and hydrophobicity were analyzed by spectroscopic methods. The chaperone-like activities of wild-type and mutant proteins were compared using alcohol dehydrogenase and citrate synthase. Subunit exchange between alphaA- and alphaB-crystallin was monitored by fluorescence resonance energy transfer (FRET). For this purpose, purified alphaB- and alphaBinvert-crystallin were labeled with Alexa fluor 350 whereas Alexa fluor 488 was used to label alphaA-crystallin.

    Results: The inversion of residues 54-60 led to homooligomers that were 38% smaller in size than their wild-type counterparts. The inversion also reduced the tryptophan fluorescence intensity by 50%, as compared to that of wild-type alphaB-crystallin. This suggests that Trp54 is less exposed than Trp60. Inversion of residues did not affect the total hydrophobicity in alphaB-crystallin. Secondary structural analysis revealed a slight increase in the alpha-helical content of alphaBinvert-crystallin protein as compared to wild-type alphaB-crystallin. Except for an increase in the ellipticity of the alphaBinvert-crystallin mutant, no change was observed in the tertiary structure, as compared with that of wild-type alphaB-crystallin. Chaperone-like function was similar in the alphaBinvert-crystallin mutant and wild-type alphaB-crystallin. The inversion of residues decreased the subunit exchange rate with alphaA-crystallin by two fold.

    Conclusions: This study establishes for the first time that proper orientation of residues contributing to RS-1 and RS-2 sites in alphaB-crystallin is important for homooligomerization and optimal subunit interaction with alphaA-crystallin.

    Funded by: NEI NIH HHS: EY 11981, EY 14795

    Molecular vision 2006;12;581-7

  • New insights into potential functions for the protein 4.1 superfamily of proteins in kidney epithelium.

    Calinisan V, Gravem D, Chen RP, Brittin S, Mohandas N, Lecomte MC and Gascard P

    Life Sciences Division, Department of Genome Biology, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.

    Members of the protein 4.1 family of adapter proteins are expressed in a broad panel of tissues including various epithelia where they likely play an important role in maintenance of cell architecture and polarity and in control of cell proliferation. We have recently characterized the structure and distribution of three members of the protein 4.1 family, 4.1B, 4.1R and 4.1N, in mouse kidney. We describe here binding partners for renal 4.1 proteins, identified through the screening of a rat kidney yeast two-hybrid system cDNA library. The identification of putative protein 4.1-based complexes enables us to envision potential functions for 4.1 proteins in kidney: organization of signaling complexes, response to osmotic stress, protein trafficking, and control of cell proliferation. We discuss the relevance of these protein 4.1-based interactions in kidney physio-pathology in the context of their previously identified functions in other cells and tissues. Specifically, we will focus on renal 4.1 protein interactions with beta amyloid precursor protein (beta-APP), 14-3-3 proteins, and the cell swelling-activated chloride channel pICln. We also discuss the functional relevance of another member of the protein 4.1 superfamily, ezrin, in kidney physio-pathology.

    Funded by: NIDDK NIH HHS: DK32094, DK56355

    Frontiers in bioscience : a journal and virtual library 2006;11;1646-66

  • Alpha B-crystallin mutation in dilated cardiomyopathy.

    Inagaki N, Hayashi T, Arimura T, Koga Y, Takahashi M, Shibata H, Teraoka K, Chikamori T, Yamashina A and Kimura A

    Department of Molecular Pathogenesis, Medical Research Institute and Laboratory of Genome Diversity, School of Biomedical Science, Tokyo Medical and Dental University, Tokyo 101-0062, Japan.

    Mutations in genes for sarcomeric proteins such as titin/connectin are known to cause dilated cardiomyopathy (DCM). However, disease-causing mutations can be identified only in a small proportion of the patients even in the familial cases, suggesting that there remains yet unidentified disease-causing gene(s) for DCM. To explore the novel disease gene for DCM, we examined CRYAB encoding alphaB-crystallin for mutation in the patients with DCM, since alphaB-crystallin was recently reported to associate with the heart-specific N2B domain and adjacent I26/I27 domain of titin/connectin, and we previously reported a N2B mutation, Gln4053ter, in DCM. A missense mutation of CRYAB, Arg157His, was found in a familial DCM patient and the mutation affected the evolutionary conserved amino acid residue among alpha-crystallins. Functional analysis revealed that the mutation decreased the binding to titin/connectin heart-specific N2B domain without affecting distribution of the mutant crystallin protein in cardiomyocytes. In contrast, another CRYAB mutation, Arg120Gly, reported in desmin-related myopathy decreased the binding to both N2B and striated muscle-specific I26/27 domains and showed intracellular aggregates of the mutant protein. These observations suggest that the Arg157His mutation may be involved in the pathogenesis of DCM via impaired accommodation to the heart-specific N2B domain of titin/connectin and its disease-causing mechanism is different from the mutation found in desmin-related myopathy.

    Biochemical and biophysical research communications 2006;342;2;379-86

  • Temperature dependence of chaperone-like activity and oligomeric state of alphaB-crystallin.

    Spinozzi F, Mariani P, Rustichelli F, Amenitsch H, Bennardini F, Mura GM, Coi A and Ganadu ML

    Dipartimento di Scienze applicate ai Sistemi Complessi, Università Politecnica delle Marche, and INFM Unità di Ancona, Via Brecce Bianche, I-60131 Ancona, Italy. f.spinozzi@alisf1.univpm.it

    The chaperone-like activity and the oligomeric state of alphaB-crystallin were studied at different temperatures and in the presence of urea and thiocyanate. The activity, assessed measuring the ability of alphaB-crystallin to prevent the aggregation of denatured insulin, strongly depends on temperature. While a significant activity increase was detected at 42 degrees C, the presence of urea and thiocyanate does not affect the protein activity in an irreversible way. In-solution SAXS measurements performed in the same experimental conditions showed that alphaB-crystallin forms near-spherical, hollowed, polydisperse oligomers, whose dimensions change above 42 degrees C. Moreover, in the presence of urea and thiocyanate, a global fit analysis confirms the high stability of alphaB-crystallin assemblies in relationship with their variable quaternary structure. In particular, the changes in the inner radius as well as the thickness and dispersion of the protein shell, account for the preservation of the chaperone-like activity.

    Biochimica et biophysica acta 2006;1764;4;677-87

  • Studies of alphaB crystallin subunit dynamics by surface plasmon resonance.

    Liu L, Ghosh JG, Clark JI and Jiang S

    Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.

    The molecular chaperone activity of alphaB crystallin, an important stress protein in humans, is regulated by physiological factors, including temperature, pH, Ca2+, and ATP. In this study, the role of these factors in regulating the subunit dynamics of human alphaB crystallin was investigated using surface plasmon resonance (SPR). SPR experiments indicate that at temperatures above 37 degrees C, where alphaB crystallin has been reported to have higher chaperone activity, the subunit dynamics of alphaB crystallin were increased with faster association and dissociation rates. SPR experiments also indicate that interactions between alphaB crystallin subunits were enhanced with much faster association and slower dissociation rates at pH values below 7.0, where alphaB crystallin has been reported to have lower chaperone activity. The results suggest that the dynamic and rapid subunit exchange rate may regulate the chaperone activity of alphaB crystallin. The effect of Ca2+ and ATP on the subunit dynamics of alphaB crystallin was minimal, suggesting that Ca2+ and ATP modulate the chaperone activity of alphaB crystallin without altering the subunit dynamics. Based on the SPR results and previously reported biochemical data for the chaperone activity of alphaB crystallin under different conditions of temperature and pH, a model for the relationship between the subunit dynamics and chaperone activity of alphaB crystallin is established. The model is consistent with previous biochemical data for the chaperone activity and subunit dynamics of small heat shock proteins (sHSPs) and establishes a working hypothesis for the relationship between complex assembly and chaperone activity for sHSPs.

    Funded by: NEI NIH HHS: EY04542, R01 EY004542

    Analytical biochemistry 2006;350;2;186-95

  • A novel alphaB-crystallin mutation associated with autosomal dominant congenital lamellar cataract.

    Liu Y, Zhang X, Luo L, Wu M, Zeng R, Cheng G, Hu B, Liu B, Liang JJ and Shang F

    Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.

    Purpose: To identify the mutation and t 1a06 he underlying mechanism of cataractogenesis in a five-generation autosomal dominant congenital lamellar cataract family.

    Methods: Nineteen mutation hot spots associated with autosomal dominant congenital cataract have been screened by PCR-based DNA sequencing. Recombinant wild-type and mutant human alphaB-crystallin were expressed in Escherichia coli and purified to homogeneity. The recombinant proteins were characterized by far UV circular dichroism, intrinsic tryptophan fluorescence, Bis-ANS fluorescence, multiangle light-scattering, and the measurement of chaperone activity.

    Results: A novel missense mutation in the third exon of the alphaB-crystallin gene (CRYAB) was found to cosegregate with the disease phenotype in a five-generation autosomal dominant congenital lamellar cataract family. The single-base substitution (G-->A) results in the replacement of the aspartic acid residue by asparagine at codon 140. Far UV circular dichroism spectra indicated that the mutation did not significantly alter the secondary structure. However, intrinsic tryptophan fluorescence spectra and Bis-ANS fluorescence spectra indicated that the mutation resulted in alterations in tertiary and/or quaternary structures and surface hydrophobicity of alphaB-crystallin. Multiangle light-scattering measurement showed that the mutant alphaB-crystallin tended to aggregate into a larger complex than did the wild-type. The mutant alphaB-crystallin was more susceptible than wild-type to thermal denaturation. Furthermore, the mutant alphaB-crystallin not only lost its chaperone-like activity, it also behaved as a dominant negative which inhibited the chaperone-like activity of wild-type alphaB-crystallin.

    Conclusions: These data indicate that the altered tertiary and/or quaternary structures and the dominant negative effect of D140N mutant alphaB-crystallin underlie the molecular mechanism of cataractogenesis of this pedigree.

    Funded by: NEI NIH HHS: EY 11717, EY 13968, R01 EY011717-07, R01 EY011717-08, R01 EY011717-09

    Investigative ophthalmology & visual science 2006;47;3;1069-75

  • The LIFEdb database in 2006.

    Mehrle A, Rosenfelder H, Schupp I, del Val C, Arlt D, Hahne F, Bechtel S, Simpson J, Hofmann O, Hide W, Glatting KH, Huber W, Pepperkok R, Poustka A and Wiemann S

    Division Molecular Genome Analysis, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany. a.mehrle@dkfz.de

    LIFEdb (http://www.LIFEdb.de) integrates data from large-scale functional genomics assays and manual cDNA annotation with bioinformatics gene expression and protein analysis. New features of LIFEdb include (i) an updated user interface with enhanced query capabilities, (ii) a configurable output table and the option to download search results in XML, (iii) the integration of data from cell-based screening assays addressing the influence of protein-overexpression on cell proliferation and (iv) the display of the relative expression ('Electronic Northern') of the genes under investigation using curated gene expression ontology information. LIFEdb enables researchers to systematically select and characterize genes and proteins of interest, and presents data and information via its user-friendly web-based interface.

    Nucleic acids research 2006;34;Database issue;D415-8

  • Lenticular chaperones suppress the aggregation of the cataract-causing mutant T5P gamma C-crystallin.

    Pigaga V and Quinlan RA

    School of Biological and Biomedical Sciences, South Road Science Site, The University, Durham DH1 3LE, UK.

    The T5P mutation in human gamma C-crystallin produces a lens cataract. Here, we have investigated the effects of the T5P mutation upon the aggregation of gamma C-crystallin in vitro and in transfected cells. By sedimentation assay and sucrose gradient centrifugation, the mutation significantly increased the aggregation of the protein and reduced dramatically its solubility in vitro. Similar effects were seen when T5P gamma C-crystallin was transfected into tissue culture cells, resulting in the formation of cytoplasmic aggregates of T5P gamma C-crystallin. Interestingly, the major lenticular protein c 1f40 haperones, alpha A- and alpha B-crystallin, increased the solubility of the T5P gamma C-crystallin both in vitro and in transfected cells. More importantly, the size of the T5P gamma C-crystallin aggregates were also significantly reduced in the presence of the lenticular chaperones. These data therefore suggest a dual role for these chaperones in maintaining transparency in the lens. The first is that these protein chaperones increase the proportion of the soluble T5P gamma C-crystallin and the second is that they also reduce light scatter by reducing the aggregate size of T5P gamma C-crystallin. Both activities could modify the cataract phenotype and help explain the observed variability reported for identical gamma-crystallin mutations, which identify cataract as a polygenic disease.

    Funded by: Wellcome Trust

    Experimental cell research 2006;312;1;51-62

  • AlphaB-crystallin is a novel oncoprotein that predicts poor clinical outcome in breast cancer.

    Moyano JV, Evans JR, Chen F, Lu M, Werner ME, Yehiely F, Diaz LK, Turbin D, Karaca G, Wiley E, Nielsen TO, Perou CM and Cryns VL

    Cell Death Regulation Laboratory, Department of Medicine, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.

    Recent gene profiling studies have identified a new breast cancer subtype, the basal-like group, which expresses genes characteristic of basal epithelial cells and is associated with poor clinical outcomes. However, the genes responsible for the aggressive behavior observed in this group are largely unknown. Here we report that the small heat shock protein alpha-basic-crystallin (alphaB-crystallin) was commonly expressed in basal-like tumors and predicted poor survival in breast cancer patients independently of other prognostic markers. We also demonstrate that overexpression of alphaB-crystallin transformed immortalized human mammary epithelial cells (MECs). In 3D basement membrane culture, alphaB-crystallin overexpression induced luminal filling and other neoplastic-like changes in mammary acini, while silencing alphaB-crystallin by RNA interference inhibited these abnormalities. alphaB-Crystallin overexpression also induced EGF- and anchorage-independent growth, increased cell migration and invasion, and constitutively activated the MAPK kinase/ERK (MEK/ERK) pathway. Moreover, the transformed phenotype conferred by alphaB-crystallin was suppressed by MEK inhibitors. In addition, immortalized human MECs overexpressing alphaB-crystallin formed invasive mammary carcinomas in nude mice that recapitulated aspects of human basal-like breast tumors. Collectively, our results indicate that alphaB-crystallin is a novel oncoprotein expressed in basal-like breast carcinomas that independently predicts shorter survival. Our data also implicate the MEK/ERK pathway as a potential therapeutic target for these tumors.

    Funded by: NCI NIH HHS: P50 CA058223, P50 CA089018, P50CA58223, P50CA89018, R01 CA097198, R01CA097198, R01CA10122701, T32 CA009560, T32CA09560; NIDDK NIH HHS: T32 DK007169, T32DK07169; NIGMS NIH HHS: T32 GM008152

    The Journal of clinical investigation 2006;116;1;261-70

  • The function of the beta3 interactive domain in the small heat shock protein and molecular chaperone, human alphaB crystallin.

    Ghosh JG, Estrada MR, Houck SA and Clark JI

    Biomolecular Structure and Design, University of Washington, Seattle, WA 98195, USA.

    Knowledge of the interactive domains on the surface of small heat shock proteins (sHSPs) is necessary for understanding the assembly of complexes and the activity as molecular chaperones. The primary sequences of 26 sHSP molecular chaperones were aligned and compared. In the interactive beta3 sequence, 73DRFSVNLDVKHFS85 of human alphaB crystallin, Ser-76, Asn-78, Lys-82, and His-83 were identified as nonconserved residues on the exposed surface of the alpha crystallin core domain. Site-directed mutagenesis produced the mutant alphaB crystallins: S76E, N78G, K82Q, and H83F. Domain swapping with homologous beta3 sequences, 32EKFEVGLDVQFFT44 from Caenorhabditis elegans sHSP12.2 or 69DKFVIFLDVKHFS81 from alphaA crystallin, resulted in the mutant alphaB crystallins, CE1 and alphaA1, respectively. Decreased chaperone activity was observed with the point mutants N78G, K82Q, and H83F and with the mutant, CE1, in aggregation assays using betaL crystallin, alcohol dehydrogenase (ADH), or citrate synthase (CS). The S76E mutant had minimal effect on chaperone activity, and domain swapping with alphaA crystallin had no effect on chaperone activity. The mutations that resulted in altered chaperone activity, produced minimal modification to the secondary, tertiary, and quaternary structure of human alphaB crystallin as determined by ultraviolet circular dichroism spectroscopy, chymotrypsin proteolysis, and size exclusion chromatography. Chaperone activity was influenced by the amount of unfolding of the target proteins and independent of complex size. The results characterized the importance of the exposed side chains of Glu-78, Lys-82, and His-83 in the interactive beta3 sequence of the alpha crystallin core domain in alphaB crystallin for chaperone function.

    Funded by: NEI NIH 1f40 HHS: EY04542; NEI NIH HHS: R01 EY004542

    Cell stress & chaperones 2006;11;2;187-97

  • AlphaB-crystallin, a small heat-shock protein, prevents the amyloid fibril growth of an amyloid beta-peptide and beta2-microglobulin.

    Raman B, Ban T, Sakai M, Pasta SY, Ramakrishna T, Naiki H, Goto Y and Rao ChM

    Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.

    AlphaB-crystallin, a small heat-shock protein, exhibits molecular chaperone activity. We have studied the effect of alphaB-crystallin on the fibril growth of the Abeta (amyloid beta)-peptides Abeta-(1-40) and Abeta-(1-42). alphaB-crystallin, but not BSA or hen egg-white lysozyme, prevented the fibril growth of Abeta-(1-40), as revealed by thioflavin T binding, total internal reflection fluorescence microscopy and CD spectroscopy. Comparison of the activity of some mutants and chimaeric alpha-crystallins in preventing Abeta-(1-40) fibril growth with their previously reported chaperone ability in preventing dithiothreitol-induced aggregation of insulin suggests that there might be both common and distinct sites of interaction on alpha-crystallin involved in the prevention of amorphous aggregation of insulin and fibril growth of Abeta-(1-40). alphaB-crystallin also prevents the spontaneous fibril formation (without externally added seeds) of Abeta-(1-42), as well as the fibril growth of Abeta-(1-40) when seeded with the Abeta-(1-42) fibril seed. Sedimentation velocity measurements show that alphaB-crystallin does not form a stable complex with Abeta-(1-40). The mechanism by which it prevents the fibril growth differs from the known mechanism by which it prevents the amorphous aggregation of proteins. alphaB-crystallin binds to the amyloid fibrils of Abeta-(1-40), indicating that the preferential interaction of the chaperone with the fibril nucleus, which inhibits nucleation-dependent polymerization of amyloid fibrils, is the mechanism that is predominantly involved. We found that alphaB-crystallin prevents the fibril growth of beta2-microglobulin under acidic conditions. It also retards the depolymerization of beta2-microglobulin fibrils, indicating that it can interact with the fibrils. Our study sheds light on the role of small heat-shock proteins in protein conformational diseases, particularly in Alzheimer's disease.

    The Biochemical journal 2005;392;Pt 3;573-81

  • Interactions of HSP22 (HSPB8) with HSP20, alphaB-crystallin, and HSPB3.

    Fontaine JM, Sun X, Benndorf R and Welsh MJ

    Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

    Seven of the 10 mammalian small heat shock proteins (sHSP) are expressed in muscle where they constitute 3% or more of total protein. sHSPs interact with one another, and these interactions are believed to be important for their functions. In cell types expressing multiple sHSPs, it is of interest to know which sHSPs interact with one another. We have previously shown that HSP22 interacts with itself as well as with HSP27, MKBP, and cvHSP. Using yeast two-hybrid assays and Förster resonance energy transfer microscopy, we now show that HSP22 also can interact with two additional members of the sHSP family, alphaB-crystallin and HSP20. We also show that HSP22 is found in HPLC fractions of primate cardiac muscle containing high molecular weight complexes that include alphaB-crystallin and HSP20. Our results suggest that a variety of oligomers composed of different proportions of different sHSPs may form in cell types expressing multiple sHSPs.

    Funded by: NIEHS NIH HHS: P01-ES11188

    Biochemical and biophysical research communications 2005;337;3;1006-11

  • Interactive domains for chaperone activity in the small heat shock protein, human alphaB crystallin.

    Ghosh JG and Clark JI

    Biomolecular Structure and Design, University of Washington, Seattle, Washington 98195-7420, USA.

    Protein pin arrays identified seven interactive sequences for chaperone activity in human alphaB crystallin using natural lens proteins, beta(H) crystallin and gammaD crystallin, and in vitro chaperone target proteins, alcohol dehydrogenase and citrate synthase. The N-terminal domain contained two interactive sequences, (9)WIRRPFFPFHSP(20) and (43)SLSPFYLRPPSFLRAP(58). The alpha crystallin core domain contained four interactive sequences, (75)FSVNLDVK(82) (beta3), (113)FISREFHR(120), (131)LTITSSLS(138) (beta8), and (141)GVLTVNGP(148) (beta9). The C-terminal domain contained one interactive sequence, (157)RTIPITRE(164), that included the highly conserved I-X-I/V motif. Two interactive sequences, (73)DRFSVNLDVKHFS(85) and (131)LTITSSLSDGV(141), belonging to the alpha crystallin core domain were synthesized as peptides and assayed for chaperone activity in vitro. Both synthesized peptides inhibited the thermal aggregation of beta(H) crystallin, alcohol dehydrogenase, and citrate synthase in vitro. Five of the seven chaperone sequences identified by the pin arrays overlapped with sequences identified previously as sequences for subunit-subunit interactions in human alphaB crystallin. The results suggested that inte 266 ractive sequences in human alphaB crystallin have dual roles in subunit-subunit assembly and chaperone activity.

    Funded by: NEI NIH HHS: EY04542, R01 EY004542

    Biochemistry 2005;44;45;14854-69

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • Alphab-crystallin-assisted reactivation of glucose-6-phosphate dehydrogenase upon refolding.

    Kumar MS, Reddy PY, Sreedhar B and Reddy GB

    National Institute of Nutrition (ICMR), Jamai Osmania, Hyderabad 500 007, India.

    Alphab-crystallin, a small heat-shock protein has been shown to prevent the aggregation of other proteins under various stress conditions. We have investigated the role of alphaB-crystallin in the reactivation of denaturant [GdmCl (guanidinium chloride)]-inactivated G6PD (glucose-6-phosphate dehydrogenase). Studies indicate that unfolding and inactivation of G6PD by GdmCl proceeds via formation of a molten globule-like state at low concentrations of GdmCl, which was characterized by having maximum surface hydrophobicity and no catalytic activity. At high concentrations of GdmCl, G6PD was completely unfolded, which upon dilution-induced refolding yielding 35% of original activity. In contrast, no activity was recovered when G6PD was refolded from a molten globule-like state. Interestingly, refolding of completely unfolded G6PD in the presence of alphaB-crystallin resulted in 70% gain of the original activity, indicating that alphaB-crystallin assisted in enhanced refolding of G6PD. Intriguingly, alphaB-crystallin was unable to reactivate G6PD from a molten globule-like state. Size-exclusion chromatography data indicate that alphaB-crystallin-assisted reactivation of completely unfolded G6PD is concomitant with the restoration of the native structure of G6PD. Nonetheless, alphaB-crystallin failed to reactivate G6PD from preformed aggregates. Moreover, methylglyoxal-modified alpha-crystallin, which occurs in aged and diabetic cataract lenses, was less efficient in the reactivation of denaturant inactivated G6PD. Diminished chaperone-like activity of alpha-crystallin due to post-translational modifications may thus result in the accumulation of aggregated/inactivated proteins.

    The Biochemical journal 2005;391;Pt 2;335-41

  • Recognition sequence 2 (residues 60-71) plays a role in oligomerization and exchange dynamics of alphaB-crystallin.

    Sreelakshmi Y and Sharma KK

    Department of Ophthalmology, University of Missouri, Columbia, Missouri 65212, USA.

    Previously, using the peptide scan method, we have determined that residues 42-57 and 60-71 in alphaB-crystallin (TSLSPFYLRPPSFLRA, named recognition sequence 1 or RS-1, and WFDTGLSEMRLE, named recognition sequence 2 or RS-2) are involved in interaction with alphaA-crystallin. To understand the significance of the RS-2 region in interactions between alphaA- and alphaB-crystallins, W60R, F61N, and S66G mutants of alphaB-crystallin were made and tested for their ability to interact with alphaA-crystallin. W60R and S66G mutations increased the oligomeric size of alphaB-crystallin by 1.6- and 2.7-fold respectively, whereas the F61N mutation had no effect. The tryptophan fluorescence intensity of alphaBS66G was 1.5-fold higher than that for the wild type. The intrinsic fluorescence of alphaBF61N was marginally lower than that of alphaB, whereas the fluorescence intensity of alphaBW60R decreased by 40% compared with that of alphaB. The relative availability of hydrophobic sites in the mutants was in the following order: alphaBS66G > alphaB = alphaBF61N = alphaBW60R. The far-UV CD profiles for the wild type and alphaB-crystallin mutants indicated no significant changes in their secondary structures, except for alphaBS66G, which showed an increase in alpha-helical content. The near-UV CD profiles of alphaBW60R and alphaBF61N were nearly similar to that of wild type alphaB. On the other hand, alphaBS66G beyond 270 nm exhibited a signature completely different from that of wild type alphaB. Mutations did not alter the chaperone-like activity of these proteins. The W60R mutation did not affect the rate of subunit exchange between alphaB- and alphaA-crystallins. On the other hand, the S66G mutation increased the subunit exchange rate by 100%, whereas the F61N mutation decreased the rate of subunit exchange between alphaB- and alphaA-crystallins by 36%. Our results establish the importance of residues 60-71 in oligomerization of alphaB-crystallin and subunit interaction between alphaB- and alphaA-crystallins.

    Funded by: NEI NIH HHS: EY 11981, EY 14795

    Biochemistry 2005;44;36;12245-52

  • Insights into hydrophobicity and the chaperone-like function of alphaA- and alphaB-crystallins: an isothermal titration calorimetric study.

    Kumar MS, Kapoor M, Sinha S and Reddy GB

    Biochemistry Division, National Institute of Nutrition, Hyderabad 500 007, India.

    Alpha-crystallin, composed of two subunits, alphaA and alphaB, has been shown to function as a molecular chaperone that prevents aggregation of other proteins under stress conditions. The exposed hydrophobic surfaces of alpha-crystallins have been implicated in this process, but their exact role has not been elucidated. In this study, we quantify the hydrophobic surfaces of alphaA- and alphaB-crystallins by isothermal titration calorimetry using 8-anilino-1-napthalenesulfonic acid (ANS) as a hydrophobic probe and analyze its correlation to the chaperone potential of alphaA- and alphaB-crystallins under various conditions. Two ANS binding sites, one with low and another with high affinity, were clearly detected, with alphaB showing a higher number of sites than alphaA at 30 degrees C. In agreement with the higher number of hydrophobic sites, alphaB-crystallin demonstrated higher chaperone activity than alphaA at this temperature. Thermodynamic analysis of ANS binding to alphaA- and alphaB-crystallins indicates that high affinity binding is driven by both enthalpy and entropy changes, with entropy dominating the low affinity binding. Interestingly, although the number of ANS binding sites was similar for alphaA and alphaB at 15 degrees C, alphaA was more potent than alphaB in preventing aggregation of the insulin B-chain. Although there was no change in the number of high affinity binding sites of alphaA and alphaB for ANS upon preheating, there was an increase in the number of low affinity sites of alphaA and alphaB. Preheated alphaA, in contrast to alphaB, exhibited remarkably enhanced chaperone activity. Our results indicate that although hydrophobicity appears to be a factor in determining the chaperone-like activity of alpha-crystallins, it does not quantitatively correlate with the chaperone function of alpha-crystallins.

    The Journal of biological chemistry 2005;280;23;21726-30

  • Identification and validation of novel ERBB2 (HER2, NEU) targets including genes involved in angiogenesis.

    Beckers J, Herrmann F, Rieger S, Drobyshev AL, Horsch M, Hrabé de Angelis M and Seliger B

    GSF-National Research Center for Environment and Health, Institute of Experimental Genetics, Neuherberg, Germany. beckers@gsf.de

    V-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2; synonyms HER2, NEU) encodes a transmembrane glycoprotein with tyrosine kinase-specific activity that acts as a major switch in different signal-transduction processes. ERBB2 amplification and overexpression have been found in a number of human cancers, including breast, ovary and kidney carcinoma. Our aim was to detect ERBB2-regulated target genes that contribute to its tumorigenic effect on a genomewide scale. The differential gene expression profile of ERBB2-transfected and wild-type mouse fibroblasts was monitored employing DNA microarrays. Regulated expression of selected genes was verified by RT-PCR and validated by Western blot analysis. Genome wide gene expression profiling identified (i) known targets of ERBB2 signaling, (ii) genes implicated in tumorigenesis but so far not associated with ERBB2 signaling as well as (iii) genes not yet associated with oncogenic transformation, including novel genes without functional annotation. We also found that at least a fraction of coexpressed genes are closely linked on the genome. ERBB2 overexpression suppresses the transcription of antiangiogenic factors (e.g., Sparc, Timp3, Serpinf1) but induces expression of angiogenic factors (e.g., Klf5, Tnfaip2, Sema3c). Profiling of ERBB2-dependent gene regulation revealed a compendium of potential diagnostic markers and putative therapeutic targets. Identification of coexpressed genes that colocalize in the genome may indicate gene regulatory mechanisms that require further study to evaluate functional coregulation. (Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.)

    International journal of cancer 2005;114;4;590-7

  • The small heat shock protein alpha B-crystallin is a novel inhibitor of TRAIL-induced apoptosis that suppresses the activation of caspase-3.

    Kamradt MC, Lu M, Werner ME, Kwan T, Chen F, Strohecker A, Oshita S, Wilkinson JC, Yu C, Oliver PG, Duckett CS, Buchsbaum DJ, LoBuglio AF, Jordan VC and Cryns VL

    Cell Death Regulation Laboratory, Department of Medicine and Cell, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611.

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor alpha family of cytokines that preferentially induces apoptosis in transformed cells, making it a promising cancer therapy. However, many neoplasms are resistant to TRAIL-induced apoptosis by mechanisms that are poorly understood. We demonstrate that the expression of the small heat shock protein alpha B-crystallin (but not other heat shock proteins or apoptosis-regulating proteins) correlates with TRAIL resistance in a panel of human cancer cell lines. Stable expression of wild-type alpha B-crystallin, but not a pseudophosphorylation mutant impaired in its assembly and chaperone function, protects cancer cells from TRAIL-induced caspase-3 activation and apoptosis in vitro. Furthermore, selective inhibition of alpha B-crystallin expression by RNA interference sensitizes cancer cells to TRAIL. In addition, wild-type alpha B-crystallin promotes xenograft tumor growth and inhibits TRAIL-induced apoptosis in vivo in nude mice, whereas a pseudophosphorylation alpha B-crystallin mutant impaired in its anti-apoptotic function inhibits xenograft tumor growth. Collectively, these findings indicate that alpha B-crystallin is a novel regulator of TRAIL-induced apoptosis and tumor growth. Moreover, these results demonstrate that targeted inhibition of alpha B-crystallin promotes TRAIL-induced apoptosis, thereby suggesting a novel strategy to overcome TRAIL resistance in cancer.

    Funded by: NCI NIH HHS: P50CA89018, T32CA70085; NIDDK NIH HHS: T32DK07169

    The Journal of biological chemistry 2005;280;12;11059-66

  • Alpha B-crystallin is a major component of glial cytoplasmic inclusions in multiple system atrophy.

    Pountn 1f40 ey DL, Treweek TM, Chataway T, Huang Y, Chegini F, Blumbergs PC, Raftery MJ and Gai WP

    Department of Human Physiology, Flinders University, Adelaide 5001, Australia.

    Multiple system atrophy (MSA) is characterized by the formation of oligodendroglial cytoplasmic inclusions (GCIs) consisting of alpha-synuclein filaments. AlphaB-crystallin, a small chaperone protein that binds to unfolded proteins and inhibits aggregation, has been documented in GCIs. We investigated the relative abundance and speciation of alphaB-crystallin in GCIs in MSA brains. We also examined the influence of alphaB-crystallin on the formation of cytoplasmic inclusions in cultured glial cells. Immunohistochemistry and confocal microscopy revealed alphaB-crystallin is a prominent component of GCIs, more abundant than in Lewy bodies in Lewy body dementia. One- and two-dimensional gel electrophoresis and mass spectrometric analysis of GCIs immunopurified from MSA brains indicated that alphaB-crystallin is a major protein component with multiple post-translationally modified species. In cultured C6 glioma cells treated with the proteasomal inhibitor, lactacystin, to induce accumulation of ubiquitinated proteins, a subset of cells showed increased cytoplasmic staining for alphaB-crystallin. Proteasome-inhibited cells transfected with GFP-tagged alpha-synuclein resulted in ubiquitin- and alphaB-crystallin-positive aggregates resembling GCIs in MSA brains. Our results indicate that alphaB-crystallin is a major chaperone in MSA, and suggest a role of the protein in the formation of inclusion bodies in glial cells.

    Neurotoxicity research 2005;7;1-2;77-85

  • Mimicking phosphorylation of the small heat-shock protein alphaB-crystallin recruits the F-box protein FBX4 to nuclear SC35 speckles.

    den Engelsman J, Bennink EJ, Doerwald L, Onnekink C, Wunderink L, Andley UP, Kato K, de Jong WW and Boelens WC

    Department of Biochemistry 161, Nijmegen Center for Molecular Life Sciences, University of Nijmegen, the Netherlands.

    The mammalian small heat shock protein alphaB-crystallin can be phosphorylated at three different sites, Ser19, Ser45 and Ser59. We compared the intracellular distribution of wild-type, nonphosphorylatable and all possible pseudophosphorylation mutants of alphaB-crystallin by immunoblot and immunocytochemical analyses of stable and transiently transfected cells. We observed that pseudophosphorylation at two (especially S19D/S45D) or all three (S19D/S45D/S59D) sites induced the partial translocation of alphaB-crystallin from the detergent-soluble to the detergent-insoluble fraction. Double immunofluorescence studies showed that the pseudophosphorylation mutants localized in nuclear speckles containing the splicing factor SC35. The alphaB-crystallin mutants in these speckles were resistant to mild detergent treatment, and also to DNase I or RNase A digestion, indicating a stable interaction with one or more speckle proteins, not dependent on intact DNA or RNA. We further found that FBX4, an adaptor protein of the ubiquitin-protein isopeptide ligase SKP1/CUL1/F-box known to interact with pseudophosphorylated alphaB-crystallin, was also recruited to SC35 speckles when cotransfected with the pseudophosphorylation mutants. Because SC35 speckles also react with an antibody against alphaB-crystallin endogenously phosphorylated at Ser45, our findings suggest that alphaB-crystallin has a phosphorylation-dependent role in the ubiquitination of a component of SC35 speckles.

    European journal of biochemistry 2004;271;21;4195-203

  • Deamidation affects structural and functional properties of human alphaA-crystallin and its oligomerization with alphaB-crystallin.

    Gupta R and Srivastava OP

    Department of Physiological Optics, University of Alabama at Birmingham, Birmingham, Alabama 35294-4390, USA.

    To determine the effects of deamidation on structural and functional properties of alphaA-crystallin, three mutants (N101D, N123D, and N101D/N123D) were generated. Deamidated alphaB-crystallin mutants (N78D, N146D, and N78D/N146D), characterized in a previous study (Gupta, R., and Srivastava, O. P. (2004) Invest. Ophthalmol. Vis. Sci. 45, 206-214) were also used. The biophysical and chaperone properties were determined in (a) homoaggregates of alphaA mutants (N101D, N123D, and N101D/N123D) and (b) reconstituted heteroaggregates of alpha-crystallin containing (i) wild type alphaA (WT-alphaA): WT-alphaB crystallins, (ii) individual alphaA-deamidated mutants:WT-alphaB crystallins, and (iii) WT-alphaA:individual alphaB-deamidated mutant crystallins. Compared with the WT-alphaA, the three alphaA-deamidated mutants showed reduced levels of chaperone activity, alterations in secondary and tertiary structures, and larger aggregates. These altered properties were relatively more pronounced in the mutant N101D compared with the mutant N123D. Further, compared with heteroagg 118a regates of WT-alphaA and WT-alphaB, the heteroaggregates containing deamidated subunits of either alphaA- or alphaB-crystallins and their counterpart WT proteins showed higher molecular mass, altered tertiary structures, lower exposed hydrophobic surfaces, and reduced chaperone activity. However, the heteroaggregate containing WT-alphaA and deamidated alphaB subunit showed lower chaperone activity, smaller oligomers, and 3-fold lower subunit exchange rate than heteroaggregate containing deamidated alphaA- and WT-alphaB subunits. Together, the results suggested that (a) both Asn residues (Asn-101 and Asn-123) are required for the structural integrity and chaperone function of alphaA-crystallin and (b) the presence of WT-alphaB in the alpha-crystallin heteroaggregate leads to packing-induced structural changes which influences the oligomerization and modulate chaperone activity.

    Funded by: NEI NIH HHS: EY-06400

    The Journal of biological chemistry 2004;279;43;44258-69

  • Developmentally dictated expression of heat shock factors: exclusive expression of HSF4 in the postnatal lens and its specific interaction with alphaB-crystallin heat shock promoter.

    Somasundaram T and Bhat SP

    Jules Stein Eye Institute, University of California, Los Angeles, California 90095-7000, USA.

    The molecular cascade of stress response in higher eukaryotes commences in the cytoplasm with the trimerization of the heat shock factor 1 (HSF1), followed by its transport to the nucleus, where it binds to the heat shock element leading to the activation of transcription from the down-stream gene(s). This well-established paradigm has been mostly studied in cultured cells. The developmental and tissue-specific control of the heat shock transcription factors (HSFs) and their interactions with heat shock promoters remain unexplored. We report here that in the rat lens, among the three mammalian HSFs, expression of HSF1 and HSF2 i bb0 s largely fetal, whereas the expression of HSF4 is predominantly postnatal. Similar pattern of expression of HSF1 and HSF4 is seen in fetal and adult human lenses. This stage-specific inverse relationship between the expression of HSF1/2 and HSF4 suggests tissue-specific management of stress depending on the presence or absence of specific HSF(s). In addition to real-time PCR and immunoblotting, gel mobility shift assays, coupled with specific antibodies and HSE probes, derived from three different heat shock promoters, establish that there is no HSF1 or HSF2 binding activity in the postnatal lens nuclear extracts. Using this unique, developmentally modulated in vivo system, we demonstrate 1) specific patterns of HSF4 binding to heat shock elements derived from alphaB-crystallin, Hsp70, and Hsp82 promoters and 2) that it is HSF4 and not HSF1 or HSF2 that interacts with the canonical heat shock element of the alphaB-crystallin gene.

    The Journal of biological chemistry 2004;279;43;44497-503

  • Small heat shock protein alphaB-crystallin is part of cell cycle-dependent Golgi reorganization.

    Gangalum RK, Schibler MJ and Bhat SP

    Jules Stein Eye Institute, University of California, Los Angeles 90095-7000, USA.

    AlphaB-crystallin is a developmentally regulated small heat shock protein known for its binding to a variety of denatured polypeptides and suppression of protein aggregation in vitro. Elevated levels of alphaB-crystallin are known to be associated with a number of neurodegenerative pathologies such as Alzheimer disease and multiple sclerosis. Mutations in alphaB-crystallin gene have been linked to desmin related cardiomyopathy and cataractogenesis. The physiological function of this protein, however, is unknown. Using discontinuous sucrose density gradient fractionation of post-nuclear supernatants, prepared from rat tissues and human glioblastoma cell line U373MG, we have identified discrete membrane-bound fractions of alphaB-crystallin, which co-sediment with the Golgi matrix protein, GM130. Confocal microscopy reveals co-localization of alphaB-crystallin with BODIPY TR ceramide and the Golgi matrix protein, GM130, in the perinuclear Golgi in human glioblastoma U373MG cells. Examination of synchronized cultures indicated that alphaB-crystallin follows disassembly of the Golgi at prometaphase and its reassembly at the completion of cytokinesis, suggesting that this small heat shock protein, with its chaperone-like activity, may have an important role in the Golgi reorganization during cell division.

    The Journal of biological chemistry 2004;279;42;43374-7

  • The reaction of alpha-crystallin with the cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate) demonstrates close proximity of the C termini of alphaA and alphaB in the native assembly.

    Swaim CL, Smith DL and Smith JB

    Department of Chemistry, University of Nebraska, Lincoln, NE 68588-0304. jsmith8@unl.edu

    Protein science : a publication of the Protein Society 2004;13;10;2832-5

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • From ORFeome to biology: a functional genomics pipeline.

    Wiemann S, Arlt D, Huber W, Wellenreuther R, Schleeger S, Mehrle A, Bechtel S, Sauermann M, Korf U, Pepperkok R, Sültmann H and Poustka A

    Molecular Genome Analysis, German Cancer Research Center, 69120 Heidelberg, Germany. s.wiemann@dkfz.de

    As several model genomes have been sequenced, the elucidation of protein function is the next challenge toward the understanding of biological processes in health and disease. We have generated a human ORFeome resource and established a functional genomics and proteomics analysis pipeline to address the major topics in the post-genome-sequencing era: the identification of human genes and splice forms, and the determination of protein localization, activity, and interaction. Combined with the understanding of when and where gene products are expressed in normal and diseased conditions, we create information that is essential for understanding the interplay of genes and proteins in the complex biological network. We have implemented bioinformatics tools and databases that are suitable to store, analyze, and integrate the different types of data from high-throughput experiments and to include further annotation that is based on external information. All information is presented in a Web database (http://www.dkfz.de/LIFEdb). It is exploited for the identification of disease-relevant genes and proteins for diagnosis and therapy.

    Genome research 2004;14;10B;2136-44

  • Interaction of the molecular chaperone alphaB-crystallin with alpha-synuclein: effects on amyloid fibril formation and chaperone activity.

    Rekas A, Adda CG, Andrew Aquilina J, Barnham KJ, Sunde M, Galatis D, Williamson NA, Masters CL, Anders RF, Robinson CV, Cappai R and Carver JA

    Department of Chemistry, University of Wollongong, Northfields Avenue, Wollongong, NSW 2522, Australia.

    alpha-Synuclein is a pre-synaptic protein, the function of which is not completely understood, but its pathological form is involved in neurodegenerative diseases. In vitro, alpha-synuclein spontaneously forms amyloid fibrils. Here, we report that alphaB-crystallin, a molecular chaperone found in Lewy bodies that are characteristic of Parkinson's disease (PD), is a potent in vitro inhibitor of alpha-synuclein fibrillization, both of wild-type and the two mutant forms (A30P and A53T) that cause familial, early onset PD. In doing so, large irregular aggregates of alpha-synuclein and alphaB-crystallin are formed implying that alphaB-crystallin redirects alpha-synuclein from a fibril-formation pathway towards an amorphous aggregation pathway, thus reducing the amount of physiologically stable amyloid deposits in favor of easily degradable amorphous aggregates. alpha-Synuclein acts as a molecular chaperone to prevent the stress-induced, amorphous aggregation of target proteins. Compared to wild-type alpha-synuclein, both mutant forms have decreased chaperone activity in vitro against the aggregation of reduced insulin at 37 degrees C and the thermally induced aggregation of betaL-crystallin at 60 degrees C. Wild-type alpha-synuclein abrogates the chaperone activity of alphaB-crystallin to prevent the precipitation of reduced insulin. Interaction between these two chaperones and formation of a complex are also indicated by NMR spectroscopy, size-exclusion chromatography and mass spectrometry. In summary, alpha-synuclein and alphaB-crystallin interact readily with each other and affect each other's properties, in particular alpha-synuclein fibril formation and alphaB-crystallin chaperone action.

    Journal of molecular biology 2004;340;5;1167-83

  • Phosphorylation of alphaB-crystallin alters chaperone function through loss of dimeric substructure.

    Aquilina JA, Benesch JL, Ding LL, Yaron O, Horwitz J and Robinson CV

    Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom.

    Phosphorylation is the most common posttranslational modification of the alpha-crystallins in the human lens. These phosphorylated forms are not only important because of their abundance in aging lenses and the implications for cataract but also because they have been identified in patients with degenerative brain disease. By using mimics corresponding to the reported in vivo phosphorylation sites in the human lens, we have examined the effects of phosphorylation upon the chaperone-like properties and structure of alphaB-crystallin. Here we show that phosphorylation of alphaB-crystallin at Ser-45 results in uncontrolled aggregation. By using an innovative tandem mass spectrometry approach, we demonstrate how this alteration in behavior stems from disruption of dimeric substructure within the polydisperse alphaB-crystallin assembly. This structural perturbation appears to disturb the housekeeping role of alphaB-crystallin and consequently has important implications for the disease states caused by protein aggregation in the lens and deposition in non-lenticular tissue.

    Funded by: NEI NIH HHS: EY 3897

    The Journal of biological chemistry 2004;279;27;28675-80

  • Human alphaA- and alphaB-crystallins bind to Bax and Bcl-X(S) to sequester their translocation during staurosporine-induced apoptosis.

    Mao YW, Liu JP, Xiang H and Li DW

    Department of Molecular Biology, University of Medicine and Dentistry of New Jersey, Stratford, NJ, USA.

    AlphaA- and alphaB-crystallins are distinct antiapoptotic regulators. Regarding the antiapoptotic mechanisms, we have recently demonstrated that alphaB-crystallin interacts with the procaspase-3 and partially processed procaspase-3 to repress caspase-3 activation. Here, we demonstrate that human alphaA- and alphaB-crystallins prevent staurosporine-induced apoptosis through interactions with members of the Bcl-2 family. Using GST pulldown assays and coimmunoprecipitations, we demonstrated that alpha-crystallins bind to Bax and Bcl-X(S) both in vitro and in vivo. Human alphaA- and alphaB-crystallins display similar affinity to both proapoptotic regulators, and so are true with their antiapoptotic ability tested in human lens epithelial cells, human retina pigment epithelial cells (ARPE-19) and rat embryonic myocardium cells (H9c2) under treatment of staurosporine, etoposide or sorbitol. Two prominent mutants, R116C in alphaA-crystallin and R120G, in alphaB-crystallin display much weaker affinity to Bax and Bcl-X(S). Through the interaction, alpha-crystallins prevent the translocation of Bax and Bcl-X(S) from cytosol into mitochondria during staurosporine-induced apoptosis. As a result, alpha-crystallins preserve the integrity of mitochondria, restrict release of cytochrome c, repress activation of caspase-3 and block degradation of PARP. Thus, our results demonstrate a novel antiapoptotic mechanism for alpha-crystallins.

    Funded by: NEI NIH HHS: EY11372, R29 EY011372

    Cell death and differentiation 2004;11;5;512-26

  • Proteins associated with type II bone morphogenetic protein receptor (BMPR-II) and identified by two-dimensional gel electrophoresis and mass spectrometry.

    Hassel S, Eichner A, Yakymovych M, Hellman U, Knaus P and Souchelnytskyi S

    Ludwig Institute for Cancer Research, Uppsala, Sweden.

    Bone morphogenetic proteins (BMP) are polypeptide growth factors that regulate cell differentiation and proliferation. BMPs bind to type I and type II serine/threonine kinase receptors to initiate intracellular signalling. BMPR-II is the type II receptor, its mutations lead to hereditary pulmonary hypertension, and knockout of Bmpr-II results in early embryonic lethality. To identify novel interacting proteins and explore signalling pathways that can be initiated by BMPR-II, we performed glutathione-S-transferase (GST) pull-down assays with BMPR-II protein constructs fused to GST and extracts of mouse myoblast C2C12 cells. We generated three constructs which contain different parts of the cytoplasmic region of BMPR-II: full-length cytoplasmic part of BMPR-II, only the kinase domain, or only the C-terminal tail of BMPR-II. Proteins which formed complexes with these BMPR-II constructs were analyzed by two-dimensional gel electrophoresis (2-D GE), and specifically interacting proteins were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). We identified 33 interacting proteins; 11 proteins interacted with the C-terminal tail of BMPR-II, 4 with full-length BMPR-II, and 18 with a short form of the receptor with a deleted tail. Fourteen proteins have assigned functions in various signalling processes, suggesting links of BMP signalling to regulation of MAP kinase pathway, apoptosis, transcription, PKCss, and PKA. Five of the identified proteins are components of the cytoskeleton, and four are enzymes involved in metabolism, e.g., processing of estrogens or lipids. We confirmed interaction of PKC beta and CtBP with BMPR-II using immunodetection. We showed that the C-terminal tail of BMPR-II provides binding sites for a number of regulatory proteins that may initiate Smad-independent signalling.

    Proteomics 2004;4;5;1346-58

  • Accumulation and aberrant modifications of alpha-crystallins in anterior polar cataracts.

    Hwang KH, Lee EH, Jho EH, Kim JH, Lee DH, Chung SK, Kim EK and Joo CK

    Department of Ophthalmology and Visual Science, Korea Eye Tissue and Gene Band, College of Medicine, The Catholic University of Korea, Seoul, Korea.

    Crystallins are the major proteins found in the lens, and the localization of specific crystallins is well known. Overexpression and accumulation of alphaB-crystallin has been observed in response to stress conditions or in certain diseases, such as brain tumors and neurodegenerative diseases. The purpose of this study was to examine whether alpha-crystallins are modified during pathological myofibroblastic changes in lens epithelial cells. Lens epithelial cells attached to the anterior capsules of patients with nuclear or anterior polar cataracts were analyzed quantitatively for alpha-crystallin proteins and mRNAs using Western blot and RT-PCR analysis., respectively. The degree of modification of alpha-crystallins was determined by 2-dimensional gel electrophoresis followed by Western blotting. Higher molecular weight protein bands that were immunoreactive to anti-alphaA- and anti-alphaB-crystallin antibodies around 45 kDa accumulated more in the anterior polar cataract samples than in those with the nuclear type of cataracts. Also monomeric alphaB-crystallins accumulated more in lens epithelial cells of patients with anterior polar cataracts. By comparison, no significant changes were found in the levels of the mRNAs encoding alphaA- and alphaB-crystallins in the different types of cataracts. Both alphaA- and alphaB-crystallin proteins seemed to undergo more extensive modification in anterior polar cataracts. Conclusion. In addition to fibrotic changes, which accompany increased levels of extracellular matrix molecules, accumulation and abnormal modification of alpha-crystallins might be implicated in the pathogenic mechanism of this type of cataract.

    Yonsei medical journal 2004;45;1;73-80

  • CD4 T-cell epitopes of human alpha B-crystallin.

    Chou YK, Burrows GG, LaTocha D, Wang C, Subramanian S, Bourdette DN and Vandenbark AA

    Department of Neurology, Oregon Health and Science University, Portland, Oregon 97201, USA. chouy@ohsu.edu

    Of potential importance to multiple sclerosis (MS), oligodendroglial alpha B-crystallin is expressed and associated with the myelin sheath at the earliest stage of MS lesion development. We selected T-cell lines specific for human alpha B-crystallin from peripheral blood mononuclear cells (PBMC) of HLA-DR2 homozygous MS patients and found that the alpha B-crystallin-specific T-cells were CD4+ and restricted by DRB1*1501, and expressed Th1 cytokines. The CD4 T-cell epitopes of human alpha B-crystallin were determined by proliferation of alpha B-crystallin-specific T-cell lines to 17 20-mer synthetic overlapping peptides spanning the entire molecule of human alpha B-crystallin. It was found that the HLA-DR2 donor-derived alpha B-crystallin-specific T-cell lines proliferated to alpha B-crystallin peptides 21-40, 41-60, and to a lesser extent, 131-150. These T-cell prolifera 49a tion responses were associated with intracellular expression of interleukin-2 (IL-2) and secretion of interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). The amino acid sequences of these peptides were compatible with predicted HLA-DR2-restricted binding motifs. PBMC of an early active MS patient proliferated to the epitope-containing peptides significantly better than did those of later stage MS patients or healthy controls. Taken together, these findings suggest that autoreactive alpha B-crystallin-specific Th1 cells may have the potential to contribute to MS pathogenesis.

    Funded by: NINDS NIH HHS: NS23221, NS41965

    Journal of neuroscience research 2004;75;4;516-23

  • Identification of metastasis-associated proteins through protein analysis of metastatic MDA-MB-435 and metastasis-suppressed BRMS1 transfected-MDA-MB-435 cells.

    Cicek M, Samant RS, Kinter M, Welch DR and Casey G

    Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.

    BRMS1 (breast cancer metastasis suppressor 1) was recently identified as a novel breast cancer metastasis suppressor gene. To further characterize BRMS1-mediated metastasis suppression, we applied two-dimensional proteomic and mass spectrometry (LC-tandem MS and MALDI-TOF) analysis to identify proteins differentially expressed between highly metastatic MDA-MB-435 cells and metastasis-suppressed BRMS1-transfected MDA-MB-435 cells. Quadruplicate independent 2D gels were run and analyzed under identical conditions. Following in-gel trypsin digestion of seven differentially expressed proteins, amino acid sequence and mass profiles of the peptides were generated. Proteins were identified from the NCBI non-redundant database using the search program TurboSequest. Differential expression was confirmed for five proteins, including annexin I and alpha B-crystallin, by Northern blot analysis and immunostaining. Furthermore, we showed that both proteins were expressed in vivo in lungs containing metastasized MDA-MB-435 cells but not expressed in normal lung tissue of athymic mice. Our results suggest that annexin I and alpha B-crystallin are important cellular proteins that are down regulated through BRMS1 mediated metastasis suppression.

    Funded by: NCI NIH HHS: CA87728, CA89019

    Clinical & experimental metastasis 2004;21;2;149-57

  • Role of the conserved SRLFDQFFG region of alpha-crystallin, a small heat shock protein. Effect on oligomeric size, subunit exchange, and chaperone-like activity.

    Pasta SY, Raman B, Ramakrishna T and Rao ChM

    Centre for Cellular and Molecular Biology, Hyderabad 500 007, India.

    Small heat shock proteins (sHsps) are necessary for several cellular functions and in stress tolerance. Most sHsps are oligomers; intersubunit interactions leading to changes in oligomeric structure and exposure of specific regions may modulate their functioning. Many sHsps, including alpha A- and alpha B-crystallin, contain a well conserved SRLFDQFFG sequence motif in the N-terminal region. Sequence-based prediction shows that it exhibits helical propensity with amphipathic character, suggesting that it plays a critical role in the structure and function of alpha-crystallins. In order to investigate the role of this motif in the structure and function of sHsps, we have made constructs deleting this sequence from alpha A- and alpha B-crystallin, overexpressed, purified, and studied these engineered proteins. Circular dichroism spectroscopic studies show changes in tertiary and secondary structure on deletion of the sequence. Glycerol density gradient centrifugation and dynamic light scattering studies show that the multimeric size of the mutant proteins is significantly reduced, indicating a role for this motif in higher order organization of the subunits. Both deletion mutants exhibit similar oligomeric size and increased chaperone-like activity. Urea-induced denaturation study shows that the SRLFDQFFG sequence contributes significantly to the structural stability. Fluorescence resonance energy transfer studies show that the rate of exchange of the subunits in the alpha Adel-crystallin oligomer is higher compared with that in the alpha A-crystallin oligomer, suggesting that this region contributes to the oligomer dynamics in addition to the higher order assembly and structural stability. Thus, our study shows that the SRLFDQFFG sequence is one of the critical motifs in structure-function regulation of alpha A- and alpha B-crystallin.

    The Journal of biological chemistry 2003;278;51;51159-66

  • Translational thermotolerance provided by small heat shock proteins is limited to cap-dependent initiation and inhibited by 2-aminopurine.

    Doerwald L, Onnekink C, van Genesen ST, de Jong WW and Lubsen NH

    Department of Biochemistry, Faculty of Science, University of Nijmegen, 6500HB Nijmegen, The Netherlands.

    Heat shock results in inhibition of general protein synthesis. In thermotolerant cells, protein synthesis is still rapidly inhibited by heat stress, but protein synthesis recovers faster than in naive heat-shocked cells, a phenomenon known as translational thermotolerance. Here we investigate the effect of overexpressing a single heat shock protein on cap-dependent and cap-independent initiation of translation during recovery from a heat shock. When overexpressing alphaB-crystallin or Hsp27, cap-dependent initiation of translation was protected but no effect was seen on cap-independent initiation of translation. When Hsp70 was overexpressed however, both cap-dependent and -independent translation were protected. This finding indicates a difference in the mechanism of protection mediated by small or large heat shock proteins. Phosphorylation of alphaB-crystallin and Hsp27 is known to significantly decrease their chaperone activity; therefore, we tested phosphorylation mutants of these proteins in this system. AlphaB-crystallin needs to be in its non-phosphorylated state to give protection, whereas phosphorylated Hsp27 is more potent in protection than the unphosphorylatable form. This indicates that chaperone activity is not a prerequisite for protection of translation by small heat shock proteins after heat shock. Furthermore, we show that in the presence of 2-aminopurine, an inhibitor of kinases, among which is double-stranded RNA-activated kinase, the protective effect of overexpressing alphaB-crystallin is abolished. The synthesis of the endogenous Hsps induced by the heat shock to test for thermotolerance is also blocked by 2-aminopurine. Most likely the protective effect of alphaB-crystallin requires synthesis of the endogenous heat shock proteins. Translational thermotolerance would then be a co-operative effect of different heat shock proteins.

    The Journal of biological chemistry 2003;278;50;49743-50

  • Myofibrillar myopathy caused by novel dominant negative alpha B-crystallin mutations.

    Selcen D and Engel AG

    Department of Neurology and Neuromuscular Research Laboratory, Mayo Clinic, Rochester, MN 55905, USA. selcen.duygu@mayo.edu

    We here report the second and third mutations in alphaB-crystallin causing myofibrillar myopathy. Two patients had adult-onset muscle weakness. Patient 1 had cervical, limb girdle, and respiratory muscle weakness and died of respiratory failure. Patient 2 had proximal and distal leg muscle weakness. Both had myopathic electromyogram with abnormal electrical irritability and muscle biopsy findings of myofibrillar myopathy and mild denervation. Myofibrillar disintegration begins at the Z-disk and results in abnormal local expression of desmin, alphaB-crystallin, dystrophin, neural cell adhesion molecule (NCAM), and CDC2 kinase. Seven to 8% of nuclei display early apoptotic changes. Both patients carry a truncating mutation in the C-terminal region of alphaB-crystallin (464delCT in Patient 1 and Q151X in Patient 2) which is crucial for the solubilization and chaperone functions of the molecule. cDNA analysis shows the same mutations and no alternatively spliced transcripts. Immunoblots of muscle demonstrate increased expression of wild-type and reduced expression of the mutant protein. Immunoblots under nondenaturing conditions show that the mutant protein forms lower than normal molecular weight multimeric complexes with wild type. We conclude that (1) despite its reduced expression, the mutant protein exerts a dominant negative effect; (2) mutations in alphaB-crystallin are an infrequent cause of myofibrillar myopathy; (3) alphaB-crystallin-related myopathies display phenotypic heterogeneity.

    Annals of neurology 2003;54;6;804-10

  • [Alpha]B-crystallin genotype has impact on the multiple sclerosis phenotype.

    van Veen T, van Winsen L, Crusius JB, Kalkers NF, Barkhof F, Peña AS, Polman CH and Uitdehaag BM

    Department of Neurology, VU University Medical Center, Amsterdam, The Netherlands. T.vanveen@VUMC.NL

    Background: Both multiple sclerosis (MS) susceptibility and MS clinical phenotype are in part genetically determined. [Alpha]B-crystallin is a candidate autoantigen in MS, and there are three polymorphisms in the promoter region of the encoding gene (CRYAB): at positions -C249G, -C650G, and -A652G.

    Methods: These polymorphisms were studied in sporadic cases of MS, assessing disease susceptibility, clinical phenotype, and MRI appearance.

    Results: The CRYAB polymorphisms influenced susceptibility as well as disease expression in MS.

    Conclusion: Carriers of the rare allele CRYAB-650*C had an increased likelihood of a noninflammatory, neurodegenerative phenotype characterized by a relatively rapid, primary progressive clinical disease course.

    Neurology 2003;61;9;1245-9

  • Alpha B-crystallin is not a dominant peripheral T-cell autoantigen in multiple sclerosis amongst Sardinians.

    Sotgiu S, Pugliatti M, Contu S, Sanna A, Sgaramella E, VanNoort JM and Rosati G

    Institute of Clinical Neurology, University of Sassari, Viale San Pietro, Sassari, Italy. stesot@hotmail.com

    The heat shock protein alpha B-crystallin appears to be the dominantly recognized autoantigen in the early demyelinative process of multiple sclerosis (MS) in brain of patients. In Sardinia, MS is linked to human leucocyte antigen (HLA)-DR alleles that might influence the production of cytokines from peripheral lymphocytes. We tested the nature of peripheral anti-alpha B-crystallin-specific T-cell response in the context of predisposing HLA haplotypes both in MS patients and healthy controls. The alpha B-crystallin specific T-cell lines were generated by using the 'split-well' technique. The results indicate that the presence of short-term T-cell lines towards alpha B-crystallin is numerically comparable between the two groups and not restricted to MS-predisposing HLA-DR alleles. As for the T-cell characterization, CD4+ anti-alpha B-crystallin T cells secreting high levels of interferon-gamma are similarly identified in MS and healthy donors. In conclusion, the peripheral response towards the myelin antigen alpha B-crystallin is neither quantitatively nor qualitatively peculiar to MS, in contrast to the theoretical paradigm suggesting peripheral activation of myelin-reactive T cells to be the prerequisite for MS induction.

    European journal of neurology 2003;10;5;583-6

  • A novel rationale for inhibition of gelatinase B in multiple sclerosis: MMP-9 destroys alpha B-crystallin and generates a promiscuous T cell epitope.

    Starckx S, Van den Steen PE, Verbeek R, van Noort JM and Opdenakker G

    Rega Institute for Medical Research, University of Leuven, 3000 Leuven, Belgium.

    The small heat shock protein alphaB-crystallin is considered as a candidate autoantigen in multiple sclerosis (MS) lesions. Gelatinase B or matrix metalloproteinase (MMP)-9 is a proteinase establishing various disease-promoting feedback loops in autoimmune diseases. Human alphaB-crystallin was digested with natural gelatinase B and all cleavage sites were identified by a combined approach of mass spectrometry and peptide sequencing analysis. Previously identified immunodominant and cryptic epitopes of alphaB-crystallin in mice and rats were generated and largely left intact by MMP-9 processing. The alphaB-crystallin peptide 1-16, generated as a remnant epitope, provoked a significant T cell response in alphaB-crystallin knockout mice. None of the remnant peptides was encephalitogenic when injected intracerebrally into mice or induced MMP-9 in vitro. Gelatinase B is thus able to release T cell epitopes from intact alphaB-crystallin, but their pathogenic role remains unclear.

    Funded by: Multiple Sclerosis Society: 651

    Journal of neuroimmunology 2003;141;1-2;47-57

  • Nuclear speckle localisation of the small heat shock protein alpha B-crystallin and its inhibition by the R120G cardiomyopathy-linked mutation.

    van den IJssel P, Wheelock R, Prescott A, Russell P and Quinlan RA

    School of Life Sciences, Medical Science Institute, The University, Dundee DD1 5EH, UK.

    In this study, the small heat shock protein (sHSP) chaperones, alpha B-crystallin and HSP27, are identified as nuclear speckle components in unstressed cells in tissue culture. This new finding suggests a constitutive function for these sHSP chaperones in the nucleus and suggests a new perspective on the cardiomyopathy-causing mutation for alpha B-crystallin that could involve transcriptional splicing effects. Both alpha B-crystallin and HSP27 were immunolocalised to nuclear speckles (interchromatin granule clusters). While alpha B-crystallin was preferentially localised to speckles as shown by colocalisation with non-snRNP, SC35, as well as the snRNP components Sm and U1A, HSP27 was also seen associated with the nucleolar compartment, indicating a subtle difference between these closely related sHSPs. Actinomycin D treatment caused the relocalisation of alpha B-crystallin along with Sm and SC35 to a smaller number of more distinct spots, suggesting a link between speckle localisation and the transcriptional status of the cells. We then examined several transformed, immortalised, and primary cells expressing endogenous alpha B-crystallin as well as some cells with ectopic alpha B-crystallin expression. All consistently showed alpha B-crystallin in nuclear speckles. The nuclear localisation of the sHSPs was also confirmed biochemically and 2D gel electrophoresis revealed that there was only one major nuclear alpha B-crystallin isoform. This suggested that phosphorylation was not required for nuclear localisation of alpha B-crystallin. This was confirmed by the transient transfection of HeLa cells with a phosphorylation-defective alpha B-crystallin. In contrast, the transfection of R120G alpha B-crystallin, the mutation that causes cardiomyopathy, inhibited the nuclear speckle localisation of alpha B-crystallin. These data suggest that the cardiomyopathy-causing mutation for alpha B-crystallin has nuclear as well as cytoplasmic consequences, suggesting an explanation for the difference in severity of the desmin and alpha B-crystallin transgenic models of their respective cardiomyopathies.

    Experimental cell research 2003;287;2;249-61

  • Gene expression analysis in a transgenic Caenorhabditis elegans Alzheimer's disease model.

    Link CD, Taft A, Kapulkin V, Duke K, Kim S, Fei Q, Wood DE and Sahagan BG

    Institute for Behavioral Genetics, University of Colorado, Campus Box 447, Boulder, CO 80309, USA. linkc@colorado.edu

    We have engineered transgenic Caenorhabditis elegans animals to inducibly express the human beta amyloid peptide (Abeta). Gene expression changes resulting from Abeta induction have been monitored by cDNA hybridization to glass slide microarrays containing probes for almost all known or predicted C. elegans genes. Using statistical criteria, we have identified 67 up-regulated and 240 down-regulated genes. Subsets of these regulated genes have been tested and confirmed by quantitative RT-PCR. To investigate whether genes identified in this model system also show gene expression changes in Alzheimer's disease (AD) brain, we have also used quantitative RT-PCR to examine in post-mortem AD brain tissue transcript levels of alphaB-crystallin (CRYAB) and tumor necrosis factor-induced protein 1 (TNFAIP1), human homologs of genes found to be robustly induced in the transgenic C. elegans model. Both CRYAB and TNFAIP1 show increased transcript levels in AD brains, supporting the validity of this approach.

    Funded by: NIA NIH HHS: AG-12423

    Neurobiology of aging 2003;24;3;397-413

  • Enhanced stability of alpha B-crystallin in the presence of small heat shock protein Hsp27.

    Fu L and Liang

    Center for Ophthalmic Research, Brigham and Women's Hospital, and Department of Ophthalmology, Harvard Medical School, Boston, MA 02115, USA.

    Lens alpha-crystallin, alpha A- and alpha B-crystallin, and Hsp27 are members of the small heat shock protein family. Both alpha A- and alpha B-crystallin are expressed in the lens and serve as structural proteins and as chaperones, but alpha B-crystallin is also expressed in nonlenticular organs where Hsp27, rather than alpha A-crystallin, is expressed along with alpha B-crystallin. It is not known what additional function Hsp27 has besides as a heat shock protein, but it may serve, as alpha A-crystallin does in the lens, to stabilize alpha B-crystallin. In this study, we investigate aspects on conformation and thermal stability for the mixture of Hsp27 and alpha B-crystallin. Size exclusion chromatography, circular dichroism (CD), and light scattering measurements indicated that Hsp27 prevented alpha B-crystallin from heat-induced structural changes and high molecular weight (HMW) aggregation. The results indicate that Hsp27 indeed promotes stability of alpha B-crystallin.

    Funded by: NEI NIH HHS: EY05803

    Biochemical and biophysical research communications 2003;302;4;710-4

  • Alteration of protein-protein interactions of congenital cataract crystallin mutants.

    Fu L and Liang JJ

    Center for Ophthalmic Research, Brigham and Women's Hospital, and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    Purpose: A recent study demonstrated the presence of protein-protein interactions among lens crystallins in a mammalian cell two-hybrid system assay and speculated about the significance of these interactions for protein solubility and lens transparency. The current study extends those findings to the following crystallin genes involved in some congenital cataracts: CRYAA (R116C), CRYAB (R120G), and CRYGC (T5P).

    Methods: A mammalian two-hybrid system was used to assay the protein-protein interactions. Congenital cataract crystallin genes were cloned and fused into the two-hybrid system vectors (target and prey proteins). Together, with the third vector containing a reporter gene, chloramphenicol acetyltransferase (CAT), they were cotransfected into human HeLa cells. The presence of protein-protein interactions and the strength of these interactions were assayed by CAT ELISA.

    Results: The pattern of changes in protein-protein interactions of those congenital cataract gene products with the three major crystallins, alphaA- or alphaB-, betaB2-, and gammaC-crystallins, differed. For the T5P gammaC-crystallin, most of the interactions were decreased; for the R116C alphaA-crystallin, the interactions with betaB2- and gammaC-crystallin decreased and those with alphaB-crystallin and heat-shock protein (Hsp)27 increased; and for the R120G alphaB-crystallin, the interactions with alphaA- and alphaB-crystallin decreased, but those with betaB2- and gammaC-crystallin increased slightly. An attempt was made to interpret the results on the basis of conformational change and disruption of dimeric interaction involving beta-strands.

    Conclusions: The results clearly indicate that crystallin mutations involved in congenital cataracts altered protein-protein interactions, which may contribute to decreased protein solubility and formation of cataract.

    Funded by: NEI NIH HHS: EY05803

    Investigative ophthalmology & visual science 2003;44;3;1155-9

  • The small heat-shock protein alpha B-crystallin promotes FBX4-dependent ubiquitination.

    den Engelsman J, Keijsers V, de Jong WW and Boelens WC

    Department of Biochemistry, Nÿmegen Center for Molecular Life Sciences, University of Nijmegen, 6500 HB Nijmegen, The Netherlands.

    AlphaB-crystallin is a small heat-shock protein in which three serine residues (positions 19, 45, and 59) can be phosphorylated under various conditions. We describe here the interaction of alphaB-crystallin with FBX4, an F-box-containing protein that is a component of the ubiquitin-protein isopeptide ligase SCF (SKP1/CUL1/F-box). The interaction with FBX4 was enhanced by mimicking phosphorylation of alphaB-crystallin at both Ser-19 and Ser-45 (S19D/S45D), but not at other combinations. Ser-19 and Ser-45 are preferentially phosphorylated during the mitotic phase of the cell cycle. Also alphaB-crystallin R120G, a mutant found to co-segregate with a desmin-related myopathy, displayed increased interaction wit 126e h FBX4. Both alphaB-crystallin S19D/S45D and R120G specifically translocated FBX4 to the detergent-insoluble fraction and stimulated the ubiquitination of one or a few yet unknown proteins. These findings implicate the involvement of alphaB-crystallin in the ubiquitin/proteasome pathway in a phosphorylation- and cell cycle-dependent manner and may provide new insights into the alphaB-crystallin-induced aggregation in desmin-related myopathy.

    The Journal of biological chemistry 2003;278;7;4699-704

  • Alpha-crystallin regions affected by adenosine 5'-triphosphate identified by hydrogen-deuterium exchange.

    Hasan A, Smith DL and Smith JB

    Department of Chemistry, University of Nebraska, Lincoln, NE 68588-0304, USA.

    ATP interaction with lens alpha-crystallins leading to enhanced chaperone activity is not yet well understood. One model for chaperone activity of small heat shock proteins proposes that ATP causes small heat shock proteins to release substrates, which are then renatured by other larger heat shock proteins. A similar role has been proposed for ATP in alpha-crystallin chaperone activity. To evaluate this model, ATP-induced structural changes of native human alpha-crystallin assemblies were determined by hydrogen-deuterium exchange. In these experiments, hydrogen-deuterium exchange, measured by mass spectrometry, gave direct evidence that ATP decreases the accessibility of amide hydrogens in multiple regions of both alphaA and alphaB. The surface encompassed by these regions is much larger than would be shielded by a single ATP, implying that multiple ATP molecules bind to each subunit and/or ATP causes a more compact alpha-crystallin structure. Such a conformational change could release a bound substrate. The regions most affected by ATP are near putative substrate binding regions of alphaA and alphaB and in the C-terminal extension of alphaB. The widespread decrease in hydrogen-deuterium exchange with particularly large decreases near substrate binding regions suggests that ATP releases substrates via both direct displacement and a global conformational change.

    Funded by: NEI NIH HHS: R01 EY 07609

    Biochemistry 2002;41;52;15876-82

  • The small heat shock protein alpha B-crystallin negatively regulates apoptosis during myogenic differentiation by inhibiting caspase-3 activation.

    Kamradt MC, Chen F, Sam S and Cryns VL

    Center for Endocrinology, Metabolism, and Molecular Medicine, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.

    Myoblasts respond to growth factor deprivation either by differentiating into multinucleated myotubes or by undergoing apoptosis; hence, the acquisition of apoptosis resistance by myogenic precursors is essential for their development. Here we demonstrate that the expression of the small heat shock protein alpha B-crystallin is selectively induced in C2C12 myoblasts that are resistant to differentiation-induced apoptosis, and we show that this induction occurs at an early stage in their differentiation in vitro. In contrast, the expression of several known anti-apoptotic proteins (FLIP, XIAP, Bcl-x(L)) was not altered during myogenesis. We also demonstrate that ectopic expression of alpha B-crystallin, but not the closely related small heat shock protein Hsp27, renders C2C12 myoblasts resistant to differentiation-induced apoptosis. Furthermore, we show that the myopathy-causing R120G alpha B-crystallin mutant is partly impaired in its cytoprotective function, whereas a pseudophosphorylation alpha B-crystallin mutant that mimics stress-induced phosphorylation is completely devoid of anti-apoptotic activity. Finally, we demonstrate that alpha B-crystallin negatively regulates apoptosis during myogenesis by inhibiting the proteolytic activation of caspase-3, whereas the R120G and pseudophosphorylation mutants are defective in this function. Taken together, our findings indicate that alpha B-crystallin is a novel negative regulator of myogenic apoptosis that directly links the differentiation program to apoptosis resistance.

    Funded by: NCI NIH HHS: 5T32-CA70085; NINDS NIH HHS: NS31957

    The Journal of biological chemistry 2002;277;41;38731-6

  • Suppression of DTT-induced aggregation of abrin by alphaA- and alphaB-crystallins: a model aggregation assay for alpha-crystallin chaperone activity in vitro.

    Reddy GB, Narayanan S, Reddy PY and Surolia I

    Biochemistry Division, National Institute of Nutrition, Hyderabad 500 007, India. geereddy@yahoo.com

    The eye lens small heat shock proteins (sHSP), alphaA- and alphaB-crystallins, have been shown to function like molecular chaperones, both in vitro and in vivo. It is essential to assess the protective effect of alphaA- and alphaB-crystallins under native conditions to extrapolate the results to in vivo conditions. Insulin and alpha-lactalbumin have widely been used to investigate the chaperone mechanism of alpha-crystallin under native conditions. Due to its smaller size, insulin B-chain may not represent the binding of putative physiological substrate proteins. As it stands, the aggregation of alpha-lactalbumin and binding of alpha-crystallin to it varies under different experimental conditions. Abrin, a ribosome inactivating protein isolated from the seeds of Abrus precatorius, consists of a 30 kDa A-chain and a lectin-like B-chain of 33 kDa joined by a single disulfide bond. Reduction of the disulfide link between the two chains of abrin leads to the aggregation of the B-chain. In this study, we demonstrate that dithiothreitol (DTT)-induced aggregation of abrin B-chain could be monitored by light scattering similar to that of insulin. Moreso, this process could be suppressed by recombinant human alphaA- and alphaB-crystallins in a concentration dependent manner, notably by binding to aggregation prone abrin B-chain. SDS-PAGE and HPLC gel filtration analysis indicate that there is a soluble complex formation between alpha-crystallin and abrin B-chain. Interestingly, in contrast to insulin, there is no significant difference between alphaA- and alphaB-crystallin in suppressing the aggregation of abrin B-chain at two different temperatures (25 and 37 degrees C). HSP26, an another small heat shock/alpha-crystallin family protein, was also able to prevent the DTT-induced aggregation of abrin. These results suggest that due to relatively larger size of its B-chain (33 kDa), compared to insulin B-chain (about 3 kDa), abrin may serve as a better model substrate for in vitro chaperone studies of alpha-crystallin and as well as other sHSP.

    FEBS letters 2002;522;1-3;59-64

  • Shotgun identification of protein modifications from protein complexes and lens tissue.

    MacCoss MJ, McDonald WH, Saraf A, Sadygov R, Clark JM, Tasto JJ, Gould KL, Wolters D, Washburn M, Weiss A, Clark JI and Yates JR

    Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

    Large-scale genomics has enabled proteomics by creating sequence infrastructures that can be used with mass spectrometry data to identify proteins. Although protein sequences can be deduced from nucleotide sequences, posttranslational modifications to proteins, in general, cannot. We describe a process for the analysis of posttranslational modifications that is simple, robust, general, and can be applied to complicated protein mixtures. A protein or protein mixture is digested by using three different enzymes: one that cleaves in a site-specific manner and two others that cleave nonspecifically. The mixture of peptides is separated by multidimensional liquid chromatography and analyzed by a tandem mass spectrometer. This approach has been applied to modification analyses of proteins in a simple protein mixture, Cdc2p protein complexes isolated through the use of an affinity tag, and lens tissue from a patient with congenital cataracts. Phosphorylation sites have been detected with known stoichiometry of as low as 10%. Eighteen sites of four different types of modification have been detected on three of the five proteins in a simple mixture, three of which were previously unreported. Three proteins from Cdc2p isolated complexes yielded eight sites containing three different types of modifications. In the lens tissue, 270 proteins were identified, and 11 different crystallins were found to contain a total of 73 sites of modification. Modifications identified in the crystallin proteins included Ser, Thr, and Tyr phosphorylation, Arg and Lys methylation, Lys acetylation, and Met, Tyr, and Trp oxidations. The method presented will be useful in discovering co- and posttranslational modifications of proteins.

    Funded by: NCI NIH HHS: R33 CA81665; NCRR NIH HHS: P41 RR011823, RR 11823-05; NEI NIH HHS: R01 EY 13288, R01 EY004542, R01 EY013288; NIDDK NIH HHS: F32 DK 59731, F32 DK059731; NIGMS NIH HHS: GM 47728, R01 GM047728

    Proceedings of the National Academy of Sciences of the United States of America 2002;99;12;7900-5

  • Inhibition of proteasomes induces accumulation, phosphorylation, and recruitment of HSP27 and alphaB-crystallin to aggresomes.

    Ito H, Kamei K, Iwamoto I, Inaguma Y, García-Mata R, Sztul E and Kato K

    Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Kamiya, Kasugai, 480-0392, Japan. itohide@inst-hsc.pref.aichi.jp

    Molecular chaperones and the ubiquitin-proteasome pathway are known to participate in the quality control of proteins in cells. In this study, we examined the responses of small heat shock proteins to proteasome inhibitors to clarify their roles under conditions where misfolded proteins are abnormally accumulated. HSP27 and alphaB-crystallin accumulated in both soluble and, more prominently, insoluble fractions after exposure to MG-132, a proteasome inhibitor. Enhanced expression of mRNAs for HSP27 and alphaB-crystallin was observed, suggesting transcriptional activation. Phosphorylation of HSP27 and alphaB-crystallin in cells treated with MG-132 was enhanced concomitantly with activation of p38 and p44/42 MAP kinase pathways. Immunofluorescence analysis revealed that exposure to proteasome inhibitors induced the formation of aggresomes in U373 MG cells, to which HSP27 and alphaB-crystallin were recruited. However, phosphorylation was not required for this accumulation in aggresomes. Thus, HSP27 and alphaB-crystallin are increased, phosphorylated and localized in aggresomes when proteasome activity is inhibited.

    Journal of biochemistry 2002;131;4;593-603

  • Detection of protein-protein interactions among lens crystallins in a mammalian two-hybrid system assay.

    Fu L and Liang JJ

    Center for Ophthalmic Research, Brigham and Women's Hospital, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    alpha-Crystallin consists of two subunits, alphaA and alphaB, and each can form an oligomer by itself or with the other. The aggregation arises from interdomain interactions. However, it is not known whether such interactions also exist among alpha-, beta-, and gamma-crystallins. This heterogeneous crystallin interaction is far weaker than the homogeneous crystallin interaction and is difficult to detect by conventional spectroscopic measurements. We used a mammalian two-hybrid system in this study. The major crystallin components, alphaA-, alphaB-, betaB2-, and gammaC-crystallin genes, were subcloned into the DNA binding domain and transcription activation domain vectors of the two-hybrid system, and they were cotransfected along with a chloramphenicol acetyltransferase (CAT) reporter vector into HeLa cells. Chloramphenicol acetyltransferase activity indicated that there were interactions between alphaA- (or alphaB-) and betaB2- or gammaC-crystallins but with an intensity of one-third that of alphaA-alphaB interactions. Hsp27, a member of the family of the small heat-shock proteins, showed a similar interaction property with alphaB-crystallin. Using the N- and C-terminal domain-truncated mutants, we demonstrated that both domains were important in the alphaA-crystallin self-interaction, but that only the C-terminal domain was important in the alphaB-crystallin self-interaction. These results show that the two-hybrid system can detect interactions among various crystallins and may be used in mapping interaction domains.

    Funded by: NEI NIH HHS: EY05803

    The Journal of biological chemistry 2002;277;6;4255-60

  • Large-scale screening for candidate genes of ossification of the posterior longitudinal ligament of the spine.

    Furushima K, Shimo-Onoda K, Maeda S, Nobukuni T, Ikari K, Koga H, Komiya S, Nakajima T, Harata S and Inoue I

    Division of Genetic Diagnosis, The Institute of Medical Science. The University of Tokyo, Japan.

    Ossification of the posterior longitudinal ligament of the spine (OPLL) is the predominant myelopathy among Japanese, and is usually diagnosed by ectopic bone formation in the paravertebral ligament in Japanese and other Asians. To detect genetic determinants associated with OPLL, we performed an extensive nonparametric linkage study with 126 affected sib-pairs using ma 1f40 rkers for various candidate genes by distinct analyses, SIBPAL and GENEHUNTER. Eighty-eight candidate genes were selected by comparing the genes identified by complementary DNA (cDNA) microarray analysis of systematic gene expression profiles during osteoblastic differentiation of human mesenchymal stem cells with the genes known to be involved in bone metabolism. Of the 24 genes regulated during osteoblastic differentiation, only one, the alpha B crystalline gene, showed evidence of linkage (p = 0.016, nonparametric linkage [NPL] score = 1.83). Of 64 genes known to be associated with bone metabolism, 7 showed weak evidence of linkage by SIBPAL analysis (p < 0.05): cadherin 13 (CDH13), bone morphogenetic protein 4 (BMP4), proteoglycan 1 (PRG1), transforming growth factor beta 3 (TGFb3), osteopontin (OPN), parathyroid hormone receptor 1 (PTHR1), and insulin-like growth factor 1 (IGF1). Among these genes, BMP4 (NPL = 2.23), CDH13 (NPL = 2.00), TGFb3 (NPL = 1.30), OPN (NPL = 1.15), and PTHR1 (NPL = 1.00) showed evidence of linkage by GENEHUNTER. Only BMP4 reached criteria of suggestive evidence of linkage. Because this gene is a well-known factor in osteogenetic function, BMP4 should be screened in further study for the polymorphism responsible.

    Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2002;17;1;128-37

  • Alpha-B crystallin gene (CRYAB) mutation causes dominant congenital posterior polar cataract in humans.

    Berry V, Francis P, Reddy MA, Collyer D, Vithana E, MacKay I, Dawson G, Carey AH, Moore A, Bhattacharya SS and Quinlan RA

    Department of Molecular Genetics, Institute of Ophthalmology, London EC1V 9EL, United Kingdom.

    Congenital cataracts are an important cause of bilateral visual impairment in infants. In a four-generation family of English descent, we mapped dominant congenital posterior polar cataract to chromosome 11q22-q22.3. The maximum LOD score, 3.92 at recombination fraction 0, was obtained for marker D11S898, near the gene that encodes crystallin alpha-B protein (CRYAB). By sequencing the coding regions of CRYAB, we found in exon 3 a deletion mutation, 450delA, that is associated with cataract in this family. The mutation resulted in a frameshift in codon 150 and produced an aberrant protein consisting of 184 residues. This is the first report of a mutation, in this gene, resulting in isolated congenital cataract.

    American journal of human genetics 2001;69;5;1141-5

  • In vivo carbamylation and acetylation of water-soluble human lens alphaB-crystallin lysine 92.

    Lapko VN, Smith DL and Smith JB

    Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304, USA.

    Several post-translational modifications of lysine residues of lens proteins have been implicated in cataractogenesis. In the present study, the molecular weight of an alpha-crystallin isolated from the water-soluble portion of a cataractous human eye lens indicated that it was a modified alphaB-crystallin. Further analysis by mass spectrometry of tryptic digests of this modified protein showed that Lys 92 was modified and that the sample was structurally heterogeneous. Lys 92 was acetylated in one population and carbamylated in another. Although carbamylation of lens crystallins has been predicted, this is the first documentation of in vivo carbamylation of a specific site. These results are also the first documentation of in vivo lysine acetylation of alphaB-crystallin. Both modifications alter the net charge on alphaB-crystallin, a feature that may have significance to cataractogenesis.

    Funded by: NEI NIH HHS: EY RO1 07609

    Protein science : a publication of the Protein Society 2001;10;6;1130-6

  • Interaction of human recombinant alphaA- and alphaB-crystallins with early and late unfolding intermediates of citrate synthase on its thermal denaturation.

    Rajaraman K, Raman B, Ramakrishna T and Rao CM

    Centre for Cellular and Molecular Biology, 500 007, Hyderabad, India.

    We have investigated the role of recombinant human alphaA- and alphaB-crystallins in the heat-induced inactivation and aggregation of citrate synthase. Homo-multimers of both alphaA- and alphaB-crystallins confer protection against heat-induced inactivation in a concentration-dependent manner and also prevent aggregation. Interaction of crystallins with early unfolding intermediates of citrate synthase reduces their partitioning into aggregation-prone intermediates. This appears to result in enhanced population of early unfolding intermediates that can be reactivated by its substrate, oxaloacetate. Both these homo-multimers do not form a stable complex with the early unfolding intermediates. However, they can form a soluble, stable complex with aggregation-prone late unfolding intermediates. This soluble complex formation prevents aggregation. Thus, it appears that the chaperone activity of alpha-crystallin involves both transient and stable interactions depending on the nature of intermediates on the unfolding pathway; one leads to reactivation of the enzyme activity while the other prevents aggregation.

    FEBS letters 2001;497;2-3;118-23

  • Ser-59 is the major phosphorylation site in alphaB-crystallin accumulated in the brains of patients with Alexander's disease.

    Kato K, Inaguma Y, Ito H, Iida K, Iwamoto I, Kamei K, Ochi N, Ohta H and Kishikawa M

    Institute for Developmental Research, Aichi Human Service Center, Aichi, Japan. kato@inst-hsc.pref.aichi.jp

    The phosphorylation state of alphaB-crystallin accumulated in the brains of two patients with Alexander's disease (one infantile and one juvenile type) was determined by means of SDS-PAGE or isoelectric focusing of soluble and insoluble fractions of brain extracts and subsequent western blot analysis with specific antibodies against alphaB-crystallin and each of three phosphorylated serine residues. The level of mammalian small heat shock protein of 25-28 kDa (Hsp27) in the same fraction was also estimated by western blot analysis. The majority of alphaB-crystallin was detected in the insoluble fraction of brain homogenates and phosphorylation was preferentially observed at Ser-59 in both cases. A significant level of phosphorylation at Ser-45 but not Ser-19 was also detected. Hsp27 was found at considerable levels in the insoluble fractions. alphaB-crystallin and phosphorylated forms were detected in the cerebrospinal fluid of patient with the juvenile type. AlphaB-crystallin and phosphorylated forms were also detectable at considerable levels in the insoluble fraction of brain homogenates from patients with Alzheimer's disease and aged controls. The phosphorylation site was mostly at Ser-59 in all cases. Immunohistochemically, alphaB-crystallin was stained in Rosenthal fibers in brains of patients with Alexander's disease and their peripheral portions were immunostained with antibodies recognizing phosphorylated Ser-59. These results indicate that the major phosphorylation site in alphaB-crystallin in brains of patients with Alexander's disease or Alzheimer's disease as well as in aged controls is Ser-59.

    Journal of neurochemistry 2001;76;3;730-6

  • Interaction between alphaB-crystallin and the human 20S proteasomal subunit C8/alpha7.

    Boelens WC, Croes Y and de Jong WW

    Department of Biochemistry, University of Nijmegen, PO Box 9101, 6500 HB, Nijmegen, The Netherlands. w.boelens@bioch.kun.nl

    alphaB-Crystallin, a member of the small heat shock protein (sHsp) family, can bind unfolding proteins, but is unable to refold them. To fulfil its protective function in vivo it is therefore likely to interact with other cellular proteins. Here we report that alphaB-crystallin binds very specifically both in vitro and in vivo to C8/alpha7, one of the 14 subunits of the 20S proteasome. The C8/alpha7 protein forms heterogeneous complexes with alphaB-crystallin of about 540 kDa. However, no strong interaction between alphaB-crystallin and 20S proteasomes was observed. Since both proteins are localized in the cytoplasm, the interaction between alphaB-crystallin and C8/alpha7 subunit might affect the assembly of the proteasome complex or facilitate the degradation of unfolded proteins bound to alphaB-crystallin.

    Biochimica et biophysica acta 2001;1544;1-2;311-9

  • DNA cloning using in vitro site-specific recombination.

    Hartley JL, Temple GF and Brasch MA

    Life Technologies, Inc., Rockville, Maryland 20850, USA. jhartley@lifetech.com

    As a result of numerous genome sequencing projects, large numbers of candidate open reading frames are being identified, many of which have no known function. Analysis of these genes typically involves the transfer of DNA segments into a variety of vector backgrounds for protein expression and functional analysis. We describe a method called recombinational cloning that uses in vitro site-specific recombination to accomplish the directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency. Numerous DNA segments can be transferred in parallel into many different vector backgrounds, providing an approach to high-throughput, in-depth functional analysis of genes and rapid optimization of protein expression. The resulting subclones maintain orientation and reading frame register, allowing amino- and carboxy-terminal translation fusions to be generated. In this paper, we outline the concepts of this approach and provide several examples that highlight some of its potential.

    Genome research 2000;10;11;1788-95

  • Self-complementary motifs (SCM) in alpha-crystallin small heat shock proteins.

    Farnsworth PN and Singh K

    Department of Pharmacology and Physiology, UMD-New Jersey Medical School, Newark, New Jersey 07103, USA. farnswor@umdnj.edu

    Small heat shock proteins (sHsp) have been implicated in many cell processes involving the dynamics of protein-protein interactions. Two unusual sequences containing self-complementary motifs (SCM) have been identified within the conserved alpha-crystallin domain of sHsps. When two SCMs are aligned in an anti-parallel direction (N to C and C to N), the charged or polar residues form either salt bridges or hydrogen bonds while the non-polar residues participate in hydrophobic interactions. When aligned in reverse order, the residues of these motifs in alpha-crystallin subunits form either hydrophobic and/or polar interactions. Homology based molecular modeling of the C-terminal domain of alpha-crystallin subunits using the crystal structure of MjHSP16.5 suggests that SCM1 and 2 participate in stabilizing secondary structure and subunit interactions. Also there is overwhelming evidence that these motifs are important in the chaperone-like activity of alpha-crystallin subunits. These sequences are conserved and appear to be characteristic of the entire sHsp superfamily. Similar motifs are also present in the Hsp70 family and the immunoglobulin superfamily.

    FEBS letters 2000;482;3;175-9

  • The effect of stress on the pattern of phosphorylation of alphaA and alphaB crystallin in the rat lens.

    Wang K, Gawinowicz MA and Spector A

    Department of Ophthalmology, Howard Hughes Medical Institute, New York, New York 10032, USA. kw32@columbia.edu

    Previously, we have shown that phosphorylation of alpha crystallin (alpha) in rat lenses can be stimulated by oxidative stress. To better understand the biological functions of the stress-induced phosphorylation of the A and B chains of alpha (alphaA and alphaB), the normal and stress-induced phosphorylation pattern of these polypeptides in the rat lens has been investigated. With either alphaA or alphaB, there is only one phosphorylation site that is significantly affected, with widely different stresses, H(2)O(2)or elevation in free Ca(++)levels. However, the phosphorylation sites are markedly different for the two polypeptides, for alphaA being on Thr-4 in the N terminal region and with alphaB on Ser-59 in the central region of the polypeptide. The difference in the sequence in the two phosphorylation regions suggests that different phosphorylation systems are probably involved. This implies that the cellular function of the phosphorylation of alphaA and alphaB may be quite different.

    Experimental eye research 2000;71;4;385-93

  • Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing.

    Simpson JC, Wellenreuther R, Poustka A, Pepperkok R and Wiemann S

    Department of Cell Biology and Biophysics, EMBL Heidelberg, Germany.

    As a first step towards a more comprehensive functional characterization of cDNAs than bioinformatic analysis, which can only make functional predictions for about half of the cDNAs sequenced, we have developed and tested a strategy that allows their systematic and fast subcellular localization. We have used a novel cloning technology to rapidly generate N- and C-terminal green fluorescent protein fusions of cDNAs to examine the intracellular localizations of > 100 expressed fusion proteins in living cells. The entire analysis is suitable for automation, which will be important for scaling up throughput. For > 80% of these new proteins a clear intracellular localization to known structures or organelles could be determined. For the cDNAs where bioinformatic analyses were able to predict possible identities, the localization was able to support these predictions in 75% of cases. For those cDNAs where no homologies could be predicted, the localization data represent the first information.

    EMBO reports 2000;1;3;287-92

  • The major in vivo modifications of the human water-insoluble lens crystallins are disulfide bonds, deamidation, methionine oxidation and backbone cleavage.

    Hanson SR, Hasan A, Smith DL and Smith JB

    Department of Chemistry, University of Nebraska, Lincoln 68588-0304, USA.

    This investigation of the water-insoluble crystallins from human lenses has used multiple chromatographic separations to obtain proteins of sufficient purity for mass spectrometric analysis. Each fraction was analysed to determine the molecular masses of the constituent proteins as well as peptides in tryptic digests of these proteins. The major components of the water-insoluble crystallins were identified as alphaA- and alphaB-crystallins. In addition, gammaS-, betaB1-, gammaD-, betaA3/A1- and betaB2-crystallins were found, in order of decreasing abundance. Although there was evidence of some backbone cleavage, the predominant forms of alphaA-, alphaB, betaB2-, gammaS- and gammaD-crystallins were the intact polypeptide chains. The major modifications distinguishing the water-soluble crystallins were increased disulfide bonding, oxidation of Met, deamidation of Gln and Asn and backbone cleavage. Of the many reactions hypothesized to lead to crystallin insolubility and cataract, these results most strongly support metal-catalysed oxidation, deamidation and truncation as initiators of conformational changes that favor aggregation.

    Funded by: NEI NIH HHS: EY RO1 07609

    Experimental eye research 2000;71;2;195-207

  • Identification and characterization of a novel protein from Sertoli cells, PASS1, that associates with mammalian small stress protein hsp27.

    Liu C, Gilmont RR, Benndorf R and Welsh MJ

    Departments of Cell and Developmental Biology and Plastic and Reconstructive Surgery, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.

    hsp27 is involved in development of tolerance to stress, possibly by its involvement in molecular chaperoning, maintenance of glutathione status, and/or modulation of microfilament structure and function. We hypothesize that hsp27 function depends on specific association with other proteins. To discover proteins that associate with hsp27, we made a differentiated rat Sertoli cell cDNA expression library and screened it using the yeast two-hybrid system. We obtained a cDNA coding for a novel protein of 428 amino acids that we have named PASS1 (protein associated with small stress proteins 1). BLAST searches did not reveal major similarity of PASS1 to any known protein, but the cDNA sequence matched several mouse EST clones and shares 34% homology with a Caenorhabditis elegans genomic sequence. In vitro, bacterially expressed glutathione S-transferase-PASS1 fusion protein bound to hsp27, and hsp27 was co-immunoprecipitated with c-Myc-tagged PASS1 overexpressed in several cell lines. The region of PASS1 responsible for association with hsp27 was identified as existing predominantly between amino acids 108 and 208 of PASS1. Northern hybridization and Western blot analysis demonstrated that PASS1 is expressed in several tissues, with the highest expression occurring in testis, primarily in Sertoli cells. The presence of a 1.4-kilobase PASS1 mRNA in kidney as well as the 1. 8-kilobase mRNA seen in other tissues suggests that alternate splicing may occur in this organ. Ectopic expression of PASS1 in two cultured cell lines was observed to inhibit the ability of hsp27 to protect cells against heat shock, indicating that PASS1 does interact with hsp27 in the live cell.

    Funded by: NCRR NIH HHS: MO1RR00042; NIEHS NIH HHS: ES06265

    The Journal of biological chemistry 2000;275;25;18724-31

  • Muscle develops a specific form of small heat shock protein complex composed of MKBP/HSPB2 and HSPB3 during myogenic differentiation.

    Sugiyama Y, Suzuki A, Kishikawa M, Akutsu R, Hirose T, Waye MM, Tsui SK, Yoshida S and Ohno S

    Department of Molecular Biology, Yokohama City University School of Medicine, Kanazawa-ku, Yokohama 236-0004, Japan.

    Previously, we identified a new mammalian sHSP, MKBP, as a myotonic dystrophy protein kinase-binding protein, and suggested its important role in muscle maintenance (Suzuki, A., Sugiyama, Y., Hayashi, Y., Nyu-i, N., Yoshida, M., Nonaka, I., Ishiura, S., Arahata, K., and Ohno, S. (1998) J. Cell Biol. 140, 1113-1124). In this paper, we develop the former work by performing extensive characterization of five of the six sHSPs so far identified, that is, HSP27, alphaB-crystallin, p20, MKBP/HSPB2, and HSPB3, omitting lens-specific alphaA-crystallin. Tissue distribution analysis revealed that although each sHSP shows differential constitutive expression in restricted tissues, tissues that express all five sHSPs are only muscle-related tissues. Especially, the expressions of HSPB3, identified for the first time as a 17-kDa protein in this paper, and MKBP/HSPB2 are distinctly specific to muscles. Moreover, these sHSPs form an oligomeric complex with an apparent molecular mass of 150 kDa that is completely independent of the oligomers formed by HSP27, alphaB-crystallin, and p20. The expressions of MKBP/HSPB2 and HSPB3 are induced during muscle differentiation under the control of MyoD, suggesting that the sHSP oligomer comprising MKBP/HSPB2 and HSPB3 represents an additional system closely related to muscle function. The functional divergence among sHSPs in different oligomers is also demonstrated in several ways: 1) an interaction with myotonic dystrophy protein kinase, which has been suggested to be important for the maintenance of myofibril integrity, was observed only for MKBP/HSPB2; 2) a myotube-specific association with actin bundles was observed for HSP27 and alphaB-crystallin, but not for MKBP/HSPB2; and 3) sHSPs whose mRNAs are induced by heat shock are alphaB-crystallin and HSP27. Taken together, the results suggest that muscle cells develop two kinds of stress response systems composed of diverged sHSP members, and 50a that these systems work independently in muscle maintenance and differentiation.

    The Journal of biological chemistry 2000;275;2;1095-104

  • Antigens recognized by autologous antibody in patients with renal-cell carcinoma.

    Scanlan MJ, Gordan JD, Williamson B, Stockert E, Bander NH, Jongeneel V, Gure AO, Jäger D, Jäger E, Knuth A, Chen YT and Old LJ

    Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. scanlanm@mskcc.org

    The screening of cDNA expression libraries derived from human tumors with autologous antibody (SEREX) is a powerful method for defining the structure of tumor antigens recognized by the humoral immune system. Sixty-five distinct antigens (NY-REN-1 to NY-REN-65) reactive with autologous IgG were identified by SEREX analysis of 4 renal cancer patients and were characterized in terms of cDNA sequence, mRNA expression pattern, and reactivity with allogeneic sera. REN-9, -10, -19, and -26 have a known association with human cancer. REN-9 (LUCA-15) and REN-10 (gene 21) map to the small cell lung cancer tumor suppressor gene locus on chromosome 3p21.3. REN-19 is equivalent to LKB1/STK11, a gene that is defective in Peutz-Jeghers syndrome and cancer. REN-26 is encoded by the bcr gene involved in the [t(9:22)] bcr/abl translocation. Genes encoding 3 of the antigens in the series showed differential mRNA expression; REN-3 displays a pattern of tissue-specific isoforms, and REN-21 and REN-43 are expressed at a high level in testis in comparison to 15 other normal tissues. The other 62 antigens were broadly expressed in normal tissues. With regard to immunogenicity, 20 of the 65 antigens reacted only with autologous sera. Thirty-three antigens reacted with sera from normal donors, indicating that their immunogenicity is not restricted to cancer. The remaining 12 antigens reacted with sera from 5-25% of the cancer patients but not with sera from normal donors. Seventy percent of the renal cancer patients had antibodies directed against one or more of these 12 antigens. Our results demonstrate the potential of the SEREX approach for the analysis of the humoral immune response against human cancer.

    International journal of cancer 1999;83;4;456-64

  • Alpha-crystallin as a molecular chaperone.

    Derham BK and Harding JJ

    Nuffield Laboratory of Ophthalmology, University of Oxford, UK.

    The role of alpha-crystallin as a molecular chaperone may explain how the lens stays transparent for so long. Alpha-crystallin prevents the aggregation of other lens crystallins and proteins that have become unfolded by "trapping" the protein in a high molecular weight complex. It also protects enzyme activities. The substrate protein may interact while in a molten globule state. Alpha-crystallin predominantly binds to proteins very early in the denaturation pathways. The amphiphilic nature of alpha-crystallin, a polar C-terminal-region and a hydrophobic N-terminal-region are all essential for chaperone function. The flexible C-terminal extension maintains solubility and can bind to opposing charged residues of unfolding proteins. Hydrophobic regions in the N-terminal region then hold the unfolded protein. Specific areas important for chaperone binding and function have been identified throughout the N-terminal-region, connecting peptide and C-terminal extension. After a substantial amount of chemical data and models, cryo-EM images of alpha-crystallin have confirmed a variable 3D surface with a hollow interior. Alpha-crystallin taken from the lens nucleus shows an age-dependent decrease in chaperone function. High molecular weight aggregates and alpha-crystallin found within the nucleus from clear and cataract lenses have reduced chaperone function. Post-translational modifications, known to occur during ageing, such as glycation, carbamylation, oxidation, phosphorylation and truncation cause a decrease in chaperone function. Alpha-crystallin is expressed outside the lens. AlphaB-crystallin can be induced by heat shock in many tissues where it is translocated from cytoplasm to nucleus. Increased expression of alphaB-crystallin has been seen in many pathological states. Conformational disorders, including cataract may have a common aetiology and potentially a common therapy.

    Progress in retinal and eye research 1999;18;4;463-509

  • A missense mutation in the alphaB-crystallin chaperone gene causes a desmin-related myopathy.

    Vicart P, Caron A, Guicheney P, Li Z, Prévost MC, Faure A, Chateau D, Chapon F, Tomé F, Dupret JM, Paulin D and Fardeau M

    Institut Pasteur, Paris, France. pvicart@pasteur.fr

    Desmin-related myopathies (DRM) are inherited neuromuscular disorders characterized by adult onset and delayed accumulation of aggregates of desmin, a protein belonging to the type III intermediate filament family, in the sarcoplasma of skeletal and cardiac muscles. In this paper, we have mapped the locus for DRM in a large French pedigree to a 26-cM interval in chromosome 11q21-23. This region contains the alphaB-crystallin gene (CRYAB), a candidate gene encoding a 20-kD protein that is abundant in lens and is also present in a number of non-ocular tissues, including cardiac and skeletal muscle. AlphaB-crystallin is a member of the small heat shock protein (shsp) family and possesses molecular chaperone activity. We identified an R120G missense mutation in CRYAB that co-segregates with the disease phenotype in this family. Muscle cell lines transfected with the mutant CRYAB cDNA showed intracellular aggregates that contain both desmin and alphaB-crystallin as observed in muscle fibers from DRM patients. These results are the first to identify a defect in a molecular chaperone as a cause for an inherited human muscle disorder.

    Nature genetics 1998;20;1;92-5

  • Large-scale concatenation cDNA sequencing.

    Yu W, Andersson B, Worley KC, Muzny DM, Ding Y, Liu W, Ricafrente JY, Wentland MA, Lennon G and Gibbs RA

    A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7-2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (> 20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (> or = 98% identity), and 16 clones generated nonexact matches (57%-97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching.

    Funded by: NHGRI NIH HHS: 1F32 HG00169-01, F32 HG000169, F33 HG000210, P30 HG00210-05, R01 HG00823, U54 HG003273

    Genome research 1997;7;4;353-8

  • Sequence analysis of betaA3, betaB3, and betaA4 crystallins completes the identification of the major proteins in young human lens.

    Lampi KJ, Ma Z, Shih M, Shearer TR, Smith JB, Smith DL and David LL

    Department of Oral Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201, USA.

    A combination of Edman sequence analysis and mass spectrometry identified the major proteins of the young human lens as alphaA, alphaB, betaA1, betaA3, betaA4, betaB1, betaB2, betaB3, gammaS, gammaC, and gammaD-crystallins and mapped their positions on two-dimensional electrophoretic gels. The primary structures of human betaA1, betaA3, betaA4, and betaB3-crystallin subunits were predicted by determining cDNA sequences. Mass spectrometric analyses of each intact protein as well as the peptides from trypsin-digested proteins confirmed the predicted amino acid sequences and detected a partially degraded form of betaA3/A1 missing either 22 or 4 amino acid residues from its N-terminal extension. These studies were a prerequisite for future studies to determine how human lens proteins are altered during aging and cataract formation.

    Funded by: NEI NIH HHS: EY07609, EY07755

    The Journal of biological chemistry 1997;272;4;2268-75

  • A "double adaptor" method for improved shotgun library construction.

    Andersson B, Wentland MA, Ricafrente JY, Liu W and Gibbs RA

    Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas, 77030, USA.

    The efficiency of shotgun DNA sequencing depends to a great extent on the quality of the random-subclone libraries used. We here describe a novel "double adaptor" strategy for efficient construction of high-quality shotgun libraries. In this method, randomly sheared and end-repaired fragments are ligated to oligonucleotide adaptors creating 12-base overhangs. Nonphosphorylated oligonucleotides are used, which prevents formation of adaptor dimers and ensures efficient ligation of insert to adaptor. The vector is prepared from a modified M13 vector, by KpnI/PstI digestion followed by ligation to oligonucleotides with ends complementary to the overhangs created in the digest. These adaptors create 5'-overhangs complementary to those on the inserts. Following annealing of insert to vector, the DNA is directly used for transformation without a ligation step. This protocol is robust and shows three- to fivefold higher yield of clones compared to previous protocols. No chimeric clones can be detected and the background of clones without an insert is <1%. The procedure is rapid and shows potential for automation.

    Funded by: NHGRI NIH HHS: R01 HG00823

    Analytical biochemistry 1996;236;1;107-13

  • Dynamic O-GlcNAcylation of the small heat shock protein alpha B-crystallin.

    Roquemore EP, Chevrier MR, Cotter RJ and Hart GW

    Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham 35294-0005, USA.

    alphaB-Crystallin, originally described as a structural lens protein, is now known to be a member of the small heat shock protein family and is expressed in a number of nonlens tissues. This highly conserved 20 kDa protein aggregates with homologous proteins, including alphaA-crystallin and the small heat shock protein HSP28, to form large heteromeric complexes. Recently, Roquemore et al. (1992) have established that both phosphorylated and unphosphorylated forms of lens alphaB-crystallin are modified with O-linked N-acetylglucosamine, a dynamic posttranslational modification abundant on nuclear and cytoplasmic proteins. In this paper, we have identified the major site of O-GlcNAcylation on lens alphaB as Thr 170. We have further shown that this modification is not restricted to lens alphaB-crystallin but occurs on alphaB isolated from rat heart tissue and human astroglioma cells. Two-dimensional electrophoresis of rat heart alphaB-crystallin revealed two O-GlcNAcylated forms with mobilities corresponding to the unphosphorylated form (alphaB2) and an unidentified, slightly more acidic form. Phosphorylated alphaB-crystallin (alphaB1) was not detected in the rat heart preparation. The major O-GlcNAcylation site on alphaB-crystallins from rat heart also appears to be at Thr 170. Metabolic pulse-chase labeling studies of U373-MG astroglioma cells indicated that turnover of the carbohydrate on alphaB-crystallin is not static but proceeds many-fold more rapidly than turnover of the protein backbone itself, consistent with a regulatory role for O-GlcNAc on this small heat shock protein.

    Funded by: NCI NIH HHS: R01 CA-42486; NIGMS NIH HHS: 5T3 GM07445

    Biochemistry 1996;35;11;3578-86

  • The major protein expression profile and two-dimensional protein database of human heart.

    Kovalyov LI, Shishkin SS, Efimochkin AS, Kovalyova MA, Ershova ES, Egorov TA and Musalyamov AK

    Research Center of Medical Genetics, Russian Academy of Medical Sciences, Moscow.

    The construction of a two-dimensional protein database of the human heart is presented. The database contains information on about 300 abundant proteins of human myocardial tissue, including approximately 40 proteins that were identified by different methods. Each protein was characterized according to several parameters, including molecular weight, isoelectric point, name, partial sequence, subcellular localization, and genetic as well as embryonic changes.

    Electrophoresis 1995;16;7;1160-9

  • Post-translational modifications of water-soluble human lens crystallins from young adults.

    Miesbauer LR, Zhou X, Yang Z, Yang Z, Sun Y, Smith DL and Smith JB

    Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907.

    Post-translational modifications of the water-soluble human lens crystallins from young adult donors were identified and located using electrospray ionization mass spectrometric analysis of the intact proteins and fast atom bombardment mass spectrometry of enzymatic digests. Peptides corresponding to all of the sequences of alpha A-, alpha B-, and beta B2-crystallins were found, permitting the entire sequences to be searched for modifications. The major portions of these three crystallins were not modified. Modifications of alpha A-crystallin that were detected included 2 phosphorylated Ser residues (1 of which appears to be unique to human lenses), deamidation at some Gln and Asn residues, a disulfide bond between Cys-131 and Cys-142, and loss of the COOH-terminal Ser residue. Three phosphorylated Ser residues, but no deamidation, were found in alpha B-crystallin. The molecular weights of neither the intact protein nor the peptides in the enzymatic digests indicated any post-translational modification of the principal beta-crystallin, beta B2. The molecular weights of the other beta- and gamma-crystallins for which sequences have been published suggested the presence of post-translational modifications or errors in the published sequences. Although enough peptides were found to establish the presence of specific proteins, peptides corresponding to all portions of these proteins were not found, and elucidation of these structures is not yet complete. This mass spectrometric characterization of the total water-soluble proteins from normal young adult lenses provides a reference data base for future investigations of the modifications present in aged and cataractous lenses.

    Funded by: NEI NIH HHS: EY RO1 07609; NIGMS NIH HHS: GM 08298

    The Journal of biological chemistry 1994;269;17;12494-502

  • Simultaneous racemization and isomerization at specific aspartic acid residues in alpha B-crystallin from the aged human lens.

    Fujii N, Ishibashi Y, Satoh K, Fujino M and Harada K

    Discovery Research Division, Takeda Chemical Industries, Ltd., Ibaraki, Japan.

    We provide evidence that the racemization and isomerization of aspartyl(Asp) residues occur simultaneously in the alpha B-crystallin in the lens of aged (mean age: 80 years) and young (age: 11 months) humans. We purified alpha B-crystallin and subjected it to tryptic digestion. The resulting peptides were separated by reverse-phase high-performance chromatography (RP-HPLC) and were characterized by amino-acid composition, sequence analysis and mass spectrometry. Two specific sites, Asp-36 (D/L of Asp: 0.92) and Asp-62(D/L of Asp: 0.57), among 13 Asp/asparginyl (Asn) residues in aged alpha B-crystallin, were found to be highly racemized and isomerized to form beta-Asp residues. The beta-Asp-containing peptides were clearly distinguished from normal Asp-containing (alpha-Asp) peptides by RP-HPLC. The racemization and isomerization of Asp residues in aged alpha B-crystallin may occur via a succinimide intermediate. In young alpha B-crystallin, we observed neither racemization nor isomerization. We also found that Met-68 was oxidized to form Met sulfoxide to a greater extent in aged alpha B-crystallin than in young alpha B-crystallin. We concluded that racemization, isomerization, and oxidation of alpha B-crystallin occur spontaneously in the aging process.

    Biochimica et biophysica acta 1994;1204;2;157-63

  • Subregional physical mapping of an alpha B-crystallin sequence and of a new expressed sequence D11S877E to human 11q.

    Jeanpierre C, Austruy E, Delattre O, Jones C and Junien C

    INSERM U73, Château de Longchamp, Bois de Boulogne, Paris, France.

    We report the regional assignment on Chromosome (Chr) 11q of two cDNA clones selected as sequences expressed in mature kidney and not expressed in Wilms' tumor. Clone T70 was identified as an alpha B-crystallin sequence (CRYA2). CRYA2 has previously been mapped to 11q22.3-23.1 by in situ hybridization. Clone 6.2 represents a new gene expressed in adult and fetal kidney, pancreas, and liver. In order to map sequences corresponding to clone 6.2 and to physically define the boundaries of the localization of CRYA2, we used somatic cell hybrids carrying either different human chromosomes or Chr 11 segments and a cell line established from a patient with an interstitial deletion of region 11q14.3-q22.1. We showed that CRYA2 lies proximal to the 11q23.2 breakpoint defined by the constitutional t(11;22) and distal to the 11q22.1 breakpoint (between D11S388 and D11S35) of a constitutional interstitial deletion. This is in agreement with previous data obtained by in situ hybridization and provides proximal and distal physical benchmarks for this localization. Clone 6.2-related sequence (D11S877E) was assigned to region 11q23.2-q24.2 defined by the breakpoints of the constitutional t(11;22) and of the Ewing's sarcoma neuroepithelioma t(11;22).

    Mammalian genome : official journal of the International Mammalian Genome Society 1993;4;2;104-8

  • Site-specific glycation of lens crystallins by ascorbic acid.

    Ortwerth BJ, Slight SH, Prabhakaram M, Sun Y and Smith JB

    Mason Institute of Ophthalmology, University of Missouri, Columbia 65212.

    The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1-14C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH3. This rate of incorporation was 6-7-fold more rapid than that obtained with [14C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the crystallin subunits. [1-14C]Ascorbic acid-label led alpha-crystallin was separated into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N epsilon-(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.

    Funded by: NEI NIH HHS: EY 07609, EY-07070

    Biochimica et biophysica acta 1992;1117;2;207-15

  • Accumulation of alpha B-crystallin in brains of patients with Alexander's disease is not due to an abnormality of the 5'-flanking and coding sequence of the genomic DNA.

    Iwaki A, Iwaki T, Goldman JE, Ogomori K, Tateishi J and Sakaki Y

    Research Laboratory for Genetic Information, Kyushu University, Fukuoka, Japan.

    alpha B-Crystallin is a major protein component of Rosenthal fibers, which massively accumulate in the brains of patients suffering from Alexander's disease. To examine whether or not accumulation of alpha B-crystallin is due to any abnormality of the gene structures, we determined the sequence of the alpha B-crystallin gene in two cases of pathologically confirmed Alexander's disease. Direct sequencing of the promoter and coding regions of the alpha B-crystallin gene in patients revealed them to have a normal sequence. Northern blotting showed a single alpha B-crystallin mRNA species expressed in the Alexander's disease brain.

    Funded by: NEI NIH HHS: EY09331; NINDS NIH HHS: NS17125

    Neuroscience letters 1992;140;1;89-92

  • Copurification of small heat shock protein with alpha B crystallin from human skeletal muscle.

    Kato K, Shinohara H, Goto S, Inaguma Y, Morishita R and Asano T

    Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Japan.

    Immunoreactive alpha B crystallin and a 28-kDa protein in an extract of human pectoral muscle were precipitated by (NH4)2SO4 at 40% saturation, and coeluted during column chromatography on DEAE-Sepharose and on Bio-Gel A-5m. The two proteins were separated on a co 1f40 lumn of S-Sepharose HP in the presence of 7 M urea. Further chromatography of each of the two resultant fractions on a column of Superdex 75 pg and on a TSK-SP 5PW column in the presence of urea yielded preparations of alpha B crystallin and the 28-kDa protein each of which gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final preparation of 28-kDa protein contained at least two subtypes, which were separable on the TSK-SP column. However, fragmentation patterns of the two major 28-kDa proteins after digestion with endoproteinase Asp-N were identical. Amino acid sequences of peptides formed by cleavage of the purified 28-kDa protein and alpha B crystallin were identical to those of particular regions of the deduced amino acid sequences of human small heat shock protein (HSP28) and lens alpha B crystallin, respectively. Using an immunoassay method, with antibodies raised in rabbits, we found that HSP28 was present in all human tissues tested and at high levels (greater than 1 micrograms/mg protein) in the heart and other tissues composed of striated and smooth muscles. HSP28, found with alpha B crystallin, in extracts of several human and bovine tissues was trapped on and coeluted with alpha B crystallin from an affinity column prepared with antibodies against alpha B crystallin. This result suggests that the two proteins are associated in cells.

    The Journal of biological chemistry 1992;267;11;7718-25

  • Human alpha B-crystallin gene and preferential promoter function in lens.

    Dubin RA, Ally AH, Chung S and Piatigorsky J

    Laboratory of Molecular and Developmental Biology, National Eye Institute, National Instit 605 utes of Health, Bethesda, Maryland 20892.

    alpha B-Crystallin, first identified as a structural component of the vertebrate eye lens, is expressed at high levels in lens and at lower levels in a number of other tissues, most notably cardiac and skeletal muscle, kidney, and brain. We have cloned and sequenced the human alpha B-crystallin gene and show that it is structurally similar to its hamster homolog. We have also identified its transcription initiation site in human lens RNA. Functional analysis of a promoter fragment extending from -537 to +21 (relative to the transcription initiation site) and fused to the bacterial chloramphenicol acetyltransferase gene suggests that this fragment contains regulatory elements that function preferentially, but not exclusively, in lens. In contrast, this fragment is apparently insufficient to promote transcription in glial cells, as this construct functioned poorly in a glioblastoma-astrocytoma cell line (U-373MG) that synthesizes high levels of the endogenous alpha B-crystallin gene product.

    Genomics 1990;7;4;594-601

  • Cellular distribution of alpha B-crystallin in non-lenticular tissues.

    Iwaki T, Kume-Iwaki A and Goldman JE

    Department of Pathology, Columbia University College of Physicians and Surgeons, New York, NY 10032.

    alpha B-Crystallin is a subunit of alpha-crystallin, a major protein component of the vertebrate lens. Recently, its expression in various extra-lenticular tissues has been demonstrated by both Western and Northern blotting. In this study, the cellular distribution of alpha B-crystallin in rat organs was examined in detail using immunohistochemistry. Positive reactions were observed in lens, iris, heart, skeletal muscle (type 1 and type 2A fibers), striated muscle in skin and esophagus, Henle's loop and medullary collecting duct of the kidney, Schwann cells of peripheral nerves, glia of the central nervous system, and decidual cells of the placenta. A close correlation with markers of oxidative activity suggests that alpha B-crystallin is expressed in cells that have high levels of oxidative function.

    Funded by: NINDS NIH HHS: NS 17125

    The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 1990;38;1;31-9

  • Assignment of the alpha B-crystallin gene to human chromosome 11.

    Ngo JT, Klisak I, Dubin RA, Piatigorsky J, Mohandas T, Sparkes RS and Bateman JB

    Vision Genetics Center, Jules Stein Eye Institute, UCLA School of Medicine 90024.

    Using a human alpha B-crystallin genomic probe and human-mouse somatic cell hybrids, the human alpha B-gene was assigned to chromosome 11 and further corroborated by in situ hybridization to normal metaphase chromosomes. This assignment confirmed and regionally mapped the locus to q22.3-23.1.

    Funded by: NEI NIH HHS: EY-06772

    Genomics 1989;5;4;665-9

  • Alpha B-crystallin is expressed in non-lenticular tissues and accumulates in Alexander's disease brain.

    Iwaki T, Kume-Iwaki A, Liem RK and Goldman JE

    Department of Pathology, Columbia University College of Physicians and Surgeons, New York State Psychiatric Institute, New York 10032.

    Rosenthal fibers (RFs) are abnormal inclusions within astrocytes, characteristic of Alexander's disease. We have previously isolated a 22 kd protein component of RFs from Alexander's disease brain. By Western blotting, we detected its equivalent in several rat organs, with the highest level in heart, and in a human astrocytoma cell line (U-373MG). A cDNA library established from U-373MG was screened with an anti-RF protein antibody. A partial cDNA clone encoding the lens protein alpha B-crystallin was isolated. The anti-RF protein antibodies react with lens alpha B-crystallin. Furthermore, the distribution of alpha B-crystallin mRNA in rat organs is consistent with the Western blots. Therefore, alpha B-crystallin is not lens-specific and it can accumulate in large amounts in astrocytes in pathological conditions.

    Funded by: NINDS NIH HHS: NS15182, NS17125

    Cell 1989;57;1;71-8

  • The primary structure of the B2 chain of human alpha-crystallin.

    Kramps JA, de Man BM and de Jong

    FEBS letters 1977;74;1;82-4

Gene lists (4)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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