G2Cdb::Gene report

Gene id
G00001832
Gene symbol
GSN (HGNC)
Species
Homo sapiens
Description
gelsolin
Orthologue
G00000583 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000020584 (Vega human gene)
Gene
ENSG00000148180 (Ensembl human gene)
2934 (Entrez Gene)
126 (G2Cdb plasticity & disease)
GSN (GeneCards)
Literature
137350 (OMIM)
Marker Symbol
HGNC:4620 (HGNC)
Protein Sequence
P06396 (UniProt)

Synonyms (1)

  • DKFZp313L0718

Diseases (2)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000268: Amyloidosis-related nephrotic syndrome N Y (16998221) Single nucleotide polymorphism (SNP) Y
D00000157: Amyloidosis N Y (16258946) Single nucleotide polymorphism (SNP) Y

References

  • Amyloidosis-related nephrotic syndrome due to a G654A gelsolin mutation: the first report from the Middle East.

    Ardalan MR, Shoja MM and Kiuru-Enari S

    Department of Nephrology, Tabriz University of Medical Sciences (TUMS), Tabriz, Iran. ardalanm@tbzmed.ac.ir

    Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 2007;22;1;272-5

  • Cardiac conduction alterations in a French family with amyloidosis of the Finnish type with the p.Asp187Tyr mutation in the GSN gene.

    Chastan N, Baert-Desurmont S, Saugier-Veber P, Dérumeaux G, Cabot A, Frébourg T and Hannequin D

    Département de Neurologie, CHU de Rouen, 76031 Rouen, France.

    Familial amyloidosis of the Finnish type (FAF) is a rare autosomal-dominant disorder caused by the accumulation of a 71-amino acid amyloidogenic fragment of mutant gelsolin, an actin-modulating protein. The main symptoms include corneal lattice dystrophy, progressive cranial and peripheral neuropathy, and skin changes. To date, only two mutations in the GSN gene have been described: the p.Asp187Asn mutation in most patients and the p.Asp187Tyr mutation in a Danish and Czech family. We report on the third family with the p.Asp187Tyr mutation and the first French FAF family. Severe cardiac conduction alterations in three patients were mainly caused by cardiac sympathetic denervation. These findings demonstrate the cardiological involvement of the FAF phenotype and suggest that cardiological follow-up is required in FAF patients.

    Muscle & nerve 2006;33;1;113-9

Literature (114)

Pubmed - human_disease

  • Amyloidosis-related nephrotic syndrome due to a G654A gelsolin mutation: the first report from the Middle East.

    Ardalan MR, Shoja MM and Kiuru-Enari S

    Department of Nephrology, Tabriz University of Medical Sciences (TUMS), Tabriz, Iran. ardalanm@tbzmed.ac.ir

    Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 2007;22;1;272-5

  • Cardiac conduction alterations in a French family with amyloidosis of the Finnish type with the p.Asp187Tyr mutation in the GSN gene.

    Chastan N, Baert-Desurmont S, Saugier-Veber P, Dérumeaux G, Cabot A, Frébourg T and Hannequin D

    Département de Neurologie, CHU de Rouen, 76031 Rouen, France.

    Familial amyloidosis of the Finnish type (FAF) is a rare autosomal-dominant disorder caused by the accumulation of a 71-amino acid amyloidogenic fragment of mutant gelsolin, an actin-modulating protein. The main symptoms include corneal lattice dystrophy, progressive cranial and peripheral neuropathy, and skin changes. To date, only two mutations in the GSN gene have been described: the p.Asp187Asn mutation in most patients and the p.Asp187Tyr mutation in a Danish and Czech family. We report on the third family with the p.Asp187Tyr mutation and the first French FAF family. Severe cardiac conduction alterations in three patients were mainly caused by cardiac sympathetic denervation. These findings demonstrate the cardiological involvement of the FAF phenotype and suggest that cardiological follow-up is required in FAF patients.

    Muscle & nerve 2006;33;1;113-9

Pubmed - other

  • The 8 and 5 kDa fragments of plasma gelsolin form amyloid fibrils by a nucleated polymerization mechanism, while the 68 kDa fragment is not amyloidogenic.

    Solomon JP, Yonemoto IT, Murray AN, Price JL, Powers ET, Balch WE and Kelly JW

    Department of Chemistry, Skaggs Institute for Chemical Biology, La Jolla, California 92037, USA.

    Familial amyloidosis of Finnish type (FAF), or gelsolin amyloidosis, is a systemic amyloid disease caused by a mutation (D187N/Y) in domain 2 of human plasma gelsolin, resulting in domain 2 misfolding within the secretory pathway. When D187N/Y gelsolin passes through the Golgi, furin endoproteolysis within domain 2 occurs as a consequence of the abnormal conformations that enable furin to bind and cleave, resulting in the secretion of a 68 kDa C-terminal fragment (amino acids 173-755, C68). The C68 fragment is cleaved upon secretion from the cell by membrane type 1 matrix metalloprotease (MT1-MMP), affording the 8 and 5 kDa fragments (amino acids 173-242 and 173-225, respectively) comprising the amyloid fibrils in FAF patients. Herein, we show that the 8 and 5 kDa gelsolin fragments form amyloid fibrils by a nucleated polymerization mechanism. In addition to demonstrating the expected concentration dependence of a nucleated polymerization reaction, the addition of preformed amyloid fibrils, or "seeds", was shown to bypass the requirement for the formation of a high-energy nucleus, accelerating 8 and 5 kDa D187N gelsolin amyloidogenesis. The C68 fragment can form small oligomers, but not amyloid fibrils, even when seeded with preformed 8 kDa fragment plasma gelsolin fibrils. Because the 68 kDa fragment of gelsolin does not form amyloid fibrils in vitro or in a recently published transgenic mouse model of FAF, we propose that administration of an MT1-MMP inhibitor could be an effective strategy for the treatment of FAF.

    Funded by: NIA NIH HHS: AG018917, R01 AG018917, R01 AG018917-08

    Biochemistry 2009;48;48;11370-80

  • Proteomics of tumor-specific proteins in cerebrospinal fluid of patients with astrocytoma: usefulness of gelsolin protein.

    Ohnishi M, Matsumoto T, Nagashio R, Kageyama T, Utsuki S, Oka H, Okayasu I and Sato Y

    Department of Molecular Diagnosis, Graduate School of Medical Sciences, Kitasato University, Kanagawa, Japan.

    Changes in cerebrospinal fluid (CSF) composition have been shown to accurately reflect pathological processes in the CNS, and are potential indicators of abnormal CNS states, such as tumor growth. To detect biomarkers in high-grade astrocytomas, the differential expression of proteins in the cerebrospinal fluid was analyzed from two cases each of diffuse astrocytoma (grade II), and glioblastoma (grade IV) using agarose 2-D gel electrophoresis (2-DE). It was found that the expression of gelsolin protein decreased with histological grade. To examine whether gelsolin is a useful indicator of tumor aggressiveness or patient outcome, its expression was further studied on immunohistochemistry in 41 formalin-fixed and paraffin-embedded astrocytomas. The positive cell rate of gelsolin in tumors was 59.4% in grade II, 30.0% in grade III and 29.4% in grade IV, respectively. Gelsolin expression was significantly lower in high-grade astrocytomas (grade III or IV) than in low-grade astrocytomas (grade II; P < 0.05). Moreover, in astrocytomas the overall survival of patients in the low-expression group was significantly poorer than in the high expression group (P < 0.05). These data suggest that gelsolin is a prognostic factor in astrocytoma.

    Pathology international 2009;59;11;797-803

  • Ca2+ binding by domain 2 plays a critical role in the activation and stabilization of gelsolin.

    Nag S, Ma Q, Wang H, Chumnarnsilpa S, Lee WL, Larsson M, Kannan B, Hernandez-Valladares M, Burtnick LD and Robinson RC

    Institute of Molecular and Cell Biology, ASTAR, 61 Biopolis Drive, Proteos, Singapore 138673.

    Gelsolin consists of six homologous domains (G1-G6), each containing a conserved Ca-binding site. Occupation of a subset of these sites enables gelsolin to sever and cap actin filaments in a Ca-dependent manner. Here, we present the structures of Ca-free human gelsolin and of Ca-bound human G1-G3 in a complex with actin. These structures closely resemble those determined previously for equine gelsolin. However, the G2 Ca-binding site is occupied in the human G1-G3/actin structure, whereas it is vacant in the equine version. In-depth comparison of the Ca-free and Ca-activated, actin-bound human gelsolin structures suggests G2 and G6 to be cooperative in binding Ca(2+) and responsible for opening the G2-G6 latch to expose the F-actin-binding site on G2. Mutational analysis of the G2 and G6 Ca-binding sites demonstrates their interdependence in maintaining the compact structure in the absence of calcium. Examination of Ca binding by G2 in human G1-G3/actin reveals that the Ca(2+) locks the G2-G3 interface. Thermal denaturation studies of G2-G3 indicate that Ca binding stabilizes this fragment, driving it into the active conformation. The G2 Ca-binding site is mutated in gelsolin from familial amyloidosis (Finnish-type) patients. This disease initially proceeds through protease cleavage of G2, ultimately to produce a fragment that forms amyloid fibrils. The data presented here support a mechanism whereby the loss of Ca binding by G2 prolongs the lifetime of partially activated, intermediate conformations in which the protease cleavage site is exposed.

    Funded by: Howard Hughes Medical Institute

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;33;13713-8

  • Gelsolin, but not its cleavage, is required for TNF-induced ROS generation and apoptosis in MCF-7 cells.

    Li Q, Ye Z, Wen J, Ma L, He Y, Lian G, Wang Z, Wei L, Wu D and Jiang B

    The Department of Biomedical Sciences, School of Life Sciences, Xiamen University, Xiamen, Fujian, China. liqinxi@xmu.edu.cn

    The tumor necrosis factor (TNF) can induce apoptosis in many cells including MCF-7 cells. To identify the genes responsible for TNF-induced apoptosis, we generated a series of TNF-resistant MCF-7 cell lines by employing retrovirus insertion-mediated random mutagenesis. In one of the resistant lines, gelsolin was found to be disrupted by viral insertion. Exogenous expression of gelsolin in this mutant cell line (Gel(mut)) restored the sensitivity to TNF-induced cell death and knock-down of gelsolin by siRNA conferred MCF-7 cells with resistance to TNF, indicating that gelsolin is required for TNF-induced apoptosis. Interestingly, the resistance of Gel(mut) cells to apoptosis induction is selective to TNF, since Gel(mut) and wild-type cells showed similar sensitivity to other death stimuli that were tested. Furthermore, TNF-induced ROS production in Gel(mut) cells was significantly decreased, demonstrating that gelsolin-mediated ROS generation plays a crucial role in TNF-induced apoptosis in MCF-7 cells. Importantly, caspase-mediated gelsolin cleavage is dispensable for TNF-triggered ROS production and subsequent apoptosis of MCF-7 cells. Our study thus provides genetic evidence linking gelsolin-mediated ROS production to TNF-induced cell death.

    Biochemical and biophysical research communications 2009;385;2;284-9

  • Secretion of amyloidogenic gelsolin progressively compromises protein homeostasis leading to the intracellular aggregation of proteins.

    Page LJ, Suk JY, Bazhenova L, Fleming SM, Wood M, Jiang Y, Guo LT, Mizisin AP, Kisilevsky R, Shelton GD, Balch WE and Kelly JW

    Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

    Familial amyloidosis of Finnish type (FAF) is a systemic amyloid disease associated with the deposition of proteolytic fragments of mutant (D187N/Y) plasma gelsolin. We report a mouse model of FAF featuring a muscle-specific promoter to drive D187N gelsolin synthesis. This model recapitulates the aberrant endoproteolytic cascade and the aging-associated extracellular amyloid deposition of FAF. Amyloidogenesis is observed only in tissues synthesizing human D187N gelsolin, despite the presence of full-length D187N gelsolin and its 68-kDa cleavage product in blood-demonstrating the importance of local synthesis in FAF. Loss of muscle strength was progressive in homozygous D187N gelsolin mice. The presence of misfolding-prone D187N gelsolin appears to exacerbate the age-associated decline in cellular protein homeostasis (proteostasis), reflected by the intracellular deposition of numerous proteins, a characteristic of the most common degenerative muscle disease of aging humans, sporadic inclusion body myositis.

    Funded by: NIA NIH HHS: AG18917, R01 AG018917; NIGMS NIH HHS: GM33301, R01 GM033301; NINDS NIH HHS: P50 NS038367, P50NS38367

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;27;11125-30

  • Plasma gelsolin and circulating actin correlate with hemodialysis mortality.

    Lee PS, Sampath K, Karumanchi SA, Tamez H, Bhan I, Isakova T, Gutierrez OM, Wolf M, Chang Y, Stossel TP and Thadhani R

    Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    Plasma gelsolin (pGSN) binds actin and bioactive mediators to localize inflammation. Low pGSN correlates with adverse outcomes in acute injury, whereas administration of recombinant pGSN reduces mortality in experimental sepsis. We found that mean pGSN levels of 150 patients randomly selected from 10,044 starting chronic hemodialysis were 140 +/- 42 mg/L, 30 to 50% lower than levels reported for healthy individuals. In a larger sample, we performed a case-control analysis to evaluate the relationship of pGSN and circulating actin with mortality; pGSN levels were significantly lower in 114 patients who died within 1 yr of dialysis initiation than in 109 survivors (117 +/- 38 mg/L versus 147 +/- 42 mg/L, P < 0.001). pGSN levels had a graded, inverse relationship with 1-yr mortality, such that patients with pGSN < 130 mg/L experienced a > 3-fold risk for mortality compared with those with pGSN > or = 150 mg/L. The 69% of patients with detectable circulating actin had lower pGSN levels than those without (127 +/- 45 mg/L versus 141 +/- 36 mg/L, P = 0.026). Compared with patients who had elevated pGSN and no detectable actin, those with low pGSN levels and detectable actin had markedly increased mortality (odds ratio 9.8, 95% confidence interval 2.9 to 33.5). Worsening renal function correlated with pGSN decline in 53 subjects with CKD not on dialysis. In summary, low pGSN and detectable circulating actin identify chronic hemodialysis patients at highest risk for 1-yr mortality.

    Funded by: Howard Hughes Medical Institute; NHLBI NIH HHS: HL093954, R13 HL093954; NIDDK NIH HHS: DK71674, R21 DK071674

    Journal of the American Society of Nephrology : JASN 2009;20;5;1140-8

  • Gelsolin regulates cardiac remodeling after myocardial infarction through DNase I-mediated apoptosis.

    Li GH, Shi Y, Chen Y, Sun M, Sader S, Maekawa Y, Arab S, Dawood F, Chen M, De Couto G, Liu Y, Fukuoka M, Yang S, Da Shi M, Kirshenbaum LA, McCulloch CA and Liu P

    Toronto General Hospital, University Health Network, Ontario, Canada.

    Gelsolin, a calcium-regulated actin severing and capping protein, is highly expressed in murine and human hearts after myocardial infarction and is associated with progression of heart failure in humans. The biological role of gelsolin in cardiac remodeling and heart failure progression after injury is not defined. To elucidate the contribution of gelsolin in these processes, we randomly allocated gelsolin knockout mice (GSN(-/-)) and wild-type littermates (GSN(+/+)) to left anterior descending coronary artery ligation or sham surgery. We found that GSN(-/-) mice have a surprisingly lower mortality, markedly reduced hypertrophy, smaller late infarct size, less interstitial fibrosis, and improved cardiac function when compared with GSN(+/+) mice. Gene expression and protein analysis identified significantly lower levels of deoxyribonuclease (DNase) I and reduced nuclear translocation and biological activity of DNase I in GSN(-/-) mice. Absence of gelsolin markedly reduced DNase I-induced apoptosis. The association of hypoxia-inducible factor (HIF)-1alpha with gelsolin and actin filaments cleaved by gelsolin may contribute to the higher activation of DNase. The expression pattern of HIF-1alpha was similar to that of gelsolin, and HIF-1alpha was detected in the gelsolin complex by coprecipitation and HIF-1alpha bound to the promoter of DNase I in both gel-shift and promoter activity assays. Furthermore, the phosphorylation of Akt at Ser473 and expression of Bcl-2 were significantly increased in GSN(-/-) mice, suggesting that gelsolin downregulates prosurvival factors. Our investigation concludes that gelsolin is an important contributor to heart failure progression through novel mechanisms of HIF-1alpha and DNase I activation and downregulation of antiapoptotic survival factors. Gelsolin inhibition may form a novel target for heart failure therapy.

    Circulation research 2009;104;7;896-904

  • Cerebral amyloid angiopathy: a common cause of cerebral hemorrhage.

    Pezzini A, Del Zotto E, Volonghi I, Giossi A, Costa P and Padovani A

    Department of Medical and Surgical Sciences, Neurology Clinic, University of Brescia, Brescia, Italy. ale_pezzini@hotmail.com

    Amyloid is a term used to describe protein deposits with circumscript physical characteristics: beta-pleated sheet configuration, apple green birefringence under polarized light after Congo red staining, fibrillary structure and high insolubility. Cerebral amyloid angiopathy (CAA) defines a clinicopathological phenomenon characterized by amyloid deposition in the walls of leptomeningeal and cortical arteries, arterioles, and, less often capillaries and veins of the central nervous system. CAAs are currently classified according to the protein deposited including amyloid beta peptide (Abeta), cystatin C (ACys C), prion protein (PrP(Sc)), ABri/ADan, transthyretin (ATTR), and gelsolin (AGel). Most often amyloid deposition occurs in sporadic forms. In less common hereditary forms, a mutated variant protein or precursor protein is abnormally metabolized by proteolytic pathways in consequence of specific gene mutations, and accumulates as amyloid. The spectrum of clinical phenotypes associated with CAA-related vasculopathic changes includes both ischemic and hemorrhagic presentations, primary intracerebral hemorrhage (PICH) being probably the most well-recognized. However, in spite of accumulating data and recent progress in understanding the pathogenesis of CAA-related hemorrhage, the exact mechanisms leading to vessel rupture in these cases are yet to be established. This represents, at present, a major limitation to the identification of reliable biomarkers and the development of disease-specific treatment options. The present paper summarizes epidemiologic and clinical aspects of CAA, and highlights the presumed pathomechanisms of amyloid deposition in both sporadic and hereditary forms.

    Current medicinal chemistry 2009;16;20;2498-513

  • Gelsolin in human colon adenocarcinoma cells with different metastatic potential.

    Litwin M, Mazur AJ, Nowak D, Mannherz HG and Malicka-Błaszkiewicz M

    Depratment of Cell Pathology, Faculty of Biotechnology, University of Wrocław, Wrocław, Poland. mmach@ibmb.uni.wroc.pl

    Gelsolin, one of a major actin-binding proteins, is involved in the regulation of actin cytoskeleton organization by its severing and capping activity towards actin filaments. Human colon adenocarcinoma cell line LS180 and its selected variants of different metastatic potential were used to check for a correlation between gelsolin level, its subcellular localization and the invasive capacity of cells. Based on immunoblotting experiments, a decreased level of gelsolin was detected in the most invasive 5W subline when compared to the parental cell line LS180. The intracellular distribution of actin filaments and gelsolin in colon adenocarcinoma cells was examined by confocal microscopy. In the 5W subline, unlike in the other examined cells, gelsolin was colocalized with filamentous actin at the cell periphery. In summary, in human colon adenocarcinoma cells, gelsolin level and its subcellular distribution seem to correlate with their metastatic potential.

    Acta biochimica Polonica 2009;56;4;739-43

  • Aldehyde dehydrogenase 1A1 and gelsolin identified as novel invasion-modulating factors in conditioned medium of pancreatic cancer cells.

    Walsh N, Dowling P, O'Donovan N, Henry M, Meleady P and Clynes M

    National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland. naomi.walsh@dcu.ie

    Conditioned medium (CM) from clonal sub-populations of the pancreatic cancer cell line, MiaPaCa-2 with differing invasive abilities, were examined for their effect on in vitro invasion. Conditioned medium from Clone #3 (CM#3) strongly promoted invasion, while CM from Clone #8 (CM#8) inhibited invasion in vitro. 2D DIGE followed by MALDI-TOF MS analysis of CM#3 and CM#8 identified 41 proteins which were differentially regulated; 27 proteins were down-regulate 1f40 d and 14 proteins up-regulated in the invasion-promoting CM#3 when compared to CM#8. Western blotting analysis confirmed the down-regulated expression of gelsolin and the up-regulation of aldehyde dehydrogenase 1A1 in CM#3. Down-regulation of aldehyde dehydrogenase 1A1 in Clone #3 CM and gelsolin levels in Clone #8 CM by siRNA transfection revealed an important involvement of these proteins in promoting and inhibiting invasion in these pancreatic cancer cell lines.

    Journal of proteomics 2008;71;5;561-71

  • Anti-amyloidogenic, anti-oxidant and anti-apoptotic role of gelsolin in Alzheimer's disease.

    Chauhan V, Ji L and Chauhan A

    New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York, 10314, USA. ved.chauhan@omr.state.ny.us

    Fibrillar amyloid beta-protein (Abeta) is a major component of amyloid plaques in the brains of individuals with Alzheimer's disease (AD) and of adults with Down syndrome (DS). Gelsolin, a cytoskeletal protein, is present both intracellularly (cytoplasmic form) and extracellularly (secretory form in biological fluids). These two forms of gelsolin differ from each other in length and in cysteinyl thiol groups. Previous studies from our and other groups have identified the anti-amyloidogenic role of gelsolin in AD. Our studies showed that both plasma and cytosolic gelsolin bind to Abeta, and that gelsolin inhibits the fibrillization of Abeta and solubilizes preformed fibrils of Abeta. Other studies have shown that peripheral administration of plasma gelsolin or transgene expression of plasma gelsolin can reduce amyloid load in the transgenic mouse model of AD. Our recent studies showed that gelsolin expression increases in cells in response to oxidative stress. Oxidative damage is considered a major feature in the pathophysiology of AD. Abeta not only can induce oxidative stress, but also its generation is increased as a result of oxidative stress. In this article, we review evidence of gelsolin as an anti-amyloidogenic agent that can reduce amyloid load by acting as an inhibitor of Abeta fibrillization, and as an antioxidant and anti-apoptotic protein.

    Funded by: NIA NIH HHS: AG020992

    Biogerontology 2008;9;6;381-9

  • Extracellular gelsolin binds lipoteichoic acid and modulates cellular response to proinflammatory bacterial wall components.

    Bucki R, Byfield FJ, Kulakowska A, McCormick ME, Drozdowski W, Namiot Z, Hartung T and Janmey PA

    University of Pennsylvania, Institute for Medicine and Engineering, Philadelphia, PA 19104, USA. buckirob@mail.med.upenn.edu

    The various functions of gelsolin in extracellular compartments are not yet clearly defined but include actin scavenging and antiinflammatory effects. Gelsolin was recently reported to bind endotoxin (LPS) from various Gram-negative bacteria with high affinity. In this study we investigate whether gelsolin also interacts with bacterial wall molecules of Gram-positive bacteria such as lipoteichoic acid (LTA) and whether gelsolin's interaction with bacterial lipids from Gram-negative or Gram-positive bacteria affects their cellular inflammatory responses. A peptide based on the PPI binding site of gelsolin (160-169) binds purified LTA at the same molecular ratio that it binds phosphatidylinositol 4,5-bisphosphate. The OD of recombinant human plasma gelsolin was found to decrease following the addition of purified LTA, and the binding of gelsolin to LTA inhibits F-actin depolymerization by gelsolin. Simultaneously, the ability of LTA to activate translocation of NF-kappaB, E-selectin expression, and adhesion of neutrophils to LTA-treated human aortic endothelial cells was compromised by gelsolin. Gelsolin was able to partially inhibit LPS- or LTA-induced release of IL-8 from human neutrophils but was unable to prevent Gram-positive Bacillus subtilis or Gram-negative Pseudomonas aeruginosa growth and had no effect on the antibacterial activity of the cathelicidin-derived antibacterial peptide LL37. These data suggest that extracellular gelsolin is involved in the host immune recognition of LTA or LPS following release of these molecules from the bacterial outer membrane during cell division or attack by drugs and immune components.

    Funded by: NIAMS NIH HHS: AR38910

    Journal of immunology (Baltimore, Md. : 1950) 2008;181;7;4936-44

  • In colon carcinogenesis, the cytoskeletal protein gelsolin is down-regulated during the transition from adenoma to carcinoma.

    Gay F, Estornes Y, Saurin JC, Joly-Pharaboz MO, Friederich E, Scoazec JY and Abello J

    Inserm, U865, Lyon, F-69372, France.

    The actin-binding protein gelsolin is involved in cell motility via the regulation of actin cytoskeleton, and its expression is modified in several human cancers. However, the potential implication of this protein in colorectal carcinogenesis is debated. By using immunohistochemistry, we studied gelsolin expression in 69 cases of colon adenocarcinomas and in 72 lesions representative of the different stages of colonic tumorigenesis. In addition, we performed Northern blot analysis of gelsolin messenger RNA in 12 paired samples of human colon cancer and normal corresponding mucosa. Gelsolin protein and messenger RNA expressions were severely down-regulated in all adenocarcinomas tested. Moreover, gelsolin protein was down-regulated in a large proportion of high-grade adenomas (14/16) before the acquisition of invasive properties but in only a small proportion of low grade adenomas and serrated adenomas (2/30) and in none of the 9 cases of nonneoplastic hyperplastic polyps tested. Our results therefore demonstrate that gelsolin down-regulation is an early and almost constant event in colon carcinogenesis and is associated with the transition from adenoma to carcinoma.

    Human pathology 2008;39;10;1420-30

  • The ubiquitin-proteasome pathway mediates gelsolin protein downregulation in pancreatic cancer.

    Ni XG, Zhou L, Wang GQ, Liu SM, Bai XF, Liu F, Peppelenbosch MP and Zhao P

    Department of Endoscopy, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

    A well-known observation with respect to cancer biology is that transformed cells display a disturbed cytoskeleton. The underlying mechanisms, however, remain only partly understood. In an effort to identify possible mechanisms, we compared the proteome of pancreatic cancer with matched normal pancreas and observed diminished protein levels of gelsolin--an actin filament severing and capping protein of crucial importance for maintaining cytoskeletal integrity--in pancreatic cancer. Additionally, pancreatic ductal adenocarcinomas displayed substantially decreased levels of gelsolin as judged by Western blot and immunohistochemical analyses of tissue micoarrays, when compared with cancerous and untransformed tissue from the same patients (P < 0.05). Importantly, no marked downregulation of gelsolin mRNA was observed (P > 0.05), suggesting that post-transcriptional mechanisms mediate low gelsolin protein levels. In apparent agreement, high activity ubiquitin-proteasome pathway in both patient samples and the BxPC-3 pancreatic cancer cell line was detected, and inhibition of the 26s proteasome system quickly restored gelsolin protein levels in the latter cell line. The status of ubiquitinated gelsolin is related to lymph node metastasis of pancreatic cancer. In conclusion, gelsolin levels are actively downregulated in pancreatic cancer and enhanced targeting of gelsolin to the ubiquitin-proteasome pathway is an important contributing factor for this effect.

    Molecular medicine (Cambridge, Mass.) 2008;14;9-10;582-9

  • Ardalan-Shoja-Kiuru syndrome--hereditary gelsolin amyloidosis plus retinitis pigmentosa.

    Shokouhi G and Khosroshahi HT

    Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 2008;23;3;1071; author reply 1071-2

  • Decreased levels of the gelsolin plasma isoform in patients with rheumatoid arthritis.

    Osborn TM, Verdrengh M, Stossel TP, Tarkowski A and Bokarewa M

    Department of Rheumatology and Inflammation Research, University of Gothenburg, Guldhedsgatan 10A, S-413 46 Gothenburg, Sweden. tmagnuson@rics.bwh.harvard.edu

    Introduction: Gelsolin is an intracellular actin-binding protein involved in cell shape changes, cell motility, and apoptosis. An extracellular gelsolin isoform, plasma gelsolin circulates in the blood of healthy individuals at a concentration of 200 +/- 50 mg/L and has been suggested to be a key component of an extracellular actin-scavenging system during tissue damage. Levels of plasma gelsolin decrease during acute injury and inflammation, and administration of recombinant plasma gelsolin to animals improves outcomes following sepsis or burn injuries. In the present study, we investigated plasma gelsolin in patients with rheumatoid arthritis.

    Methods: Circulating and intra-articular levels of plasma gelsolin were measured in 78 patients with rheumatoid arthritis using a functional (pyrene-actin nucleation) assay and compared with 62 age- and gender-matched healthy controls.

    Results: Circulating plasma gelsolin levels were significantly lower in patients with rheumatoid arthritis compared with healthy controls (141 +/- 32 versus 196 +/- 40 mg/L, P = 0.0002). The patients' intra-articular plasma gelsolin levels were significantly lower than in the paired plasma samples (94 +/- 24 versus 141 +/- 32 mg/L, P = 0.0001). Actin was detected in the synovial fluids of all but four of the patients, and immunoprecipitation experiments identified gelsolin-actin complexes.

    Conclusions: The plasma isoform of gelsolin is decreased in the plasma of patients with rheumatoid arthritis compared with healthy controls. The reduced plasma concentrations in combination with the presence of actin and gelsolin-actin complexes in synovial fluids suggest a local consumption of this potentially anti-inflammatory protein in the inflamed joint.

    Arthritis research & therapy 2008;10;5;R117

  • Plasma gelsolin depletion and circulating actin in sepsis: a pilot study.

    Lee PS, Patel SR, Christiani DC, Bajwa E, Stossel TP and Waxman AB

    Pulmonary and Critical Care Division, Brigham and Women's Hospital, Boston, MA, USA. plee4@partners.org

    Background: Depletion of the circulating actin-binding protein, plasma gelsolin (pGSN) has been described in septic patients and animals. We hypothesized that the extent of pGSN reduction correlates with outcomes of septic patients and that circulating actin is a manifestation of sepsis.

    We assayed pGSN in plasma samples from non-surgical septic patients identified from a pre-existing database which prospectively enrolled patients admitted to adult intensive care units at an academic hospital. We identified 21 non-surgical septic patients for the study. Actinemia was detected in 17 of the 21 patients, suggesting actin released into circulation from injured tissues is a manifestation of sepsis. Furthermore, we documented the depletion of pGSN in human clinical sepsis, and that the survivors had significantly higher pGSN levels than the non-survivors (163+/-47 mg/L vs. 89+/-48 mg/L, p = 0.01). pGSN levels were more strongly predictive of 28-day mortality than APACHE III scores. For every quartile reduction in pGSN, the odds of death increased 3.4-fold.

    Conclusion: We conclude that circulating actin and pGSN deficiency are associated with early sepsis. The degree of pGSN deficiency correlates with sepsis mortality. Reversing pGSN deficiency may be an effective treatment for sepsis.

    Funded by: NHLBI NIH HHS: 5R01 HL074859-04, R01 HL074859

    PloS one 2008;3;11;e3712

  • Concurrent down-regulation of Egr-1 and gelsolin in the majority of human breast cancer cells.

    Liu J, Liu YG, Huang R, Yao C, Li S, Yang W, Yang D and Huang RP

    Department of Gynecology and Obstetrics, Emory University School of Medicine, Atlanta, GA 30322, USA.

    Unlabelled: A growing body of evidence suggests that early growth response-1 (Egr-1), a transcription factor, may function as a tumor suppressor. The aim of this study was to gain more evidence to support the role of Egr-1 in the suppression of cancer cell growth and to examine the potential correlation between Egr-1 and gelsolin.

    Histochemical staining coupled with breast cancer tissue arrays were used to examine the expression levels of Egr-1 and gelsolin. Reporter assays and gel shift were used to study the transcriptional activity of Egr-1 on the regulation of gelsolin.

    Results: Our data showed that most normal mammary tissues expressed high levels of Egr-1, while the majority of breast cancer tissues expressed very small amounts of Egr-1. The expression pattern of Egr-1 in human breast cancer tissues was highly correlated with gelsolin expression. Induction of Egr-1 by serum stimulation accompanied the increase of gelsolin expression. In cells lacking the induction of Egr-1 in response to serum stimulation, gelsolin expression remained unchanged. Furthermore, gelsolin promoter activity was profoundly reduced in Egr-1 null mouse embryonic fibroblasts compared to Egr-1 wild-type mouse embryonic fibroblasts. Gel shift experiments indicated that Egr-1 can directly bind to the gelsolin promoter.

    Conclusion: Our results suggest that Egr-1 may be an important breast cancer marker and that an as yet uncharacterized pathway involved in Egr-1 and gelsolin expression exists which leads to breast cancer cell development.

    Funded by: NCI NIH HHS: CA107783

    Cancer genomics & proteomics 2007;4;6;377-85

  • Genomic and proteomic profiles reveal the association of gelsolin to TP53 status and bladder cancer progression.

    Sanchez-Carbayo M, Socci ND, Richstone L, Corton M, Behrendt N, Wulkfuhle J, Bochner B, Petricoin E and Cordon-Cardo C

    Tumor Markers Group, Spanish National Cancer Center, Madrid, Spain. mscarbayo@cnio.es

    Bladder cancer transformation and immortalization require the inactivation of key regulatory genes, including TP53. Genotyping of a large cohort of bladder cancer patients (n = 256) using the TP53 GeneChip showed mutations in 103 cases (40.2%), the majority of them mapping to the DNA-binding core domain. TP53 mutation status was significantly associated with tumor stage (P = 0.0001) and overall survival for patients with advanced disease (P = 0.01). Transcript profiling using oligonucleotide arrays was performed on a subset of these cases (n = 46). Supervised analyses identified genes differentially expressed between invasive bladder tumors with wild-type (n = 24) and mutated TP53 (n = 22). Pathway analyses of top-ranked genes supported the central role of TP53 in the functional network of such gene patterns. A proteomic strategy using reverse phase arrays with protein extracts of bladder cancer cell lines validated the association of identified differentially expressed genes, such as gelsolin, to TP53 status. Immunohistochemistry on tissue microarrays (n = 294) revealed that gelsolin was associated with tumor stage and overall survival, correlating positively with TP53 status in a subset of these patients. This study further reveals that TP53 mutations are frequent events in bladder cancer progression and identified gelsolin related to TP53 status, tumor staging, and clinical outcome by independent high-throughput strategies.

    The American journal of pathology 2007;171;5;1650-8

  • Functional specialization of beta-arrestin interactions revealed by proteomic analysis.

    Xiao K, McClatchy DB, Shukla AK, Zhao Y, Chen M, Shenoy SK, Yates JR and Lefkowitz RJ

    Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.

    Beta-arrestins are cytosolic proteins that form complexes with seven-transmembrane receptors after agonist stimulation and phosphorylation by the G protein-coupled receptor kinases. They play an essential role in receptor desensitization and endocytosis, and they also serve as receptor-regulated signaling scaffolds and adaptors. Moreover, in the past decade, a growing list of protein-protein interactions of beta-arrestins pertinent to these functions has been documented. The discovery of several novel functions of beta-arrestins stimulated us to perform a global proteomics analysis of beta-arrestin-interacting proteins (interactome) as modulated by a model seven-transmembrane receptor, the angiotensin II type 1a receptor, in an attempt to assess the full range of functions of these versatile molecules. As determined by LC tandem MS, 71 proteins interacted with beta-arrestin 1, 164 interacted with beta-arrestin 2, and 102 interacted with both beta-arrestins. Some proteins bound only after agonist stimulation, whereas others dissociated. Bioinformatics analysis of the data indicates that proteins involved in cellular signaling, organization, and nucleic acid binding are the most highly represented in the beta-arrestin interactome. Surprisingly, both S-arrestin (visual arrestin) and X-arrestin (cone arrestin) were also found in heteromeric complex with beta-arrestins. The beta-arrestin interactors distribute not only in the cytoplasm, but also in the nucleus as well as other subcellular compartments. The binding of 16 randomly selected newly identified beta-arrestin partners was validated by coimmunoprecipitation assays in HEK293 cells. This study provides a comprehensive analysis of proteins that bind beta-arrestin isoforms and underscores their potentially broad regulatory roles in mammalian cellular physiology.

    Funded by: NCRR NIH HHS: P41 RR011823, P41 RR11823; NHLBI NIH HHS: HL16037, R01 HL016037, R01 HL070631; NIMH NIH HHS: 5R01MH067880-02, R01 MH067880

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;29;12011-6

  • Two distinct sites of interaction form the calponin: gelsolin complex and two calcium switches control its activity.

    Ferjani I, Fattoum A, Maciver SK, Manai M, Benyamin Y and Roustan C

    DIMNP, University of Montpellier 2 and 1, CNRS Montpellier, Place E, Bataillon, CC107, 34095 Montpellier Cedex 5, France.

    Gelsolin and calponin are well characterized actin-binding proteins that form a tight gelsolin:calponin complex (GCC). We show here that the GCC is formed through two distinct interfaces. One of these is formed between 144-182 of calponin and 25-150 of gelsolin (G1). The second is a calcium-sensitive site centred on calponin's CH domain, and the C-terminal half of gelsolin (G4-6). The behaviour of this second interface is dependent on the presence of calcium and so it is possible that potential GCC-binding partners may be selected by calcium availability. Actin is one such GCC-binding partner and we show that a larger complex is formed with monomeric actin in calcium. The stoichiometry of this complex is determined to be 1 gelsolin/1 calponin/2 G-actins (GCA(2)). Both actin monomers bind the GCC through gelsolin. Both calponin and gelsolin are reported to play signaling roles in addition to their better-characterized actin-binding properties and it is possible that the GCC regulates both of these functions.

    Biochimica et biophysica acta 2007;1774;7;952-8

  • Oncogenic K-RAS subverts the antiapoptotic role of N-RAS and alters modulation of the N-RAS:gelsolin complex.

    Keller JW, Haigis KM, Franklin JL, Whitehead RH, Jacks T and Coffey RJ

    Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.

    Activating mutations in members of the RAS family of genes are among the most common genetic events in human tumorigenesis. Once thought to be functionally interchangeable, it is increasingly recognized that the classical members of this protein family (H-RAS, N-RAS and K-RAS4B) exhibit unique and shared functions that are highly context-dependent. Herein, we demonstrate that the presence of an oncogenic KRAS allele results in elevated levels of GTP-bound N-RAS (N-RAS.GTP) in two human colorectal cancer cell lines, HCT 116 and DLD-1, compared to their isogenic counterparts in which the mutant KRAS allele has been disrupted by homologous recombination. N-RAS subserves an antiapoptotic role in cells expressing wild-type K-RAS; this function is compromised, however, by the presence of mutant K-RAS, and these cells display increased sensitivity to apoptotic stimuli. We additionally identify a physical interaction between N-RAS and gelsolin, a factor that has been shown to promote survival and show that the N-RAS:gelsolin complex is modulated differently in wild-type and mutant K-RAS environments following apoptotic challenge. These findings represent the first biochemical evidence of a functional relationship between endogenous RAS proteins and identify a dynamic physical interaction between endogenous N-RAS and gelsolin that correlates with survival.

    Funded by: NCI NIH HHS: CA046413-19, CA084239-09, CA095103-06, P50 CA095103, P50 CA095103-06, R01 CA046413, R01 CA046413-19, R01 CA46413, U01 CA084239, U01 CA084239-09; PHS HHS: P50 95103, U01 084239

    Oncogene 2007;26;21;3051-9

  • Lattice corneal dystrophy type II: clinical, pathologic, and molecular study in a Spanish family.

    Huerva V, Velasco A, Sánchez MC, Mateo AJ and Matías-Guiu X

    Ophthalmology Department, Universitary Hospital Arnau de Vilanova, IRB Lleida, University of Lleida, Lleida, Spain. vhuerva@mixmail.com

    Purpose: To report a family with lattice corneal dystrophy type II (LCD II) associated with systemic amyloidosis type V.

    Methods: A 69-year-old woman presented a LCD II and marked dermachalasis. A lower blepharoplasty was performed. Two years later a penetrating keratoplasty was performed in her left eye. Three children of the patient were studied. Subtle manifestations of LCD were identified in two of them. Pathologic study of the excised skin and corneal button was made. DNA from peripheral blood was obtained, and was subjected to amplification of exon 5 of the gelsolin.

    Results: Pathologic examination of the skin of blepharoplasty specimen demonstrated the presence of amyloid. Microscopic examination of the corneal button showed the presence of amyloid deposits beneath the normal-appearing Bowman layer and also within the stroma. Immunostaining for S-100 protein did not demonstrate a significant relationship between amyloid deposits and corneal nerves. Electron microscopic evaluation demonstrated the presence of amyloid fibrils. No clear relationship was found between amyloid deposits and corneal nerves. These findings confirm LCD type II or Meretoja syndrome. A mutation analysis of the gelsolin gene demonstrated the presence of G to A transition at nucleotide 654. Two children with manifestations of LCD also showed the identical mutation in gelsolin gene.

    Conclusions: A new family with Meretoja syndrome is reported. This is the first documented family with Meretoja syndrome in Spain and in the Mediterranean countries. The molecular study shows the same mutation of reported families from Finland, Japan, the United States, and the United Kingdom.

    European journal of ophthalmology 2007;17;3;424-9

  • HDM2-binding partners: interaction with translation elongation factor EF1alpha.

    Frum R, Busby SA, Ramamoorthy M, Deb S, Shabanowitz J, Hunt DF and Deb SP

    Department of Biochemistry and the Massey Cancer Center, Virginia Commonwealth University, Richmond, Virginia 23298, USA.

    To understand the cellular functions of HDM2, we attempted to identify novel HDM2-interacting proteins by proteomic analysis. Along with previously identified interactions with the ribosomal proteins, our analysis reveals interactions of HDM2 with the ribosomal translation elongation factor EF1alpha, 40S ribosomal protein S20, tubulins, glyceraldehyde 3-phosphate dehydrogenase, and a proteolysis-inducing factor dermicidin in the absence of tumor suppressor p53. Because a CTCL tumor antigen HD-CL-08 has high degree of homology with EF1alpha, we confirmed interaction of HDM2 with EF1alpha by immunoprecipitation and Western blot analysis in transformed as well as near normal diploid cells. Endogenous HDM2- EF1alpha complex was detected in cancer cells overexpressing HDM2, suggesting a possible role of this interaction in HDM2-mediated oncogenesis. Consistent with their interaction, colocalization of HDM2 and EF1alpha can be detected in the cytoplasm of normal or transformed cells. Amino acid residues 1-58 and 221-325 of HDM2 were found to be essential for its interaction with EF1alpha, suggesting that the interaction is independent of its other ribosomal interacting proteins L5, L11, and L23. Overexpression of HDM2 did not affect translation. Because EF1alpha has been implicated in DNA replication and severing of microtubules, interaction of HDM2 with EF1alpha may signify a p53-independent cell growth regulatory role of HDM2.

    Funded by: NCI NIH HHS: CA70712, CA74172; NIGMS NIH HHS: R01 GM037537

    Journal of proteome research 2007;6;4;1410-7

  • Gelsolin overexpression alters actin dynamics and tyrosine phosphorylation of lipid raft-associated proteins in Jurkat T cells.

    Morley SC, Sung J, Sun GP, Martelli MP, Bunnell SC and Bierer BE

    Laboratory of Lymphocyte Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. morley_c@kids.wustl.edu

    Upon T cell receptor engagement, both the actin cytoskeleton and substrates of tyrosine phosphorylation are remodeled to create a signaling complex at the interface of the antigen-presenting cell and responding T cell. While T cell signaling has been shown to regulate actin reorganization, the mechanisms by which changes in actin dynamics affect early T cell signaling have not been fully explored. Using gelsolin, an actin-binding protein with capping and severing activities, and latrunculin, an actin-depolymerizing agent, we have further investigated the interplay between actin dynamics and the regulation of T cell signaling. Overexpression of gelsolin altered actin dynamics in Jurkat T cells, and alteration of actin dynamics correlated with dysregulation of tyrosine phosphorylation of raft-associated substrates. This perturbation of tyrosine phosphorylation was correlated with inhibition of activation-dependent signaling pathways regulating Erk-1/2 phosphorylation, NF-AT transcriptional activation and IL-2 production. Modification of actin by the depolymerizing agent latrunculin also altered the tyrosine phosphorylation patterns of proteins associated with lipid rafts, and pre-treatment with latrunculin inhibited anti-CD3 mAb-mediated NF-AT activation. Thus, our data indicate that actin cytoskeletal dynamics modulate the tyrosine phosphorylation of raft-associated proteins and subsequent downstream signal transduction.

    Funded by: Intramural NIH HHS; NCI NIH HHS: T32 CA009141, T32 CA009141-23, T32 CA009141-24, T32 CA09141

    Molecular immunology 2007;44;9;2469-80

  • Calcium-induced conformational changes in the amino-terminal half of gelsolin.

    Roustan C, Ferjani I, Maciver SK, Fattoum A, Rebière B and Benyamin Y

    UMR 5539 (CNRS) Laboratoire de motilité cellulaire (Ecole Pratique des Hautes Etudes), Université de Montpellier 2, Place E. Bataillon, CC107, 34095 Montpellier Cedex 5, France.

    Gelsolin is an actin-binding protein that is regulated by the occupancy of multiple calcium-binding sites. We have studied calcium induced conformational changes in the G1-2 and G1-3 sub-domains, and report the binding affinities for the three type II sites. A new probe for G3 has been produced and a K(d) of 5 microM has been measured for calcium in the context of G1-3. The two halves of gelsolin, G1-3 and G4-6 bind weakly with or without calcium, suggesting that once separated by apoptotic proteolysis, G1-3 and G4-6 remain apart allowing G1-3 to sever actin in a calcium free manner.

    FEBS letters 2007;581;4;681-6

  • Gel 1f40 solin segment 5 inhibits HIV-induced T-cell apoptosis via Vpr-binding to VDAC.

    Qiao H and McMillan JR

    Department of Dermatology, Hokkaido University Graduate School of Medicine, Kita-15, Nishi-7, Kita-Ku, Sapporo 060-0815, Japan. qiao@igm.hokudai.ac.jp

    Viral protein R (Vpr) from the human immunodeficiency virus induces cell cycle arrest in proliferating cells, stimulates virus transcription, and regulates activation and apoptosis of infected T-lymphocytes. We report that Jurkat cells overexpressing full-length gelsolin show resistance to Vpr-induced T-cell apoptosis with abrogation of mitochondrial membrane potential loss and the release of cytochrome c. Co-immunoprecipitation assays in HEK293T cells demonstrated that overexpression of full-length or segment 5 (G5) but not G5-deleted gelsolin (DeltaG5) bound to the voltage-dependent anion channel (VDAC), and that the G5 subunit can inhibit HIV-1-Vpr-binding to VDAC. We also confirmed that full-length gelsolin has the same effect in Jurkat cells. Clonogenic analysis showed that transfection of G5 but not DeltaG5 cDNA protects Jurkat T cells from HIV-Vpr-Tet induced T-cell apoptosis and promoted cell survival, as did full-length gelsolin. These results suggest that the gelsolin G5 domain inhibits HIV-Vpr-induced T-cell apoptosis by blocking the interaction between Vpr and VDAC, and might be used as a protective treatment against HIV-Vpr-induced T-cell apoptosis.

    FEBS letters 2007;581;3;535-40

  • Large-scale mapping of human protein-protein interactions by mass spectrometry.

    Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, Bukhman YV, Ethier M, Sheng Y, Vasilescu J, Abu-Farha M, Lambert JP, Duewel HS, Stewart II, Kuehl B, Hogue K, Colwill K, Gladwish K, Muskat B, Kinach R, Adams SL, Moran MF, Morin GB, Topaloglou T and Figeys D

    Protana, Toronto, Ontario, Canada.

    Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

    Molecular systems biology 2007;3;89

  • Prognostic significance of MCM2, Ki-67 and gelsolin in non-small cell lung cancer.

    Yang J, Ramnath N, Moysich KB, Asch HL, Swede H, Alrawi SJ, Huberman J, Geradts J, Brooks JS and Tan D

    Roswell Park Cancer Institute, Buffalo, NY 14263, USA. Jun.Yang@RoswellPark.org

    Background: Uncontrolled proliferation and increased motility are hallmarks of neoplastic cells, therefore markers of proliferation and motility may be valuable in assessing tumor progression and prognosis. MCM2 is a member of the minichromosome maintenance (MCM) protein family. It plays critical roles in the initiation of DNA replication and in replication fork movement, and is intimately related to cell proliferation. Ki-67 is a proliferation antigen that is expressed during all but G0 phases of the cell cycle. Gelsolin is an actin-binding protein that regulates the integrity of the actin cytoskeletal structure and facilitates cell motility. In this study, we assessed the prognostic significance of MCM2 and Ki-67, two markers of proliferation, and gelsolin, a marker of motility, in non-small cell lung cancer (NSCLC).

    Methods: 128 patients with pathologically confirmed, resectable NSCLC (stage I-IIIA) were included. Immunohistochemistry was utilized to measure the expressions of these markers in formalin-fixed, paraffin-embedded tumor tissues. Staining and scoring of MCM2, Ki-67 and gelsolin was independently performed. Analyses were performed to evaluate the prognostic significance of single expression of each marker, as well as the prognostic significance of composite expressions of MCM2 and gelsolin. Cox regression and Kaplan-Meier survival analysis were used for statistical analysis.

    Results: Of the three markers, higher levels of gelsolin were significantly associated with an increased risk of death (adjusted RR = 1.89, 95% CI = 1.17-3.05, p = 0.01), and higher levels of MCM2 were associated with a non-significant increased risk of death (adjusted RR = 1.36, 95% CI = 0.84-2.20, p = 0.22). Combined, adjusted analyses revealed a significantly poor prognostic effect for higher expression of MCM2 and gelsolin compared to low expression of both biomarkers (RR = 2.32, 95% CI = 1.21-4.45, p = 0.01). Ki-67 did not display apparent prognostic effect in this study sample.

    Conclusion: The results suggest that higher tumor proliferation and motility may be important in the prognosis of NSCLC, and composite application of biomarkers might be of greater value than single marker application in assessing tumor prognosis.

    Funded by: NCI NIH HHS: CA016056, CA72768, P30 CA016056; NIGMS NIH HHS: GM49294

    BMC cancer 2006;6;203

  • Posttranslational N-myristoylation is required for the anti-apoptotic activity of human tGelsolin, the C-terminal caspase cleavage product of human gelsolin.

    Sakurai N and Utsumi T

    Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan.

    Protein N-myristoylation has been recognized as a cotranslational protein modification. Recently, it was demonstrated that protein N-myristoylation could occur posttranslationally, as in the case of the pro-apoptotic protein BID and cytoskeletal actin. Our previous study showed that the N-terminal nine residues of the C-terminal caspase cleavage product of human gelsolin, an actin-regulatory protein, efficiently direct the protein N-myristoylation. In this study, to analyze the posttranslational N-myristoylation of gelsolin during apoptosis, metabolic labeling of gelsolin and its caspase cleavage products expressed in COS-1 cells with [3H]myristic acid was performed. It was found that the C-terminal caspase cleavage product of human gelsolin (tGelsolin) was efficiently N-myristoylated. When COS-1 cells transiently transfected with gelsolin cDNA were treated with etoposide or staurosporine, apoptosis-inducing agents, N-myristoylated tGelsolin was generated, as demonstrated by in vivo metabolic labeling. The generation of posttranslationally N-myristoylated tGelsolin during apoptosis was also observed on endogenous gelsolin expressed in HeLa cells. Immunofluorescence staining and subcellular fractionation experiment revealed that exogenously expressed tGelsolin did not localize to mitochondria but rather was diffusely distributed in the cytoplasm. To study the role of this modification in the anti-apoptotic activity of tGelsolin, we constructed the bicistronic expression plasmid tGelsolin-IRES-EGFP capable of overexpressing tGelsolin concomitantly with EGFP. Overexpression of N-myristoylated tGelsolin in COS-1 cells using this plasmid significantly inhibited etoposide-induced apoptosis, whereas overexpression of the non-myristoylated tGelsolinG2A mutant did not cause resistance to apoptosis. These results indicate that posttranslational N-myristoylation of tGelsolin does not direct mitochondrial targeting, but this modification is involved in the anti-apoptotic activity of tGelsolin.

    The Journal of biological chemistry 2006;281;20;14288-95

  • siRNA gelsolin knockdown induces epithelial-mesenchymal transition with a cadherin switch in human mammary epithelial cells.

    Tanaka H, Shirkoohi R, Nakagawa K, Qiao H, Fujita H, Okada F, Hamada J, Kuzumaki S, Takimoto M and Kuzumaki N

    Division of Cancer Gene Regulation, Research Section of Disease Control, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan.

    Epithelial-mesenchymal transition (EMT) describes a process occurring during development and oncogenesis by which epithelial cells obtain fibroblast-like properties and show reduced cell adhesion and increased motility. In this report, we demonstrated typical EMT in human mammary epithelial MCF10A small interfering (si)RNA gelsolin-knockdown cells. EMT was characterized by fibroblastic morphology, loss of contact inhibition and focus formation in monolayer growth, enhanced motility and invasiveness in vitro, increased actin filaments, overexpression of RAC, activation of both extracellular signal-regulated kinase and AKT, inactivation of glycogen synthase kinase-3, conversion of cadherin from the E- to N-type and induction of the transcription factor Snail. These results suggested that gelsolin functions as a switch that controls E- and N-cadherin conversion via Snail, and demonstrated that its knockdown leads to EMT in human mammary epithelial cells and possibly to the development of human mammary tumors.

    International journal of cancer 2006;118;7;1680-91

  • Modifications in the human T cell proteome induced by intracellular HIV-1 Tat protein expression.

    Coiras M, Camafeita E, Ureña T, López JA, Caballero F, Fernández B, López-Huertas MR, Pérez-Olmeda M and Alcamí J

    AIDS Immunopathology Unit, National Centre of Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.

    The effects of the human immunodeficiency virus type 1 (HIV-1) Tat protein on cellular gene expression were analysed using a Jurkat cell line that was stably transfected with tat gene in a doxycycline-repressible expression system. Expressed Tat protein (aa 1-101) was proved to present basically a nuclear localisation, and to be fully functional to induce HIV LTR transactivation. Tat expression also resulted in protection from Tunicamycin-induced apoptosis as determined by DNA staining and TUNEL assays. We applied proteomics methods to investigate changes in differential protein expression in the transfected Jurkat-Tat cells. Protein identification was performed using 2-D DIGE followed by MS analysis. We identified the down-regulation of several cytoskeletal proteins such as actin, beta-tubulin, annexin II, as well as gelsolin, cofilin and the Rac/Rho-GDI complex. Down-expression of these proteins could be involved in the survival of long-term reservoirs of HIV-infected CD4+ T cells responsible for continuous viral production. In conclusion, in addition to its role in viral mRNA elongation, the proteomic approach has provided insight into the way that Tat modifies host cell gene expression.

    Proteomics 2006;6 Suppl 1;S63-73

  • Heparin accelerates gelsolin amyloidogenesis.

    Suk JY, Zhang F, Balch WE, Linhardt RJ and Kelly JW

    Department of Chemistry and The Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

    The chemical environment of the extracellular matrix may influence the tissue-selective deposition observed there in gelsolin amyloid disease. Previously, we have identified the proteases that generate the amyloidogenic fragments from the full-length gelsolin variants and demonstrated that heparin is capable of accelerating gelsolin amyloidogenesis. Herein, we identify the structural features of heparin that promote the 8 kDa disease-associated gelsolin fragments (residues 173-243) generated at the cell surface to form amyloid. In conjunction with electron microscopy analyses, our kinetic studies demonstrate that heparin efficiently accelerates the formation of gelsolin amyloid by enabling intermolecular beta-sheet formation. The use of heparin analogues reveals that sulfation is important in accelerating amyloidogenesis and that the extent of acceleration is proportional to the molecular weight of heparin. In addition, heparin accelerated aggregation at both early and late stages of amyloidogenesis. Dynamic light scattering coupled to size exclusion chromatography showed that heparin promotes the formation of soluble aggregates. Collectively, these data reveal that heparin templates fibril formation and affords solubility to the aggregating peptides through its sulfated structure. By extension, the biochemical results herein suggest that tissue-selective deposition characteristic of the gelsolin amyloidoses is likely influenced by the extracellular localization of distinct glycosaminoglycans.

    Funded by: NHLBI NIH HHS: HL52622, HL62244, R01 HL052622, R01 HL052622-08, R01 HL062244, R01 HL062244-06; NIA NIH HHS: AG18917, R01 AG018917, R01 AG018917-02; NIGMS NIH HHS: GM038060, R01 GM038060, R01 GM038060-18

    Biochemistry 2006;45;7;2234-42

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T 5a8 , Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • Inactivation of endotoxin by human plasma gelsolin.

    Bucki R, Georges PC, Espinassous Q, Funaki M, Pastore JJ, Chaby R and Janmey PA

    Department of Physiology and Institute for Medicine and Engineering, University of Pennsylvania, 1010 Vagelos Research Laboratories, 3340 Smith Walk, Philadelphia, Pennsylvania 19104, USA. buckirob@ mail.med.upenn.edu

    Septic shock from bacterial endotoxin, triggered by the release of lipopolysaccharide (LPS) molecules from the outer wall of Gram-negative bacteria, is a major cause of human death for which there is no effective treatment once the complex inflammatory pathways stimulated by these small amphipathic molecules are activated. Here we report that plasma gelsolin, a highly conserved human protein, binds LPS from various bacteria with high affinity. Solid-phase binding assays, fluorescence measurements, and functional assays of actin depolymerizing effects show that gelsolin binds more tightly to LPS than it does to its other known lipid ligands, phosphatidylinositol 4,5-bisphosphate and lysophosphatidic acid. Gelsolin also competes with LPS-binding protein (LBP), a high-affinity carrier for LPS. One result of gelsolin-LPS binding is inhibition of the actin binding activity of gelsolin as well as the actin depolymerizing activity of blood serum. Simultaneously, effects of LPS on cellular functions, including cytoskeletal actin remodeling, and collagen-induced platelet activation by pathways independent of toll-like receptors (TLRs) are neutralized by gelsolin and by a peptide based on gelsolin residues 160-169 (GSN160-169) which comprise part of gelsolin's phosphoinositide binding site. Additionally, TLR-dependent NF-kappaB translocation in astrocytes appears to be blocked by gelsolin. These results show a strong effect of LPS on plasma gelsolin function and suggest that some effects of endotoxin in vivo may be mediated or inhibited by plasma gelsolin.

    Funded by: NHLBI NIH HHS: HL67286; NIAMS NIH HHS: AR38910

    Biochemistry 2005;44;28;9590-7

  • Mechanisms of gelsolin-dependent and -independent EGF-stimulated cell motility in a human lung epithelial cell line.

    Lader AS, Lee JJ, Cicchetti G and Kwiatkowski DJ

    Hematology Division, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA. alader@rics.bwh.harvard.edu

    Acquisition of motility is an important step in malignant progression of tumor cells and involves dynamic changes in actin filament architecture orchestrated by many actin binding proteins. A role for the actin-binding protein gelsolin has been demonstrated in fibroblast motility. In this report, we investigated the role of gelsolin in bronchial epithelial cell motility. The non-tumorigenic bronchial epithelial cell line, NL20 migrated towards EGF in a modified Boyden chamber cell motility assay. However, the tumorigenic NL20-TA cell line derived from the NL20 cells and lacking gelsolin, did not migrate towards EGF. Ectopic expression of gelsolin in NL20-TA cells restored the EGF response, while motility of NL20-TA derived cells towards serum, PDGF, and fibronectin was independent of gelsolin expression. PI3-kinase inhibition failed to block EGF-stimulated motility in gelsolin transfected NL20-TA cells. Furthermore, EGF stimulated a motility response in cells lacking gelsolin in the presence of fibronectin or fibrinogen that was blocked with PI3-kinase inhibition. Thus, EGF-stimulated motility in NL20 cells and its derivatives are gelsolin dependent and PI3-kinase independent, while fibronectin and fibrinogen enhances EGF-stimulated motility through a pathway independent of gelsolin and dependent upon PI3-kinase.

    Funded by: NCI NIH HHS: R21 CA93486; NHLBI NIH HHS: R01 HL 54188

    Experimental cell research 2005;307;1;153-63

  • Gelsolin binds to polyphosphoinositide-free lipid vesicles and simultaneously to actin microfilaments.

    Méré J, Chahinian A, Maciver SK, Fattoum A, Bettache N, Benyamin Y and Roustan C

    UMR 5539, CNRS Laboratoire de motilité cellulaire, Ecole Pratique des Hautes Etudes, Université de Montpellier 2, Place E. Bataillon, CC107, 34095 Montpellier Cedex 5, France.

    Gelsolin is a calcium-, pH- and lipid-dependent actin filament severing/capping protein whose main function is to regulate the assembly state of the actin cytoskeleton. Gelsolin is associated with membranes in cells, and it is generally assumed that this interaction is mediated by PPIs (polyphosphoinositides), since an interaction with these lipids has been characterized in vitro. We demonstrate that non-PPI lipids also bind gelsolin, especially at low pH. The data suggest further that gelsolin becomes partially buried in the lipid bilayer under mildly acidic conditions, in a manner that is not dependent of the presence of PPIs. Our data also suggest that lipid binding involves a number of sites that are spread throughout the gelsolin molecule. Linker regions between gelsolin domains have been implicated by other work, notably the linker between G1 and G2 (gelsolin domains 1 and 2 respectively), and we postulate that the linker region between the N-terminal and C-terminal halves of gelsolin (between G3 and G4) is also involved in the interaction with lipids. This region is compatible with other studies in which additional binding sites have been located within G4-6. The lipid-gelsolin interactions reported in the present paper are not calcium-dependent, and are likely to involve significant conformational changes to the gelsolin molecule, as the chymotryptic digest pattern is altered by the presence of lipids under our conditions. We also report that vesicle-bound gelsolin is capable of binding to actin filaments, presumably through barbed end capping. Gelsolin bound to vesicles can nucleate actin assembly, but is less active in severing microfilaments.

    The Biochemical journal 2005;386;Pt 1;47-56

  • Topological assignment of the N-terminal extension of plasma gelsolin to the gelsolin surface.

    Fock U, Jockusch BM, Schubert WD and Hinssen H

    Cell Biology, Zoological Institute, Technical University of Braunschweig, D-38106 Braunschweig, Germany.

    The actin-binding protein gelsolin is highly conserved in vertebrates and exists in two isoforms, a cytoplasmic and an extracellular variant, generated by alternative splicing. In mammals, these isoforms differ only by an N-terminal extension in plasma gelsolin, a short sequence of up to 25 amino acids. Cells and tissues may contain both variants, as plasma gelsolin is secreted by many cell types. The tertiary structure of equine plasma gelsolin has been elucidated, but without any information on the N-terminal extension. In this paper, we present topographical data on the N-terminal extension, derived using a biochemical and immunological approach. For this purpose, a monoclonal antibody was generated that exclusively recognizes cytoplasmic gelsolin but not the extracellular variant and thus allows isoform-specific immunodetection and quantification of cytoplasmic gelsolin in the presence of plasma gelsolin. Using limited proteolysis and pepscan analysis, we mapped the binding epitope and localized it within two regions in segment 1 of the cytoplasmic gelsolin sequence: Tyr34-Ile45 and Leu64-Ile78. In the tertiary structure of the cytoplasmic variant, these sequences are mutually adjacent and located in the proximity of the N-terminus. We therefore conclude that the binding site of the antibody is covered by the N-terminal extension in plasma gelsolin and thus sterically hinders antibody binding. Our results allow for a topological model of the N-terminal extension on the surface of the gelsolin molecule, which was unknown previously.

    The Biochemical journal 2005;385;Pt 3;659-65

  • 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

  • Apoptosis induced by interferon-alpha and antagonized by EGF is regulated by caspase-3-mediated cleavage of gelsolin in human epidermoid cancer cells.

    Boccellino M, Giuberti G, Quagliuolo L, Marra M, D'Alessandro AM, Fujita H, Giovane A, Abbruzzese A and Caraglia M

    Dipartimento di Biochimica e Biofisica, Seconda Università di Napoli, Naples, Italy.

    We have previously reported that interferon-alpha (IFNalpha) induces apoptosis and EGF can antagonize this effect in human epidermoid cancer KB cells. Since apoptosis occurs together with cytoskeleton reorganization we have evaluated if IFNalpha and EGF could modulate cell remodeling in our experimental conditions. We have found that 48 h 1,000 IU/ml IFNalpha induced structural reorganization of stress fibers and membrane delocalization and partial capping of the actin severing protein gelsolin. The transfection of KB cells with both a wild type (WT) or a C-terminal truncated form of gelsolin caused overexpression of the protein and an increase of both the spontaneous and IFNalpha-induced apoptosis and cell cytoskeletal modifications. In fact, after 48 h of treatment IFNalpha induced 45% of apoptotic cell death in parental cells while an approximately 80% of cell population was apoptotic in transfected cells. These effects occurred together with an increase of the expression and consequent degradation of gelsolin. Again the addition of EGF to IFNalpha-treated transfected cells caused a recovery of the apoptosis. Notably, IFNalpha and EGF did not modify the expression of other molecules associated to cytoskeleton such as focal adhesion kinase and vinculin. In the same experimental conditions IFNalpha induced also gelsolin cleavage that occurred together with caspase-3 activation and release of cytochrome c. All these effects were antagonized by the exposure of IFNalpha-treated KB to 10 nM EGF for the last 12 h. Moreover, the specific inhibition of caspase-3 with 20 microM DEVD completely abrogated apoptosis and gelsolin cleavage induced by IFNalpha. In conclusion, our data are the first demonstration that IFNalpha can induce morphological cell changes that are peculiar of apoptosis onset through the caspase-3-mediated cleavage of gelsolin. Furthermore, we have demonstrated that EGF is able to antagonize these effects through the inhibition of caspase-3 activation.

    Journal of cellular physiology 2004;201;1;71-83

  • Gelsolin superfamily proteins: key regulators of cellular functions.

    Silacci P, Mazzolai L, Gauci C, Stergiopulos N, Yin HL and Hayoz D

    Laboratory of Hemodynamics and Cardiovascular Technology, Swiss Federal Institute of Technology, Lausanne, Switzerland. paolo.silacci@epfl.ch

    Cytoskeletal rearrangement occurs in a variety of cellular processes and involves a wide spectrum of proteins. Among these, the gelsolin superfamily proteins control actin organization by severing filaments, capping filament ends and nucleating actin assembly [1]. Gelsolin is the founding member of this family, which now contains at least another six members: villin, adseverin, capG, advillin, supervillin and flightless I. In addition to their respective role in actin filament remodeling, these proteins have some specific and apparently non-overlapping particular roles in several cellular processes, including cell motility, control of apoptosis and regulation of phagocytosis (summarized in table 1). Evidence suggests that proteins belonging to the gelsolin superfamily may be involved in other processes, including gene expression regulation. This review will focus on some of the known functions of the gelsolin superfamily proteins, thus providing a basis for reflection on other possible and as yet incompletely understood roles for these proteins.

    Funded by: NIGMS NIH HHS: GM066110, GM21681

    Cellular and molecular life sciences : CMLS 2004;61;19-20;2614-23

  • Oncogenic Ras promotes butyrate-induced apoptosis through inhibition of gelsolin expression.

    Klampfer L, Huang J, Sasazuki T, Shirasawa S and Augenlicht L

    Department of Oncology, Albert Einstein Cancer Center, Montefiore Medical Center, 111 E. 210th Street, Bronx, NY 10467, USA. lklampf@aecom.yu.edu

    Activation of Ras promotes oncogenesis by altering a multiple of cellular processes, such as cell cycle progression, differentiation, and apoptosis. Oncogenic Ras can either promote or inhibit apoptosis, depending on the cell type and the nature of the apoptotic stimuli. The response of normal and transformed colonic epithelial cells to the short chain fatty acid butyrate, a physiological regulator of epithelial cell maturation, is also divergent: normal epithelial cells proliferate, and transformed cells undergo apoptosis in response to butyrate. To investigate the role of k-ras mutations in butyrate-induced apoptosis, we utilized HCT116 cells, which harbor an oncogenic k-ras mutation and two isogenic clones with targeted inactivation of the mutant k-ras allele, Hkh2, and Hke-3. We demonstrated that the targeted deletion of the mutant k-ras allele is sufficient to protect epithelial cells from butyrate-induced apoptosis. Consistent with this, we showed that apigenin, a dietary flavonoid that has been shown to inhibit Ras signaling and to reverse transformation of cancer cell lines, prevented butyrate-induced apoptosis in HCT116 cells. To investigate the mechanism whereby activated k-ras sensitizes colonic cells to butyrate, we performed a genome-wide analysis of Ras target genes in the isogenic cell lines HCT116, Hkh2, and Hke-3. The gene exhibiting the greatest down-regulation by the activating k-ras mutation was gelsolin, an actin-binding protein whose expression is frequently reduced or absent in colorectal cancer cell lines and primary tumors. We demonstrated that silencing of gelsolin expression by small interfering RNA sensitized cells to butyrate-induced apoptosis through amplification of the activation of caspase-9 and caspase-7. These data therefore demonstrate that gelsolin protects cells from butyrate-induced apoptosis and suggest that Ras promotes apoptosis, at least in part, through its ability to down-regulate the expression of gelsolin.

    Funded by: NCI NIH HHS: CA 88104; PHS HHS: P0 13330

    The Journal of biological chemistry 2004;279;35;36680-8

  • Structure of the N-terminal half of gelsolin bound to actin: roles in severing, apoptosis and FAF.

    Burtnick LD, Urosev D, Irobi E, Narayan K and Robinson RC

    Department of Chemistry and Centre for Blood Research, The University of British Columbia, Vancouver, BC, Canada.

    The actin filament-severing functionality of gelsolin resides in its N-terminal three domains (G1-G3). We have determined the structure of this fragment in complex with an actin monomer. The structure reveals the dramatic domain rearrangements that activate G1-G3, which include the replacement of interdomain interactions observed in the inactive, calcium-free protein by new contacts to actin, and by a novel G2-G3 interface. Together, these conformational changes are critical for actin filament severing, and we suggest that their absence leads to the disease Finnish-type familial amyloidosis. Furthermore, we propose that association with actin drives the calcium-independent activation of isolated G1-G3 during apoptosis, and that a similar mechanism operates to activate native gelsolin at micromolar levels of calcium. This is the first structure of a filament-binding protein bound to actin and it sets stringent, high-resolution limitations on the arrangement of actin protomers within the filament.

    The EMBO journal 2004;23;14;2713-22

  • Analysis of erythrocyte and platelet membrane proteins in various forms of beta-thalassemia.

    Alekperova GA, Orudzhev AG and Javadov SA

    Azerbaijan Medical University, Baku 370022, Azerbaijan.

    Major membrane proteins have been quantitatively analyzed in erythrocytes and platelets from patients with homozygous (splenectomized and non-splenectomized) and heterozygous forms of beta-thalassemia depending on severity of clinical manifestation of this disease. Quantitative analysis of erythrocyte membrane proteins revealed increase in alpha- and beta-spectrin. (In non-splenectomized patients with homozygous beta-thalassemia the amount of this protein was lower than in corresponding controls.) Besides spectrin, the increase of 2.1-2.3 fractions of ankyrin, and the decrease of band 3 protein (anion-transport protein), 4.1, palladin, and glyceraldehyde-3-phosphate dehydrogenase were also found. Analysis of major platelet membrane proteins revealed significant increase in gelsolin. This increase was found in all forms of beta-thalassemia irrespective of gender. Significant changes in platelet membrane protein fractions were found in patients (especially non-splenectomized) with homozygous beta-thalassemia. These included significant decrease in myosin, profilin, and gamma-actin and increase in actin-binding protein in both male and female patients. The content of other protein fractions (alpha-actinin, tubulin, tropomyosin) remained unchanged. Changes in protein fractions of erythrocytes and platelets correlated with severity of clinical manifestation of the disease.

    Biochemistry. Biokhimiia 2004;69;7;748-53

  • The human plasma proteome: a nonredundant list developed by combination of four separate sources.

    Anderson NL, Polanski M, Pieper R, Gatlin T, Tirumalai RS, Conrads TP, Veenstra TD, Adkins JN, Pounds JG, Fagan R and Lobley A

    The Plasma Proteome Institute, Washington DC 20009-3450, USA. leighanderson@plasmaproteome.org

    We have merged four different views of the human plasma proteome, based on different methodologies, into a single nonredundant list of 1175 distinct gene products. The methodologies used were 1) literature search for proteins reported to occur in plasma or serum; 2) multidimensional chromatography of proteins followed by two-dimensional electrophoresis and mass spectroscopy (MS) identification of resolved proteins; 3) tryptic digestion and multidimensional chromatography of peptides followed by MS identification; and 4) tryptic digestion and multidimensional chromatography of peptides from low-molecular-mass plasma components followed by MS identification. Of 1,175 nonredundant gene products, 195 were included in more than one of the four input datasets. Only 46 appeared in all four. Predictions of signal sequence and transmembrane domain occurrence, as well as Genome Ontology annotation assignments, allowed characterization of the nonredundant list and comparison of the data sources. The "nonproteomic" literature (468 input proteins) is strongly biased toward signal sequence-containing extracellular proteins, while the three proteomics methods showed a much higher representation of cellular proteins, including nuclear, cytoplasmic, and kinesin complex proteins. Cytokines and protein hormones were almost completely absent from the proteomics data (presumably due to low abundance), while categories like DNA-binding proteins were almost entirely absent from the literature data (perhaps unexpected and therefore not sought). Most major categories of proteins in the human proteome are represented in plasma, with the distribution at successively deeper layers shifting from mostly extracellular to a distribution more like the whole (primarily cellular) proteome. The resulting nonredundant list confirms the presence of a number of interesting candidate marker proteins in plasma and serum.

    Molecular & cellular proteomics : MCP 2004;3;4;311-26

  • Protein profile of tax-associated complexes.

    Wu K, Bottazzi ME, de la Fuente C, Deng L, Gitlin SD, Maddukuri A, Dadgar S, Li H, Vertes A, Pumfery A and Kashanchi F

    Department of Biochemistry and Molecular Biology, School of Medicine and Health Sciences, The George Washington University, Washington, DC 20037, USA.

    Infection with human T-cell leukemia virus type 1 (HTLV-1) results in adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. Tax, a 40-kDa protein, regulates viral and cellular transcription, host signal transduction, the cell cycle, and apoptosis. Tax has been shown to modulate cellular CREB and NFkappaB pathways; however, to date, its role in binding to various host cellular proteins involved in tumorigenesis has not been fully described. In this study, we describe the Tax-associated proteins and their functions in cells using several approaches. Tax eluted from a sizing column mostly at an apparent molecular mass of 1800 kDa. Following Tax immunoprecipitation, washes with high salt buffer, two-dimensional gel separation, and mass spectrometric analysis, a total of 32 proteins was identified. Many of these proteins belong to the signal transduction and cytoskeleton pathways and transcription/chromatin remodeling. A few of these proteins, including TXBP151, have been shown previously to bind to Tax. The interaction of Tax with small GTPase-cytoskeleton proteins, such as ras GAP1m, Rac1, Cdc42, RhoA, and gelsolin, indicates how Tax may regulate migration, invasion, and adhesion in T-cell cancers. Finally, the physical and functional association of Tax with the chromatin remodeling SWI/SNF complex was assessed using in vitro chromatin remodeling assays, chromatin remodeling factor BRG1 mutant cells, and RNA interference experiments. Collectively, Tax is able to bind and regulate many cellular proteins that regulate transcription and cytoskeletal related pathways, which might explain the pleiotropic effects of Tax leading to T-cell transformation and leukemia in HTLV-1-infected patients.

    Funded by: NIAID NIH HHS: AI43894, AI44357; PHS HHS: 13969

    The Journal of biological chemistry 2004;279;1;495-508

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • Towards gelsolin amyloid formation.

    Liepina I, Janmey P, Czaplewski C and Liwo A

    Latvian Institute of Organic Synthesis, Aizkraukles str. 21, Riga, LV1006, Latvia. inta@osi.lv

    Amyloid diseases result from protein misfolding and aggregation into fibrils. Some features of gelsolin amyloidogenic fragments comprised of residues 173-243 (G173-243) and residues 173-202 (G173-202) were investigated by the method of molecular dynamics (MD). The alpha-helical structure of G173-243 present in the whole protein unwinds during the course of MD simulation of the fragment G173-243, suggesting that the G173-243 structure is not stable and could unfold before becoming involved in gelsolin amyloid fibril formation. Twelve fragments of G173-202 were used to build a possible beta-fibril. During the course of the simulation, G173-202 fragments formed hydrogen bonds and tended to turn by an angle of 10 degrees -20 degrees towards each other.

    Biopolymers 2004;76;6;543-8

  • Aging-associated increase of gelsolin for apoptosis resistance.

    Ahn JS, Jang IS, Kim DI, Cho KA, Park YH, Kim K, Kwak CS and Chul Park S

    Department of Biochemistry and Molecular Biology, The Aging and Apoptosis Research Center, Seoul National University College of Medicine, Seoul, South Korea.

    Gelsolin, a Ca(2+)-dependent actin regulatory protein, was recently suggested to participate in apoptosis regulation. In this study, we found that the level of gelsolin is elevated in senescent human diploid fibroblasts (HDFs) and also in the tissues of old rats, i.e., in the liver, kidney, heart, spleen, stomach, and brain, etc. The ubiquitous increase of gelsolin in the aged organs and cells led us to assume that it might be related with one of the cardinal senescent phenotypes, aging-associated apoptosis resistency. Thus, we tested the sensitivity of senescent cells to apoptosis by menadione, an apoptosis-inducing agent, before and after the down-regulation of gelsolin. The down-regulation of gelsolin in senescent HDFs, independently of Bcl-2 family expression, resulted in an increased sensitivity to menadione-induced apoptotic cell death. The observed ubiquitous increase of gelsolin in the senescent states of cells and tissues, and the increased sensitivity to apoptosis-induction by gelsolin down-regulation, suggests that gelsolin would be partly responsible for age-related apoptosis resistance.

    Biochemical and biophysical research communications 2003;312;4;1335-41

  • Characterization of the interaction of the stress kinase SPAK with the Na+-K+-2Cl- cotransporter in the nervous system: evidence for a scaffolding role of the kinase.

    Piechotta K, Garbarini N, England R and Delpire E

    Department of Anesthesiology and Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.

    Activity of heterologously expressed NKCC1 was analyzed under basal and activated conditions in the presence and absence of binding of Ste20-related proline-alanine-rich kinase (SPAK). Mutant NKCC1 that lacks the ability to bind to this kinase showed K+ transport function identical to wild-type NKCC1. Thus, preventing the binding of the kinase to the cotransporter does not affect cotransporter function. In contrast, several experiments suggest a possible role for SPAK as a scaffolding protein. First, Western blot analysis revealed the presence, and in some tissues abundance, of truncated forms of SPAK and OSR1 in which the kinase domains are affected and thus lack kinase activity. Second, a yeast two-hybrid screen of proteins that interact with the regulatory (binding) domain of SPAK identified several proteins all involved in cellular stress pathways. Third, p38, one of the three major MAPKs, can be coimmunoprecipitated with SPAK and with NKCC1 in an activity-dependent manner. The amount of p38 coimmunoprecipitated with the kinase and the cotransporter significantly decreases upon cellular stress, whereas the interaction of the kinase with NKCC1 remains unchanged. These findings suggest that cation-chloride cotransporters might act as "sensors" for cellular stress, and SPAK, by interacting with the cotransporter, serves as an intermediate in the response to cellular stress.

    Funded by: NINDS NIH HHS: NS36758

    The Journal of biological chemistry 2003;278;52;52848-56

  • Gelsolin for senescence-associated resistance to apoptosis.

    Ahn JS, Jang IS, Rhim JH, Kim K, Yeo EJ and Park SC

    Department of Biochemistry, Seoul National University College of Medicine, Seoul, South Korea.

    One of the characteristics of the senescent cell is apoptotic resistance. Gelsolin, a Ca(2+)-dependent actin regulatory protein, is believed to regulate the intracellular movements which are necessary for cell growth, proliferation, and differentiation. Recently, gelsolin was suggested to play a role in apoptotic resistance, which led us to examine its involvement in the apoptotic resistance of senescent cells. We found that the protein and mRNA levels of gelsolin were increased in senescent human diploid fibroblasts (HDFs). Gelsolin was intracellularly co-localized to the actin stress fiber and distributed to the nucleus and mitochondria in old HDFs. To examine the anti-apoptotic function of gelsolin in senescent HDFs, we tried to downregulate the expression of gelsolin by using antisense oligonucleotide in old HDFs. We then treated the senescent HDFs with the apoptosis-inducing agent menadione. Downregulation of gelsolin in senescent HDFs resulted in increased sensitivity to menadione-induced apoptotic cell death. This suggests that gelsolin plays a role in the apoptotic resistance observed in senescent HDFs.

    Annals of the New York Academy of Sciences 2003;1010;493-5

  • Gelsolin domain 2 Ca2+ affinity determines susceptibility to furin proteolysis and familial amyloidosis of finnish type.

    Huff ME, Page LJ, Balch WE and Kelly JW

    Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute BCC265, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

    Mutation of aspartic acid 187 to asparagine (D187N) or tyrosine (D187Y) in domain 2 of the actin-modulating protein gelsolin causes the neurodegenerative disease familial amyloidosis of Finnish type (FAF). These mutations render plasma gelsolin susceptible to aberrant proteolysis by furin in the trans-Golgi network, the initial proteolytic event in the formation of 71 and 53 residue fragments that assemble into amyloid fibrils. Ca(2+) binding stabilizes wild-type domain 2 gelsolin against denaturation and proteolysis, but the FAF variants are unable to bind and be stabilized by Ca(2+). Though the chain of events initiating FAF has been elucidated recently, uncertainty remains about the mechanistic details that allow the FAF variants to be processed. To test the hypothesis that impaired Ca(2+) binding in the D187 variants, but not other factors specific to residue 187, increases susceptibility to aberrant proteolysis and subsequent amyloidogenesis, we designed the gelsolin variant E209Q to remove a different Ca(2+) ligand from the same Ca(2+) site that is affected in the FAF variants. Here, we show that E209Q domain 2 does not bind Ca(2+) and is not stabilized against denaturation or furin proteolysis, analogous to the behavior exhibited by the FAF variants. Transfection of full-length E209Q into COS cells results in secretion of both the full-length and furin-processed fragments, as observed with D187N and D187Y. Mutation of the furin consensus sequence in D187N and E209Q gelsolin prevents cleavage during secretion, indicating that inhibition of proprotein convertases (furin) represents a viable therapeutic approach for the treatment of FAF. Mutations that diminish domain 2 Ca(2+) binding allow furin access to an otherwise protected cleavage site, initiating the proteolytic cascade that leads to gelsolin amyloidogenesis and FAF.

    Funded by: NIA NIH HHS: AG18917

    Journal of molecular biology 2003;334;1;119-27

  • Structural analysis of gelsolin using synchrotron protein footprinting.

    Kiselar JG, Janmey PA, Almo SC and Chance MR

    Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York, 10461-1602, USA.

    Protein footprinting provides detailed structural information on protein structure in solution by directly identifying accessible and hydroxyl radical-reactive side chain residues. Radiolytic generation of hydroxyl radicals using millisecond pulses of a synchrotron "white" beam results in the formation of stable side chain oxidation products, which can be digested with proteases for mass spectrometry (MS) analysis. Liquid chromatography-coupled MS and tandem MS methods allow for the quantitation of the ratio of modified and unmodified peptides and identify the specific side chain probes that are oxidized, respectively. The ability to monitor the changes in accessibility of multiple side chain probes by monitoring increases or decreases in their oxidation rates as a function of ligand binding provides an efficient and powerful tool for analyzing protein structure and dynamics. In this study, we probe the detailed structural features of gelsolin in its "inactive" and Ca2+-activated state. Oxidation rate data for 81 peptides derived from the trypsin digestion of gelsolin are presented; 60 of these peptides were observed not to be oxidized, and 21 had detectable oxidation rates. We also report the Ca2+-dependent changes in oxidation for all 81 peptides. Fifty-nine remained unoxidized, five increased their oxidation rate, and two experienced protections. Tandem mass spectrometry was used to identify the specific side chain probes responsible for the Ca2+-insensitive and Ca2+-dependent responses. These data are consistent with crystallographic data for the inactive form of gelsolin in terms of the surface accessibility of reactive residues within the protein. The results demonstrate that radiolytic protein footprinting can provide detailed structural information on the conformational dynamics of ligand-induced structural changes, and the data provide a detailed model for gelsolin activation.

    Funded by: NCI NIH HHS: R33-CA-84714; NIBIB NIH HHS: P41-EB-01979; NIGMS NIH HHS: R01-GM-53807

    Molecular & cellular proteomics : MCP 2003;2;10;1120-32

  • Activation in isolation: exposure of the actin-binding site in the C-terminal half of gelsolin does not require actin.

    Narayan K, Chumnarnsilpa S, Choe H, Irobi E, Urosev D, Lindberg U, Schutt CE, Burtnick LD and Robinson RC

    Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, 751 23 Uppsala, Sweden.

    Gelsolin requires activation to carry out its severing and capping activities on F-actin. Here, we present the structure of the isolated C-terminal half of gelsolin (G4-G6) at 2.0 A resolution in the presence of Ca(2+) ions. This structure completes a triptych of the states of activation of G4-G6 that illuminates its role in the function of gelsolin. Activated G4-G6 displays an open conformation, with the actin-binding site on G4 fully exposed and all three type-2 Ca(2+) sites occupied. Neither actin nor the type-l Ca(2+), which normally is sandwiched between actin and G4, is required to achieve this conformation.

    FEBS letters 2003;552;2-3;82-5

  • From the first to the second domain of gelsolin: a common path on the surface of actin?

    Irobi E, Burtnick LD, Urosev D, Narayan K and Robinson RC

    Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, 751 23 Uppsala, Sweden.

    We present the 2.6 A resolution crystal structure of a complex formed between G-actin and gelsolin fragment Met25-Gln160 (G1+). The structure differs from those of other gelsolin domain 1 (G1) complexes in that an additional six amino acid residues from the crucial linker region into gelsolin domain 2 (G2) are visible and are attached securely to the surface of actin. The linker segment extends away from G1 up the face of actin in a direction that infers G2 will bind along the same long-pitch helical strand as the actin bound to G1. This is consistent with a mechanism whereby G2 attaches gelsolin to the side of a filament and then directs G1 toward a position where it would disrupt actin-actin contacts. Alignment of the sequence of the structurally important residues within the G1-G2 linker with those of WH2 (WASp homology domain 2) domain protein family members (e.g. WASp (Wiscott-Aldridge syndrome protein) and thymosin beta4) suggests that the opposing activities of filament assembly and disassembly may exploit a common patch on the surface of actin.

    FEBS letters 2003;552;2-3;86-90

  • Modulation of androgen receptor transactivation by gelsolin: a newly identified androgen receptor coregulator.

    Nishimura K, Ting HJ, Harada Y, Tokizane T, Nonomura N, Kang HY, Chang HC, Yeh S, Miyamoto H, Shin M, Aozasa K, Okuyama A and Chang C

    Department of Urology, Graduate School of Medicine, Osaka University, Yamadaoka, Suita 565-0871, Japan.

    The partial agonist effect of antiandrogens has been well documented, and such effect is amplified by derived mutant androgen receptors (ARs) in prostate cancer cells. Here we report the identification of gelsolin (GSN) as an AR-associated protein. Hydroxyflutamide (HF), as well as androgens, can promote the interaction between AR and GSN in a dose-dependent manner. GSN interacts with AR DNA-binding domain and ligand-binding domain via its COOH-terminal domain. Immunolocalization studies show that GSN interacts with AR during nuclear translocation. Functional analyses additionally demonstrate that GSN enhances AR activity in the presence of either androgen or HF. Two peptides representing partial regions of the AR DNA-binding domain and the ligand-binding domain can block the GSN-enhanced AR activity. The expression of GSN is enhanced in LNCaP cells, LNCaP xenografts, and human prostate tumors after androgen depletion. Increasing expression of GSN enhances the AR activity in the presence of HF. Together, these data suggest that the weak androgenic effect of HF may be amplified by increasing the amount of GSN after androgen ablation treatment. Therefore, blockage of the interaction between AR and GSN could become a potential therapeutic target for the treatment of prostate cancer.

    Funded by: NIDDK NIH HHS: DK60905, DK60948

    Cancer research 2003;63;16;4888-94

  • The structure of nonvertebrate actin: implications for the ATP hydrolytic mechanism.

    Vorobiev S, Strokopytov B, Drubin DG, Frieden C, Ono S, Condeelis J, Rubenstein PA and Almo SC

    Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

    The structures of Saccharomyces cerevisiae, Dictyostelium, and Caenorhabditis elegans actin bound to gelsolin segment-1 have been solved and refined at resolutions between 1.9 and 1.75 A. These structures reveal several features relevant to the ATP hydrolytic mechanism, including identification of the nucleophilic water and the roles of Gln-137 and His-161 in positioning and activating the catalytic water, respectively. The involvement of these residues in the catalytic mechanism is consistent with yeast genetics studies. This work highlights both structural and mechanistic similarities with the small and trimeric G proteins and restricts the types of mechanisms responsible for the considerable enhancement of ATP hydrolysis associated with actin polymerization. The conservation of functionalities involved in nucleotide binding and catalysis also provide insights into the mechanistic features of members of the family of actin-related proteins.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;10;5760-5

  • Co-operation of domain-binding and calcium-binding sites in the activation of gelsolin.

    Lagarrigue E, Maciver SK, Fattoum A, Benyamin Y and Roustan C

    UMR 5539 (CNRS), Laboratoire de motilité cellulaire (Ecole Pratique des Hautes Etudes), Université de Montpellier 2, France.

    Gelsolin is an abundant calcium dependent actin filament severing and capping protein. In the absence of calcium the molecule is compact but in the presence of calcium, as its six similar domains alter their relative position, a generally more open configuration is adopted to reveal the three actin binding sites. It is generally held that a 'helical-latch' at the C-terminus of gelsolin's domain 6 (G6), binds domain 2 (G2) to keep gelsolin in the calcium-free compact state, and that the crutial calcium binding site(s) reside in the C-terminal half of gelsolin perhaps involving the C-terminal helix itself has to be bound to release this latch. Here we provide evidence for a calcium dependent conformational change within G2 (Kd = approximately 15 micro m). We also report a calcium dependent binding site for the C-terminus (G4-6) within G2 and delimit this further to a specific region formed by residues 203-225 and 159-193. It is known that the activation of gelsolin involves multiple calcium binding events (around 6) the first of which (in G6) may release the latch. We propose that the calcium-dependent conformational change in G2 may be a subsequent step that is necessary for the dissociation of G2 from G4-6, and that this movement occurs in sympathy with calcium induced conformational changes within G6 by the physical coupling of the two calcium binding sites within G2 and G6. Additional calcium binding in other domains then result in the complete opening and activation of the gelsolin molecule.

    European journal of biochemistry 2003;270;10;2236-43

  • Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.

    Gevaert K, Goethals M, Martens L, Van Damme J, Staes A, Thomas GR and Vandekerckhove J

    Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium. kris.gevaert@rug.ac.be

    Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.

    Nature biotechnology 2003;21;5;566-9

  • Visualizing the Ca2+-dependent activation of gelsolin by using synchrotron footprinting.

    Kiselar JG, Janmey PA, Almo SC and Chance MR

    Department of Physiology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461-1602, USA.

    Radiolytic protein footprinting with a synchrotron source is used to reveal detailed structural changes that occur in the Ca(2+)-dependent activation of gelsolin. More than 80 discrete peptides segments within the structure, covering 95% of the sequence in the molecule, were examined by footprinting and mass spectrometry for their solvent accessibility as a function of Ca(2+) concentration in solution. Twenty-two of the peptides exhibited detectable oxidation; for seven the oxidation extent was seen to be Ca(2+) sensitive. Ca(2+)titration isotherms monitoring the oxidation within residues 49-72 (within subdomain S1), 121-135 (S1), 162-166 (S2), and 722-748 (S6) indicate a three-state activation process with a intermediate that was populated at a Ca(2+) concentration of 1-5 microM that is competent for capping and severing activity. A second structural transition with a midpoint of approximately 60-100 microM, where the accessibility of the above four peptides is further increased, is also observed. Tandem mass spectrometry showed that buried residues within the helical "latch" of S6 (including Pro-745) that contact an F-actin-binding site on S2 and buried F-actin-binding residues within S2 (including Phe-163) are unmasked in the submicromolar Ca(2+) transition. However, residues within S4 that are part of an extended beta-sheet with S6 (including Tyr-453) are revealed only in the subsequent transition at higher Ca(2+) concentrations; the disruption of this extended contact between S4 and S6 (and likely the analogous contact between S1 and S3) likely results in an extended structure permitting additional functions consistent with the fully activated gelsolin molecule.

    Funded by: NCI NIH HHS: R33-CA-84713; NIBIB NIH HHS: P41 EB001979, P41-EB-01979; NIGMS NIH HHS: R01 GM053807, R01-GM-53807

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;7;3942-7

  • Gelsolin and plasminogen activator inhibitor-1 are Ap3A-binding proteins.

    Vartanian AA

    Engelhardt Institute of Molecular Biology, Moscow, Russia. vartan@imb.ac.ru

    Previous data on the accumulation of diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) in cells in response to various physiological factors raised the issue of identification of Ap3A binding proteins as potential targets for Ap3A. Ap3A binding proteins were isolated from a human leukocyte lysate by affinity chromatography through Ap3A-aga-rose. Two proteins, gelsolin and plasminogen activator inhibitor-1 (PAI-1), were tentatively identified by in-gel tryptic digestion and mass fingerprint analysis by MALDI-TOF mass spectrometry. The ability of the pure proteins to bind Ap3A was confirmed. Scatchard analysis of [3H]Ap3A binding data yielded dissociation constants of 0.3 microM for gelsolin and 4.1 microM for PAI-1. Binding was saturable at 0.78 mol Ap3A/mol of gelsolin and 0.68 mol Ap3A/mol PAI-1. The binding was non-covalent and insensitive to the presence of divalent metal ions. In neither case was binding affected by a 100-fold molar excess of ATP, ADP and AMP or Ap4A, suggesting a high degree of specificity for Ap3A. Ap3A produced significant effects on cell morphology when added at 10 microM to reversibly permeabilized CEM-SS cells, suggesting that it might influence cytoskeletal disruption by activating gelsolin. Ap3A added externally to HL60 promyelocytic cells reduced the inhibitory effect of PAI-1 on VP16-induced apoptosis. These findings provide new information about intra- and extracellular targets of Ap3A.

    The Italian journal of biochemistry 2003;52;1;9-16

  • Gelsolin suppresses tumorigenicity through inhibiting PKC activation in a human lung cancer cell line, PC10.

    Sagawa N, Fujita H, Banno Y, Nozawa Y, Katoh H and Kuzumaki N

    Division of Cancer Gene Regulation, Research Section of Disease Control, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan.

    Gelsolin expression is frequently downregulated in lung cancer and several types of different human cancers. To examine the effects of gelsolin restoration on tumorigenicity, we here stably expressed various levels of gelsolin via gene transfer in lung cancer cells (squamous cell carcinoma line, PC10). We observed the alterations in tumorigenicity in vivo when implanted in nude mice, and the changes in growth properties in vitro. As compared to parental cells and control clones, gelsolin transfectants highly reduced tumorigenicity and repressed cell proliferation. Moreover, we investigated bradykinin-induced responses in gelsolin-overexpressing clones, because agonist-stimulated activation of the phospholipases C (PLC)/protein kinase C (PKC) signal transduction pathway is critical for cell growth and tumorigenicity. Bradykinin promotes phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by PLC and translocation of various PKC isoforms from the cytosolic fraction to the particulate fraction. Bradykinin treatment did not increase inositoltriphosphate (IP3) production and induce the membrane fractions of PKC alpha and PKC gamma in gelsolin tranfectants, while it induced PIP2 hydrolysis and increased the fractions in parental and control clones. These results suggest that gelsolin suppressed the activation of PKCs involved in phospholipid signalling pathways, inhibiting cell proliferation and tumorigenicity.

    British journal of cancer 2003;88;4;606-12

  • Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin.

    Wang Q, Xie Y, Du QS, Wu XJ, Feng X, Mei L, McDonald JM and Xiong WC

    Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

    Osteoclast activation is important for bone remodeling and is altered in multiple bone disorders. This process requires cell adhesion and extensive actin cytoskeletal reorganization. Proline-rich tyrosine kinase 2 (PYK2), a major cell adhesion-activated tyrosine kinase in osteoclasts, plays an important role in regulating this event. The mechanisms by which PYK2 regulates actin cytoskeletal organization and osteoclastic activation remain largely unknown. In this paper, we provide evidence that PYK2 directly interacts with gelsolin, an actin binding, severing, and capping protein essential for osteoclastic actin cytoskeletal organization. The interaction is mediated via the focal adhesion-targeting domain of PYK2 and an LD motif in gelsolin's COOH terminus. PYK2 phosphorylates gelsolin at tyrosine residues and regulates gelsolin bioactivity, including decreasing gelsolin binding to actin monomer and increasing gelsolin binding to phosphatidylinositol lipids. In addition, PYK2 increases actin polymerization at the fibroblastic cell periphery. Finally, PYK2 interacts with gelsolin in osteoclasts, where PYK2 activation is required for the formation of actin rings. Together, our results suggest that PYK2 is a regulator of gelsolin, revealing a novel PYK2-gelsolin pathway in regulating actin cytoskeletal organization in multiple cells, including osteoclasts.

    Funded by: NIAMS NIH HHS: AR43225, AR46031, AR47830, AR48120, P30 AR046031, P30AR46031, R01 AR043225, R01 AR047830, R01 AR048120; NINDS NIH HHS: NS40480, R01 NS040480

    The Journal of cell biology 2003;160;4;565-75

  • [The mechanism for reduced expression of gelsolin, tumor suppressor protein, in bladder cancer].

    Haga K

    Renal and Genito-urinary Surgery, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan.

    [Hokkaido igaku zasshi] The Hokkaido journal of medical science 2003;78;1;29-37

  • The calcium activation of gelsolin: insights from the 3A structure of the G4-G6/actin complex.

    Choe H, Burtnick LD, Mejillano M, Yin HL, Robinson RC and Choe S

    Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92186-5800, USA.

    Gelsolin participates in the reorganization of the actin cytoskeleton that is required during such phenomena as cell movement, cytokinesis, and apoptosis. It consists of six structurally similar domains, G1-G6, which are arranged at resting intracellular levels of calcium ion so as to obscure the three actin-binding surfaces. Elevation of Ca(2+) concentrations releases latches within the constrained structure and produces large shifts in the relative positioning of the domains, permitting gelsolin to bind to and sever actin filaments. How Ca(2+) is able to activate gelsolin has been a major question concerning the function of this protein. We present the improved structure of the C-terminal half of gelsolin bound to monomeric actin at 3.0 A resolution. Two classes of Ca(2+)-binding site are evident on gelsolin: type 1 sites share coordination of Ca(2+) with actin, while type 2 sites are wholly contained within gelsolin. This structure of the complex reveals the locations of two novel metal ion-binding sites in domains G5 and G6, respectively. We identify both as type 2 sites. The absolute conservation of the type 2 calcium-ligating residues across the six domains of gelsolin suggests that this site exists in each of the domains. In total, gelsolin has the potential to bind eight calcium ions, two type 1 and six type 2. The function of the type 2 sites is to facilitate structural rearrangements within gelsolin as part of the activation and actin-binding and severing processes. We propose the novel type 2 site in G6 to be the critical site that initiates overall activation of gelsolin by releasing the tail latch that locks calcium-free gelsolin in a conformation unable to bind actin.

    Journal of molecular biology 2002;324;4;691-702

  • Dissecting the gelsolin-polyphosphoinositide interaction and engineering of a polyphosphoinositide-sensitive gelsolin C-terminal half protein.

    Xian W and Janmey PA

    Institute for Medicine and Engineering, University of Pennsylvania, 1010 Vagelos Laboratories, 3340 Smith Walk, Philadelphia 19104, USA. wxian@mail.med.upenn.edu

    Gelsolin and other proteins in the villin/gelsolin family are regulated by polyphosphoinositides (PPIs), and manipulation of cellular PIP(2) levels alters the structure of the actin cytoskeleton coincident with the dissociation of gelsolin-actin complexes. This work explores the structure-function relationship of the gelsolin-PPI interaction. Circular dichroism experiments show that upon binding to PPIs, the PPI-sensitive N-terminal half of gelsolin undergoes significant secondary and tertiary structural changes that do not occur in the structurally homologous but PPI-insensitive C-terminal half. Secondary structure modeling algorithms predict an alpha-helical conformation for one of the gelsolin PPI-binding sites, P2, which differs from the conformation of P2 in the structure of gelsolin determined by X-ray crystallography, whereas structure prediction of the C-terminal homolog of P2 agrees well with the X-ray crystallography structure. Simulation of a change to helical conformation for P2 using molecular modeling indicates that such a structural transition will destabilize the F-actin-binding sites in domain 2. A hypothesis is proposed that PPIs initiate conformational changes at the PPI-binding site(s) that destabilize the protein structure, and subsequently disrupt the actin-binding sites. To further evaluate the role of P2 in the gelsolin-PPI interaction, a Ct mutant P2Ct is constructed by inserting P2 in place of its C-terminal homologous site. P2Ct interacts with actin in the same way as the wild-type protein. In contrast to Ct, however, P2Ct interacts strongly with PPIs, and its monomeric actin-binding activity becomes regulated by PPIs. It is concluded that the P2 site is sufficient for PPI-sensitivity in gelsolin. Furthermore, the P2 site in P2Ct and the actin-binding sites of Ct do not overlap, suggesting that PPIs regulate actin binding of P2Ct through induction of structural changes, rather than through direct competition.

    Funded by: NHLBI NIH HHS: T32 HL079054; NIAMS NIH HHS: R01 AR38910

    Journal of molecular biology 2002;322;4;755-71

  • Molecular mechanism of transcriptional repression of gelsolin in human breast cancer cells.

    Dong Y, Asch HL, Ying A and Asch BB

    Division of Experimental Pathology, Roswell Park Cancer Institute (RPCI), Buffalo, New York 14263, USA.

    Loss of gelsolin, a tumor suppressor, is one of the most frequently occurring molecular defects in breast cancers of diverse etiologies and across at least three animal species: human, mouse, and rat. Our previous analysis of breast cancer cells demonstrated that the deficiency is not due to mutation of the gelsolin gene, but instead to epigenetic factors, including decreased transcription of the gene. The study described herein provides the first functional characterization of the human gelsolin promoter and reveals a mechanistic basis for the reduced gelsolin transcription. In reporter gene assays, the gelsolin promoter was less active in low-gelsolin-expressing breast cancer cells. A cis-element mediating this reduced promoter activity was defined as a 27-bp sequence located approximately 135 bp upstream of the transcription start site. Gel shift and supershift assays and Southwestern blotting analysis indicated that activating transcription factor-1 (ATF-1) and a protein of approximately 100 kDa may have cancer cell-specific DNA-binding activity to the 27-bp gelsolin cis-element. Although the ATF-1 protein was highly expressed in both benign and tumorigenic breast cells, its DNA-binding activity was selectively abundant in the cancer cells and correlated inversely with the gelsolin mRNA level. Thus, our results suggest a role for ATF-1 in gelsolin promoter silencing in contrast to its transactivating effect on various other promoters.

    Funded by: NCI NIH HHS: CA16056, CA72768

    Experimental cell research 2002;276;2;328-36

  • [An actin-regulatory protein, gelsolin functions as a regulator of cell growth and apoptosis].

    Fujita H

    Division of Cancer Gene Regulation, Research Section of Disease Control, Institute of Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815.

    Seikagaku. The Journal of Japanese Biochemical Society 2002;74;2;135-9

  • Loss of a metal-binding site in gelsolin leads to familial amyloidosis-Finnish type.

    Kazmirski SL, Isaacson RL, An C, Buckle A, Johnson CM, Daggett V and Fersht AR

    Cambridge University Chemical Laboratory and Cambridge Centre for Protein Engineering, MRC Centre, Hills Road, Cambridge CB2 2QH, UK.

    Mutations in domain 2 (D2, residues 151-266) of the actin-binding protein gelsolin cause familial amyloidosis-Finnish type (FAF). These mutations, D187N or D187Y, lead to abnormal proteolysis of plasma gelsolin at residues 172-173 and a second hydrolysis at residue 243, resulting in an amyloidogenic fragment. Here we present the structure of human gelsolin D2 at 1.65 A and find that Asp 187 is part of a Cd2+ metal-binding site. Two Ca2+ ions are required for a conformational transition of gelsolin to its active form. Differential scanning calorimetry (DSC) and molecular dynamics (MD) simulations suggest that the Cd2+-binding site in D2 is one of these two Ca2+-binding sites and is essential to the stability of D2. Mutation of Asp 187 to Asn disrupts Ca2+ binding in D2, leading to instabilities upon Ca2+ activation. These instabilities make the domain a target for aberrant proteolysis, thereby enacting the first step in the cascade leading to FAF.

    Nature structural biology 2002;9;2;112-6

  • Phosphatidylinositol 3,4,5-trisphosphate directs association of Src homology 2-containing signaling proteins with gelsolin.

    Chellaiah MA, Biswas RS, Yuen D, Alvarez UM and Hruska KA

    Department of Oral and Craniofacial Biological Sciences, University of Maryland, 666 W. Baltimore St., Baltimore, MD 21201, USA. mac001@dental.umaryland.edu

    Podosomes are adhesion structures in osteoclasts and are structurally related to focal adhesions mediating cell motility during bone resorption. Here we show that gelsolin coprecipitates some of the focal adhesion-associated proteins such as c-Src, phosphoinositide 3-kinase (PI3K), p130(Cas), focal adhesion kinase, integrin alpha(v)beta(3), vinculin, talin, and paxillin. These proteins were inducibly tyrosine-phosphorylated in response to integrin activation by osteopontin. Previous studies have defined unique biochemical properties of gelsolin related to phosphatidylinositol 3,4,5-trisphosphate in osteoclast podosomes, and here we demonstrate phosphatidylinositol 3,4,5-trisphosphate/gelsolin function in mediating organization of the podosome signaling complex. Overlay and GST pull-down assays demonstrated strong phosphatidylinositol 3,4,5-trisphosphate-PI3K interactions based on the Src homology 2 domains of PI3K. Furthermore, lipid extraction of lysates from activated osteoclasts eliminated interaction between gelsolin, c-Src, PI3K, and focal adhesion kinase despite equal amounts of gelsolin in both the lipid-extracted and unextracted experiment. The cytoplasmic protein tyrosine phosphatase (PTP)-proline-glutamic acid-serine-threonine amino acid sequences (PEST) was also found to be associated with gelsolin in osteoclast podosomes and with stimulation of alpha(v)beta(3)-regulated phosphorylation of PTP-PEST. We conclude that gelsolin plays a key role in recruitment of signaling proteins to the plasma membrane through phospholipid-protein interactions and by regulation of their phosphorylation status through its association with PTP-PEST. Because both gelsolin deficiency and PI3K inhibition impair bone resorption, we conclude that phosphatidylinositol 3,4,5-trisphosphate-based protein interactions are critical for osteoclast function.

    Funded by: NIAMS NIH HHS: AR 41677, AR 46292; NIDDK NIH HHS: DK 09976

    The Journal of biological chemistry 2001;276;50;47434-44

  • Binding of gelsolin domain 2 to actin. An actin interface distinct from that of gelsolin domain 1 and from ADF/cofilin.

    Renoult C, Blondin L, Fattoum A, Ternent D, Maciver SK, Raynaud F, Benyamin Y and Roustan C

    UMR 5539 (CNRS) Laboratoire de Motilité Cellulaire (Ecole Pratique des Hautes Etudes), Université de Montpellier, France.

    It is generally assumed that of the six domains that comprise gelsolin, domain 2 is primarily responsible for the initial contact with the actin filament that will ultimately result in the filament being severed. Other actin-binding regions within domains 1 and 4 are involved in gelsolin's severing and subsequent capping activity. The overall fold of all gelsolin repeated domains are similar to the actin depolymerizing factor (ADF)/cofilin family of actin-binding proteins and it has been proposed that there is a similarity in the actin-binding interface. Gelsolin domains 1 and 4 bind G-actin in a similar manner and compete with each other, whereas domain 2 binds F-actin at physiological salt concentrations, and does not compete with domain 1. Here we investigate the domain 2 : actin interface and compare this to our recent studies of the cofilin : actin interface. We conclude that important differences exist between the interfaces of actin with gelsolin domains 1 and 2, and with ADF/cofilin. We present a model for F-actin binding of domain 2 with respect to the F-actin severing and capping activity of the whole gelsolin molecule.

    European journal of biochemistry 2001;268;23;6165-75

  • LIM-kinase 2 induces formation of stress fibres, focal adhesions and membrane blebs, dependent on its activation by Rho-associated kinase-catalysed phosphorylation at threonine-505.

    Amano T, Tanabe K, Eto T, Narumiya S and Mizuno K

    Biological Institute, Graduate School of Science, Tohoku University, Sendai 980-8578, and Department of Biology, Graduate School of Science, Kyushu University, Fukuoka 812-8581, Japan.

    LIM-kinase 1 and 2 (LIMK1 and LIMK2) phosphorylate cofilin and induce actin cytoskeletal reorganization. LIMK1 is activated by Rho-associated, coiled-coil-forming protein kinase (ROCK) and p21-activated kinase 1 (PAK1), but activation mechanisms and cellular functions of LIMK2 have remained to be determined. We report here that LIMK1 and LIMK2 phosphorylate both cofilin and actin-depolymerizing factor (ADF) specifically at Ser-3 and exhibit partially distinct substrate specificity when tested using site-directed cofilin mutants as substrates. We also show that LIMK2 is activated by ROCK by phosphorylation at Thr-505 within the activation loop. Wild-type LIMK2, but not its mutant (T505V) with replacement of Thr-505 by Val, was activated by ROCK in vitro and in vivo. LIMK2 mutants with replacement of Thr-505 by one or two Glu residues (T505E or T505EE) increased the kinase activity about 3.6-fold but were not further activated by ROCK. When expressed in HeLa cells, wild-type LIMK2, but not the T505V mutant, induced the formation of stress fibres, focal adhesions and membrane blebs. Furthermore, inhibitors of Rho and ROCK significantly suppressed LIMK2-induced stress fibres and membrane blebs. These results suggest that LIMK2 functions downstream of the Rho-ROCK signalling pathway and plays a role in reorganization of actin filaments and membrane structures, by phosphorylating cofilin/ADF proteins.

    The Biochemical journal 2001;354;Pt 1;149-59

  • VASP protects actin filaments from gelsolin: an in vitro study with implications for platelet actin reorganizations.

    Bearer EL, Prakash JM, Manchester RD and Allen PG

    Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island 02912, USA. Elaine_Bearer@Brown.edu

    An initial step in platelet shape change is disassembly of actin filaments, which are then reorganized into new actin structures, including filopodia and lamellipodia. This disassembly is thought to be mediated primarily by gelsolin, an abundant actin filament-severing protein in platelets. Shape change is inhibited by VASP, another abundant actin-binding protein. Paradoxically, in vitro VASP enhances formation of actin filaments and bundles them, activities that would be expected to increase shape change, not inhibit it. We hypothesized that VASP might inhibit shape change by stabilizing filaments and preventing their disassembly by gelsolin. Such activity would explain VASP's known physiological role. Here, we test this hypothesis in vitro using either purified recombinant or endogenous platelet VASP by fluorescence microscopy and biochemical assays. VASP inhibited gelsolin's ability to disassemble actin filaments in a dose-dependent fashion. Inhibition was detectable at the low VASP:actin ratio found inside the platelet (1:40 VASP:actin). Gelsolin bound to VASP-actin filaments at least as well as to actin alone. VASP inhibited gelsolin-induced nucleation at higher concentrations (1:5 VASP:actin ratios). VASP's affinity for actin (K(d) approximately 0.07 microM) and its ability to promote polymerization (1:20 VASP actin ratio) were greater with Ca(++)-actin than with Mg(++)-actin (K(d) approximately 1 microM and 1:1 VASP), regardless of the presence of gelsolin. By immunofluorescence, VASP and gelsolin co-localized in the filopodia and lamellipodia of platelets spreading on glass, suggesting that these in vitro interactions could take place within the cell as well. We conclude that VASP stabilizes actin filaments to the severing effects of gelsolin but does not inhibit gelsolin from binding to the filaments. These results suggest a new concept for actin dynamics inside cells: that bundling proteins protect the actin superstructure from disassembly by severing, thereby preserving the integrity of the cytoskeleton.

    Funded by: NIGMS NIH HHS: GM47368, GM57256, R01 GM047368, R01 GM047368-05, R01 GM057256

    Cell motility and the cytoskeleton 2000;47;4;351-64

  • A novel model system for characterization of phagosomal maturation, acidification, and intracellular collagen degradation in fibroblasts.

    Arora PD, Manolson MF, Downey GP, Sodek J and McCulloch CA

    Medical Research Council Group in Periodontal Physiology, the Faculty of Dentistry, and the Faculty of Medicine, Division of Respirology, University of Toronto, Toronto M5S 3E8, Ontario, Canada.

    Intracellular collagen degradation by fibroblasts is an important but poorly understood pathway for the physiological remodeling of mature connective tissues. The objective of this study was to determine whether gingival fibroblasts that express endogenous alpha(2)beta(1) integrin, the collagen receptor, would exhibit the cellular machinery required for phagosomal maturation and collagen degradation. There was a time-dependent increase of collagen bead internalization and a time-dependent decrease of bead-associated alpha(2)beta(1) integrin after initial bead binding. beta-Actin and gelsolin associated transiently with beads (0-30 min) followed by LAMP-2 (60-240 min) and cathepsin B (30-240 min). Cytochalasin D prevented phagosome formation and also prevented the sequential fusion of early endosomes with lysosomes. Collagen bead-associated pH was progressively reduced from 7.25 to 5.4, which was contemporaneous with progressive increases in degradation of bead-associated collagen (30-120 min). Concanamycin blocked acidification of phagolysosomes and collagen degradation but not phagosome maturation. Phagosomal acidification was partly dependent on elevated intracellular calcium. These studies demonstrate that the cellular machinery required for intracellular collagen degradation in fibroblasts closely resembles the vacuolar system in macrophages.

    The Journal of biological chemistry 2000;275;45;35432-41

  • Human gelsolin prevents apoptosis by inhibiting apoptotic mitochondrial changes via closing VDAC.

    Kusano H, Shimizu S, Koya RC, Fujita H, Kamada S, Kuzumaki N and Tsujimoto Y

    Osaka University Graduate School of Medicine, Biomedical Research Center, Department of Medical Genetics, Suita, Japan.

    Gelsolin is a Ca2+-dependent actin-regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility through modulation of the actin network. Gelsolin was also recently suggested to be involved in the regulation of apoptosis: human gelsolin (hGsn) has anti-apoptotic activity, whereas mouse gelsolin (mGsn) exerts either proapoptotic or anti-apoptotic activity depending on different cell types. Here, we studied the basis of anti-apoptotic activity of hGsn. We showed that both endogenous and overexpressed hGsn has anti-apoptotic activity, that depends on its C-terminal half. We also found that hGsn and its C-terminal half but not mGsn could prevent apoptotic mitochondrial changes such as Apsi loss and cytochrome c release in isolated mitochondria to a similar extent as Bcl-xL, indicating that hGsn targets the mitochondria to prevent apoptosis via its C-terminal half. In the same way as anti-apoptotic Bcl-xL, which we recently found to prevent apoptotic mitochondrial changes by binding and closing the voltage-dependent anion channel (VDAC), hGsn and its C-terminal half inhibited the activity of VDAC on liposomes through direct binding in a Ca2+-dependent manner. These results suggest that hGsn inhibits apoptosis by blocking mitochondrial VDAC activity.

    Oncogene 2000;19;42;4807-14

  • Gelsolin inhibits apoptosis by blocking mitochondrial membrane potential loss and cytochrome c release.

    Koya RC, Fujita H, Shimizu S, Ohtsu M, Takimoto M, Tsujimoto Y and Kuzumaki N

    Division of Cancer Gene Regulation, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-Ku, Sapporo 060-0815, Japan.

    Apoptotic cell death, characterized by chromatin condensation, nuclear fragmentation, cell membrane blebbing, and apoptotic body formation, is also accompanied by typical mitochondrial changes. The latter includes enhanced membrane permeability, fall in mitochondrial membrane potential (Deltapsi(m)) and release of cytochrome c into the cytosol. Gelsolin, an actin regulatory protein, has been shown to inhibit apoptosis, but when cleaved by caspase-3, a fragment that is implicated as an effector of apoptosis is generated. The mechanism by which the full-length form of gelsolin inhibits apoptosis is unclear. Here we show that the overexpression of gelsolin inhibits the loss of Deltapsi(m) and cytochrome c release from mitochondria resulting in the lack of activation of caspase-3, -8, and -9 in Jurkat cells treated with staurosporine, thapsigargin, and protoporphyrin IX. These effects were corroborated in vitro using recombinant gelsolin protein on isolated rat mitochondria stimulated with Ca(2+), atractyloside, or Bax. This protective function of gelsolin, which was not due to simple Ca(2+) sequestration, was inhibited by polyphosphoinositide binding. In addition we confirmed that gelsolin, besides its localization in the cytosol, is also present in the mitochondrial fraction of cells. Gelsolin thus acts on an early step in the apoptotic signaling at the level of mitochondria.

    The Journal of biological chemistry 2000;275;20;15343-9

  • Domain 2 of gelsolin binds directly to tropomyosin.

    Maciver SK, Ternent D and McLaughlin PJ

    Genes and Development Group, Department of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, UK. skm@srv4.med.ed.ac.uk

    Gelsolin is an actin filament severing protein composed of six similar structured domains that differ with respect to actin, calcium and polyphospho-inositide binding. Previous work has established that gelsolin binds tropomyosin [Koepf, E.K. and Burtnick, L.D. (1992) FEBS Lett. 309, 56-58]. We have produced various specific gelsolin domains in Escherichia coli in order to establish which of the six domains binds tropomyosin. Gelsolin domains 1-3 (G1-3), G1-2 and G2 all bind tropomyosin in a pH and calcium insensitive manner whereas binding of G4-6 to tropomyosin was barely detectable under the conditions tested. We conclude that gelsolin binds tropomyosin via domain 2 (G2).

    FEBS letters 2000;473;1;71-5

  • Identification of a novel member of the chloride intracellular channel gene family (CLIC5) that associates with the actin cytoskeleton of placental microvilli.

    Berryman M and Bretscher A

    Department of Biomedical Sciences, Ohio University College of Osteopathic Medicine, Athens, Ohio 45701, USA. berryman@ohiou.edu

    The chloride intracellular channel (CLIC) gene family has been implicated in chloride ion transport within various subcellular compartments. We report here the molecular, biochemical, and cellular characterization of a new member of this gene family termed CLIC5. CLIC5 was isolated from extracts of placental microvilli as a component of a multimeric complex consisting of several known cytoskeletal proteins, including actin, ezrin, alpha-actinin, gelsolin, and IQGAP1. We cloned human cDNAs and generated antibodies specific for CLIC5, CLIC1/NCC27, and CLIC4/huH1/p64H1. CLIC5 shares 52-76% overall identity with human CLIC1, CLIC2, CLIC3, and CLIC4. Northern blot analysis showed that CLIC5 has a distinct pattern of expression compared with CLIC1 and CLIC4. Immunoblot analysis of extracts from placental tissues demonstrated that CLIC4 and CLIC5 are enriched in isolated placental microvilli, whereas CLIC1 is not. Moreover, in contrast to CLIC1 and CLIC4, CLIC5 is associated with the detergent-insoluble cytoskeletal fraction of microvilli. Indirect immunofluorescence microscopy revealed that CLIC4 and CLIC5 are concentrated within the apical region of the trophoblast, whereas CLIC1 is distributed throughout the cytoplasm. These studies suggest that CLIC1, CLIC4, and CLIC5 play distinct roles in chloride transport and that CLIC5 interacts with the cortical actin cytoskeleton in polarized epithelial cells.

    Funded by: NIGMS NIH HHS: GM14352, GM36652, R01 GM036652

    Molecular biology of the cell 2000;11;5;1509-21

  • Domain movement in gelsolin: a calcium-activated switch.

    Robinson RC, Mejillano M, Le VP, Burtnick LD, Yin HL and Choe S

    Structural Biology Laboratory, Salk Institute for Biological Studies, Post Office Box 85800, San Diego, CA 92186-5800, USA.

    The actin-binding protein gelsolin is involved in remodeling the actin cytoskeleton during growth-factor signaling, apoptosis, cytokinesis, and cell movement. Calcium-activated gelsolin severs and caps actin filaments. The 3.4 angstrom x-ray structure of the carboxyl-terminal half of gelsolin (G4-G6) in complex with actin reveals the basis for gelsolin activation. Calcium binding induces a conformational rearrangement in which domain G6 is flipped over and translated by about 40 angstroms relative to G4 and G5. The structural reorganization tears apart the continuous beta sheet core of G4 and G6. This exposes the actin-binding site on G4, enabling severing and capping of actin filaments to proceed.

    Science (New York, N.Y.) 1999;286;5446;1939-42

  • TNIK, a novel member of the germinal center kinase family that activates the c-Jun N-terminal kinase pathway and regulates the cytoskeleton.

    Fu CA, Shen M, Huang BC, Lasaga J, Payan DG and Luo Y

    Rigel, Inc., South San Francisco, California 94080, USA.

    Germinal center kinases (GCKs) compose a subgroup of the Ste20 family of kinases. Here we describe the cloning and characterization of a novel GCK family kinase, Traf2- and Nck-interacting kinase (TNIK) that interacts with both Traf2 and Nck. TNIK encodes a polypeptide of 1360 amino acids with eight spliced isoforms. It has 90% amino acid identity to the Nck-interacting kinase in both the N-terminal kinase domain and the C-terminal germinal center kinase homology region. The homology drops to 53% in the intermediate region. TNIK specifically activates the c-Jun N-terminal kinase pathway when transfected into Phoenix-A cells (derivatives of 293 cells), similar to many GCKs. However, in contrast to other GCKs, this activation is mediated solely by the GCK homology region of TNIK. In addition, in Phoenix-A, NIH-3T3, and Hela cells, overexpression of wild type TNIK, but not the kinase mutant form of TNIK, results in the disruption of F-actin structure and the inhibition of cell spreading. Furthermore, TNIK can phosphorylate Gelsolin in vitro. This is the first time that a GCK family kinase is shown to be potentially involved in the regulation of cytoskeleton.

    The Journal of biological chemistry 1999;274;43;30729-37

  • Binding of gelsolin, a secretory protein, to amyloid beta-protein.

    Chauhan VP, Ray I, Chauhan A and Wisniewski HM

    New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York 10314-6399, USA. chauhanvps@aol.com

    Soluble amyloid beta-protein (Abeta) is normally present in the cerebrospinal fluid (CSF) and plasma. However, it is fibrillized and deposited as plaques in the brains of patients with Alzheimer's disease. Cerebrospinal fluid (CSF) contains several circulating proteins (apolipoprotein E, apolipoprotein J, and transthyretin) that bind to Abeta. We report here that gelsolin, a secretory protein, also binds to Abeta in a concentration-dependent manner. Under similar conditions, other proteins such as G-actin, protein kinase C, polyglutamic acid, and gelatin did not bind to Abeta. Solid phase binding assays showed two Abeta binding sites on gelsolin that have dissociation constants (Kd) of 1.38 and 2.55 microM. Abeta was found to co-immunoprecipitate along with gelsolin from the plasma, suggesting that gelsolin-Abeta complex exists under physiological conditions. The gelsolin-Abeta complex was sodium dodecyl sulfate (SDS)stable in the absence of reducing agent, but was dissociated when the SDS stop solution contained dithiothreitol (reducing agent). This study suggests that the function of secretory gelsolin in the CSF and plasma is to bind and sequester Abeta.

    Funded by: NIA NIH HHS: AG 14199

    Biochemical and biophysical research communications 1999;258;2;241-6

  • Identification of Tyr438 as the major in vitro c-Src phosphorylation site in human gelsolin: a mass spectrometric approach.

    De Corte V, Demol H, Goethals M, Van Damme J, Gettemans J and Vandekerckhove J

    Flanders Interuniversity Institute for Biotechnology, Department of Biochemistry, Faculty of Medicine, Universiteit Gent, Ghent, Belgium.

    Gelsolin is an actin-binding protein (82 kDa) consisting of six repeated segments (S1-S6), each approximately 120 residues long. It interacts with phospholipids and we previously showed that phosphatidylinositol 4,5-bisphosphate promotes phosphorylation of gelsolin by the tyrosine kinase c-Src. We used a combination of different methods, such as thin-layer chromatography and anti-phosphotyrosine-agarose immunoprecipitation of phosphopeptides combined with matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) and post source decay (PSD) analysis, to identify the phosphorylation sites in gelsolin. The major phosphorylation site (Tyr438) was located in subdomain 4 (S4). Phosphorylation of gelsolin in the gelsolin-actin2 complex was inhibited by 90%. Gelsolin phosphorylation by c-Src in the presence of lysophosphatidic acid also revealed Tyr438 as the most prominent site. Additional minor sites were found using the anti-phosphotyrosine bead immunoprecipitation method followed by MALDI-MS and PSD analysis. These sites, representing approximately 5% of the total phosphate incorporation, were identified as Tyr59, Tyr382, Tyr576, and Tyr624. Based on these results we generated antibodies which specifically recognize Tyr438 phosphorylated gelsolin.

    Protein science : a publication of the Protein Society 1999;8;1;234-41

  • The actin-binding proteins adseverin and gelsolin are both highly expressed but differentially localized in kidney and intestine.

    Lueck A, Brown D and Kwiatkowski DJ

    Brigham and Women's Hospital, Harvard Medical School, Department of Medicine, Division of Experimental Medicine, Boston, MA 02115, USA.

    To understand the distinct functions of the closely related actin-severing proteins adseverin and gelsolin, we examined the expression of these proteins in detail during mouse and human development using a new highly sensitive and specific set of antibody reagents. Immunoblot analysis demonstrated that adseverin was highly expressed in mouse kidney and intestine at all stages of development and in human fetal and adult kidney. In contrast and as reported previously, gelsolin was expressed much more widely in both murine and human tissues. Immunohistochemistry on murine kidney sections revealed a predominantly differential localization of adseverin and gelsolin. Adseverin was expressed in peripolar cells, thin limbs, thick ascending limbs, and principal cells of cortical and medullary collecting ducts where it was diffusely localized in the cytoplasm. Gelsolin was expressed in the distal convoluted tubule, intercalated cells and principal cells of cortical and medullary collecting ducts, and in ureter. In the distal convoluted tubule, gelsolin showed a diffuse distribution and in principal cells of collecting ducts a localization at the basolateral pole. In intercalated cells, gelsolin localization was heterogeneous, either at the apical pole or diffusely in the cytoplasm. In human fetal and adult kidney, adseverin was expressed only in collecting ducts whereas gelsolin was expressed in thick ascending limbs and collecting ducts. In mouse and human intestine adseverin was expressed in enterocytes with a gradient of increasing expression from the duodenum to the colon, and from the crypt to the villus. The observations indicate high level expression of adseverin in specific cells of the kidney and colon, and suggest a previously unrecognized function of adseverin in epithelial cell function.

    Journal of cell science 1998;111 ( Pt 24);3633-43

  • A cloning method for caspase substrates that uses the yeast two-hybrid system: cloning of the antiapoptotic gene gelsolin.

    Kamada S, Kusano H, Fujita H, Ohtsu M, Koya RC, Kuzumaki N and Tsujimoto Y

    Department of Medical Genetics, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

    Caspase-mediated proteolysis is a critical and central element of the apoptotic process; therefore, it is important to identify the downstream molecular targets of caspases. We established a method for cloning the genes of caspase substrates by two major modifications of the yeast two-hybrid system: (i) both large and small subunits of active caspases were expressed in yeast under ADH1 promoters and the small subunit was fused to the LexA DNA-binding domain; and (ii) a point mutation was introduced that substituted serine for the active site cysteine and thereby prevented proteolytic cleavage of the substrates, possibly stabilizing the enzyme-substrate complexes in yeast. After screening a mouse embryo cDNA expression library by using the bait plasmid for caspase-3, we obtained 13 clones that encoded proteins binding to caspase-3, and showed that 10 clones including gelsolin, an actin-regulatory protein implicated in apoptosis, were cleaved by recombinant caspase-3 in vitro. Using the same bait, we also isolated human gelsolin cDNA from a human thymus cDNA expression library. We showed that human gelsolin was cleaved during Fas-mediated apoptosis in vivo and that the caspase-3 cleavage site of human gelsolin was at D352 of DQTD352G, findings consistent with previous observations on murine gelsolin. In addition, we ascribed the antiapoptotic activity of gelsolin (which we previously reported) to prevention of a step leading to cytochrome c release from the mitochondria into the cytosol. Our results indicate that this cloning method is useful for identification of the substrates of caspases and possibly also of other enzymes.

    Proceedings of the National Academy of Sciences of the United States of America 1998;95;15;8532-7

  • Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis.

    Kothakota S, Azuma T, Reinhard C, Klippel A, Tang J, Chu K, McGarry TJ, Kirschner MW, Koths K, Kwiatkowski DJ and Williams LT

    Chiron Corporation, Emeryville, CA 94608, USA.

    The caspase-3 (CPP32, apopain, YAMA) family of cysteinyl proteases has been implicated as key mediators of apoptosis in mammalian cells. Gelsolin was identified as a substrate for caspase-3 by screening the translation products of small complementary DNA pools for sensitivity to cleavage by caspase-3. Gelsolin was cleaved in vivo in a caspase-dependent manner in cells stimulated by Fas. Caspase-cleaved gelsolin severed actin filaments in vitro in a Ca2+-independent manner. Expression of the gelsolin cleavage product in multiple cell types caused the cells to round up, detach from the plate, and undergo nuclear fragmentation. Neutrophils isolated from mice lacking gelsolin had delayed onset of both blebbing and DNA fragmentation, following apoptosis induction, compared with wild-type neutrophils. Thus, cleaved gelsolin may be one physiological effector of morphologic change during apoptosis.

    Funded by: NHLBI NIH HHS: P01 HL48743, R01 HL54188

    Science (New York, N.Y.) 1997;278;5336;294-8

  • Thrombin activation of human platelets dissociates a complex containing gelsolin and actin from phosphatidylinositide-specific phospholipase Cgamma1.

    Baldassare JJ, Henderson PA, Tarver A and Fisher GJ

    Department of Pharmacological and Physiological Science, St. Louis Health Science Center, St. Louis, MO 63110, USA.

    We have examined the association of two cytoskeleton proteins, gelsolin and actin, with phosphatidylinositide-specific phospholipase Cgamma1 (PLCgamma1) in resting and thrombin-stimulated human platelets. In unstimulated platelets, gelsolin, actin and PLCgamma1 were immunoprecipitated as a complex by a polyclonal antibody to PLCgamma1. The association of gelsolin and actin was specific for PLCgamma1 because immunoprecipitates of PLCs beta2, beta3, gamma2 and delta1, which are also expressed in human platelets, did not contain detectable gelsolin or actin. Activation with thrombin resulted in platelet aggregation and the dissociation of gelsolin and actin from PLCgamma1. Inhibition of thrombin-induced platelet aggregation blocked the dissociation of gelsolin and actin from PLCgamma1. After stimulation with thrombin, PLCgamma1 activity in immunoprecipitates was increased 2-3-fold. This elevation in PLCgamma1 activity in response to thrombin activation was not observed when platelet aggregation was blocked. Although PLCgamma1 is tyrosine phosphorylated in response to many agonists, we could not detect, by Western analysis with anti-phosphotyrosine antibodies, tyrosine phosphorylation of PLCgamma1 immunoprecipitated from thrombin-stimulated platelets. These results demonstrate that PLCgamma1 is associated with gelsolin and actin in resting platelets, and that thrombin-induced platelet aggregation results in the dissociation of PLCgamma1 from gelsolin and actin, and the stimulation of PLCgamma1 activity.

    Funded by: PHS HHS: R01-40901

    The Biochemical journal 1997;324 ( Pt 1);283-7

  • Tissue distribution and levels of gelsolin mRNA in normal individuals and patients with gelsolin-related amyloidosis.

    Paunio T, Kangas H, Kiuru S, Palo J, Peltonen L and Syvänen AC

    Department of Human Molecular Genetics, National Public Health Institute, Helsinki, Finland. tiina.paunio@ktl.fi

    We measured quantitatively the mRNA levels of intracellular and secretory forms of gelsolin, an actin-modulating protein, in human tissues from subjects of different ages. The intracellular gelsolin mRNA constituted the major type of gelsolin steady-state mRNA in all tissues analyzed. Both forms of gelsolin were expressed in most adult tissues, with particularly high mRNA levels in all types of muscle and interestingly in skin. Between the adult and infantile tissues the most striking difference in expression levels was found in liver, as the adult liver contained only a subtle amount of gelsolin mRNA. Skin and muscle samples from patients with gelsolin-related amyloidosis (FAF), with significantly increased concentrations of serum gelsolin, did not reveal an increased expression of the gene, and both mutant and wild-type alleles were expressed in equal amounts. The high level of expression of the gelsolin gene in the skin in general could locally contribute to the characteristic skin amyloidosis in FAF patients.

    FEBS letters 1997;406;1-2;49-55

  • Functional consequences of disulfide bond formation in gelsolin.

    Allen PG

    Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA. pallen@calvin.bwh.harvard.edu

    Gelsolin is an actin monomer binding and filament severing protein synthesized in plasma and cytoplasmic forms differing by an N-terminal amino acid extension and a disulfide bond between Cys-188 and Cys-201. To determine whether this bond altered gelsolin regulation or function, oxidized and reduced plasma gelsolins were assayed for severing, monomer binding and nucleation activity at a variety of rate-limiting calcium concentrations. The results indicate that the disulfide bond in domain 2 of gelsolin influences the transmission of information from C-terminal regulatory sites to functional sites in the N-terminus.

    FEBS letters 1997;401;1;89-94

  • The plasma and cytoplasmic forms of human gelsolin differ in disulfide structure.

    Wen D, Corina K, Chow EP, Miller S, Janmey PA and Pepinsky RB

    Biogen, Inc., Cambridge, Massachusetts 02142, USA.

    Gelsolin is a widely distributed actin binding protein that regulates actin filament length. It exists in both an intracellular and an extracellular form that is derived from a single gene by alternative splicing. Both forms contain the six homologous domains that are responsible for function. Little is known regarding differences between the forms. We have used a combination of cysteine-specific modification with 4-vinylpyridine, HPLC peptide mapping methods, and mass spectrometry to analyze the disulfide structures of human plasma and cytoplasmic gelsolin. Of the five Cys residues in the human gelsolin sequence, all were present in the free thiol form in human cytoplasmic gelsolin, while only three of them were free thiols in the human plasma form. Cys residues 188 and 201 in domain 2 of plasma gelsolin were disulfide linked. Recombinant human plasma gelsolin that had been expressed intracellularly in Escherichia coli and as a secreted protein from Cos green monkey cells was also investigated. The E. coli product lacked the disulfide but could be converted to the plasma-like structure with mild oxidation while the mammalian product formed the correct disulfide prior to isolation. Structural differences were also detected by limited proteolysis with plasmin. The differences in proteolytic susceptibility were also due to perturbations in domain 2. These studies demonstrate that the intracellular and extracellular gelsolins are structurally distinct and suggest that at least some of the preparations of recombinant gelsolin that are being used to study structure/function may be improperly folded. The experiments also demonstrate a general method for the location of disulfide bonds in proteins.

    Funded by: NHLBI NIH HHS: HL54253

    Biochemistry 1996;35;30;9700-9

  • Spectroscopic studies of a phosphoinositide-binding peptide from gelsolin: behavior in solutions of mixed solvent and anionic micelles.

    Xian W, Vegners R, Janmey PA and Braunlin WH

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

    The peptide G(150-169) corresponds to a phosphatidylinositol 4,5-bisphosphate (PIP2) and filamentous actin (F-actin) binding site on gelsolin (residues 150-169, with the sequence KHVVPNEVVVQRLFQVKGRR). The conformation of this peptide in trifluoroethanol (TFE) aqueous solution was determined by 1H nuclear magnetic resonance as the first step toward understanding the structural aspects of the interaction of G(150-169) and PIP2. The circular dichroism experiments show that G(150-169) adopts a predominantly alpha-helical form in both 50% TFE aqueous solution and in the presence of PIP2 micelles, therefore establishing a connection between the two conformations. 1H nuclear magnetic resonance experiments of G(150-169) in TFE co-solvent show that the helical region extends from Pro-154 to Lys-166. The amphiphilic nature of this helical structure may be the key to understanding the binding of the peptide to lipids. Sodium dodecyl sulfate micelle solution is used as a model for anionic lipid environments. Preliminary studies of the conformation of G(150-169) in sodium dodecyl sulfate micelle solution show that the peptide forms an alpha-helix similar to but with some structural differences from that in TFE co-solvent. Fluorescence experiments provide evidence of peptide clustering over a narrow range of peptide/PIP2 ratios, which is potentially relevant to the biological function of PIP2.

    Funded by: NIAMS NIH HHS: AR38910

    Biophysical journal 1995;69;6;2695-702

  • Asp187Asn mutation of gelsolin in an American kindred with familial amyloidosis, Finnish type (FAP IV).

    Steiner RD, Paunio T, Uemichi T, Evans JP and Benson MD

    Department of Medicine, University of Washington, School of Medicine, Seattle 98195.

    Familial amyloidosis, Finnish type (FAP-IV) was identified clinically in an American kindred with Scandinavian ancestry. A polymerase chain reaction (PCR)-based DNA diagnostic assay was used to identify a G-to-A mutation at position 654 of the gelsolin cDNA (G654A) in this family. Molecular diagnostic testing demonstrated the mutation in individuals in three generations--the clinically affected proband, here deceased clinically affected father, and her presumably affected presymptomatic child. This report represents a rare example of FAP IV and the G654A mutation identified in a family outside Finland. The disease-associated haplotype was similar to that observed in Finnish FAP IV families (suggesting common distant ancestry).

    Funded by: NCRR NIH HHS: RR-00750; NIDDK NIH HHS: NIDDK-34881; NIGMS NIH HHS: GM 07454; ...

    Human genetics 1995;95;3;327-30

  • Localization of the calcium-sensitive actin monomer binding site in gelsolin to segment 4 and identification of calcium binding sites.

    Pope B, Maciver S and Weeds A

    MRC Laboratory of Molecular Biology, Cambridge, U.K.

    Gelsolin is composed of six repeating segments of sequence (G1-6) and contains three distinct actin binding sites, two that bind to G-actin and one that binds to filaments. The calcium-dependent actin monomer binding site present in the carboxyl-terminal half of the protein (G4-6) plays a critical role both in the cooperative binding of actin by gelsolin and in its nucleating activity. Here we have localized this actin binding site to segment 4 (G4) by expressing the segments G4, G4-5, G5, and G5-6 in Escherichia coli and analyzing their actin binding properties. In addition we have measured their calcium binding. G4-5 and G5-6 each bind a single calcium ion, but there is no binding by G4 or G5. The affinity of binding by G5-6 is 10 times higher than that of G4-5, and calcium binding by G4-6 shows two sites of different affinity. Thus each actin binding site of gelsolin is restricted to a single segment (G1, G2, and G4), but the nonbinding segments G5 and G6 play an important role in the calcium regulation of actin binding and other activities of gelsolin.

    Biochemistry 1995;34;5;1583-8

  • Haplotype analysis in gelsolin-related amyloidosis reveals independent origin of identical mutation (G654A) of gelsolin in Finland and Japan.

    Paunio T, Sunada Y, Kiuru S, Makishita H, Ikeda S, Weissenbach J, Palo J and Peltonen L

    Department of Human Molecular Genetics, National Public Health Institute, Helsinki, Finland.

    Familial amyloidosis, Finnish type (FAF) (gelsolin-related amyloidosis) is an autosomal dominant form of systemic amyloidosis characterized by corneal lattice dystrophy and peripheral polyneuropathy. The accumulating protein in FAF consists of fragments of gelsolin, an actin-modulating protein. The gelsolin mutation G654A has been found in both Finnish and Japanese patients. To study the origin of the gelsolin mutation in these patients we performed haplotype analysis in 10 Finnish and 2 Japanese FAF families. Poymorphic DNA markers GSN, D9S103, AFMa061xd9, and AFMa139xb9 revealed a uniform disease haplotype in all the disease-associated chromosomes of the Finnish FAF families, which was different from the one observed in the Japanese families. The present results and the previously detected gelsolin mutation G654T in Czech and Danish FAF patients suggest that nucleotide 654 may represent a mutation hot spot in the gelsolin gene. The DNA markers studied here will be useful in future genealogical analyses of FAF.

    Human mutation 1995;6;1;60-5

  • Reduction in viscosity of cystic fibrosis sputum in vitro by gelsolin.

    Vasconcellos CA, Allen PG, Wohl ME, Drazen JM, Janmey PA and Stossel TP

    Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston, MA 02115.

    Obstruction of airways by viscous sputum causes lung damage in patients with cystic fibrosis (CF). Sputum samples from CF patients were shown to contain filamentous actin. Human plasma gelsolin, a protein that severs actin filaments, rapidly decreased the viscosity of CF sputum samples in vitro. Gc globulin and deoxyribonuclease I, proteins that sequester monomeric actin but do not sever actin filaments, were less efficient than gelsolin in diminishing sputum viscosity. These results suggest that gelsolin may have therapeutic potential as a mucolytic agent in CF patients.

    Funded by: NHLBI NIH HHS: HL19170, HL19429; NIAMS NIH HHS: AR38910; ...

    Science (New York, N.Y.) 1994;263;5149;969-71

  • Structure of gelsolin segment 1-actin complex and the mechanism of filament severing.

    McLaughlin PJ, Gooch JT, Mannherz HG and Weeds AG

    MRC Laboratory of Molecular Biology, Cambridge, UK.

    The structure of the segment 1 domain of gelsolin, a protein that fragments actin filaments in cells, is reported in complex with actin. Segment 1 binds monomer using an apolar patch rimmed by hydrogen bonds in a cleft between actin domains. On the actin filament model it binds tangentially, disrupting only those contacts between adjacent subunits in one helical strand. The segment 1 fold is general for all segments of the gelsolin family because the conserved residues form the core of the structure. It also provides a basis for understanding the origin of an amyloidosis caused by a gelsolin variant.

    Nature 1993;364;6439;685-92

  • Gelsolin-derived familial amyloidosis caused by asparagine or tyrosine substitution for aspartic acid at residue 187.

    de la Chapelle A, Tolvanen R, Boysen G, Santavy J, Bleeker-Wagemakers L, Maury CP and Kere J

    Department of Medical Genetics, University of Helsinki, Finland.

    Dominantly inherited familial amyloidosis, Finnish type (FAF) is caused by the accumulation of a 71-amino acid amyloidogenic fragment of mutant gelsolin (GSN). FAF is common in Finland but is very rare elsewhere. In Finland and in two American families, the mutation is a G654A transition leading to an Asp to Asn substitution at residue 187. We found the same mutation in a Dutch family but a Danish FAF family had a G654T mutation, predicting Asp to Tyr at residue 187. We also found the G654T transversion in a Czech family. Using GSN polymorphisms, different haplotypes were found in the Danish and Czech families. We conclude that substitution of the uncharged Asn or Tyr for the acidic Asp at residue 187 creates a conformation that may be preferentially amyloidogenic for GSN.

    Nature genetics 1992;2;2;157-60

  • Identification of a polyphosphoinositide-binding sequence in an actin monomer-binding domain of gelsolin.

    Yu FX, Sun HQ, Janmey PA and Yin HL

    Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235.

    Gelsolin is an actin filament-severing and -capping protein that has profound effects on actin filament organization and assembly. It is activated by Ca2+ and inhibited by polyphosphoinositides (PPI). We have previously shown that PPI inhibit actin filament severing by the amino-terminal half of gelsolin and hypothesized that this is mediated through inhibition of actin filament side binding (by domains II-III of gelsolin), a requisite first step in severing. In this paper, we report that the subsequent step in severing, which is mediated by an actin monomer binding site located in domain I of gelsolin, is also regulated by PPI. We used deletional mutagenesis and a synthetic peptide to locate the sequence required for high affinity PPI binding in domain I. Our results show that the PPI-binding sequence has a basic charge distribution that is also present in the PPI-regulated actin filament side binding domain, and the two gelsolin PPI-binding sites have similar PPI-binding affinities. In addition, a similar motif is present in several other PPI-binding proteins, including a highly conserved region in the phospholipase C family. We propose that the sequences identified in gelsolin may represent a consensus for PPI binding in a variety of proteins.

    Funded by: NHLBI NIH HHS: HL29113; NIAMS NIH HHS: AR38910

    The Journal of biological chemistry 1992;267;21;14616-21

  • Familial amyloidosis, Finnish type: G654----a mutation of the gelsolin gene in Finnish families and an unrelated American family.

    de la Chapelle A, Kere J, Sack GH, Tolvanen R and Maury CP

    Department of Medical Genetics, University of Helsinki, Finland.

    The Finnish type of familial amyloid polyneuropathy (FAF) is an autosomal dominant form of systemic amyloidosis caused by a mutation in the gelsolin gene. The mutation leads to the expression of amyloidogenic mutant Asp187----Asn gelsolin, an actin-modulating protein. We previously developed a DNA test based on amplification by the polymerase chain reaction followed by allele-specific oligonucleotide hybridization that identifies the base substitution adenine for guanine at nucleotide 654 in the gelsolin gene causing the disease. We show here that the same mutation is present in members of six apparently unrelated Finnish families and in a member of an unrelated American family. These results, taken together with previously published findings in nine additional Finnish families and another unrelated American family, indicate that most, perhaps all, FAF patients in Finland and possibly worldwide carry the same mutation. We suggest two alternative explanations: (i) the mutation arose in a very early common ancestor or (ii) the Asn187 mutation is particularly, perhaps uniquely, amyloidogenic.

    Genomics 1992;13;3;898-901

  • Solid-phase minisequencing test reveals Asp187----Asn (G654----A) mutation of gelsolin in all affected individuals with Finnish type of familial amyloidosis.

    Paunio T, Kiuru S, Hongell V, Mustonen E, Syvänen AC, Bengström M, Palo J and Peltonen L

    Department of Neurology, University of Helsinki, Finland.

    Genomics 1992;13;1;237-9

  • Gelsolin gene mutation--at codon 187--in familial amyloidosis, Finnish: DNA-diagnostic assay.

    Haltia M, Levy E, Meretoja J, Fernandez-Madrid I, Koivunen O and Frangione B

    Department of Pathology, New York University Medical Center, New York.

    Familial amyloidosis, Finnish (FAF), is an autosomal dominant form of systemic amyloidosis with lattice corneal dystrophy and progressive cranial neuropathy as principal clinical manifestations. We have shown that the novel amyloid fibril protein found in these patients is an internal degradation fragment of gelsolin, an actin-binding protein, and that it contains an amino acid substitution, asparagine for aspartic acid at position 15, that is due to a guanine-to-adenine transversion corresponding to codon 187 of human plasma gelsolin cDNA. To test that this mutation cosegregates with the disease high-molecular-weight genomic DNA was isolated from autopsied tissues or lymphocytes of 23 patients, 6 healthy relatives and 20 unrelated healthy control persons. Specific fragments were amplified with the polymerase chain reaction for oligonucleotide hybridization analysis using the slot-blot technique. The guanine-to-adenine transversion was found in all FAF patients tested, but in none of the control subjects. Our results show that the mutation (G to A) cosegregates with the disease phenotype, and that the slot-blot analysis can be used as a diagnostic assay, including prenatal evaluation.

    Funded by: NIA NIH HHS: AG-05891, AG-08721; NIAMS NIH HHS: AR-02594

    American journal of medical genetics 1992;42;3;357-9

  • Amyloidosis due to a mutation of the gelsolin gene in an American family with lattice corneal dystrophy type II.

    Gorevic PD, Munoz PC, Gorgone G, Purcell JJ, Rodrigues M, Ghiso J, Levy E, Haltia M and Frangione B

    Department of Medicine, State University of New York, Stony Brook.

    Funded by: NIAMS NIH HHS: AR02594

    The New England journal of medicine 1991;325;25;1780-5

  • Finnish type of familial amyloidosis: cosegregation of Asp187----Asn mutation of gelsolin with the disease in three large families.

    Hiltunen T, Kiuru S, Hongell V, Heliö T, Palo J and Peltonen L

    Department of Neurology, University of Helsinki, Finland.

    Familial amyloidosis of Finnish type (FAF) is one of the familial amyloidotic polyneuropathy (FAP) syndromes, a group of inherited disorders characterized by extracellular accumulation of amyloid and by clinical symptoms and signs of polyneuropathy. FAF, an autosomal dominant trait, belongs to those rare monogenic disorders which occur with increased frequency in the Finnish population: only single FAF cases have been reported from other populations. In most types of FAP syndromes the accumulating protein is a transthyretin variant. However, recent evidence has suggested that the amyloid peptides in FAF are related to gelsolin, an actin modulating protein. The gelsolin fragments isolated from at least one patient with amyloidosis have been reported to have an amino acid substitution, with asparagine replacing aspartic acid at position 187 of the plasma gelsolin. In this study allele-specific oligonucleotides were used to analyze three large FAF families with multiple affected individuals as well as healthy family members. We found the corresponding G-A mutation in nucleotide 654 of the plasma gelsolin gene to cosegregate with the disease. The result was confirmed by sequencing and strongly suggests that the mutation has caused all the FAF cases of these families. Since the disease is clustered in restricted areas on the southern coast of Finland, this mutation most probably causes the majority, if not all, of FAF cases in Finland.

    American journal of human genetics 1991;49;3;522-8

  • Gelsolin variant (Asn-187) in familial amyloidosis, Finnish type.

    Ghiso J, Haltia M, Prelli F, Novello J and Frangione B

    Department of Pathology, New York University Medical Center, NY 10016.

    Familial amyloidosis, Finnish type (FAF), is an inherited form of systemic amyloidosis clinically characterized by cranial neuropathy and lattice corneal dystrophy. We have demonstrated that the protein subunit isolated from amyloid fibrils shows considerable sequence identity with gelsolin, an actin-binding protein. We have purified the amyloid subunit from a second case and further analysed different fractions from the previous one. Sequence analysis shows that, in both cases, the amyloid subunit starts at position 173 of the mature molecule; it has a heterogeneous N-terminus and contains one amino acid substitution, namely asparagine for aspartic acid, at position 15 (gelsolin residue 187), that is due to a guanine-to-adenine transversion corresponding to nucleotide-654 of human plasma gelsolin cDNA. The substitution maps in a fragment with actin-binding activity and is located in a repetitive motif highly conserved among species. Thus FAF is the first human disease known to be caused by an internal abnormal degradation of a gelsolin variant. We designate this variant of gelsolin-associated amyloidosis 'Agel Asn-187'.

    Funded by: NIA NIH HHS: AG 05891, AG 08721; NIAMS NIH HHS: AR 02594

    The Biochemical journal 1990;272;3;827-30

  • Mutation in gelsolin gene in Finnish hereditary amyloidosis.

    Levy E, Haltia M, Fernandez-Madrid I, Koivunen O, Ghiso J, Prelli F and Frangione B

    Department of Pathology, New York University Medical Center, New York 10016.

    Familial amyloidosis, Finnish type (FAF), is an autosomal dominant form of familial amyloid polyneuropathy. The novel amyloid fibril protein found in these patients is a degradation fragment of gelsolin, an actin-binding protein. We found a mutation (adenine for guanine) at nucleotide 654 of the gelsolin gene in genomic DNA isolated from five FAF patients. This site is polymorphic since the normal allele was also present in all the patients tested. This mutation was not found in two unaffected family members and 11 normal controls. The A for G transition causes an amino acid substitution (asparagine for aspartic acid) that was found at position 15 of the amyloid protein. The mutation and consequent amino acid substitution may lead to the development of FAF.

    Funded by: NIA NIH HHS: AG-05891; NIAMS NIH HHS: AR-02594

    The Journal of experimental medicine 1990;172;6;1865-7

  • Amyloid protein in familial amyloidosis (Finnish type) is homologous to gelsolin, an actin-binding protein.

    Haltia M, Prelli F, Ghiso J, Kiuru S, Somer H, Palo J and Frangione B

    Department of Pathology, New York University Medical Center, New York 10016.

    Familial amyloidosis, Finnish type, is clinically characterized by cranial neuropathy and lattice corneal dystrophy. It is an autosomal dominant form of systemic amyloidosis with small deposits of congophilic material occurring in most tissues, particularly in association with blood vessel walls and basement membranes. Amyloid fibrils were extracted from the kidney of patient VUO, and rabbit antiserum raised against the 12 kDa purified amyloid subunit displayed strong immunohistochemical reactivity with the amyloid deposits. The amino terminal sequence of this 12 kDa amyloid protein (ATEVPVSWESFNNGD) showed homology with gelsolin (or actin depolymerizing factor), a 93 kDa plasma protein. The amyloid peptide is a degradation product, starting at position 173, of the gelsolin molecule.

    Funded by: NIA NIH HHS: AG 05891; NIAMS NIH HHS: AR01431

    Biochemical and biophysical research communications 1990;167;3;927-32

  • Finnish hereditary amyloidosis. Amino acid sequence homology between the amyloid fibril protein and human plasma gelsoline.

    Maury CP, Alli K and Baumann M

    Fourth Department of Medicine, University of Helsinki, Finland.

    Amyloid fibrils were isolated from the kidney of a patient with Finnish hereditary amyloidosis. After solubilization of the fibrils in guanidine-HCl, fractionation by gel filtration, and purification by reverse-phase high-performance liquid chromatography, a homogeneous amyloid protein with an apparent Mr of 9000 was obtained. The protein was subjected to enzymatic digestion by trypsin and endoproteinase Lys-C. The amino acid sequences were determined for 6 of the released peptides and they were all found to be identical to the reported, deduced primary structure of human plasma gelsoline in the region of amino acids 235-269. The results show that the amyloid fibril protein in Finnish hereditary amyloidosis represents a new type of amyloid protein that shows amino acid sequence homology with gelsoline, an actin-modulating protein.

    FEBS letters 1990;260;1;85-7

  • Localization of gelsolin proximal to ABL on chromosome 9.

    Kwiatkowski DJ, Westbrook CA, Bruns GA and Morton CC

    Department of Medicine, Massachusetts General Hospital, Boston 02114.

    Gelsolin is a plasma and cytoskeletal protein that severs actin filaments and is regulated by both Ca+2 and polyphosphoinositides. The two forms of gelsolin are encoded by a single gene and derived through alternative message splicing. By Southern blot analysis of somatic cell hybrids and in situ chromosomal localization, we demonstrate that the gelsolin gene is present on human chromosome 9 in bands q32-q34. In situ hybridization of gelsolin to cells containing a Philadelphia chromosome [(9;22)(q34;q11)], as well as Southern blot analysis of K562 cell DNA, indicates that gelsolin is centromeric to the ABL locus in 9q34. Southern blot analysis of NotI-digested, pulsed-field gel electrophoresis-separated DNA indicates the gelsolin gene is greater than or equal to 40 kb centromeric to ABL. These studies and standard Southern blot analysis of digested DNA also indicate that the NotI restriction site contained in the gelsolin gene is uncleavable in DNA from white blood cells and hematopoietic cell lines.

    Funded by: NCI NIH HHS: CA44700-01; NHLBI NIH HHS: HL01582; NICHD NIH HHS: HD18658

    American journal of human genetics 1988;42;4;565-72

  • Plasma and cytoplasmic gelsolins are encoded by a single gene and contain a duplicated actin-binding domain.

    Kwiatkowski DJ, Stossel TP, Orkin SH, Mole JE, Colten HR and Yin HL

    Gelsolin is representative of a class of actin-modulating proteins found in lower eukaryotes to mammals, which sever actin filaments. Gelsolin found in the cytoplasm of cells is functionally similar to a mammalian plasma protein of similar size, originally called ADF or brevin. Human plasma and rabbit macrophage gelsolins differ by the presence of a 25-amino-acid residue extension on plasma gelsolin which appears to account for the difference in relative molecular mass (Mr) between the proteins as assessed by SDS-polyacrylamide gel electrophoresis (PAGE), 93,000 (93K) and 90K, respectively. Here we report the isolation of full-length human plasma gelsolin complementary DNA clones from a HepG2 library. The inferred amino-acid sequence reveals the presence of a signal peptide, a long tandem repeat that matches the actin-binding domains of gelsolin, a tetrapeptide present in actin and extended regions of identical sequence with rabbit macrophage gelsolin. Southern blot analysis indicates that a single gene in the haploid genome encodes both protein forms.

    Funded by: NHLBI NIH HHS: HL01582, HL19429, HL29113; ...

    Nature 1986;323;6087;455-8

  • Human plasma gelsolin binds to fibronectin.

    Lind SE and Janmey PA

    Human plasma gelsolin, a 93,000-dalton actin-binding protein binds to human plasma fibronectin. Qualitative data obtained from experiments employing quasi-elastic light scattering, sucrose gradient sedimentation, gel filtration chromatography, and fibronectin polymerization indicate that gelsolin and fibronectin form a complex in solution. Solid-phase binding studies show that both human plasma and rabbit macrophage gelsolin bind to immobilized fibronectin with a Kd of about 1 microM in a 1:1 complex. The ability of gelsolin to interact with actin was not affected by the presence of fibronectin. Fibronectin also increased the amount of gelsolin binding to fibrin clots. Binding of gelsolin to fibronectin may serve to localize plasma gelsolin in regions where fibronectin is deposited, such as inflammatory sites.

    Funded by: NHLBI NIH HHS: HL 01063, HL 23591; NIGMS NIH HHS: GM 09523

    The Journal of biological chemistry 1984;259;21;13262-6

  • Human platelets contain gelsolin. A regulator of actin filament length.

    Lind SE, Yin HL and Stossel TP

    Morphologic and biochemical studies suggest that actin in human platelets polymerizes in response to various stimuli and that shortening of actin filaments can be regulated by calcium. We report that human platelets contain gelsolin, a protein of Mr 91,000 that binds reversibly to actin in the presence of calcium. Platelet gelsolin exhibits immunologic crossreactivity with rabbit macrophage gelsolin and shortens actin filaments as demonstrated by viscosity measurements and gel point determinations. Gelsolin is active in micromolar calcium concentrations and its effects upon actin filaments are reversible. Gelsolin may be a dynamic regulator of actin filament length in the human platelet.

    Funded by: NCI NIH HHS: CA 09321

    The Journal of clinical investigation 1982;69;6;1384-7

Gene lists (7)

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
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000034 G2C Homo sapiens Pocklington H3 Human orthologues of cluster 3 (mouse) from Pocklington et al (2006) 30
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
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© 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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