G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase)
G00000376 (Mus musculus)

Databases (8)

ENSG00000154277 (Ensembl human gene)
7345 (Entrez Gene)
801 (G2Cdb plasticity & disease)
UCHL1 (GeneCards)
191342 (OMIM)
Marker Symbol
HGNC:12513 (HGNC)
Protein Expression
2580 (human protein atlas)
Protein Sequence
P09936 (UniProt)

Synonyms (2)

  • PGP9.5
  • Uch-L1

Literature (104)

Pubmed - other

  • Analysis of the UCHL1 genetic variant in Parkinson's disease among Chinese.

    Tan EK, Lu CS, Peng R, Teo YY, Wu-Chou YH, Chen RS, Weng YH, Chen CM, Fung HC, Tan LC, Zhang ZJ, An XK, Lee-Chen GJ, Lee MC, Fook-Chong S, Burgunder JM, Wu RM and Wu YR

    Department of Neurology, Singapore General Hospital, National Neuroscience Institute, Singapore. gnrtek@sgh.com.sg

    The inverse association of the functional ubiquitin carboxy-terminal hydrolase L1 (UCHL1) S18Y variant with Parkinson's disease (PD) among Caucasian populations has been debated. We conducted a large-scale analysis to investigate the age-of-onset effect of the UCHL1 variant in PD among ethnic Chinese. Individual data sets from 5 centers comprising a total of 4088 study subjects were analyzed. In the univariate analysis, only data from 1 center showed a trend towards a protective effect among young subjects. However, in the combined analysis, no significant association between the UCHL1 variant and PD was detected (A allele frequency 0.531 vs. 0.528, p=0.87, OR 1.01, 95% CI 0.92-1.1). Among subjects less than 60 years old, the OR is 0.99 (95% CI 0.84-1.16, p=0.88). A multivariate logistic regression analysis showed that family history, UCHL1 variant and the interaction of UCHL1 variant and age at onset (p=0.816) were not significantly associated with PD.

    Neurobiology of aging 2010;31;12;2194-6

  • Ubiquitin carboxyl terminal hydrolase L1 negatively regulates TNFalpha-mediated vascular smooth muscle cell proliferation via suppressing ERK activation.

    Ichikawa T, Li J, Dong X, Potts JD, Tang DQ, Li DS and Cui T

    Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29208, USA.

    Deubiquitinating enzymes (DUBs) appear to be critical regulators of a multitude of processes such as proliferation, apoptosis, differentiation, and inflammation. We have recently demonstrated that a DUB of ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) inhibits vascular lesion formation via suppressing inflammatory responses in vasculature. However, the precise underlying mechanism remains to be defined. Herein, we report that a posttranscriptional up-regulation of UCH-L1 provides a negative feedback to tumor necrosis factor alpha (TNFalpha)-mediated activation of extracellular signal-regulated kinases (ERK) and proliferation in vascular smooth muscle cells (VSMCs). In rat adult VSMCs, adenoviral over-expression of UCH-L1 inhibited TNFalpha-induced activation of ERK and DNA synthesis. In contrast, over-expression of UCH-L1 did not affect platelet derived growth factor (PDGF)-induced VSMC proliferation and activation of growth stimulating cascades including ERK. TNFalpha hardly altered UCH-L1 mRNA expression and stability; however, up-regulated UCH-L1 protein expression via increasing UCH-L1 translation. These results uncover a novel mechanism by which UCH-L1 suppresses vascular inflammation.

    Funded by: NCRR NIH HHS: 5P20 RR016434-09, P20 RR016434, P20 RR016434-09; NHLBI NIH HHS: R01 HL033756

    Biochemical and biophysical research communications 2010;391;1;852-6

  • Association between the ubiquitin carboxyl-terminal esterase L1 gene (UCHL1) S18Y variant and Parkinson's Disease: a HuGE review and meta-analysis.

    Ragland M, Hutter C, Zabetian C and Edwards K

    Department of Epidemiology, University of Washington, Center for Genomics and Public Health, Box 354921, 6200 NE 74th Street, Building 29, Suite 250, Seattle, WA 98115, USA.

    The ubiquitin carboxyl-terminal esterase L1 gene, UCHL1, located on chromosome 4p14, has been studied as a potential candidate gene for Parkinson's disease risk. The authors conducted a Human Genome Epidemiology review and meta-analysis of published case-control studies of the UCHL1 S18Y variant and Parkinson's disease in Asian and Caucasian samples. The meta-analysis of studies in populations of Asian ancestry showed a statistically significant association between the Y allele and reduced risk of Parkinson's disease under a recessive model (odds ratio (OR) for YY vs. SY + SS = 0.79, 95% confidence interval (CI): 0.67, 0.94; P = 0.006). For a dominant model, the association was not significant in Asian populations (OR for YY + SY vs. SS = 0.88, 95% CI: 0.68, 1.14; P = 0.33). For populations of European ancestry, the meta-analysis showed a significant association between the Y allele and decreased risk of Parkinson's disease under a dominant model (OR = 0.89, 95% CI: 0.81, 0.98; P = 0.02) but not under a recessive model (OR = 0.92, 95% CI: 0.66, 1.30; P = 0.65). Using the Venice criteria, developed by the Human Genome Epidemiology Network Working Group on the assessment of cumulative evidence, the authors concluded that moderate evidence exists for an association between the S18Y variant and Parkinson's disease.

    American journal of epidemiology 2009;170;11;1344-57

  • Polymorphism of HD and UCHL-1 genes in Huntington's disease.

    Xu EH, Tang Y, Li D and Jia JP

    Department of Neurology, Xuanwu Hospital, Capital University of Medical Sciences, Beijing 100053, China. xuerhe@163.com

    This study analyzed the association between the polymorphism of the Huntington's disease (HD) and ubiquitin carboxyl-terminal hydrolase L1 (UCHL-1) genes and the age of HD onset. We examined the size of trinucleotide CAG repeats in the HD gene of 53 individuals from families with a history of HD, six unrelated HD patients, and 51 healthy controls. Polymerase chain reaction and restriction fragment length polymorphism was performed to examine UCHL-1 S18Y polymorphism prevalence in this group. We identified five HD patients in the families and four pre-clinical HD patients in their high-risk offspring. The differences in S18Y allele prevalence between families and healthy controls were not statistically significant. The SY genotype was identified most frequently (prevalence >50%). The YY genotype was not identified in non-related HD patients, and the SS genotype had a higher prevalence than the SY genotype. The S allele was identified more frequently than the Y allele, and the difference with healthy controls was significant. Multiple linear regression analysis revealed that UCHL-1 S18Y polymorphism accounted for 15.6% of variance in the age of disease onset among 11 patients. The number of CAG repeats accounted for 71.4% of the variance. The size of CAG repeats in the HD gene is an important factor affecting the age at disease onset, but is not the only factor. UCHL-1 S18Y polymorphism is a modifier of HD with a modest regulatory role in the age at disease onset, suggesting that UCHL-1 may be involved in HD pathogenesis.

    Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia 2009;16;11;1473-7

  • Interaction of the ubiquitin carboxyl terminal esterase L1 with alpha(2)-adrenergic receptors inhibits agonist-mediated p44/42 MAP kinase activation.

    Weber B, Schaper C, Wang Y, Scholz J and Bein B

    Department of Anesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany. brendiee@web.de

    Neuroprotective effects of alpha(2)-adrenergic receptor (AR) agonists are mediated via the alpha(2A)AR subtype, but the molecular mechanisms underlying these actions are still not elucidated. A two-hybrid screen was performed to identify new proteins that may control alpha(2)AR receptor function and trafficking. This screen identified the ubiquitin carboxyl-terminal hydrolase-L1 (Uch-L1), a protein associated with Parkinson's disease, as alpha(2)AR interacting protein. This interaction was confirmed and evaluated by GST pull down assays demonstrating that Uch-L1 binds preferentially to the alpha(2A)AR subtype and only with less affinity to alpha(2B)AR and alpha(2C)AR. Co-immunoprecipitation of epitope-tagged proteins confirmed the specificity of this interaction in vivo. Moreover, co-transfection of a truncated G-protein coupled receptor kinase-DNA preventing alpha(2)AR phosphorylation led to an increased signal-strength of coimmunoprecipitated Uch-L1. Confocal laser microscopy showed that interaction of alpha(2A)AR and Uch-L1 occurred in the cytoplasm. alpha(2)AR agonist mediated activation of p44/42 MAP Kinase was drastically decreased in the presence of Uch-L1 indicating a functional relevance of this interaction. These findings may present a mechanism contributing to subtype-specific alpha(2)AR trafficking and a potential pathway for the neuroprotective effects of alpha(2)AR agonists.

    Cellular signalling 2009;21;10;1513-21

  • PGP 9.5 expression in cutaneous keratoacanthomas and squamous cell carcinomas.

    Mastoraki A, Ioannidis E, Patsouris E, Safioleas M and Aroni K

    4th Department of Surgery, Attikon University Hospital, Medical School, Athens University, 12462 Chaidari, Athens, Greece. dr_kamast@yahoo.gr

    The aim of the study was to investigate the expression of PGP 9.5 in cutaneous keratoacanthomas (KAs) and squamous cell carcinomas (SCCs). Thirty-one cases of KA (10 in the growth stage, 9 in the mature phase and 12 in the involution stage) and 36 SCCs including 13 well differentiated cases, 12 moderately differentiated tumors, 7 poorly differentiated lesions and 4 pseudoadenoid entities were investigated. PGP 9.5 expression was positively correlated with tumor stage (P < 0.001) and potential perineural invasion (P < 0.001). There was no significant difference in the distribution of patients presenting variable levels of PGP 9.5 staining with regard to maximal tumor size and the extent and degree of stromal invasion. PGP 9.5 expression proved closely associated with tumor aggressiveness and is classified as a marker for predicting the outcome of resection-treated skin cancer patients.

    Archives of dermatological research 2009;301;9;653-8

  • PGP9.5 was less frequently methylated in advanced gastric carcinoma.

    Mizukami H, Goto T, Kitamura Y, Sakata M, Saito M, Ishibashi K, Kigawa G, Nemoto H, Sanada Y and Hibi K

    Department of Surgery, Showa University Fujigaoka Hospital, Aoba-ku, Yokohama 227-8501, Japan.

    Recently, it was examined the methylation status of the Protein Gene Product 9.5 (PGP9.5) gene in primary tumors derived from 49 patients with colorectal cancer and evaluated the correlation between the methylation status and the clinicopathological findings. A significant difference was observed in lymph node metastasis (p = 0.029), suggesting that PGP9.5 was less frequently methylated in metastatic colorectal cancer. This result prompted us to examine the methylation status of the PGP9.5 gene in gastric cancers.

    Methodology: It was examined the methylation status of the PGP9.5 gene in primary tumors derived from 30 patients with gastric cancer using qMSP and evaluated the correlation between the methylation status and the clinicopathological findings.

    Results: Aberrant methylation of the PGP9.5 gene was detected in 5 of 30 (17%) primary gastric cancers. The present results suggested that the aberrant methylation of the PGP9.5 gene was frequently observed in gastric cancers. Subsequently, clinicopathological data were correlated with the methylation results. A significant difference was observed in extent of tumor, lymph node metastasis, and TNM stage (p = 0.034, 0.015, and 0.028, respectively).

    Conclusion: PGP9.5 was less frequently methylated in advanced gastric cancer compared to earlier one.

    Hepato-gastroenterology 2009;56;94-95;1576-9

  • Expression and functional studies of ubiquitin C-terminal hydrolase L1 regulated genes.

    Bheda A, Shackelford J and Pagano JS

    Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, USA.

    Deubiquitinating enzymes (DUBs) have been increasingly implicated in regulation of cellular processes, but a functional role for Ubiquitin C-terminal Hydrolases (UCHs), which has been largely relegated to processing of small ubiquitinated peptides, remains unexplored. One member of the UCH family, UCH L1, is expressed in a number of malignancies suggesting that this DUB might be involved in oncogenic processes, and increased expression and activity of UCH L1 have been detected in EBV-immortalized cell lines. Here we present an analysis of genes regulated by UCH L1 shown by microarray profiles obtained from cells in which expression of the gene was inhibited by RNAi. Microarray data were verified with subsequent real-time PCR analysis. We found that inhibition of UCH L1 activates genes that control apoptosis, cell cycle arrest and at the same time suppresses expression of genes involved in proliferation and migration pathways. These findings are complemented by biological assays for apoptosis, cell cycle progression and migration that support the data obtained from microarray analysis, and suggest that the multi-functional molecule UCH L1 plays a role in regulating principal pathways involved in oncogenesis.

    Funded by: NCI NIH HHS: 2-P01-CA19014-26, P01 CA019014; NIAID NIH HHS: R03 AI085545

    PloS one 2009;4;8;e6764

  • Defining the human deubiquitinating enzyme interaction landscape.

    Sowa ME, Bennett EJ, Gygi SP and Harper JW

    Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

    Funded by: NIA NIH HHS: AG085011, R01 AG011085, R01 AG011085-16; NIDDK NIH HHS: K01 DK098285; NIGMS NIH HHS: GM054137, GM67945, R01 GM054137, R01 GM054137-14, R01 GM067945

    Cell 2009;138;2;389-403

  • Positive reciprocal regulation of ubiquitin C-terminal hydrolase L1 and beta-catenin/TCF signaling.

    Bheda A, Yue W, Gullapalli A, Whitehurst C, Liu R, Pagano JS and Shackelford J

    Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

    Deubiquitinating enzymes (DUBs) are involved in the regulation of distinct critical cellular processes. Ubiquitin C-terminal Hydrolase L1 (UCH L1) has been linked to several neurological diseases as well as human cancer, but the physiological targets and the regulation of UCH L1 expression in vivo have been largely unexplored. Here we demonstrate that UCH L1 up-regulates beta-catenin/TCF signaling: UCH L1 forms endogenous complexes with beta-catenin, stabilizes it and up-regulates beta-catenin/TCF-dependent transcription. We also show that, reciprocally, beta-catenin/TCF signaling up-regulates expression of endogenous UCH L1 mRNA and protein. Moreover, using ChIP assay and direct mutagenesis we identify two TCF4-binding sites on the uch l1 promoter that are involved in this regulation. Since the expression and deubiquitinating activity of UCH L1 are required for its own basic promoter activity, we propose that UCH L1 up-regulates its expression by activation of the oncogenic beta-catenin/TCF signaling in transformed cells.

    Funded by: NCI NIH HHS: 2-P01-CA19014-26, P01 CA019014, T32 CA009156; NIAID NIH HHS: R03 AI085545

    PloS one 2009;4;6;e5955

  • Regulation of synaptic structure by ubiquitin C-terminal hydrolase L1.

    Cartier AE, Djakovic SN, Salehi A, Wilson SM, Masliah E and Patrick GN

    Section of Neurobiology, Department of Biological Sciences, University of California, San Diego, La Jolla, California 92093-0347, USA.

    Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function. Here, we show that UCH-L1 functions in maintaining normal synaptic structure in hippocampal neurons. We found that UCH-L1 activity is rapidly upregulated by NMDA receptor activation, which leads to an increase in the levels of free monomeric ubiquitin. Conversely, pharmacological inhibition of UCH-L1 significantly reduces monomeric ubiquitin levels and causes dramatic alterations in synaptic protein distribution and spine morphology. Inhibition of UCH-L1 activity increases spine size while decreasing spine density. Furthermore, there is a concomitant increase in the size of presynaptic and postsynaptic protein clusters. Interestingly, however, ectopic expression of ubiquitin restores normal synaptic structure in UCH-L1-inhibited neurons. These findings point to a significant role of UCH-L1 in synaptic remodeling, most likely by modulating free monomeric ubiquitin levels in an activity-dependent manner.

    Funded by: NIA NIH HHS: AG10435, AG18440, AG22074, P01 AG010435, P01 AG010435-160006, P01 AG022074, P01 AG022074-070009, R01 AG018440, R37 AG018440, R37 AG018440-08; NINDS NIH HHS: R01 NS047533, R01 NS047533-07, R21 NS054732, R21 NS054732-01A2

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;24;7857-68

  • Association study between single-nucleotide polymorphisms in 199 drug-related genes and commonly measured quantitative traits of 752 healthy Japanese subjects.

    Saito A, Kawamoto M and Kamatani N

    Division of Genomic Medicine, Department of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, Shinjuku-ku, Tokyo, Japan. a-saito@horae.dti.ne.jp

    With dense single-nucleotide polymorphism (SNP) maps for 199 drug-related genes, we examined associations between 4190 SNPs and 38 commonly measured quantitative traits using data from 752 healthy Japanese subjects. On analysis, we observed a strong association between five SNPs within the uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) gene and serum total bilirubin levels (minimum P-value in Mann-Whitney test=1.82 x 10(10)). UGT1A1 catalyzes the conjugation of bilirubin with glucuronic acid, thus enhancing bilirubin elimination. This enzyme is known to play an important role in the variation of serum bilirubin levels. The five SNPs, including a nonsynonymous SNP-rs4148323 (211G>A or G71R variant allele known as UGT1A1*6)-showed strong linkage disequilibrium with each other. No other genes were clearly associated with serum total bilirubin levels. Results of linear multiple regression analysis on serum total bilirubin levels followed by analysis of variance showed that at least 13% of the variance in serum total bilirubin levels could be explained by three haplotype-tagging SNPs in the UGT1A1 gene.

    Journal of human genetics 2009;54;6;317-23

  • Do polymorphisms in the familial Parkinsonism genes contribute to risk for sporadic Parkinson's disease?

    Sutherland GT, Halliday GM, Silburn PA, Mastaglia FL, Rowe DB, Boyle RS, O'Sullivan JD, Ly T, Wilton SD and Mellick GD

    Eskitis Institute for Cell and Molecular Therapies, School of Biomolecular and Physical Sciences, Griffith University, Brisbane, Queensland, Australia.

    Recent whole genome association studies provided little evidence that polymorphisms at the familial Parkinsonism loci influence the risk for Parkinson's disease (PD). However, these studies are not designed to detect the types of subtle effects that common variants may impose. Here, we use an alternative targeted candidate gene approach to examine common variation in 11 genes related to familial Parkinsonism. PD cases (n = 331) and unaffected control subjects (n = 296) were recruited from three specialist movement disorder clinics in Brisbane, Australia and the Australian Electoral Roll. Common genetic variables (76 SNPs and 1 STR) were assessed in all subjects and haplotype, genotype, and allele associations explored. Modest associations (uncorrected P < 0.05) were observed for common variants around SNCA, UCHL1, MAPT, and LRRK2 although none were of sufficient magnitude to survive strict statistical corrections for multiple comparisons. No associations were seen for PRKN, PINK1, GBA, ATP13A2, HTRA2, NR4A2, and DJ1. Our findings suggest that common genetic variables of selected PD-related loci contribute modestly to PD risk in Australians.

    Movement disorders : official journal of the Movement Disorder Society 2009;24;6;833-8

  • Membrane-associated farnesylated UCH-L1 promotes alpha-synuclein neurotoxicity and is a therapeutic target for Parkinson's disease.

    Liu Z, Meray RK, Grammatopoulos TN, Fredenburg RA, Cookson MR, Liu Y, Logan T and Lansbury PT

    Center for Neurologic Diseases, Brigham and Women's Hospital and Department of Neurology, Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, USA.

    Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is linked to Parkinson's disease (PD) and memory and is selectively expressed in neurons at high levels. Its expression pattern suggests a function distinct from that of its widely expressed homolog UCH-L3. We report here that, in contrast to UCH-L3, UCH-L1 exists in a membrane-associated form (UCH-L1(M)) in addition to the commonly studied soluble form. C-terminal farnesylation promotes the association of UCH-L1 with cellular membranes, including the endoplasmic reticulum. The amount of UCH-L1(M) in transfected cells is shown to correlate with the intracellular level of alpha-synuclein, a protein whose accumulation is associated with neurotoxicity and the development of PD. Reduction of UCH-L1(M) in cell culture models of alpha-synuclein toxicity by treatment with a farnesyltransferase inhibitor (FTI-277) reduces alpha-synuclein levels and increases cell viability. Proteasome function is not affected by UCH-L1(M), suggesting that it may negatively regulate the lysosomal degradation of alpha-synuclein. Therefore, inhibition of UCH-L1 farnesylation may be a therapeutic strategy for slowing the progression of PD and related synucleinopathies.

    Funded by: Intramural NIH HHS; NIA NIH HHS: Z01 AG000953

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;12;4635-40

  • Accuracy of combined protein gene product 9.5 and parafibromin markers for immunohistochemical diagnosis of parathyroid carcinoma.

    Howell VM, Gill A, Clarkson A, Nelson AE, Dunne R, Delbridge LW, Robinson BG, Teh BT, Gimm O and Marsh DJ

    Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia.

    Context: Parafibromin, encoded by HRPT2, is the first marker with significant benefit in the diagnosis of parathyroid carcinoma. However, because parafibromin is only involved in up to 70% of parathyroid carcinomas and loss of parafibromin immunoreactivity may not be observed in all cases of HRPT2 mutation, a complementary marker is needed.

    Objective: We sought to determine the efficacy of increased expression of protein gene product 9.5 (PGP9.5), encoded by ubiquitin carboxyl-terminal esterase L1 (UCHL1) as an additional marker to loss of parafibromin immunoreactivity for the diagnosis of parathyroid carcinoma.

    Design: In total, 146 parathyroid tumors and nine normal tissues were analyzed for the expression of parafibromin and PGP9.5 by immunohistochemistry and for UCHL1 by quantitative RT-PCR. These samples included six hyperparathyroidism-jaw tumor syndrome-related tumors and 24 sporadic carcinomas.

    Results: In tumors with evidence of malignancy, strong staining for PGP9.5 had a sensitivity of 78% for the detection of parathyroid carcinoma and/or HRPT2 mutation and a specificity of 100%. Complete lack of nuclear parafibromin staining had a sensitivity of 67% and a specificity of 100%. PGP9.5 was positive in a tumor with the HRPT2 mutation L64P that expressed parafibromin. Furthermore, UCHL1 was highly expressed in the carcinoma/hyperparathyroidism-jaw tumor syndrome group compared to normal (P < 0.05) and benign specimens (P < 0.001).

    Conclusion: These results suggest that positive staining for PGP9.5 has utility as a marker for parathyroid malignancy, with a slightly superior sensitivity (P = 0.03) and similar high specificity to that of parafibromin.

    The Journal of clinical endocrinology and metabolism 2009;94;2;434-41

  • Effects of UCH-L1 on alpha-synuclein over-expression mouse model of Parkinson's disease.

    Yasuda T, Nihira T, Ren YR, Cao XQ, Wada K, Setsuie R, Kabuta T, Wada K, Hattori N, Mizuno Y and Mochizuki H

    Research Institute for Diseases of Old Age, Juntendo University, Tokyo, Japan.

    The rare inherited form of Parkinson's disease (PD), PARK5, is caused by a missense mutation in ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) gene, resulting in Ile93Met substitution in its gene product (UCH-L1(Ile93Met)). PARK5 is inherited in an autosomal-dominant mode, but whether the Ile93Met mutation gives rise to a gain-of-toxic-function or loss-of-function of UCH-L1 protein remains controversial. Here, we investigated the selective vulnerabilities of dopaminergic (DA) neurons in UCH-L1-transgenic (Tg) and spontaneous UCH-L1-null gracile axonal dystrophy mice to an important PD-causing insult, abnormal accumulation of alpha-synuclein (alphaSyn). Immunohistochemistry of midbrain sections of a patient with sporadic PD showed alphaSyn- and UCH-L1-double-positive Lewy bodies in nigral DA neurons, suggesting physical and/or functional interaction between the two proteins in human PD brain. Recombinant adeno-associated viral vector-mediated over-expression of alphaSyn for 4 weeks significantly enhanced the loss of nigral DA cell bodies in UCH-L1(Ile93Met)-Tg mice, but had weak effects in age-matched UCH-L1(wild-type)-Tg mice and non-Tg littermates. In contrast, the extent of alphaSyn-induced DA cell loss in gracile axonal dystrophy mice was not significantly different from wild-type littermates at 13-weeks post-injection. Our results support the hypothesis that PARK5 is caused by a gain-of-toxic-function of UCH-L1(Ile93Met) mutant, and suggest that regulation of UCH-L1 in nigral DA cells could be a future target for treatment of PD.

    Journal of neurochemistry 2009;108;4;932-44

  • Ubiquitin C-terminal hydrolase-L1 is a key regulator of tumor cell invasion and metastasis.

    Kim HJ, Kim YM, Lim S, Nam YK, Jeong J, Kim HJ and Lee KJ

    The Center for Cell Signaling & Drug Discovery Research, College of Pharmacy and Division of Life & Pharmaceutical Sciences, Ewha Womans University, Seoul, Republic of Korea.

    Ubiquitin C-terminal hydrolase-L1 (UCH-L1) catalyses the hydrolysis of ubiquitin ester and amide mainly in neuronal cells. Recently it was proposed as a marker with a potential role in carcinogenesis. However, the molecular mechanism underlying the biological function of UCH-L1 in tumor cells is poorly understood. We found that UCH-L1 is highly expressed in non-small lung cancer cell line H157, having high invasive potential, and that the expression of UCH-L1 in tumor cells enhances their invasive potential in vitro and in vivo. UCH-L1 changes cell morphology by regulating cell adhesion through Akt-mediated pathway. Suppressing UCH-L1 expression by RNAi significantly suppressed the invasion in vitro and in vivo, and the activation of Akt and downstream mitogen activated protein kinases c-Jun N-terminal kinases and p38, but not ERK. In Akt-negative mutants, overexpression of UCH-L1 does not affect the invasion and migration capability of H157 cells. These results suggest that UCH-L1 is a key molecule to regulate tumor-cell invasion by upstream activation of Akt.

    Oncogene 2009;28;1;117-27

  • Immunohistochemical PGP 9.5 positivity in human osteoblasts may indicate that compensatory and dysplastic craniofacial growth are under control by peripheral nerves.

    Kjaer I and Nolting D

    Department of Orthodontics, School of Dentistry, University of Copenhagen, Copenhagen, Denmark. ik@odont.ku.dk

    Objectives: The purpose was to examine human osteoblasts immunohistochemically in order to clarify the significance of the innervation for alveolar bone growth.

    Unstained sections available from 21 normal human mandibles (foetal gestational ages: 14-22 weeks).

    Before sectioning in 1980-1990, the mandibular tissue had been fixed in 4% neutral-buffered formaldehyde for 5 days. Tissue blocks were then decalcified in equal parts of 2% citric acid and 20% sodium citrate for 7-15 days, dehydrated, embedded in paraffin, and sagittally cut into 4-mum-thick serial sections and mounted on Superfrost Plus microscope slides. Sections were dried overnight at 40 degrees C. In the present study, paraffin sections were deparaffinized and treated with Tris-EDTA (Merck, Germany), pH 9.0, and immunohistochemically tested with polyclonal rabbit anti-PGP 9.5, and the EnVision +/HRP dual link (K4065; DAKO Denmark A/S, Denmark) method.

    Results: A pronounced protein gene product (PGP) 9.5 activity was registered in osteoblasts from alveolar bone in all specimens. In all cases, the activity was intense at the top of and labially to the alveolar bone, while less or no activity was observed on the inner lingual aspects of the alveolar processes. Osteoclasts and osteocytes reacted vaguely or negatively.

    Conclusion: As the present study has demonstrated that human osteoblast activity in the alveolar bone seemingly responds to innervation, it is suggested that the peripheral nervous system via the trigeminal ganglion regulates compensatory and dysplastic alveolar bone formation.

    Orthodontics & craniofacial research 2008;11;4;196-200

  • Over-expression of ubiquitin carboxy terminal hydrolase-L1 induces apoptosis in breast cancer cells.

    Wang WJ, Li QQ, Xu JD, Cao XX, Li HX, Tang F, Chen Q, Yang JM, Xu ZD and Liu XP

    Department of Pathology, Shanghai Medical College, Fudan University, Shanghai 200032, PR China.

    Ubiquitin carboxy terminal hydrolase-L1 (UCH-L1) belongs to the UCH proteases family that deubiquitinates ubiquitin-protein conjugates in the ubiquitin-proteasome system. Previous research showed that UCH-L1 was expressed in mouse retinal cells and testicular germ cells, and its function was associated with apoptosis. But it is still unclear whether UCH-L1 is concerned with apoptosis in tumor cells. In order to clarify the role of UCH-L1 in tumor cells, multi-drug resistance (MDR) human breast carcinoma cell line MCF7/Adr, that expresses relatively high UCH-L1, and its parental cell line MCF7, that expresses relatively low UCH-L1, were chosen for this study. We transfected pcDNA3.1-UCH-L1 plasmid and UCH-L1 siRNA into MCF7 and MCF7/Adr cells, respectively. Using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, western blot, Hoechst 33258 staining assay and flow cytometry, we found that over-expression of UCH-L1 in MCF7 cells induced apoptosis. On the other hand, silencing of UCH-L1 in MCF7/Adr cells led to the opposite effect. Moreover, to explore the mechanism underling these observations, we further investigated the expression of phospho-Akt and its downstream signal phospho-IkB-alpha and other signal molecules including Fas, Fas-L, Trail, DR4, DR5, Bax, cytochrome C, active caspase-3, phospho-p53, phospho-Mdm-2, Bcl-2, Bcl-xL, p21 and p27. The results indicated that the process of apoptosis triggered by UCH-L1 is, at least in part, probably through Phosphoinositide 3-kinase (PI3K)/Akt signal pathway. Our findings suggest that modulating the ubiquitination and deubiquitination pathway could be a novel method for tumor therapy.

    Funded by: NCI NIH HHS: CA 66077, CA109371, R01 CA066077, R01 CA066077-10, R01 CA066077-11, R21 CA109371, R21 CA109371-01, R21 CA109371-02

    International journal of oncology 2008;33;5;1037-45

  • [Association of the ubiquitin carboxy-terminal hydrolase-L1 genetic polymorphism with the susceptibility of Parkinson's disease].

    Xiao Y and Zhang B

    Department of Neurology, the General Hospital of Tianjin Medical University, Tianjin 300052, P.R. China. xxy.jun@ eyou.com

    Objective: To study the association of two polymorphisms of ubiquitin carboxy-terminal hydrolase-L1 gene(UCH-L1), the 54C/A in exon 3 and the 277C/G in exon 4, with sporadic Parkinson's disease(PD) in Hans from North China.

    Methods: Polymerase chain reaction-restriction fragment length polymorphism was used to investigate the genotype and allele frequencies of the UCH-L1 C/A and C/G loci, in a case-control study including 75 sporadic PD and 100 randomly selected healthy control subjects.

    Results: (1)There was significant difference between PD patients and controls in the frequencies of UCH-L1 genotype and C/A allele(P<0.05). The frequencies of allele A and genotype AA were both significantly lower in PD patients than that in the controls(P<0.05).(2)There was no polymorphism in the UCH-L1 C/G locus in all cases and controls.

    Conclusion: (1)There might be association between the polymorphisms of UCH-L1 C/A locus and sporadic PD in Han population from North China.(2)There is no polymorphism in the UCH-L1 C/G locus in Hans from this region.

    Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 2008;25;5;586-7

  • Lack of evidence for association of a UCH-L1 S18Y polymorphism with Parkinson's disease in a Han-Chinese population.

    Zhang ZJ, Burgunder JM, An XK, Wu Y, Chen WJ, Zhang JH, Wang YC, Xu YM, Gou YR, Yuan GG, Mao XY and Peng R

    Department of Neurology, West China Hospital, Sichuan University, Chengdu 610041, Chengdu, China.

    Mutation in UCH-L1 has been reported as a rare cause of autosomal dominant Parkinson's disease (PD). A S18Y polymorphism in the same gene has been associated with sporadic PD. We investigated the frequency of this polymorphism among the Han-Chinese ethnic population in a case-control study. A total of 600 patients with PD and 334 unrelated healthy controls were genotyped using PCR-restriction fragment length polymorphism analysis. We did not observe any difference in allele or genotype frequencies between the cases and the controls (P>0.05). Our results do not support a role for this variant in sporadic PD.

    Neuroscience letters 2008;442;3;200-2

  • PGP9.5 methylation as a marker for metastatic colorectal cancer.

    Mizukami H, Shirahata A, Goto T, Sakata M, Saito M, Ishibashi K, Kigawa G, Nemoto H, Sanada Y and Hibi K

    Department of Surgery, Showa University Fujigaoka Hospital, Yokohama 227-8501, Japan.

    Background: Recently, it has been proven that protein gene product 9.5 (PGP9.5) hypomethylation might play an important role in re-expression of the PGP9.5 gene in gallbladder cancer. We previously examined the expression of PGP9.5 in primary colorectal cancer using immunohistochemistry and found that PGP9.5 expression is related to tumor progression and may be useful as a marker for invasive colorectal cancer. These results prompted us to examine the methylation status of the PGP9.5 gene in colorectal cancer.

    The methylation status of the PGP9.5 gene in primary tumors derived from 49 patients with colorectal cancer using a quantitative methylation-specific polymerase chain reaction (qMSP) and the association between the methylation status and the clinicopathological findings was evaluated.

    Results: An aberrant methylation of the PGP9.5 gene was detected in 36 out of 49 (73%) primary colon cancer samples. Subsequently, clinicopathological data were tested for their association with the methylation results. Lymph node metastasis was significantly associated with a lower frequency of methylation (p=0.029).

    Conclusion: These findings indicated that PGP9.5 was less frequently methylated in metastatic colorectal cancer, suggesting that PGP9.5 hypomethylation might play an important role in re-expression of the PGP9.5 gene in colorectal cancer.

    Anticancer research 2008;28;5A;2697-700

  • Aberrant interaction between Parkinson disease-associated mutant UCH-L1 and the lysosomal receptor for chaperone-mediated autophagy.

    Kabuta T, Furuta A, Aoki S, Furuta K and Wada K

    Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo, Japan. kabuta@ncnp.go.jp

    Parkinson disease (PD) is the most common neurodegenerative movement disorder. An increase in the amount of alpha-synuclein protein could constitute a cause of PD. Alpha-synuclein is degraded at least partly by chaperone-mediated autophagy (CMA). The I93M mutation in ubiquitin C-terminal hydrolase L1 (UCH-L1) is associated with familial PD. However, the relationship between alpha-synuclein and UCH-L1 in the pathogenesis of PD has remained largely unclear. In this study, we found that UCH-L1 physically interacts with LAMP-2A, the lysosomal receptor for CMA, and Hsc70 and Hsp90, which can function as components of the CMA pathway. These interactions were abnormally enhanced by the I93M mutation and were independent of the monoubiquitin binding of UCH-L1. In a cell-free system, UCH-L1 directly interacted with the cytosolic region of LAMP-2A. Expression of I93M UCH-L1 in cells induced the CMA inhibition-associated increase in the amount of alpha-synuclein. Our findings may provide novel insights into the molecular links between alpha-synuclein and UCH-L1 and suggest that aberrant interaction of mutant UCH-L1 with CMA machinery, at least partly, underlies the pathogenesis of PD associated with I93M UCH-L1.

    The Journal of biological chemistry 2008;283;35;23731-8

  • The role of PGP9.5 as a tumor suppressor gene in human cancer.

    Tokumaru Y, Yamashita K, Kim MS, Park HL, Osada M, Mori M and Sidransky D

    Department of Otolaryngology, Head and Neck Cancer Research Institute, Johns Hopkins University, Baltimore, MD, USA.

    PGP9.5 is a controversial molecule from an oncologic point of view. We recently identified frequent methylation of PGP9.5 gene exclusively in primary head and neck squamous cell carcinoma (HNSCC), suggesting that it could be a tumor suppressor gene. On the other hand, PGP9.5 was reported to be overexpressed in a subset of human cancers presumably due to intrinsic oncogenic properties or as a result of transformation. To demonstrate that PGP9.5 possesses tumor suppressive activity, we examined forced expression by stable transfection of PGP9.5 in 4 HNSCC cell lines. Although all 4 cell lines demonstrated reduced log growth rates in culture after transfection, only 2 cell lines with wild type p53 (011, 022) demonstrated decreased growth in soft agar. In 2 cell lines with mutant p53 (013, 019), we observed no altered growth in soft agar and increased sensitivity to UV irradiation. We then tested for and found a high frequency of promoter methylation in a larger panel of primary tumors including HNSCC, esophageal SCC, gastric, lung, prostate and hepatocellular carcinoma. Our data support the notion that PGP9.5 is a tumor suppressor gene that is inactivated by promoter methylation or gene deletion in several types of human cancers.

    Funded by: NCI NIH HHS: P50 CA096784, P50 CA096784-01, U01 CA084986, U01 CA084986-05; NIDCR NIH HHS: R01 DE013561, R01 DE013561-05A1

    International journal of cancer 2008;123;4;753-9

  • Epigenetic identification of ubiquitin carboxyl-terminal hydrolase L1 as a functional tumor suppressor and biomarker for hepatocellular carcinoma and other digestive tumors.

    Yu J, Tao Q, Cheung KF, Jin H, Poon FF, Wang X, Li H, Cheng YY, Röcken C, Ebert MP, Chan AT and Sung JJ

    Department of Medicine, Institute of Digestive Disease, Chinese University of Hong Kong, Hong Kong.

    Unlabelled: The ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a carboxyl-terminal ubiquitin hydrolase regulating cellular ubiquitin levels, recently suggested as a tumor suppressor. However, the role of UCHL1 in hepatocellular carcinoma (HCC) is not clear. We investigated the expression and DNA methylation of the UCHL1 in primary HCC, liver metastases from digestive carcinomas, and primary digestive cancers. UCHL1 is expressed in all normal tissues and immortalized normal epithelial cell lines, but was low or silenced in 77% (10/13) of HCC cell lines, which is well correlated with its promoter methylation status. Methylation was further detected in 44% (12/27) of HCCs, but less in metastatic tumors generated from colorectal and stomach in the liver (19%, 3/16; P < 0.05). Methylation was also detected in primary digestive tumors, including 71% (22/31) of colon, 77% (53/69) of gastric, and 40% (18/45) of esophageal carcinomas, but none or occasionally in paired adjacent nontumor tissues. Detailed methylation analysis of 49 CpG sites at a 540-bp promoter region by bisulfite genomic sequencing confirmed the methylation. UCHL1 silencing could be reversed by chemical or genetic demethylation of the promoter, indicating direct epigenetic silencing. Restoring UCHL1 expression in silenced cell lines significantly inhibited their growth and colony formation ability by inhibiting cell proliferation, causing cell cycle arrest in G2/M phase and inducing apoptosis through the intrinsic caspase-dependent pathway. Moreover, UCHL1 directly interacts with p53 and stabilizes p53 through the ubiquitination pathway.

    Conclusion: Epigenetic inactivation of UCHL1 is common in primary HCCs and other digestive tumors. UCHL1 appears to be a functional tumor suppressor involved in the tumorigenesis of HCCs and other digestive cancers.

    Hepatology (Baltimore, Md.) 2008;48;2;508-18

  • The S18Y polymorphic variant of UCH-L1 confers an antioxidant function to neuronal cells.

    Kyratzi E, Pavlaki M and Stefanis L

    Division of Basic Neurosciences, Biomedical Research Foundation, Academy of Athens, Athens, Greece.

    A number of studies have associated the S18Y polymorphic variant of UCH-L1 with protection from sporadic Parkinson's Disease (PD). The mechanism involved in this protective function is unknown, but has generally been assumed to be linked to the ubiquitin-proteasome system (UPS). In the current study, we have investigated the effects of overexpression of UCH-L1 and its variants, including S18Y, in neuronal cells. We find that S18Y, but not WT, UCH-L1 confers a specific antioxidant protective function when expressed at physiological levels in human neuroblastoma cells and primary cortical neurons. In contrast, neither WT nor S18Y UCH-L1 appear to directly impact the proteasome, although they both lead to stabilization of free ubiquitin. Lack of WT mouse UCH-L1 in neurons derived from gad mice led to a decrease of free ubiquitin, but no overall decrease in UPS function or enhanced sensitivity to oxidative stress. We conclude that the S18Y variant of UCH-L1 confers a novel antioxidant function that is not present in the WT form and that this function may underlie the protective effects of this variant in certain PD populations. Our results furthermore provide indirect evidence for the importance of oxidative stress as a pathogenetic factor in certain forms of sporadic PD.

    Human molecular genetics 2008;17;14;2160-71

  • CpG hypermethylation of the UCHL1 gene promoter is associated with pathogenesis and poor prognosis in renal cell carcinoma.

    Kagara I, Enokida H, Kawakami K, Matsuda R, Toki K, Nishimura H, Chiyomaru T, Tatarano S, Itesako T, Kawamoto K, Nishiyama K, Seki N and Nakagawa M

    Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.

    Purpose: Aberrant DNA hypermethylation has been reported in renal cell carcinoma. We performed microarray analysis in the renal cancer cell line ACHN treated with the demethylating agent 5-aza-2'-deoxycytidine and investigated the UCHL1 gene involved in the regulation of cellular ubiquitin levels.

    We subjected 131 renal cell carcinoma and 61 corresponding normal kidney tissue samples to real-time reverse transcriptase-polymerase chain reaction, quantitative methylation specific polymerase chain reaction and immunohistochemistry. We also established a stable UCHL1 transfectant to evaluate cell growth.

    Results: We identified 10 genes that were up-regulated more than 2.5-fold in 5-aza-2'-deoxycytidine treated vs untreated ACHN cells. UCHL1 expression was increased 3.41-fold by 5-aza-2'-deoxycytidine treatment. In clinical samples the UCHL1 methylation index was significantly higher in renal cell carcinoma than in normal kidney tissue (p = 0.011). Conversely UCHL1 mRNA expression was significantly lower in renal cell carcinoma than in normal kidney tissue (p <0.0001). There was a negative correlation between mRNA expression and the UCHL1 methylation index (p = 0.017). The immunostaining score for UCHL1 was significantly higher in normal kidney tissue than in renal cell carcinoma (p <0.0001). Kaplan-Meier analysis showed that a positive UCHL1 methylation index had a significant adverse effect on prognosis (p = 0.048). Significant growth inhibition in UCHL1 transfectant compared to that in WT ACHN (p <0.0001) suggests that UCHL1 functions as a potential tumor suppressor gene in human renal cell carcinoma.

    Conclusions: To our knowledge we report the first study demonstrating that the mechanism of UCHL1 down-regulation in renal cell carcinoma is through CpG hypermethylation of the promoter region and methylation of the UCHL1 gene is associated with a poor prognosis in patients with renal cell carcinoma.

    The Journal of urology 2008;180;1;343-51

  • [Association of two polymorphisms in ubiquitin carboxy-terminal hydrolase-L1 gene with Parkinson's disease in Shanghai].

    Hao YX, Zhang J, Fang CP, Zhang Q, Zhang JR and Shen Q

    Department of Laboratory Diagnosis, Changhai Hospital, the Second Military Medical University, Shanghai, 200433 People's Republic of China. hao.yixin@ yahoo.com.cn

    Objective: To explore the relationship of polymorphisms in the ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) gene with Parkinson's disease(PD)in Shanghai Han Nationality.

    Methods: The distribution of a Serine18Tyrosine polymorphism in exon 3(C/A) and a Serine89Phenylalanine polymorphism in exon 4(C/T)of UCH-L1 gene were detected in 164 PD cases and 172 healthy controls, using polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method.

    Results: (1)The C allelic frequency in exon 3 of UCH-L1 gene in PD patients(62.2%) was significantly higher than that of the healthy controls(51.7%) (OR=1.53, P=0.006), as was the C/C genotype(OR=1.90, P=0.008). (2)There was no significant difference in the distribution of the C/T allele and genotypes in exon 4 between PD patients and healthy controls.

    Conclusion: The C allele in exon 3 of UCH-L1 gene might be one of the risk factors for PD in Shanghai Han Nationality, but the polymorphisms of C/T in exon 4 showed no association with the onset of PD.

    Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 2008;25;3;272-5

  • Downregulation of two isoforms of ubiquitin carboxyl-terminal hydrolase isozyme L1 correlates with high metastatic potentials of human SN12C renal cell carcinoma cell clones.

    Tanaka T, Kuramitsu Y, Fujimoto M, Naito S, Oka M and Nakamura K

    Department of Digestive Surgery and Surgical Oncology Department of Surgery, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan.

    Proteomic differential display analysis was performed on human renal cell carcinoma cell SN12C clones having different metastatic potentials by using 2-DE and LC-MS/MS. The SN12C cell clones were SN12C parent cell line, SN12C-clone 2, SN12C-clone 4, and SN12C-PM6. The SN12C parent cell line was established from an HRCC surgical specimen. SN12C-clone 4 has lower, and SN12C-clone 2 and SN12C-PM6 have higher metastatic potential than SN12C parent cells. We found eight protein spots whose expression level was different between low metastatic clones and high metastatic clones. The protein expression of three appeared to be higher in high metastatic clones than low metastatic clones, and that of other five protein spots appeared to be lower in high metastatic clones than low metastatic clones. These spots were selected, digested and analyzed by LC-MS/MS analysis, and they were identified by peptide sequencing tag. In high metastatic potential clones, two isoforms of ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1) were downregulated. These results suggest that UCH-L1 expression seems to be associated with the metastatic potential of HRCC SN12C cell clones.

    Electrophoresis 2008;29;12;2651-9

  • Aberrant molecular properties shared by familial Parkinson's disease-associated mutant UCH-L1 and carbonyl-modified UCH-L1.

    Kabuta T, Setsuie R, Mitsui T, Kinugawa A, Sakurai M, Aoki S, Uchida K and Wada K

    Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo 187-8502, Japan. kabuta@ncnp.go.jp

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons. The I93M mutation in ubiquitin C-terminal hydrolase L1 (UCH-L1) is associated with familial PD, and we have previously shown that the I93M UCH-L1-transgenic mice exhibit dopaminergic cell loss. Over 90% of neurodegenerative diseases, including PD, occur sporadically. However, the molecular mechanisms underlying sporadic PD as well as PD associated with I93M UCH-L1 are largely unknown. UCH-L1 is abundant (1-5% of total soluble protein) in the brain and is a major target of oxidative/carbonyl damage associated with sporadic PD. As well, abnormal microtubule dynamics and tubulin polymerization are associated with several neurodegenerative diseases including frontotemporal dementia and parkinsonism linked to chromosome 17. Here we show that familial PD-associated mutant UCH-L1 and carbonyl-modified UCH-L1 display shared aberrant properties: compared with wild-type UCH-L1, they exhibit increased insolubility and elevated interactions with multiple proteins, which are characteristics of several neurodegenerative diseases-linked mutants. Circular dichroism analyses suggest similar structural changes in both UCH-L1 variants. We further report that one of the proteins interacting with UCH-L1 is tubulin, and that aberrant interaction of mutant or carbonyl-modified UCH-L1 with tubulin modulates tubulin polymerization. These findings may underlie the toxic gain of function by mutant UCH-L1 in familial PD. Our results also suggest that the carbonyl modification of UCH-L1 and subsequent abnormal interactions of carbonyl-modified UCH-L1 with multiple proteins, including tubulin, constitute one of the causes of sporadic PD.

    Human molecular genetics 2008;17;10;1482-96

  • Toward a confocal subcellular atlas of the human proteome.

    Barbe L, Lundberg E, Oksvold P, Stenius A, Lewin E, Björling E, Asplund A, Pontén F, Brismar H, Uhlén M and Andersson-Svahn H

    Department of Biotechnology, AlbaNova University Center, Royal Institute of Technology, SE-106 91 Stockholm, Sweden.

    Information on protein localization on the subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here we report on a pilot study of 466 proteins in three human cell lines aimed to allow large scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated, and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.

    Molecular & cellular proteomics : MCP 2008;7;3;499-508

  • Lack of evidence for an association between UCHL1 S18Y and Parkinson's disease.

    Hutter CM, Samii A, Factor SA, Nutt JG, Higgins DS, Bird TD, Griffith A, Roberts JW, Leis BC, Montimurro JS, Kay DM, Edwards KL, Payami H and Zabetian CP

    Department of Epidemiology, University of Washington, Seattle, WA, USA.

    UCHL1 has been proposed as a candidate gene for Parkinson's disease (PD). A meta-analysis of white and Asian subjects reported an inverse association between the non-synonymous UCHL1 S18Y polymorphism and PD risk. However, this finding was not replicated in a large case-control study and updated meta-analysis restricted to white subjects. We performed a case-control study of 1757 PD patients recruited from movement disorder clinics and 2016 unrelated controls from four regions of the United States. All subjects self-reported as white. We did not observe evidence for an association between S18Y genotypes and PD (overall P-value for association: P = 0.42). After adjustment for age, sex, and recruitment region, the odds ratio for Y/S versus S/S was 0.91 (95% CI: 0.78-1.06) and for Y/Y versus S/S was 0.87 (95% CI: 0.58-1.29). We also did not observe a significant association for recessive or dominant models of inheritance, or after stratification by age at onset, age at blood draw, sex, family history of PD, or recruitment region. Our results suggest that UCHL1 S18Y is not a major susceptibility factor for PD in white populations although we cannot exclude the possibility that the S18Y variant exerts weak effects on risk, particularly in early-onset disease.

    Funded by: NCI NIH HHS: R01 CA149051; NIA NIH HHS: P30 AG008017; NINDS NIH HHS: K08 NS044138, K08 NS044138-05, R01 NS036960

    European journal of neurology 2008;15;2;134-9

  • Human prostate cancer cells express neuroendocrine cell markers PGP 9.5 and chromogranin A.

    Leiblich A, Cross SS, Catto JW, Pesce G, Hamdy FC and Rehman I

    Section of Oncology, Academic Urology Unit, University of Sheffield, Royal Hallamshire Hospital, Sheffield, UK.

    Background: A proportion of men with prostate cancer will progress to develop metastatic disease involving the lymph-nodes and bone. To identify novel candidates associated with metastatic progression, we compared the proteomic profiles of LNCaP (lymph-node metastatic, androgen-dependant) and PC-3 (bone metastatic, androgen-independent), human prostate cancer cells.

    Methods: Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by electrospray ionisation tandem mass spectrometry (ESI-MS/MS), was used to identify differentially expressed proteins. Western blotting was used to validate the identity of any candidates. Immunohistochemistry was used to assess tissue expression.

    Results: 2D-PAGE followed by ESI-MS/MS analyses identified the expression of glutathione S-transferase-pi (GST-pi) and protein gene product 9.5 (PGP 9.5) in PC-3 cells, but absent expression in LNCaP cells. PGP 9.5 expression in PC-3 cells was confirmed by Western blotting, in addition to expression in DU145 cells. Analysis of cell conditioned media showed that PGP 9.5 was secreted. Sequencing of the PGP 9.5 gene promoter region in bisulfite modified DNA, suggested that the regulation of expression involves promoter hypermethylation. RT-PCR analysis for Chromogranin A (ChA) mRNA (a marker of neuroendocrine cells), showed expression in PC-3 and DU145 cells but was undetectable in LNCaP cells. Immunohistochemistry localised PGP 9.5 expression exclusively within neuroendocrine cells and nerve fibres.

    Conclusions: Our unexpected finding that the neuroendocrine cell markers PGP 9.5 and ChA are expressed by PC-3 and DU145 cells, suggests that these cells may have been derived from metastatic adenocarcinomas which had undergone neuroendocrine differentiation or alternatively the expression occurred ectopically as a result of cell culture.

    Funded by: Medical Research Council: G0500966

    The Prostate 2007;67;16;1761-9

  • Identification of methylation-silenced genes in colorectal cancer cell lines: genomic screening using oligonucleotide arrays.

    Fukutomi S, Seki N, Koda K and Miyazaki M

    Department of General Surgery, Chiba University Graduate School of Medicine, Chuoh-ku, Chiba, Japan.

    Objective: Aberrant methylation of promoter CpG islands is associated with the loss of expression of tumor suppressor genes in human cancers. The purpose of this study was to examine methylation-silenced genes in colorectal cancer (CRC) cell lines.

    Using an oligonucleotide array, we undertook a genome-wide search for genes upregulated following treatment with a demethylating agent (5-aza-2'-deoxycytidine) in two CRC cell lines, DLD-1 and HT29. Promoter methylation status was determined in 12 CRC cell lines and 11 CRC tissues by methylation-specific polymerase chain reaction (MSP).

    Results: After treatment, 350 genes were up-regulated 1.5-fold or more. Six genes (PAGE-5, VCX, MAEL, GAGED2, UCHL1, and GAGE7), which contained putative 5' CpG islands in their promoter regions, were confirmed to be silenced in CRC cell lines. UCHL1 (also known as PGP9.5) is involved in regulation of cellular ubiquitin levels, and its promoter methylation was detected in 10 out of 12 CRC cell lines. The level of methylation of UCHL1 was significantly higher in tumors than in corresponding normal mucosae (p = 0.005).

    Conclusions: Chemical genomic screening led to the identification of a specific promoter subject to hypermethylation in CRC. These results suggest that aberrant promoter methylation is the primary mechanism of transcriptional silencing of the UCHL1 gene and that methylation of the UCHL1 gene promoter increases during the development and progression of CRC.

    Scandinavian journal of gastroenterology 2007;42;12;1486-94

  • Ubiquitin carboxyl-terminal hydrolase L1, a novel deubiquitinating enzyme in the vasculature, attenuates NF-kappaB activation.

    Takami Y, Nakagami H, Morishita R, Katsuya T, Cui TX, Ichikawa T, Saito Y, Hayashi H, Kikuchi Y, Nishikawa T, Baba Y, Yasuda O, Rakugi H, Ogihara T and Kaneda Y

    Department of Geriatric Medicine, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita 565-0871, Japan.

    Objective: We identified a ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) gene, which encodes a deubiquitinating enzyme and is expressed in the vasculature, by functional screening of a human endothelial cell (EC) cDNA library. UCHL1 is expressed in neurons, and abnormalities in UCHL1 are responsible for inherited Parkinson's disease via its effects on the ubiquitin-proteasome system. Therefore, the goal of present study was to clarify the role of the UCHL1 gene in vascular remodeling by evaluating nuclear factor-kappaB (NF-kappaB) inactivation in ECs and vascular smooth muscle cells (VSMCs).

    From Northern blot and immunohistochemical analysis, the UCHL1 gene was endogenously expressed in vascular ECs, VSMCs, and brain tissue. Expression of UCHL1 was markedly increased in the neointima of the balloon-injured carotid artery and was also present in atherosclerotic lesions from human carotid arteries. Overexpression of the UCHL1 gene significantly attenuated tumor necrosis factor (TNF)-alpha-induced NF-kappaB activity in vascular cells and increased inhibitor of kappa B-alpha (IkappaB-alpha), possibly through the attenuation of IkappaB-alpha ubiquitination, leading to decreased neointima in the balloon-injured artery. In contrast, knockdown of UCHL1 by small interfering RNA resulted in increased NF-kappaB activity in VSMCs.

    Conclusions: These data suggest that UCHL1 may partially attenuate vascular remodeling through inhibition of NF-kappaB activity.

    Arteriosclerosis, thrombosis, and vascular biology 2007;27;10;2184-90

  • DJ-1 and UCH-L1 gene activity patterns in the brains of controls, Parkinson and schizophrenia patients and in rodents.

    Galter D, Westerlund M, Belin AC and Olson L

    Karolinska Institutet, Department of Neuroscience, Retzius väg 8 B2:4, S-171 77 Stockholm, Sweden.

    DJ-1 (PARK7) has been implicated in early onset and familial cases of Parkinson's disease (PD). We therefore mapped cellular activity patterns of the DJ-1 gene in human and rodent brain tissue with radioactive in-situ hybridization. In all three mammals mRNA expression was restricted mainly to neurons in all regions analyzed. White matter, such as crus cerebri and capsula interna appeared negative, suggesting that glial cells express DJ-1 at levels below the detection limit of our method. We compared DJ-1 mRNA expression to the neuronal marker UCH-L1, which has also been implicated in PD, and found lower levels for DJ-1 but very similar patterns of expression. Measurement of the signal intensity revealed that human frontal cortex of control cases expressed DJ-1 mRNA more abundantly than other regions such as substantia nigra in the midbrain. Comparing DJ-1 expression in dopamine neurons on hemi-sections from controls and patients we could not detect any difference between 14 controls, 8 idiopathic Parkinson and 5 schizophrenia cases. Of note, DJ-1 is expressed in several other tissues such as the liver, gastrointestinal tract, adrenal and pituitary gland and during embryonic development, while UCH-L1 has a strictly neuronal expression also outside the CNS. We conclude that DJ-1 and UCH-L1, like other genes linked to PD, are not expressed specifically in DA neurons, but instead generally in neurons. The abundant expression of DJ-1 in certain peripheral tissues and of UCH-L1 in peripheral neurons may also be of relevance for the spectrum of symptoms in different forms of PD.

    Funded by: OMHHE CDC HHS: R24-MN069955

    Physiology & behavior 2007;92;1-2;46-53

  • The role of ubiquitin C-terminal hydrolase L1 in neurodegenerative disorders.

    Gong B and Leznik E

    Taub Institute for Research on Alzheimer's disease and the Aging Brain, Department of Pathology, Columbia University, New York, New York, USA. bg2058@columbia.edu

    Impairment of the ubiquitin-proteasome system (UPS) results in the failure to remove and degrade misfolded proteins and consequently causes the accumulation of misfolded proteins in the cell. The aberrant interactions between misfolded proteins and normal intracellular proteins are thought to underlie the pathogenesis in many neurodegenerative diseases. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is an important component of the UPS. Its major function is related to mono-ubiquitin recycling and thereby, sustaining protein degradation. Mutations of the UCH-L1 gene and alterations of its proteins' activity have been found to associate with several neurodegenerative disorders. In this review, we will discuss a link between UCH-L1 and Parkinson's, Huntington's and Alzheimer's diseases. We will also present a potential strategy for the treatment of Alzheimer's disease by boosting endogenous UCH-L1 activity.

    Drug news & perspectives 2007;20;6;365-70

  • S18Y in ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) associated with decreased risk of Parkinson's disease in Sweden.

    Carmine Belin A, Westerlund M, Bergman O, Nissbrandt H, Lind C, Sydow O and Galter D

    Department of Neuroscience, Karolinska Institutet, Retzius väg 8 B2:4, 171 77 Stockholm, Sweden. Andrea.Carmine.Belin@ki.se

    Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) is a neuron-specific enzyme that removes ubiquitin from the C-terminal end of substrates and a component of the ubiquitin-proteasome system. A protective effect of a UCH-L1 variant, S18Y, was suggested since the common variant was found to be inversely associated with sporadic Parkinson's disease (PD). We investigated the association of S18Y in our Swedish PD material. The tyrosine variant was significantly inversely associated with PD (P=0.049) and with a low age of onset (50 years) (P=0.017) in the case-control material, supporting the hypothesis of a protective function.

    Parkinsonism & related disorders 2007;13;5;295-8

  • Altered gene expression in cells from patients with lysosomal storage disorders suggests impairment of the ubiquitin pathway.

    Bifsha P, Landry K, Ashmarina L, Durand S, Seyrantepe V, Trudel S, Quiniou C, Chemtob S, Xu Y, Gravel RA, Sladek R and Pshezhetsky AV

    Sainte-Justine Hospital, Department of Pediatrics and Biochemistry, University of Montreal, Montreal, Canada.

    By comparing mRNA profiles in cultured fibroblasts from patients affected with lysosomal storage diseases, we identified differentially expressed genes common to these conditions. These studies, confirmed by biochemical experiments, demonstrated that lysosomal storage is associated with downregulation of ubiquitin C-terminal hydrolase, UCH-L1 in the cells of eight different lysosomal disorders, as well as in the brain of a mouse model of Sandhoff disease. Induction of lysosomal storage by the cysteine protease inhibitor E-64 also reduced UCH-L1 mRNA, protein level and activity. All cells exhibiting lysosomal storage contained ubiquitinated protein aggregates and showed reduced levels of free ubiquitin and decreased proteasome activity. The caspase-mediated apoptosis in E-64-treated fibroblasts was reversed by transfection with a UCH-L1 plasmid, and increased after downregulation of UCH-L1 by siRNA, suggesting that UCH-L1 deficiency and impairment of the ubiquitin-dependent protein degradation pathway can contribute to the increased cell death observed in many lysosomal storage disorders.

    Cell death and differentiation 2007;14;3;511-23

  • Ubiquitin COOH-terminal hydrolase 1: a biomarker of renal cell carcinoma associated with enhanced tumor cell proliferation and migration.

    Seliger B, Fedorushchenko A, Brenner W, Ackermann A, Atkins D, Hanash S and Lichtenfels R

    Institute of Medical Immunology, Martin-Luther-University Halle-Wittenberg, Halle, Germany. Barbara.Seliger@medizin.uni-halle.de

    Purpose: Renal cell carcinoma (RCC) accounts for 2% to 3% of all malignancies. It represents one of the most radiation- and chemotherapy-resistant tumors and surgical resections are only effective in organ-defined disease. However, RCC is an immunogenic tumor with response rates to immunotherapies between 10% and 20% of the treated patients. Due to the currently inefficient therapies and the low 5-year survival rates of RCC patients, novel diagnostic, prognostic, and therapeutic markers are urgently needed for this disease.

    Proteome-based approaches were used to identify (a) differentially expressed proteins in RCC compared with normal kidney epithelium and (b) proteins that are able to induce an antibody response in RCC patients. Based on these experiments, a promising candidate was subsequently validated by reverse transcription-PCR, Western blot analyses, and immunohistochemistry. In addition, functional assays were done in generated transfectants.

    Results: The ubiquitin COOH-terminal hydrolase L1 (UCHL1) was found to be differentially expressed in both RCC lesions and RCC cell lines and immunoreactive using patients' sera. UCHL1 expression was often down-regulated in primary RCC when compared with normal kidney epithelium but dependent on the RCC subtype, the von Hippel-Lindau phenotype, and the tumor grading. Moreover, the frequency and the level of UCHL1 expression were higher in metastases when compared with primary RCC lesions. Gain-of-function transfectants exhibited a significant higher proliferation and migration rate than UCHL1-negative RCC cells.

    Conclusions: UCHL1 expression seems to be associated with the metastatic phenotype of RCC and therefore might serve as potential biomarker for the diagnosis and prognosis of RCC patients.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2007;13;1;27-37

  • Substrate recognition and catalysis by UCH-L1.

    Luchansky SJ, Lansbury PT and Stein RL

    Center for Neurologic Diseases, Harvard Medical School and Brigham and Women's Hospital and Laboratory for Drug Discovery in Neurodegeneration, Harvard Center for Neurodegeneration and Repair, Cambridge, Massachusetts 02139, USA. sarah.luchansky@gmail.com

    Deubiquitinating enzymes regulate essential cellular processes, and their dysregulation is implicated in multiple disease states. Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) has garnered attention for its links with Parkinson's disease and cancer; however, the mechanism of action of this enzyme in cells remains poorly understood. In order to advance our understanding of UCH-L1 function, we have been developing small molecule modulators of the enzyme for use as tools to probe its role in cells. In support of these efforts, an investigation of the mechanism of UCH-L1 catalysis was previously reported. Here, we extend this mechanistic evaluation and examine substrate recognition by UCH-L1. We developed a panel of ubiquitin fusions to test the contribution of specific residues of ubiquitin to binding and catalysis by the enzyme, and determined the activation parameters of selected variants to gain additional mechanistic insight. Ubiquitin side chains critical for establishing the Michaelis complex and enabling catalysis were identified, and features of this complex that differ between UCH-L1 and a homologue, UCH-L3, were revealed. These data provide dramatic examples of differences in substrate specificity between these enzymes. The implications of our experiments with UCH-L1 for selective inhibitor design and the relationship to disease are discussed.

    Biochemistry 2006;45;49;14717-25

  • Up-regulation of expression of the ubiquitin carboxyl-terminal hydrolase L1 gene in human airway epithelium of cigarette smokers.

    Carolan BJ, Heguy A, Harvey BG, Leopold PL, Ferris B and Crystal RG

    Department of Genetic Medicine and Division of Pulmonary and Critical Care Medicine, Weill Medical College of Cornell University, New York, New York, USA.

    Neuroendocrine differentiation is a common feature of lung cancer and increased numbers of neuroendocrine cells and their peptides have been described in chronic smokers. To understand the effects of cigarette smoking on the gene expression profile of neuroendocrine cells, microarray analysis with TaqMan confirmation was used to assess airway epithelial samples obtained by fiberoptic bronchoscopy from 81 individuals [normal nonsmokers, normal smokers, smokers with early chronic obstructive lung disease (COPD), and smokers with established COPD]. Of 11 genes considered to be neuroendocrine cell specific, only ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a member of the ubiquitin proteasome pathway, was consistently up-regulated in smokers compared with nonsmokers. Up-regulation of UCHL1 at the protein level was observed with immunohistochemical analysis of bronchial biopsies of smokers compared with nonsmokers. UCHL1 expression was evident only in neuroendocrine cells of the airway epithelium in nonsmokers; however, UCHL1 was also expressed in ciliated epithelial cells in smokers. This observation may add further weight to recent observations that ciliated cells are capable of transdifferentiating to other airway epithelial cells. In the context that UCHL1 is involved in the degradation of unwanted, misfolded, or damaged proteins within the cell and is overexpressed in >50% of lung cancers, its overexpression in chronic smokers may represent an early event in the complex transformation from normal epithelium to overt malignancy.

    Funded by: NCRR NIH HHS: M01RR00047; NHLBI NIH HHS: R01 HL074326

    Cancer research 2006;66;22;10729-40

  • Case-control study of UCHL1 S18Y variant in Parkinson's disease.

    Tan EK, Puong KY, Fook-Chong S, Chua E, Shen H, Yuen Y, Pavanni R, Wong MC, Puvan K and Zhao Y

    Department of Neurology, Singapore General Hospital, Singapore. gnrtek@sgh.com.sg

    A recent meta-analysis observed a greater significant inverse association of the ubiquitin carboxy-terminal hydrolase L1 (UCHL1) S18Y variant with Parkinson's disease (PD) for Asian (predominantly Japanese) populations compared with Caucasian populations. We performed an independent case-control study in 335 PD and 341 control subjects with data from a Chinese population to investigate the age-of-onset effect of the UCHL1 variant in PD. The Y/Y and Y/S genotypes were less frequent in the PD young-onset group than in controls and the frequency of the Y alleles was higher in young controls compared to young-onset PD (age at examination <or= 65 years; P = 0.003). Multivariate analysis revealed the Y/Y genotype was significantly lower (P = 0.008) in the young-onset PD (Y/Y vs. S/S: odds ratio [OR]: 0.42; 95% confidence interval [CI]: 0.24, 0.74; S/Y vs. S/S: OR: 0.66, 95% CI: 0.41, 1.08) compared with controls, but this difference was not seen for the late-onset PD. Kaplan-Meier analysis carried out on PD subjects demonstrated that the Y/Y genotype was associated with a later onset of PD than Y/S plus S/S genotypes (P = 0.05). We provided an independent confirmation of the protective effect of the UCHL1 S18Y variant (Y/Y genotype) against PD in young Chinese subjects. Further functional studies of the S18Y variant in both cell and animal models will be of interest.

    Movement disorders : official journal of the Movement Disorder Society 2006;21;10;1765-8

  • Silencing of the UCHL1 gene in human colorectal and ovarian cancers.

    Okochi-Takada E, Nakazawa K, Wakabayashi M, Mori A, Ichimura S, Yasugi T and Ushijima T

    Carcinogenesis Division, National Cancer Center Research Institute, Tokyo, Japan.

    Aberrant DNA methylation is associated with many types of human cancers. To identify genes silenced in human colorectal cancers, we performed a microarray analysis for genes whose expression was induced by treatment of HCT116 human colon cancer cells with a demethylating agent, 5-aza-2'-deoxycitidine (5-aza-dC). Seven known genes were identified as being upregulated (> or =8-fold) and expressed at more than twice as high as the average level. Among these was the UCHL1 gene (also known as PGP9.5), which is involved in regulation of cellular ubiquitin levels. A dense CpG island in its promoter region was completely methylated in HCT116 cells, and no mRNA was detected. 5-Aza-dC treatment of HCT116 cells induced dose-dependent demethylation of the CpG island, and restored UCHL1 mRNA and protein expression. UCHL1 silencing was observed in 11 of 12 human colorectal cancer cell lines, and its methylation was detected in 8 of 17 primary colorectal cancers. Further, UCHL1 silencing was observed in 6 of 13 ovarian cancer cell lines, and its methylation was detected in 1 of 17 primary ovarian cancers. These results showed that UCHL1 is inactivated in human colorectal and ovarian cancers by its promoter methylation, and suggest that disturbance of cellular ubiquitin levels is present.

    International journal of cancer. Journal international du cancer 2006;119;6;1338-44

  • Analysis of functional polymorphisms in three synaptic plasticity-related genes (BDNF, COMT AND UCHL1) in Alzheimer's disease in Colombia.

    Forero DA, Benítez B, Arboleda G, Yunis JJ, Pardo R and Arboleda H

    Grupo de Neurociencias, Facultad de Medicina e Instituto de Genética, Universidad Nacional de Colombia, Bogotá, Colombia.

    In recent years, it has been proposed that synaptic dysfunction may be an important etiological factor for Alzheimer's disease (AD). This hypothesis has important implications for the analysis of AD genetic risk in case-control studies. In the present work, we analyzed common functional polymorphisms in three synaptic plasticity-related genes (brain-derived neurotrophic factor, BDNF Val66Met; catechol-O-methyl transferase, COMT Val158; ubiquitin carboxyl-terminal hydroxylase, UCHL1 S18Y) in a sample of 102 AD cases and 168 age and sex matched controls living in Bogotá, Colombia. There was not association between UCHL1 polymorphism and AD in our sample. We have found an initial association with BDNF polymorphism in familial cases and with COMT polymorphism in male and sporadic patients. These initial associations were lost after Bonferroni correction for multiple testing. Unadjusted results may be compatible with the expected functional effect of variations in these genes on pathological memory and cognitive dysfunction, as has been implicated in animal and cell models and also from neuropsychological analysis of normal subjects carriers of the AD associated genotypes. An exploration of functional variants in these and in other synaptic plasticity-related genes (a synaptogenomics approach) in independent larger samples will be important to discover new genes associated with AD.

    Neuroscience research 2006;55;3;334-41

  • Genetic association between Ubiquitin Carboxy-terminal Hydrolase-L1 gene S18Y polymorphism and sporadic Alzheimer's disease in a Chinese Han population.

    Xue S and Jia J

    Department of Neurology, Xuanwu Hospital, Capital University of Medical Sciences, 45 Changchun Street, 100053 Beijing, China.

    Increasing evidence indicates that the dysfunction of ubiquitin-proteasome system (UPS) is associated with Alzheimer's disease (AD). In the ubiquitin-proteasome pathway, Ubiquitin Carboxy-terminal Hydrolase-L1 (UCH-L1) plays an important role for the cellular clearance of abnormal proteins. Since a substitution of serine by tyrosine at codon 18, exon 3 (S18Y polymorphism) of the UCH-L1 gene exhibits a protective effect against the development of degenerative disease such as sporadic Parkinson's disease (PD) in several different ethnic groups, we hypothesized that UCH-L1 gene S18Y polymorphism may have that same effect on the pathologic process of AD. We examined UCH-L1 S18Y polymorphism genotypes of 116 sporadic AD patients and 123 healthy subjects in Chinese Han population using PCR-restriction fragment length polymorphism (RFLP) analysis. The allele and genotype data as well as data after stratification by age of onset failed to demonstrate any association between AD and S18Y polymorphism. However, after stratification by gender, female AD patients showed significantly less frequencies of Y allele and YY genotype in S18Y polymorphism than female controls (P = 0.003 and P = 0.015 respectively). We conclude that Y allele and YY genotype of S18Y in the UCH-L1 gene may have a protective effect against sporadic AD in female subjects, probably due to altering the function of UCH-L1 and the interactions among different risk factors.

    Brain research 2006;1087;1;28-32

  • Evidence for a role of the ubiquitin-proteasome pathway in pancreatic islets.

    López-Avalos MD, Duvivier-Kali VF, Xu G, Bonner-Weir S, Sharma A and Weir GC

    Section on Islet Transplantation and Cell Biology, Joslin Diabetes Center, Harvard Medical School, Boston, MA, USA. lopezavalos@uma.es

    The ubiquitin-proteasome pathway is crucial for protein turnover. Part of the pathway involves deubiquitination, which is carried out by cystein proteases known as ubiquitin COOH-terminal hydrolases. The isoform Uch-L1 was found to be present in large amounts in rat islets by immunostaining, Western blot analysis, and RT-PCR. Culturing islets in high glucose concentrations (16.7 mmol/l) for 24 h led to decreased gene expression. Exposure to chronic hyperglycemia following 90% partial pancreatectomy also led to reduced Uch-L1 expression. Expression of other members of the ubiquitin-proteasome pathway studied after culturing islets at high glucose concentrations revealed little change except for modest declines in parkin, human ubiquitin-conjugating enzyme 5 (UbcH5), and beta-TRCP (transducin repeat-containing protein). With the pancreatectomy model, expression of polyubiquitin-B and c-Cbl were increased and E6-associated protein was reduced. Further insight about the proteasome pathway was obtained with the proteasome inhibitor lactacystin, which in short-term 2-h experiments enhanced glucose-induced insulin secretion. An important role for the ubiquitin-proteasome pathways in beta-cells is suggested by the findings that changes in glucose concentration influence expression of genes in the pathway and that blockade of the proteasome degradation machinery enhances glucose-stimulated insulin secretion.

    Funded by: NIDDK NIH HHS: DK 35449, P30 DK 36836-16

    Diabetes 2006;55;5;1223-31

  • Reduced ubiquitin C-terminal hydrolase-1 expression levels in dementia with Lewy bodies.

    Barrachina M, Castaño E, Dalfó E, Maes T, Buesa C and Ferrer I

    Institut de Neuropatologia, Servei Anatomia Patològica, IDIBELL-Hospital Universitari de Bellvitge, L'Hospitalet de Llobregat, Spain.

    Parkinson disease (PD) and dementia with Lewy bodies (DLB) are characterized by the accumulation of abnormal alpha-synuclein and ubiquitin in protein aggregates conforming Lewy bodies and Lewy neurites. Ubiquitin C-terminal hydrolase-1 (UCHL-1) disassembles polyubiquitin chains to increase the availability of free monomeric ubiquitin to the ubiquitin proteasome system (UPS) thus favoring protein degradation. Since mutations in the UCHL-1 gene, reducing UPS activity by 50%, have been reported in autosomal dominant PD, and UCHL-1 inhibition results in the formation of alpha-synuclein aggregates in mesencephalic cultured neurons, the present study was initiated to test UCHL-1 mRNA and protein levels in post-mortem frontal cortex (area 8) of PD and DLB cases, compared with age-matched controls. TaqMan PCR assays, and Western blots demonstrated down-regulation of UCHL-1 mRNA and UCHL-1 protein in the cerebral cortex in DLB (either in pure forms, not associated with Alzheimer disease: AD, and in common forms, with accompanying AD changes), but not in PD, when compared with age-matched controls. Interestingly, UCHL-1 mRNA and protein expressions were reduced in the medulla oblongata in the same PD cases. Moreover, UCHL-1 protein was decreased in the substantia nigra in cases with Lewy body pathology. UCHL-1 down-regulation was not associated with reduced protein levels of several proteasomal subunits, including 20SX, 20SY, 19S and 11Salpha. Yet UCHL-3 expression was reduced in the cerebral cortex of PD and DLB patients. Together, these observations show reduced UCHL-1 expression as a contributory factor in the abnormal protein aggregation in DLB, and points UCHL-1 as a putative therapeutic target in the treatment of DLB.

    Neurobiology of disease 2006;22;2;265-73

  • Activity profiling of deubiquitinating enzymes in cervical carcinoma biopsies and cell lines.

    Rolén U, Kobzeva V, Gasparjan N, Ovaa H, Winberg G, Kisseljov F and Masucci MG

    Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, Sweden.

    Ubiquitin specific proteases (USPs) regulate the production and recycling of ubiquitin and are thereby critically involved in the control of cell growth, differentiation, and apoptosis. Increasing evidence implicates deregulation of USPs in malignant transformation but there is very little information on the overall and specific activity of USPs in normal and tumor tissues. We have used a chemistry-based functional proteomics approach to profile the activities of individual USPs in biopsies of human papillomavirus (HPV) carrying cervical carcinoma and adjacent normal tissue. To assess the contribution of HPV proteins, USP activity was also compared in HPV positive and negative cervical carcinoma cell lines and HPV E6/E7 immortalized human keratinocytes. The activity of the C-terminal hydrolases UCH-L3 and UCH37 was upregulated in the majority of tumor tissues compared to the adjacent normal tissues. UCH-L1 activity was lower in a significant proportion of the tumors but to a less extent in advanced tumors. In accordance with the relatively low UCH-L1 activity in tumor biopsies, UCH-L1 was detected only in one out of eight cervical carcinoma lines. UCH-L1, UCH-L3, USP7, and USP9X activity was upregulated following HPV E6/E7 immortalization of keratinocytes, suggesting a role of these enzymes in growth transformation.

    Molecular carcinogenesis 2006;45;4;260-9

  • UCHL-1 is not a Parkinson's disease susceptibility gene.

    Healy DG, Abou-Sleiman PM, Casas JP, Ahmadi KR, Lynch T, Gandhi S, Muqit MM, Foltynie T, Barker R, Bhatia KP, Quinn NP, Lees AJ, Gibson JM, Holton JL, Revesz T, Goldstein DB and Wood NW

    Department of Molecular Neuroscience, Institute of Neurology, University College London, London, United Kingdom. danhealy@doctors.org.uk

    Objective: The UCHL-1 gene is widely cited as a susceptibility factor for sporadic Parkinson's disease (PD). The strongest evidence comes from a meta-analysis of small studies that reported the S18Y polymorphism as protective against PD, after pooling studies of white and Asian subjects. Here, we present data that challenge this association.

    Methods: In a new large case-control study in white individuals (3,023 subjects), the S18Y variant was not protective against PD under any genetic model of inheritance. Similarly, a more powerful haplotype-tagging approach did not detect other associated variants.

    Results: Finally, in an updated S18Y-PD meta-analysis (6,594 subjects), no significant association was observed under additive, recessive, or dominant models (odds ratio = 1.00 [95% confidence interval: 0.74-1.33]; odds ratio = 1.01 [95% confidence interval: 0.76-1.35]; and odds ratio = 0.96 [95% confidence interval: 0.86-1.08], respectively), and a cumulative meta-analysis showed a trend toward a null effect.

    Interpretation: Based on the current evidence, the UCHL-1 gene does not exhibit a protective effect in PD.

    Funded by: Parkinson's UK: G-4029

    Annals of neurology 2006;59;4;627-33

  • Structural basis for conformational plasticity of the Parkinson's disease-associated ubiquitin hydrolase UCH-L1.

    Das C, Hoang QQ, Kreinbring CA, Luchansky SJ, Meray RK, Ray SS, Lansbury PT, Ringe D and Petsko GA

    Department of Chemistry and Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454-9110, USA.

    The ubiquitin C-terminal hydrolase UCH-L1 (PGP9.5) comprises >1% of total brain protein but is almost absent from other tissues [Wilkinson, K. D., et al. (1989) Science 246, 670-673]. Mutations in the UCH-L1 gene have been reported to be linked to susceptibility to and protection from Parkinson's disease [Leroy, E., et al. (1998) Nature 395, 451-452; Maraganore, D. M., et al. (1999) Neurology 53, 1858-1860]. Abnormal overexpression of UCH-L1 has been shown to correlate with several forms of cancer [Hibi, K., et al. (1998) Cancer Res. 58, 5690-5694]. Because the amino acid sequence of UCH-L1 is similar to that of other ubiquitin C-terminal hydrolases, including the ubiquitously expressed UCH-L3, which appear to be unconnected to neurodegenerative disease, the structure of UCH-L1 and the effects of disease associated mutations on the structure and function are of considerable importance. We have determined the three-dimensional structure of human UCH-L1 at 2.4-A resolution by x-ray crystallography. The overall fold resembles that of other ubiquitin hydrolases, including UCH-L3, but there are a number of significant differences. In particular, the geometry of the catalytic residues in the active site of UCH-L1 is distorted in such a way that the hydrolytic activity would appear to be impossible without substrate induced conformational rearrangements.

    Funded by: NCI NIH HHS: CO-1020; NIGMS NIH HHS: GM-1104

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;12;4675-80

  • The S18Y polymorphism in the UCHL1 gene is a genetic modifier in Huntington's disease.

    Metzger S, Bauer P, Tomiuk J, Laccone F, Didonato S, Gellera C, Soliveri P, Lange HW, Weirich-Schwaiger H, Wenning GK, Melegh B, Havasi V, Balikó L, Wieczorek S, Arning L, Zaremba J, Sulek A, Hoffman-Zacharska D, Basak AN, Ersoy N, Zidovska J, Kebrdlova V, Pandolfo M, Ribaï P, Kadasi L, Kvasnicova M, Weber BH, Kreuz F, Dose M, Stuhrmann M and Riess O

    Department of Medical Genetics, University of Tübingen, Calwerstrasse 7, 72076 Tübingen, Germany.

    An expanded polyglutamine stretch in the huntingtin protein has been identified as the pathogenetic cause of Huntington's disease (HD). Although the length of the expanded polyglutamine repeat is inversely correlated with the age-at-onset, additional genetic factors are thought to modify the variance in the disease onset. As linkage analysis suggested a modifier locus on chromosome 4p, we investigated the functional relevance of S18Y polymorphism of the ubiquitin carboxy-terminal hydrolase L1 in 946 Caucasian HD patients. In this group, the allelic variation on locus S18Y is responsible for 1.1% of the variance in the HD age-at-onset, and the rare Y allele is associated with younger-aged cases.

    Neurogenetics 2006;7;1;27-30

  • Characterization of multimetric variants of ubiquitin carboxyl-terminal hydrolase L1 in water by small-angle neutron scattering.

    Naito S, Mochizuki H, Yasuda T, Mizuno Y, Furusaka M, Ikeda S, Adachi T, Shimizu HM, Suzuki J, Fujiwara S, Okada T, Nishikawa K, Aoki S and Wada K

    High Energy Accelerator Research Organization, 1-1 Oho, Tsukuba-shi, Ibaraki 305-0801, Japan. sachio@post.kek.jp

    Here, we illustrated that the morphological structures of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) variants and Parkinson's disease (PD) exhibit good pathological correlation by a small-angle neutron scattering (SANS). UCH-L1 is a neuro-specific multiple functional enzyme, deubiquitinating, ubiquityl ligase, and also involved in stabilization of mono-ubiquitin. To examine the relationship between multiple functions of UCH-L1 and the configuration of its variants [wild-type, I93M (linked to familial Parkinson's disease), and S18Y (linked to reduced risk of Parkinson's disease)], in this report, we proposed that these were all self-assembled dimers by an application of a rotating ellipsoidal model; the configurations of these dimers were quite different. The wild-type was a rotating ellipsoidal. The globular form of the monomeric component deformed by the I93M mutation. Conversely, the S18Y polymorphism promoted the globularity. Thus, the multiple functional balance is closely linked to the intermolecular interactions between the UCH-L1 monomer and the final dimeric configuration.

    Biochemical and biophysical research communications 2006;339;2;717-25

  • Ubiquitin C-terminal hydrolase L1 regulates the morphology of neural progenitor cells and modulates their differentiation.

    Sakurai M, Ayukawa K, Setsuie R, Nishikawa K, Hara Y, Ohashi H, Nishimoto M, Abe T, Kudo Y, Sekiguchi M, Sato Y, Aoki S, Noda M and Wada K

    Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, 187-8502, Japan.

    Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a component of the ubiquitin system, which has a fundamental role in regulating various biological activities. However, the functional role of the ubiquitin system in neurogenesis is not known. Here we show that UCH-L1 regulates the morphology of neural progenitor cells (NPCs) and mediates neurogenesis. UCH-L1 was expressed in cultured NPCs as well as in embryonic brain. Its expression pattern in the ventricular zone (VZ) changed between embryonic day (E) 14 and E16, which corresponds to the transition from neurogenesis to gliogenesis. At E14, UCH-L1 was highly expressed in the ventricular zone, where neurogenesis actively occurs; whereas its expression was prominent in the cortical plate at E16. UCH-L1 was very weakly detected in the VZ at E16, which corresponds to the start of gliogenesis. In cultured proliferating NPCs, UCH-L1 was co-expressed with nestin, a marker of undifferentiated cells. In differentiating cells, UCH-L1 was highly co-expressed with the early neuronal marker TuJ1. Furthermore, when UCH-L1 was induced in nestin-positive progenitor cells, the number and length of cellular processes of the progenitors decreased, suggesting that the progenitor cells were differentiating. In addition, NPCs derived from gad (UCH-L1-deficient) mice had longer processes compared with controls. The ability of UCH-L1 to regulate the morphology of nestin-positive progenitors was dependent on its binding affinity for ubiquitin but not on hydrolase activity; this result was also confirmed using gad-mouse-derived NPCs. These results suggest that UCH-L1 spatially mediates and enhances neurogenesis in the embryonic brain by regulating progenitor cell morphology.

    Journal of cell science 2006;119;Pt 1;162-71

  • Overexpression of ubiquitin carboxyl-terminal hydrolase L1 arrests spermatogenesis in transgenic mice.

    Wang YL, Liu W, Sun YJ, Kwon J, Setsuie R, Osaka H, Noda M, Aoki S, Yoshikawa Y and Wada K

    Department of Degenerative Neurological Diseases, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan.

    Ubiquitin carboxyl-terminal hydrolase 1 (UCH-L1) can be detected in mouse testicular germ cells, mainly spermatogonia and somatic Sertoli cells, but its physiological role is unknown. We show that transgenic (Tg) mice overexpressing EF1alpha promoter-driven UCH-L1 in the testis are sterile due to a block during spermatogenesis at an early stage (pachytene) of meiosis. Interestingly, almost all spermatogonia and Sertoli cells expressing excess UCH-L1, but little PCNA (proliferating cell nuclear antigen), showed no morphological signs of apoptosis or TUNEL-positive staining. Rather, germ cell apoptosis was mainly detected in primary spermatocytes having weak or negative UCH-L1 expression but strong PCNA expression. These data suggest that overexpression of UCH-L1 affects spermatogenesis during meiosis and, in particular, induces apoptosis in primary spermatocytes. In addition to results of caspases-3 upregulation and Bcl-2 downregulation, excess UCH-L1 influenced the distribution of PCNA, suggesting a specific role for UCH-L1 in the processes of mitotic proliferation and differentiation of spermatogonial stem cells during spermatogenesis.

    Molecular reproduction and development 2006;73;1;40-9

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

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

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

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

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

    Nature 2005;437;7062;1173-8

  • Proteomics of human umbilical vein endothelial cells applied to etoposide-induced apoptosis.

    Bruneel A, Labas V, Mailloux A, Sharma S, Royer N, Vinh J, Pernet P, Vaubourdolle M and Baudin B

    Service de Biochimie A, Hôpital Saint-Antoine, AP-HP, Paris, France. arnaud.bruneel@sat.ap-hop-paris.fr

    We have undertaken to continue the proteomic study of human umbilical vein endothelial cells (HUVECs) using the combination of 2-DE, automated trypsin digestion, and PMF analysis after MALDI-TOF MS and peptide sequencing using nano LC-ESI-MS/MS. The overall functional characterization of the 162 identified proteins from primary cultures of HUVECs confirms the metabolic capabilities of endothelium and illustrates various cellular functions more related to cell motility and angiogenesis, protein folding, anti-oxidant defenses, signal transduction, proteasome pathway and resistance to apoptosis. In comparison with controls cells, the differential proteomic analysis of HUVECs treated by the pro-apoptotic topoisomerase inhibitor etoposide further revealed the variation of eight proteins, namely, GRP78, GRP94, valosin-containing protein, proteinase inhibitor 9, cofilin, 37-kDa laminin receptor protein, bovine apolipoprotein, and tropomyosin. These data suggest that etoposide-induced apoptosis of human vascular endothelial cells results from the intricate involvement of multiple apoptosis processes including at least the mitochondrial and the ER stress pathways. The presented 2-D pattern and protein database, as well as the data related to apoptosis of HUVECs, are available at http://www.huvec.com.

    Proteomics 2005;5;15;3876-84

  • UCHL-1 gene in multiple system atrophy: a haplotype tagging approach.

    Healy DG, Abou-Sleiman PM, Quinn N, Ahmadi KR, Ozawa T, Kamm C, Wullner U, Oertel WH, Burk K, Dupont E, Pellecchia MT, Tolosa E, Gasser T, Holton JL, Revesz T, Goldstein DB, Lees AJ, Wood NW and European MSA Study Group

    Department of Molecular Neuroscience, Institute of Neurology, Queen Square, London United Kingdom.

    To date, the etiology of multiple system atrophy (MSA) has proved impenetrable. We investigated the role of genetic variation in the UCHL-1 gene in MSA and looked for the presence of disease susceptibility alleles. We determined the linkage disequilibrium structure of the gene and employed a haplotype tagging strategy with power to represent 95% of the haplotype diversity. This approach was performed using a set of tagging single nucleotide polymorphisms (SNPs) that can infer the allelic state of all the common SNPs in UCHL-1 with a high coefficient of determination. This strategy enabled us to scan across the gene and maintain the power to detect signal(s) from any potential functional variant(s). In 257 Gilman-probable or -definite MSA subjects and 1,536 controls, we did not detect a case-control frequency difference for either the tagged haplotypes or for individual tagging SNPs. This search included the S18Y variant of UCHL-1, which has been reported to be protective in Parkinson's disease.

    Funded by: Parkinson's UK: G-4062

    Movement disorders : official journal of the Movement Disorder Society 2005;20;10;1338-43

  • 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

  • UCHL1 is associated with Parkinson's disease: a case-unaffected sibling and case-unrelated control study.

    Facheris M, Strain KJ, Lesnick TG, de Andrade M, Bower JH, Ahlskog JE, Cunningham JM, Lincoln S, Farrer MJ, Rocca WA and Maraganore DM

    Department of Neurology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA.

    To avoid the possible confounding effect of population stratification, we employed a discordant sibling study design and a liberalization of the sibling transmission disequilibrium test to confirm the association of the S18Y variant of the ubiquitin carboxi-terminal hydrolase L1 (UCHL1) gene with Parkinson's disease (PD). The study included 497 case-control pairs (427 case-unaffected sibling pairs and 70 case-unrelated control pairs). Analyses confirmed a significant inverse association of the UCHL1 S18Y polymorphism with PD overall (OR=0.18, 95% CI=0.05-0.64, p=0.002, recessive model) and in several strata.

    Funded by: NIEHS NIH HHS: R01 ES10751; NINDS NIH HHS: P01 NS40256, R01 NS33978

    Neuroscience letters 2005;381;1-2;131-4

  • Stereological quantification of nerve fibers immunoreactive to PGP 9.5, NPY, and VIP in rat prostate during postnatal development.

    Rodríguez R, Pozuelo JM, Martín R, Arriazu R and Santamaria L

    Department of Physiology, Morphology, and Nutritional Sciences, San Pablo University, Madrid, Spain.

    This work was undertaken to study prostate innervation during the postnatal development of rats. It deals with the quantification of nervous fibers throughout all the regions of the rat prostate during the postnatal development using a general marker for nervous tissue, protein gene product 9.5, and 2 neuropeptides (NPY and VIP). Forty male Wistar rats (prepubertals, pubertals, young, and aged adults) were studied for immunohistochemistry of protein gene product (PGP 9.5), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP). They were also evaluated for length density of nerve fibers (L(V) PGP 9.5, L(V) NPY, L(V) VIP). Nerve fibers immunoreactive to the 3 antigens studied were detected in all the groups and in all the prostate zones. Periductal L(V) NPY evidenced a significant increase in the pubertal group, maintained throughout adult life. Periductal L(V) VIP showed a significant increase in young adults. The length densities of VIP and NPY fibers were significantly higher in periductal and ampular locations in comparison with dorsal and ventral sites. It can be concluded that the relative amount of nerve fibers in rat prostate, detected by PGP 9.5, does not change during postnatal development. There were significant changes in NPY and VIP fibers, showing an increase in periurethral ducts at puberty. The abundance of peptidergic innervation around the excretory ducts is related to their contractility. The development of innervation of periurethral ducts is regulated by androgens.

    Journal of andrology 2005;26;2;197-204

  • 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

  • Genetic causes of Parkinson's disease: UCHL-1.

    Healy DG, Abou-Sleiman PM and Wood NW

    Department of Molecular Neuroscience, Institute of Neurology, Queen Square, WC1N 3BG, London, UK. d.healy@ion.ucl.ac.uk

    The ubiquitin proteasome system is an important cellular pathway that ubiquitinates damaged proteins and degrades them via the 26S proteasome. Abnormalities of this pathway can result in molecular protein aggregation and have been associated with Parkinson's disease (PD). UCHL-1, an enzyme central to the system, possesses catalytic hydrolase activity that can hydrolyze peptide-ubiquitin bonds and recycle ubiquitin monomers for re-use in the same process. Recently, UCHL-1 has been shown to possess a second dimerisation-dependent ligase activity and, at least in vitro, this ligase activity promotes alpha synuclein aggregation. UCHL-1 was first implicated in PD by the discovery of an I93M mutation identified in a German sib-pair with probable autosomal dominant PD. Although no further UCHL-1 mutations have been identified, a common non-synonymous S18Y polymorphism has been suggested to reduce disease susceptibility in non-mendelian forms of PD. In vitro functional data support this protective effect, with evidence that S18Y possesses reduced ligase activity compared with wild type UCHL-1. One study has found increased hydrolase activity associated with S18Y, although another study has not. Important issues regarding UCHL-1 and its role in PD remain inconclusive, especially regarding the pathogenicity of the mendelian I93M mutation. This review tries to address some of these uncertainties.

    Cell and tissue research 2004;318;1;189-94

  • Sequence comparison of human and mouse genes reveals a homologous block structure in the promoter regions.

    Suzuki Y, Yamashita R, Shirota M, Sakakibara Y, Chiba J, Mizushima-Sugano J, Nakai K and Sugano S

    Human Genome Center, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, 108-8639, Japan. ysuzuki@ims.u-tokyo.ac.jp

    Comparative sequence analysis was carried out for the regions adjacent to experimentally validated transcriptional start sites (TSSs), using 3324 pairs of human and mouse genes. We aligned the upstream putative promoter sequences over the 1-kb proximal regions and found that the sequence conservation could not be further extended at, on average, 510 bp upstream positions of the TSSs. This discontinuous manner of the sequence conservation revealed a "block" structure in about one-third of the putative promoter regions. Consistently, we also observed that G+C content and CpG frequency were significantly different inside and outside the blocks. Within the blocks, the sequence identity was uniformly 65% regardless of their length. About 90% of the previously characterized transcription factor binding sites were located within those blocks. In 46% of the blocks, the 5' ends were bounded by interspersed repetitive elements, some of which may have nucleated the genomic rearrangements. The length of the blocks was shortest in the promoters of genes encoding transcription factors and of genes whose expression patterns are brain specific, which suggests that the evolutional diversifications in the transcriptional modulations should be the most marked in these populations of genes.

    Genome research 2004;14;9;1711-8

  • Delta12-Prostaglandin J2 inhibits the ubiquitin hydrolase UCH-L1 and elicits ubiquitin-protein aggregation without proteasome inhibition.

    Li Z, Melandri F, Berdo I, Jansen M, Hunter L, Wright S, Valbrun D and Figueiredo-Pereira ME

    Department of Biological Sciences, Hunter College of City University of New York, New York, NY 10021, USA.

    To investigate molecular mechanisms linking inflammation with neurodegeneration, we treated neuronal cultures with prostaglandins (PGs), which are mediators of inflammation. PGA1, D2, J2, and Delta12-PGJ2, but not PGE2, reduced the viability and raised the levels of ubiquitinated proteins in the neuronal cells. PGJ2 and its metabolite, Delta12-PGJ2, were the most potent of the four neurotoxic PGs tested in inducing both effects. To address the mechanism by which these agents lead to the accumulation of ubiquitinated proteins, we tested their effects on neuronal ubiquitin hydrolases UCH-L1 and UCH-L3 as well as on proteasome activity. Notably, Delta12-PGJ2 inhibited the activities of UCH-L1 (K(i) approximately 3.5 microM) and UCH-L3 (K(i) approximately 8.1 microM) without affecting proteasome activity. Intracellular aggregates containing ubiquitinated proteins were detected in Delta12-PGJ2-treated cells, indicating that these aggregates can form independently of proteasome inhibition. In conclusion, impairment of ubiquitin hydrolase activity, such as triggered by Delta12-PGJ2, may be an important contributor to neurodegeneration associated with accumulation of ubiquitinated proteins and inflammation.

    Funded by: NCRR NIH HHS: RR03037; NINDS NIH HHS: NS34018

    Biochemical and biophysical research communications 2004;319;4;1171-80

  • Granular cell tumor: immunohistochemical assessment of inhibin-alpha, protein gene product 9.5, S100 protein, CD68, and Ki-67 proliferative index with clinical correlation.

    Le BH, Boyer PJ, Lewis JE and Kapadia SB

    Department of Pathology, Penn State University College of Medicine, Hershey, Pa, USA. brianle@psu.edu

    Context: Granular cell tumor (GCT) is a rare tumor of nerve sheath origin with a predilection for upper aerodigestive tract, skin, and soft tissue. The neoplastic cells typically express S100 and CD68 (KP-1), the latter due to cytoplasmic lysosome content. However, the histogenesis of this tumor is unknown. Additionally, distinction between benign and malignant GCT is difficult because of histologic similarity and lack of reliable criteria that can predict clinical behavior.

    Objective: To perform a comparative, side-by-side immunohistochemical assessment of the traditional immunohistochemical markers for GCTs (S100, CD68), along with the newer markers (inhibin-alpha, protein gene product 9.5) for these tumors.

    Design: To address diagnostic and prognostic issues, we studied 30 specimens of GCT (27 primary and 3 recurrent tumors, 2 of which occurred consecutively in the same patient) for (1) nuclear pleomorphism, prominent nucleoli, necrosis, spindling, high nuclear-cytoplasmic ratio, and mitoses; (2) immunohistochemical expression of inhibin-alpha, protein gene product 9.5, S100, CD68 (KP-1), and Ki-67 using the avidin-biotin complex method on formalin-fixed, paraffin-embedded sections; and (3) correlation between tumor grade, proliferative fraction, and clinical data.

    Results: Twenty-seven of 27 primary GCTs and 1 of 3 recurrent GCTs had typical histologic features, while the 2 consecutive recurrent GCT specimens from the same patient were atypical (moderate nuclear atypia and prominent nucleoli alone). The mean age for primary GCT was 37.3 years (range, 5-67 years), and mean size was 1.89 cm. None of the cases metastasized. All 30 specimens showed diffuse (2+ to 3+) staining for S100, CD68, and inhibin-alpha, and 3+ staining for protein gene product 9.5; pseudoepitheliomatous hyperplasia was nonreactive. The Ki-67 proliferative index was less than 1% to 20% in typical nonrecurrent cases, 1% in the typical recurrent case, and 1% and 10% in 2 sequential recurrences of the atypical case.

    Conclusion: Our study expands the immunophenotype of GCT (S100, CD68, protein gene product 9.5, and inhibin-alpha) regardless of location and supports a neural origin. Intensity of immunohistochemical staining had no prognostic significance. Although 1 of the 2 recurrent GCTs had atypical features, the Ki-67 proliferative index did not distinguish reliably between typical (nonrecurrent) and atypical or recurrent GCTs. The significance of inhibin expression with regard to cell differentiation and pathogenesis is unclear and warrants further investigation.

    Archives of pathology & laboratory medicine 2004;128;7;771-5

  • UCH-L1 aggresome formation in response to proteasome impairment indicates a role in inclusion formation in Parkinson's disease.

    Ardley HC, Scott GB, Rose SA, Tan NG and Robinson PA

    Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St. James's University Hospital, Leeds, UK. h.c.ardley@leeds.ac.uk

    Aggresomes are associated with many neurodegenerative disorders, including Parkinson's disease, and polyglutamine disorders such as Huntington's disease. These inclusions commonly contain ubiquitylated proteins. The stage at which these proteins are ubiquitylated remains unclear. A malfunction of the ubiquitin/proteasome system (UPS) may be associated with their formation. Conversely, it may reflect an unsuccessful attempt by the cell to remove them. Previously, we demonstrated that overexpression of Parkin, a ubiquitin-protein ligase associated with autosomal recessive juvenile Parkinsonism, generates aggresome-like inclusions in UPS compromised cells. Mutations in the de-ubiquitylating enzyme, UCH-L1, cause a rare form of Parkinsonism. We now demonstrate that overexpression of UCH-L1 also forms ribbon-like aggresomes in response to proteasomal inhibition. Disease-associated mutations, which affect enzymatic activities, significantly increased the number of inclusions. UCH-L1 aggresomes co-localized with ubiquitylated proteins, HSP70, gamma-tubulin and, to a lesser extent, the 20S proteasome and the chaperone BiP. Similar to Parkin inclusions, we found UCH-L1 aggresomes to be surrounded by a tubulin rather than a vimentin cage-like structure. Furthermore, UCH-L1 aggregates with Parkin and alpha-synuclein in some, but not all inclusions, suggesting the heterogeneous nature of these inclusion bodies. This study provides additional evidence that aggregation-prone proteins are likely to recruit UPS components in an attempt to clear proteins from failing proteasomes. Furthermore, UCH-L1 accumulation is likely to play a pathological role in inclusion formation in Parkinson's disease.

    Journal of neurochemistry 2004;90;2;379-91

  • UCHL1 is a Parkinson's disease susceptibility gene.

    Maraganore DM, Lesnick TG, Elbaz A, Chartier-Harlin MC, Gasser T, Krüger R, Hattori N, Mellick GD, Quattrone A, Satoh J, Toda T, Wang J, Ioannidis JP, de Andrade M, Rocca WA, Toda T and UCHL1 Global Genetics Consortium

    Department of Neurology, Mayo Clinic, Rochester, MN, USA. dmaraganore@mayo.edu

    The reported inverse association between the S18Y variant of the ubiquitin carboxy-terminal hydrolase L1 (UCHL1) gene and Parkinson's disease (PD) has strong biological plausibility. If confirmed, genetic association of this variant with PD may support molecular targeting of the UCHL1 gene and its product as a therapeutic strategy for PD. In this light, we performed a collaborative pooled analysis of individual-level data from all 11 published studies of the UCHL1 S18Y gene variant and PD. There were 1,970 cases and 2,224 unrelated controls. We found a statistically significant inverse association of S18Y with PD. Carriers of the variant allele (Y/Y plus Y/S vs S/S) had an odds ratio (OR) of 0.84 (95% confidence interval [CI], 0.73-0.95) and homozygotes for the variant allele (Y/Y vs S/S plus Y/S) had an OR of 0.71 (95% CI, 0.57-0.88). There was a linear trend in the log OR consistent with a gene dose effect (p = 0.01). The inverse association was most apparent for young cases compared with young controls. There was no evidence for publication bias and the associations remained significant after excluding the first published, hypothesis-generating study. These findings confirm that UCHL1 is a susceptibility gene for PD and a potential target for disease-modifying therapies.

    Funded by: NIEHS NIH HHS: ES10751; NINDS NIH HHS: NS33978

    Annals of neurology 2004;55;4;512-21

  • Oxidative modifications and down-regulation of ubiquitin carboxyl-terminal hydrolase L1 associated with idiopathic Parkinson's and Alzheimer's diseases.

    Choi J, Levey AI, Weintraub ST, Rees HD, Gearing M, Chin LS and Li L

    Department of Pharmacology, Center for Neurodegenerative Disease, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA.

    Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative diseases that occur either in relatively rare, familial forms or in common, sporadic forms. The genetic defects underlying several monogenic familial forms of AD and PD have recently been identified, however, the causes of other AD and PD cases, particularly sporadic cases, remain unclear. To gain insights into the pathogenic mechanisms involved in AD and PD, we used a proteomic approach to identify proteins with altered expression levels and/or oxidative modifications in idiopathic AD and PD brains. Here, we report that the protein level of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), a neuronal de-ubiquitinating enzyme whose mutation has been linked to an early-onset familial PD, is down-regulated in idiopathic PD as well as AD brains. By using a combination of two-dimensional gel electrophoresis and mass spectrometry, we have identified three human brain UCH-L1 isoforms, a full-length form and two amino-terminally truncated forms. Our proteomic analyses reveal that the full-length UCH-L1 is a major target of oxidative damage in AD and PD brains, which is extensively modified by carbonyl formation, methionine oxidation, and cysteine oxidation. Furthermore, immunohistochemical studies show that prominent UCH-L1 immunostaining is associated with neurofibrillary tangles and that the level of soluble UCH-L1 protein is inversely proportional to the number of tangles in AD brains. Together, these results provide evidence supporting a direct link between oxidative damage to the neuronal ubiquitination/de-ubiquitination machinery and the pathogenesis of sporadic AD and PD.

    Funded by: NCI NIH HHS: CA 54174; NIA NIH HHS: AG 021489, AG 10130; NIEHS NIH HHS: ES 12068; NINDS NIH HHS: NS 047199

    The Journal of biological chemistry 2004;279;13;13256-64

  • 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

  • Ubiquitin carboxy-terminal hydrolase L1 binds to and stabilizes monoubiquitin in neuron.

    Osaka H, Wang YL, Takada K, Takizawa S, Setsuie R, Li H, Sato Y, Nishikawa K, Sun YJ, Sakurai M, Harada T, Hara Y, Kimura I, Chiba S, Namikawa K, Kiyama H, Noda M, Aoki S and Wada K

    Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, 187-8502, Japan.

    Mammalian neuronal cells abundantly express a deubiquitylating enzyme, ubiquitin carboxy-terminal hydrolase 1 (UCH L1). Mutations in UCH L1 are linked to Parkinson's disease as well as gracile axonal dystrophy (gad) in mice. In contrast to the UCH L3 isozyme that is universally expressed in all tissues, UCH L1 is expressed exclusively in neurons and testis/ovary. We found that UCH L1 associates and colocalizes with monoubiquitin and elongates ubiquitin half-life. The gad mouse, in which the function of UCH L1 is lost, exhibited a reduced level of monoubiquitin in neurons. In contrast, overexpression of UCH L1 caused an increase in the level of ubiquitin in both cultured cells and mice. These data suggest that UCH L1, with avidity and affinity for ubiquitin, insures ubiquitin stability within neurons. This study is the first to show the function of UCH L1 in vivo.

    Human molecular genetics 2003;12;16;1945-58

  • Complex interactions in Parkinson's disease: a two-phased approach.

    Maraganore DM, de Andrade M, Lesnick TG, Farrer MJ, Bower JH, Hardy JA and Rocca WA

    Department of Neurology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA. dmaraganore@mayo.edu

    The identification of pathogenic mutations in the three genes alpha-synuclein, parkin, and ubiquitin carboxy-terminal hydrolase L1 (UCHL1) has elucidated the ubiquitin proteasome system (UPS) and its potential role as a causal pathway in Parkinson's disease (PD). In addition, polymorphisms of these three genes have been shown to be independently associated with PD. In a sample of 298 unrelated PD cases and 185 controls, we applied a two-phased approach of recursive partitioning and logistic regression analyses to explore complex interactions. For women only, we observed an epistatic interaction of UCHL1 and alpha-synuclein genotypes with significant effects on PD risk (odds ratio = 2.42; P = 0.003). Our findings are consistent with the hypothesis that PD is a multigenic disorder of the UPS.

    Funded by: NIEHS NIH HHS: ES10751; NINDS NIH HHS: NS33978, NS40256

    Movement disorders : official journal of the Movement Disorder Society 2003;18;6;631-6

  • Alterations of structure and hydrolase activity of parkinsonism-associated human ubiquitin carboxyl-terminal hydrolase L1 variants.

    Nishikawa K, Li H, Kawamura R, Osaka H, Wang YL, Hara Y, Hirokawa T, Manago Y, Amano T, Noda M, Aoki S and Wada K

    Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, 187-8502, Tokyo, Japan.

    Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a neuron-specific ubiquitin recycling enzyme. A mutation at residue 93 and polymorphism at residue 18 within human UCH-L1 are linked to familial Parkinson's disease and a decreased Parkinson's disease risk, respectively. Thus, we constructed recombinant human UCH-L1 variants and examined their structure (using circular dichroism) and hydrolase activities. We confirmed that an I93M substitution results in a decrease in kcat (45.6%) coincident with an alteration in alpha-helical content. These changes may contribute to the pathogenesis of Parkinson's disease. In contrast, an S18Y substitution results in an increase in kcat (112.6%) without altering the circular dichroistic spectrum. These data suggest that UCH-L1 hydrolase activity may be inversely correlated with Parkinson's disease risk and that the hydrolase activity is protective against the disease. Furthermore, we found that oxidation of UCH-L1 by 4-hydroxynonenal, a candidate for endogenous mediator of oxidative stress-induced neuronal cell death, results in a loss of hydrolase activity. Taken together, these results suggest that further studies of altered UCH-L1 hydrolase function may provide new insights into a possible common pathogenic mechanism between familial and sporadic Parkinson's disease.

    Biochemical and biophysical research communications 2003;304;1;176-83

  • An Ile93Met substitution in the UCH-L1 gene is not a disease-causing mutation for idiopathic Parkinson's disease.

    Shi Q and Tao E

    Department of Internal Medicine, Second Affiliated Hospital, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China.

    Objective: To ascertain whether a coding mutation (Ile93Met) in ubiquitin carboxy-terminal hydrolase (UCH-L1) gene plays a role in idiopathic Parkinson's disease (IPD).

    Methods: Polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP) was used to distinguish the wild-type (two DNA fragments of 34 and 126 bp) from the variant allele (three fragments of 34, 60 and 66 bp) because the mutation created a new site for restriction endonuclease BsmF1. DNA was isolated from various blood samples using a phenolchloroform extraction.

    Results: Ile93Met substitution was found neither in PD patients nor in controls.

    Conclusions: Our study suggested that Ile93Met of UCH-L1 gene did not influence risk of IPD.

    Chinese medical journal 2003;116;2;312-3

  • S18Y polymorphism in the UCH-L1 gene and Parkinson's disease: evidence for an age-dependent relationship.

    Elbaz A, Levecque C, Clavel J, Vidal JS, Richard F, Corrèze JR, Delemotte B, Amouyel P, Alpérovitch A, Chartier-Harlin MC and Tzourio C

    Institut National de la Santé et de la Recherche Médicale, Unit 360, Hôpital de la Salpêtrière, Paris, France. alexis.elbaz@chups.jussieu.fr

    We studied the relationship between Parkinson's disease (PD) and the S18Y polymorphism in the UCH-L1 gene and the effect on this relationship of age at onset, smoking, and pesticides. Patients requested free health coverage for PD to the Mutualité Sociale Agricole (MSA), the French health insurance organization for people whose work is related to agriculture. Controls requested reimbursement of health expenses to the MSA. A maximum of three controls were matched to each case. Analyses included participants with both parents born in Europe. There were no differences in S18Y genotypes between patients (n = 209; 67% SS, 32% SY, 1% YY) and controls (n = 488; 66% SS, 30% SY, 4% YY). The relationship between PD and S18Y was modified by age at onset (P = 0.03). The Y allele was inversely associated with PD for patients with onset before 61 years (odds ratio [OR] = 0.53; 95% confidence interval [CI], 0.29-0.99); there was no association for older patients (62-68 years: OR = 1.21; 95% CI, 0.67-2.20; >68 years: OR = 1.24; 95% CI, 0.67-2.31). Among patients, Y carriers had a later onset than noncarriers (P = 0.04). These findings were not modified or confounded by smoking and pesticides. In this community-based case-control study, carriers of the Y allele were at decreased risk of developing PD at a young age, independently of pesticides and smoking.

    Movement disorders : official journal of the Movement Disorder Society 2003;18;2;130-7

  • The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson's disease susceptibility.

    Liu Y, Fallon L, Lashuel HA, Liu Z and Lansbury PT

    Center for Neurologic Diseases, Brigham and Women's Hospital, Department of Neurology, Harvard Medical School, Cambridge, MA 02139, USA.

    The assumption that each enzyme expresses a single enzymatic activity in vivo is challenged by the linkage of the neuronal enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1) to Parkinson's disease (PD). UCH-L1, especially those variants linked to higher susceptibility to PD, causes the accumulation of alpha-synuclein in cultured cells, an effect that cannot be explained by its recognized hydrolase activity. UCH-L1 is shown here to exhibit a second, dimerization-dependent, ubiquityl ligase activity. A polymorphic variant of UCH-L1 that is associated with decreased PD risk (S18Y) has reduced ligase activity but comparable hydrolase activity as the wild-type enzyme. Thus, the ligase activity as well as the hydrolase activity of UCH-L1 may play a role in proteasomal protein degradation, a critical process for neuronal health.

    Funded by: NINDS NIH HHS: NS38375

    Cell 2002;111;2;209-18

  • Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part I: creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1.

    Castegna A, Aksenov M, Aksenova M, Thongboonkerd V, Klein JB, Pierce WM, Booze R, Markesbery WR and Butterfield DA

    Department of Chemistry, Center of Membrane Sciences, University of Kentucky, Lexington 40506-0055, USA.

    Oxidative alterations of proteins by reactive oxygen species (ROS) have been implicated in the progression of aging and age-related neurodegenerative disorders such as Alzheimer's disease (AD). Protein carbonyls, a marker of protein oxidation, are increased in AD brain, indicating that oxidative modification of proteins is relevant in AD. Oxidative damage can lead to several events such as loss in specific protein function, abnormal protein clearance, depletion of the cellular redox-balance and interference with the cell cycle, and, ultimately, to neuronal death. Identification of specific targets of protein oxidation represents a crucial step in establishing a relationship between oxidative modification and neuronal death in AD, and was partially achieved previously in our laboratory through immunochemical detection of creatine kinase BB and beta-actin as specifically oxidized proteins in AD brain versus control brain. However, this process is laborious, requires the availability of specific antibodies, and, most importantly, requires a reasonable guess as to the identity of the protein in the first place. In this study, we present the first proteomics approach to identify specifically oxidized proteins in AD, by coupling 2D fingerprinting with immunological detection of carbonyls and identification of proteins by mass spectrometry. The powerful techniques, emerging from application of proteomics to neurodegenerative disease, reveal the presence of specific targets of protein oxidation in Alzheimer's disease (AD) brain: creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1. These results are discussed with reference to potential involvement of these oxidatively modified proteins in neurodegeneration in AD brain. Proteomics offers a rapid means of identifying oxidatively modified proteins in aging and age-related neurodegenerative disorders without the limitations of the immunochemical detection method.

    Funded by: NHLBI NIH HHS: R01 HL66358-01; NIA NIH HHS: 5 P30 AG-05144, AG-05119, AG-10836, AG-12423

    Free radical biology & medicine 2002;33;4;562-71

  • Mutation analysis and association studies of the ubiquitin carboxy-terminal hydrolase L1 gene in Huntington's disease.

    Nazé P, Vuillaume I, Destée A, Pasquier F and Sablonnière B

    Laboratoire de Biochimie et de Biologie Moléculaire, Unité Fonctionnelle de Neurobiologie, Hôpital R. Salengro, Centre Hospitalier Régional et Universitaire de Lille, 59037 Lille Cedex, France.

    Huntington's disease (HD) is attributed to a triplet CAG repeat mutation, and about 70% of the variance in age-at-onset can be explained by the size of the repeat expansion. Among potential candidates as modifier genes, we investigated the role of ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) gene. We examined the association of HD with the I93M mutation and S18Y polymorphism in 138 HD patients and 136 control subjects, but we did not identify the I93M mutation. The S18Y polymorphism was present in 17% of HD patients. Of the variance in the age-at-onset that was not accounted for by the CAG repeat, 13% could be attributed to S18Y polymorphism. We sequenced the entire coding region of the UCH-L1 gene in seven HD patients with unexplained older or younger onset age. The S18Y polymorphism was found in three out of the four patients presenting with a later age-at-onset. We conclude that the UCH-L1 gene may be a genetic factor that influences the variability in age-at-onset of HD.

    Neuroscience letters 2002;328;1;1-4

  • ACT and UCH-L1 polymorphisms in Parkinson's disease and age of onset.

    Wang J, Zhao CY, Si YM, Liu ZL, Chen B and Yu L

    Institute of Genetics, School of Life Science, Fudan University, Shanghai, People's Republic of China. wangj@pharm.sunysb.edu

    alpha1-Antichymotrypsin (ACT) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) have been suggested as susceptibility factors for Parkinson's disease (PD). We replicated these findings in a Chinese case-control sample consisting of 160 PD cases and 160 carefully matched control subjects. Genotypes were determined using polymerase chain reaction and BstN1 or Rsa1 restriction enzyme assay. Analysis showed no significant difference between PD patients and controls for genotype or allele frequencies of the ACT and UCH-L1 S18Y polymorphisms. UCH-L1 S18Y polymorphism carriers, however, were found to be significantly less frequent in early-onset PD patients with a reduced risk of 0.557 (95% C.I. = 0.314-0.985; P = 0.043). These data suggest that ACT polymorphism does not influence the risk for developing PD. UCH-L1 S18Y polymorphism, however, may be a weak protective factor against early-onset PD.

    Movement disorders : official journal of the Movement Disorder Society 2002;17;4;767-71

  • The TRC8 hereditary kidney cancer gene suppresses growth and functions with VHL in a common pathway.

    Gemmill RM, Bemis LT, Lee JP, Sozen MA, Baron A, Zeng C, Erickson PF, Hooper JE and Drabkin HA

    Division of Medical Oncology, University of Colorado Health Sciences Center, 4200 E. 9th Avenue, Denver, Colorado, CO 80262, USA. robert.gemmill@uchsc.edu

    VHL is part of an SCF related E3-ubiquitin ligase complex with 'gatekeeper' function in renal carcinoma. However, no mutations have been identified in VHL interacting proteins in wild type VHL tumors. We previously reported that the TRC8 gene was interrupted by a t(3;8) translocation in a family with hereditary renal and non-medullary thyroid cancer. TRC8 encodes a multi-membrane spanning protein containing a RING-H2 finger with in vitro ubiquitin ligase activity. We isolated the Drosophila homologue, DTrc8, and studied its function by genetic manipulations and a yeast 2-hybrid screen. Human and Drosophila TRC8 proteins localize to the endoplasmic reticulum. Loss of either DTrc8 or DVhl resulted in an identical ventral midline defect. Direct interaction between DTrc8 and DVhl was confirmed by GST-pulldown and co-immunoprecipitation experiments. CSN-5/JAB1 is a component of the COP9 signalosome, recently shown to regulate SCF function. We found that DTrc8 physically interacts with CSN-5 and that human JAB1 localization is dependent on VHL mutant status. Lastly, overexpression of DTrc8 inhibited growth consistent with its presumed role as a tumor suppressor gene. Thus, VHL, TRC8, and JAB1 appear to be linked both physically and functionally and all three may participate in the development of kidney cancer.

    Funded by: NCI NIH HHS: CA46934, CA76035

    Oncogene 2002;21;22;3507-16

  • Interaction and colocalization of PGP9.5 with JAB1 and p27(Kip1).

    Caballero OL, Resto V, Patturajan M, Meerzaman D, Guo MZ, Engles J, Yochem R, Ratovitski E, Sidransky D and Jen J

    Department of Otolaryngology-Head and Neck Surgery, Division of Head and Neck Cancer Research, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    PGP9.5 (UCH-L1) is a member of the ubiquitin C-terminal hydrolase (UCH) family of proteins that is expressed in neuronal tissues. Our previous studies have shown that PGP9.5 was highly expressed in primary lung cancers and lung cancer cell lines. Additionally, the frequency of PGP9.5 over expression increases with tumor stage, indicating that PGP9.5 may play a role in lung cancer tumorigenesis. We used the yeast two-hybrid system to identify proteins that interact with PGP9.5. We show that PGP9.5 interacts with at least three proteins, one of which is JAB1, a Jun activation domain binding protein that can bind to p27(Kip1) and is involved in the cytoplasmic transportation of p27(Kip1) for its degradation. We also show that PGP9.5 is associated with JAB1 in vitro and in vivo; and that both proteins can be a part of a heteromeric complex containing p27(Kip1) in the nucleus in lung cancer cells. Furthermore, under serum-restimulation, nuclear translocation of both PGP9.5 and JAB1 coincides with a reduced level of p27(Kip1) in the nucleus. In contrast, when cells are contact inhibited, both PGP9.5 and JAB1 became more perinuclear and cytoplasmic in localization while p27(Kip1) was present only in the nucleus. Therefore, PGP9.5 may contribute to p27(Kip1) degradation via its interaction and nuclear translocation with JAB1.

    Funded by: NCI NIH HHS: CA 58184

    Oncogene 2002;21;19;3003-10

  • Alpha-synuclein has an altered conformation and shows a tight intermolecular interaction with ubiquitin in Lewy bodies.

    Sharma N, McLean PJ, Kawamata H, Irizarry MC and Hyman BT

    Department of Neurology, Massachusetts General Hospital East, Charlestown 02129, USA.

    Alpha-synuclein, a protein in which two mutations have been identified that cause autosomal dominant Parkinson's disease, is thought to serve as a nidus for the development of a Lewy body. We hypothesized that alpha-synuclein would display different intra- and intermolecular associations in Lewy bodies than it does in its normal intracellular compartments. Using sensitive fluorescence resonance energy transfer (FRET) techniques, we found evidence that alpha-synuclein is more compact and in closer association with other alpha-synuclein molecules in Lewy bodies than it is in the neuropil. In addition, we found evidence of a close, direct intermolecular interaction between the N terminus of alpha-synuclein and ubiquitin. These observations provide support for the hypothesis that in Lewy bodies alpha-synuclein adopts an altered three-dimensional structure and undergoes N-terminal ubiquitination.

    Funded by: NIMH NIH HHS: MH/NS 31862

    Acta neuropathologica 2001;102;4;329-34

  • A polymorphic variation of serine to tyrosine at codon 18 in the ubiquitin C-terminal hydrolase-L1 gene is associated with a reduced risk of sporadic Parkinson's disease in a Japanese population.

    Satoh J and Kuroda Y

    Division of Neurology, Department of Internal Medicine, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan. satoj1@post.saga-med.ac.jp

    Recent studies suggest that ubiquitin C-terminal hydrolase-L1 (UCH-L1), a neuronal deubiquitinating enzyme, represents a candidate gene responsible for either the development of familial Parkinson's disease (PD) or the protection against sporadic PD in Caucasian populations, although these findings are not fully verified in non-Caucasian populations. To determine an association of the variations in the UCH-L1 gene with development of sporadic PD in a Japanese population, a Ser18Tyr polymorphism and an Ile93Met mutation were studied by PCR-RFLP analysis in 74 Japanese patients with sporadic PD and 155 age-matched non-PD controls. The frequency of 18Tyr allele was significantly lower in PD patients than the controls (38.5% vs. 53.5%) (chi(2)=9.064, p=0.0026; the odds ratio=1.84, 95% confident interval=1.23-2.74). Furthermore, the frequency of 18Tyr/Tyr homozygotes was significantly lower in PD patients than the controls (14.9% vs. 33.5%), compared with that of two other genotypes combined (chi(2)=8.767, p=0.0031; the odds ratio=0.35, 95% confident interval=0.27-0.45). The Ile93Met substitution was not detected in any Japanese subjects examined. These results indicate that the presence of 18Tyr allele and 18Tyr/Tyr homozygosity in the UCH-L1 gene is associated with a reduced risk for development of sporadic PD in a Japanese population, supporting the previous observations on sporadic PD in Caucasian populations.

    Journal of the neurological sciences 2001;189;1-2;113-7

  • Lack of association between ubiquitin carboxy-terminal hydrolase L1 gene polymorphism and PD.

    Savettieri G, De Marco EV, Civitelli D, Salemi G, Nicoletti G, Annesi G, Cirò Candiano IC and Quattrone A

    Institute of Neurology, University of Palermo, Italy.

    Neurology 2001;57;3;560-1

  • No genetic association of the ubiquitin carboxy-terminal hydrolase-L1 gene S18Y polymorphism with familial Parkinson's disease.

    Levecque C, Destée A, Mouroux V, Becquet E, Defebvre L, Amouyel P and Chartier-Harlin MC

    INSERM 508, Institut Pasteur de Lille, France.

    Parkinson's disease (PD) is a neurodegenerative disorder for which genetic susceptibility has been documented in sporadic and familial cases. Recently, a polymorphism located in exon 3 at codon 18 (S18Y) of the Ubiquitin Carboxy-terminal Hydrolase-L1 (UCH-L1) gene has been associated with the disease in 2 populations of German origin and also in a Japanese population. We tested the impact of this polymorphism in a French sample of familial PD patients (n = 114) and controls (n = 93). No association was observed, indicating that this polymorphism did not confer susceptibility for familial PD in our population, even among the youngest age of onset group. This observation suggests that the previous positive results obtained may reflect mechanisms restricted to the sporadic form of the disease or to a founder effect of the disease susceptibility.

    Journal of neural transmission (Vienna, Austria : 1996) 2001;108;8-9;979-84

  • The ubiquitin carboxy-terminal hydrolase-L1 gene S18Y polymorphism does not confer protection against idiopathic Parkinson's disease.

    Mellick GD and Silburn PA

    Department of Medicine, University of Queensland, Princess Alexandra Hospital, Queensland, 4102, Woolloongabba, Australia. gmellick@medicine.pa.uq.edu.au

    The ubiquitin carboxy-terminal hydrolase L1gene (UCH-L1) has been implicated in the aetiology of Parkinson's disease (PD). A rare Ile93Met mutation in UCH-L1 in a German PD sib-pair has been reported. Recently, a S18Y (C54A) polymorphism in exon 3 of UCH-L1 was found to be under-represented in PD patients compared to controls. To test the reproducibility of this negative association, we conducted an allele-association study of the S18Y polymorphism in an Australian case-control sample consisting of 142 PD cases and 142 closely matched control subjects. Genotypes were determined using polymerase chain reaction and RsaI restriction enzyme assay. Analysis revealed no significant difference between PD patients and controls for genotype or allele frequencies of the S18Y polymorphism. The frequency of the S18Y allele in Australian subjects is similar to that reported elsewhere. This study suggests that the S18Y polymorphism in UCH-L1 does not influence the risk for developing PD.

    Neuroscience letters 2000;293;2;127-30

  • Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice.

    Saigoh K, Wang YL, Suh JG, Yamanishi T, Sakai Y, Kiyosawa H, Harada T, Ichihara N, Wakana S, Kikuchi T and Wada K

    Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, 187-8502, Japan.

    The gracile axonal dystrophy (gad) mouse is an autosomal recessive mutant that shows sensory ataxia at an early stage, followed by motor ataxia at a later stage. Pathologically, the mutant is characterized by 'dying-back' type axonal degeneration and formation of spheroid bodies in nerve terminals. Recent pathological observations have associated brain ageing and neurodegenerative diseases with progressive accumulation of ubiquitinated protein conjugates. In gad mice, accumulation of amyloid beta-protein and ubiquitin-positive deposits occur retrogradely along the sensory and motor nervous systems. We previously reported that the gad mutation was transmitted by a gene on chromosome 5 (refs 10,11). Here we find that the gad mutation is caused by an in-frame deletion including exons 7 and 8 of Uchl1, encoding the ubiquitin carboxy-terminal hydrolase (UCH) isozyme (Uch-l1) selectively expressed in the nervous system and testis. The gad allele encodes a truncated Uch-l1 lacking a segment of 42 amino acids containing a catalytic residue. As Uch-l1 is thought to stimulate protein degradation by generating free monomeric ubiquitin, the gad mutation appears to affect protein turnover. Our data suggest that altered function of the ubiquitin system directly causes neurodegeneration. The gad mouse provides a useful model for investigating human neurodegenerative disorders.

    Nature genetics 1999;23;1;47-51

  • The Ile93Met mutation in the ubiquitin carboxy-terminal-hydrolase-L1 gene is not observed in European cases with familial Parkinson's disease.

    Harhangi BS, Farrer MJ, Lincoln S, Bonifati V, Meco G, De Michele G, Brice A, Dürr A, Martinez M, Gasser T, Bereznai B, Vaughan JR, Wood NW, Hardy J, Oostra BA and Breteler MM

    Department of Epidemiology & Biostatistics, Erasmus University Medical School, Rotterdam, The Netherlands.

    Recently an Ile93Met mutation in the ubiquitin-carboxy-terminal-hydrolase-L1 gene (UCH-L1) has been described in a German family with Parkinson's disease (PD). The authors showed that this mutation is responsible for an impaired proteolytic activity of the UCH-L1 protein and may lead to an abnormal aggregation of proteins in the brain. In order to determine the importance of this or any other mutation in the coding region of the UCH-L1 gene in PD, we performed mutation analysis on Caucasian families with at least two affected sibs. We did not detect any mutations in the UCH-L1 gene, however, we cannot exclude mutations in the regulatory or intronic regions of the UCH-L1 gene since these regions were not sequenced. We conclude that the UCH-L1 gene is not a major gene responsible for familial PD.

    Neuroscience letters 1999;270;1;1-4

  • Low frequency of pathogenic mutations in the ubiquitin carboxy-terminal hydrolase gene in familial Parkinson's disease.

    Lincoln S, Vaughan J, Wood N, Baker M, Adamson J, Gwinn-Hardy K, Lynch T, Hardy J and Farrer M

    Mayo Clinic Jacksonville, FL 32224, USA.

    A coding substitution (I93M) in the ubiquitin carboxy-terminal L1 (UCH-L1) gene has recently been identified in a German family with Parkinson's disease. We have sequenced the entire coding region of the gene in 11 families who have a pattern of disease consistent with autosomal dominant inheritance. We found a polymorphism (S18Y) in exon 3, two polymorphisms in the 5' non-coding region, upstream of the transcription start, and an insertion/deletion polymorphism in intron 4. The S18Y allele is present on approximately 20% of chromosomes in a Caucasian population. These changes are, therefore, unlikely to be pathogenic. We conclude that the I93M variant must either be a rare cause of disease or a harmless substitution whose occurrence in the family reflects a chance co-occurrence.

    Neuroreport 1999;10;2;427-9

  • Intron-exon structure of ubiquitin c-terminal hydrolase-L1.

    Leroy E, Boyer R and Polymeropoulos MH

    Genetic Disease Research Branch, NHGRI, NIH, Bethesda, MD 20892, USA. eleroy@nhgri.nih.gov

    DNA research : an international journal for rapid publication of reports on genes and genomes 1998;5;6;397-400

  • Cleavage of the C-terminus of NEDD8 by UCH-L3.

    Wada H, Kito K, Caskey LS, Yeh ET and Kamitani T

    Division of Molecular Medicine, Department of Internal Medicine, and Research Center for Cardiovascular Diseases, Institute of Molecular Medicine for the Prevention of Human Diseases, The University of Texas- Houston Health Science Center, Houston,

    NEDD8 is a novel ubiquitin-like protein that has been shown to conjugate to nuclear proteins in a manner analogous to ubiquitination and sentrinization. To identify proteins that are involved in the NEDD8-conjugation and de-conjugation pathway, the yeast two-hybrid system was used to screen a human heart cDNA library using NEDD8 as a bait. Seven strongly positive clones were found to contain a cDNA insert encoding the ubiquitin C-terminal hydrolase, UCH-L3. In vitro GST pull-down assay demonstrated that UCH-L3 bound to both NEDD8 and ubiquitin. In contrast, UCH-L3 did not bind to sentrin-1, sentrin-2, or sentrin-3. Recombinant UCH-L3, but not UCH-L1, was able to cleave the C-terminus of NEDD8. Thus, UCH-L3 can function as a C-terminal hydrolase for both NEDD8 and ubiquitin. UCH-L3 may play a physiologically significant role in the cleavage of the C-terminus of NEDD8, which is required for NEDD8 to conjugate to target proteins.

    Funded by: NHLBI NIH HHS: HL-45851

    Biochemical and biophysical research communications 1998;251;3;688-92

  • The ubiquitin pathway in Parkinson's disease.

    Leroy E, Boyer R, Auburger G, Leube B, Ulm G, Mezey E, Harta G, Brownstein MJ, Jonnalagada S, Chernova T, Dehejia A, Lavedan C, Gasser T, Steinbach PJ, Wilkinson KD and Polymeropoulos MH

    Nature 1998;395;6701;451-2

  • Substrate specificity of deubiquitinating enzymes: ubiquitin C-terminal hydrolases.

    Larsen CN, Krantz BA and Wilkinson KD

    Department of Biochemistry, Emory University, Atlanta, Georgia 30322, USA.

    Ubiquitin C-terminal hydrolases (UCH) are deubiquitinating enzymes which hydrolyze C-terminal esters and amides of ubiquitin. Here we report the processing of a number of ubiquitin derivatives by two human UCH isozymes (isozymes L1 and L3) and find that these enzymes show little discrimination based on the P1' amino acid, except that proline is cleaved slowly. Ubiquitinyllysine derivatives linked by the alpha- or epsilon-amino group are hydrolyzed at identical rates. Isozyme-specific hydrolytic preferences are only evident when the leaving group is large. The ubiquitin gene products can be cotranslationally processed by one or both of these UCH isozymes, and purified UbCEP52 can be hydrolyzed by UCH isozyme L3. Binding of nucleic acid by UbCEP52 converts it to a form resistant to processing by these enzymes, apparently because of the formation of a larger, more tightly folded substrate. Consistent with this postulate is the observation that these enzymes do not hydrolyze large ubiquitin derivatives such as N epsilon-ubiquitinyl-cytochrome-c, N epsilon-K48polyubiquitinyl-lysozyme, or an N alpha-ubiquitinyl-beta-galactosidase fusion protein. Thus, these enzymes rapidly and preferentially cleave small leaving groups such as amino acids and oligopeptides from the C-terminus of ubiquitin, but not larger leaving groups such as proteins. These data suggest that the physiological role of UCH is to hydrolyze small adducts of ubiquitin and to generate free monomeric ubiquitin from ubiquitin proproteins, but not to deubiquitinate ubiquitin-protein conjugates or disassemble polyubiquitin chains.

    Funded by: NIGMS NIH HHS: GM30308, R01 GM030308, T32-GM08367

    Biochemistry 1998;37;10;3358-68

  • The immunolocalization of PGP 9.5 in normal human kidney and renal cell carcinoma.

    D'Andrea V, Malinovsky L, Berni A, Biancari F, Biassoni L, Di Matteo FM, Corbellini L, Falvo L, Santoni F, Spyrou M and De Antoni E

    3rd Department of General Surgery, University La Sapienza Rome, Italy.

    The Authors studied the localization of protein gene product (PGP) 9.5-like immunoreactivity in normal human kidney tissue and compared the results with the same immunostaining in renal cell carcinoma. PGP 9.5-like immunoreactivity was found in cells of distal convoluted tubules and in some glomerular capillaries. The cells of proximal convoluted tubules did not show any immunostaining. Sections from renal cell carcinoma showed a very low immunostaining or were negative for PGP 9.5. As PGP 9.5 is a marker of the diffuse endocrine system, the Authors believe that the stained cells of distal tubules should be considered as neuroendocrine cells. The negative reaction to PGP 9.5 antibodies in renal cell carcinoma is rather surprising since not only tumours of neuroectodermal origin, but also tumours of other origin and tissues from some chronic degenerative diseases show a positive reaction. The explication of a negative reaction in renal cell carcinoma remains open: one of the possible explanations could be the specific histogenesis of this tumour.

    Il Giornale di chirurgia 1997;18;10;521-4

  • Substrate binding and catalysis by ubiquitin C-terminal hydrolases: identification of two active site residues.

    Larsen CN, Price JS and Wilkinson KD

    Department of Biochemistry, Emory University, Atlanta, Georgia 30322, USA.

    Ubiquitin C-terminal hydrolases (UCH's) are a newly-defined class of thiol proteases implicated in the proteolytic processing of polymeric ubiquitin. They are important for the generation of monomeric ubiquitin, the active component of the eukaryotic ubiquitin-dependent proteolytic system. There are at least three mammalian isozymes which are tissue specific and developmentally regulated. To study the structure and functional roles of these highly homologous enzymes, we have subcloned and overexpressed two of these isozymes, UCH-L1 and UCH-L3. Here, we report their purification, physical characteristics, and the mutagenesis of UCH-L1. Site-directed mutagenesis of UCH-L1 reveals that C90 and H161 are involved in catalytic rate enhancement. Data from circular dichroic and Raman spectroscopy, as well as secondary structure prediction algorithms, indicate that both isozymes have a significant amount of alpha-helix (> 35%), and contain no disulfide bonds. Both enzymes are reasonably stable, undergoing a reversible thermal denaturation at 52 degrees C. These transitions are characterized by thermodynamic parameters typical of single domain globular proteins. Substrate binding affinity to UCH-L3 was directly measured by equilibrium gel filtration (Kd = 0.5 microM), and the results are similar to the kinetically determined Km for ubiquitin ethyl ester (o.6 microM). The binding is primarily electrostatic in nature and indicates the existence of a specific and extensive binding site for ubiquitin on the surface of the enzyme.

    Funded by: NIGMS NIH HHS: 5-T32-GM08367, GM30308, R01 GM030308

    Biochemistry 1996;35;21;6735-44

  • Localization and characterization of white blood cell populations within the human ovary throughout the menstrual cycle and menopause.

    Best CL, Pudney J, Welch WR, Burger N and Hill JA

    Fearing Research Laboratory, Department of Obstetrics, Gynaecology, and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    The purpose of this investigation was to localize and characterize white blood cell populations in the human ovary through its physiological life cycle. Ovaries from 30 women of reproductive age and from three post-menopausal women were embedded in paraffin or frozen. Clinical information and pathology review were used to obtain accurate menstrual cycle information and to ensure the absence of ovarian disease. Tissue sections were stained for leukocyte phenotypes and the numbers of white blood cells in the ovary were semiquantitatively assessed by two separate examiners using a 0-3 plus (+) scoring system. Our results demonstrated that macrophages and T lymphocytes were the primary immune cells of the ovary, the concentrations of which were dependent on the location and stage of development of the structures containing leukocytes. Developing follicles contained few (+) macrophages located in the theca, while atretic follicles possessed moderate (+2) numbers in the granulosa and few (+) to moderate (+2) numbers in the theca. Newly formed corpora lutea contained few (+) macrophages, while regressing corpora lutea contained abundant (+3) numbers. Human leukocyte antigen (HLA)-DR positive cells were located predominantly at sites where macrophages were present. T lymphocytes were generally not present in the developing follicle but focal, small (+) numbers were observed in blood vessels of the theca. Atretic follicles contained few (+) T lymphocytes in the granulosa and few (+) to moderate (+2) numbers in the theca. Few (+) T lymphocytes were present in new corpora lutea, while moderate (+2) to abundant (+3) numbers were present in regressing corpora lutea. T lymphocytes at all sites were UCHL1 positive. The CD4 (T helper) to CD8 (T suppressor) ration in the corpus luteum was 1:1. B-lymphocytes and natural killer cells were generally absent in the pre-menopausal ovary. The post-menopausal ovary, in contrast, only contained few (+) macrophages, T lymphocytes and natural killer cells in the stroma. In conclusion, our results indicate that the human ovary is an immunologically dynamic tissue containing activated macrophages and T lymphocytes which provide an anatomical basis for immunoendocrine interactions within the ovary.

    Funded by: NICHD NIH HHS: HD00815, HD23547

    Human reproduction (Oxford, England) 1996;11;4;790-7

  • Human TRE17 oncogene is generated from a family of homologous polymorphic sequences by single-base changes.

    Onno M, Nakamura T, Mariage-Samson R, Hillova J and Hill M

    Laboratory of Cellular and Molecular Biology, Centre National de la Recherche Scientifique, Villejuif, France.

    The tre oncogenic locus was identified in transformants receiving human DNA from Ewing's sarcoma cells EW1. Genetic elements of tre originate from chromosomes 5, 18, and 17. The TRE17 oncogene is consistently transcribed in various human cancer cells and proves oncogenic (onc) in expression vector-based assays. Here, the nucleotide sequence of TRE17 with defined noncoding and two coding exons (4,426 nucleotides) was compared with sequences cloned from placental DNA library or generated by polymerase chain reaction (PCR) from EW1 and independent healthy individuals. Cloned sequences displayed restriction site polymorphism, with different patterns for EW1 and normal tissues. Sequence analysis revealed that they originate from a family of homologous sequences alpha, beta, and gamma. TRE17 alpha and less frequent TRE17 beta (similarity score approximately equal to 88%) were found in both normal and EW1 cells. oncTRE17, classified as TRE17 beta, differed from the wild-type TRE17 beta, besides a few intronic changes, by a single-base frameshift insertion in one of the coding exons. TRE17 gamma, so far identified in EW1 but not in normal somatic cells, diverged from oncTRE17 by 6% nucleotide substitutions and by stop codons in each reading frame. The results are consistent with the possibility that TRE17 sequences other than oncTRE17 are translated if alternatively spliced. Expression of TRE17 in normal somatic cells was, however, not yet reported.

    DNA and cell biology 1993;12;2;107-18

  • Microsequences of 145 proteins recorded in the two-dimensional gel protein database of normal human epidermal keratinocytes.

    Rasmussen HH, van Damme J, Puype M, Gesser B, Celis JE and Vandekerckhove J

    Institute of Medical Biochemistry, Aarhus University, Denmark.

    Microsequencing of proteins recovered from two-dimensional (2-D) gels is being used systematically to identify proteins in the master human keratinocyte 2-D gel database. To date, about 250 protein spots recorded in human 2-D gel databases have been microsequenced and, of these, 145 are recorded in the keratinocyte database under the entry partial amino acid sequence. Coomassie Brilliant Blue-stained protein spots cut from several (up to 40) dry gels were concentrated by elution-concentration gel electrophoresis, electroblotted onto PVDF membranes and digested in situ with trypsin. Eluting peptides were separated by reversed-phase HPLC, collected individually and sequenced. Computer search using the FASTA and TFASTA programs from Genetics Computer Group indicated that 110 of the microsequenced polypeptides shared significant similarity with proteins contained in the PIR, Mipsx or GenEMBL databases. Only 35 polypeptides corresponded to hitherto unknown proteins. Peptide sequences of all 145 proteins are listed together with their coordinates (apparent molecular weight and pI) in the keratinocyte database.

    Electrophoresis 1992;13;12;960-9

  • The gene for human neurone specific ubiquitin C-terminal hydrolase (UCHL1, PGP9.5) maps to chromosome 4p14.

    Edwards YH, Fox MF, Povey S, Hinks LJ, Thompson RJ and Day IN

    M.R.C. Human Biochemical Genetics Unit, Galton Laboratory, London.

    Ubiquitin carboxy terminal hydrolase 1, UCHL1, is a neurone-specific protein involved in the ubiquitin-mediated proteolytic pathway. The gene for human UCHL1 has been mapped to chromosome 4 using the polymerase chain reaction to amplify specifically the human UCHL1 sequences in rodent/human somatic cell hybrid DNA. A regional assignment of this locus to 4p14 has been made by in situ hybridization to metaphase chromosomes using both tritium and fluorescently labelled probes.

    Funded by: Wellcome Trust

    Annals of human genetics 1991;55;4;273-8

  • Neuronal protein gene product 9.5 (IEF SSP 6104) is expressed in cultured human MRC-5 fibroblasts of normal origin and is strongly down-regulated in their SV40 transformed counterparts.

    Honoré B, Rasmussen HH, Vandekerckhove J and Celis JE

    Institute of Medical Biochemistry, Aarhus University, Denmark.

    Neuronal protein gene product 9.5 (PGP 9.5) most likely identical to ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1) has been reported to be expressed almost exclusively in neuronal and neuroendocrine tissues. By two-dimensional (2D) immunoblotting, comigration and microsequencing of proteins recovered from 2D gels we have identified PGP 9.5 UCH-L1 as polypeptide IEF SSP 6104 (Mr = 27,000, pI = 5.49) in the comprehensive 2D gel cellular protein database of human embryonal lung MRC-5 fibroblasts [(1989) Electrophoresis 10, 76 115; (1990) Electrophoresis 11, 1072 1113]. This protein is expressed at high levels in quiescent and proliferating cultured normal fibroblasts and is strongly down-regulated (about 10 times) in their transformed counterparts.

    FEBS letters 1991;280;2;235-40

  • The structure of the human gene encoding protein gene product 9.5 (PGP9.5), a neuron-specific ubiquitin C-terminal hydrolase.

    Day IN, Hinks LJ and Thompson RJ

    University Department of Clinical Biochemistry, Southampton General Hospital, U.K.

    Database search using a bovine thymus ubiquitin C-terminal hydrolase sequence indicated 54% sequence identity with the abundant human neuron-specific protein gene product 9.5 (PGP9.5), which was then shown to possess the same activity [Wilkinson, Lee, Deshpande, Duerksen-Hughes, Boss & Pohl (1989) Science 246, 670-673]. A yeast counterpart of the enzyme is also known. The human PGP9.5 gene, described here, spans 10 kb, contains nine exons and displays 5' features some common to many genes and some common with neurofilament neuron-specific enolase and Thy-1-antigen gene 5' regions.

    Funded by: Wellcome Trust

    The Biochemical journal 1990;268;2;521-4

  • Molecular cloning of cDNA coding for human PGP 9.5 protein. A novel cytoplasmic marker for neurones and neuroendocrine cells.

    Day IN and Thompson RJ

    The co-ordinate sequencing of the human neuronal and neuroendocrine marker protein PGP 9.5 and its cDNA is described. The cDNA encodes the complete protein (212 amino acids), and the 340 nucleotide 3'-noncoding region including the polyadenylation signal, indicating an mRNA slightly larger than 1 kb in size. Protein sequencing of 50% of PGP 9.5 confirms the deduced protein sequence.

    FEBS letters 1987;210;2;157-60

  • Isolation of PGP 9.5, a new human neurone-specific protein detected by high-resolution two-dimensional electrophoresis.

    Doran JF, Jackson P, Kynoch PA and Thompson RJ

    Protein gene product (PGP) 9.5 is a new brain-specific protein originally detected by high-resolution two-dimensional electrophoresis of the soluble proteins of human brain and other organs. We have purified this protein from human brain and raised a rabbit antihuman PGP 9.5 antiserum. The protein has a monomer molecular weight of approximately 27,000 and is present in brain at concentrations at least 50 times greater than in other organs. Immunoperoxidase labelling has localised PGP 9.5 to neurones in the human cerebral cortex with no evidence of staining of glial elements. PGP 9.5 is estimated to be present in brain at concentrations of 200-500 micrograms/g wet weight and represents a major protein component of neuronal cytoplasm. This new neurone-specific cytoplasmic marker may prove useful in studies of neuronal development and in the detection of neuronal damage in disease of the nervous system.

    Journal of neurochemistry 1983;40;6;1542-7

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
L00000011 G2C Homo sapiens Human clathrin Human orthologues of mouse clathrin coated vesicle genes adapted from Collins et al (2006) 150
L00000012 G2C Homo sapiens Human Synaptosome Human orthologues of mouse synaptosome adapted from Collins et al (2006) 152
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
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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