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1.  A central role for ubiquitination within a circadian clock protein modification code 
Circadian rhythms, endogenous cycles of about 24 h in physiology, are generated by a master clock located in the suprachiasmatic nucleus of the hypothalamus and other clocks located in the brain and peripheral tissues. Circadian disruption is known to increase the incidence of various illnesses, such as mental disorders, metabolic syndrome, and cancer. At the molecular level, periodicity is established by a set of clock genes via autoregulatory translation–transcription feedback loops. This clock mechanism is regulated by post-translational modifications such as phosphorylation and ubiquitination, which set the pace of the clock. Ubiquitination in particular has been found to regulate the stability of core clock components but also other clock protein functions. Mutation of genes encoding ubiquitin ligases can cause either elongation or shortening of the endogenous circadian period. Recent research has also started to uncover roles for deubiquitination in the molecular clockwork. Here, we review the role of the ubiquitin pathway in regulating the circadian clock and we propose that ubiquitination is a key element in a clock protein modification code that orchestrates clock mechanisms and circadian behavior over the daily cycle.
doi:10.3389/fnmol.2014.00069
PMCID: PMC4124793  PMID: 25147498
circadian clock; clock gene; ubiquitin; ubiquitin ligase; deubiquitinase; stability
2.  Regulation of behavioral circadian rhythms and clock protein PER1 by the deubiquitinating enzyme USP2 
Biology Open  2012;1(8):789-801.
Summary
Endogenous 24-hour rhythms are generated by circadian clocks located in most tissues. The molecular clock mechanism is based on feedback loops involving clock genes and their protein products. Post-translational modifications, including ubiquitination, are important for regulating the clock feedback mechanism. Previous work has focused on the role of ubiquitin ligases in the clock mechanism. Here we show a role for the rhythmically-expressed deubiquitinating enzyme ubiquitin specific peptidase 2 (USP2) in clock function. Mice with a deletion of the Usp2 gene (Usp2 KO) display a longer free-running period of locomotor activity rhythms and altered responses of the clock to light. This was associated with altered expression of clock genes in synchronized Usp2 KO mouse embryonic fibroblasts and increased levels of clock protein PERIOD1 (PER1). USP2 can be coimmunoprecipitated with several clock proteins but directly interacts specifically with PER1 and deubiquitinates it. Interestingly, this deubiquitination does not alter PER1 stability. Taken together, our results identify USP2 as a new core component of the clock machinery and demonstrate a role for deubiquitination in the regulation of the circadian clock, both at the level of the core pacemaker and its response to external cues.
doi:10.1242/bio.20121990
PMCID: PMC3507220  PMID: 23213472
Circadian clock; Locomotor activity rhythms; PER1; Ubiquitin; USP2
3.  Identification of Distinctive Patterns of USP19-Mediated Growth Regulation in Normal and Malignant Cells 
PLoS ONE  2011;6(1):e15936.
We previously reported that the USP19 deubiquitinating enzyme positively regulates proliferation in fibroblasts by stabilizing KPC1, a ubiquitin ligase for p27Kip1. To explore whether this role of USP19 extends to other cellular systems, we tested the effects of silencing of USP19 in several human prostate and breast models, including carcinoma cell lines. Depletion of USP19 inhibited proliferation in prostate cancer DU145, PC-3 and 22RV1 cells, which was similar to the pattern established in fibroblasts in that it was due to decreased progression from G1 to S phase and associated with a stabilization of the cyclin-dependent kinase inhibitor p27Kip1. However, in contrast to previous findings in fibroblasts, the stabilization of p27Kip1 upon USP19 depletion was not associated with changes in the levels of the KPC1 ligase. USP19 could also regulate the growth of immortalized MCF10A breast epithelial cells through a similar mechanism. This regulatory pattern was lost, though, in breast cancer MCF7 and MDA-MB-231 cells and in prostate carcinoma LNCaP cells. Of interest, the transformation of fibroblasts through overexpression of an oncogenic form of Ras disrupted the USP19-mediated regulation of cell growth and of levels of p27Kip1 and KPC1. Thus, the cell context appears determinant for the ability of USP19 to regulate cell proliferation and p27Kip1 levels. This may occur through both KPC1 dependent and independent mechanisms. Moreover, a complete loss of USP19 function on cell growth may arise as a result of oncogenic transformation of cells.
doi:10.1371/journal.pone.0015936
PMCID: PMC3022023  PMID: 21264218
5.  USP19 Deubiquitinating Enzyme Supports Cell Proliferation by Stabilizing KPC1, a Ubiquitin Ligase for p27Kip1▿  
Molecular and Cellular Biology  2008;29(2):547-558.
p27Kip1 is a cyclin-dependent kinase inhibitor that regulates the G1/S transition. Increased degradation of p27Kip1 is associated with cellular transformation. Previous work demonstrated that the ubiquitin ligases KPC1/KPC2 and SCFSkp2 ubiquitinate p27Kip1 in G1 and early S, respectively. The regulation of these ligases remains unclear. We report here that the USP19 deubiquitinating enzyme interacts with and stabilizes KPC1, thereby modulating p27Kip1 levels and cell proliferation. Cells depleted of USP19 by RNA interference exhibited an inhibition of cell proliferation, progressing more slowly from G0/G1 to S phase, and accumulated p27Kip1. This increase in p27Kip1 was associated with normal levels of Skp2 but reduced levels of KPC1. The overexpression of KPC1 or the use of p27−/− cells inhibited significantly the growth defect observed upon USP19 depletion. KPC1 was ubiquitinated in vivo and stabilized by proteasome inhibitors and by overexpression of USP19, and it also coimmunoprecipitated with USP19. Our results identify USP19 as the first deubiquitinating enzyme that regulates the stability of a cyclin-dependent kinase inhibitor and demonstrate that progression through G1 to S phase is, like the metaphase-anaphase transition, controlled in a hierarchical, multilayered fashion.
doi:10.1128/MCB.00329-08
PMCID: PMC2612522  PMID: 19015242
6.  The UPS in diabetes and obesity 
BMC Biochemistry  2008;9(Suppl 1):S6.
Type 2 diabetes is caused by defects in both insulin signaling and insulin secretion. Though the role of the ubiquitin proteasome system (UPS) in the pathogenesis of type 2 diabetes remains largely unexplored, the few examples present in the literature are interesting and suggest targets for drug development. Studies indicate that insulin resistance can be induced by stimulating the degradation of important molecules in the insulin signaling pathway, in particular the insulin receptor substrate proteins IRS1, IRS2 and the kinase AKT1 (Akt). In addition, a defect in insulin secretion could occur due to UPS-mediated degradation of IRS2 in the β-cells of the pancreas. The UPS also appears to be involved in regulating lipid synthesis in adipocytes and lipid production by the liver and could influence the development of obesity. Other possible mechanisms for inducing defects in insulin signaling and secretion remain to be explored, including the role of ubiquitylation in insulin receptor internalization and trafficking.
Republished from Current BioData's Targeted Proteins database (TPdb; ).
doi:10.1186/1471-2091-9-S1-S6
PMCID: PMC2582800  PMID: 19007436
8.  Mice Lacking the UBC4-testis Gene Have a Delay in Postnatal Testis Development but Normal Spermatogenesis and Fertility 
Molecular and Cellular Biology  2005;25(15):6346-6354.
Activation of ubiquitination occurs during spermatogenesis and is dependent on the induction of isoforms of the UBC4 family of ubiquitin-conjugating enzymes. The UBC4-testis isoform is testis specific, is induced in round spermatids, and demonstrates biochemical functions distinct from a ubiquitously expressed isoform UBC4-1. To explore further the function of UBC4-testis, mice bearing inactivation of this gene were produced. Homozygous (−/−) mice showed normal body growth and fertility. Although testis weight and morphology were normal in testes from adult mice, examination of young mice during the first wave of spermatogenesis revealed that testes were ∼10% smaller in weight at 40 and 45 days of age but had become normal at 65 days of age. Overall protein content, levels of ubiquitinated proteins, and ubiquitin-conjugating activity did not differ between wild-type and homozygous (−/−) mice. Spermatid number, as well as the motility of spermatozoa isolated from the epididymis, was also normal in homozygous (−/−) mice. To determine whether the germ cells lacking UBC4-testis might be more sensitive to stress, testes from wild-type and knockout mice were exposed to heat stress by implantation in the abdominal cavity. Testes from both strains of mice showed similar rates of degeneration in response to heat. The lack of an obvious phenotype did not appear to be due to induction of other UBC4 isoforms, as shown by two-dimensional gel immunoblotting. Our data indicate that UBC4-testis plays a role in early maturation of the testis and suggest that the many UBC4 isoforms have mixed redundant and specific functions.
doi:10.1128/MCB.25.15.6346-6354.2005
PMCID: PMC1190331  PMID: 16024774
9.  Characterization of E3Histone, a Novel Testis Ubiquitin Protein Ligase Which Ubiquitinates Histones 
Molecular and Cellular Biology  2005;25(7):2819-2831.
During spermatogenesis, a large fraction of cellular proteins is degraded as the spermatids evolve to their elongated mature forms. In particular, histones must be degraded in early elongating spermatids to permit chromatin condensation. Our laboratory previously demonstrated the activation of ubiquitin conjugation during spermatogenesis. This activation is dependent on the ubiquitin-conjugating enzyme (E2) UBC4, and a testis-particular isoform, UBC4-testis, is induced when histones are degraded. Therefore, we tested whether there are UBC4-dependent ubiquitin protein ligases (E3s) that can ubiquitinate histones. Indeed, a novel enzyme, E3Histone, which could conjugate ubiquitin to histones H1, H2A, H2B, H3, and H4 in vitro, was found. Only the UBC4/UBC5 family of E2s supported E3Histone-dependent ubiquitination of histone H2A, and of this family, UBC4-1 and UBC4-testis are the preferred E2s. We purified this ligase activity 3,600-fold to near homogeneity. Mass spectrometry of the final material revealed the presence of a 482-kDa HECT domain-containing protein, which was previously named LASU1. Anti-LASU1 antibodies immunodepleted E3Histone activity. Mass spectrometry and size analysis by gel filtration and glycerol gradient centrifugation suggested that E3Histone is a monomer of LASU1. Our assays also show that this enzyme is the major UBC4-1-dependent histone-ubiquitinating E3. E3Histone is therefore a HECT domain E3 that likely plays an important role in the chromatin condensation that occurs during spermatid maturation.
doi:10.1128/MCB.25.7.2819-2831.2005
PMCID: PMC1061639  PMID: 15767685
10.  S-Nitrosylation of IRP2 Regulates Its Stability via the Ubiquitin-Proteasome Pathway 
Molecular and Cellular Biology  2004;24(1):330-337.
Nitric oxide (NO) is an important signaling molecule that interacts with different targets depending on its redox state. NO can interact with thiol groups resulting in S-nitrosylation of proteins, but the functional implications of this modification are not yet fully understood. We have reported that treatment of RAW 264.7 cells with NO caused a decrease in levels of iron regulatory protein 2 (IRP2), which binds to iron-responsive elements present in untranslated regions of mRNAs for several proteins involved in iron metabolism. In this study, we show that NO causes S-nitrosylation of IRP2, both in vitro and in vivo, and this modification leads to IRP2 ubiquitination followed by its degradation in the proteasome. Moreover, mutation of one cysteine (C178S) prevents NO-mediated degradation of IRP2. Hence, S-nitrosylation is a novel signal for IRP2 degradation via the ubiquitin-proteasome pathway.
doi:10.1128/MCB.24.1.330-337.2004
PMCID: PMC303342  PMID: 14673166
12.  Divergent N-Terminal Sequences Target an Inducible Testis Deubiquitinating Enzyme to Distinct Subcellular Structures 
Molecular and Cellular Biology  2000;20(17):6568-6578.
Ubiquitin-specific processing proteases (UBPs) presently form the largest enzyme family in the ubiquitin system, characterized by a core region containing conserved motifs surrounded by divergent sequences, most commonly at the N-terminal end. The functions of these divergent sequences remain unclear. We identified two isoforms of a novel testis-specific UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini, thereby permitting dissection of the functions of these two regions. Both isoforms were germ cell specific and developmentally regulated. Immunocytochemistry revealed that UBP-t1 was induced in step 16 to 19 spermatids while UBP-t2 was expressed in step 18 to 19 spermatids. Immunoelectron microscopy showed that UBP-t1 was found in the nucleus while UBP-t2 was extranuclear and was found in residual bodies. For the first time, we show that the differential subcellular localization was due to the distinct N-terminal sequences. When transfected into COS-7 cells, the core region was expressed throughout the cell but the UBP-t1 and UBP-t2 isoforms were concentrated in the nucleus and the perinuclear region, respectively. Fusions of each N-terminal end with green fluorescent protein yielded the same subcellular localization as the native proteins, indicating that the N-terminal ends were sufficient for determining differential localization. Interestingly, UBP-t2 colocalized with anti-γ-tubulin immunoreactivity, indicating that like several other components of the ubiquitin system, a deubiquitinating enzyme is associated with the centrosome. Regulated expression and alternative N termini can confer specificity of UBP function by restricting its temporal and spatial loci of action.
PMCID: PMC86134  PMID: 10938131

Results 1-12 (12)