Diabetes, antidiabetic medications and risk of dementia: A systematic umbrella review and meta-analysis.

AIMS: The objective of this umbrella review and meta-analysis was to evaluate the effect of diabetes on risk of dementia, as well as the mitigating effect of antidiabetic treatments. MATERIALS AND METHODS: We conducted a systematic umbrella review on diabetes and its treatment, and a meta-analysis focusing on treatment. We searched MEDLINE/PubMed, Embase, PsycINFO, CINAHL and the Cochrane Library for systematic reviews and meta-analyses assessing the risk of cognitive decline/dementia in individuals with diabetes until 2 July 2023. We conducted random-effects meta-analyses to obtain risk ratios and 95% confidence intervals estimating the association of metformin, thiazolidinediones, pioglitazone, dipeptidyl peptidase-4 inhibitors, α-glucosidase inhibitors, meglitinides, insulin, sulphonylureas, glucagon-like peptide-1 receptor agonists (GLP1RAs) and sodium-glucose cotransporter-2 inhibitors (SGLT2is) with risk of dementia from cohort/case-control studies. The subgroups analysed included country and world region. Risk of bias was assessed with the AMSTAR tool and Newcastle-Ottawa Scale. RESULTS: We included 100 reviews and 27 cohort/case-control studies (N = 3 046 661). Metformin, thiazolidinediones, pioglitazone, GLP1RAs and SGLT2is were associated with significant reduction in risk of dementia. When studies examining metformin were divided by country, the only significant effect was for the United States. Moreover, the effect of metformin was significant in Western but not Eastern populations. No significant effect was observed for dipeptidyl peptidase-4 inhibitors, α-glucosidase inhibitors, or insulin, while meglitinides and sulphonylureas were associated with increased risk. CONCLUSIONS: Metformin, thiazolidinediones, pioglitazone, GLP1RAs and SGLT2is were associated with reduced risk of dementia. More longitudinal studies aimed at determining their relative benefit in different populations should be conducted.

USP19 deubiquitinase inactivation regulates α-synuclein ubiquitination and inhibits accumulation of Lewy body-like aggregates in mice.

The USP19 deubiquitinase is found in a locus associated with Parkinson's Disease (PD), interacts with chaperonins, and promotes secretion of α-synuclein (α-syn) through the misfolding-associated protein secretion (MAPS) pathway. Since these processes might modulate the processing of α-syn aggregates in PD, we inactivated USP19 (KO) in mice expressing the A53T mutation of α-syn and in whom α-syn preformed fibrils (PFF) had been injected in the striatum. Compared to WT, KO brains showed decreased accumulation of phospho-synuclein (pSyn) positive aggregates. This improvement was associated with less activation of microglia and improved performance in a tail-suspension test. Exposure of primary neurons from WT and KO mice to PFF in vitro also led to decreased accumulation of pSyn aggregates. KO did not affect uptake of PFF nor propagation of aggregates in the cultured neurons. We conclude that USP19 instead modulates intracellular dynamics of aggregates. At an early time following PFF injection when the number of pSyn-positive neurons were similar in WT and KO brains, the KO neurons contained less aggregates. KO brain aggregates stained more intensely with anti-ubiquitin antibodies. Immunoprecipitation of soluble proteins from WT and KO brains with antibodies to pSyn showed higher levels of ubiquitinated oligomeric species in the KO samples. We propose that the improved pathology in USP19 KO brains may arise from decreased formation or enhanced clearance of the more ubiquitinated aggregates and/or enhanced disassembly towards more soluble oligomeric species. USP19 inhibition may represent a novel therapeutic approach that targets the intracellular dynamics of α-syn complexes.

Association of Low Muscle Mass With Cognitive Function During a 3-Year Follow-up Among Adults Aged 65 to 86 Years in the Canadian Longitudinal Study on Aging.

Importance: Cross-sectional studies have shown that combined low muscle mass and strength are associated with cognitive impairment. Whether low muscle mass, reflective of physiologic reserve, is independently associated with faster cognitive decline remains unknown. Objective: To investigate the associations between low muscle mass and cognitive decline in 3 distinct domains among adults aged at least 65 years. Design, Setting, and Participants: The Canadian Longitudinal Study on Aging is a prospective population-based cohort study of community-dwelling adults. Enrollment occurred from 2011 to 2015 with a 3-year follow-up. Analyses for this study were conducted on those aged at least 65 years from April 24 to August 12, 2020. Exposure: Appendicular lean soft tissue mass (ALM) was assessed by dual energy x-ray absorptiometry. Low ALM was identified using the sex-specific Canadian cut points. Main Outcomes and Measures: Memory was assessed using the Rey auditory verbal learning test. Executive function was assessed using the mental alternation test, Stroop high interference (words/dot) test, the animal fluency test, and the controlled oral word association test. Psychomotor speed was assessed using computer-administered choice reaction time. Composite scores by domain were created. Results: Of 8279 participants, 4003 (48%) were female, 8005 (97%) were White, and the mean (SD) age was 72.9 (5.6) years. A total of 1605 participants (19.4%) had low ALM at baseline. Participants with low ALM were older, had lower body mass index and physical activity level. The presence of low ALM at baseline was associated with faster 3-year cognitive decline in executive functions and psychomotor speed from multiple linear regressions. After adjusting for covariates including age, level of education, percentage body fat, and handgrip strength, low ALM remained independently associated with executive function decline (standardized ß: -0.032; P = .03) only. Low ALM was not associated with memory. Conclusions and Relevance: This cohort study found longitudinal associations between low ALM and cognition in aging. Identification of older adults with low muscle mass, a targetable modifiable factor, may help estimate those at risk for accelerated executive function decline. Further longer-term investigation of associations is warranted.

Interactions of the super complexes: When mTORC1 meets the proteasome.

Homeostatic regulation of energy and metabolic status requires that anabolic and catabolic signaling pathways be precisely regulated and coordinated. Mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is a mega protein complex that promotes energy-consuming anabolic processes of protein and nucleic acid synthesis as well lipogenesis in times of energy and nutrient abundance. However, it is best characterized as the regulator of steps leading to protein synthesis. The ubiquitin-proteasome proteolytic system (UPS) is a major intracellular proteolytic system whose activity is increased during periods of nutrient scarcity and in muscle wasting conditions such as cachexia. Recent studies have examined the impact of mTORC1 on levels and functions of the 26S proteasome, the mega protease complex of the UPS. Here we first briefly review current understanding of the regulation of mTORC1, the UPS, and the 26S proteasome complex. We then review evidence of the effect of each complex on the abundance and functions of the other. Given the fact that drugs that inhibit either complex are either in clinical trials or are approved for treatment of cancer, a muscle wasting condition, we identify studying the effect of combinatory mTORC1-proteasome inhibition on skeletal muscle mass and health as a critical area requiring investigation.

Physical function-derived cut-points for the diagnosis of sarcopenia and dynapenia from the Canadian longitudinal study on aging.

BACKGROUND: Aging is associated with sarcopenia (low muscle mass) and dynapenia (low muscle strength) leading to disability and mortality. Widely used previous cut-points for sarcopenia were established from dated, small, or pooled cohorts. We aimed to identify cut-points of low strength as a determinant of impaired physical performance and cut-points of low appendicular lean mass (ALM) as a predictor of low strength in a single, large, and contemporary cohort of community-dwelling older adults and compare these criteria with others. METHODS: Cross-sectional analyses were conducted on baseline data from 4725 and 4363 community-dwelling men and women (65-86 years, 96.8% Caucasian) of the Canadian longitudinal study on aging comprehensive cohort. Physical performance was evaluated from gait speed, timed up-and-go, chair rise, and balance tests; a weighted-sum score was computed using factor analysis. Strength was measured by handgrip dynamometry; ALM, by dual-energy X-ray absorptiometry and ALM index (ALMI; kg/m2 ), was calculated. Classification and regression tree analyses determined optimal sex-specific cut-points of ALMI predicting low strength and of strength predicting impaired physical performance (score < 1.5 SD below the sex-specific mean). RESULTS: Modest associations were found between ALMI and strength and between strength and physical performance score in both sexes. ALMI was not an independent predictor of physical performance score. Cut-points of <33.1 and <20.4 kg were found to define dynapenia in men and in women, respectively, corresponding to 21.5% and 24.0% prevalence rates. Sarcopenia cut-points were <7.76 kg/m2 in men and <5.72 kg/m2 in women; prevalence rates of 21.7% and 13.7%. Overall, 8.3% of men and 5.5% of women had sarco-dynapenia. Sarcopenic were older and had lower fat mass and body mass index (BMI) than non-sarcopenic participants. While the agreement between current criteria and the updated European Working Group for Sarcopenia in Older Persons recommendations was fair, we found only slight agreement with the Foundation for the National Institute of Health sarcopenia project. Older persons identified with sarcopenia as per the Foundation for the National Institute of Health criteria (using ALM/BMI as the index) have higher BMI and fat mass compared with non-sarcopenic and have normal ALMI as per our criteria. CONCLUSIONS: The proposed function-derived cut-points established from this single, large, and contemporary Canadian cohort should be used for the identification of sarcopenia and dynapenia in Caucasian older adults. We advise on using criteria based on ALMI in the diagnosis of sarcopenia. The modest agreement between sarcopenia and dynapenia denotes potential distinct health implications justifying to study both components separately.

The deubiquitinating enzyme USP19 modulates adipogenesis and potentiates high-fat-diet-induced obesity and glucose intolerance in mice.

AIMS/HYPOTHESIS: Elucidating the molecular mechanisms of fat accumulation and its metabolic consequences is crucial to understanding and treating obesity, an epidemic disease. We have previously observed that Usp19 deubiquitinating enzyme-null mice (Usp19-/-) have significantly lower fat mass than wild-type (WT) mice. Thus, this study aimed to provide further understanding of the role of ubiquitin-specific peptidase 19 (USP19) in fat development, obesity and diabetes. METHODS: In this study, the metabolic phenotypes of WT and Usp19-/- mice were compared. The stromal vascular fractions (SVFs) of inguinal fat pads from WT and Usp19-/- mice were isolated and cells were differentiated into adipocytes in culture to assess their adipogenic capacity. Mice were fed a high-fat diet (HFD) for 18 weeks. Body composition, glucose metabolism and metabolic variables were assessed. In addition, following insulin injection, signalling activity was analysed in the muscle, liver and adipose tissue. Finally, the correlation between the expression of Usp19 mRNA and adipocyte function genes in human adipose tissue was analysed. RESULT: Upon adipogenic differentiation, SVF cells from Usp19-/- failed to accumulate lipid and upregulate adipogenic genes, unlike cells from WT mice. Usp19-/- mice were also found to have smaller fat pads throughout the lifespan and a higher percentage of lean mass, compared with WT mice. When fed an HFD, Usp19-/- mice were more glucose tolerant, pyruvate tolerant and insulin sensitive than WT mice. Moreover, HFD-fed Usp19-/- mice had enhanced insulin signalling in the muscle and the liver, but not in adipose tissue. Finally, USP19 mRNA expression in human adipose tissue was positively correlated with the expression of important adipocyte genes in abdominal fat depots, but not subcutaneous fat depots. CONCLUSIONS/INTERPRETATION: USP19 is an important regulator of fat development. Its inactivation in mice exerts effects on multiple tissues, which may protect against the negative metabolic effects of high-fat feeding. These findings suggest that inhibition of USP19 could have therapeutic potential to protect from the deleterious consequences of obesity and diabetes.

Knockout of USP19 Deubiquitinating Enzyme Prevents Muscle Wasting by Modulating Insulin and Glucocorticoid Signaling.

Muscle atrophy arises because of many chronic illnesses, as well as from prolonged glucocorticoid treatment and nutrient deprivation. We previously demonstrated that the USP19 deubiquitinating enzyme plays an important role in chronic glucocorticoid- and denervation-induced muscle wasting. However, the mechanisms by which USP19 exerts its effects remain unknown. To explore this further, we fasted mice for 48 hours to try to identify early differences in the response of wild-type and USP19 knockout (KO) mice that could yield insights into the mechanisms of USP19 action. USP19 KO mice manifested less myofiber atrophy in response to fasting due to increased rates of protein synthesis. Insulin signaling was enhanced in the KO mice, as revealed by lower circulating insulin levels, increased insulin-stimulated glucose disposal and phosphorylation of Akt and S6K in muscle, and improved overall glucose tolerance. Glucocorticoid signaling, which is essential in many conditions of atrophy, was decreased in KO muscle, as revealed by decreased expression of glucocorticoid receptor (GR) target genes upon both fasting and glucocorticoid treatment. This decreased GR signaling was associated with lower GR protein levels in the USP19 KO muscle. Restoring the GR levels in USP19-deficient muscle was sufficient to abolish the protection from myofiber atrophy. Expression of GR target genes also correlated with that of USP19 in human muscle samples. Thus, USP19 modulates GR levels and in so doing may modulate both insulin and glucocorticoid signaling, two critical pathways that control protein turnover in muscle and overall glucose homeostasis.

The AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), but not metformin, prevents inflammation-associated cachectic muscle wasting.

Activation of AMPK has been associated with pro-atrophic signaling in muscle. However, AMPK also has anti-inflammatory effects, suggesting that in cachexia, a syndrome of inflammatory-driven muscle wasting, AMPK activation could be beneficial. Here we show that the AMPK agonist AICAR suppresses IFNγ/TNFα-induced atrophy, while the mitochondrial inhibitor metformin does not. IFNγ/TNFα impair mitochondrial oxidative respiration in myotubes and promote a metabolic shift to aerobic glycolysis, similarly to metformin. In contrast, AICAR partially restored metabolic function. The effects of AICAR were prevented by the AMPK inhibitor Compound C and were reproduced with A-769662, a specific AMPK activator. AICAR and A-769662 co-treatment was found to be synergistic, suggesting that the anti-cachectic effects of these drugs are mediated through AMPK activation. AICAR spared muscle mass in mouse models of cancer and LPS induced atrophy. Together, our findings suggest a dual function for AMPK during inflammation-driven atrophy, wherein it can play a protective role when activated exogenously early in disease progression, but may contribute to anabolic suppression and atrophy when activated later through mitochondrial dysfunction and subsequent metabolic stress.

FAM210A is a novel determinant of bone and muscle structure and strength.

Osteoporosis and sarcopenia are common comorbid diseases, yet their shared mechanisms are largely unknown. We found that genetic variation near FAM210A was associated, through large genome-wide association studies, with fracture, bone mineral density (BMD), and appendicular and whole body lean mass, in humans. In mice, Fam210a was expressed in muscle mitochondria and cytoplasm, as well as in heart and brain, but not in bone. Grip strength and limb lean mass were reduced in tamoxifen-inducible Fam210a homozygous global knockout mice (TFam210a-/- ), and in tamoxifen-inducible Fam210 skeletal muscle cell-specific knockout mice (TFam210aMus-/- ). Decreased BMD, bone biomechanical strength, and bone formation, and elevated osteoclast activity with microarchitectural deterioration of trabecular and cortical bones, were observed in TFam210a-/- mice. BMD of male TFam210aMus-/- mice was also reduced, and osteoclast numbers and surface in TFam210aMus-/- mice increased. Microarray analysis of muscle cells from TFam210aMus-/- mice identified candidate musculoskeletal modulators. FAM210A, a novel gene, therefore has a crucial role in regulating bone structure and function, and may impact osteoporosis through a biological pathway involving muscle as well as through other mechanisms.

Ubiquitin Ligase Huwe1 Modulates Spermatogenesis by Regulating Spermatogonial Differentiation and Entry into Meiosis.

Spermatogenesis consists of a series of highly regulated processes that include mitotic proliferation, meiosis and cellular remodeling. Although alterations in gene expression are well known to modulate spermatogenesis, posttranscriptional mechanisms are less well defined. The ubiquitin proteasome system plays a significant role in protein turnover and may be involved in these posttranscriptional mechanisms. We previously identified ubiquitin ligase Huwe1 in the testis and showed that it can ubiquitinate histones. Since modulation of histones is important at many steps in spermatogenesis, we performed a complete characterization of the functions of Huwe1 in this process by examining the effects of its inactivation in the differentiating spermatogonia, spermatocytes and spermatids. Inactivation of Huwe1 in differentiating spermatogonia led to their depletion and formation of fewer pre-leptotene spermatocytes. The cell degeneration was associated with an accumulation of DNA damage response protein γH2AX, impaired downstream signalling and apoptosis. Inactivation of Huwe1 in spermatocytes indicated that Huwe1 is not essential for meiosis and spermiogenesis, but can result in accumulation of γH2AX. Collectively, these results provide a comprehensive survey of the functions of Huwe1 in spermatogenesis and reveal Huwe1's critical role as a modulator of the DNA damage response pathway in the earliest steps of spermatogonial differentiation.

Huwe1 Regulates the Establishment and Maintenance of Spermatogonia by Suppressing DNA Damage Response.

Spermatogenesis is sustained by a heterogeneous population of spermatogonia that includes the spermatogonial stem cells. However, the mechanisms underlying their establishment from gonocyte embryonic precursors and their maintenance thereafter remain largely unknown. In this study, we report that inactivation of the ubiquitin ligase Huwe1 in male germ cells in mice led to the degeneration of spermatogonia in neonates and resulted in a Sertoli cell-only phenotype in the adult. Huwe1 knockout gonocytes showed a decrease in mitotic re-entry, which inhibited their transition to spermatogonia. Inactivation of Huwe1 in primary spermatogonial culture or the C18-4 cell line resulted in cell degeneration. Degeneration of Huwe1 knockout spermatogonia was associated with an increased level of histone H2AX and an elevated DNA damage response that led to apparent mitotic catastrophe but not apoptosis or senescence. Blocking this increase in H2AX prevented the degeneration of Huwe1-depleted cells. Taken together, these results reveal a previously undefined role of Huwe1 in orchestrating the physiological DNA damage response in the male germline that contributes to the establishment and maintenance of spermatogonia.

Role of the deubiquitinating enzyme ubiquitin-specific protease-14 in proteostasis in renal cells.

Kidney cell injury may be associated with protein misfolding and induction of endoplasmic reticulum (ER) stress. Examples include complement-induced glomerular epithelial cell (GEC)/podocyte injury in membranous nephropathy and ischemia-reperfusion injury. Renal cell injury can also result from mutations in integral proteins, which lead to their misfolding and accumulation. Certain nephrin missense mutants misfold, accumulate in the ER, and induce ER stress. We examined if enhancement of ubiquitin-proteasome system function may facilitate proteostasis and confer protection against injury. Ubiquitin-specific protease 14 (Usp14) is reported to retard proteasomal protein degradation. Thus inhibition of Usp14 may enhance degradation of misfolded proteins and attenuate cell injury. In GEC, the reporter proteins GFPu (a "misfolded" protein) and CD3δ (an ER-associated degradation substrate) undergo time-dependent proteasomal degradation. Complement did not affect degradation of CD3δ-yellow fluorescent protein (YFP), but accelerated degradation of GFPu, and the Usp14-directed inhibitor IU1 further accelerated this degradation. Conversely, overexpression of Usp14 reduced degradation of GFPu and CD3δ-YFP. In 293T cells, IU1 did not enhance degradation of disease-associated nephrin missense mutants I171N and S724C, whereas overexpression of Usp14 reduced degradation. IU1 was cytoprotective after injury induced by the ER stressor tunicamycin and in vitro ischemia-reperfusion, but did not affect complement-induced cytotoxicity. In conclusion, Usp14 controls proteasomal degradation of some misfolded proteins. In addition, a Usp14-directed inhibitor reduces cytotoxicity in the context of global protein misfolding during certain types of renal cell injury.

Deubiquitinating enzymes in skeletal muscle atrophy-An essential role for USP19.

The ubiquitin proteasome system is well recognized to be involved in mediating muscle atrophy in response to diverse catabolic conditions. To date, almost all of the genes that have been implicated are ubiquitin ligases. Although ubiquitination is modulated also by deubiquitinating enzymes, the roles of these enzymes in muscle wasting remains largely unexplored. In this article, the potential roles of deubiquitinating enzymes in regulating muscle size are discussed. This is followed by a review of the roles described for USP19, the deubiquitinating enzyme that has been most studied in muscle wasting. This enzyme is upregulated in muscle in many catabolic conditions and its inactivation leads to protection from muscle loss induced by stimuli that are common in many illnesses causing cachexia. It can regulate both protein synthesis and protein degradation as well as myogenesis, thereby modulating the key processes that control muscle mass. Roles for other deubiquitinating enzymes remain possible and to be explored.

The ubiquitin proteasome system in atrophying skeletal muscle: roles and regulation.

Muscle atrophy complicates many diseases as well as aging, and its presence predicts both decreased quality of life and survival. Much work has been conducted to define the molecular mechanisms involved in maintaining protein homeostasis in muscle. To date, the ubiquitin proteasome system (UPS) has been shown to play an important role in mediating muscle wasting. In this review, we have collated the enzymes in the UPS whose roles in muscle wasting have been confirmed through loss-of-function studies. We have integrated information on their mechanisms of action to create a model of how they work together to produce muscle atrophy. These enzymes are involved in promoting myofibrillar disassembly and degradation, activation of autophagy, inhibition of myogenesis as well as in modulating the signaling pathways that control these processes. Many anabolic and catabolic signaling pathways are involved in regulating these UPS genes, but none appear to coordinately regulate a large number of these genes. A number of catabolic signaling pathways appear to instead function by inhibition of the insulin/IGF-I/protein kinase B anabolic pathway. This pathway is a critical determinant of muscle mass, since it can suppress key ubiquitin ligases and autophagy, activate protein synthesis, and promote myogenesis through its downstream mediators such as forkhead box O, mammalian target of rapamycin, and GSK3ß, respectively. Although much progress has been made, a more complete inventory of the UPS genes involved in mediating muscle atrophy, their mechanisms of action, and their regulation will be useful for identifying novel therapeutic approaches to this important clinical problem.

The business of deubiquitination - location, location, location.

A majority of proteins in the cell can be modified by ubiquitination, thereby altering their function or stability. This ubiquitination is controlled by both ubiquitinating and deubiquitinating enzymes (DUBs). The number of ubiquitin ligases exceeds that of DUBs by about eightfold, indicating that DUBs may have much broader substrate specificity. Despite this, DUBs have been shown to have quite specific physiological functions. This functional specificity is likely due to very precise regulation of activity arising from the sophisticated use of all mechanisms of enzyme regulation. In this commentary, we briefly review key features of DUBs with more emphasis on regulation. In particular, we focus on localization of the enzymes as a critical regulatory mechanism which when integrated with control of expression, substrate activation, allosteric regulation, and post-translational modifications results in precise spatial and temporal deubiquitination of proteins and therefore specific physiological functions. Identification of compounds that target the structural elements in DUBs that dictate localization may be a more promising approach to development of drugs with specificity of action than targeting the enzymatic activity, which for most DUBs is dependent on a thiol group that can react non-specifically with many compounds in large-scale screening.

Inactivation of the ubiquitin-specific protease 19 deubiquitinating enzyme protects against muscle wasting.

The ubiquitin system plays a critical role in muscle wasting. Previous work has focused on the roles of ubiquitination. However, a role for deubiquitination in this process has not been established. Because ubiquitin-specific protease (USP)19 deubiquitinating enzyme is induced in skeletal muscle in many catabolic conditions, we generated USP19 knockout (KO) mice. These mice lost less muscle mass than wild-type (WT) animals in response to glucocorticoids, a common systemic cause of muscle atrophy as well as in response to denervation, a model of disuse atrophy. KO mice retained more strength and had less myofiber atrophy with both type I and type IIb fibers being protected. Rates of muscle protein synthesis were similar in WT and KO mice, suggesting that the sparing of atrophy was attributed to suppressed protein degradation. Consistent with this, expression of the ubiquitin ligases MuRF1 and MAFbx/atrogin-1 as well as several autophagy genes was decreased in the muscles of catabolic KO mice. Expression of USP19 correlates with that of MuRF1 and MAFbx/atrogin-1 in skeletal muscles from patients with lung cancer or gastrointestinal cancer, suggesting that USP19 is involved in human muscle wasting. Inhibition of USP19 may be a useful approach to the treatment of many muscle-wasting conditions.

USP19 deubiquitinating enzyme inhibits muscle cell differentiation by suppressing unfolded-protein response signaling.

The USP19 deubiquitinating enzyme modulates the expression of myogenin and myofibrillar proteins in L6 muscle cells. This raised the possibility that USP19 might regulate muscle cell differentiation. We therefore tested the effects of adenoviral-mediated overexpression or small interfering RNA (siRNA)-mediated silencing of either the cytoplasmic or endoplasmic reticulum (ER)-localized isoforms of USP19. Only the ER-localized isoform of USP19 (USP19-ER) modulated myoblast fusion as well as the expression of myogenin and myofibrillar proteins, and these effects were also dependent on USP19 catalytic activity. USP19-ER inhibited muscle cell differentiation and the induction of CHOP, a transcription factor in the unfolded-protein response (UPR) that is activated during differentiation. Inducing the UPR by creating mild ER stress with thapsigargin was able to reverse the defect in myoblast fusion caused by the overexpression of USP19-ER, suggesting strongly that USP19 exerts its effects on fusion through its effects on UPR signaling. USP19 also functions similarly in vivo, as USP19(-/-) mice display improved muscle regeneration concomitant with enhanced expression of CHOP. Collectively these results implicate a deubiquitinating enzyme as a regulator of the UPR. They also suggest that inhibition of USP19 may be a therapeutic approach for the enhancement of muscle growth following injury.

USP2 regulates the intracellular localization of PER1 and circadian gene expression.

Endogenous 24-h rhythms in physiology are driven by a network of circadian clocks located in most tissues. The molecular clock mechanism is based on feedback loops involving clock genes and their protein products. Posttranslational modifications, including ubiquitination, are important for regulating the clock feedback mechanism. Recently, we showed that the deubiquitinating enzyme ubiquitin-specific peptidase 2 (USP2) associates with clock proteins and deubiquitinates PERIOD1 (PER1) but does not affect its overall stability. Mice devoid of USP2 display defects in clock function. Here, we show that USP2 regulates nucleocytoplasmic shuttling and nuclear retention of PER1 and its repressive role on the clock transcription factors CLOCK and BMAL1. The rhythm of nuclear entry of PER1 in Usp2 knockout mouse embryonic fibroblasts (MEFs) was advanced but with reduced nuclear accumulation of PER1. Although Per1 mRNA expression rhythm remained intact in the Usp2 KO MEFs, the expression profiles of other core clock genes were altered. This was also true for the expression of clock-controlled genes (e.g., Dbp, Tef, Hlf, E4bp4). A similar phase advance of PER1 nuclear localization rhythm and alteration of clock gene expression profiles were also observed in livers of Usp2 KO mice. Taken together, our results demonstrate a novel function of USP2 in the molecular clock in which it regulates PER1 function by gating its nuclear entry and accumulation.

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.