USP4 inhibits p53 through deubiquitinating and stabilizing ARF-BP1

Tumour suppressor p53 levels in the cell are tightly regulated by controlled degradation through ubiquitin ligases including Mdm2, COP1, Pirh2, and ARF-BP1. The ubiquitination process is reversible via deubiquitinating enzymes, such as ubiquitin-specific peptidases (USPs). In this study, we identified ubiquitin-specific peptidase 4 (USP4) as an important regulator of p53. USP4 interacts directly with and deubiquitinates ARF-BP1, leading to the stabilization of ARF-BP1 and subsequent reduction of p53 levels. Usp4 knockout mice are viable and developmentally normal, but showed enhanced apoptosis in thymus and spleen in response to ionizing radiation. Compared with wild-type mouse embryonic fibroblasts (MEFs), Usp4-/- MEFs exhibited retarded growth, premature cellular senescence, resistance to oncogenic transformation, and hyperactive DNA damage checkpoints, consistent with upregulated levels and activity of p53 in the absence of USP4. Finally, we showed that USP4 is overexpressed in several types of human cancer, suggesting that USP4 is a potential oncogene.     

USP22在转录调控及肿瘤发生中的作用

泛素特异性蛋白酶22（ubiquitin specific protease 22,USP22）是一种组蛋白去泛素化酶,在核受体介导的基因转录调控中起增强转录活性的作用。USP22在肿瘤发生、发展中发挥着重要作用,影响癌基因c-Myc介导的基因转录,从而促进肿瘤的增殖和生长;另一方面,USP22可改变远上游识别序列结合蛋白1（far upstream element—binding protein1,FBP1）的泛素化水平,从而影响FBP1下游的抑癌基因p21的表达。USP22与胃癌、大肠癌、乳腺癌及膀胱癌等肿瘤的病理进程相关。此外,USP22在端粒稳态的维持中起作用。本文主要综述USP22参与核受体介导的基因转录调控的表观遗传学机制及其在肿瘤发生中的作用。     

USP7 limits CDK1 activity throughout the cell cycle

Chemical inhibitors of the deubiquitinase USP7 are currently being developed as anticancer agents based on their capacity to stabilize P53. Regardless of this activity, USP7 inhibitors also generate DNA damage in a p53‐independent manner. However, the mechanism of this genotoxicity and its contribution to the anticancer effects of USP7 inhibitors are still under debate. Here we show that, surprisingly, even if USP7 inhibitors stop DNA replication, they also induce a widespread activation of CDK1 throughout the cell cycle, which leads to DNA damage and is toxic for mammalian cells. In addition, USP7 interacts with the phosphatase PP2A and supports its active localization in the cytoplasm. Accordingly, inhibition of USP7 or PP2A triggers very similar changes of the phosphoproteome, including a widespread increase in the phosphorylation of CDK1 targets. Importantly, the toxicity of USP7 inhibitors is alleviated by lowering CDK1 activity or by chemical activation of PP2A. Our work reveals that USP7 limits CDK1 activity at all cell cycle stages, providing a novel mechanism that explains the toxicity of USP7 inhibitors through untimely activation of CDK1.     

USP7 controls Chk1 protein stability by direct deubiquitination

Chk1, an essential checkpoint kinase in the DNA damage response pathway (DDR), is tightly regulated by both ATR-dependent phosphorylation and proteasome-mediated degradation. Here we identify ubiquitin hydrolase USP7 as a novel regulator of Chk1 protein stability. USP7 was shown before to regulate other DDR proteins such as p53, Hdm2 and Claspin, an adaptor protein in the ATR-Chk1 pathway required for Chk1 activation. Depletion or inhibition of USP7 leads to lower Chk1 levels. The decreased Chk1 protein after USP7 knock down cannot be rescued by simultaneously elevating Claspin levels, demonstrating that the effect of USP7 on Chk1 is independent of its known effect on Claspin. Conversely, overexpression of USP7 wild type, but not a catalytic mutant version, elevates Chk1 levels and increases the half-life of Chk1 protein. Importantly, wild type, but not catalytic mutant USP7 can deubiquitinate Chk1 in vivo and in vitro, confirming that USP7 directly regulates Chk1 protein levels. Finally we show that USP7 catalytic mutant is (mono-)ubiquitinated, which suggests auto-deubiquitination by this ubiquitin hydrolase, possibly important for its regulation.     

大肠癌组织中联合检测USP22和BMI-1表达的临床意义

目的:探讨大肠癌组织中联合检测USP22和BMI-1表达的临床意义。方法:应用定量RT-PCR检测82例大肠癌手术切除标本及相应的癌旁组织中USP22及BMI-1 mRNA的表达水平,并分析其表达的差异,研究其表达水平与大肠癌患者临床病理特征及预后的关系。结果:USP22、BMI-1 mRNA在大肠癌及癌旁组织中均可被检出,大肠癌组织中USP22、BMI-1 mRNA的相对表达水平均显著高于癌旁组织(P0.01),且二者之间呈显著正相关(r=0.708,P0.01)。USP22、BMI-1 mRNA的高表达均与大肠癌的美国癌症分期联合委员会(AJCC)分期密切相关(P0.01)。COX回归分析显示USP22及BMI-1的共表达可作为大肠癌患者预后的独立预测因素(P0.01)。结论:USP22和BMI-1的共同激活可促进大肠癌的进展,并预示预后不良。     

The deubiquitinating enzyme USP37 enhances CHK1 activity to promote the cellular response to replication stress

The deubiquitinating enzyme USP37 is known to contribute to timely onset of S phase and progression of mitosis. However, it is not clear if USP37 is required beyond S-phase entry despite expression and activity of USP37 peaking within S phase. We have utilized flow cytometry and microscopy to analyze populations of replicating cells labeled with thymidine analogs and monitored mitotic entry in synchronized cells to determine that USP37-depleted cells exhibited altered S-phase kinetics. Further analysis revealed that cells depleted of USP37 harbored increased levels of the replication stress and DNA damage markers γH2AX and 53BP1 in response to perturbed replication. Depletion of USP37 also reduced cellular proliferation and led to increased sensitivity to agents that induce replication stress. Underlying the increased sensitivity, we found that the checkpoint kinase 1 is destabilized in the absence of USP37, attenuating its function. We further demonstrated that USP37 deubiquitinates checkpoint kinase 1, promoting its stability. Together, our results establish that USP37 is required beyond S-phase entry to promote the efficiency and fidelity of replication. These data further define the role of USP37 in the regulation of cell proliferation and contribute to an evolving understanding of USP37 as a multifaceted regulator of genome stability.     

The deubiquitinating enzyme USP20 stabilizes ULK1 and promotes autophagy initiation

Autophagy begins with the formation of autophagosomes, a process that depends on the activity of the serine/threonine kinase ULK1 (hATG1). Although earlier studies indicated that ULK1 activity is regulated by dynamic polyubiquitination, the deubiquitinase involved in the regulation of ULK1 remained unknown. In this study, we demonstrate that ubiquitin‐specific protease 20 (USP20) acts as a positive regulator of autophagy initiation through stabilizing ULK1. At basal state, USP20 binds to and stabilizes ULK1 by removing the ubiquitin moiety, thereby interfering with the lysosomal degradation of ULK1. The stabilization of basal ULK1 protein levels is required for the initiation of starvation‐induced autophagy, since the depletion of USP20 by RNA interference inhibits LC3 puncta formation, a marker of autophagic flux. At later stages of autophagy, USP20 dissociates from ULK1, resulting in enhanced ULK1 degradation and apoptosis. Taken together, our findings provide the first evidence that USP20 plays a crucial role in autophagy initiation by maintaining the basal expression level of ULK1.     

The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins

Initially described as a nonspecific degradation process induced upon starvation, autophagy is now known also to be involved in the degradation of specific ubiquitinated substrates such as mitochondria, bacteria and aggregated proteins, ensuring crucial functions in cell physiology and immunity. We report here that the deubiquitinating enzyme USP36 controls selective autophagy activation in Drosophila and in human cells. We show that dUsp36 loss of function autonomously inhibits cell growth while activating autophagy. Despite the phenotypic similarity, dUSP36 is not part of the TOR signaling pathway. Autophagy induced by dUsp36 loss of function depends on p62/SQSTM1, an adaptor for delivering cargo marked by polyubiquitin to autophagosomes. Consistent with p62 requirement, dUsp36 mutant cells display nuclear aggregates of ubiquitinated proteins, including Histone H2B, and cytoplasmic ubiquitinated proteins; the latter are eliminated by autophagy. Importantly, USP36 function in p62-dependent selective autophagy is conserved in human cells. Our work identifies a novel, crucial role for a deubiquitinating enzyme in selective autophagy.     

USP52 inhibits cell proliferation by stabilizing PTEN protein in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer. Ubiquitination is closely related to the development of lung cancer. However, the biological importance of newly discovered ubiquitin-specific peptidase (USP) 52 (USP52) in NSCLC remained unclear. Here, our findings identify USP52 as a novel tumor suppressor of NSCLC, the low expression of USP52 predicts a poor prognosis for NSCLC patients. The present study demonstrates that USP52 inhibits cancer cell proliferation through down-regulation of cyclin D1 (CCND1) as well as AKT/mTOR signaling pathway inhibition. Meanwhile, USP25 also suppresses NSCLC progression via enhancing phosphatase and tensin homolog (PTEN) stability in cancer cells, which further indicates the significance/importance of USP52 in NSCLC suppression.     

USP8 regulates mitophagy by removing K6‐linked ubiquitin conjugates from parkin

Mutations in the Park2 gene, encoding the E3 ubiquitin‐ligase parkin, are responsible for a familial form of Parkinson's disease (PD). Parkin‐mediated ubiquitination is critical for the efficient elimination of depolarized dysfunctional mitochondria by autophagy (mitophagy). As damaged mitochondria are a major source of toxic reactive oxygen species within the cell, this pathway is believed to be highly relevant to the pathogenesis of PD. Little is known about how parkin‐mediated ubiquitination is regulated during mitophagy or about the nature of the ubiquitin conjugates involved. We report here that USP8/UBPY, a deubiquitinating enzyme not previously implicated in mitochondrial quality control, is critical for parkin‐mediated mitophagy. USP8 preferentially removes non‐canonical K6‐linked ubiquitin chains from parkin, a process required for the efficient recruitment of parkin to depolarized mitochondria and for their subsequent elimination by mitophagy. This work uncovers a novel role for USP8‐mediated deubiquitination of K6‐linked ubiquitin conjugates from parkin in mitochondrial quality control.     

AS160 controls eukaryotic cell cycle and proliferation by regulating the CDK inhibitor p21

AS160 (TBC1D4) has been implicated in multiple biological processes. However, the role and the mechanism of action of AS160 in the regulation of cell proliferation remain unclear. In this study, we demonstrated that AS160 knockdown led to blunted cell proliferation in multiple cell types, including fibroblasts and cancer cells. The results of cell cycle analysis showed that these cells were arrested in the G1 phase. Intriguingly, this inhibition of cell proliferation and the cell cycle arrest caused by AS160 depletion were glucose independent. Moreover, AS160 silencing led to a marked upregulation of the expression of the cyclin-dependent kinase inhibitor p21. Furthermore, whereas AS160 overexpression resulted in p21 downregulation and rescued the arrested cell cycle in AS160-depeleted cells, p21 silencing rescued the inhibited cell cycle and proliferation in the cells. Thus, our results demonstrated that AS160 regulates glucose-independent eukaryotic cell proliferation through p21-dependent control of the cell cycle, and thereby revealed a molecular mechanism of AS160 modulation of cell cycle and proliferation that is of general physiological significance.