USP21 regulates Hippo signaling to promote radioresistance by deubiquitinating FOXM1 in cervical cancer.

The ectopic expression of ubiquitin-specific peptidase 21 (USP21) is common in different types of cancer. However, its relationship with radio-sensitivity in cervical cancer (CC) remains unclear. In this study, we aimed to uncover the effect of USP21 on CC radio-resistance and its underlying mechanism. Our results showed that the expression of USP21 was markedly increased in CC tissues of radio-resistant patients and CC cells treated with radiation. Besides, knockdown of USP21 restrained the survival fractions, and facilitated apoptosis of CC cells in the absence or presence of radiation. Additionally, USP21 in combination with FOXM1 regulated the stability and ubiquitination of FOXM1. However, FOXM1 reversed the effects of USP21 knockdown on the radio-resistance of CC cells. Furthermore, FOXM1 knockdown activated the Hippo pathway by inhibiting the nuclear translocation of Yes-associated protein 1 (YAP1), and FOXM1 knockdown attenuated the radio-resistance of CC cells via inhibiting the Hippo-YAP1 pathway. USP21 activated the Hippo pathway by mediating FOXM1. Knockdown of USP21 enhanced the radio-sensitivity of CC cells in vivo. In summary, USP21 contributed to the radio-resistance of CC cells via FOXM1/Hippo signaling, and may serve as a promising target for radio-sensitizers in the radiotherapy of CC.

Loss of Ubiquitin Carboxy-Terminal Hydrolase L1 Impairs Long-Term Differentiation Competence and Metabolic Regulation in Murine Spermatogonial Stem Cells.

Spermatogonia are stem and progenitor cells responsible for maintaining mammalian spermatogenesis. Preserving the balance between self-renewal of spermatogonial stem cells (SSCs) and differentiation is critical for spermatogenesis and fertility. Ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) is highly expressed in spermatogonia of many species; however, its functional role has not been identified. Here, we aimed to understand the role of UCH-L1 in murine spermatogonia using a Uch-l1-/- mouse model. We confirmed that UCH-L1 is expressed in undifferentiated and early-differentiating spermatogonia in the post-natal mammalian testis. The Uch-l1-/- mice showed reduced testis weight and progressive degeneration of seminiferous tubules. Single-cell transcriptome analysis detected a dysregulated metabolic profile in spermatogonia of Uch-l1-/- compared to wild-type mice. Furthermore, cultured Uch-l1-/- SSCs had decreased capacity in regenerating full spermatogenesis after transplantation in vivo and accelerated oxidative phosphorylation (OXPHOS) during maintenance in vitro. Together, these results indicate that the absence of UCH-L1 impacts the maintenance of SSC homeostasis and metabolism and impacts the differentiation competence. Metabolic perturbations associated with loss of UCH-L1 appear to underlie a reduced capacity for supporting spermatogenesis and fertility with age. This work is one step further in understanding the complex regulatory circuits underlying SSC function.

The deubiquitinase USP10 restores PTEN activity and inhibits non-small cell lung cancer cell proliferation.

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein is a key player in tumorigenesis of non-small cell lung cancer (NSCLC) and was recently found to be inactivated by tripartite motif containing 25 (TRIM25)-mediated K63-linked polyubiquitination. However, the deubiquitinase (Dub) coordinate TRIM25 in PTEN ubiquitination is still elusive. In the present study, we found that this K63-linked polyubiquitination could be ablated by the ubiquitin-specific protease 10 (USP10) in a screen against a panel of Dubs. We found using coimmununoprecipitation/immunoblotting that USP10 interacted with PTEN and reduced the K63-linked polyubiquitination of PTEN mediated by TRIM25 in NSCLC cells. Moreover, USP10, but not its inactive C424A deubiquitinating mutant or other Dubs, abolished PTEN from K63-linked polyubiquitination mediated by TRIM25. In contrast to TRIM25, USP10 restored PTEN phosphatase activity and reduced the production of the secondary messenger phosphatidylinositol-3,4,5-trisphosphate, thereby inhibiting AKT/mammalian target of rapamycin progrowth signaling transduction in NSCLC cells. Moreover, USP10 was downregulated in NSCLC cell lines and primary tissues, whereas TRIM25 was upregulated. Consistent with its molecular activity, re-expression of USP10 suppressed NSCLC cell proliferation and migration, whereas knockout of USP10 promoted NSCLC cell proliferation and migration. In conclusion, the present study demonstrates that USP10 coordinates TRIM25 to modulate PTEN activity. Specifically, USP10 activates PTEN by preventing its K63-linked polyubiquitination mediated by TRIM25 and suppresses the AKT/mammalian target of rapamycin signaling pathway, thereby inhibiting NSCLC proliferation, indicating that it may be a potential drug target for cancer treatment.

UCHL3 promotes aerobic glycolysis of pancreatic cancer through upregulating LDHA expression

Background Aerobic glycolysis has a pivotal role in the carcinogenic process. The current understanding of the functional role and mechanism of UCHL3-related aerobic glycolysis in pancreatic cancer is far from comprehensive, therefore requires an in-depth analysis on this aspect. Methods In the present research, the expressions of ubiquitin carboxyl-terminal hydrolase L3 (UCHL3), lactate dehydrogenase A (LDHA) and Forkhead box protein M1 (FOXM1) were detected by qRT-PCR, Western blot and immunohistochemistry. The effects of UCHL3 knockdown or overexpression on pancreatic cancer cells were examined by determining cell viability and colony formation. Aerobic glycolysis was assessed according to glucose uptake, lactic acid production, and lactate dehydrogenase (LDH) activity. Dual-luciferase reporter assay was performed to detect LDHA promoter activity. Results The results showed that UCHL3 expression was significantly increased in the pancreatic cancer tissues and cells, and that knocking down UCHL3 noticeably inhibited cell viability and aerobic glycolysis. Further investigations revealed that LDHA expression was promoted by UCHL3 and could be reduced by shFOXM1, and that low-expressed LDHA partly reversed the inhibition of aerobic glycolysis induced by overexpressed UCHL3. Conclusions To conclude, this study demonstrates that UCHL3 plays a carcinogenic role by promoting aerobic glycolysis in pancreatic cancer, suggesting that UCHL3 may be a potential diagnostic and therapeutic target for the treatment of cancer (AU)

BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation.

BAP1 is mutated or deleted in many cancer types, including mesothelioma, uveal melanoma, and cholangiocarcinoma. It is the catalytic subunit of the PR-DUB complex, which removes PRC1-mediated H2AK119ub1, essential for maintaining transcriptional repression. However, the precise relationship between BAP1 and Polycombs remains elusive. Using embryonic stem cells, we show that BAP1 restricts H2AK119ub1 deposition to Polycomb target sites. This increases the stability of Polycomb with their targets and prevents diffuse accumulation of H2AK119ub1 and H3K27me3. Loss of BAP1 results in a broad increase in H2AK119ub1 levels that is primarily dependent on PCGF3/5-PRC1 complexes. This titrates PRC2 away from its targets and stimulates H3K27me3 accumulation across the genome, leading to a general chromatin compaction. This provides evidence for a unifying model that resolves the apparent contradiction between BAP1 catalytic activity and its role in vivo, uncovering molecular vulnerabilities that could be useful for BAP1-related pathologies.

Ubiquitin-specific protease 22 ameliorates chronic alcohol-associated liver disease by regulating BRD4.

Alcohol-associated liver disease (ALD) is a liver system disease caused by alcohol abuse, and it involves complex processes ranging from steatosis to fibrosis, cirrhosis and hepatocellular carcinoma. Steatosis and inflammation are the main phenomena involved in ALD. Ubiquitin-specific protease 22 (USP22) plays an important role in liver steatosis; however, its functional contribution to ALD remains unclear. USP22-silenced mice were fed a Lieber-DeCarli liquid diet. AML-12 and HEK293T cells were used to detect the interaction between USP22 and BRD4. Here, we report that hepatic USP22 expression was dramatically upregulated in mice with ALD. Inflammation and steatosis were significantly ameliorated following USP22 silencing in vivo, as indicated by decreased IL-6 and IL-1ß levels. We further showed that the overexpression of USP22 increased inflammation, while knocking down BRD4 suppressed the inflammatory response in AML-12 cells. Notably, USP22 functioned as a BRD4 deubiquitinase to facilitate BRD4 inflammatory functions. More importantly, the expression levels of USP22 and BRD4 in patients with ALD were significantly increased. In conclusion, USP22 acts a key pathogenic factor in ALD by deubiquitinating BRD4, which facilitates the inflammatory response and aggravates ALD.

The ubiquitin isopeptidase USP10 deubiquitinates LC3B to increase LC3B levels and autophagic activity.

Components of the autophagy machinery are subject to regulation by various posttranslational modifications. Previous studies showed that monoubiquitination of LC3B catalyzed by the ubiquitin-activating enzyme UBA6 and ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 targets LC3B for proteasomal degradation, thus reducing LC3B levels and autophagic activity under conditions of stress. However, mechanisms capable of counteracting this process are not known. Herein, we report that LC3B ubiquitination is reversed by the action of the deubiquitinating enzyme USP10. We identified USP10 in a CRISPR-Cas9 knockout screen for ubiquitination-related genes that regulate LC3B levels. Biochemical analyses showed that silencing of USP10 reduces the levels of both the LC3B-I and LC3B-II forms of LC3B through increased ubiquitination and proteasomal degradation. In turn, the reduced LC3B levels result in slower degradation of the autophagy receptors SQSTM1 and NBR1 and an increased accumulation of puromycin-induced aggresome-like structures. Taken together, these findings indicate that the levels of LC3B and autophagic activity are controlled through cycles of LC3B ubiquitination and deubiquitination.

UCHL3 promotes aerobic glycolysis of pancreatic cancer through upregulating LDHA expression.

BACKGROUND: Aerobic glycolysis has a pivotal role in the carcinogenic process. The current understanding of the functional role and mechanism of UCHL3-related aerobic glycolysis in pancreatic cancer is far from comprehensive, therefore requires an in-depth analysis on this aspect. METHODS: In the present research, the expressions of ubiquitin carboxyl-terminal hydrolase L3 (UCHL3), lactate dehydrogenase A (LDHA) and Forkhead box protein M1 (FOXM1) were detected by qRT-PCR, Western blot and immunohistochemistry. The effects of UCHL3 knockdown or overexpression on pancreatic cancer cells were examined by determining cell viability and colony formation. Aerobic glycolysis was assessed according to glucose uptake, lactic acid production, and lactate dehydrogenase (LDH) activity. Dual-luciferase reporter assay was performed to detect LDHA promoter activity. RESULTS: The results showed that UCHL3 expression was significantly increased in the pancreatic cancer tissues and cells, and that knocking down UCHL3 noticeably inhibited cell viability and aerobic glycolysis. Further investigations revealed that LDHA expression was promoted by UCHL3 and could be reduced by shFOXM1, and that low-expressed LDHA partly reversed the inhibition of aerobic glycolysis induced by overexpressed UCHL3. CONCLUSIONS: To conclude, this study demonstrates that UCHL3 plays a carcinogenic role by promoting aerobic glycolysis in pancreatic cancer, suggesting that UCHL3 may be a potential diagnostic and therapeutic target for the treatment of cancer.

Ubiquitin-Specific Protease 6 Functions as a Tumor Suppressor in Ewing Sarcoma through Immune Activation.

Ewing sarcoma is the second most common pediatric bone cancer, with a 5-year survival rate for metastatic disease of only 20%. Recent work indicates that survival is strongly correlated with high levels of tumor-infiltrating lymphocytes (TIL), whose abundance is associated with IFN-inducible chemokines CXCL10 and CCL5. However, the tumor-intrinsic factors that drive chemokine production and TIL recruitment have not been fully elucidated. We previously showed that ubiquitin-specific protease 6 (USP6) directly deubiquitinates and stabilizes Jak1, thereby inducing an IFN signature in Ewing sarcoma cells. Here, we show that this gene set comprises chemokines associated with immunostimulatory, antitumorigenic functions, including CXCL10 and CCL5. USP6 synergistically enhanced chemokine production in response to exogenous IFN by inducing surface upregulation of IFNAR1 and IFNGR1. USP6-expressing Ewing sarcoma cells stimulated migration of primary human monocytes and T lymphocytes and triggered activation of natural killer (NK) cells in vitro. USP6 inhibited Ewing sarcoma xenograft growth in nude but not NSG mice and was accompanied by increased intratumoral chemokine production and infiltration and activation of NK cells, dendritic cells, and macrophages, consistent with a requirement for innate immune cells in mediating the antitumorigenic effects of USP6. High USP6 expression in patients with Ewing sarcoma was associated with chemokine production, immune infiltration, and improved survival. This work reveals a previously unrecognized tumor-suppressive function for USP6, which engenders an immunostimulatory microenvironment through pleiotropic effects on multiple immune lineages. This further raises the possibility that USP6 activity may be harnessed to create a "hot" tumor microenvironment in immunotherapy. SIGNIFICANCE: This study reveals a novel tumor-suppressive function for USP6 by inducing an immunostimulatory microenvironment, suggesting that USP6 activity may be exploited to enhance immunotherapy regimens.

USP14 negatively regulates RIG-I-mediated IL-6 and TNF-α production by inhibiting NF-κB activation.

Ubiquitin specific protease 14 (USP14) is a regulator of protein deubiquitination and proteasome activation, and has been implicated in negative regulation of type I IFN signaling pathway. However, the effect of USP14 on RNA virus-related inflammatory response has not been studied. Retinoic acid-inducible gene I (RIG-I) is the important pattern recognition receptor of the innate immunity to detect RNA viruses or intracellular Poly(I:C)-LMW. Here, we reported that USP14 knockdown increased pro-inflammatory cytokines production in macrophages upon VSV infection or intracellular Poly(I:C)-LMW stimulation. USP14-overexpressed HeLa cells exhibited a decrease in RIG-I-mediated IL-6 and TNF-α expression. IU1, USP14 inhibitor, significantly promotes pro-inflammatory cytokines production in VSV-infected mice in vivo. Furthermore, USP14 was also found to inhibit the RIG-I-triggered NF-κB activation by deubiquitinating K63-linked RIG-I. Thus, our results demonstrate that USP14 is a negative regulator of RIG-I-mediated inflammatory response.

Deubiquitinase Ubiquitin-Specific Protease 10 Deficiency Regulates Sirt6 signaling and Exacerbates Cardiac Hypertrophy.

Background Cardiac hypertrophy (CH) is a physiological response that compensates for blood pressure overload. Under pathological conditions, hypertrophy can progress to heart failure as a consequence of the disorganized growth of cardiomyocytes and cardiac tissue. USP10 (ubiquitin-specific protease 10) is a member of the ubiquitin-specific protease family of cysteine proteases, which are involved in viral infection, oxidative stress, lipid drop formation, and heat shock. However, the role of USP10 in CH remains largely unclear. Here, we investigated the roles of USP10 in CH. Methods and Results Cardiac-specific USP10 knockout (USP10-CKO) mice and USP10-transgenic (USP10-TG) mice were used to examined the role of USP10 in CH following aortic banding. The specific functions of USP10 were further examined in isolated cardiomyocytes. USP10 expression was increased in murine hypertrophic hearts following aortic banding and in isolated cardiomyocytes in response to hypertrophic agonist. Mice deficient in USP10 in the heart exhibited exaggerated cardiac hypertrophy and fibrosis following pressure overload stress, which resulted in worsening of cardiac contractile function. In contrast, cardiac overexpression of USP10 protected against pressure overload-induced maladaptive CH. Mechanistically, we demonstrated that USP10 activation and interaction with Sirt6 in response to angiotensin II led to a marked increase in the ubiquitination of Sirt6 and resulted in Akt signaling downregulation and attenuation of cardiomyocyte hypertrophy. Accordingly, inactivation of USP10 reduced Sirt6 abundance and stability and diminished Sirt6-induced downstream signaling in cardiomyocytes. Conclusions USP10 functions as a Sirt6 deubiquitinase that induces cardiac myocyte hypertrophy and triggers maladaptive CH.

Deubiquitylase UCHL3 regulates bi-orientation and segregation of chromosomes during mitosis.

Equal segregation of chromosomes during mitosis ensures euploidy of daughter cells. Defects in this process may result in an imbalance in the chromosomal composition and cellular transformation. Proteolytic and non-proteolytic ubiquitylation pathways ensure directionality and fidelity of mitotic progression but specific mitotic functions of deubiquitylating enzymes (DUBs) remain less studied. Here we describe the role of the DUB ubiquitin carboxyl-terminal hydrolase isozyme L3 (UCHL3) in the regulation of chromosome bi-orientation and segregation during mitosis. Downregulation or inhibition of UCHL3 leads to chromosome alignment defects during metaphase. Frequent segregation errors during anaphase are also observed upon inactivation of UCHL3. Mechanistically, UCHL3 interacts with and deubiquitylates Aurora B, the catalytic subunit of chromosome passenger complex (CPC), known to be critically involved in the regulation of chromosome alignment and segregation. UCHL3 does not regulate protein levels of Aurora B or the binding of Aurora B to other CPC subunits. Instead, UCHL3 promotes localization of Aurora B to kinetochores, suggesting its role in the error correction mechanism monitoring bi-orientation of chromosomes during metaphase. Thus, UCHL3 contributes to the regulation of faithful genome segregation and maintenance of euploidy in human cells.

Typically inhibiting USP14 promotes autophagy in M1-like macrophages and alleviates CLP-induced sepsis.

Macrophages, with diverse functions and variable phenotypes, are considered as an important executor of inflammatory diseases. And it has been proved that autophagy is deeply connected with the development of inflammation, while the exact regulatory mechanism still remains unclear, and the application of autophagy regulators in anti-inflammation needs to be further confirmed. Here, we firstly verified that neochromine S5 (hereinafter referred to as S5) significantly inhibited M1-like macrophage polarization with decrease of the proinflammatory cytokines and downregulation of NF-κB and STAT1 signals. Then, in vivo experiments demonstrated S5 improved cecal ligation and puncture (CLP)-induced sepsis specially based on the regulation of M1-like macrophages. Mechanistic studies indicated that S5 treatment dramatically upregulated cellular autophagy in M1-like macrophage. Furthermore, by multiple methods, S5 was revealed to directly bind with ubiquitin-specific proteases 14 (USP14) at Ser404, Phe405, and Cys414 by hydrogen bond to inhibit its deubiquitinating activity, and block USP14-TRAF6 (TNF receptor associated factor 6) interaction, subsequently promoting ubiquitination of Beclin1, interrupting Beclin1-Bcl2 interaction, and accumulating the autophagosome in macrophages, which finally resulted in the blockade of M1-like macrophage polarization. Animal experiments also confirmed the protection of S5 in CLP mice was dependent on activation of macrophage autophagy. What's more, as a novel USP14 inhibitor, S5 exhibited higher efficiency and safety than IU1, the known USP14 inhibitor. Therefore, this study has demonstrated that typically inhibiting USP14 promotes autophagy in M1-like macrophages and alleviates CLP-induced sepsis. Moreover, we provide a new candidate compound, S5, for sensitizing autophagy to interfere with the macrophage inflammation.

The evolutionarily conserved deubiquitinase UBH1/UCH-L1 augments DAF7/TGF-ß signaling, inhibits dauer larva formation, and enhances lung tumorigenesis.

Modification of the transforming growth factor ß (TGF-ß) signaling components by (de)ubiquitination is emerging as a key regulatory mechanism that controls cell signaling responses in health and disease. Here, we show that the deubiquitinating enzyme UBH-1 in Caenorhabditis elegans and its human homolog, ubiquitin C-terminal hydrolase-L1 (UCH-L1), stimulate DAF-7/TGF-ß signaling, suggesting that this mode of regulation of TGF-ß signaling is conserved across animal species. The dauer larva-constitutive C. elegans phenotype caused by defective DAF-7/TGF-ß signaling was enhanced and suppressed, respectively, by ubh-1 deletion and overexpression in the loss-of-function genetic backgrounds of daf7, daf-1/TGF-ßRI, and daf4/R-SMAD, but not of daf-8/R-SMAD. This suggested that UBH-1 may stimulate DAF-7/TGF-ß signaling via DAF-8/R-SMAD. Therefore, we investigated the effect of UCH-L1 on TGF-ß signaling via its intracellular effectors, i.e. SMAD2 and SMAD3, in mammalian cells. Overexpression of UCH-L1, but not of UCH-L3 (the other human homolog of UBH1) or of the catalytic mutant UCH-L1C90A, enhanced TGF-ß/SMAD-induced transcriptional activity, indicating that the deubiquitination activity of UCH-L1 is indispensable for enhancing TGF-ß/SMAD signaling. We also found that UCH-L1 interacts, deubiquitinates, and stabilizes SMAD2 and SMAD3. Under hypoxia, UCH-L1 expression increased and TGF-ß/SMAD signaling was potentiated in the A549 human lung adenocarcinoma cell line. Notably, UCH-L1-deficient A549 cells were impaired in tumorigenesis, and, unlike WT UCH-L1, a UCH-L1 variant lacking deubiquitinating activity was unable to restore tumorigenesis in these cells. These results indicate that UCH-L1 activity supports DAF-7/TGF-ß signaling and suggest that UCH-L1's deubiquitination activity is a potential therapeutic target for managing lung cancer.

USP32 promotes tumorigenesis and chemoresistance in gastric carcinoma via upregulation of SMAD2.

USP32, a member of the ubiquitin-specific proteases family, has been implicated in the development of breast cancer and small lung cancer. However, its biological functions and clinical significance in gastric cancer (GC) remain unclear. In the present study, we reported that knockdown or depletion of USP32 significantly inhibited GC cell proliferation and migration in vitro and in vivo, indicating that USP32 functions as an oncogene in GC. Importantly, results from immunohistochemical staining in a tissue microarray revealed that USP32 was upregulated in GC tissues compared with paracancerous tissues. Further analyses showed that high expression of USP32 was closely related with high T-staging and poor outcomes of GC patients. Mechanistically, USP32 silencing caused a decrease in the expression of SMAD2, which resulted in the inhibitory effects of GC cells on growth, motility, and chemoresistance to cisplatin. Therefore, our findings strongly suggest the involvement of USP32 in GC progression and provide a potential target for future therapy of GC.

Inhibition of the deubiquitinase USP10 induces degradation of SYK.

BACKGROUND: There is growing evidence that spleen tyrosine kinase (SYK) is critical for acute myeloid leukaemia (AML) transformation and maintenance of the leukemic clone in AML patients. It has also been found to be over-expressed in AML patients, with activating mutations in foetal liver tyrosine kinase 3 (FLT3), particularly those with internal tandem duplications (FLT3-ITD), where it transactivates FLT3-ITD and confers resistance to treatment with FLT3 tyrosine kinase inhibitors (TKIs). METHODS: We have previously described a pharmacological approach to treating FLT3-ITD-positive AML that relies on proteasome-mediated FLT3 degradation via inhibition of USP10, the deubiquitinating enzyme (DUB) responsible for cleaving ubiquitin from FLT3. RESULTS: Here, we show that USP10 is also a major DUB required for stabilisation of SYK. We further demonstrate that degradation of SYK can be induced by USP10-targeting inhibitors. USP10 inhibition leads to death of cells driven by active SYK or oncogenic FLT3 and potentiates the anti-leukemic effects of FLT3 inhibition in these cells. CONCLUSIONS: We suggest that USP10 inhibition is a novel approach to inhibiting SYK and impeding its role in the pathology of AML, including oncogenic FLT3-positive AML. Also, given the significant transforming role SYK in other tumours, targeting USP10 may have broader applications in cancer.

Deubiquitinating enzyme USP33 restrains docetaxel-induced apoptosis via stabilising the phosphatase DUSP1 in prostate cancer.

The treatment of castration-resistant prostate cancer (CRPC) still faces many challenges. Docetaxel is a chemotherapeutic drug commonly used in CRPC patients. However, docetaxel-based chemotherapy usually causes docetaxel resistance, partially due to the resistance of CRPC cells to docetaxel-induced apoptosis. Here, we report that the deubiquitinating enzyme ubiquitin-specific protease 33 (USP33) inhibits docetaxel-induced apoptosis of prostate cancer cells, including androgen-independent prostate cancer cells. USP33 is overexpressed in prostate cancer cells and tissues. We found that knockdown or knockout of USP33 enhanced docetaxel-induced apoptosis of prostate cancer cells, accompanied by increased phosphorylation of the cJUN NH2-terminal kinase (JNK). After blocking docetaxel-induced JNK activation using the JNK inhibitor SP600125 or siRNA targeting JNK, the USP33 knockout-enhanced apoptosis was reversed. Furthermore, we found that USP33 could interact with the phosphatase DUSP1 to negatively regulate the activation of JNK, while USP33 knockdown promoted the proteasomal degradation of DUSP1. Mechanistically, we found that USP33 could inhibit the Lys48 (K48)-linked polyubiquitination of DUSP1. More importantly, DUSP1 overexpression could reverse the USP33 knockdown-induced JNK activation and apoptosis in docetaxel-treated prostate cancer cells. Therefore, USP33 overexpression in prostate cancer may contribute to docetaxel resistance by inhibiting the degradation of its partner DUSP1, leading to impaired JNK activation and apoptosis. Our study suggests that USP33-DUSP1-JNK may be a key signalling module mediating the docetaxel resistance of CRPC, indicating that USP33 is a potential novel therapeutic target in CRPC.

Ubiquitin-specific protease 8 (USP8/UBPy): a prototypic multidomain deubiquitinating enzyme with pleiotropic functions.

Protein modification by ubiquitin is one of the most versatile posttranslational regulations and counteracted by almost 100 deubiquitinating enzymes (DUBs). USP8 was originally identified as a growth regulated ubiquitin-specific protease and is like many other DUBs characterized by its multidomain architecture. Besides the catalytic domain, specific protein-protein interaction modules were characterized which contribute to USP8 substrate recruitment, regulation and targeting to distinct protein complexes. Studies in mice and humans impressively showed the physiological relevance and non-redundant function of USP8 within the context of the whole organism. USP8 knockout (KO) mice exhibit early embryonic lethality while induced deletion in adult animals rapidly causes lethal liver failure. Furthermore, T-cell specific ablation disturbs T-cell development and function resulting in fatal autoimmune inflammatory bowel disease. In human patients, somatic mutations in USP8 were identified as the underlying cause of adrenocorticotropic hormone (ACTH) releasing pituitary adenomas causing Cushing's disease (CD). Here we provide an overview of the versatile molecular, cellular and pathology associated function and regulation of USP8 which appears to depend on specific protein binding partners, substrates and the cellular context.

Versatility of USP18 in physiology and pathophysiology.

Ubiquitin-specific peptidase 18 (USP18) is a multifunctional protein and its roles are still being investigated. This enzyme removes ubiquitin-like molecules from their substrates and the only known interferon-stimulated gene 15 (ISG15) specific protease. Apart from its enzymatic function, it also inhibits interferon type I and III signalling pathways. USP18 is known to regulate multiple processes, such as: cell cycle, cell signalling and response to viral and bacterial infections. Moreover, it contributes to the development of several autoimmune diseases and carcinogenesis, and recently was described as a cardiac remodelling inhibitor. This review summarizes the current knowledge on USP18 functions, highlighting its contribution to the development of heart failure, given the fact that this disease's etiology is now considered to be inflammatory in nature.

UCHL1 regulates muscle fibers and mTORC1 activity in skeletal muscle.

AIMS: Skeletal muscle wasting is associated with many chronic diseases. Effective prevention and treatment of muscle wasting remain as a challenging task due to incomplete understanding of mechanisms by which muscle mass is maintained and regulated. This study investigated the functional role of Ubiquitin C-terminal hydrolase L1 (UCHL1) in skeletal muscle. MAIN METHODS: Mice with skeletal muscle specific gene knockout of UCHL1 and C2C12 myoblast cells with UCHL1 knockdown were used. Muscle fiber types and size were measured using tissue or cell staining. The mammalian target of rapamycin complex 1 (mTORC1) and mTORC2 activities were assessed with the phosphorylation of their downstream targets. KEY FINDINGS: In mouse skeletal muscle, UCHL1 was primarily expressed in slow twitch muscle fibers. Mice with skeletal muscle specific knockout (skmKO) of UCHL1 exhibited enlarged muscle fiber sizes in slow twitch soleus but not fast twitch extensor digitorum longus (EDL) muscle. Meanwhile, UCHL1 skmKO enhanced mTORC1 activity and reduced mTORC2 activity in soleus but not in EDL. Consistently, in C2C12 cells, UCHL1 knockdown increased the myotube size, enhanced mTORC1 activity, and reduced mTORC2 activities as compared with control cells. UCHL1 knockdown did not change the major proteins of mTOR complex but decreased the protein turnover of PRAS40, an inhibitory factor of mTORC1. SIGNIFICANCE: These data revealed a novel function of UCHL1 in regulation of mTORC1 activity and skeletal muscle growth in slow twitch skeletal muscle. Given the upregulation of UCHL1 in denervation and spinal muscle atrophy, our finding advances understanding of regulators that are involved in muscle wasting.