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.     

USP28 contributes to the proliferation and metastasis of gastric cancer

USP28, a member of the deubiquitinating enzymes family, plays a vital role in the physiological process of cell proliferation, differentiation and apoptosis, DNA repair, immune response, and stress response. USP28 has been reported to be overexpressed in bladder cancer, colon cancer, breast carcinomas, and so on. Nevertheless, the role of USP28 in gastric cancer has not yet been investigated. In our study, we examined the USP28 expression in 87 paired samples of gastric cancer and normal gastric tissues. We found that USP28 was overexpressed in gastric cancer compared with normal gastric tissues (P < 0.01), and its overexpression was related to the degree of differentiation and metastases. Inhibiting USP28 expression in vitro suppressed the proliferation and invasion of gastric cancer cells by downregulating lysine specific demethylase 1. On the basis of our data, it can be concluded that USP28 may be a novel therapeutic target for gastric cancer.     

Pharmacological inhibition of USP7 suppresses growth and metastasis of melanoma cells in vitro and in vivo

Melanoma is a highly aggressive type of skin cancer. The development of diverse resistance mechanisms and severe adverse effects significantly limit the efficiency of current therapeutic approaches. Identification of the new therapeutic targets involved in the pathogenesis will benefit the development of novel therapeutic strategies. The deubiquitinase ubiquitin–specific protease-7, a potential target for cancer treatment, is deregulated in types of cancer, but its role in melanoma is still unclear. We investigated the role and the inhibitor P22077 of ubiquitin-specific protease-7 in melanoma treatment. We found that ubiquitin-specific protease-7 was overexpressed and correlated with poor prognosis in melanoma. Further, pharmacological inhibition of ubiquitin-specific protease-7 by P22077 can effectively inhibit proliferation, and induce cell cycle arrest and apoptosis via ROS accumulation–induced DNA damage in melanoma cells. Inhibition of ubiquitin-specific protease-7 by P22077 also inhibits melanoma tumour growth in vivo. Moreover, inhibition of ubiquitin-specific protease-7 prevented migration and invasion of melanoma cells in vitro and in vivo by decreasing the Wnt/β-catenin signalling pathway. Taken together, our study revealed that ubiquitin-specific protease-7 acted as an oncogene involved in melanoma cell proliferation and metastasis. Therefore, ubiquitin-specific protease-7 may serve as potential candidates for the treatment of melanoma.     

TRIM32/USP11 Balances ARID1A Stability and the Oncogenic/Tumor-Suppressive Status of Squamous Cell Carcinoma

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The emerging role of USP29 in cancer and other diseases

Reversible protein ubiquitination is a key process for maintaining cellular homeostasis. Deubiquitinases, which can cleave ubiquitin from substrate proteins, have been reported to be deeply involved in disease progression ranging from oncology to neurological diseases. The human genome encodes approximately 100 deubiquitinases, most of which are poorly characterized. One of the well-characterized deubiquitases is ubiquitin-specific protease 29 (USP29), which is often upregulated in pathological tissues and plays important roles in the progression of different diseases. Moreover, several studies have shown that deletion of Usp29 in mice does not cause visible growth and developmental defects, indicating that USP29 may be an ideal therapeutic target. In this review, we provide a comprehensive summary of the important roles and regulatory mechanisms of USP29 in cancer and other diseases, which may help us better understand its biological functions and improve future studies to construct suitable USP29-targeted therapy systems.     

USP44 suppresses proliferation and enhances apoptosis in colorectal cancer cells by inactivating the Wnt/β‐catenin pathway via Axin1 deubiquitination

Colorectal cancer (CRC) is the leading cause of cancer death, and its 5‐year survival rate remains unsatisfactory. Recent studies have revealed that ubiquitin‐specific protease 44 (USP44) is a cancer suppressor or oncogene depending on the type of neoplasm. However, its role in CRC remains unclear. Here, we found that the USP44 expression level was markedly decreased in CRC, and USP44 overexpression inhibited proliferation while enhancing apoptosis in CRC cells, suggesting that USP44 is a cancer suppressor in CRC. We then investigated if USP44 functioned through regulating the Wnt/β‐catenin pathway. We found that USP44 overexpression increased the Axin1 protein while decreasing β‐catenin, c‐myc, and cyclin D1 proteins, suggesting that USP44 inhibited the activation of the Wnt/β‐catenin pathway. Moreover, we found that two Wnt/β‐catenin activators, LiCl and SKL2001, both attenuated oeUSP44‐mediated proliferation and apoptosis in CRC cells. Collectively, these data points indicated that USP44 inhibited proliferation while promoting apoptosis in CRC cells by inhibiting the Wnt/β‐catenin pathway. Interestingly, we observed that USP44 overexpression did not affect the Axin1 mRNA level. Further study uncovered that USP44 interacted with Axin1 and reduced the ubiquitination of Axin1. Furthermore, Axin1 knock‐down abolished the effects of oeUSP44 on proliferation, apoptosis, and Wnt/β‐catenin activity in CRC cells. Taken together, this study demonstrates that USP44 inhibits proliferation while enhancing apoptosis in CRC cells by inactivating the Wnt/β‐catenin pathway via Axin1 deubiquitination. USP44 is a cancer suppressor in CRC and a potential target for CRC therapy.     

ERK-mediated Cytoplasmic Retention of USP11 Contributes to Breast Cancer Cell Proliferation by Stabilizing Cytoplasmic p21

Breast cancer ranks as the most frequently diagnosed cancer among women worldwide. Elevated cytoplasmic p21 levels are often found in breast cancer tissues and related to a poor prognosis. However, the underlying mechanisms that lead to the stabilization of cytoplasmic p21 protein, which normally has a very short half-life, remain obscure. In this study, we found that there was a strong correlation between p21 and USP11 in the cytoplasm of breast cancer tissues and cells. Furthermore, we revealed that ERK1/2 phosphorylated USP11 at the Ser905 site, which promoted the cytoplasmic localization of USP11. In the cytoplasm, USP11 colocalized and interacted with p21. As a result, USP11 catalyzed the removal of polyubiquitin chains bound to cytoplasmic p21 and resulted in its stabilization. Functionally, USP11-mediated stabilization of cytoplasmic p21 induced breast cancer cell proliferation in vitro and in vivo. Our findings provide the first evidence that ubiquitinated p21 in the cytoplasm can be recycled through USP11-mediated deubiquitination, and we identified the USP11-p21 axis in the cytoplasm as a potential therapeutic target for breast cancer control.     

The role of ubiquitination and deubiquitination in the regulation of cell junctions

Maintenance of cell junctions plays a crucial role in the regulation of cellular functions including cell proliferation, permeability, and cell death. Disruption of cell junctions is implicated in a variety of human disorders, such as inflammatory diseases and cancers. Understanding molecular regulation of cell junctions is important for development of therapeutic strategies for intervention of human diseases. Ubiquitination is an important type of post-translational modification that primarily regulates endogenous protein stability, receptor internalization, enzyme activity, and protein-protein interactions. Ubiquitination is tightly regulated by ubiquitin E3 ligases and can be reversed by deubiquitinating enzymes. Recent studies have been focusing on investigating the effect of protein stability in the regulation of cell-cell junctions. Ubiquitination and degradation of cadherins, claudins, and their interacting proteins are implicated in epithelial and endothelial barrier disruption. Recent studies have revealed that ubiquitination is involved in regulation of Rho GTPases’ biological activities. Taken together these studies, ubiquitination plays a critical role in modulating cell junctions and motility. In this review, we will discuss the effects of ubiquitination and deubiquitination on protein stability and expression of key proteins in the cell-cell junctions, including junction proteins, their interacting proteins, and small Rho GTPases. We provide an overview of protein stability in modulation of epithelial and endothelial barrier integrity and introduce potential future search directions to better understand the effects of ubiquitination on human disorders caused by dysfunction of cell junctions.     

USP7 promotes cell proliferation through the stabilization of Ki-67 protein in non-small cell lung cancer cells

The Ki-67 antigen (Ki-67) is the most reliable immunohistochemical marker for evaluation of cell proliferation in non-small cell lung cancer. However, the mechanisms underlying the regulation of protein levels of Ki-67 in non-small cell lung cancer have remained elusive. In this study, we found that Ki-67 and ubiquitin-specific processing protease 7 (USP7) protein were highly expressed in the nucleus of non-small cell lung cancer cells. Furthermore, statistical analysis uncovered the existence of a strong correlation between Ki-67 and USP7 levels. We could also show that the protein levels of Ki-67 in non-small cell lung cancer cells significantly decreased after treatment with P22077, a selective chemical inhibitor of USP7, while the Ki-67 mRNA levels were unperturbed. Similar results were obtained by knocking down USP7 using short hairpin RNA (shRNA) in lung cancer cells. Interestingly, we noticed that ubiquitination levels of Ki-67 increased dramatically in USP7-silenced cells. The tests in vitro and vivo showed a significant delay in tumor cell growth upon knockdown of USP7. Additionally, drug sensitivity tests indicated that USP7-silenced A549 cells had enhanced sensitivity to paclitaxel and docetaxel, while there was no significant change in sensitivity toward carboplatin and cisplatin. Taken together, these data strongly suggest that the overexpression of USP7 might promote cell proliferation by deubiquitinating Ki-67 protein, thereby maintaining its high levels in the non-small cell lung cancer. Our study also hints potential for the development of deubiquitinase-based therapies, especially those targeting USP7 to improve the condition of patients diagnosed with non-small cell lung cancer.     

The ubiquitin E3 ligase MARCH7 is differentially regulated by the deubiquitylating enzymes USP7 and USP9X

Protein modification by one or more ubiquitin chains serves a critical signalling function across a wide range of cellular processes. Specificity within this system is conferred by ubiquitin E3 ligases, which target the substrates. Their activity is balanced by deubiquitylating enzymes (DUBs), which remove ubiquitin from both substrates and ligases. The RING-CH ligases were initially identified as viral immunoevasins involved in the downregulation of immunoreceptors. Their cellular orthologues, the Membrane-Associated RING-CH (MARCH) family represent a subgroup of the classical RING genes. Unlike their viral counterparts, the cellular RING-CH proteins appear highly regulated, and one of these in particular, MARCH7, was of interest because of a potential role in neuronal development and lymphocyte proliferation. Difficulties in detection and expression of this orphan ligase lead us to search for cellular cofactors involved in MARCH7 stability. In this study, we show that MARCH7 readily undergoes autoubiquitylation and associates with two deubiquitylating enzymes – ubiquitin-specific protease (USP)9X in the cytosol and USP7 in the nucleus. Exogenous expression and short interfering RNA depletion experiments demonstrate that MARCH7 can be stabilized by both USP9X and USP7, which deubiquitylate MARCH7 in the cytosol and nucleus, respectively. We therefore demonstrate compartment-specific regulation of this E3 ligase through recruitment of site-specific DUBs.     

The ubiquitin-specific protease USP2a enhances tumor progression by targeting cyclin A1 in bladder cancer

The deubiquitinating enzyme USP2a has shown oncogenic properties in many cancer types by impairing ubiquitination of FASN, MDM2, MDMX or Aurora A. Aberrant expression of USP2a has been linked to progression of human tumors, particularly prostate cancer. However, little is known about the role of USP2a or its mechanism of action in bladder cancer. Here, we provide evidence that USP2a is an oncoprotein in bladder cancer cells. Enforced expression of USP2a caused enhanced proliferation, invasion, migration and resistance to several chemotherapeutic reagents, while USP2a loss resulted in slower proliferation, greater chemosensitivity and reduced migratory/invasive capability compared with control cells. USP2a, but not a catalytically inactive mutant, enhanced proliferation in immortalized TRT-HU1 normal human bladder epithelial cells. USP2a bound to cyclin A1 and prevented cyclin A1 ubiquitination, leading to accumulation of cyclin A1 by a block in degradation. Enforced expression of wild type USP2a, but not an inactive USP2a mutant, resulted in cyclin A1 accumulation and increased cell proliferation. We conclude that USP2a impairs ubiquitination and stabilizes an important cell cycle regulator, cyclin A1, raising the possibility of USP2a targeting as a therapeutic strategy against bladder tumors in combination with chemotherapy.     

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.     

Deubiquitylation and stabilization of ARMC5 by ubiquitin-specific processing protease 7 (USP7) are critical for RCC proliferation

The ubiquitin-proteasome system is an essential regulator of ARMC5, which serves as a new tumour suppressor protein for inhibiting meningiomas and hereditary adrenocortical tumorigenesis. However, the precise mechanism for the deubiquitination of ARMC5 is still not fully understood. A Western blot analysis of ARMC5 was performed and showed that the expression of ARMC5 was decreased in the renal cancer cell tissues and lines. By screening a deubiquitinase library, we identified USP7 as a potential ARMC5 associated deubiquitinase. In this paper, we demonstrated that there was an interaction between USP7 and ARMC5 in vivo and in vitro. Employing the overexpression and knockdown assay indicated that USP7 could greatly increase the steady state of ARMC5 through the ubiquitin-proteasome pathway and regulate ARMC5 ubiquitination. Moreover, USP7 altered cell cycle G1/S phases and regulated renal cancer cell proliferation by targeting ARMC5. Together, these results suggest that USP7 plays an important role in the RCC proliferation through modulating ARMC5 stability.     

USP24-GSDMB complex promotes bladder cancer proliferation via activation of the STAT3 pathway

Background: Bladder cancer is the fourth and tenth most common malignancy in men and women worldwide, respectively. One of the main reasons for the unsatisfactory therapeutic control of bladder cancer is that the molecular biological mechanism of bladder cancer is complex. Gasdermin B (GSDMB) is one member of the gasdermin family and participates in the regulation of cell pyroptosis. The role of GSDMB in bladder cancer has not been studied to date.Methods: TCGA database was used to exam the clinical relevance of GSDMB. Functional assays such as MTT assay, Celigo fluorescent cell-counting assay, Annexin V-APC assay and xenografts were used to evaluate the biological role of GSDMB in bladder cancer. Mass spectrometry and immunoprecipitation were used to detect the protein interaction between GSDMB and STAT3, or GSDMB and USP24. Western blot and immunohistochemistry were used to study the relationship between USP24, GSDMB and STAT3.Results: In this study, bioinformatics analysis indicated that the mRNA expression level of GSDMB in bladder cancer tissues was higher than that in adjacent normal tissues. Then, we showed that GSDMB promoted bladder cancer progression. Furthermore, we demonstrated that GSDMB interacted with STAT3 to increase the phosphorylation of STAT3 and modulate the glucose metabolism and promote tumor growth in bladder cancer cells. Besides, we also showed that USP24 stabilized GSDMB to activate STAT3 signaling, which was blocked by the USP24 inhibitor.Conclusions: We suggested that aberrantly up-regulated GSDMB was responsible for enhancing the growth and invasion ability of bladder cancer cells. Then, we showed that GSDMB could bind to STAT3 and activate STAT3 signaling in bladder cancer. Furthermore, we also demonstrated that USP24 interacted with GSDMB and prevented GSDMB from degradation in bladder cancer cells. Therefore, the USP24/GSDMB/STAT3 axis may be a new targetable signaling pathway for bladder cancer treatment.     

Protein stability of mitochondrial superoxide dismutase SOD2 is regulated by USP36

SOD2 is a key mitochondrial antioxidant enzyme and its perturbation leads to oxidative cell death, which results in various disorders. In this study, we identified a deubiquitinating enzyme USP36 that regulates the protein stability of SOD2. The regulatory effect of USP36 on SOD2 was initially identified by 2-DE and MALDI-TOF/MS analyses. In addition, endogenous USP36 and SOD2 were shown to interact in an immunoprecipitation assay, which was verified using the yeast two-hybrid system. Furthermore, we demonstrated that SOD2 binds with ubiquitin molecules to form polyubiquitination chains and undergoes degradation through the ubiquitin-proteasomal pathway. Finally, USP36 was shown to be a specific deubiquitinating enzyme that reduces the ubiquitination level of SOD2 and was involved in SOD2 protein stability by extending its half-life.     

Propofol suppresses proliferation,invasion, and migration of human melanoma cells via regulating microRNA-137 and fibroblast growth factor 9

Propofol is an intravenous anesthetic widely used in clinical surgeries, such as tumor resection. Propofol affects the growth of many cancers, though its effect on melanoma is unknown. Our study aimed to explore how propofol affects melanoma cells. Melanoma cells A2058 and WM793B were cultured with propofol for 24 hr. Propofol significantly suppressed proliferation, migration, and invasion of A2058 and WM793B cells. Lower miR-137 level was observed in A2058 and WM793B cells, compared with normal human epidermal melanocyte HEMa-LP cells. Propofol-induced miR-137 upregulation and decreased proliferation, invasive ability, and migrated ability of A2058 and WM793B cells. Transfection with the miR-137 inhibitor reversed these effects. Additionally, miR-137 was verified to target and negatively regulate fibroblast growth factor 9 (FGF9) expression. Propofol efficiently downregulated FGF9 protein expression by upregulating miR-137. Furthermore, FGF9 overexpression abrogated propofol's repressive effects on the malignant potential of A2058 and WM793B cells. These findings indicate that propofol suppressed melanoma cell proliferation, invasion, and migration by regulating miR-137 and FGF9.     

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.