A protein sequence-based deep transfer learning framework for identifying human proteome-wide deubiquitinase-substrate interactions.

Protein ubiquitination regulates a wide range of cellular processes. The degree of protein ubiquitination is determined by the delicate balance between ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase (DUB)-mediated deubiquitination. In comparison to the E3-substrate interactions, the DUB-substrate interactions (DSIs) remain insufficiently investigated. To address this challenge, we introduce a protein sequence-based ab initio method, TransDSI, which transfers proteome-scale evolutionary information to predict unknown DSIs despite inadequate training datasets. An explainable module is integrated to suggest the critical protein regions for DSIs while predicting DSIs. TransDSI outperforms multiple machine learning strategies against both cross-validation and independent test. Two predicted DUBs (USP11 and USP20) for FOXP3 are validated by "wet lab" experiments, along with two predicted substrates (AR and p53) for USP22. TransDSI provides new functional perspective on proteins by identifying regulatory DSIs, and offers clues for potential tumor drug target discovery and precision drug application.

Discovery of an OTUD3 inhibitor for the treatment of non-small cell lung cancer.

The ubiquitin-proteasome system (UPS) controls protein turnover, and its dysfunction contributes to human diseases including cancer. Deubiquitinating enzymes (DUBs) remove ubiquitin from proteins to maintain their stability. Inhibition of DUBs could induce the degradation of selected oncoproteins and has therefore become a potential therapeutic strategy for cancer. The deubiquitylase OTUD3 was reported to promote lung tumorigenesis by stabilizing oncoprotein GRP78, implying that inhibition of OTUD3 may be a therapeutic strategy for lung cancer. Here, we report a small-molecule inhibitor of OTUD3 (named OTUDin3) by computer-aided virtual screening and biological experimental verification. OTUDin3 exhibited pronounced antiproliferative and proapoptotic effects by inhibiting deubiquitinating activity of OTUD3 in non-small-cell lung cancer (NSCLC) cell lines. Moreover, OTUDin3 efficaciously inhibited growth of lung cancer xenografts in mice. In summary, our results support OTUDin3 as a potent inhibitor of OTUD3, the inhibition of which may be a promising therapeutic strategy for NSCLC.

All-Trans Retinoic Acid Promotes a Tumor Suppressive OTUD6B-ß-TrCP-SNAIL Axis in Esophageal Squamous Cell Carcinoma and Enhances Immunotherapy.

ß-TrCP is an E3 ubiquitin ligase that plays important roles in multiple human cancers including esophageal squamous cell carcinoma (ESCC). Analysis of ESCC patient samples reveal that only protein level but not transcript level of ß-TrCP associated with patient prognosis, suggesting regulators of ß-TrCP protein stability play an essential role in ESCC progression and may be novel targets to develop ESCC therapies. Although ß-TrCP stability is known to be mediated by the ubiquitin-proteasome system, it is unclear which enzymes play a major role to determine ß-TrCP stability in the context of ESCC. In this study, OTUD6B is identified as a potent deubiquitinase of ß-TrCP that suppress ESCC progression through the OTUD6B-ß-TrCP-SNAIL axis. Low OTUD6B expression is associated with a poor prognosis of ESCC patients. Importantly, all-trans retinoic acid (ATRA) is found to promote OTUD6B translation and thus suppress ESCC tumor growth and enhance the response of ESCC tumors to anti-PD-1 immunotherapies. These findings demonstrate that OTUD6B is a crucial deubiquitinase of ß-TrCP in ESCC and suggest combination of ATRA and anti-PD-1 immune checkpoint inhibitor may benefit a cohort of ESCC patients.

OTUB1 promotes osteoblastic bone formation through stabilizing FGFR2.

Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Dysregulation of this process leads to multiple diseases, including osteoporosis. However, the underlying molecular mechanisms are not fully understood. Here, we show that the global and conditional osteoblast knockout of a deubiquitinase Otub1 result in low bone mass and poor bone strength due to defects in osteogenic differentiation and mineralization. Mechanistically, the stability of FGFR2, a crucial regulator of osteogenesis, is maintained by OTUB1. OTUB1 attenuates the E3 ligase SMURF1-mediated FGFR2 ubiquitination by inhibiting SMURF1's E2 binding. In the absence of OTUB1, FGFR2 is ubiquitinated excessively by SMURF1, followed by lysosomal degradation. Consistently, adeno-associated virus serotype 9 (AAV9)-delivered FGFR2 in knee joints rescued the bone mass loss in osteoblast-specific Otub1-deleted mice. Moreover, Otub1 mRNA level was significantly downregulated in bones from osteoporotic mice, and restoring OTUB1 levels through an AAV9-delivered system in ovariectomy-induced osteoporotic mice attenuated osteopenia. Taken together, our results suggest that OTUB1 positively regulates osteogenic differentiation and mineralization in bone homeostasis by controlling FGFR2 stability, which provides an optical therapeutic strategy to alleviate osteoporosis.

Destabilization of TP53 by USP10 is essential for neonatal autophagy and survival.

Autophagy is essential for the maintenance of energy homeostasis and for survival during the neonatal starvation period. At birth, the trans-placental nutrient supply is suddenly interrupted, and neonates adapt to this adverse circumstance by activating autophagy. However, the mechanisms underlying the precise regulation of neonatal autophagy remain undefined. Here, we show that the destabilization of TP53 by the deubiquitylase ubiquitin-specific peptidase 10 (USP10) is essential for neonatal autophagy and survival. Usp10 deficiency results in decreased E3 ligase activity of MDM2 and accumulation of cytoplasmic TP53, which interferes with the conjugation of ATG12 and ATG5, the key autophagy-related genes, and ultimately inhibits autophagy in neonatal mice. Combined deletion of Tp53 and Usp10 recovers the nutrition supply and rescues the death phenotype of Usp10-deficient neonates. These findings reveal a role of the USP10-MDM2-TP53 axis in nutrient homeostasis and neonatal viability and provide insights into the long-perplexing mechanism by which cytoplasmic TP53 inhibits autophagy.

Deubiquitylase OTUD6B stabilizes the mutated pVHL and suppresses cell migration in clear cell renal cell carcinoma.

Von Hippel-Lindau (VHL) is an important tumor suppressor, and its inactivation is a hallmark of inherited VHL disease and most sporadic clear cell renal cell carcinoma (ccRCC). VHL protein (pVHL) with missense point mutations are unstable and degraded by the proteasome because of the disruption of elongin binding. Deubiquitylase ovarian tumor domain-containing 6B (OTUD6B) had been documented to couple pVHL and elongin B to form stable VHL - elonginB - elonginC complex, which protects pVHL from degradation. However, whether OTUD6B governs the stability of pVHL wild type and the missense mutants in ccRCC remains largely elusive. Here, we reported that low OTUD6B level predicted poorer survival in ccRCC patients with VHL missense mutation, but not frameshift deletion and nonsense mutation. OTUD6B is able to interact with wild type pVHL and tumor-derived pVHL missense mutants, except for pVHL I151T, and decrease their ubiquitylation and proteasomal degradation in ccRCC cells. Functionally, we revealed that OTUD6B depletion enhanced cell migration and HIF-2α level in ccRCC cells in a pVHL dependent manner. In addition, OTUD6B depletion reduced the inhibitory effects of ectopic pVHL missense mutants on cell migration and HIF-2α level, except for pVHL I151T. Thus, we speculated that I151 residue might be one of key sites of pVHL binding to OTUD6B. These results suggested that OTUD6B is an important regulator for the stability of pVHL missense mutants, which provides a potential therapeutic strategy for ccRCC with VHL mutations.

The deubiquitinase OTUD1 inhibits colonic inflammation by suppressing RIPK1-mediated NF-κB signaling.

The E3 ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase (DUB)-mediated deubiquitination processes are closely associated with the occurrence and development of colonic inflammation. Ovarian tumor deubiquitinase 1 (OTUD1) is involved in immunoregulatory functions linked to infectious diseases. However, the effect of OTUD1 on intestinal immune responses during colonic inflammatory disorders such as inflammatory bowel disease (IBD) remains unclear. Here, we show that loss of OTUD1 in mice contributes to the pathogenesis of dextran sulfate sodium (DSS)-induced colitis via excessive release of proinflammatory cytokines. In addition, bone marrow transplantation experiments revealed that OTUD1 in hematopoietic cells plays a dominant role in protection against colitis. Mechanistically, OTUD1 physically interacts with receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and selectively cleaves K63-linked polyubiquitin chains from RIPK1 to inhibit the recruitment of NF-κB essential modulator (NEMO). Moreover, the expression of OTUD1 in mucosa samples from ulcerative colitis (UC) patients was lower than that in mucosa samples from healthy controls. Furthermore, we demonstrate that the UC-associated OTUD1 G430V mutation abolishes the ability of OTUD1 to inhibit RIPK1-mediated NF-κB activation and intestinal inflammation. Taken together, our study unveils a previously unexplored role of OTUD1 in moderating intestinal inflammation by inhibiting RIPK1-mediated NF-κB activation, suggesting that the OTUD1-RIPK1 axis could be a potential target for the treatment of IBD.

Splicing factor Srsf5 deletion disrupts alternative splicing and causes noncompaction of ventricular myocardium.

The serine/arginine-rich (SR) family of splicing factors plays important roles in mRNA splicing activation, repression, export, stabilization, and translation. SR-splicing factor 5 (SRSF5) is a glucose-inducible protein that promotes tumor cell growth. However, the functional role of SRSF5 in tissue development and disease remains unknown. Here, Srsf5 knockout (Srsf5 -/- ) mice were generated using CRISPR-Cas9. Mutant mice were perinatally lethal and exhibited cardiac dysfunction with noncompaction of the ventricular myocardium. The left ventricular internal diameter and volume were increased in Srsf5 -/- mice during systole. Null mice had abnormal electrocardiogram patterns, indicative of a light atrioventricular block. Mechanistically, Srsf5 promoted the alternative splicing of Myom1 (myomesin-1), a protein that crosslinks myosin filaments to the sarcomeric M-line. The switch between embryonic and adult isoforms of Myom1 could not be completed in Srsf5-deficient heart. These findings indicate that Srsf5-regulated alternative splicing plays a critical role during heart development.

OTULIN allies with LUBAC to govern angiogenesis by editing ALK1 linear polyubiquitin.

OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.

Neddylation of PTEN regulates its nuclear import and promotes tumor development.

PTEN tumor suppressor opposes the PI3K/Akt signaling pathway in the cytoplasm and maintains chromosomal integrity in the nucleus. Nucleus-cytoplasm shuttling of PTEN is regulated by ubiquitylation, SUMOylation and phosphorylation, and nuclear PTEN has been proposed to exhibit tumor-suppressive functions. Here we show that PTEN is conjugated by Nedd8 under high glucose conditions, which induces PTEN nuclear import without effects on PTEN stability. PTEN neddylation is promoted by the XIAP ligase and removed by the NEDP1 deneddylase. We identify Lys197 and Lys402 as major neddylation sites on PTEN. Neddylated PTEN accumulates predominantly in the nucleus and promotes rather than suppresses cell proliferation and metabolism. The nuclear neddylated PTEN dephosphorylates the fatty acid synthase (FASN) protein, inhibits the TRIM21-mediated ubiquitylation and degradation of FASN, and then promotes de novo fatty acid synthesis. In human breast cancer tissues, neddylated PTEN correlates with tumor progression and poor prognosis. Therefore, we demonstrate a previously unidentified pool of nuclear PTEN in the Nedd8-conjugated form and an unexpected tumor-promoting role of neddylated PTEN.

Ubiquitin ligase CHIP regulates OTUD3 stability and suppresses tumour metastasis in lung cancer.

Ovarian tumour domain-containing protein 3 (OTUD3), a key OTU (ovarian tumour protease) family deubiquitylase, plays context-dependent roles in cancers. It suppresses tumorigenesis in breast, colon, liver and cervical cancer through stabilizing PTEN (phosphatase and tension homologue deleted on chromosome 10) while promotes lung tumorigenesis through stabilizing GRP78 (The glucose-regulated protein 78 kDa). The regulation especially post-translational modification of OTUD3 remains unclear. Here, we report that the carboxyl terminus of Hsc70-interacting protein (CHIP) is a ubiquitin ligase for OTUD3. CHIP interacts with, polyubiquitylates OTUD3 and promotes OTUD3 degradation. Knockdown of CHIP stabilizes OTUD3 which leads to elevated GRP78 levels in lung cancer cells. CHIP-knockdown lung cancer cells exhibit increased invasion in OTUD3 and GRP78 dependent manner. Further study demonstrates that CHIP-knockdown lung cancer cells are more prone to metastasize to mice lung when injected intravenously or subcutaneously. Moreover, the expression of CHIP is low in human lung cancer tissues and inversely correlates with OTUD3 expression and GRP78 expression. Furthermore, we identified CHIP mutations in human lung cancers, which reduce CHIP catalytic activity. These findings demonstrate that CHIP is a negative regulator of OTUD3 and CHIP suppresses lung cancer metastasis through inhibiting OTUD3-GRP78 signaling axis.

Deubiquitylase OTUD6B Governs pVHL Stability in an Enzyme-Independent Manner and Suppresses Hepatocellular Carcinoma Metastasis.

Hypoxia inducible factors (HIFs) are the key transcription factors that allow cancer cells to survive hypoxia. HIF's stability is mainly controlled by von Hippel-Lindau (pVHL)-mediated ubiquitylation. Unlike sporadic clear-cell renal carcinomas, VHL mutation is rarely observed in hepatocellular carcinoma (HCC) and the regulatory mechanisms of pVHL-HIF signaling remain elusive. Here, it is shown that deubiquitylase ovarian tumor domain-containing 6B (OTUD6B) suppresses HCC metastasis through inhibiting the HIF activity. OTUD6B directly interacts with pVHL, decreases its ubiquitylation and proteasomal degradation to reduce HIF-1α accumulation in HCC cells under hypoxia. Surprisingly, OTUD6B limits the ubiquitylation of pVHL independent of its deubiquitylase activity. OTUD6B couples pVHL and elongin B/C to form more CBCVHL ligase complex, which protects pVHL from proteasomal degradation. Depletion of OTUD6B results in the dissociation of CBCVHL complex and the degradation of pVHL by Trp Asp repeat and suppressors of cytokine signaling box-containing protein 1 (WSB1). In human HCC tissues, the protein level of OTUD6B is positively correlated with pVHL, but negatively with HIF-1α and vascular endothelial growth factor. Low expression of OTUD6B predicts poor patient survival. Furthermore, OTUD6B gene is a direct transcriptional target of HIF-1α and upregulated upon hypoxia. These results indicate a previously unrecognized feedback loop consisting of OTUD6B, pVHL, and HIF-1α, and provide insights into the targeted hypoxic microenvironment for HCC therapy.

Regulation of Stem Cells by Cullin-RING Ligase.

Stem cells can remain quiescent, self-renewal, and differentiate into many types of cells and even cancer stem cells. The coordination of these complex processes maintains the homeostasis of the organism. Ubiquitination is an important posttranslational modification process that regulates protein stability and activity. The ubiquitination levels of stem cell-associated proteins are closely related with stem cell characteristics. Cullin-RING Ligases (CRLs) are the largest family of E3 ubiquitin ligases, accounting for approximately 20% of proteins degraded by proteasome. In this review, we discuss the role of CRLs in stem cell homeostasis, self-renewal, and differentiation and expound their ubiquitination substrates. In addition, we also discuss the effect of CRLs on the formation of cancer stem cells that may provide promising therapy strategies for cancer.

The functions and regulation of Smurfs in cancers.

Smad ubiquitination regulatory factor 1 (Smurf1) and Smurf2 are HECT-type E3 ubiquitin ligases, and both Smurfs were initially identified to regulate Smad protein stability in the TGF-ß/BMP signaling pathway. In recent years, Smurfs have exhibited E3 ligase-dependent and -independent activities in various kinds of cells. Smurfs act as either potent tumor promoters or tumor suppressors in different tumors by regulating biological processes, including metastasis, apoptosis, cell cycle, senescence and genomic stability. The regulation of Smurfs activity and expression has therefore emerged as a hot spot in tumor biology research. Further, the Smurf1- or Smurf2-deficient mice provide more in vivo clues for the functional study of Smurfs in tumorigenesis and development. In this review, we summarize these milestone findings and, in turn, reveal new avenues for the prevention and treatment of cancer by regulating Smurfs.

Functional analysis of deubiquitylating enzymes in tumorigenesis and development.

Deubiquitylating enzymes (DUBs) are proteases that remove the ubiquitin moiety from ubiquitylated substrates to antagonize the modification mediated by E3 ubiquitin ligases. Currently, DUBs have been found to play critical roles in the regulation of various physiological or pathological processes, such as embryogenesis, immune homeostasis, tumorigenesis and neurodegenerative diseases. Accumulating evidences have suggested that different DUBs exert distinct function such as oncogenic, tumor-suppressive or context-dependent roles in tumorigenesis, mainly by affecting the protein stability, enzymatic activity or subcellular localization of its substrates. Importantly, multiple potent inhibitors targeting the enzymatic activity of oncogenic DUBs have been developed and show promising anti-cancer efficacy in preclinical models. Thus, exploring the unique role of DUB enzymes and their downstream effectors will provide novel insights into the molecular basis of cancer development. Here, we review and summarize recent progress on DUB functional annotation, as well as its biochemical regulation, to provide a better understanding for cancer therapies by targeting DUBs.

Hepatopoietin Cn (HPPCn) Generates Protective Effects on Acute Liver Injury.

Objective: To observe the protective role of hapatopoietin Cn (HPPcn) on acute liver injury. Methods: Six hours after 10 mmol/L CCl4, 150 mmol/L ethanol, or 0.6 mmol/L H2O2 treatment, SMMC7721 human hepatoma cells were incubated with 10, 100, or 200 ng/ml recombinant human HPPCn protein (rhHPPCn) for an additional 24 h. The cell survival rate was analyzed using the CCK-8 assay. The CCl4-induced apoptosis of SMMC7721 cells was detected by flow cytometry. Then, the levels of glutamic oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), malondialdehyde (MDA), lactate dehydrogenase (LDH), glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD) in SMMC7721 cell lysates and cell culture supernatant were detected. SMMC7721 cells were treated with different concentrations of rhHPPCn (0, 10, and 100 ng/ml). The cell proliferation indexes (BrdU incorporation and PCNA expression) were detected by immunohistochemistry (IHC). An acute liver injury mouse model was established by a one-time intraperitoneal injection of 20% CCl4 at a volume of 5 ml/kg body weight. One hour after CCl4 injection, 1.25 or 2.5 mg rhHPPCn/12 h/kg body weight was injected via the tail vein. The serum levels of GOT and GPT were detected at different time points. Pathological changes in the liver were evaluated. PCNA expression levels were observed by IHC. Results: rhHPPCn increased the survival rate of SMMC7721 cells and inhibited chemical toxicity-induced cell apoptosis. The levels of GOT, GPT, MDA, and LDH in the cell supernatant were significantly reduced, while GSH-PX and SOD were significantly increased after rhHPPCn treatment in the CCl4-treated SMMC7721 cells. BrdU incorporation and PCNA expression increased in a concentration-dependent manner, indicating that rhHPPCn promotes cell proliferation. The results showed that rhHPPCn significantly reduced the serum levels of GOT and GPT in CCl4-induced acute liver injury mice. rhHPPCn alleviated the tissue damage and increased PCNA expression, indicating the promotion of proliferation after acute injury. Conclusion: rhHPPCn protects hepatocytes from chemical toxins by promoting proliferation and inhibiting apoptosis in vivo and in vitro. Our study provides new insights for the clinical treatment of acute liver injury.

The deubiquitylase OTUD3 stabilizes GRP78 and promotes lung tumorigenesis.

The deubiquitylase OTUD3 plays a suppressive role in breast tumorigenesis through stabilizing PTEN protein, but its role in lung cancer remains unclear. Here, we demonstrate that in vivo deletion of OTUD3 indeed promotes breast cancer development in mice, but by contrast, it slows down KrasG12D-driven lung adenocarcinoma (ADC) initiation and progression and markedly increases survival in mice. Moreover, OTUD3 is highly expressed in human lung cancer tissues and its higher expression correlates with poorer survival of patients. Further mechanistic studies reveal that OTUD3 interacts with, deubiquitylates and stabilizes the glucose-regulated protein GRP78. Knockdown of OTUD3 results in a decrease in the level of GRP78 protein, suppression of cell growth and migration, and tumorigenesis in lung cancer. Collectively, our results reveal a previously unappreciated pro-oncogenic role of OTUD3 in lung cancer and indicate that deubiquitylases could elicit tumor-suppressing or tumor-promoting activities in a cell- and tissue-dependent context.

VCP/p97 increases BMP signaling by accelerating ubiquitin ligase Smurf1 degradation.

The bone morphogenetic protein (BMP)-Smad signaling pathway plays a crucial role in the control of bone homeostasis by regulating osteoblast activity. It is known that the ubiquitin ligase Smad ubiquitination regulatory factor (Smurf)1 is a master negative regulator of BMP signaling, but how its stability and activity are regulated remains poorly understood. Our study showed that valosin-containing protein/p97, the mutations of which lead to rare forms of Paget's disease of bone (PDB)-like syndrome-such as inclusion body myopathy (IBM) associated with Paget's disease of bone and frontotemporal dementia (IBM-PFD)-together with its adaptor nuclear protein localization (NPL)4, specifically interact with Smurf1 and deliver the ubiquitinated Smurf1 for degradation. Depletion of either p97 or NPL4 resulted in the elevation of Smurf1 protein level and decreased BMP signaling accordingly. Mechanically, a typical proline, glutamic acid, serine, and threonine motif specifically existing in Smurf1 is necessary for its recognition and degradation by p97, and this process is dependent on p97 ATPase activity. More importantly, compared with p97 WT, PDB-associated mutation of p97 (mainly A232E) harboring the higher ATPase activity of p97 further promoted Smurf1 degradation, thus increasing BMP signaling activity. Our findings first establish a link between p97 and Smurf1, providing an in-depth understanding of how Smurf1 is regulated, as well as the mechanism of p97-related bone diseases.-Li, H., Cui, Y., Wei, J., Liu, C., Chen, Y., Cui, C.-P., Li, L., Zhang, X., Zhang, L. VCP/p97 increases BMP signaling by accelerating ubiquitin ligase Smurf1 degradation.