RNA binding protein ELAVL1-mediated USP33 stabilizes HIF1A to promote pathological proliferation, migration and angiogenesis of RECs.

BACKGROUND: Dysfunction of retinal vascularization plays pathogenic roles in retinopathy of prematurity (ROP). Hypoxia-inducible factor 1 alpha (HIF1A) is activated by hypoxia and contributes to ROP progression. Herein, we clarified the mechanism underlying HIF1A activation in human retinal vascular endothelial cells (HRECs) under hypoxia. METHODS: Protein expression was assayed by immunoblot analysis. Cell migration, microtubule formation, invasion, proliferation, and viability were detected by wound-healing, tube formation, transwell, EdU, and CCK-8 assays, respectively. Bioinformatics was used to predict the deubiquitinase-HIF1A interactions and RNA binding proteins (RBPs) bound to USP33. The impact of USP33 on HIF1A deubiquitination was validated by immunoprecipitation (IP) assay. RNA stability analysis was performed with actinomycin D (Act D) treatment. The ELAVL1/USP33 interaction was assessed by RNA immunoprecipitation experiment. RESULTS: In hypoxia-exposed HRECs, HIF1A and USP33 protein levels were upregulated. Deficiency of HIF1A or USP33 suppressed cell migration, proliferation and microtubule formation of hypoxia-exposed HRECs. Mechanistically, USP33 deficiency led to an elevation in HIF1A ubiquitination and degradation. USP33 deficiency reduced HIF1A protein levels to suppress the proliferation and microtubule formation of hypoxia-induced HRECs. Moreover, the RBP ELAVL1 stabilized USP33 mRNA to increase USP33 protein levels. ELAVL1 decrease repressed the proliferation and microtubule formation of hypoxia-induced HRECs by reducing USP33. CONCLUSION: Our study identifies a novel ELAVL1/USP33/HIF1A regulatory cascade with the ability to affect hypoxia-induced pathological proliferation, angiogenesis, and migration in HRECs.

The deubiquitinase OTUB1 inhibits gluconeogenesis by stabilizing YWHAB.

Hepatic gluconeogenesis plays a crucial role in maintaining glucose homeostasis and serves as a potential therapeutic target for type 2 diabetes, while its underlying mechanisms are not fully understood. This study elucidates the role of the deubiquitinase OTU domain-containing ubiquitin aldehyde binding protein 1 (OTUB1) in gluconeogenesis. We found that hepatic OTUB1 expression is reduced in both db/db mice and patients with type 2 diabetes. Deletion of hepatic OTUB1 significantly elevates fasting blood glucose levels and increases the expression of key gluconeogenic genes. Conversely, overexpression of OTUB1 in hepatocytes mitigates diabetic hyperglycemia and enhances insulin sensitivity. It is known that the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein ß (YWHAB) functions as an inhibitor of hepatic gluconeogenesis by interacting with forkhead box protein O (FOXO1) and glucagon receptor (GPCR), but its own modification mechanism remains unclear. Our findings indicate that OTUB1 interacts with YWHAB and deubiquitinates it through a catalytic process, which in turn suppresses gluconeogenesis. Therefore, OTUB1 plays a pivotal role in inhibiting hepatic gluconeogenesis, highlighting its potential as a therapeutic target for type 2 diabetes.

The ubiquitin-specific protease 21 is critical for cancer cell mitochondrial function and regulates proliferation and migration.

Ubiquitin-Specific Peptidases (USPs) are the main members of deubiquitinases (DUBs) that catalyze removing ubiquitin chains from target proteins, thereby modulating their half-life and function. Enzymatic activity of USP21 regulates protein degradation which is critical for maintaining cell homeostasis. USP21 determines the stability of oncogenic proteins and therefore is implicated in carcinogenesis. In this study, we investigated the effect of USP21 deletion on cancer cell metabolism. Transcriptomic and proteomic analysis of USP21 knockout HAP-1 cells revealed that endogenous USP21 is critical for the expression of genes and proteins involved in mitochondrial function. Additionally, we have found that deletion of USP21 reduced STAT3 activation and STAT3-dependent gene and protein expression in cancer cells. Genetic deletion of USP21 impaired mitochondrial respiration and disturbed ATP production. This resulted in cellular consequences such as inhibition of cell proliferation and migration. Presented results provide new insights into the biology of USP21, suggesting novel mechanisms for controlling STAT3 activity and mitochondrial function in tumor cells. Taken together, our findings indicate that targeting USP21 dysregulates the energy status of cancer cells offering new perspectives for anti-cancer therapy.

Protein ubiquitination in ovarian cancer immunotherapy: The progress and therapeutic strategy.

Ovarian cancer is a common cancer for females, and the incidence and mortality rates are on the rise. Many treatment strategies have been developed for ovarian cancer, including chemotherapy and immunotherapy, but they are often ineffective and prone to drug resistance. Protein ubiquitination is an important class of post-translation modifications that have been found to be associated with various human diseases and cancer development. Recent studies have revealed that protein ubiquitination is involved in the progression of ovarian cancer and plays an important role in the tumor immune process. Moreover, the combination of ubiquitinase/deubiquitinase inhibitors and cancer immunotherapy approaches can effectively reduce treatment resistance and improve treatment efficacy, which provides new ideas for cancer treatment. Herein, we review the role of protein ubiquitination in relation to ovarian cancer immunotherapy and recent advances in the use of ubiquitinase/deubiquitinase inhibitors in combination with cancer immunotherapy.

USP11 deubiquitinates E-cadherin and maintains luminal fate of mammary tumor cells to suppress breast cancer.

Basal-like breast cancer may originate from luminal epithelial or cancerous cells. Inadequately repaired DNA damage impairs luminal differentiation and promotes aberrant luminal to basal trans-differentiation in mammary epithelial cells (MECs). Ubiquitin-specific peptidase 11 (USP11), a deubiquitinase, plays a critical role in DNA damage repair. The role of USP11 in controlling mammary cell differentiation and tumorigenesis remains poorly understood. We generated Usp11 knockout mice and breast cancer cell lines expressing wild-type (WT) and mutant form of USP11. By using these mutant mice, cell lines, and human USP11-deficient and -proficient breast cancer tissues, we tested how USP11 controls mammary cell fate. We generated Usp11 knock-out mice and found that deletion of Usp11 reduced the expression of E-cadherin and promoted DNA damage in MECs. Overexpression of WT USP11, but not a deubiquitinase-inactive mutant form of USP11, promoted luminal differentiation, enhanced DNA damage repair, and suppressed tumorigenesis in mice. Mechanistically, we found that USP11 enhanced the protein expression of E-cadherin dependent on its deubiquitinase activity, and that USP11 deubiquitinated E-cadherin at K738. We discovered that USP11 bound to E-cadherin through its C-terminal region. In human breast cancers, expression of USP11 was positively correlated with that of E-cadherin, and high USP11 predicted better recurrence-free survival. Our findings provide compelling genetic and biochemical evidence that USP11 not only promotes DNA damage repair but also deubiquitinates E-cadherin and maintains the luminal feature of mammary tumor cells, thereby suppressing luminal breast cancer.

Role of UCHL3 in health and disease.

Ubiquitin C-terminal hydrolase 3 (UCHL3) is a cysteine protease that plays a crucial role in cell cycle regulation, DNA repair, and apoptosis by carrying out deubiquitination and deneddylation activities. It has emerged as a promising therapeutic target for certain cancers due to its ability to stabilize oncoproteins. The dysregulation of UCHL3 also has been associated with neurodegenerative diseases, underscoring its significance in maintaining protein homeostasis within cells. Research on UCHL3, including studies on Uchl3 knockout mice, has revealed its involvement in learning deficits, cellular stress responses, and retinal degeneration. This review delves into the cellular processes controlled by UCHL3 and its role in health and disease progression, as well as the development of UCHL3 inhibitors. Further investigation into the molecular mechanisms and physiological functions of UCHL3 is crucial for a comprehensive understanding of its impact on health and disease.

The plant extract PNS mitigates atherosclerosis via promoting Nrf2-mediated inhibition of ferroptosis through reducing USP2-mediated Keap1 deubiquitination.

BACKGROUND AND PURPOSE: Atherosclerosis is the basis of cardiovascular disease. Ferroptosis is a form of programmed cell death characterized by lipid peroxidation, which contributes to atherogenesis. The plant extract PNS (Panax notoginseng saponins), containing the main active ingredients of Panax notoginseng, exhibits anti-atherogenic properties. Herein, we determined whether PNS and its major components could attenuate atherosclerosis by suppressing ferroptosis and revealed the underlying mechanism(s). EXPERIMENTAL APPROACH: The anti-atherogenic effects of PNS and their association with inhibition of ferroptosis was determined in apoE-/- mice. In vitro, the anti-ferroptotic effect and mechanism(s) of PNS components were demonstrated in the presence of ferroptosis inducers. Expression of ferroptosis markers and the ubiquitination of Keap1 were evaluated in USP2-/- macrophages. Finally, the anti-atherogenic effect of USP2 knockout was determined by using USP2-/- mice treated with high-fat diet (HFD) and AAV-PCSK9. KEY RESULTS: PNS inhibited ferroptosis and atherosclerosis in vivo. PNS suppressed ferroptosis and ferroptosis-aggravated foam cell formation and inflammation in vitro. Mechanistically, PNS and its components activated Nrf2 by antagonizing Keap1, which was attributed to the inhibition of USP2 expression. USP2 knockout antagonized ferroptosis and ferroptosis-aggravated foam cell formation and inflammation, thus mitigating atherosclerosis. USP2 knockout abolished inhibitory effects of PNS on foam cell formation and inflammation in vitro. CONCLUSION AND IMPLICATIONS: PNS reduced USP2-mediated Keap1 de-ubiquitination and promoted Keap1 degradation, thereby activating Nrf2, improving iron metabolism and reducing lipid peroxidation, thus contributing to an anti-atherosclerotic outcome. Our study revealed the mechanism(s) underlying inhibition of ferroptosis and atherosclerosis by PNS.

Recruitment of USP10 by GCS1 to deubiquitinate GRP78 promotes the progression of colorectal cancer via alleviating endoplasmic reticulum stress.

BACKGROUND: Long-term accumulation of misfolded proteins leads to endoplasmic reticulum (ER) stress in colorectal cancer (CRC). However, the precise pathways controlling the decision between survival and apoptosis in CRC are unclear. Therefore, in this study, we investigated the function and molecular mechanism of glucosidase I (GCS1) in regulating ER stress in CRC. METHODS: A public database was used to confirm the expression level of GCS1 in CRC and normal tissues. Clinical samples from our center were used to confirm the mRNA and protein expression levels of GCS1. Cell proliferation, migration, invasion, and apoptosis assays revealed the biological role of GCS1. Immunohistochemical techniques were used to evaluate the expression of key proteins in subcutaneous implanted tumors in nude mice, which provided further evidence for the biological function of GCS1 in promoting cancer in vivo. The results of coimmunoprecipitation-mass spectrometry analysis and immunofluorescence colocalization analysis the interaction between GCS1 and GRP78. In addition, the mechanism of action of USP10, GRP78, and GCS1 at the post- translational level was investigated. Finally, a tissue microarray was used to examine the connection between GCS1 and GRP78 expression and intracellular localization of these proteins using immunohistochemistry and immunofluorescence. RESULTS: The experimental results revealed that GCS1 was substantially expressed in CRC, with higher expression indicating a worse prognosis. Thus, GCS1 can enhance the proliferation and metastasis while inhibiting the apoptosis of CRC cells both in vivo and in vitro. Mechanistically, GCS1 binds to GRP78, recruits USP10 for deubiquitination of GRP78 to promote its degradation, and decreases ER stress-mediated apoptosis, increasing CRC cell proliferation and metastasis. CONCLUSIONS: In summary, GCS1 stimulates CRC growth and migration and reduces ER stress-mediated apoptosis via USP10-mediated deubiquitination of GRP78. Our findings identify a possible therapeutic target for CRC.

Ubiquitination and De-Ubiquitination in the Synthesis of Cow Milk Fat: Reality and Prospects.

Ubiquitination modifications permit the degradation of labelled target proteins with the assistance of proteasomes and lysosomes, which is the main protein degradation pathway in eukaryotic cells. Polyubiquitination modifications of proteins can also affect their functions. De-ubiquitinating enzymes reverse the process of ubiquitination via cleavage of the ubiquitin molecule, which is known as a de-ubiquitination. It was demonstrated that ubiquitination and de-ubiquitination play key regulatory roles in fatty acid transport, de novo synthesis, and desaturation in dairy mammary epithelial cells. In addition, natural plant extracts, such as stigmasterol, promote milk fat synthesis in epithelial cells via the ubiquitination pathway. This paper reviews the current research on ubiquitination and de-ubiquitination in dairy milk fat production, with a view to providing a reference for subsequent research on milk fat and exploring new directions for the improvement of milk quality.

The Role of Deubiquitinating Enzymes in Primary Bone Cancer.

Bone is a living, intricate, and dynamic tissue providing locomotion and protection of the body. It also performs hematopoiesis and mineral homeostasis. Osteosarcoma (OS), Ewing sarcoma (ES), and chondrosarcoma (CS) are primary bone cancers. OS and ES mostly develop in younger individuals, and CS generally develops in adults. Ubiquitination regulates numerous cellular processes. The deubiquitinating enzymes (DUBs) detach the ubiquitin molecules from the ubiquitin labeled substrate, altering ubiquitinated protein functions and regulating protein stability via various signaling pathways. Protein homeostasis and bone remodeling are both crucially influenced by the UPS. Recently, there have been several reports on DUBs involved in bone homeostasis and various bone disorders through the regulation of osteoblasts and osteoclasts via NF-κB, Wnt/ß-catenin, TRAF6, TGFß, ERK1/2, and PI3K/Akt pathways. However, DUBs regulating function in bone homeostasis is still in its infancy. Here, we summarized several recent identifications on DUBs, with a focus on their role in bone cancer progression. Therefore, the study attempts to summarize association with the expression level of DUBs as key factors driving bone cancers and also provide new insights on DUBs as key pharmacologic targets for bone cancer therapeutics.

ALDH2 deficiency augments atherosclerosis through the USP14-cGAS-dependent polarization of proinflammatory macrophages.

The aldehyde dehydrogenase 2 (ALDH2) rs671 polymorphism commonly exists in the East Asian populations and is associated with high risks of cardiovascular disease (CVD). However, the cellular and molecular mechanisms that underlie the ALDH2 rs671 mutant-linked high CVD remain elusive. Here, we show that macrophages derived from human ALDH2 rs671 carriers and ALDH2 knockout mice exhibited an enhanced pro-inflammatory macrophage phenotype and an impaired anti-inflammatory macrophage phenotype. Transplanting bone marrow from ALDH2-/-ApoE-/- to ApoE-/- mice significantly increased atherosclerotic plaque growth and pro-inflammatory macrophage polarization in vivo. Mechanistically, ALDH2 inhibited activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway in macrophages. Pharmacological inhibition of cGAS by RU.521 completely neutralized ALDH2-deficiency-induced macrophage polarization. In-depth mechanistic investigation showed that ALDH2 accelerated cGAS K48-linked polyubiquitination degradation at lysine 282 in macrophages by reducing the interaction between ubiquitin-specific protease 14 (USP14) and cGAS, mainly through its enzymatic role in mitigating 4-hydroxy-2-nonenal (4-HNE) accumulation. Consistently, USP14 knockdown in bone marrow cells alleviated proinflammatory responses in macrophages and protected against atherosclerosis. Our findings provide new mechanistic insights of ALDH2 deficiency-associated proinflammation and atherosclerosis and new therapeutic and preventive paradigms for treatment of atherosclerosis-associated CVD.

Target deubiquitinase OTUB1 as a therapeatic strategy for BLCA via ß-catenin/necroptosis signal pathway.

Ubiquitination, a prevalent and highly dynamic reversible post-translational modification, is tightly regulated by the deubiquitinating enzymes (DUBs) superfamily. Among them, OTU Domain-Containing Ubiquitin Aldehyde-Binding Protein 1 (OTUB1) stands out as a critical member of the OTU deubiquitinating family, playing a pivotal role as a tumor regulator across various cancers. However, its specific involvement in BLCA (BLCA) and its clinical significance have remained ambiguous. This study aimed to elucidate the biofunctions of OTUB1 in BLCA and its implications for clinical prognosis. Our investigation revealed heightened OTUB1 expression in BLCA, correlating with unfavorable clinical outcomes. Through in vivo and in vitro experiments, we demonstrated that increased OTUB1 levels promote BLCA tumorigenesis and progression, along with conferring resistance to cisplatin treatment. Notably, we established a comprehensive network involving OTUB1, ß-catenin, necroptosis, and BLCA, delineating their regulatory interplay. Mechanistically, we uncovered that OTUB1 exerts its influence by deubiquitinating and stabilizing ß-catenin, leading to its nuclear translocation. Subsequently, nuclear ß-catenin enhances the transcriptional activity of c-myc and cyclin D1 while suppressing the expression of RIPK3 and MLKL, thereby fostering BLCA progression and cisplatin resistance. Importantly, our clinical data suggest that the OTUB1/ß-catenin/RIPK3/MLKL axis holds promise as a potential biomarker for BLCA.

Crosstalk of ubiquitin system and non-coding RNA in fibrosis.

Chronic tissue injury triggers changes in the cell type and microenvironment at the site of injury and eventually fibrosis develops. Current research suggests that fibrosis is a highly dynamic and reversible process, which means that human intervention after fibrosis has occurred has the potential to slow down or cure fibrosis. The ubiquitin system regulates the biological functions of specific proteins involved in the development of fibrosis, and researchers have designed small molecule drugs to treat fibrotic diseases on this basis, but their therapeutic effects are still limited. With the development of molecular biology technology, researchers have found that non-coding RNA (ncRNA) can interact with the ubiquitin system to jointly regulate the development of fibrosis. More in-depth explorations of the interaction between ncRNA and ubiquitin system will provide new ideas for the clinical treatment of fibrotic diseases.

The Deubiquitinase USP22-Stabilized COL17A1 Promotes Lung Adenocarcinoma Progression.

BACKGROUND: Lung adenocarcinoma (LUAD) is a highly aggressive and rapidly fatal malignancy worldwide. Collagen XVII (COL17A1) has been implicated in various protumorigenic processes. However, the functions and mechanisms of COL17A1 in LUAD progression still remain elusive. METHODS: COL17A1 and ubiquitin-specific protease 22 (USP22) mRNA analysis was performed by quantitative PCR, and their protein levels were detected by immunoblotting and immunohistochemistry. The functional influence was evaluated by determining cell viability, proliferation, apoptosis, invasion, migration, and ferroptosis in vitro, as well as xenograft growth in vivo. Co-immunoprecipitation (Co-IP) and IP experiments were used to examine the USP22/COL17A1 interaction and COL17A1 deubiquitination. Cycloheximide treatment was used to analyze COL17A1 protein stability. RESULTS: COL17A1 and USP22 were upregulated in human LUAD tissues and cell lines. Functionally, COL17A1 knockdown acted for the suppression of LUAD cell growth, invasion, and migration as well as promotion of cell apoptosis and ferroptosis in vitro. COL17A1 knockdown could diminish the tumorigenicity of LUAD cells in vivo. Mechanistically, USP22 stabilized and upregulated COL17A1 by enhancing the deubiquitination of COL17A1. Additionally, reexpression of COL17A1 could reverse USP22 silencing-induced phenotype changes of LUAD cells in vitro. CONCLUSION: Our findings demonstrate that USP22-stabilized COL17A1 possesses oncogenic activity in LUAD. We propose that USP22 and COL17A1 would be potential targets for the establishment of therapeutic approaches against LUAD.

Hydrogen sulfide inhibits skeletal muscle ageing by up-regulating autophagy through promoting deubiquitination of adenosine 5'-monophosphate (AMP)-activated protein kinase α1 via ubiquitin specific peptidase 5.

BACKGROUND: Hydrogen sulfide (H2S), the third gasotransmitter discovered, regulates a variety of physiological functions. Whether H2S alleviates skeletal muscle ageing by regulating autophagy has not been reported. METHODS: Mice were administered 150 mg/kg/day of D-galactose ( D-gal), and C2C12 myotubes were cultured in 20 g/L D-gal to induce ageing. Sodium hydrosulfide (NaHS) was employed as an exogenous donor in the treatment group. The intracellular concentration of H2S was quantified by the 7-azido-4-methylcoumarin fluorescence probe. The proteins involved in the ubiquitin-mediated degradation of AMPKα1 were detected by liquid chromatography tandem mass spectrometry (LC-MS/MS) and co-immunoprecipitation (Co-IP). S-sulfhydration of USP5 was tested by a biotin-switch assay. Associated proteins were analysed by western blot. RESULTS: NaHS was found to effectively restore the H2S content in both ageing gastrocnemius (+91.89%, P < 0.001) and C2C12 myotubes (+27.55%, P < 0.001). In comparison to the D-gal group, NaHS was observed to increase the mean cross-sectional area of muscle fibres (+44.91%, P < 0.001), to decrease the collagen volume fraction of gastrocnemius (-81.32%, P = 0.001) and to reduce the ß-galactosidase-positive area of C2C12 myotubes (-28.74%, P < 0.001). NaHS was also found to reverse the expression of muscle atrophy F box protein (MAFbx), muscle-specific RING finger protein 1 (MuRF1), Cyclin D1 and p21 in the ageing gastrocnemius tissue (MAFbx: -31.73%, P = 0.008; MuRF1: -32.37%, P = 0.003; Cyclin D1: +45.34%, P = 0.010; p21: -25.53%, P = 0.022) and C2C12 myotubes (MAFbx: -16.38%, P < 0.001; MuRF1: -16.45%, P = 0.003; Cyclin D1: +40.23%, P < 0.001; p21: -35.85%, P = 0.026). The AMPKα1-ULK1 pathway was activated and autophagy was up-regulated in NaHS-treated gastrocnemius tissue (p-AMPKα1: +61.61%, P = 0.018; AMPKα1: +30.64%, P = 0.010; p-ULK1/ULK1: +85.87%, P = 0.005; p62: -29.07%, P < 0.001; Beclin1: +24.75%, P = 0.007; light chain 3 II/I [LC3 II/I]: +55.78%, P = 0.004) and C2C12 myotubes (p-AMPKα1: +77.49%, P = 0.018; AMPKα1: +26.18%, P = 0.022; p-ULK1/ULK1: +38.34%, P = 0.012; p62: -9.02%, P = 0.014; Beclin1: +13.36%, P < 0.001; LC3 II/I: +79.38%, P = 0.017; autophagy flux: +24.88%, P = 0.034) compared with the D-gal group. The effects of NaHS on autophagy were comparable to those of acadesine and LYN-1604, and chloroquine could reverse its effects on ageing. LC-MS/MS and Co-IP experiments demonstrated that USP5 is a deubiquitinating enzyme of AMPKα1. Following the knockdown of USP5, the activation of AMPKα1 was decreased (p-AMPKα1: -42.10%, P < 0.001; AMPKα1: -43.93%, P < 0.001), autophagy was inhibited (p-ULK1/ULK1: -27.51, P = 0.001; p62: +36.00, P < 0.001; Beclin1: -22.15%, P < 0.001) and NaHS lost its ability to up-regulate autophagy. NaHS was observed to restore the expression (gastrocnemius: +62.17%, P < 0.001; C2C12 myotubes: +37.51%, P = 0.003) and S-sulfhydration (+53.07%, P = 0.009) of USP5 and reduce the ubiquitination of AMPKα1. CONCLUSIONS: H2S promotes the deubiquitination of AMPKα1 by increasing the expression and S-sulfhydration of USP5, thereby up-regulating autophagy and alleviating skeletal muscle ageing.

ATAD5 functions as a regulatory platform for Ub-PCNA deubiquitination.

Ubiquitination status of proliferating cell nuclear antigen (PCNA) is crucial for regulating DNA lesion bypass. After the resolution of fork stalling, PCNA is subsequently deubiquitinated, but the underlying mechanism remains undefined. We found that the N-terminal domain of ATAD5 (ATAD5-N), the largest subunit of the PCNA-unloading complex, functions as a scaffold for Ub-PCNA deubiquitination. ATAD5 recognizes DNA-loaded Ub-PCNA through distinct DNA-binding and PCNA-binding motifs. Furthermore, ATAD5 forms a heterotrimeric complex with UAF1-USP1 deubiquitinase, facilitating the deubiquitination of DNA-loaded Ub-PCNA. ATAD5 also enhances the Ub-PCNA deubiquitination by USP7 and USP11 through specific interactions. ATAD5 promotes the distinct deubiquitination process of UAF1-USP1, USP7, and USP11 for poly-Ub-PCNA. Additionally, ATAD5 mutants deficient in UAF1-binding had increased sensitivity to DNA-damaging agents. Our results ultimately reveal that ATAD5 and USPs cooperate to efficiently deubiquitinate Ub-PCNA prior to its release from the DNA in order to safely deactivate the DNA repair process.

The deubiquitinase BAP1 and E3 ligase UBE3C sequentially target IRF3 to activate and resolve the antiviral innate immune response.

Ubiquitination is essential for the proteasomal turnover of IRF3, the central factor mediating the antiviral innate immune response. However, the spatiotemporal regulation of IRF3 ubiquitination for the precise activation and timely resolution of innate immunity remains unclear. Here, we identified BRCA1-associated protein-1 (BAP1) and ubiquitin-protein ligase E3C (UBE3C) as the key deubiquitinase and ubiquitinase for temporal control of IRF3 stability during viral infection. In the early stage, BAP1 dominates and removes K48-linked ubiquitination of IRF3 in the nucleus, preventing its proteasomal degradation and facilitating efficient interferon (IFN)-ß production. In the late stage, E3 ligase UBE3C, induced by IFN-ß, specifically mediates IRF3 ubiquitination and promotes its proteasomal degradation. Overall, the sequential interactions with BAP1 and UBE3C govern IRF3 stability during innate response, ensuring effective viral clearance and inflammation resolution. Our findings provide insights into the temporal control of innate signaling and suggest potential interventions in viral infection.

Ubiquitin-specific protease UBP14 stabilizes HY5 by deubiquitination to promote photomorphogenesis in Arabidopsis thaliana.

Transcription factor ELONGATED HYPOCOTYL5 (HY5) is the central hub for seedling photomorphogenesis. E3 ubiquitin (Ub) ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) inhibits HY5 protein accumulation through ubiquitination. However, the process of HY5 deubiquitination, which antagonizes E3 ligase-mediated ubiquitination to maintain HY5 homeostasis has never been studied. Here, we identified that Arabidopsis thaliana deubiquitinating enzyme, Ub-SPECIFIC PROTEASE 14 (UBP14) physically interacts with HY5 and enhances its protein stability by deubiquitination. The da3-1 mutant lacking UBP14 function exhibited a long hypocotyl phenotype, and UBP14 deficiency led to the failure of rapid accumulation of HY5 during dark to light. In addition, UBP14 preferred to stabilize nonphosphorylated form of HY5 which is more readily bound to downstream target genes. HY5 promoted the expression and protein accumulation of UBP14 for positive feedback to facilitate photomorphogenesis. Our findings thus established a mechanism by which UBP14 stabilizes HY5 protein by deubiquitination to promote photomorphogenesis in A. thaliana.

Amelioration of nonalcoholic fatty liver disease by inhibiting the deubiquitylating enzyme RPN11.

Nonalcoholic fatty liver disease (NAFLD), including its more severe manifestation nonalcoholic steatohepatitis (NASH), is a global public health challenge. Here, we explore the role of deubiquitinating enzyme RPN11 in NAFLD and NASH. Hepatocyte-specific RPN11 knockout mice are protected from diet-induced liver steatosis, insulin resistance, and steatohepatitis. Mechanistically, RPN11 deubiquitinates and stabilizes METTL3 to enhance the m6A modification and expression of acyl-coenzyme A (CoA) synthetase short-chain family member 3 (ACSS3), which generates propionyl-CoA to upregulate lipid metabolism genes via histone propionylation. The RPN11-METTL3-ACSS3-histone propionylation pathway is activated in the livers of patients with NAFLD. Pharmacological inhibition of RPN11 by Capzimin ameliorated NAFLD, NASH, and related metabolic disorders in mice and reduced lipid contents in human hepatocytes cultured in 2D and 3D. These results demonstrate that RPN11 is a novel regulator of NAFLD/NASH and that suppressing RPN11 has therapeutic potential for the treatment.

Autoinhibition of ubiquitin-specific protease 8: insights into domain interactions and mechanisms of regulation.

Ubiquitin-specific proteases (USPs) are a family of multi-domain deubiquitinases (DUBs) with variable architectures, some containing regulatory auxiliary domains. Among the USP family, all occurrences of intramolecular regulation presently known are autoactivating. USP8 remains the sole exception as its putative WW-like domain, conserved only in vertebrate orthologs, is autoinhibitory. Here, we present a comprehensive structure-function analysis describing the autoinhibition of USP8 and provide evidence of the physical interaction between the WW-like and catalytic domains. The solution structure of full-length USP8 reveals an extended, monomeric conformation. Coupled with DUB assays, the WW-like domain is confirmed to be the minimal autoinhibitory unit. Strikingly, autoinhibition is only observed with the WW-like domain in cis and depends on the length of the linker tethering it to the catalytic domain. Modelling of the WW:CD complex structure and mutagenesis of interface residues suggests a novel binding site in the S1 pocket. To investigate the interplay between phosphorylation and USP8 autoinhibition, we identify AMP-activated protein kinase as a highly selective modifier of S718 in the 14-3-3 binding motif. We show that 14-3-3γ binding to phosphorylated USP8 potentiates autoinhibition in a WW-like domain-dependent manner by stabilizing an autoinhibited conformation. These findings provide mechanistic details on the autoregulation of USP8 and shed light on its evolutionary significance.