K63-linked Ubiquitin Chains Research Articles

VCP enhances autophagy-related osteosarcoma progression by recruiting USP2 to inhibit ubiquitination and degradation of FASN

Osteosarcoma (OS) is a highly aggressive malignant tumor with a high rate of disability and mortality rates, and dysregulated autophagy is a crucial factor in cancer. However, the molecular mechanisms that regulate autophagy in OS remain unclear. This study aimed to explore key molecules that affect autophagy in OS and their regulatory mechanisms. We found that fatty acid synthase (FASN) was significantly increased in activated autophagy models of OS and promoted OS proliferation in an autophagy-dependent manner, as detected by LC3 double-labeled fluorescence confocal microscopy, western blotting, transmission electron microscopy (TEM), and cell functional experiments. Furthermore, co-immunoprecipitation combined with mass spectrometry (Co-IP/MS), ubiquitination modification, molecular docking, and protein truncation methods were used to identify FASN-interacting proteins and analyze their effects on OS. Valosin-containing protein (VCP) enhanced the FASN stability by recruiting ubiquitin specific peptidase-2 (USP2) to remove the K48-linked ubiquitin chains from FASN; domain 2 of VCP and the amino acid sequence () of USP2 were critical for their interactions. Gain- and loss-of-function experiments showed that the inhibition of FASN or USP2 attenuated the stimulatory effect of VCP overexpression on autophagy and the malignant phenotypes of OS cells in vitro and in vivo. Notably, micro-CT indicated that VCP induced severe bone destruction in nude mice, which was abrogated by FASN or USP2 downregulation. In summary, VCP recruits USP2 to stabilize FASN by deubiquitylation, thereby activating autophagy and promoting OS progression. The identification of the VCP/USP2/FASN axis, which mediates autophagy regulation, provides important insights into the underlying mechanisms of OS and offers potential diagnostic and therapeutic strategies for patients with OS.

Linkage-specific ubiquitin binding interfaces modulate the activity of the chlamydial deubiquitinase Cdu1 towards poly-ubiquitin substrates.

The chlamydial deubiquitinase Cdu1 of the obligate intracellular human pathogenic bacterium Chlamydia trachomatis plays important roles in the maintenance of chlamydial infection. Despite the structural similarities shared with its homologue Cdu2, both DUBs display remarkable differences in their enzymatic activity towards poly-UB chain substrates. Whereas Cdu1 is highly active towards K48- and K63- poly-UB chains, Cdu2 activity is restricted mostly to mono-UB substrates. Here, we shed light on the molecular mechanisms of the differential activity and the substrate specificity of Cdu1 to better understand the cellular processes it is involved in, including infection-related events. We found that the strikingly elevated activity of Cdu1 relative to its paralogue Cdu2 can be attributed to an N-terminally extended α-helix, which has not been observed in Cdu2. Moreover, by employing isothermal titration calorimetry and nuclear magnetic resonance spectroscopy, we demonstrate the differential recognition of K48- and K63-linked poly-UB substrates by Cdu1. Whereas K63-linked poly-UB substrates appear to be recognized by Cdu1 with only two independent ubiquitin interaction sites, up to four different binding interfaces are present for K48-linked ubiquitin chains. Combined, our data suggest that Cdu1 possesses a poly-UB chain directed activity that may enable its function as a multipurpose DUB with a broad substrate specificity.

Promotion of RNF168-Mediated Nucleosomal H2A Ubiquitylation by Structurally Defined K63-Polyubiquitylated Linker Histone H1.

The chemical synthesis of histones with homogeneous modifications is a powerful approach for quantitatively deciphering the functional crosstalk between different post-translational modifications (PTMs). In this study, we developed an expedient site-specific (poly)ubiquitylation strategy (CAEPL, Cysteine Aminoethylation coupled with Enzymatic Protein Ligation), which integrates the Cys-aminoethylation reaction with the process of ubiquitin-activating enzyme UBA1-assisted native chemical ligation. Using this strategy, we successfully prepared monoubiquitylated and K63-linked di- and tri-ubiquitylated linker histone H1.0 proteins, which were incorporated into individual chromatosomes. Quantitative biochemical analysis of different RNF168 constructs on H1 ubiquitylated chromatosomes with different ubiquitin chain lengths demonstrated that K63-linked polyubiquitylated H1.0 could directly stimulate RNF168 ubiquitylation activity by enhancing the affinity between RNF168 and the chromatosome. Subsequent cryo-EM structural analysis of the RNF168/UbcH5c-Ub/H1.0-K63-Ub3 chromatosome complex revealed the potential recruitment orientation between RNF168 UDM1 domain and K63-linked ubiquitin chain on H1.0. Finally, we explored the impact of H1.0 ubiquitylation on RNF168 activity in the context of asymmetric H1.0-K63-Ub3 di-nucleosome substrate, revealing a comparable stimulation effect of both the inter- and intra-nucleosomal crosstalk. Overall, our study highlights the significance of access to structurally defined polyubiquitylated H1.0 by the CAEPL strategy, enabling in-depth mechanistic investigations of in-trans PTM crosstalk between linker histone H1.0 and core histone H2A ubiquitylation.

USP33 facilitates the ovarian cancer progression via deubiquitinating and stabilizing CBX2.

Post-translational modifications of proteins play a pivotal role in both the initiation and progression of ovarian cancer. Despite the recognition of USP33 as a significant factor in various cancers, its specific function and underlying mechanisms in ovarian cancer remain elusive. Proteomics and ubiquitinomics approaches were coupled to screen novel substrate proteins directly regulated by USP33. Our findings unveil that USP33 was observed to eliminate K27- and K48-linked ubiquitin chains from CBX2 at the K277 position. Notably, acetylation of CBX2 at K199, catalyzed by lysine acetyltransferase GCN5, was found to enhance its interaction with USP33, subsequently promoting further deubiquitination and stabilization. Functionally, our experiments demonstrate that USP33 significantly enhances ovarian cancer proliferation and metastasis in a CBX2-dependent manner. Furthermore, analysis revealed a direct positive correlation between the expression levels of USP33 and CBX2 proteins in human specimens, with elevated levels being associated with reduced survival rates in ovarian cancer patients. These findings elucidate the mechanism by which USP33 augments ovarian cancer progression through the stabilization of CBX2, underscoring the USP33-CBX2 axis as a promising therapeutic target in ovarian cancer management.

Transcriptome-Wide Association Study Reveals New Molecular Interactions Associated with Melanoma Pathogenesis.

A transcriptome-wide association study (TWAS) was conducted on genome-wide association study (GWAS) summary statistics of malignant melanoma of skin (UK Biobank dataset) and The Cancer Genome Atlas-Skin Cutaneous Melanoma (TCGA-SKCM) gene expression weights to identify melanoma susceptibility genes. The GWAS included 2465 cases and 449,799 controls, while the gene expression testing was conducted on 103 cases. Afterward, a gene enrichment analysis was applied to identify significant TWAS associations. The melanoma's gene-microRNA (miRNA) regulatory network was constructed from the TWAS genes and their corresponding miRNAs. At last, a disease enrichment analysis was conducted on the corresponding miRNAs. The TWAS detected 27 genes associated with melanoma with p-values less than 0.05 (the top three genes are LOC389458 (RBAK), C16orf73 (MEIOB), and EIF3CL). After the joint/conditional test, one gene (AMIGO1) was dropped, resulting in 26 significant genes. The Gene Ontology (GO) biological process associated the extended gene set (76 genes) with protein K11-linked ubiquitination and regulation of cell cycle phase transition. K11-linked ubiquitin chains regulate cell division. Interestingly, the extended gene set was related to different skin cancer subtypes. Moreover, the enriched pathways were nsp1 from SARS-CoV-2 that inhibit translation initiation in the host cell, cell cycle, translation factors, and DNA repair pathways full network. The gene-miRNA regulatory network identified 10 hotspot genes with the top three: TP53, BRCA1, and MDM2; and four hotspot miRNAs: mir-16, mir-15a, mir-125b, and mir-146a. Melanoma was among the top ten diseases associated with the corresponding (106) miRNAs. Our results shed light on melanoma pathogenesis and biologically significant molecular interactions.

Targeting the TRIM14/USP14 Axis Enhances Immunotherapy Efficacy by Inducing Autophagic Degradation of PD-L1.

Immunotherapy has greatly improved cancer treatment in recent years by harnessing the immune system to target cancer cells. The first immunotherapeutic agent approved by the FDA was IFNα. Treatment with IFNα can lead to effective immune activation and attenuate tumor immune evasion, but persistent treatment has been shown to elicit immunosuppressive effects. Here, we identified an autophagy-dependent mechanism by which IFNα triggers tumor immune evasion by upregulating PD-L1 to suppress the antitumor activity of CD8+ T cells. Mechanistically, IFNα increased the transcription of TRIM14, which recruited the deubiquitinase USP14 to inhibit the autophagic degradation of PD-L1. USP14 removed K63-linked ubiquitin chains from PD-L1, impairing its recognition by the cargo receptor p62 (also known as SQSTM1) for subsequent autophagic degradation. Combining the USP14 inhibitor IU1 with IFNα and anti-CTLA4 treatment effectively suppressed tumor growth without significant toxicity. This work suggests a strategy for targeting selective autophagy to abolish PD-L1-mediated cancer immune evasion. Significance: IFNα-induced TRIM14 transcription suppresses antitumor immunity by recruiting USP14 to inhibit autophagic degradation of PD-L1, indicating that targeting this axis could be an effective immunotherapeutic approach for treating cancer.

Localized K63 ubiquitin signaling is regulated by VCP/p97 during oxidative stress.

Under stress conditions, cells reprogram their molecular machineries to mitigate damage and promote survival. Ubiquitin signaling is globally increased during oxidative stress, controlling protein fate and supporting stress defenses at several subcellular compartments. However, the rules driving subcellular ubiquitin localization to promote these concerted response mechanisms remain understudied. Here, we show that K63-linked ubiquitin chains, known to promote proteasome-independent pathways, accumulate primarily in non-cytosolic compartments during oxidative stress induced by sodium arsenite in mammalian cells. Our subcellular ubiquitin proteomic analyses of non-cytosolic compartments expanded 10-fold the pool of proteins known to be ubiquitinated during arsenite stress (2,046) and revealed their involvement in pathways related to immune signaling and translation control. Moreover, subcellular proteome analyses revealed proteins that are recruited to non-cytosolic compartments under stress, including a significant enrichment of helper ubiquitin-binding adaptors of the ATPase VCP that processes ubiquitinated substrates for downstream signaling. We further show that VCP recruitment to non-cytosolic compartments under arsenite stress occurs in a ubiquitin-dependent manner mediated by its adaptor NPLOC4. Additionally, we show that VCP and NPLOC4 activities are critical to sustain low levels of non-cytosolic K63-linked ubiquitin chains, supporting a cyclical model of ubiquitin conjugation and removal that is disrupted by cellular exposure to reactive oxygen species. This work deepens our understanding of the role of localized ubiquitin and VCP signaling in the basic mechanisms of stress response and highlights new pathways and molecular players that are essential to reshape the composition and function of the human subcellular proteome under dynamic environments.

The deubiquitinase USP2a promotes tumor immunosuppression by stabilizing immune checkpoint B7–H4 in lung adenocarcinoma harboring EGFR-activating mutants

B7–H4 is an immune checkpoint crucial for inhibiting CD8+ T-cell activity. A clinical trial is underway to investigate B7–H4 as a potential immunotherapeutic agent. However, the regulatory mechanism of B7–H4 degradation via the ubiquitin-proteasome pathway (UPP) remains poorly understood. In this study, we discovered that proteasome inhibitors effectively increased B7–H4 expression, while EGFR-activating mutants promoted B7–H4 expression through the UPP. We screened B7–H4 binding proteins by co-immunoprecipitation and mass spectrometry and found that USP2a acted as a deubiquitinase of B7–H4 by removing K48- and K63-linked ubiquitin chains from B7–H4, leading to a reduction in B7–H4 degradation. EGFR mutants enhanced B7–H4 stability by upregulating USP2a expression. We further investigated the role of USP2a in tumor growth in vivo. Depletion of USP2a in L858R/LLC cells inhibited tumor cell proliferation, consequently suppressing tumor growth in immune-deficient nude mice by destabilizing downstream molecules such as Cyclin D1. In an immune-competent C57BL/6 mouse tumor model, USP2a abrogation facilitated infiltration of CD95+CD8+ effector T cells and hindered infiltration of Tim-3+CD8+ and LAG-3+CD8+ exhausted T cells by destabilizing B7–H4. Clinical lung adenocarcinoma samples showed a significant correlation between B7–H4 abundance and USP2a expression, indicating the contribution of the EGFR/USP2a/B7–H4 axis to tumor immunosuppression. In summary, this study elucidates the dual effects of USP2a in tumor growth by stabilizing Cyclin D1, promoting tumor cell proliferation, and stabilizing B7–H4, contributing to tumor immunosuppression. Therefore, USP2a represents a potential target for tumor therapy.

K48- and K63-linked ubiquitin chain interactome reveals branch- and length-specific ubiquitin interactors.

The ubiquitin (Ub) code denotes the complex Ub architectures, including Ub chains of different lengths, linkage types, and linkage combinations, which enable ubiquitination to control a wide range of protein fates. Although many linkage-specific interactors have been described, how interactors are able to decode more complex architectures is not fully understood. We conducted a Ub interactor screen, in humans and yeast, using Ub chains of varying lengths, as well as homotypic and heterotypic branched chains of the two most abundant linkage types-lysine 48-linked (K48) and lysine 63-linked (K63) Ub. We identified some of the first K48/K63-linked branch-specific Ub interactors, including histone ADP-ribosyltransferase PARP10/ARTD10, E3 ligase UBR4, and huntingtin-interacting protein HIP1. Furthermore, we revealed the importance of chain length by identifying interactors with a preference for Ub3 over Ub2 chains, including Ub-directed endoprotease DDI2, autophagy receptor CCDC50, and p97 adaptor FAF1. Crucially, we compared datasets collected using two common deubiquitinase inhibitors-chloroacetamide and N-ethylmaleimide. This revealed inhibitor-dependent interactors, highlighting the importance of inhibitor consideration during pulldown studies. This dataset is a key resource for understanding how the Ub code is read.

USP20 deubiquitinates and stabilizes the reticulophagy receptor RETREG1/FAM134B to drive reticulophagy

ABSTRACT The endoplasmic reticulum (ER) serves as a hub for various cellular processes, and maintaining ER homeostasis is essential for cell function. Reticulophagy is a selective process that removes impaired ER subdomains through autophagy-mediatedlysosomal degradation. While the involvement of ubiquitination in autophagy regulation is well-established, its role in reticulophagy remains unclear. In this study, we screened deubiquitinating enzymes (DUBs) involved in reticulophagy and identified USP20 (ubiquitin specific peptidase 20) as a key regulator of reticulophagy under starvation conditions. USP20 specifically cleaves K48- and K63-linked ubiquitin chains on the reticulophagy receptor RETREG1/FAM134B (reticulophagy regulator 1), thereby stabilizing the substrate and promoting reticulophagy. Remarkably, despite lacking a transmembrane domain, USP20 is recruited to the ER through its interaction with VAPs (VAMP associated proteins). VAPs facilitate the recruitment of early autophagy proteins, including WIPI2 (WD repeat domain, phosphoinositide interacting 2), to specific ER subdomains, where USP20 and RETREG1 are enriched. The recruitment of WIPI2 and other proteins in this process plays a crucial role in facilitating RETREG1-mediated reticulophagy in response to nutrient deprivation. These findings highlight the critical role of USP20 in maintaining ER homeostasis by deubiquitinating and stabilizing RETREG1 at distinct ER subdomains, where USP20 further recruits VAPs and promotes efficient reticulophagy. Abbreviations: ACTB actin beta; ADRB2 adrenoceptor beta 2; AMFR/gp78 autocrine motility factor receptor; ATG autophagy related; ATL3 atlastin GTPase 3; BafA1 bafilomycin A1; BECN1 beclin 1; CALCOCO1 calcium binding and coiled-coil domain 1; CCPG1 cell cycle progression 1; DAPI 4’,6-diamidino-2-phenylindole; DTT dithiothreitol; DUB deubiquitinating enzyme; EBSS Earle’s Balanced Salt Solution; FFAT two phenylalanines (FF) in an acidic tract; GABARAP GABA type A receptor-associated protein; GFP green fluorescent protein; HMGCR 3-hydroxy-3-methylglutaryl-CoA reductase; IL1B interleukin 1 beta; LIR LC3-interacting region; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; PIK3C3/Vps34 phosphatidylinositol 3-kinase catalytic subunit type 3; RB1CC1/FIP200 RB1 inducible coiled-coil 1; RETREG1/FAM134B reticulophagy regulator 1; RFP red fluorescent protein; RHD reticulon homology domain; RIPK1 receptor interacting serine/threonine kinase 1; RTN3L reticulon 3 long isoform; SEC61B SEC61 translocon subunit beta; SEC62 SEC62 homolog, preprotein translocation factor; SIM super-resolution structured illumination microscopy; SNAI2 snail family transcriptional repressor 2; SQSTM1/p62 sequestosome 1; STING1/MITA stimulator of interferon response cGAMP interactor 1; STX17 syntaxin 17; TEX264 testis expressed 264, ER-phagy receptor; TNF tumor necrosis factor; UB ubiquitin; ULK1 unc-51 like autophagy activating kinase 1; USP20 ubiquitin specific peptidase 20; USP33 ubiquitin specific peptidase 33; VAMP8 vesicle associated membrane protein 8; VAPs VAMP associated proteins; VMP1 vacuole membrane protein 1; WIPI2 WD repeat domain, phosphoinositide interacting 2; ZFYVE1/DFCP1 zinc finger FYVE-type containing 1.

Rainbow trout DUBA inhibits type I interferon signaling by deubiquitinating TRAF3

Deubiquitinating enzyme A (DUBA), a member of the ovarian tumor (OTU) subfamily of deubiquitinases (DUBs), is recognized for its negative regulatory role in type I interferon (IFN) expression downstream of Toll-like receptor 3 (TLR3). However, its involvement in the TLR3 signaling pathway in fish remains largely unexplored. In this study, we investigated the regulatory role of DUBA (OmDUBA) in the TLR3 response in rainbow trout (Oncorhynchus mykiss). OmDUBA features a conserved OTU domain, and its expression increased in RTH-149 cells following stimulation with the TLR3 agonist poly(I:C). Gain- and loss-of-function experiments demonstrated that OmDUBA attenuated the activation of TANK-binding kinase 1 (TBK1), resulting in a subsequent reduction in type I IFN expression and IFN-stimulated response element (ISRE) activation in poly(I:C)-stimulated cells. OmDUBA interacted with TRAF3, a crucial mediator in TLR3-mediated type I IFN production. Under poly(I:C) stimulation, there was an augmentation in the K63-linked polyubiquitination of TRAF3, a process significantly inhibited upon OmDUBA overexpression. These findings suggest that OmDUBA may function similarly to its mammalian counterparts in downregulating the poly(I:C)-induced type I IFN response in rainbow trout by removing the K63-linked ubiquitin chain on TRAF3. Our study provides novel insights into the role of fish DUBA in antiviral immunity.

GSK3β and UCHL3 govern RIPK4 homeostasis via deubiquitination to enhance tumor metastasis in ovarian cancer.

Receptor-interacting protein kinase 4 (RIPK4) is increasingly recognized as a pivotal player in ovarian cancer, promoting tumorigenesis and disease progression. Despite its significance, the posttranslational modifications dictating RIPK4 stability in ovarian cancer remain largely uncharted. In this study, we first established that RIPK4 levels are markedly higher in metastatic than in primary ovarian cancer tissues through single-cell sequencing. Subsequently, we identified UCHL3 as a key deubiquitinase that regulates RIPK4. We elucidate the mechanism that UCHL3 interacts with and deubiquitinates RIPK4 at the K469 site, removing the K48-linked ubiquitin chain and thus enhancing RIPK4 stabilization. Intriguingly, inhibition of UCHL3 activity using TCID leads to increased RIPK4 ubiquitination and degradation. Furthermore, we discovered that GSK3β-mediated phosphorylation of RIPK4 at Ser420 enhances its interaction with UCHL3, facilitating further deubiquitination and stabilization. Functionally, RIPK4 was found to drive the proliferation and metastasis of ovarian cancer in a UCHL3-dependent manner both in vitro and in vivo. Importantly, positive correlations between RIPK4 and UCHL3 protein expression levels were observed, with both serving as indicators of poor prognosis in ovarian cancer patients. Overall, this study uncovers a novel pathway wherein GSK3β-induced phosphorylation of RIPK4 strengthens its interaction with UCHL3, leading to increased deubiquitination and stabilization of RIPK4, thereby promoting ovarian cancer metastasis. These findings offer new insights into the molecular underpinnings of ovarian cancer and highlight potential therapeutic targets for enhancing antitumor efficacy.

TRIM28 facilitates type I interferon activation by targeting TBK1.

Type I interferons play a fundamental role in innate host defense against viral infections by eliciting the induction of an antiviral gene program that serves to inhibit viral replication. Activation of type I interferon is regulated by the IRF3 transcription factor, which undergoes phosphorylation-dependent activation by the upstream kinase, TBK1, during viral infection. However, the mechanisms by which TBK1 achieves activation to support signaling to IRF3 remain incompletely understood. Here we identified the E3 ubiquitin ligase, tripartite motif containing 28 (TRIM28), as a positive regulator of type I interferon activation by facilitating TBK1 signaling. Genetic deletion of TRIM28 via CRISPR-Cas9 editing resulted in impaired type I interferon activation upon both RNA and DNA virus challenge, corresponding with increased susceptibility to virus infections in TRIM28 knockout cells. Mechanistically, TRIM28 interacted with TBK1 and mediated the assembly of K63-linked ubiquitin chains onto TBK1, a post-translational modification shown to augment TBK1 signal transmission events. TRIM28 knockout cells further displayed defective TBK1 phosphorylation and complex assembly with IRF3, resulting in impaired IRF3 phosphorylation. Altogether, our data demonstrate TBK1 to be a novel substrate for TRIM28 and identify TRIM28 as an essential regulatory factor in controlling innate antiviral immune responses.

Proteasome condensate formation is driven by multivalent interactions with shuttle factors and ubiquitin chains

Stress conditions can cause the relocalization of proteasomes to condensates in yeast and mammalian cells. The interactions that facilitate the formation of proteasome condensates, however, are unclear. Here, we show that the formation of proteasome condensates in yeast depends on ubiquitin chains together with the proteasome shuttle factors Rad23 and Dsk2. These shuttle factors colocalize to these condensates. Strains deleted for the third shuttle factor gene, DDI1, show proteasome condensates in the absence of cellular stress, consistent with the accumulation of substrates with long K48-linked ubiquitin chains that accumulate in this mutant. We propose a model where the long K48-linked ubiquitin chains function as a scaffold for the ubiquitin-binding domains of the shuttle factors and the proteasome, allowing for the multivalent interactions that further drive condensate formation. Indeed, we determined different intrinsic ubiquitin receptors of the proteasome-Rpn1, Rpn10, and Rpn13-and the Ubl domains of Rad23 and Dsk2 are critical under different condensate-inducing conditions. In all, our data support a model where the cellular accumulation of substrates with long ubiquitin chains, potentially due to reduced cellular energy, allows for proteasome condensate formation. This suggests that proteasome condensates are not simply for proteasome storage, but function to sequester soluble ubiquitinated substrates together with inactive proteasomes.

USP8 prevents aberrant NF-κB and Nrf2 activation by counteracting ubiquitin signals from endosomes.

K63-linked ubiquitin chains attached to plasma membrane proteins serve as tags for endocytosis and endosome-to-lysosome sorting. USP8 is an essential deubiquitinase for the maintenance of endosomal functions. Prolonged depletion of USP8 leads to cell death, but the major effects on cellular signaling pathways are poorly understood. Here, we show that USP8 depletion causes aberrant accumulation of K63-linked ubiquitin chains on endosomes and induces immune and stress responses. Upon USP8 depletion, two different decoders for K63-linked ubiquitin chains, TAB2/3 and p62, were recruited to endosomes and activated the TAK1-NF-κB and Keap1-Nrf2 pathways, respectively. Oxidative stress, an environmental stimulus that potentially suppresses USP8 activity, induced accumulation of K63-linked ubiquitin chains on endosomes, recruitment of TAB2, and expression of the inflammatory cytokine. The results demonstrate that USP8 is a gatekeeper of misdirected ubiquitin signals and inhibits immune and stress response pathways by removing K63-linked ubiquitin chains from endosomes.

The deubiquitinating enzyme USP19 facilitates hepatocellular carcinoma progression through stabilizing YAP

Hippo pathway plays a crucial role in the progression of hepatocellular carcinoma (HCC), and yes-associated protein (YAP) is one of the major factors of the Hippo pathway. However, the mechanism of abnormal YAP activation in HCC has not been well elucidated. Here, we screened a Deubiquitinating enzymes’ (DUB) siRNA library targeting DUBs, and identified Ubiquitin Specific Peptidase 19 (USP19) as a specific deubiquitinating enzyme of YAP in HCC, which could stabilize YAP at K76 and K90 sites via removing the K48- and K11-linked ubiquitin chains. USP19 knockdown decreased the expression of YAP protein and its target gene (CTGF, CYR61, ANKRD1) expression. Through substantial in vivo and in vitro experiments, we prove that USP19 facilities the proliferation and migration of HCC. More importantly, we found that USP19 was upregulated in HCC tissues and associated with poor prognosis. In general, our research revealed a novel post-translational mechanism between USP19 and YAP in HCC, suggesting that USP19 may be a pivotal therapeutic target for HCC treatment.

FBXO7 Confers Mesenchymal Properties and Chemoresistance in Glioblastoma by Controlling Rbfox2-Mediated Alternative Splicing.

Mesenchymal glioblastoma (GBM) is highly resistant to radio-and chemotherapy and correlates with worse survival outcomes in GBM patients; however, the underlying mechanism determining the mesenchymal phenotype remains largely unclear. Herein, it is revealed that FBXO7, a substrate-recognition component of the SCF complex implicated in the pathogenesis of Parkinson's disease, confers mesenchymal properties and chemoresistance in GBM by controlling Rbfox2-mediated alternative splicing. Specifically, FBXO7 ubiquitinates Rbfox2 Lys249 through K63-linked ubiquitin chains upon arginine dimethylation at Arg341 and Arg441 by PRMT5, leading to Rbfox2 stabilization. FBXO7 controls Rbfox2-mediated splicing of mesenchymal genes, including FoxM1, Mta1, and Postn. FBXO7-induced exon Va inclusion of FoxM1 promotes FoxM1 phosphorylation by MEK1 and nuclear translocation, thereby upregulates CD44, CD9, and ID1 levels, resulting in GBM stem cell self-renewal and mesenchymal transformation. Moreover, FBXO7 is stabilized by temozolomide, and FBXO7 depletion sensitizes tumor xenografts in mice to chemotherapy. The findings demonstrate that the FBXO7-Rbfox2 axis-mediated splicing contributes to mesenchymal transformation and tumorigenesis, and targeting FBXO7 represents a potential strategy for GBM treatment.

Ubiquitinated H2A.X-Induced RNF168 Condensation Promotes DNA Double-Strand Break Repair and Tumor Radioresistance

Ubiquitination of histone is an essential process involved in DNA damage response (DSB) serving as scaffolds for DNA repair proteins, but how these factors are recruited so quickly and regulated in a spatiotemporal manner remains poorly understood. Liquid-liquid phase separation (LLPS) has recently emerged as a mechanism for membraneless condensation driven by multivalent interactions. In this study, we aimed to investigate the LLPS potential of RNF168, an E3 ligase essential for DSB repair, and the mechanism underlying its-mediated tumor radio-resistance. The intrinsic disordered domain (IDR) of RNF168 was determined by the PONDR website. The LLPS properties were validated by droplet formation in vivo and in vitro. RNF168-mEGFP were expressed in Escherichia coli and purified with GST tag. The synthesized K63-linked ubiquitin chains were added to mimic the interactions between RNF168 and radiation-induced ubiquitinated-histone. Effects of RNF168 LLPS on downstream proteins were verified by immunofluorescence. RNF168-mEGFP recombinant protein formed liquid-like droplets in vivo and co-localized with γ-H2A.X foci after irradiation. The droplet's fluorescence recovered quickly after photobleaching, which could be abolished by 1,6-hexanediol treatment or ATP deprivation. Purified RNF168-mEGFP protein also condensed in vitro, and the size and number of droplets were related to protein concentration, salt concentration, pH, and temperature. Condensation of RNF168 was dependent on the IDR (323-459 amino acid), and more importantly, enhanced by synthesized K63-linked ubiquitin chains. LLPS of RNF168 was required for recruitment of RNF168 to DSB and RNF168-mediated γ-H2A.X ubiquitination. LLPS deficiency of RNF168 resulted in decreased recruitment of 53BP1, BRCA1, and RAP80 proteins, resulting in impaired DSB repair and genomic instability. Notably, higher expression of RNF168 was correlated with a poorer response to neoadjuvant radiochemotherapy in rectal cancer patients. Finally, RNF168 condensate-induced tumor radioresistance was further verified in the xenograft model. RNF168 undergoes LLPS at the DSB site, which is determined by both the IDR domain and the interaction with K63-linked ubiquitin chains. Radiation-induced RNF168 condensation accelerates the accumulation of RNF168 and promotes the recruitment of downstream effectors to DSB, resulting in enhanced DSB repair and tumor radioresistance.

Pristimerin suppresses AIM2 inflammasome by modulating AIM2-PYCARD/ASC stability via selective autophagy to alleviate tendinopathy

ABSTRACT Macroautophagy/autophagy plays an important role in regulating cellular homeostasis and influences the pathogenesis of degenerative diseases. Tendinopathy is characterized by tendon degeneration and inflammation. However, little is known about the role of selective autophagy in tendinopathy. Here, we find that pristimerin (PM), a quinone methide triterpenoid, is more effective in treating tendinopathy than the first-line drug indomethacin. PM inhibits the AIM2 inflammasome and alleviates inflammation during tendinopathy by promoting the autophagic degradation of AIM2 through a PYCARD/ASC-dependent manner. A mechanistic study shows that PM enhances the K63-linked ubiquitin chains of PYCARD/ASC at K158/161, which serves as a recognition signal for SQSTM1/p62-mediated autophagic degradation of the AIM2-PYCARD/ASC complex. We further identify that PM binds the Cys53 site of deubiquitinase USP50 through the Michael-acceptor and blocks the binding of USP50 to PYCARD/ASC, thereby reducing USP50-mediated cleavage of K63-linked ubiquitin chains of PYCARD/ASC. Finally, PM treatment in vivo generates an effect comparable to inflammasome deficiency in alleviating tendinopathy. Taken together, these findings demonstrate that PM alleviates the progression of tendinopathy by modulating AIM2-PYCARD/ASC stability via SQSTM1/p62-mediated selective autophagic degradation, thus providing a promising autophagy-based therapeutic for tendinopathy. Abbreviations: 3-MA: 3-methyladenine; AIM2: absent in melanoma 2; AT: Achilles tenotomy; ATP: adenosine triphosphate; BMDMs: bone marrow-derived macrophages; CHX: cycloheximide; Col3a1: collagen, type III, alpha 1; CQ: chloroquine; Cys: cysteine; DARTS: drug affinity responsive target stability; DTT: dithiothreitol; DUB: deubiquitinase; gDNA: genomic DNA; GSH: glutathione; His: histidine; IL1B/IL-1β: interleukin 1 beta; IND: indomethacin; IP: immunoprecipitation; LPS: lipopolysaccharide; MMP: mitochondrial membrane potential; NLRP3: NLR family, pyrin domain containing 3; PM: pristimerin; PYCARD/ASC: PYD and CARD domain containing; SN: supernatants; SOX9: SRY (sex determining region Y)-box 9; SQSTM1: sequestosome 1; Tgfb: transforming growth factor, beta; TIMP3: tissue inhibitor of metalloproteinase 3; TNMD: tenomodulin; TRAF6: TNF receptor-associated factor 6; Ub: ubiquitin; USP50: ubiquitin specific peptidase 50; WCL: whole cell lysates.

Structural snapshots along K48-linked ubiquitin chain formation by the HECT E3 UBR5.

Ubiquitin (Ub) chain formation by homologous to E6AP C-terminus (HECT)-family E3 ligases regulates vast biology, yet the structural mechanisms remain unknown. We used chemistry and cryo-electron microscopy (cryo-EM) to visualize stable mimics of the intermediates along K48-linked Ub chain formation by the human E3, UBR5. The structural data reveal a ≈ 620 kDa UBR5 dimer as the functional unit, comprising a scaffold with flexibly tethered Ub-associated (UBA) domains, and elaborately arranged HECT domains. Chains are forged by a UBA domain capturing an acceptor Ub, with its K48 lured into the active site by numerous interactions between the acceptor Ub, manifold UBR5 elements and the donor Ub. The cryo-EM reconstructions allow defining conserved HECT domain conformations catalyzing Ub transfer from E2 to E3 and from E3. Our data show how a full-length E3, ubiquitins to be adjoined, E2 and intermediary products guide a feed-forward HECT domain conformational cycle establishing a highly efficient, broadly targeting, K48-linked Ub chain forging machine.