K63 ubiquitination in immune signaling.

Ubc13-catalyzed K63 ubiquitination is a major control point for immune signaling. Recent evidence has shown that the control of multiple immune functions, including chronic inflammation, pathogen responses, lymphocyte activation, and regulatory signaling, is altered by K63 ubiquitination. In this review, we detail the novel cellular sensors that are dependent on K63 ubiquitination for their function in the immune signaling network. Many pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can target K63 ubiquitination to inhibit pathogen immune responses; we describe novel details of the pathways involved and summarize recent clinically relevant SARS-CoV-2-specific responses. We also discuss recent evidence that regulatory T cell (Treg) versus T helper (TH) 1 and TH17 cell subset regulation might involve K63 ubiquitination. Knowledge gaps that merit future investigation and clinically relevant pathways are also addressed.

Dysregulation of innate immune signaling in animal models of spinal muscular atrophy.

BACKGROUND: Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by hypomorphic loss of function in the survival motor neuron (SMN) protein. SMA presents across a broad spectrum of disease severity. Unfortunately, genetic models of intermediate SMA have been difficult to generate in vertebrates and are thus unable to address key aspects of disease etiology. To address these issues, we developed a Drosophila model system that recapitulates the full range of SMA severity, allowing studies of pre-onset biology as well as late-stage disease processes. RESULTS: Here, we carried out transcriptomic and proteomic profiling of mild and intermediate Drosophila models of SMA to elucidate molecules and pathways that contribute to the disease. Using this approach, we elaborated a role for the SMN complex in the regulation of innate immune signaling. We find that mutation or tissue-specific depletion of SMN induces hyperactivation of the immune deficiency (IMD) and Toll pathways, leading to overexpression of antimicrobial peptides (AMPs) and ectopic formation of melanotic masses in the absence of an external challenge. Furthermore, the knockdown of downstream targets of these signaling pathways reduced melanotic mass formation caused by SMN loss. Importantly, we identify SMN as a negative regulator of a ubiquitylation complex that includes Traf6, Bendless, and Diap2 and plays a pivotal role in several signaling networks. CONCLUSIONS: In alignment with recent research on other neurodegenerative diseases, these findings suggest that hyperactivation of innate immunity contributes to SMA pathology. This work not only provides compelling evidence that hyperactive innate immune signaling is a primary effect of SMN depletion, but it also suggests that the SMN complex plays a regulatory role in this process in vivo. In summary, immune dysfunction in SMA is a consequence of reduced SMN levels and is driven by cellular and molecular mechanisms that are conserved between insects and mammals.

SET7 methylates the deubiquitinase OTUB1 at Lys 122 to impair its binding to E2 enzyme UBC13 and relieve its suppressive role on ferroptosis.

The deubiquitinating enzyme OTUB1 possesses canonical deubiquitinase (DUB) activity and noncanonical, catalytic-independent activity, which has been identified as an essential regulator of diverse physiological processes. Posttranslational modifications of OTUB1 affect both its DUB activity and its noncanonical activity of binding to the E2 ubiquitin-conjugation enzyme UBC13, but further investigation is needed to characterize the full inventory of modifications to OTUB1. Here, we demonstrate that SET7, a lysine monomethylase, directly interacts with OTUB1 to catalyze OTUB1 methylation at lysine 122. This modification does not affect DUB activity of OTUB1 but impairs its noncanonical activity, binding to UBC13. Moreover, we found using cell viability analysis and intracellular reactive oxygen species assay that SET7-mediated methylation of OTUB1 relieves its suppressive role on ferroptosis. Notably, the methylation-mimic mutant of OTUB1 not only loses the ability to bind to UBC13 but also relieves its suppressive role on Tert-Butyl hydroperoxide-induced cell death and Cystine starvation/Erastin-induced cellular reactive oxygen species. Collectively, our data identify a novel modification of OTUB1 that is critical for inhibiting its noncanonical activity.

Functional Characterization of Potato UBC13-UEV1s Genes Required for Ubiquitin Lys63 Chain to Polyubiquitination.

Ubiquitin-conjugating enzymes (E2s/UBC) are components of the ubiquitin proteasome system (UPS), and the ubiquitin-conjugating enzyme variant (UEV) is one of E2s (ubiquitin-conjugating enzymes, UBC) subfamily. The UEVs and UBC13 play an auxiliary role in mediating Lys63-linked polyUb chain assembly, which is correlated with target protein non-proteolytic functions, such as DNA repair or response to stress. However, the collaborative mechanism of StUBC13 (homologue of AtUBC13) and StUEVs (the UEVs in potato) involved in potato are not fully understood understood. Here, we identified two StUBC13 and seven StUEVs from potato genome. We analyzed protein motif and conserved domain, gene structure, phylogenetic features, cis-acting elements of StUBC13 and StUEVs. Subsequently, we screened StUBC13 partners protein and verified interaction between StUBC13 and StUEVs using yeast two-hybrid, split luciferase complementation (SLC) and bimolecular fluorescence complementation (BiFC) approach. The expression profile and qRT-PCR analysis suggested that StUBC13 and StUEVs gene exhibited a tissue-specific expression and were induced by different stress. Overall, this investigative study provides a comprehensive reference and view for further functional research on StUBC13 and StUEV1s in potato.

Functions and mechanisms of the Ubc13-UEV complex and lysine 63-linked polyubiquitination in plants.

Ubiquitination is one of the best-known post-translational modifications in eukaryotes, in which different linkage types of polyubiquitination result in different outputs of the target proteins. Distinct from the well-characterized K48-linked polyubiquitination that usually serves as a signal for degradation of the target protein, K63-linked polyubiquitination often requires a unique E2 heterodimer Ubc13-UEV and alters the target protein activity instead of marking it for degradation. This review focuses on recent advances on the roles of Ubc13-UEV-mediated K63-linked polyubiquitination in plant growth, development, and response to environmental stresses.

Genetic rescue of lineage-balanced blood cell production reveals a crucial role for STAT3 antiinflammatory activity in hematopoiesis.

Blood cell formation must be appropriately maintained throughout life to provide robust immune function, hemostasis, and oxygen delivery to tissues, and to prevent disorders that result from over- or underproduction of critical lineages. Persistent inflammation deregulates hematopoiesis by damaging hematopoietic stem and progenitor cells (HSPCs), leading to elevated myeloid cell output and eventual bone marrow failure. Nonetheless, antiinflammatory mechanisms that protect the hematopoietic system are understudied. The transcriptional regulator STAT3 has myriad roles in HSPC-derived populations and nonhematopoietic tissues, including a potent antiinflammatory function in differentiated myeloid cells. STAT3 antiinflammatory activity is facilitated by STAT3-mediated transcriptional repression of Ube2n, which encodes the E2 ubiquitin-conjugating enzyme Ubc13 involved in proinflammatory signaling. Here we demonstrate a crucial role for STAT3 antiinflammatory activity in preservation of HSPCs and lineage-balanced hematopoiesis. Conditional Stat3 removal from the hematopoietic system led to depletion of the bone marrow lineage- Sca-1+ c-Kit+ CD150+ CD48- HSPC subset (LSK CD150+ CD48- cells), myeloid-skewed hematopoiesis, and accrual of DNA damage in HSPCs. These responses were accompanied by intrinsic transcriptional alterations in HSPCs, including deregulation of inflammatory, survival and developmental pathways. Concomitant Ube2n/Ubc13 deletion from Stat3-deficient hematopoietic cells enabled lineage-balanced hematopoiesis, mitigated depletion of bone marrow LSK CD150+ CD48- cells, alleviated HSPC DNA damage, and corrected a majority of aberrant transcriptional responses. These results indicate an intrinsic protective role for STAT3 in the hematopoietic system, and suggest that this is mediated by STAT3-dependent restraint of excessive proinflammatory signaling via Ubc13 modulation.

Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity.

Constitutive NF-κB signaling represents a hallmark of chronic inflammation and autoimmune diseases. The E3 ligase TNF receptor-associated factor 6 (TRAF6) acts as a key regulator bridging innate immunity, pro-inflammatory cytokines, and antigen receptors to the canonical NF-κB pathway. Structural analysis and point mutations have unraveled the essential role of TRAF6 binding to the E2-conjugating enzyme ubiquitin-conjugating enzyme E2 N (Ubc13 or UBE2N) to generate Lys63-linked ubiquitin chains for inflammatory and immune signal propagation. Genetic mutations disrupting TRAF6-Ubc13 binding have been shown to reduce TRAF6 activity and, consequently, NF-κB activation. However, to date, no small-molecule modulator is available to inhibit the TRAF6-Ubc13 interaction and thereby counteract NF-κB signaling and associated diseases. Here, using a high-throughput small-molecule screening approach, we discovered an inhibitor of the TRAF6-Ubc13 interaction that reduces TRAF6-Ubc13 activity both in vitro and in cells. We found that this compound, C25-140, impedes NF-κB activation in various immune and inflammatory signaling pathways also in primary human and murine cells. Importantly, C25-140 ameliorated inflammation and improved disease outcomes of autoimmune psoriasis and rheumatoid arthritis in preclinical in vivo mouse models. Hence, the first-in-class TRAF6-Ubc13 inhibitor C25-140 expands the toolbox for studying the impact of the ubiquitin system on immune signaling and underscores the importance of TRAF6 E3 ligase activity in psoriasis and rheumatoid arthritis. We propose that inhibition of TRAF6 activity by small molecules represents a promising novel strategy for targeting autoimmune and chronic inflammatory diseases.

A conserved asparagine in a ubiquitin-conjugating enzyme positions the substrate for nucleophilic attack.

The mechanism used by the ubiquitin-conjugating enzyme, Ubc13, to catalyze ubiquitination is probed with three computational techniques: Born-Oppenheimer molecular dynamics, single point quantum mechanics/molecular mechanics energies, and classical molecular dynamics. These simulations support a long-held hypothesis and show that Ubc13-catalyzed ubiquitination uses a stepwise, nucleophilic attack mechanism. Furthermore, they show that the first step-the formation of a tetrahedral, zwitterionic intermediate-is rate limiting. However, these simulations contradict another popular hypothesis that supposes that the negative charge on the intermediate is stabilized by a highly conserved asparagine (Asn79 in Ubc13). Instead, calculated reaction profiles of the N79A mutant illustrate how charge stabilization actually increases the barrier to product formation. Finally, an alternate role for Asn79 is suggested by simulations of wild-type, N79A, N79D, and H77A Ubc13: it stabilizes the motion of the electrophile prior to the reaction, positioning it for nucleophilic attack. © 2019 Wiley Periodicals, Inc.

Emerging roles of Lys63-linked polyubiquitylation in immune responses.

Among all the E2 ubiquitin-conjugating enzymes, Ubc13, which heterodimerizes with Uev1a, specifically mediates lysine 63 (K63)-linked protein polyubiquitylation, a process that does not lead to proteasomal degradation of its substrates. Instead, it plays a key role in signal transduction. Numerous roles of Lys63-linked polyubiquitylation in immune responses have emerged, indicating the importance of this regulatory strategy. Here, we summarize some of the signaling pathways that depend on Lys63-linked polyubiquitylation during innate and adaptive immune responses, with a focus on the underlying molecular mechanisms. In addition, we describe how Ubc13 itself is regulated and outline its function in transforming growth factor ß signaling. We discuss the current progress in pharmacological targeting of Ubc13 in inflammatory and autoimmune diseases as well as cancer therapy.

RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment.

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. Here, we defined the activated RNF8-Ubc13∼ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13 active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. These findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.

Fertilization-induced K63-linked ubiquitylation mediates clearance of maternal membrane proteins.

In Caenorhabditis elegans, fertilization triggers endocytosis and rapid turnover of maternal surface membrane proteins in lysosomes, although the precise mechanism of this inducible endocytosis is unknown. We found that high levels of K63-linked ubiquitin chains transiently accumulated on endosomes upon fertilization. Endocytosis and the endosomal accumulation of ubiquitin were both regulated downstream of the anaphase-promoting complex, which drives the oocyte's meiotic cell cycle after fertilization. The clearance of maternal membrane proteins and the accumulation of K63-linked ubiquitin on endosomes depended on UBC-13 and UEV-1, which function as an E2 complex that specifically mediates chain elongation of K63-linked polyubiquitin. CAV-1-GFP, an endocytic cargo protein, was modified with K63-linked polyubiquitin in a UBC-13/UEV-1-dependent manner. In ubc-13 or uev-1 mutants, CAV-1-GFP and other membrane proteins were internalized from the plasma membrane normally after fertilization. However, they were not efficiently targeted to the multivesicular body (MVB) pathway but recycled to the cell surface. Our results suggest that UBC-13-dependent K63-linked ubiquitylation is required for proper MVB sorting rather than for internalization. These results also demonstrate a developmentally controlled function of K63-linked ubiquitylation.

Characterization of four rice UEV1 genes required for Lys63-linked polyubiquitination and distinct functions.

BACKGROUND: The error-free branch of the DNA-damage tolerance (DDT) pathway is orchestrated by Lys63-linked polyubiquitination of proliferating cell nuclear antigen (PCNA), and this polyubiquitination is mediated by a Ubc13-Uev complex in yeast. We have previously cloned OsUBC13 from rice, whose product functions as an E2 to promote Lys63-linked ubiquitin chain assembly in the presence of yeast or human Uev. RESULTS: Here we identify four highly conserved UEV1 genes in rice whose products are able to form stable heterodimers with OsUbc13 and mediate Lys63-linked ubiquitin chain assembly. Expression of OsUEV1s is able to rescue the yeast mms2 mutant from death caused by DNA-damaging agents. Interestingly, OsUev1A contains a unique C-terminal tail with a conserved prenylation site not found in the other three OsUev1s, and this post-translational modification appears to be required for its unique subcellular distribution and association with the membrane. The analysis of OsUEV1 expression profiles obtained from the Genevestigator database indicates that these genes are differentially regulated. CONCLUSIONS: We speculate that different OsUev1s play distinct roles by serving as a regulatory subunit of the Ubc13-Uev1 complex to respond to diverse cellular, developmental and environmental signals.

Regulation of IL-1ß-induced NF-κB by hydroxylases links key hypoxic and inflammatory signaling pathways.

Hypoxia is a prominent feature of chronically inflamed tissues. Oxygen-sensing hydroxylases control transcriptional adaptation to hypoxia through the regulation of hypoxia-inducible factor (HIF) and nuclear factor κB (NF-κB), both of which can regulate the inflammatory response. Furthermore, pharmacologic hydroxylase inhibitors reduce inflammation in multiple animal models. However, the underlying mechanism(s) linking hydroxylase activity to inflammatory signaling remains unclear. IL-1ß, a major proinflammatory cytokine that regulates NF-κB, is associated with multiple inflammatory pathologies. We demonstrate that a combination of prolyl hydroxylase 1 and factor inhibiting HIF hydroxylase isoforms regulates IL-1ß-induced NF-κB at the level of (or downstream of) the tumor necrosis factor receptor-associated factor 6 complex. Multiple proteins of the distal IL-1ß-signaling pathway are subject to hydroxylation and form complexes with either prolyl hydroxylase 1 or factor inhibiting HIF. Thus, we hypothesize that hydroxylases regulate IL-1ß signaling and subsequent inflammatory gene expression. Furthermore, hydroxylase inhibition represents a unique approach to the inhibition of IL-1ß-dependent inflammatory signaling.

Activation of the canonical IKK complex by K63/M1-linked hybrid ubiquitin chains.

Polyubiquitin (pUb) chains formed between the C terminus of ubiquitin and lysine 63 (K63) or methionine 1 (M1) of another ubiquitin have been implicated in the activation of the canonical IκB kinase (IKK) complex. Here, we demonstrate that nearly all of the M1-pUb chains formed in response to interleukin-1, or the Toll-Like Receptors 1/2 agonist Pam3CSK4, are covalently attached to K63-pUb chains either directly as K63-pUb/M1-pUb hybrids or indirectly by attachment to the same protein. Interleukin-1 receptor (IL-1R)-associated kinase (IRAK) 1 is modified first by K63-pUb chains to which M1-pUb linkages are added subsequently, and myeloid differentiation primary response gene 88 (MyD88) and IRAK4 are also modified by both K63-pUb and M1-pUb chains. We show that the heme-oxidized IRP2 ubiquitin ligase 1 interacting protein (HOIP) component of the linear ubiquitin assembly complex catalyzes the formation of M1-pUb chains in response to interleukin-1, that the formation of K63-pUb chains is a prerequisite for the formation of M1-pUb chains, and that HOIP interacts with K63-pUb but not M1-pUb linkages. These findings identify K63-Ub oligomers as a major substrate of HOIP in cells where the MyD88-dependent signaling network is activated. The TGF-beta-activated kinase 1 (TAK1)-binding protein (TAB) 2 and TAB3 components of the TAK1 complex and the NFκB Essential Modifier (NEMO) component of the canonical IKK complex bind to K63-pUb chains and M1-pUb chains, respectively. The formation of K63/M1-pUb hybrids may therefore provide an elegant mechanism for colocalizing both complexes to the same pUb chain, facilitating the TAK1-catalyzed activation of IKKα and IKKß. Our study may help to resolve the debate about the relative importance of K63-pUb and M1-pUb chains in activating the canonical IKK complex.

Lysine 63-linked polyubiquitination is dispensable for Parkin-mediated mitophagy.

PINK1/Parkin-mediated mitophagy is thought to ensure mitochondrial quality control in neurons as well as other cells. Upon the loss of mitochondrial membrane potential (ΔΨm), Lys-63-linked polyubiquitin chains accumulate on the mitochondrial outer membrane in a Parkin-dependent manner. However, the physiological significance of Lys-63-linked polyubiquitination during mitophagy is not fully understood. Here, we report that the suppression of Lys-63-linked polyubiquitination through the removal of Ubc13 activity essentially affects neither PINK1 activation nor the degradation of depolarized mitochondria. Moreover, the inactivation of Ubc13 did not modulate the mitochondrial phenotypes of PINK1 knockdown Drosophila. Our data indicate that the formation of Lys-63-linked polyubiquitin chains on depolarized mitochondria is not a key factor for the PINK1-Parkin pathway as was once thought.

UBC13, an E2 enzyme for Lys63-linked ubiquitination, functions in root development by affecting auxin signaling and Aux/IAA protein stability.

Unlike conventional lysine (K) 48-linked polyubiquitination, K63-linked polyubiquitination plays signaling roles in yeast and animals. Thus far, UBC13 is the only known ubiquitin-conjugating enzyme (E2) specialized in K63-linked polyubiquitination. Previous identification of Arabidopsis genes encoding UBC13 as well as its interacting partner UEV1 indicates that the UBC13-mediated ubiquitination pathway is conserved in plants; however, little is known about functions and signaling mediated through K63-linked polyubiquitination in plants. To address the functions of UBC13-mediated ubiquitination in plants, we created Arabidopsis ubc13 null mutant lines in which the two UBC13 genes were disrupted. The double mutant displayed altered root development, including shorter primary root, fewer lateral roots and only a few short root hairs in comparison with the wild type and single mutant plants, indicating that UBC13 activity is critical for all major aspects of root development. The double mutant plants were insensitive to auxin treatments, suggesting that the strong root phenotypes do not simply result from a reduced level of auxin. Instead, the ubc13 mutant had a reduced auxin response, as indicated by the expression of an auxin-responsive DR5 promoter-GFP. Furthermore, both the enzymatic activity and protein level of an AXR3/IAA17-GUS reporter were greatly increased in the ubc13 mutant, whereas the induction of many auxin-responsive genes was suppressed. Collectively, these results suggest that Aux/IAA proteins accumulate in the ubc13 mutant, resulting in a reduced auxin response and defective root development. Hence, this study provides possible mechanistic links between UBC13-mediated protein ubiquitination, root development and auxin signaling.

The structural network of inflammation and cancer: merits and challenges.

Inflammation, the first line of defense against pathogens can contribute to all phases of tumorigenesis, including tumor initiation, promotion and metastasis. Within this framework, the Toll-like receptor (TLR) pathway plays a central role in inflammation and cancer. Although extremely useful, the classical representation of this, and other pathways in the cellular network in terms of nodes (proteins) and edges (interactions) is incomplete. Structural pathways can help complete missing parts of such diagrams: they demonstrate in detail how signals coming from different upstream pathways merge and propagate downstream, how parallel pathways compensate each other in drug resistant mutants, how multi-subunit signaling complexes form and in particular why they are needed and how they work, how allosteric events can control these proteins and their pathways, and intricate details of feedback loops and how kick in. They can also explain the mechanisms of some oncogenic SNP mutations. Constructing structural pathways is a challenging task. Here, our goal is to provide an overview of inflammation and cancer from the structural standpoint, focusing on the TLR pathway. We use the powerful PRISM (PRotein Interactions by Structural Matching) tool to reveal important structural information of interactions in and within key orchestrators of the TLR pathway, such as MyD88.

Helicobacter pylori activates NF-κB by inducing Ubc13-mediated ubiquitination of lysine 158 of TAK1.

The Helicobacter pylori virulence factor CagA targets a variety of host proteins to alter different cellular responses, including the induction of pro-inflammatory cytokines. We have previously shown that CagA-facilitated lysine 63-linked ubiquitination of TAK1 is essential for the H. pylori-induced NF-κB activation and the expression of proinflammatory cytokines. However, the molecular mechanism for TAK1 ubiquitination and activation in H. pylori-mediated NF-κB activation remains elusive. Here, we identify lysine 158 of TAK1 as the key residue undergoing lysine 63-linked ubiquitination in response to H. pylori infection. Mutation of lysine 158 to arginine prevents the ubiquitination of TAK1 and impairs H. pylori-induced TAK1 and NF-κB activation. Moreover, we demonstrate that E2 ubiquitin conjugating enzyme Ubc13 is involved in H. pylori-mediated TAK1 ubiquitination. Suppressing the activity of Ubc13 by a dominant-negative mutant or siRNA abolishes CagA-facilitated and H. pylori-induced TAK1 and NF-κB activation. These findings further underscore the importance of lysine 63-linked ubiquitination of TAK1 in H. pylori-induced NF-κB activation and NF-κB-mediated inflammatory response.

Structural mechanisms underlying signaling in the cellular response to DNA double strand breaks.

DNA double strand breaks (DSBs) constitute one of the most dangerous forms of DNA damage. In actively replicating cells, these breaks are first recognized by specialized proteins that initiate a signal transduction cascade that modulates the cell cycle and results in the repair of the breaks by homologous recombination (HR). Protein signaling in response to double strand breaks involves phosphorylation and ubiquitination of chromatin and a variety of associated proteins. Here we review the emerging structural principles that underlie how post-translational protein modifications control protein signaling that emanates from these DNA lesions.

The deubiquitinating enzyme 13 retards non-alcoholic steatohepatitis via blocking inactive rhomboid protein 2-dependent pathway.

Nowadays potential preclinical drugs for the treatment of nonalcoholic steatohepatitis (NASH) have failed to achieve expected therapeutic efficacy because the pathogenic mechanisms are underestimated. Inactive rhomboid protein 2 (IRHOM2), a promising target for treatment of inflammation-related diseases, contributes to deregulated hepatocyte metabolism-associated nonalcoholic steatohepatitis (NASH) progression. However, the molecular mechanism underlying Irhom2 regulation is still not completely understood. In this work, we identify the ubiquitin-specific protease 13 (USP13) as a critical and novel endogenous blocker of IRHOM2, and we also indicate that USP13 is an IRHOM2-interacting protein that catalyzes deubiquitination of Irhom2 in hepatocytes. Hepatocyte-specific loss of the Usp13 disrupts liver metabolic homeostasis, followed by glycometabolic disorder, lipid deposition, increased inflammation, and markedly promotes NASH development. Conversely, transgenic mice with Usp13 overexpression, lentivirus (LV)- or adeno-associated virus (AAV)-driven Usp13 gene therapeutics mitigates NASH in 3 models of rodent. Mechanistically, in response to metabolic stresses, USP13 directly interacts with IRHOM2 and removes its K63-linked ubiquitination induced by ubiquitin-conjugating enzyme E2N (UBC13), a ubiquitin E2 conjugating enzyme, and thus prevents its activation of downstream cascade pathway. USP13 is a potential treatment target for NASH therapy by targeting the Irhom2 signaling pathway.