-
International Journal of Molecular... Apr 2021Ubiquitylation and deubiquitylation are reversible protein post-translational modification (PTM) processes involving the regulation of protein degradation under... (Review)
Review
Ubiquitylation and deubiquitylation are reversible protein post-translational modification (PTM) processes involving the regulation of protein degradation under physiological conditions. Loss of balance in this regulatory system can lead to a wide range of diseases, such as cancer and inflammation. As the main members of the deubiquitinases (DUBs) family, ubiquitin-specific peptidases (USPs) are closely related to biological processes through a variety of molecular signaling pathways, including DNA damage repair, p53 and transforming growth factor-β (TGF-β) pathways. Over the past decade, increasing attention has been drawn to USPs as potential targets for the development of therapeutics across diverse therapeutic areas. In this review, we summarize the crucial roles of USPs in different signaling pathways and focus on advances in the development of USP inhibitors, as well as the methods of screening and identifying USP inhibitors.
Topics: Deubiquitinating Enzymes; Drug Evaluation, Preclinical; Humans; Neoplasms; Protein Processing, Post-Translational; Proteolysis; Signal Transduction; Ubiquitin; Ubiquitin-Specific Proteases; Ubiquitination
PubMed: 33925279
DOI: 10.3390/ijms22094546 -
Nature Chemical Biology Apr 2022Many diseases are driven by proteins that are aberrantly ubiquitinated and degraded. These diseases would be therapeutically benefited by targeted protein stabilization...
Many diseases are driven by proteins that are aberrantly ubiquitinated and degraded. These diseases would be therapeutically benefited by targeted protein stabilization (TPS). Here we present deubiquitinase-targeting chimeras (DUBTACs), heterobifunctional small molecules consisting of a deubiquitinase recruiter linked to a protein-targeting ligand, to stabilize the levels of specific proteins degraded in a ubiquitin-dependent manner. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48-ubiquitin-specific deubiquitinase OTUB1. We showed that a DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR), robustly stabilized ΔF508-CFTR protein levels, leading to improved chloride channel conductance in human cystic fibrosis bronchial epithelial cells. We also demonstrated stabilization of the tumor suppressor kinase WEE1 in hepatoma cells. Our study showcases covalent chemoproteomic approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.
Topics: Chimera; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Deubiquitinating Enzymes; Humans; Ligands; Ubiquitin
PubMed: 35210618
DOI: 10.1038/s41589-022-00971-2 -
Nature Communications Nov 2020NOD-like receptor protein 3 (NLRP3) detects microbial infections or endogenous danger signals and activates the NLRP3 inflammasome, which has important functions in host...
NOD-like receptor protein 3 (NLRP3) detects microbial infections or endogenous danger signals and activates the NLRP3 inflammasome, which has important functions in host defense and contributes to the pathogenesis of inflammatory diseases, and thereby needs to be tightly controlled. Deubiquitination of NLRP3 is considered a key step in NLRP3 inflammasome activation. However, the mechanisms by which deubiquitination controls NLRP3 inflammasome activation are unclear. Here, we show that the UAF1/USP1 deubiquitinase complex selectively removes K48-linked polyubiquitination of NLRP3 and suppresses its ubiquitination-mediated degradation, enhancing cellular NLRP3 levels, which are indispensable for subsequent NLRP3 inflammasome assembly and activation. In addition, the UAF1/USP12 and UAF1/USP46 complexes promote NF-κB activation, enhance the transcription of NLRP3 and proinflammatory cytokines (including pro-IL-1β, TNF, and IL-6) by inhibiting ubiquitination-mediated degradation of p65. Consequently, Uaf1 deficiency attenuates NLRP3 inflammasome activation and IL-1β secretion both in vitro and in vivo. Our study reveals that the UAF1 deubiquitinase complexes enhance NLRP3 and pro-IL-1β expression by targeting NLRP3 and p65 and licensing NLRP3 inflammasome activation.
Topics: Animals; Cytokines; Deubiquitinating Enzymes; Endopeptidases; HEK293 Cells; Humans; Inflammasomes; Inflammation; Intracellular Signaling Peptides and Proteins; Macrophages, Peritoneal; Mice, Inbred C57BL; Mice, Knockout; NLR Family, Pyrin Domain-Containing 3 Protein; Protein Binding; Protein Stability; Transcription, Genetic; Ubiquitin Thiolesterase; Ubiquitination
PubMed: 33247121
DOI: 10.1038/s41467-020-19939-8 -
Cell Death & Disease Aug 2022Deubiquitinating enzymes (DUBs) regulate numerous cellular functions by removing ubiquitin modifications. We examined the effects of 88 human DUBs on linear ubiquitin...
Deubiquitinating enzymes (DUBs) regulate numerous cellular functions by removing ubiquitin modifications. We examined the effects of 88 human DUBs on linear ubiquitin chain assembly complex (LUBAC)-induced NF-κB activation, and identified OTUD1 as a potent suppressor. OTUD1 regulates the canonical NF-κB pathway by hydrolyzing K63-linked ubiquitin chains from NF-κB signaling factors, including LUBAC. OTUD1 negatively regulates the canonical NF-κB activation, apoptosis, and necroptosis, whereas OTUD1 upregulates the interferon (IFN) antiviral pathway. Mass spectrometric analysis showed that OTUD1 binds KEAP1, and the N-terminal intrinsically disordered region of OTUD1, which contains an ETGE motif, is indispensable for the KEAP1-binding. Indeed, OTUD1 is involved in the KEAP1-mediated antioxidant response and reactive oxygen species (ROS)-induced cell death, oxeiptosis. In Otud1-mice, inflammation, oxidative damage, and cell death were enhanced in inflammatory bowel disease, acute hepatitis, and sepsis models. Thus, OTUD1 is a crucial regulator for the inflammatory, innate immune, and oxidative stress responses and ROS-associated cell death pathways.
Topics: Animals; Cell Death; Deubiquitinating Enzymes; Humans; Kelch-Like ECH-Associated Protein 1; Mice; NF-E2-Related Factor 2; NF-kappa B; Reactive Oxygen Species; Ubiquitin; Ubiquitin-Specific Proteases; Ubiquitination
PubMed: 35941131
DOI: 10.1038/s41419-022-05145-5 -
The EMBO Journal Mar 2019Deubiquitination of NLRP3 has been suggested to contribute to inflammasome activation, but the roles and molecular mechanisms are still unclear. We here demonstrate that...
Deubiquitination of NLRP3 has been suggested to contribute to inflammasome activation, but the roles and molecular mechanisms are still unclear. We here demonstrate that ABRO1, a subunit of the BRISC deubiquitinase complex, is necessary for optimal NLRP3-ASC complex formation, ASC oligomerization, caspase-1 activation, and IL-1β and IL-18 production upon treatment with NLRP3 ligands after the priming step, indicating that efficient NLRP3 activation requires ABRO1. Moreover, we report that ABRO1 deficiency results in a remarkable attenuation in the syndrome severity of NLRP3-associated inflammatory diseases, including MSU- and Alum-induced peritonitis and LPS-induced sepsis in mice. Mechanistic studies reveal that LPS priming induces ABRO1 binding to NLRP3 in an S194 phosphorylation-dependent manner, subsequently recruiting the BRISC to remove K63-linked ubiquitin chains of NLRP3 upon stimulation with activators. Furthermore, deficiency of BRCC3, the catalytically active component of BRISC, displays similar phenotypes to ABRO1 knockout mice. Our findings reveal an ABRO1-mediated regulatory signaling system that controls activation of the NLRP3 inflammasome and provide novel potential targets for treating NLRP3-associated inflammatory diseases.
Topics: Animals; Deubiquitinating Enzymes; Female; Inflammasomes; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; NLR Family, Pyrin Domain-Containing 3 Protein; Nuclear Matrix-Associated Proteins; Peritonitis; Phosphorylation; Proteolysis; Reactive Oxygen Species; Signal Transduction; Ubiquitin-Specific Proteases; Ubiquitination; Ubiquitins
PubMed: 30787184
DOI: 10.15252/embj.2018100376 -
Trends in Cancer Oct 2019Alterations in protein ubiquitylation and hypoxia are commonly associated with cancer. Ubiquitylation is carried out by three sequentially acting ubiquitylating enzymes... (Review)
Review
Alterations in protein ubiquitylation and hypoxia are commonly associated with cancer. Ubiquitylation is carried out by three sequentially acting ubiquitylating enzymes and can be opposed by deubiquitinases (DUBs), which have emerged as promising drug targets. Apart from protein localization and activity, ubiquitylation regulates degradation of proteins, among them hypoxia-inducible factors (HIFs). Thereby, various E3 ubiquitin ligases and DUBs regulate HIF abundance. Conversely, several E3s and DUBs are regulated by hypoxia. While hypoxia is a powerful HIF regulator, less is known about hypoxia-regulated DUBs and their impact on HIFs. Here, we review current knowledge about the relationship of E3s, DUBs, and hypoxia signaling. We also discuss the reciprocal regulation of DUBs by hypoxia and use of DUB-specific drugs in cancer.
Topics: Animals; Deubiquitinating Enzymes; Homeostasis; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Neoplasms; Oxygen; Protein Transport; Proteolysis; Signal Transduction; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 31706510
DOI: 10.1016/j.trecan.2019.08.005 -
Nature Communications Feb 2023Deubiquitinating enzymes (DUBs) are an emerging drug target class of ~100 proteases that cleave ubiquitin from protein substrates to regulate many cellular processes. A...
Deubiquitinating enzymes (DUBs) are an emerging drug target class of ~100 proteases that cleave ubiquitin from protein substrates to regulate many cellular processes. A lack of selective chemical probes impedes pharmacologic interrogation of this important gene family. DUBs engage their cognate ligands through a myriad of interactions. We embrace this structural complexity to tailor a chemical diversification strategy for a DUB-focused covalent library. Pairing our library with activity-based protein profiling as a high-density primary screen, we identify selective hits against 23 endogenous DUBs spanning four subfamilies. Optimization of an azetidine hit yields a probe for the understudied DUB VCPIP1 with nanomolar potency and in-family selectivity. Our success in identifying good chemical starting points as well as structure-activity relationships across the gene family from a modest but purpose-build library challenges current paradigms that emphasize ultrahigh throughput in vitro or virtual screens against an ever-increasing scope of chemical space.
Topics: Ubiquitin; Endopeptidases; Peptide Hydrolases; Structure-Activity Relationship; Deubiquitinating Enzymes; Ubiquitination
PubMed: 36754960
DOI: 10.1038/s41467-023-36246-0 -
Journal of Molecular Biology Nov 2017Post-translational modification of cellular proteins by ubiquitin regulates numerous cellular processes, including innate and adaptive immune responses.... (Review)
Review
Post-translational modification of cellular proteins by ubiquitin regulates numerous cellular processes, including innate and adaptive immune responses. Ubiquitin-mediated control over these processes can be reversed by cellular deubiquitinating enzymes (DUBs), which remove ubiquitin from cellular targets and depolymerize polyubiquitin chains. The importance of protein ubiquitination to host immunity has been underscored by the discovery of viruses that encode proteases with deubiquitinating activity, many of which have been demonstrated to actively corrupt cellular ubiquitin-dependent processes to suppress innate antiviral responses and promote viral replication. DUBs have now been identified in diverse viral lineages, and their characterization is providing valuable insights into virus biology and the role of the ubiquitin system in host antiviral mechanisms. Here, we provide an overview of the structural biology of these fascinating viral enzymes and their role innate immune evasion and viral replication.
Topics: Deubiquitinating Enzymes; Host-Pathogen Interactions; Immune Evasion; Immunity, Innate; Viral Proteins; Viruses
PubMed: 28625850
DOI: 10.1016/j.jmb.2017.06.010 -
Advanced Science (Weinheim,... Dec 2023Deubiquitylating enzymes (DUBs) maintain relative homeostasis of the cellular ubiquitome by removing the post-translational modification ubiquitin moiety from... (Review)
Review
Deubiquitylating enzymes (DUBs) maintain relative homeostasis of the cellular ubiquitome by removing the post-translational modification ubiquitin moiety from substrates. Numerous DUBs have been demonstrated specificity for cleaving a certain type of ubiquitin linkage or positions within ubiquitin chains. Moreover, several DUBs perform functions through specific protein-protein interactions in a catalytically independent manner, which further expands the versatility and complexity of DUBs' functions. Dysregulation of DUBs disrupts the dynamic equilibrium of ubiquitome and causes various diseases, especially cancer and immune disorders. This review summarizes the Janus-faced roles of DUBs in cancer including proteasomal degradation, DNA repair, apoptosis, and tumor metastasis, as well as in immunity involving innate immune receptor signaling and inflammatory and autoimmune disorders. The prospects and challenges for the clinical development of DUB inhibitors are further discussed. The review provides a comprehensive understanding of the multi-faced roles of DUBs in cancer and immunity.
Topics: Humans; Ubiquitin; Protein Processing, Post-Translational; Neoplasms; Deubiquitinating Enzymes
PubMed: 37888853
DOI: 10.1002/advs.202303807 -
Nature Nov 2020The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and...
The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.
Topics: Animals; COVID-19; Coronavirus Papain-Like Proteases; Cytokines; Deubiquitinating Enzymes; Humans; Immunity, Innate; Interferon Regulatory Factor-3; Interferons; Mice; Models, Molecular; Molecular Dynamics Simulation; NF-kappa B; Protein Binding; SARS-CoV-2; Ubiquitination; Ubiquitins; COVID-19 Drug Treatment
PubMed: 32726803
DOI: 10.1038/s41586-020-2601-5