The Role of E3 Ubiquitin Ligases and Deubiquitinases in Inflammatory Bowel Disease: Friend or Foe?

Inflammatory bowel disease (IBD), which include Crohn's disease (CD) and ulcerative colitis (UC), exhibits a complex multifactorial pathogenesis involving genetic susceptibility, imbalance of gut microbiota, mucosal immune disorder and environmental factors. Recent studies reported associations between ubiquitination and deubiquitination and the occurrence and development of inflammatory bowel disease. Ubiquitination modification, one of the most important types of post-translational modifications, is a multi-step enzymatic process involved in the regulation of various physiological processes of cells, including cell cycle progression, cell differentiation, apoptosis, and innate and adaptive immune responses. Alterations in ubiquitination and deubiquitination can lead to various diseases, including IBD. Here, we review the role of E3 ubiquitin ligases and deubiquitinases (DUBs) and their mediated ubiquitination and deubiquitination modifications in the pathogenesis of IBD. We highlight the importance of this type of posttranslational modification in the development of inflammation, and provide guidance for the future development of targeted therapeutics in IBD.

An Integrated View of Deubiquitinating Enzymes Involved in Type I Interferon Signaling, Host Defense and Antiviral Activities.

Viral infectious diseases pose a great challenge to human health around the world. Type I interferons (IFN-Is) function as the first line of host defense and thus play critical roles during virus infection by mediating the transcriptional induction of hundreds of genes. Nevertheless, overactive cytokine immune responses also cause autoimmune diseases, and thus, tight regulation of the innate immune response is needed to achieve viral clearance without causing excessive immune responses. Emerging studies have recently uncovered that the ubiquitin system, particularly deubiquitinating enzymes (DUBs), plays a critical role in regulating innate immune responses. In this review, we highlight recent advances on the diverse mechanisms of human DUBs implicated in IFN-I signaling. These DUBs function dynamically to calibrate host defenses against various virus infections by targeting hub proteins in the IFN-I signaling transduction pathway. We also present a future perspective on the roles of DUB-substrate interaction networks in innate antiviral activities, discuss the promises and challenges of DUB-based drug development, and identify the open questions that remain to be clarified. Our review provides a comprehensive description of DUBs, particularly their differential mechanisms that have evolved in the host to regulate IFN-I-signaling-mediated antiviral responses.

Targeting ubiquitin signaling for cancer immunotherapy.

Cancer immunotherapy has become an attractive approach of cancer treatment with tremendous success in treating various advanced malignancies. The development and clinical application of immune checkpoint inhibitors represent one of the most extraordinary accomplishments in cancer immunotherapy. In addition, considerable progress is being made in understanding the mechanism of antitumor immunity and characterizing novel targets for developing additional therapeutic approaches. One active area of investigation is protein ubiquitination, a post-translational mechanism of protein modification that regulates the function of diverse immune cells in antitumor immunity. Accumulating studies suggest that E3 ubiquitin ligases and deubiquitinases form a family of potential targets to be exploited for enhancing antitumor immunity in cancer immunotherapy.

Excessive deubiquitination of NLRP3-R779C variant contributes to very-early-onset inflammatory bowel disease development.

BACKGROUND: Very-early-onset inflammatory bowel disease (VEOIBD) is a chronic inflammatory disease of the gastrointestinal tract occurring during infancy or early childhood. NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome has emerged as a crucial regulator of intestinal homeostasis; however, whether NLRP3 variants may modify VEOIBD risk is unknown. OBJECTIVE: We sought to investigate whether and how a rare NLRP3 variant, found in 3 patients with gastrointestinal symptoms, contributes to VEOIBD development. METHODS: Whole-exome sequencing and bioinformatic analysis were performed to screen disease-associated NLRP3 variants from a cohort of children with VEOIBD. Inflammasome activation was determined in reconstituted HEK293T human embryonic kidney cells with NLRP3 inflammasome components, doxycycline-inducible NLRP3 macrophages, as well as PBMCs and biopsies from patients with NLRP3 variants. Pathogenesis of the variants was determined using a dextran sulfate sodium-induced acute colitis model. RESULTS: We identified a dominant gain-of-function missense variant of NLRP3, encoded by rs772009059 (R779C), in 3 patients with gastrointestinal symptoms. Functional analysis revealed that R779C increased NLRP3 inflammasome activation and pyroptosis in macrophages. This was mediated by enhanced deubiquitination of NLRP3 via binding with deubiquitinases BRCC3 and JOSD2, which are highly expressed in myeloid cells. In a dextran sulfate sodium-induced acute colitis model, NLRP3-R779C in hematopoietic cells resulted in more severe colitis, which can be ameliorated via knockdown of BRCC3 or JOSD2. CONCLUSIONS: BRCC3 and JOSD2 mediate NLRP3-R779C deubiquitination, which promotes NLRP3 inflammasome activation and the risk of developing VEOIBD.

The BRCC3 regulated by Cdk5 promotes the activation of neuronal NLRP3 inflammasome in Parkinson's disease models.

BRCA1-BRCA2-containing complex subunit 3 (BRCC3), a Lys-63-specific deubiquitinase, is a member of the JAMM/MPN family of zinc metalloproteases. BRCC3 have been shown to promote the inflammasome activation by deubiquitinating NOD-like receptor containing pyrin domain 3 (NLRP3). We reported the involvement of neuronal inflammasome in Parkinson's Disease (PD), but the molecular mechanism remains unknown. In this study, we showed that BRCC3 expression was increased in PD models. Knock-down of BRCC3 with shRNA lentivirus decreased NLRP3 neuronal inflammasome. Interestingly, upregulating cyclin-dependent kinase 5 (Cdk5) increased the expression of BRCC3 in HEK293 cell, while inhibition of Cdk5 decreased the upregulated BRCC3 level in MPP+-induced PD cell model. The interaction between Cdk5 and BRCC3 was further confirmed by immunoprecipitation. Moreover, inhibition of Cdk5 suppressed the expression of NLRP3, pro-caspase-1, the adaptor molecule apoptosis-associated speck-like protein containing a CARD (ASC) and interleukin-1 beta (IL-1ß). Besides, inhibition of BRCC3 blocked the increased secretion of IL-1ß. Together, these results suggest that Cdk5-mediated BRCC3 expression may play a critical role in neuronal inflammation by regulating the NLRP3 inflammasome in PD.

Structural Basis of BRCC36 Function in DNA Repair and Immune Regulation.

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.

Regulation of T cell differentiation and function by ubiquitin-specific proteases.

T cells play critical roles in immune responses to pathogens, autoimmunity, and antitumor immunity. During the past few decades, increasing numbers of studies have demonstrated the significance of protein ubiquitination in T cell-mediated immunity. Several E3 ubiquitin ligases and deubiquitinases (DUBs) have been identified as either positive or negative regulators of T cell development and function. In this review, we mainly focus on the roles of DUBs (especially ubiquitin-specific proteases (USPs)) in modulating T cell differentiation and function, as well as the molecular mechanisms. Understanding how T cell development and function is regulated by ubiquitination and deubiquitination will provide novel strategies for treating infection, autoimmune diseases, and cancer.

Viral deubiquitinases: role in evasion of anti-viral innate immunity.

Host anti-viral innate-immune signalling pathways are regulated by a variety of post-translation modifications including ubiquitination, which is critical to regulate various signalling pathways for synthesis of anti-viral molecules. A homeostasis of host immune responses, induced due to viral infection and further ubiquitination, is maintained by the action of deubiquitinases (DUB). Infecting viruses utilize the process of deubiquitination for tricking host immune system wherein viral DUBs compete with host DUBs for inhibition of innate-immune anti-viral signalling pathways, which instead of maintaining an immune homeostasis bring about virus-mediated pathogenesis. This suggests that viruses co-evolve with their hosts to acquire similar machinery for tricking immune surveillance and establishing infection.

Essential role of HCMV deubiquitinase in promoting oncogenesis by targeting anti-viral innate immune signaling pathways.

Cancer is a multifactorial disease and virus-mediated carcinogenesis is one of the crucial factors, which is poorly understood. Human cytomegalovirus (HCMV) is a herpesvirus and its components have been evidenced to be associated with cancer of different tissue origin. However, its role in cancer remains unknown. Here, we identified a conserved herpesviral tegument protein known as pUL48 of HCMV, encoding deubiquitinase enzyme, as having a key role in carcinogenesis. We show using deubiquitinase sufficient- and deficient-HCMV that HCMV deubiquitinase is a key in inducing enhanced cellular metabolic activity through upregulation of several anti-apoptotic genes and downregulation of several pro-apoptotic genes expression. Furthermore, HCMV deubiquitinase acquires pro-tumor functions by inhibiting PRR-mediated type I interferon via deubiquitination of TRAF6, TRAF3, IRAK1, IRF7 and STING. Taken together, our results suggest that HCMV infection may promote oncogenesis by inhibiting innate immunity of the host.

NLRP3 Phosphorylation Is an Essential Priming Event for Inflammasome Activation.

Many infections and stress signals can rapidly activate the NLRP3 inflammasome to elicit robust inflammatory responses. This activation requires a priming step, which is thought to be mainly for upregulating NLRP3 transcription. However, recent studies report that the NLRP3 inflammasome can be activated independently of transcription, suggesting that the priming process has unknown essential regulatory steps. Here, we report that JNK1-mediated NLRP3 phosphorylation at S194 is a critical priming event and is essential for NLRP3 inflammasome activation. We show that NLRP3 inflammasome activation is disrupted in NLRP3-S194A knockin mice. JNK1-mediated NLRP3 S194 phosphorylation is critical for NLRP3 deubiquitination and facilitates its self-association and the subsequent inflammasome assembly. Importantly, we demonstrate that blocking S194 phosphorylation prevents NLRP3 inflammasome activation in cryopyrin-associated periodic syndromes (CAPS). Thus, our study reveals a key priming molecular event that is a prerequisite for NLRP3 inflammasome activation. Inhibiting NLRP3 phosphorylation could be an effective treatment for NLRP3-related diseases.

Novel therapeutic targets in Waldenstrom macroglobulinemia.

Understanding of molecular mechanisms that drive Waldenstrom macroglobulinemia (WM) cell survival are rapidly evolving. This review briefly highlights emerging "WM-relevant" targets; for which therapeutic strategies are currently being investigated in preclinical and clinical studies. With the discovery of MYD88L265P signaling and remarkable activity of ibrutinib in WM, other targets within the B-cell receptor pathway are now being focused on for therapeutic intervention. Additional targets which play a role in WM cell survival include TLR7, 8 and 9, proteasome-associated deubiquitinating enzymes (USP14 and UCHL5), XPO1/CRM1 and AURKA. New drugs for established targets are also discussed. Lastly, we spotlight 3 highly innovative WM-specific therapies: MYD88 peptide inhibitors, MYD88L265P-directed immune activation and CD19-directed chimeric antigen receptor T-cell therapy, which are in various stages of development. Indeed, treatment of WM is poised to undergo a paradigm shift in the coming years towards highly disease-driven and more personalized therapeutic modalities with curative intent.