Rare germline heterozygous missense variants in BRCA1-associated protein 1, BAP1, cause a syndromic neurodevelopmental disorder.

Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes.

Fragment-Sized and Bidentate (Immuno)Proteasome Inhibitors Derived from Cysteine and Threonine Targeting Warheads.

Constitutive- and immunoproteasomes are part of the ubiquitin-proteasome system (UPS), which is responsible for the protein homeostasis. Selective inhibition of the immunoproteasome offers opportunities for the treatment of numerous diseases, including inflammation, autoimmune diseases, and hematologic malignancies. Although several inhibitors have been reported, selective nonpeptidic inhibitors are sparse. Here, we describe two series of compounds that target both proteasomes. First, benzoxazole-2-carbonitriles as fragment-sized covalent immunoproteasome inhibitors are reported. Systematic substituent scans around the fragment core of benzoxazole-2-carbonitrile led to compounds with single digit micromolar inhibition of the ß5i subunit. Experimental and computational reactivity studies revealed that the substituents do not affect the covalent reactivity of the carbonitrile warhead, but mainly influence the non-covalent recognition. Considering the small size of the inhibitors, this finding emphasizes the importance of the non-covalent recognition step in the covalent mechanism of action. As a follow-up series, bidentate inhibitors are disclosed, in which electrophilic heterocyclic fragments, i.e., 2-vinylthiazole, benzoxazole-2-carbonitrile, and benzimidazole-2-carbonitrile were linked to threonine-targeting (R)-boroleucine moieties. These compounds were designed to bind both the Thr1 and ß5i-subunit-specific residue Cys48. However, inhibitory activities against (immuno)proteasome subunits showed that bidentate compounds inhibit the ß5, ß5i, ß1, and ß1i subunits with submicromolar to low-micromolar IC50 values. Inhibitory assays against unrelated enzymes showed that compounds from both series are selective for proteasomes. The presented nonpeptidic and covalent derivatives are suitable hit compounds for the development of either ß5i-selective immunoproteasome inhibitors or compounds targeting multiple subunits of both proteasomes.

Arkadia-SKI/SnoN signaling differentially regulates TGF-ß-induced iTreg and Th17 cell differentiation.

TGF-ß signaling is fundamental for both Th17 and regulatory T (Treg) cell differentiation. However, these cells differ in requirements for downstream signaling components, such as SMAD effectors. To further characterize mechanisms that distinguish TGF-ß signaling requirements for Th17 and Treg cell differentiation, we investigated the role of Arkadia (RNF111), an E3 ubiquitin ligase that mediates TGF-ß signaling during development. Inactivation of Arkadia in CD4+ T cells resulted in impaired Treg cell differentiation in vitro and loss of RORγt+FOXP3+ iTreg cells in the intestinal lamina propria, which increased susceptibility to microbiota-induced mucosal inflammation. In contrast, Arkadia was dispensable for Th17 cell responses. Furthermore, genetic ablation of two Arkadia substrates, the transcriptional corepressors SKI and SnoN, rescued Arkadia-deficient iTreg cell differentiation both in vitro and in vivo. These results reveal distinct TGF-ß signaling modules governing Th17 and iTreg cell differentiation programs that could be targeted to selectively modulate T cell functions.

Comprehensive analysis of ubiquitin-proteasome system genes related to prognosis and immunosuppression in head and neck squamous cell carcinoma.

The ubiquitin-proteasome system (UPS) with a capacity of degrading multiple intracellular proteins is an essential regulator in tumor immunosurveillance. Tumor cells that escape from recognition and destruction of immune system have been consistently characterized an important hallmark in the setting of tumor progression. Little know about the exact functions of UPS-related genes (UPSGs) and their relationships with antitumor immunity in head and neck squamous cell carcinoma (HNSCC) patients. In this study, for the first time, we comprehensively identified 114 differentially expressed UPSGs (DEUPSGs) and constructed a prognostic risk model based on the eight DEUPSGs (BRCA1, OSTM1, PCGF2, PSMD2, SOCS1, UCHL1, UHRF1, and USP54) in the TCGA-HNSCC database. This risk model was validated using multiple data sets (all P < 0.05). The high-risk score was found to be an independently prognostic factor in HNSCC patients and was significantly correlated with T cells suppression. Accordingly, our risk model can act as a prognostic signature and provide a novel concept for improving the precise immunotherapy for patients with HNSCC.

The ubiquitylation of IL-1ß limits its cleavage by caspase-1 and targets it for proteasomal degradation.

Interleukin-1ß (IL-1ß) is activated by inflammasome-associated caspase-1 in rare autoinflammatory conditions and in a variety of other inflammatory diseases. Therefore, IL-1ß activity must be fine-tuned to enable anti-microbial responses whilst limiting collateral damage. Here, we show that precursor IL-1ß is rapidly turned over by the proteasome and this correlates with its decoration by K11-linked, K63-linked and K48-linked ubiquitin chains. The ubiquitylation of IL-1ß is not just a degradation signal triggered by inflammasome priming and activating stimuli, but also limits IL-1ß cleavage by caspase-1. IL-1ß K133 is modified by ubiquitin and forms a salt bridge with IL-1ß D129. Loss of IL-1ß K133 ubiquitylation, or disruption of the K133:D129 electrostatic interaction, stabilizes IL-1ß. Accordingly, Il1bK133R/K133R mice have increased levels of precursor IL-1ß upon inflammasome priming and increased production of bioactive IL-1ß, both in vitro and in response to LPS injection. These findings identify mechanisms that can limit IL-1ß activity and safeguard against damaging inflammation.

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.

Discovery of linear ubiquitination, a crucial regulator for immune signaling and cell death.

Ubiquitination is a reversible post-translational modification that regulates function of conjugated proteins by decorating with ubiquitin chains-polymer of ubiquitin-in most cases. The discovery of linear ubiquitin chains and the linear ubiquitin chain assembly complex (LUBAC) ubiquitin ligase complex can be considered as paradigm shift in the ubiquitin research because the linear ubiquitin chain is generated via the N-terminal Met of ubiquitin, although the other ubiquitin chains are generated via one of seven Lys residues in ubiquitin. Moreover, ubiquitination is distributed throughout eukaryotic kingdoms; however, no linear ubiquitination could be found in lower eukaryotes including yeasts. Although the involvement of ubiquitination in proteolysis is well-documented, linear ubiquitination plays crucial roles in immune signaling and cell death regulation. Moreover, dysregulation of LUBAC-mediated linear ubiquitination underlies various human diseases including autoinflammation and cancer. Here, I introduce how linear ubiquitination was discovered and outline a brief history of linear ubiquitination research.

E3 ubiquitin ligase Casitas B lineage lymphoma-b and its potential therapeutic implications for immunotherapy.

The distinction of self from non-self is crucial to prevent autoreactivity and ensure protection from infectious agents and tumors. Maintaining the balance between immunity and tolerance of immune cells is strongly controlled by several sophisticated regulatory mechanisms of the immune system. Among these, the E3 ligase ubiquitin Casitas B cell lymphoma-b (Cbl-b) is a newly identified component in the ubiquitin-dependent protein degradation system, which is thought to be an important negative regulator of immune cells. An update on the current knowledge and new concepts of the relevant immune homeostasis program co-ordinated by Cbl-b in different cell populations could pave the way for future immunomodulatory therapies of various diseases, such as autoimmune and allergic diseases, infections, cancers and other immunopathological conditions. In the present review, the latest findings are comprehensively summarized on the molecular structural basis of Cbl-b and the suppressive signaling mechanisms of Cbl-b in physiological and pathological immune responses, as well as its emerging potential therapeutic implications for immunotherapy in animal models and human diseases.

Ubiquitination of MHC Class II Is Required for Development of Regulatory but Not Conventional CD4+ T Cells.

MHC class II (MHC II) displays peptides at the cell surface, a process critical for CD4+ T cell development and priming. Ubiquitination is a mechanism that dictates surface MHC II with the attachment of a polyubiquitin chain to peptide-loaded MHC II, promoting its traffic away from the plasma membrane. In this study, we have examined how MHC II ubiquitination impacts the composition and function of both conventional CD4+ T cell and regulatory T cell (Treg) compartments. Responses were examined in two models of altered MHC II ubiquitination: MHCIIKRKI /KI mice that express a mutant MHC II unable to be ubiquitinated or mice that lack membrane-associated RING-CH 8 (MARCH8), the E3 ubiquitin ligase responsible for MHC II ubiquitination specifically in thymic epithelial cells. Conventional CD4+ T cell populations in thymus, blood, and spleen of MHCIIKRKI/KI and March8 -/- mice were largely unaltered. In MLRs, March8 -/-, but not MHCIIKRKI/KI, CD4+ T cells had reduced reactivity to both self- and allogeneic MHC II. Thymic Treg were significantly reduced in MHCIIKRKI/KI mice, but not March8 -/- mice, whereas splenic Treg were unaffected. Neither scenario provoked autoimmunity, with no evidence of immunohistopathology and normal levels of autoantibody. In summary, MHC II ubiquitination in specific APC types does not have a major impact on the conventional CD4+ T cell compartment but is important for Treg development.

Legionella pneumophila Excludes Autophagy Adaptors from the Ubiquitin-Labeled Vacuole in Which It Resides.

Xenophagy targets intracellular pathogens for destruction by the host autophagy pathway. Ubiquitin chains are conjugated to xenophagic targets and recruit multiple autophagy adaptors. The intracellular pathogen Legionella pneumophila resides in a vacuole that is ubiquitinated; however, this pathogen avoids xenophagic detection. Here, the mechanisms by which L. pneumophila can prevent the host xenophagy pathway from targeting the vacuole in which it resides were examined. Ubiquitin-labeled vacuoles containing L. pneumophila failed to recruit autophagy adaptors by a process that was independent of RavZ function. Coinfection studies were conducted using a strain of Listeria monocytogenes that served as a robust xenophagic target. Legionella pneumophila infection blocked xenophagic targeting of L. monocytogenes by a RavZ-dependent mechanism. Importantly, when coinfection studies were conducted with a RavZ-deficient strain of L. pneumophila, L. monocytogenes was targeted by the host xenophagy system but vacuoles containing L. pneumophila avoided targeting. Enhanced adaptor recruitment to the vacuole was observed by using a strain of L. pneumophila in which all of the effector proteins in the SidE family were deleted; however, this strain was still not targeted by the host autophagy pathway. Thus, there are at least two pathways by which L. pneumophila can disrupt xenophagic targeting of the vacuole in which it resides. One mechanism involves global disruption of the host autophagy machinery by the effector protein RavZ. A second cis-acting mechanism prevents the binding of autophagy adaptors to the ubiquitin-decorated surface of the L. pneumophila-containing vacuole.

The Ubiquitin-Modifying Enzyme A20 Terminates C-Type Lectin Receptor Signals and Is a Suppressor of Host Defense against Systemic Fungal Infection.

C-type lectin receptors (CLRs) play key roles in antifungal defense. CLR-induced NF-κB is central to CLR functions in immunity, and thus, molecules that control the amplitude of CLR-induced NF-κB could profoundly influence host defense against fungal pathogens. However, little is known about the mechanisms that negatively regulate CLR-induced NF-κB, and molecules which act on the CLR family broadly and which directly regulate acute CLR-signaling cascades remain unidentified. Here, we identify the ubiquitin-editing enzyme A20 as a negative regulator of acute NF-κB activation downstream of multiple CLR pathways. Absence of A20 suppression results in exaggerated CLR responses in cells which are A20 deficient and also cells which are A20 haplosufficient, including multiple primary immune cells. Loss of a single allele of A20 results in enhanced defense against systemic Candida albicans infection and prolonged host survival. Thus, A20 restricts CLR-induced innate immune responses in vivo and is a suppressor of host defense against systemic fungal infection.

A nanobody that recognizes a 14-residue peptide epitope in the E2 ubiquitin-conjugating enzyme UBC6e modulates its activity.

A substantial fraction of eukaryotic proteins is folded and modified in the endoplasmic reticulum (ER) prior to export and secretion. Proteins that enter the ER but fail to fold correctly must be degraded, mostly in a process termed ER-associated degradation (ERAD). Both protein folding in the ER and ERAD are essential for proper immune function. Several E2 and E3 enzymes localize to the ER and are essential for various aspects of ERAD, but their functions and regulation are incompletely understood. Here we identify and characterize single domain antibody fragments derived from the variable domain of alpaca heavy chain-only antibodies (VHHs or nanobodies) that bind to the ER-localized E2 UBC6e, an enzyme implicated in ERAD. One such VHH, VHH05 recognizes a 14 residue stretch and enhances the rate of E1-catalyzed ubiquitin E2 loading in vitroand interferes with phosphorylation of UBC6e in response to cell stress. Identification of the peptide epitope recognized by VHH05 places it outside the E2 catalytic core, close to the position of activation-induced phosphorylation on Ser184. Our data thus suggests a site involved in allosteric regulation of UBC6e's activity. This VHH should be useful not only to dissect the participation of UBC6e in ERAD and in response to cell stress, but also as a high affinity epitope tag-specific reagent of more general utility.

Ubiquitin-Dependent and -Independent Roles of E3 Ligase RIPLET in Innate Immunity.

The conventional view posits that E3 ligases function primarily through conjugating ubiquitin (Ub) to their substrate molecules. We report here that RIPLET, an essential E3 ligase in antiviral immunity, promotes the antiviral signaling activity of the viral RNA receptor RIG-I through both Ub-dependent and -independent manners. RIPLET uses its dimeric structure and a bivalent binding mode to preferentially recognize and ubiquitinate RIG-I pre-oligomerized on dsRNA. In addition, RIPLET can cross-bridge RIG-I filaments on longer dsRNAs, inducing aggregate-like RIG-I assemblies. The consequent receptor clustering synergizes with the Ub-dependent mechanism to amplify RIG-I-mediated antiviral signaling in an RNA-length dependent manner. These observations show the unexpected role of an E3 ligase as a co-receptor that directly participates in receptor oligomerization and ligand discrimination. It also highlights a previously unrecognized mechanism by which the innate immune system measures foreign nucleic acid length, a common criterion for self versus non-self nucleic acid discrimination.

Regulation of host cell pyroptosis and cytokines production by Mycobacterium tuberculosis effector PPE60 requires LUBAC mediated NF-κB signaling.

Tuberculosis, caused by Mycobacterium tuberculosis infection, remains a global public health threat. The success of M. tuberculosis largely contributes to its manipulation of host cell fate. The role of M. tuberculosis PE/PPE family effectors in the host destiny was intensively explored. In this study, the role of PPE60 (Rv3478) was characterized by using Rv3478 recombinant M. smegmatis. PPE60 can promote host cell pyroptosis via caspases/NLRP3/gasdermin. The production of pro-inflammatory cytokines, such as IL-1ß, IL-6, IL-12p40 and TNF-α was altered by PPE60. We found that LUBAC was involved in PPE60-elicited NF-κB signaling by using Linear Ubiquitin Chain Assembly Complex (LUBAC)-specific inhibitor gliotoxin. The PPE60 recombinant M. smegmatis survival rate within macrophages is increased, as well as elevated resistance to stresses such as low pH, surface stresses and antibiotics exposure. For a first time it is firstly reported that M. tuberculosis effector PPE60 can modulate the host cell fate via LUBAC-mediated NF-κB signaling.

Hydrazide Mimics for Protein Lysine Acylation To Assess Nucleosome Dynamics and Deubiquitinase Action.

A range of acyl-lysine (acyl-Lys) modifications on histones and other proteins have been mapped over the past decade but for most, their functional and structural significance remains poorly characterized. One limitation in the study of acyl-Lys containing proteins is the challenge of producing them or their mimics in site-specifically modified forms. We describe a cysteine alkylation-based method to install hydrazide mimics of acyl-Lys post-translational modifications (PTMs) on proteins. We have applied this method to install mimics of acetyl-Lys, 2-hydroxyisobutyryl-Lys, and ubiquityl-Lys that could be recognized selectively by relevant acyl-Lys modification antibodies. The acyl-Lys modified histone H3 proteins were reconstituted into nucleosomes to study nucleosome dynamics and stability as a function of modification type and site. We also installed a ubiquityl-Lys mimic in histone H2B and generated a diubiquitin analog, both of which could be cleaved by deubiquitinating enzymes. Nucleosomes containing the H2B ubiquityl-Lys mimic were used to study the SAGA deubiquitinating module's molecular recognition. These results suggest that acyl-Lys mimics offer a relatively simple and promising strategy to study the role of acyl-Lys modifications in the function, structure, and regulation of proteins and protein complexes.

Checkpoints in TNF-Induced Cell Death: Implications in Inflammation and Cancer.

Tumor necrosis factor (TNF) is a proinflammatory cytokine that coordinates tissue homeostasis by regulating cytokine production, cell survival, and cell death. However, how life and death decisions are made in response to TNF is poorly understood. Many inflammatory pathologies are now recognized to be driven by aberrant TNF-induced cell death, which, in most circumstances, depends on the kinase Receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Recent advances have identified ubiquitin (Ub)-mediated phosphorylation of RIPK1 as belonging to crucial checkpoints for cell fate in inflammation and infection. A better understanding of these checkpoints might lead to new approaches for the treatment of chronic inflammatory diseases fueled by aberrant RIPK1-induced cell death, and/or reveal novel strategies for anticancer immunotherapies, harnessing the ability of RIPK1 to trigger immunogenic cell death.

Integrin beta 1 inhibition alleviates the chronic hyperproliferative dermatitis phenotype of SHARPIN-deficient mice.

SHARPIN (Shank-Associated RH Domain-Interacting Protein) is a component of the linear ubiquitin chain assembly complex (LUBAC), which enhances TNF-induced NF-κB activity. SHARPIN-deficient (Sharpincpdm/cpdm) mice display multi-organ inflammation and chronic proliferative dermatitis (cpdm) due to TNF-induced keratinocyte apoptosis. In cells, SHARPIN also inhibits integrins independently of LUBAC, but it has remained enigmatic whether elevated integrin activity levels in the dermis of Sharpincpdm/cpdm mice is due to increased integrin activity or is secondary to inflammation. In addition, the functional contribution of increased integrin activation to the Sharpincpdm/cpdm phenotype has not been investigated. Here, we find increased integrin activity in keratinocytes from Tnfr1-/- Sharpincpdm/cpdm double knockout mice, which do not display chronic inflammation or proliferative dermatitis, thus suggesting that SHARPIN indeed acts as an integrin inhibitor in vivo. In addition, we present evidence for a functional contribution of integrin activity to the Sharpincpdm/cpdm skin phenotype. Treatment with an integrin beta 1 function blocking antibody reduced epidermal hyperproliferation and epidermal thickness in Sharpincpdm/cpdm mice. Our data indicate that, while TNF-induced cell death triggers the chronic inflammation and proliferative dermatitis, absence of SHARPIN-dependent integrin inhibition exacerbates the epidermal hyperproliferation in Sharpincpdm/cpdm mice.

Viral oncoproteins and ubiquitination: accessing a cellular toolbox for modifying protein function.

Human Papillomavirus oncoproteins directly target components of the Ubiquitin Proteasome System (UPS) and allow perturbation of multiple cellular processes linked to the control of apoptosis, transcription and innate immunity. This activity primarily creates an environment favourable for viral replication, but can contribute towards malignancy, thus highlighting the roles that manipulation of the UPS can play in the development of human cancers.