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Deubiquitinating enzymes (DUBs) are key regulators of cellular homoeostasis, and their dysregulation is associated with several human diseases. The ovarian tumour protease (OTU) family of DUBs are biochemically well-characterised and of therapeutic interest, yet only a few tool compounds exist to study their cellular function and therapeutic potential. Here we present a chemoproteomics fragment screening platform for identifying novel DUB-specific hit matter, that combines activity-based protein profiling with high-throughput chemistry direct-to-biology optimisation to enable rapid elaboration of initial fragment hits against OTU DUBs. Applying these approaches, we identify an enantioselective covalent fragment for OTUD7B, and validate it using chemoproteomics and biochemical DUB activity assays. The ovarian tumour protease (OTU) family of deubiquitinating enzymes (DUBs) are biochemically well-characterised and of therapeutic interest, however, only a few tool compounds exist to study their cellular function and therapeutic potential. Here, the authors present a chemoproteomics fragment screening platform for identifying DUB-specific hit matter, which combines activity-based protein profiling with high-throughput optimisation to enable rapid elaboration of initial fragment hits against OTU DUBs, and identify an enantioselective chloroacetamide-based covalent fragment for OTUD7B. [ABSTRACT FROM AUTHOR]

Identifying pharmacological probes for human proteins represents a key opportunity to accelerate the discovery of new therapeutics. High-content screening approaches to expand the ligandable proteome offer the potential to expedite the discovery of novel chemical probes to study protein function. Screening libraries of reactive fragments by chemoproteomics offers a compelling approach to ligand discovery, however, optimising sample throughput, proteomic depth, and data reproducibility remains a key challenge. We report a versatile, label-free quantification proteomics platform for competitive profiling of cysteine-reactive fragments against the native proteome. This high-throughput platform combines SP4 plate-based sample preparation with rapid chromatographic gradients. Data-independent acquisition performed on a Bruker timsTOF Pro 2 consistently identified ~23,000 cysteine sites per run, with a total of ~32,000 cysteine sites profiled in HEK293T and Jurkat lysate. Crucially, this depth in cysteinome coverage is met with high data completeness, enabling robust identification of liganded proteins. In this study, 80 reactive fragments were screened in two cell lines identifying >400 ligand-protein interactions. Hits were validated through concentration-response experiments and the platform was utilised for hit expansion and live cell experiments. This label-free platform represents a significant step forward in high-throughput proteomics to evaluate ligandability of cysteines across the human proteome.This paper presents a label-free chemoproteomics platform using data-independent acquisition to profile covalent fragment binding across the human proteome. The platform offers high reproducibility and data completeness, identifying >400 protein-ligand interactions for probe development. [ABSTRACT FROM AUTHOR]

Protein modification with ubiquitin is a key mechanism for the regulation of numerous cellular functions (8). The transfer of ubiquitin onto a substrate is catalysed by E3 ligases, which can [...], Linear ubiquitin chains are important regulators of cellular signalling pathways that control innate immunity and inflammation through nuclear factor (NF)-κB activation and protection against tumour necrosis factor-α-induced apoptosis (1-5). They are synthesized by HOIP, which belongs to the RBR (RING-between-RING) family of E3 ligases and is the catalytic component of LUBAC (linear ubiquitin chain assembly complex), a multisubunit E3 ligase (6). RBR family members act as RING/HECT hybrids, employing RING1 to recognize ubiquitin-loaded E2 while a conserved cysteine in RING2 subsequently forms a thioester intermediate with the transferred or 'donor' ubiquitin (7). Here we report the crystal structure of the catalytic core of HOIP in its apo form and in complex with ubiquitin. The carboxy-terminal portion of HOIP adopts a novel fold that, together with a zinc-finger, forms a ubiquitin-binding platform that orients the acceptor ubiquitin and positions its α-amino group for nucleophilic attack on the E3~ubiquitin thioester. The C-terminal tail of a second ubiquitin molecule is located in close proximity to the catalytic cysteine, providing a unique snapshot of the ubiquitin transfer complex containing both donor and acceptor ubiquitin. These interactions are required for activation of the (NF)-κB pathway in vivo, and they explain the determinants of linear ubiquitin chain specificity by LUBAC.

In addition to the established roles of K63- and K48-linked ubiquitin chains, linear ubiquitin chains have recently emerged as important regulators of the NF-κB pathway, controlling immune responses, as well [...], SHARPIN is a ubiquitin-binding and ubiquitin-like-domain-containing protein which, when mutated in mice, results in immune system disorders and multi-organ inflammation (1,2). Here we report that SHARPIN functions as a novel component of the linear ubiquitin chain assembly complex (LUBAC) and that the absence of SHARPIN causes dysregulation of NF-κB and apoptotic signalling pathways, explaining the severe phenotypes displayed by chronic proliferative dermatitis (cpdm) in SHARPIN-deficient mice. Upon binding to the LUBAC subunit HOIP (also known as RNF31), SHARPIN stimulates the formation of linear ubiquitin chains in vitro and in vivo. Coexpression of SHARPIN and HOIP promotes linear ubiquitination of NEMO (also known as IKBKG), an adaptor of the IκB kinases (IKKs) and subsequent activation of NF-κB signalling, whereas SHARPIN deficiency in mice causes an impaired activation of the IKK complex and NF-κB in B cells, macrophages and mouse embryonic fibroblasts (MEFs). This effect is further enhanced upon concurrent downregulation of HOIL-1L (also known as RBCK1), another HOIP-binding component of LUBAC. In addition, SHARPIN deficiency leads to rapid cell death upon tumour-necrosis factor α (TNF-α) stimulation via FADD- and caspase-8-dependent pathways. SHARPIN thus activates NF-κB and inhibits apoptosis via distinct pathways in vivo.

14-3-3 proteins mediate signal transduction by binding to proteins containing phosphoserine. RSXpSXP and RXY/FXpSXP, binding motifs, have been identified as being present in most known 14-3-3 binding proteins. The crystal structure of 14-3-3zeta was complexed with a phosphoserine motif in polyoma middle-T, and determined to 2.6 A resolution. It is shown that the 14-3-3 dimer tightly binds to single molecules, reflecting bidentate association as a signaling mechanism.

Research into whether GAPs stimulate the intrinsic GTPase activity of the Ras superfamily of proteins has involved determining the structure of p50rhoGAP alone and as a complex with Cdc42Hs.GMPPNP. This made it possible to identify residues that could play a role in G-protein binding and GTPase activation. It was established that the conformational rearrangement that takes place in progression from the ground state (GMPPNP) to the transition state (GDP.A1F(super -)(sub 4)) seems to be vital for GTPase activation.