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The NLRP3 inflammasome responds to infection and tissue damage, and rapidly escalates the intensity of inflammation by activating interleukin (IL)-1β, IL-18 and cell death by pyroptosis. How the NLRP3 inflammasome is negatively regulated is poorly understood. Here we show that NLRP3 inflammasome activation is suppressed by sumoylation. NLRP3 is sumoylated by the SUMO E3-ligase MAPL, and stimulation-dependent NLRP3 desumoylation by the SUMO-specific proteases SENP6 and SENP7 promotes NLRP3 activation. Defective NLRP3 sumoylation, either by NLRP3 mutation of SUMO acceptor lysines or depletion of MAPL, results in enhanced caspase-1 activation and IL-1β release. Conversely, depletion of SENP7 suppresses NLRP3-dependent ASC oligomerisation, caspase-1 activation and IL-1β release. These data indicate that sumoylation of NLRP3 restrains inflammasome activation, and identify SUMO proteases as potential drug targets for the treatment of inflammatory diseases. The NLRP3 inflammasome is an important component of inflammatory responses, but how it is negatively regulated is still unclear. Here the authors show that post-translational modification of NLRP3 by sumoylation suppresses inflammasome activity, and that desumoylation of NLRP3 by the SENP6 and SENP7 proteases promotes NLRP3 activation.

Small ubiquitin-like modifier (SUMO) typically conjugates to target proteins through isopeptide linkage to the ε-amino group of lysine residues. This posttranslational modification (PTM) plays pivotal roles in modulating protein function. Cofilins are key regulators of actin cytoskeleton dynamics and are well-known to undergo several different PTMs. Here, we show that cofilin-1 is conjugated by SUMO1 both in vitro and in vivo. Using mass spectrometry and biochemical and genetic approaches, we identify the N-terminal α-amino group as the SUMO-conjugation site of cofilin-1. Common to conventional SUMOylation is that the N-α-SUMOylation of cofilin-1 is also mediated by SUMO activating (E1), conjugating (E2), and ligating (E3) enzymes and reversed by the SUMO deconjugating enzyme, SENP1. Specific to the N-α-SUMOylation is the physical association of the E1 enzyme to the substrate, cofilin-1. Using F-actin co-sedimentation and actin depolymerization assays in vitro and fluorescence staining of actin filaments in cells, we show that the N-α-SUMOylation promotes cofilin-1 binding to F-actin and cofilin-induced actin depolymerization. This covalent conjugation by SUMO at the N-α amino group of cofilin-1, rather than at an internal lysine(s), serves as an essential PTM to tune cofilin-1 function during regulation of actin dynamics. SUMOylation plays a key role in modulating protein function. Here, the authors uncover a form of SUMOylation, termed N-αSUMOylation, where SUMO1 attaches to the N-terminus of cofilin1. This SUMOylation promotes cofilin-1 binding to F-actin and cofilin-induced actin depolymerization.

Crosstalk between the SUMO and ubiquitin pathways has recently been reported. However, no approach currently exists to determine the interrelationship between these modifications. Here, we report an optimized immunoaffinity method that permits the study of both protein ubiquitylation and SUMOylation from a single sample. This method enables the unprecedented identification of 10,388 SUMO sites in HEK293 cells. The sequential use of SUMO and ubiquitin remnant immunoaffinity purification facilitates the dynamic profiling of SUMOylated and ubiquitylated proteins in HEK293 cells treated with the proteasome inhibitor MG132. Quantitative proteomic analyses reveals crosstalk between substrates that control protein degradation, and highlights co-regulation of SUMOylation and ubiquitylation levels on deubiquitinase enzymes and the SUMOylation of proteasome subunits. The SUMOylation of the proteasome affects its recruitment to promyelocytic leukemia protein (PML) nuclear bodies, and PML lacking the SUMO interacting motif fails to colocalize with SUMOylated proteasome further demonstrating that this motif is required for PML catabolism. Ubiquitylation and SUMOylation are two important related post-translational modifications. Here the authors present an approach for the simultaneous identification and quantification of protein-wide SUMO and ubiquitin sites from a single sample, uncovering widespread crosstalk between the two modifications.

SUMOylation is a dynamic protein post-translational modification that regulates many eukaryotic proteins. Now a methodology using commercially available monoclonal antibodies coupled to MS analysis leads to the enrichment and identification of endogenous targets for SUMO1 and for SUMO2/3 in HeLa cells and mouse liver. This protocol can be adapted for other tissues and organs. SUMOylation is an essential modification that regulates hundreds of proteins in eukaryotic cells. Owing to its dynamic nature and low steady-state levels, endogenous SUMOylation is challenging to detect. Here, we present a method that allows efficient enrichment and identification of endogenous targets of SUMO1 and the nearly identical SUMO2 and 3 (SUMO 2/3) from vertebrate cells and complex organ tissue. Using monoclonal antibodies for which we mapped the epitope, we enriched SUMOylated proteins by immunoprecipitation and peptide elution. We used this approach in combination with MS to identify SUMOylated proteins, which resulted in the first direct comparison of the endogenous SUMO1- and SUMO2/3-modified proteome in mammalian cells, to our knowledge. This protocol provides an affordable and feasible tool to investigate endogenous SUMOylation in primary cells, tissues and organs, and it will facilitate understanding of SUMO's role in physiology and disease.

GABA A receptors (GABA A Rs) mediate the majority of fast inhibitory neurotransmission in the brain via synergistic association with the postsynaptic scaffolding protein gephyrin and its interaction partners. However, unlike their counterparts at glutamatergic synapses, gephyrin and its binding partners lack canonical protein interaction motifs; hence, the molecular basis for gephyrin scaffolding has remained unclear. In this study, we identify and characterize two new posttranslational modifications of gephyrin, SUMOylation and acetylation. We demonstrate that crosstalk between SUMOylation, acetylation and phosphorylation pathways regulates gephyrin scaffolding. Pharmacological intervention of SUMO pathway or transgenic expression of SUMOylation-deficient gephyrin variants rescued gephyrin clustering in CA1 or neocortical neurons of Gabra2 -null mice, which otherwise lack gephyrin clusters, indicating that gephyrin SUMO modification is an essential determinant for scaffolding at GABAergic synapses. Together, our results demonstrate that concerted modifications on a protein scaffold by evolutionarily conserved yet functionally diverse signalling pathways facilitate GABAergic transmission. Gephyrin is a cytoplasmic scaffolding protein that selectively forms postsynaptic scaffolds at GABAergic and glycinergic synapses. Here the authors characterize regulatory mechanisms determining gephyrin scaffolding and GABAA receptor synaptic transmission that involve acetylation, SUMOylation and phosphorylation.

Substantial increases in the conjugation of the main human SUMO paralogs, SUMO1, SUMO2, and SUMO3, are observed upon exposure to different cellular stressors, and such increases are considered important to facilitate cell survival to stress. Despite their critical cellular role, little is known about how the levels of the SUMO modifiers are regulated in the cell, particularly as it relates to the changes observed upon stress. Here we characterize the contribution of alternative splicing towards regulating the expression of the main human SUMO paralogs under normalcy and three different stress conditions, heat-shock, cold-shock, and Influenza A Virus infection. Our data reveal that the normally spliced transcript variants are the predominant mature mRNAs produced from the SUMO genes and that the transcript coding for SUMO2 is by far the most abundant of all. We also provide evidence that alternatively spliced transcripts coding for protein isoforms of the prototypical SUMO proteins, which we refer to as the SUMO alphas, are also produced, and that their abundance and nuclear export are affected by stress in a stress- and cell-specific manner. Additionally, we provide evidence that the SUMO alphas are actively synthesized in the cell as their coding mRNAs are found associated with translating ribosomes. Finally, we provide evidence that the SUMO alphas are functionally different from their prototypical counterparts, with SUMO1α and SUMO2α being non-conjugatable to protein targets, SUMO3α being conjugatable but targeting a seemingly different subset of protein from those targeted by SUMO3, and all three SUMO alphas displaying different cellular distributions from those of the prototypical SUMOs. Thus, alternative splicing appears to be an important contributor to the regulation of the expression of the SUMO proteins and the cellular functions of the SUMOylation system.

Centromeres are defined by a self-propagating chromatin structure based on stable inheritance of CENP-A containing nucleosomes. Here, we present a genetic screen coupled to pulse-chase labeling that allow us to identify proteins selectively involved in deposition of nascent CENP-A or in long-term transmission of chromatin-bound CENP-A. These include factors with known roles in DNA replication, repair, chromatin modification, and transcription, revealing a broad set of chromatin regulators that impact on CENP-A dynamics. We further identify the SUMO-protease SENP6 as a key factor, not only controlling CENP-A stability but virtually the entire centromere and kinetochore. Loss of SENP6 results in hyper-SUMOylation of CENP-C and CENP-I but not CENP-A itself. SENP6 activity is required throughout the cell cycle, suggesting that a dynamic SUMO cycle underlies a continuous surveillance of the centromere complex that in turn ensures stable transmission of CENP-A chromatin. Centromeres are a self-propagating chromatin structure that feature nucleosomes containing histone H3 variant CENP-A. Here, the authors screen for factors that play a role in CENP-A chromatin maintenance, finding that SUMO-protease SENP6 controls inheritance of chromatin bound CENP-A and is required for the maintenance of the centromere and kinetochore complex.

Targeting tumor proteostasis has emerged as a promising strategy in anticancer therapy, particularly through Hsp90 inhibition, which has shown clinical potential. However, the efficacy of Hsp90 inhibitors is limited by the activation of HSF1, a master regulator of the heat shock response (HSR), which mitigates proteotoxic stress by inducing protective chaperones. To address this limitation, we investigated the role of HSF1 SUMOylation in modulating its activity and its impact on Hsp90 inhibitor efficacy. We generated HSF1 mutants with lysine-to-arginine substitutions at five SUMOylation sites and studied their function in H1299 lung carcinoma cells with HSF1/HSF2 knockout, which lack a functional HSR. Unexpectedly, these mutants retained full transcriptional activity during the early phase of the heat shock response, mimicking the initial stress response of wild-type HSF1. SUMOylation inhibition using Subasumstat also led to altered nuclear stress bodies morphology but did not impair Hsp70 induction or enhance Hsp90 inhibitor cytotoxicity. Our findings reveal that SUMOylation is dispensable for HSF1 activation and transactivation capacity during the early phase of HSR. These results refine our understanding of HSF1 regulation and suggest that alternative strategies targeting HSF1 stability and degradation may enhance the therapeutic efficacy of proteostasis-targeting cancer therapies.

Rap2b, a proto-oncogene upregulated in colorectal cancer (CRC), undergoes protein S-palmitoylation at specific C-terminus sites (C176/C177). These palmitoylation sites are crucial for Rap2b localization on the plasma membrane (PM), as mutation of C176 or C177 results in cytosolic relocation of Rap2b. Our study demonstrates that Rap2b influences cell migration and invasion in CRC cells, independent of proliferation, and this activity relies on its palmitoylation. We identify ABHD17a as the depalmitoylating enzyme for Rap2b, altering PM localization and inhibiting cell migration and invasion. EGFR/PI3K signaling regulates Rap2b palmitoylation, with PI3K phosphorylating ABHD17a to modulate its activity. These findings highlight the potential of targeting Rap2b palmitoylation as an intervention strategy. Blocking the C176/C177 sites using an interacting peptide attenuates Rap2b palmitoylation, disrupting PM localization, and suppressing CRC metastasis. This study offers insights into therapeutic approaches targeting Rap2b palmitoylation for the treatment of metastatic CRC, presenting opportunities to improve patient outcomes.

E1 enzymes activate ubiquitin (Ub) and ubiquitin-like (Ubl) proteins in two steps by carboxy-terminal adenylation and thioester bond formation to a conserved catalytic cysteine in the E1 Cys domain. The structural basis for these intermediates remains unknown. Here we report crystal structures for human SUMO E1 in complex with SUMO adenylate and tetrahedral intermediate analogues at 2.45 and 2.6 Å, respectively. These structures show that side chain contacts to ATP·Mg are released after adenylation to facilitate a 130 degree rotation of the Cys domain during thioester bond formation that is accompanied by remodelling of key structural elements including the helix that contains the E1 catalytic cysteine, the crossover and re-entry loops, and refolding of two helices that are required for adenylation. These changes displace side chains required for adenylation with side chains required for thioester bond formation. Mutational and biochemical analyses indicate these mechanisms are conserved in other E1s. Conformational change in SUMO E1 Post-translational modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins such as SUMO regulate a broad array of cellular processes. Prior to conjugation, E1 enzymes must first activate Ub/Ubls in two steps. In the first step, E1s utilize ATP and magnesium to adenylate the C-terminal Ub/Ubl glycine, releasing pyrophosphate. In the second step, the Ub/Ubl adenylate is attacked by a conserved E1 cysteine, resulting in release of AMP and formation of a thioester bond between the C-terminal Ub/Ubl glycine and E1. Here, Olsen et al . use a combination of chemistry and structural biology to trap the E1 bound to mimics of the Ub/Ubl adenylate and thioester intermediates. These structures reveal that the E1 undergoes dramatic conformational changes and structural remodelling to create distinct active sites that propel the reaction forward. This study provides the most complete insight into the details of the E1 catalytic cycle yet observed. The post-translational modification of cellular proteins by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins — such as SUMO — regulates a broad array of cellular processes. E1 enzymes activate Ub and Ubl in two steps, by carboxy-terminal adenylation and thioester bond formation to a catalytic cysteine, but the structural basis for the intermediates remains unknown. Crystal structures for SUMO E1 in complex with SUMO adenylate and tetrahedral intermediate analogues are now reported and analysed.