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NLRP3 is a molecular sensor recognizing a wide range of danger signals. Its activation leads to the assembly of an inflammasome that allows for activation of caspase-1 and subsequent maturation of IL-1β and IL-18, as well as cleavage of Gasdermin-d and pyroptotic cell death. The NLRP3 inflammasome has been implicated in a plethora of diseases including gout, type 2 diabetes, atherosclerosis, Alzheimer's disease, and cancer. In this publication, we describe the discovery of a novel, tricyclic, NLRP3-binding scaffold by high-throughput screening. The hit ( 1 ) could be optimized into an advanced compound NP3-562 demonstrating excellent potency in human whole blood and full inhibition of IL-1β release in a mouse acute peritonitis model at 30 mg/kg po dose. An X-ray structure of NP3-562 bound to the NLRP3 NACHT domain revealed a unique binding mode as compared to the known sulfonylurea-based inhibitors. In addition, NP3-562 shows also a good overall development profile.

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is essential for immune responses triggered by antigen receptors but the contribution of its paracaspase activity is not fully understood. Here, we studied how MALT1 proteolytic function regulates T-cell activation and fate after engagement of the T-cell receptor pathway. We show that MLT-827, a potent and selective MALT1 paracaspase inhibitor, does not prevent the initial phase of T-cell activation, in contrast to the pan-protein kinase C inhibitor AEB071. However, MLT-827 strongly impacted cell expansion after activation. We demonstrate this is the consequence of profound inhibition of IL-2 production as well as reduced expression of the IL-2 receptor alpha subunit (CD25), resulting from defective canonical NF-κB activation and accelerated mRNA turnover mechanisms. Accordingly, MLT-827 revealed a unique transcriptional fingerprint of MALT1 protease activity, providing evidence for broad control of T-cell signaling pathways. Altogether, this first report with a potent and selective inhibitor elucidates how MALT1 paracaspase activity integrates several T-cell activation pathways and indirectly controls gamma-chain receptor dependent survival, to impact on T-cell expansion., (© 2017 Australasian Society for Immunology Inc.)

The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed that TRAF6 can activate MALT1 independently of the CBM. Intramolecular cleavage at R781/R770 removes a C-terminal TRAF6-binding site in both MALT1 isoforms, leaving MALT1B devoid of the two key interaction sites with TRAF6. A previously identified auto-proteolysis site of MALT1 at R149 leads to deletion of the death-domain, thereby abolishing interaction with BCL10. By using MALT1 isoforms and cleaved fragments thereof, as well as TRAF6 WT and mutant forms, this work shows that TRAF6 induces N-terminal auto-proteolytic cleavage of MALT1 at R149 and accelerates MALT1 protein turnover. The MALT1 fragment generated by N-terminal self-cleavage at R149 was labile and displayed enhanced signaling properties that required an intact K644 residue, previously shown to be a site for mono-ubiquitination of MALT1. Conversely, C-terminal self-cleavage at R781/R770 hampered the ability for self-cleavage at R149 and stabilized MALT1 by hindering interaction with TRAF6. C-terminal self-cleavage had limited impact on MALT1A but severely reduced MALT1B proteolytic and signaling functions. It also abrogated NF-κB activation by N-terminally cleaved MALT1A. Altogether, this study provides further insights into mechanisms that regulate the scaffolding and activation cycle of MALT1. It also emphasizes the reduced functional capacity of MALT1B as compared to MALT1A., Competing Interests: All co-authors work or used to work for Novartis. However, this does not alter our adherence to PLoS ONE policies on sharing data and materials.

The MALT1 paracaspase has arginine-directed proteolytic activity. A20 is a dual ubiquitin-editing enzyme involved in termination of NF-κB signaling. Upon T- or B-cell receptor engagement human (h) A20 is cleaved by MALT1 after arginine 439, yielding an N-terminal fragment (hA20p50) and a C-terminal one (hA20p37). The hA20p50 fragment has never been detected directly, thus limiting insight into the functional consequences of MALT1-mediated cleavage of A20. Here, various antibodies were tested, including newly generated hA20p50 and hA20p37 specific antibodies, leading to detection of the hA20p50 fragment produced after MALT1-mediated cleavage of ectopically expressed as well as endogenous A20 proteins. The properties of both A20 fragments, generated upon co-expression with a constitutively active MALT1 protein, were further studied by sub-cellular fractionation and fluorescence microscopy. In contrast to full-length A20 which is particulate and insoluble, we found hA20p50 to be soluble and readily released into the cytosol whereas hA20p37 was partially soluble, thus suggesting loss of compartmentalization as a possible mechanism for MALT1-mediated dampening of A20 function., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Mutations in the ubiquitous factor eIF2B involved in protein synthesis and its regulation have been reported in human brain genetic disorders. In order to analyse the functional consequences of the mutations and to find specific biomarkers of eIF2B-related disorders, proteomics and peptidomics studies were performed on lymphoblasts from eIF2B-mutated patients versus healthy patients. Curiously, following two-dimensional gel electrophoresis and mass fingerprints, mutations in the eIF2B complex did not significantly affect the proteome of the mutated lymphoblasts extracts. However, liquid chromatography based peptidomics studies revealed one apparently instable candidate compound in five out of the six mutated lymphoblastoid cell lines investigated.