【兴大报告 Xing Da Lecture 554】

题  目：N-terminomics. How TAILS Positional Proteomics Mechanistically Deciphers Pathology and led to an Allosteric Molecular Corrector Rescuing Function in an Immunodeficient Patient 

报告人: 

Prof. Christopher M. Overall

Centre for Blood Research, University of British Columbia, Canada

时　间：2018年10月19日(星期五)下午2:00

地　点：化学楼A204/206

主请人：刘小云

报告摘要：

Tospecifically enrich for mature protein N-termini and neo-N-termini of proteinswe developed 6 and 10-plex TMT TAILS (Terminal Amine Isotopic Labeling ofSubstrates) (Nature Biotech 28, 281-288 (2010);Nature Protocols 6, 1578-1611 (2011). In analyzing the N-terminome of normalhuman tissues we find that the N-termini of protein chains in vivo can commence at many points C terminal to the predictedstart site and result from proteolytic processing to generate stable proteinchains: Proteolytic processing generates new protein species withcharacteristic neo-N termini that are frequently accompanied by alteredhalf-lives, function, interactions and location. We used TAILS tomechanistically dissect a severe human immunodeficiency disease.

    The paracaspase MALT1proteolytic activity and its molecular scaffolding are central for transducinglymphocyte antigen receptor activation of NF-κB with unregulated MALT1 activityleading to B cell lymphomas and autoimmune disease. We developed nanomolar,selective allosteric inhibitors of MALT1 paracaspase activity that bind byreplacing the side chain of Trp580 and locking the protease in an inactiveconformation. Interestingly, we had previously identified a patient homozygousfor a hypomorphic MALT1 mutationsuffering from combined immunodeficiency who carries a serine mutation atTrp580. We used TAILS, the N-terminal positionalproteomics approach to compare lymphocytes from the MALT1^mut/mut patient with healthyMALT1^+/mut family membersand normal individuals using 10-plex Tandem Mass Tag in the N-terminomics TAILSapproach with MS3 synchronous peak selection quantification, with and without BCell receptor stimulation. Fromthe MALT1 cleaved neo-N terminal peptide (prime side) and the natural Nterminus of HOIL1 identified by TAILS, and the nonprime side of the HOIL1cleavage site identified by preTAILS shotgun proteomics, we identified HOIL1 ofthe linear ubiquitin chain assembly complex (LUBAC) as a novel MALT1 substrate.Upon B and T cell receptor stimulation HOIL1 cleavage resulted in disassemblyof LUBAC and loss of linear ubiquitination in T and B cells that preventedreactivation of NF-kB signaling. However,in this immunodeficiency, the Trp580Ser mutation weakened theinteraction between the paracaspase and C-terminal immunoglobulin domainsresulting in protein instability (Tm 46.1°C versus52.3°C) and consequently reduced MALT1 function and protein levels. The newallosteric inhibitors also bind MALT1-Trp580Ser, stabilizing the mutant proteinand increasing the Tm to that of wild-type MALT1. Allosteric, but not MALT1active site inhibition of patient MALT1^mut/mutlymphocytes restored MALT1 protein levels invivo and rescued NF-κB and JNK signaling. Following compound washout, MALT1substrate cleavage was also rescued. Thus, a low molecular weight compound canrescue an enzyme deficiency by substituting for the mutated residue, inspiringpotential novel precision therapies to increase mutant enzyme activity.