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-jB 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 gammachain receptor dependent survival, to impact on T-cell expansion.
Activity-based proteomics is a methodology that is used to quantify the catalytically active subfraction of enzymes present in complex mixtures such as lysates or living cells. To apply this approach for in-cell selectivity profiling of inhibitors of serine proteases, we designed a novel activity-based probe (ABP). This ABP consists of (i) a fluorophosphonate-reactive group, directing the probe toward serine hydrolases or proteases and (ii) an alkyne functionality that can be specifically detected at a later stage with an azide-functionalized reporter group through a Cu(I)-catalyzed coupling reaction ("click chemistry"). This novel ABP was shown to label the active site of several serine proteases with greater efficiency than a previously reported fluorophosphonate probe. More importantly, our probe was cell-permeable and achieved labeling of enzymes within living cells with efficiency similar to that observed for the corresponding lysate fraction. Several endogenous serine hydrolases whose activities were detected upon in-cell labeling were identified by two-dimensional gel and MS analyses. As a proof of principle, cell-permeable inhibitors of an endogenous serine protease (prolyl endopeptidase) were assessed for their potency and specificity in competing for the in situ labeling of the selected enzyme. Altogether these results open new perspectives for safety profiling studies in uncovering potential cellular "side effects" of drugs (unanticipated off-target inhibition or activation) that may be overlooked by standard selectivity profiling methods. Molecular & Cellular Proteomics 7:1241-1253, 2008.Activity-based proteomics, in comparison with classic genomics and proteomics approaches, has been specially devised to enable the detection of active enzymes. Such a methodology is of particular relevance for example in the protease field where only a subfraction of the total enzyme pool (having undergone successive translocation, post-translational modifications, and proteolytic activation and having escaped binding of endogenous inhibitors) effectively participates in the cellular processes. To profile enzymatic activities in biological samples, activity-based proteomics relies on small reactive marker molecules called activity-based probes (ABPs), 1 which covalently and specifically label the accessible active sites of catalytic enzymes (1-3). So far, several directed and non-directed ABPs have been described that allow the monitoring of more than 20 enzyme classes (for extensive reviews, see Refs. 2-5).The detection of the population of active enzymes is of primary relevance for biological and in particular for pharmaceutical research because it could lead to the discovery of new targets for drug development. Indeed by comparing the activity profile of enzymes under physiological versus pathological conditions (e.g. of normal cells versus parasite-infected cells (6) or versus cancer cells (7-11)), several groups have identified up-regulated active enzymes potentially involved in the development and/or the mainte...
Summary SDZ 280-446 is a semi-synthetic derivative of a natural cyclic peptolide. Its ability to sensitise in vitro tumour cells whose resistance is due to P-glycoprotein-mediated anticancer-drug efflux was shown using four different pairs of parental drug-sensitive (Par-) and multidrug-resistant (MDR-) cell lines, from three different species (mouse, human, Chinese hamster) representing four different cell lineages (monocytic leukaemia, nasopharyngeal epithelial carcinoma, colon epithelial carcinoma, ovary fibroblastoid carcinoma), and using four different drug classes (colchicine, vincristine, daunomycin/doxorubicin and etoposide). By measuring its capacity to restore normal drug sensitivity of MDR-cells in culture in vitro, it appeared that SDZ 280-446 belongs to the same class of very potent chemosensitisers as the cyclosporin derivative SDZ PSC 833: both are about one order of magnitude more active than cyclosporin A (CsA), which is itself about one order of magnitude more active than other known chemosensitisers (including verapamil, quinidine and amiodarone which have already entered clinical trials in MDR reversal). Low concentrations of SDZ 280-446 could also restore cellular daunomycin retention in MDR-P388 cells to the levels found in the Par-P388 cells. SDZ 280-446 was also effective as a chemosensitiser when given orally in vivo. In a syngeneic mouse model, combined therapy with vinca alkaloids given i.p. and SDZ 280-446 given per os for 5 consecutive days significantly prolonged the survival of MDR-P388 tumour-bearing mice, when compared with mice receiving vinca alkaloids alone. Another protocol, using three cycles of i.p. doxorubicin at 4 day intervals, could also not increase MDR-P388 tumour-bearing mouse survival unless the mice received SDZ 280-446 orally 4h before each doxorubicin injection. Though only very few combined therapy treatment protocols have been tested so far, clear increases in survival time of MDR-tumour-bearing mice were regularly obtained, leaving hope for major improvement of the therapy using other dosing schedules.
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