Figure 5. MALT1 auto-proteolysis in activated B cells.

<p>A) ABC-DLBCL cell lines HBL-1 and OCI-Ly3 were treated with 50 µM z-VRPR-fmk (36 hrs hrs) and lysates were analysed for the presence of MALT1 and BCL10 cleavage fragments with a-MALT1, a-Cleaved BCL10 and a-Tubulin (loading control). B-C) The GCB-DLBCL cell lines BJAB and Raji were left untreated or stimulated with PMA/ionomycin (30 min min) with or without pre-treatment with 50 µM z-VRPR-fmk (30 min min). Lysates were analysed for the presence of MALT1 and BCL10 cleavage fragments, for p-ERK (activation control) and tubulin (loading control). D) Immunoblot of lysates of SSK41 cells and SSK41 cells with ectopic expression of the API2-MALT1 fusion variants A7M3 and A7M8, or the L232LI mutant of Card11 (C11m) respectively, with antibodies against the MALT1 C-terminus, the p76 neo-epitope, the CYLD C-terminus, the NIK C-terminus and Flag (ectopic A7M3, A7M8 and C11m). Numbers below blots depict band intensities of MALT1, p76 and the CYLD p70 fragment relative to lane 1. *  =  non-specific band. LC: loading control, a non-specific band obtained with the p76 neo-epitope antibody was used.</p

BCL10 mediates cleavage of MALT1 at R149 in 293T cells.

<p>A) Immunoblot of lysates of 293T cells transiently expressing MALT1 alone or in combination with BCL10 with antibodies against MALT1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103774#pone.0103774-Rebeaud1" target="_blank">[31]</a>, BCL10 and tubulin. B) Immunoblot of streptavidin pull-downs (bio-IPs) of Avi-tagged MALT1 and its mutants co-expressed with BCL10 in 293T cells as specified. AS: a-specific band. Arrows indicate the N-terminal p19 cleavage fragment. All molecular mass standards are in kDa.</p

MALT1 auto-proteolysis is required for IL-2 production by Jurkat T cells.

<p>A) Jurkat T cells were left untreated or stimulated with P/I for 30 min, with or without pre min, with or without pre-treatment with 50 µM z-VRPR-fmk for 30 min. Lysates were analysed for the presence of the cleavage fragments for BCL10 and MALT1 p19, for p min. Lysates were analysed for the presence of the cleavage fragments for BCL10 and MALT1 p19, for p-ERK (activation control) and tubulin (loading control). B) IL-2 production (ELISA) of Jurkat T cells stably expressing MALT1, MALT1-R149A, MALT1-C464A or MALT1-RACA, either untreated (-) or stimulated for 18 hrs with PMA hrs with PMA/ionomycin (P/I). Data shown as mean +/- S.D. (n = 3). Inset: Immunoblot with a-MALT1-C and a-Flag showing expression of ectopic MALT1 and mutants relative to endogenous MALT1 (lane 1). Numbers indicate fold overexpression relative to endogenous MALT1. AS: a-specific band obtained with a-Flag that serves as loading control. C) Immunoblot of cell lysates (top) and bio-IPs (bottom) from Jurkat T cells and Jurkat T cells with stable expression of Avi-tagged MALT1 or MALT1 mutants R149A, C464A and RACA with indicated antibodies.</p

MALT1 undergoes auto-proteolysis <i>in vitro</i>.

<p>A) Features of F-STII-MALT1 and LZ-MALT1. F: Flag epitope, STII: StrepII-tag, 6H: 6-Histidine tag, LZ: Leuzine zipper. B) Top: <i>In vitro</i> cleavage of the fluorogenic tetratpeptide substrate Ac-LVSR-AMC (50 µM) by F-STII-MALT1 in increasing concentrations of the cosmotropic salt NH4-citrate (0.2, 0.4, 0.6, and 0.8 M M), by F-STII-MALT1 in 0.8M NH₄-citrate buffer in the presence of the MALT1 protease inhibitors z-VRPR-fmk (10 µM) and z-LVSR-fmk (10 µM) and by LZ-MALT1 or LZ-MALT1-C464A in 0.8 M NH M NH₄-citrate buffer. The barchart shows cleavage activity as Fluorescence Units (FU) increase/min. Results are expressed as means ± SD (n = 3). Bottom: enzymatic reactions were analysed by immunoblotting with a-MALT1-C, a-p76 neo-epitope and a-MALT1-N.</p

Targeting mp-MALT1 to DRMs induces its proteolysis at R149 in 293T cells.

<p>A) Features of mp-MALT1 (mp: myristoylation-palmitoylation sequence) and Ub-p76 (Ubiquitin-p76 fusion protein). R149: MALT1 cleavage site. Flag: Flag epitope, DD: Death Domain, Ig: immunoglobulin-like domain, p20: caspase p20-like domain, C464: MALT1 catalytic cysteine, T6-Ig and T6-C: TRAF6 binding site in second Ig domain and C-terminus, respectively. Ub: Ubiquitin. B) NF-κB-reporter assays of 293T cells transiently expressing wild-type MALT1, mp-MALT1 or empty vector (mock). NF-κB-dependent luciferase activity is shown as fold induction of vector-transfected cells and represents the mean +/- S.D. of at least three independent experiments (n = 3). Cell lysates were immunoblotted with a-Flag, a non-specific band was used as loading control (LC). C) Lysates of 293T cells transiently transfected with mp-MALT1 were subjected to sucrose density gradient centrifugation and aliquots of the serial fractions (1-12 from top to bottom) were immunoblotted with a-MALT1-N, a-Lck, a kinase residing in the Detergent Resistant Membrane (DRM) fractions, and a-GAPDH, a cytosolic marker. D-E) Immunoblot of lysates of 293T cells transiently expressing wild-type MALT1, mp-MALT1 and its mutants or Ubiquitin-p76 as specified with indicated antibodies. eMALT1: endogenous MALT1. β-actin (D) and LC: non-specific band (E) are loading controls. Arrows (panel C, D, E)) indicate the N-terminal p19 or the C-terminal p76 cleavage fragment respectively. All molecular mass standards are in kDa.</p

Design of Selective sPLA₂‑X Inhibitor (−)-2-{2-[Carbamoyl-6-(trifluoromethoxy)‑1<i>H</i>‑indol-1-yl]pyridine-2-yl}propanoic Acid

A lead generation campaign identified indole-based sPLA₂-X inhibitors with a promising selectivity profile against other sPLA₂ isoforms. Further optimization of sPLA₂ selectivity and metabolic stability resulted in the design of (−)-<b>17</b>, a novel, potent, and selective sPLA₂-X inhibitor with an exquisite pharmacokinetic profile characterized by high absorption and low clearance, and low toxicological risk. Compound (−)-<b>17</b> was tested in an ApoE^–/– murine model of atherosclerosis to evaluate the effect of reversible, pharmacological sPLA₂-X inhibition on atherosclerosis development. Despite being well tolerated and achieving adequate systemic exposure of mechanistic relevance, (−)-<b>17</b> did not significantly affect circulating lipid and lipoprotein biomarkers and had no effect on coronary function or histological markers of atherosclerosis

Discovery of a Series of Indole‑2 Carboxamides as Selective Secreted Phospholipase A₂ Type X (sPLA₂‑X) Inhibitors

In order to assess the potential of sPLA₂-X as a therapeutic target for atherosclerosis, novel sPLA₂ inhibitors with improved type X selectivity are required. To achieve the objective of identifying such compounds, we embarked on a lead generation effort that resulted in the identification of a novel series of indole-2-carboxamides as selective sPLA2-X inhibitors with excellent potential for further optimization