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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Peptide Inhibitors of the Intrinsic Pathway of ApoptosisTargeting CARD-CARD InteractionsYadira Palacios-Rodríguez 1,2 , Guillermo García-Lainez 1 ,Mar Orzáez 1 , and Enrique Pérez-Payá 1,21 Laboratory of Peptide and Protein Chemistry, Centro de Investigación Príncipe Felipe,Valencia, E-46012, Valencia, Spain; 2 Instituto de Biomedicina de Valencia IBV –CSIC,E-46010, Valencia, SpainIntroductionThe caspase recruitment domain (CARD) is a module present in proteins that play pivotalroles in programmed cell death (apoptosis) and inflammation. In the apoptotic intrinsicpathway, the interaction between the CARD domain of Apaf-1 (apoptotic proteaseactivatingfactor) and the CARD domain of procaspase 9 (PC9) is essential for theapoptosome activation [1]. In inflammation, proteins as those of the NLR family inparticular NOD-1, NOD-2 and Nalp-1 also allow binding to a downstream effectormolecule through CARD-CARD interaction [2,3]. Although the topology between CARDdomains is identical, the primary sequence conservation is generally low; however, as someinterface resident amino acids show a high degree of conservation, they were selected asbasis for the synthesis of peptide apoptosome inhibitors.Results and DiscussionPeptides derived from Apaf-1, PC9 and the counterpart sequences of NOD-1 and Nalp-1CARD were synthesized by Fmoc chemistry, characterized by mass spectrometry andpurified by HPLC. We have selected the helices interacting between Apaf-1 CARD andPC9 CARD which include strongly conserved amino acids between several CARDs. Thepanel of four peptides homologues to CARD from Apaf-1 and PC9 were inhibitors ofapoptosome in two in-vitro assay formats (Table 1), the first of them was with therecombinant active mini Apaf-1 depleted from WD40 domain [4] and determining the PC9activity by Ac-LEHD-afc substrate degradation. In the second assay format, cytosolicextracts of 293 cells were depleted from endogenous Apaf-1 by chromatography(FT fraction) and the effect of each peptide was evaluated with recombinant Apaf-1 fulllength supplemented externally, the result is reported as effect of caspase-3 activityinhibition by Ac-DEVD-afc substrate degradation (Figure 1A and 1B).A. B. C.% Caspase 9 activity inhibition1008060402002.63 3.65 1.66 4.67 2.75 3.76 3.78Peptide 100 μM% Caspase 3 activity inhibition1008060402002.63 3.65 1.66Peptide 100 μM02.63:3.65 50:50 50:25 25:50 25:251.66:4.67 - - - -Peptide proportion μMFig. 1. Peptide inhibitory effect. A. Caspase-9 activity inhibition using rApaf-1 amino acids1-591 and rPC9 in presence of dATP. B. Caspase-3 activity on FT extracts reconstitutedwith rApaf-1-XL. C. Synergic peptide effect. The peptides were combined at differentproportions in micro molar range. The inhibitory effect was potentiated only by synergismbetween Apaf-1 CARD-derived peptides (2.63 and 3.65).% Caspase 3 activity inhibition (%)10080604020-50:50552