Abstract 582: Determining and Modeling of the Cardiac Protein-Protein Interaction Network of the Inotropic Factor S100A1 by AP-MS/MS

Abstract

Background: S100A1 is an EF-hand calcium (Ca 2+ ) sensor protein with predominant expression in the mammalian heart. Although its outstanding influence on cardiac performance is well described, only few target molecules are known so far, such as the sarcoplasmic reticulum Ca 2+ ATPase (SERCA2a). Objective and Results: The discrepancy between the known interactors and the diversity of S100A1`s functions brings up the question which key targets are undetected so far and can, in addition to the known ones, explain the importance of S100A1 for the heart function. S100A1 knock-out (SKO -/- ) mouse heart extracts and lysates of isolated SKO -/- cardiomyocytes were subjected to HPLC S100A1 affinity-purification (AP) columns coupled to tandem mass spectrometry (MS) followed by network modeling of the interactome, to identify novel and to confirm previously known targets in the heart. Human recombinant S100A1 protein AP columns were used to trap EGTA- and Ca 2+ -dependent interactions. Subsequent MS and computational analyses revealed more than 90 novel cardiac interactors for S100A1 in a strict Ca 2+ -dependent manner, while previously known targets were confirmed. We showed that helix 4 of S100A1 (S100A1ct) alone can improve main parameters for the heart performance and might thereby function as a therapeutic biologic. Investigating the molecular mechanisms of S100A1`s interactions with its target molecules might help to understand this effect and unveil relevant regions for other therapeutic effects. In order to uncover the mechanisms of the various interactions, docking experiments were performed to gain an understanding, which areas of S100A1 and S100A1ct might be relevant for the biological functions on selected targets. Thus we detected a region on SERCA2a where S100A1 as well as S100A1ct might compete with the SERCA2a inhibitor phospholamban and by that may improve the SERCA2a activity. These in silico findings will be combined with experimental investigations of the mode of interaction by Cross-link MS experiments. Conclusion: Combining target screening via AP-MS/MS with computer-supported epitope mapping provides a powerful strategy to understand the molecular action of known therapies and by that helps to develop new treatments against heart failure.