Abstract 445: Essential Role of the S100A1-C-terminus Domain in Maintaining Cardiac-inotrope S100A1 Protein Stability

Abstract

Introduction: S100A1 is a type of S100 protein with two EF-hand motif. It has a unique characteristic of interacting with multiple proteins of contractile machinery & mitochondria. S100A1 protein able to govern proper systolic & diastolic performance of cardiomyocyte interacting, respectively, with SERCA2A & RYR2. S100A1 level decreases dramatically as a consequence of cellular cues such as oxidative-stress or activation of cellular proteasomes. Selective reconstitution of reduced S100A1 level by AAV9-gene transfer rescued failing heart. Because of the unique ability of S100A1 to interact with myriads of proteins with multiple function, we aimed to dissect the structure-function relationship of the domains of S100A1. Methods & results: The C-terminus domain of S100A1 (S100A1ct) equally recapitulated the inotropic & other benefits of the S100A1-FL in experimental model of heart failure & identified as the bioactive lead domain of S100A1. To elucidate the function of the S100A1 without the C-terminus, here after, referred as S100A1ΔCT, we adenoviraly expressed S100A1ΔCT, & as a control, either expressing GFP or S100A1-FL. We, inadvertently, discovered that S100A1ΔCT could not be detected at protein level. PCR analysis & sequencing proved the presence S100A1ΔCT mRNA transcript. Bioinformatics revealed the presence of SUMO interaction motif (SIM) with in the C-terminus & multiple ubiquitination residues on S100A1ΔCT, suggesting the SIM domain of the S100A1 is crucial for stability. Inhibition of cellular proteasome lead to the detection of the S100A1ΔCT protein in cells over expressing S100A1ΔCT. We overexpressed S100a1ΔCT, deletion mutants having part of the SIM motif (S100a1-SIM - ), the entire motif (S100a1-SIM + ) & the S100a1-FL. Whereas the overexpression of S100A1-SIM + & S100A1-FL were detected, S100A1ΔCT & S100A1-SIM - could not be detected at protein level. Conclusion: The C-terminus of S100A1 is essential for stability of S100A1, besides, its potent inotropic function. Taken together, we deciphered the previously unrecognized posttranslational modification governed S100A1 regulation, thus providing new insight into the development of novel therapeutic strategies for heart failure via posttranslational manipulation of S100A1.