Poison peptides interactions with the sarcoplasmic reticulum Ca-ATPase (SERCA).

Abstract

Maintaining cellular integrity requires maintenance of protein quality, so when damaged/misfolded proteins fail to be repaired, they are degraded via proteolysis. Proteolysis increases in heart disease, overwhelming the proteosome, resulting in an accumulation of protein fragments in the cell. We propose that undegraded transmembrane protein fragments found in heart failure may persist in the membrane and interact with SERCA, competing with and displacing the physiological regulatory micropeptide phospholamban. These interactions with “poison peptides” may contribute to calcium mishandling in heart failure. To test this hypothesis, we performed mass spectrometry of membrane fractions isolated from failing human myocardium. We detected approximately 1000 protein fragments of interest in the failing heart, then narrowed our selection to high-abundance transmembrane protein fragments ranging from 20-40 amino acids. We used fluorescence resonance energy transfer (FRET) to test whether seven candidate poison proteins could interact with SERCA. Some peptides did not interact with SERCA at all, some bound weakly, and some bound with a high affinity. The highest affinity candidates bound SERCA as avidly as some native regulatory micropeptides. The candidate peptides tested here include transmembrane fragments of phospholamban (PLB), phospholemman (PLM), calcium voltage-gated channel subunit alpha1 E (CACNA1E) and sarcolemma associated protein (SLMAP). We also tested whether the putative poison peptides altered the structure of SERCA, using time-correlated single photon counting to quantify intramolecular FRET in a “2-color SERCA” construct labeled with mCyRFP-1 and mMaroon1. Overall, the results suggest that transmembrane peptide fragments generated in heart failure can interact with SERCA and may disrupt calcium handling in the failing heart.