Neprilysin 4 modulates SERCA activity via cleavage of Sarcolamban A

PAULULAT A; SCHIEMANN R; FERRERO P; HARTEN H; STUKE M

Filadelfia

Congreso; 59th Annual Drosophila Research Conference; 2018

Sociedad Americana de Genética

393 Neprilysin 4 modulates SERCA activity via cleavage of Sarcolamban A. R. Schiemann1, M. Stuke1, P. Ferrero2, A. Paululat1, H. Harten1 1) Zoology and Developmental Biology, Osnabrueck University, Osnabrueck, Lower Saxony, DE; 2) Cardiovascular Research Center, National University of La Plata, La Plata, Argentina. Muscle contraction represents a highly conserved and well characterized molecular process that requires proper regulationof the intracellular Ca2+ concentration. By transporting Ca2+ from the cytosol into the sarcoplasmic reticulum (SR), the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) constitutes a major player regulating this concentration. Dysregulation of SERCA severely affects initiation of muscle relaxation, which renders tight regulation of SERCA activity crucial to proper heart and muscle function. As known for vertebrates, SERCA activity is regulated by binding of certain peptides, e.g. Phospholamban or Sarcolipin. In Drosophila melanogaster, at least two peptides are known to mediate this functionality, Sarcolamban A (SclA) and Sarcolamban B (SclB). By binding to SERCA, the Scl peptides significantly reduce its activity (Magny et al., 2013). Consequently, Scl Loss-of function mutants exhibit impaired Ca2+ transients in heart cells, concomitant with severe heart arrhythmia. Up to now, it is largely unknown how turnover of such peptides within membranes of the SR is regulated. Interestingly, we found that increased expression of the peptidase Neprilysin 4 (Nep4), which also localizes to the SR membrane, phenocopies these effects on heart physiology. In addition, SERCA-activity is considerably elevated in corresponding animals, which presumably represents the physiological explanation for the phenotypes. Ectopic expression of catalytically inactive Nep4 is without consequences, which confirms that impaired catalytic activity, and thus abnormal peptide hydrolysis, represents a causative factor. In order to characterize the molecular mechanism by which Nep4 regulates SERCA activity, we initially assessed orientation of Nep4 in the SR membrane. Based on the results we performed cleavage assays, which confirmed that Nep4 is able to hydrolyze SclA at its C-terminus. Heart-specific expression of the resulting truncated peptide will reveal the physiological relevance of the cleavage event. In addition, simultaneous overexpression of SclA and Nep4, combined with super resolution microscopy and pull-down assays, will help to further elucidate the Nep4-Scl-SERCA interaction. Our detailed analysis of the Nep4 mediated regulation of SERCA activity will significantly advance the current understanding of muscle physiology and functionality.