SERCA regulation by Peptides
Project Title: Functional characterization of a novel mechanism regulating SERCA activity and Ca2+ homeostasis in Drosophila melanogaster
Funding Source: DFG SFB944, TP21
Principle Investigator: Heiko Harten with Annika Buhr, Ronja Schiemann
I am using Drosophila melanogaster as a model organism to understand the physiological functionality of metalloproteases in general and of neprilysins in particular. In addition to investigating their functional relevance during development, we are interested in understanding the regulatory impact of neprilysins on distinct physiological processes, such as muscle and heart contraction or energy metabolism.
Muscle contraction represents a well characterized molecular process that is, to a considerable extent, regulated by variable Ca2+ concentrations within the cytosol. A major player responsible for modulating this concentration is the Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase (SERCA) that transports Ca2+ from the cytosol into the sarcoplasmic reticulum (SR). This activity significantly reduces cytosolic Ca2+ levels and thus marks the beginning of the muscle relaxation phase. Accordingly, tight regulation of SERCA-activity is critical to proper functionality of muscle tissue.
As known for vertebrates and insects, activity of SERCA is regulated by peptides that bind to the enzyme and thereby modulate its activity. Vertebrate and fly loss-of-function mutants for corresponding peptides exhibit impaired Ca2+ transients in heart cells as well as severe heart arrhythmia. Up to now, it is largely unknown how homeostasis and turnover of such peptides are regulated.
We found that increased expression of the peptidase Neprilysin 4 (Nep4) phenocopied the depicted effects on heart physiology. Significantly, Nep4 catalytic activity was essential, thus confirming abnormal peptide hydrolysis as a causative factor. By combining in vitro and in vivo analyses, we identified Nep4 mediated hydrolysis of SERCA regulatory Sarcolamban peptides as the critical physiological event. Furthermore, Nep4 and SERCA coprecipitated and exhibited substantial colocalization with Sarcolamban in membranes of the SR, indicating that close spatial proximity is of high mechanistic relevance.
We currently focus on in-depth analyses of the suborganellar localization and interaction dynamics between Nep4, Sarcolamban, and SERCA. Due to a high evolutionary conservation of involved factors, the project has significant potential to advance the current understanding of muscle physiology and function in health and disease.
STED-based triple labeling of Nepilysin4, SERCA, and Sarcolamban in 3rd instar larval body wall muscles confirms co-localization of all factors in distinct membrane microcompartments of the sarcoplasmic reticulum, particularly around the nucleus (N).