Neprilysin 4 regulates muscle contraction and male fertility in Drosophila melanogaster

Abstract

Neprilysins belong to the highly conserved M13 family of zinc metalloendopeptidases. They are expressed in various tissues and predominantly occur as membrane-bound ectoenzymes. Based on their broad substrate specificity, neprilysins are involved in numerous physiological processes by hydrolyzing and thus inactivating signaling peptides. As neprilysin-mediated cleavage is critical to peptide activity and homeostasis, impaired neprilysin activity is linked to several human diseases. However, despite their physiological relevance and their therapeutic potential, the physiological functions of many neprilysin family members are only partially understood. This thesis focuses on the Drosophila melanogaster Neprilysin 4 (Nep4) and aims to expand the current knowledge on its physiological impact. In the first project, Nep4 was identified as an essential regulator of the sarcoplasmic and endoplasmic reticulum Ca2+-ATPase (SERCA) in myocytes. In this context, Nep4 localizes to the sarcoplasmic reticulum (SR) membrane and hydrolyzes two SERCA-inhibitory micropeptides: Sarcolamban A and Sarcolamban B. The Nep4-mediated hydrolysis affects the SR membrane integral peptides on different levels. On the one hand, cleaved micropeptides lose their membrane anchoring, thus likely becoming unable to interact with SERCA. On the other hand, cleaved peptides lose the ability to form oligomeric species, which is an additional regulatory feature in their interaction with SERCA. Strikingly, the analysis of corresponding human factors indicates an evolutionary conservation of the underlying regulatory mechanism. In the second project of this thesis, the impact of the Nep4 peptidase on male fertility in Drosophila was investigated. In sperm, Nep4 localizes to the acrosome, an organelle that is highly conserved and essential for successful fertilization. The analysis shows that Nep4 activity is critical during spermatogenesis by maintaining the structural integrity of the sperm head and, particularly, the acrosome. Impaired Nep4 activity resulted in deformed acrosomes, which are shorter and more spherical in the anterior region when compared to wildtype organelles. Furthermore, impaired Nep4 function disrupted the structural organization of the sperm head, as indicated by an altered distribution of specific glycoproteins at the surface of the sperm tip and a less pronounced acrosome compartmentalization. Ultimately, these defects lead to male sterility. In summary, the present work describes two novel and crucial functions of Nep4 and contributes to a more comprehensive understanding of the functionality of neprilysins in general. It is noteworthy that these functions rely on the subcellular localization of Nep4 to internal membranes, rather than on the typical neprilysin localization at the cell surface. This observation uncovers novel sites of action for neprilysins and further expands their broad physiological relevance