Neprilysins: An Evolutionarily Conserved Family of Metalloproteases That Play Important Roles in Reproduction in Drosophila

Abstract

Members of the M13 class of metalloproteases have been implicated in diseases and in reproductive fitness. Nevertheless, their physiological role remains poorly understood. To obtain a tractable model with which to analyze this protein family's function, we characterized the gene family in Drosophila melanogaster and focused on reproductive phenotypes. The D. melanogaster genome contains 24 M13 class protease homologs, some of which are orthologs of human proteases, including neprilysin. Many are expressed in the reproductive tracts of either sex. Using RNAi we individually targeted the five Nep genes most closely related to vertebrate neprilysin, Nep1-5, to investigate their roles in reproduction. A reduction in Nep1, Nep2, or Nep4 expression in females reduced egg laying. Nep1 and Nep2 are required in the CNS and the spermathecae for wild-type fecundity. Females that are null for Nep2 also show defects as hosts of sperm competition as well as an increased rate of depletion for stored sperm. Furthermore, eggs laid by Nep2 mutant females are fertilized normally, but arrest early in embryonic development. In the male, only Nep1 was required to induce normal patterns of female egg laying. Reduction in the expression of Nep2-5 in the male did not cause any dramatic effects on reproductive fitness, which suggests that these genes are either nonessential for male fertility or perform redundant functions. Our results suggest that, consistent with the functions of neprilysins in mammals, these proteins are also required for reproduction in Drosophila, opening up this model system for further functional analysis of this protein class and their substrates. P ROTEASES play key roles in diverse physiological systems. One such family of metalloproteases, the M13 class of neutral endopeptidases, consists mainly of membranebound zinc proteases that are involved in the processing of neuropeptides and peptide hormones (reviewed in Turner et al. 2000;Turner et al. 2001;Bland et al. 2008). In mammals, seven members of this family have been identified, of which neprilysin (NEP) and endothelin converting enzyme (ECE) are the best studied. These proteins have been implicated in various diseases including cardiovascular disease (Segura and Ruilope 2011;Wick et al. 2011), Alzheimer's disease (Mulder et al. 2012;Klein et al. 2013), inflammation and inflammatory disorders (Wong et al. 2011), and cancer (Smollich et al. 2007;Maguer-Satta et al. 2011).In addition to their role in disease, NEPs are essential for development and reproduction in mammals. The mammalian Neprilysin-2, called NL1 in mice, is highly expressed in the testis. NL1-deficient males sire fewer pups, even though spermatogenesis appears to be unaffected (Carpentier et al. 2004). In females, NEP expression in the uterus is modulated by estrogen treatment in rats (Pinto et al. 1999) and during the estrogen/progesterone cycle in humans (Head et al. 1993). In female rats and mice, controlled degradation of tachykinins, particularly substance-P, by NEP in the uterus ...

Critical roles of long noncoding RNAs in Drosophila spermatogenesis

“…Some testis-specific lncRNAs are required for male fertility and late spermatogenesis Given the testis-specific expression of the lncRNAs we identified, we examined the fertility of knockout (KO) mutant males using a standard fertility test (Sitnik et al 2014). After virgin females mated with wild-type control or lncRNA knockout males, the numbers of progeny from each female fly were counted daily over a 15-d period.…”

“…For each lncRNA knockout mutant, one lncRNA homozygous mutant virgin male was placed in a vial with one wild-type virgin female at 27.5°C. For the next 15 d, the flies from each mating were transferred to new vials every 24 h. Upon eclosion, all progeny from each vial were counted (Sitnik et al 2014). The average number of flies per parental pair and standard errors were calculated for each combination of genotypes.…”

Drosophila neprilysins control insulin signaling and food intake via cleavage of regulatory peptides

“…In Drosophila melanogaster , at least five neprilysin genes are expressed (Meyer et al, 2011; Sitnik et al, 2014), two of the corresponding protein products, Nep2 and Nep4, were reported to be enzymatically active (Bland et al, 2007; Meyer et al, 2009; Thomas et al, 2005). With respect to Nep4, a critical function of the enzyme's non-catalytic intracellular N-terminus has been demonstrated: when present in excess, the domain induces severe muscle degeneration concomitant with lethality during late larval development.…”

“…In addition, Nep2 has been implicated in the regulation of locomotion and geotactic behavior (Bland et al, 2009), and neprilysin activity in general appears to be critical to the formation of middle- and long-term memory (Turrel et al, 2016), as well as to the regulation of pigment dispersing factor (PDF) signaling within circadian neural circuits (Isaac et al, 2007). However, despite these experiments and recent findings that suggest a critical role of neprilysins in reproduction (Sitnik et al, 2014), the physiological functionality of these enzymes is still far from being understood. In this respect, the lack of identified substrates with in vivo relevance is a major hindrance.…”

Effects of temperature on transcriptome and cuticular hydrocarbon expression in ecologically differentiated populations of desert Drosophila

“…The first cluster was enriched for 43 proteolysis/peptidase genes including trypsin encoding genes and the ortholog Ance , a member of the peptidase M2 family. There were also members of the M13 class of metalloproteases including neprilysins, involved with peptide hormone metabolism in Drosophila , and male and female reproduction including egg laying and sperm use in mated females (Sitnik et al., ). Some of these endopeptidase genes are expressed in the female reproductive tracts of D. mojavensis and D. arizonae (Kelleher & Markow, ; Kelleher & Pennington, ).…”