Identification and bioinformatic analysis of neprilysin and neprilysin-like metalloendopeptidases in Drosophila melanogaster.

The neprilysin (M13) family of metalloendopeptidases comprises highly conserved ectoenzymes that cleave and thereby inactivate many physiologically relevant peptides in the extracellular space. Impaired neprilysin activity is associated with numerous human diseases. Here, we present a comprehensive list and classification of M13 family members in Drosophila melanogaster. Seven Neprilysin (Nep) genes encode active peptidases, while 21 Neprilysin-like (Nepl) genes encode proteins predicted to be catalytically inactive. RNAseq data demonstrate that all 28 genes are expressed during development, often in a tissue-specific pattern. Most Nep proteins possess a transmembrane domain, whereas almost all Nepl proteins are predicted to be secreted

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

Insulin and IGF signaling are critical to numerous developmental and physiological processes, with perturbations being pathognomonic of various diseases, including diabetes. Although the functional roles of the respective signaling pathways have been extensively studied, the control of insulin production and release is only partially understood. Herein, we show that in Drosophila expression of insulin-like peptides is regulated by neprilysin activity. Concomitant phenotypes of altered neprilysin expression included impaired food intake, reduced body size, and characteristic changes in the metabolite composition. Ectopic expression of a catalytically inactive mutant did not elicit any of the phenotypes, which confirms abnormal peptide hydrolysis as a causative factor. A screen for corresponding substrates of the neprilysin identified distinct peptides that regulate insulin-like peptide expression, feeding behavior, or both. The high functional conservation of neprilysins and their substrates renders the characterized principles applicable to numerous species, including higher eukaryotes and humans. DOI: http://dx.doi.org/10.7554/eLife.19430.00

Functional in vivo characterization of Neprilysin as a central regulator of insulin signaling and muscle contraction in Drosophila melanogaster

Peptides play pivotal roles in the regulation of various physiological processes. As neuropeptides or peptide hormones, they can bind to a range of receptors and thereby trigger the activation of different pathways, including insulin signaling. Another central functionality is facilitated by the action of the as regulins summarized transmembrane micropeptides. By binding to the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), the regulins control Ca2+ homeostasis and muscle contraction. With the ongoing identification of novel modulatory micropeptides encoded by small open reading frames, the urgency to understand peptide-dependent regulatory networks rises. In this regard, especially impact and physiological relevance exerted by the enzymatic inactivation of the mature, biologically active peptides are far from completely understood. Neprilysins are metalloendopeptidases expressed throughout the animal kingdom. Based on their broad substrate specificity, the activity of neprilysins is crucial for the modulation of multiple peptide-dependent processes. This work aimed to identify new peptide substrates of the Drosophila melanogaster Neprilysin 4 (Nep4) and investigate the enzyme's physiological impact on the affected regulatory mechanisms. The first part of the work could identify 16 novel Nep4 peptide substrates that play essential roles in insulin signaling and the regulation of food intake: allatostatin A1-A4, adipokinetic hormone, corazonin, diuretic hormone 31, drosulfakinin 1 and 2, leucokinin, two short neuropeptide F peptides, and tachykinin 1-4. Thereby, aberrant expression of Nep4 leads to severe phenotypes linked to misregulation of insulin signaling, including reduced body size and weight, compromised food intake, and a characteristic shift in metabolomic composition. To further investigate and understand the complex functionality of the newly discovered Nep4 substrates, these peptides were tested for their ability to modulate the Drosophila heartbeat. A combined in vitro/in vivo screen revealed that the tested substrates exert chronotropic as well as inotropic effects, rendering the peptides as essential novel modulators of the heartbeat in Drosophila. The main project of this thesis was based on the initial finding that animals with Nep4 overexpression exhibit severe impairments of body wall muscle and heart functionality. By applying various experiments, including analyses of muscle and heart contraction, measurement of Ca2+ transients, pull-down studies, STED super-resolution microscopy, and mass spectrometry, Neprilysin 4 was identified as a novel modulator of SERCA activity. The molecular underpinning of this regulatory mechanism is the Nep4 mediated cleavage and inactivation of Drosophila SERCA-inhibitory Sarcolamban micropeptides SCLA and SCLB. Strikingly, cleavage experiments using the mammalian neprilysin and apparent colocalization of Neprilysin and SERCA in human heart tissue indicate evolutionary conservation of this mechanism. In summary, this work could identify a range of so far unknown Nep4 substrates and thereby point out the critical roles these class of enzymes plays in insulin signaling as well as the physiology of muscle and heart contraction

Identification and In Vivo Characterisation of Cardioactive Peptides in <i>Drosophila melanogaster</i>

Neuropeptides and peptide hormones serve as critical regulators of numerous biological processes, including development, growth, reproduction, physiology, and behaviour. In mammals, peptidergic regulatory systems are complex and often involve multiple peptides that act at different levels and relay to different receptors. To improve the mechanistic understanding of such complex systems, invertebrate models in which evolutionarily conserved peptides and receptors regulate similar biological processes but in a less complex manner have emerged as highly valuable. Drosophila melanogaster represents a favoured model for the characterisation of novel peptidergic signalling events and for evaluating the relevance of those events in vivo. In the present study, we analysed a set of neuropeptides and peptide hormones for their ability to modulate cardiac function in semi-intact larval Drosophila melanogaster. We identified numerous peptides that significantly affected heart parameters such as heart rate, systolic and diastolic interval, rhythmicity, and contractility. Thus, peptidergic regulation of the Drosophila heart is not restricted to chronotropic adaptation but also includes inotropic modulation. By specifically interfering with the expression of corresponding peptides in transgenic animals, we assessed the in vivo relevance of the respective peptidergic regulation. Based on the functional conservation of certain peptides throughout the animal kingdom, the identified cardiomodulatory activities may be relevant not only to proper heart function in Drosophila, but also to corresponding processes in vertebrates, including humans

Interplay between SERCA, 4E-BP, and eIF4E in the Drosophila heart.

Appropriate cardiac performance depends on a tightly controlled handling of Ca2+ in a broad range of species, from invertebrates to mammals. The role of the Ca2+ ATPase, SERCA, in Ca2+ handling is pivotal, and its activity is regulated, inter alia, by interacting with distinct proteins. Herein, we give evidence that 4E binding protein (4E-BP) is a novel regulator of SERCA activity in Drosophila melanogaster during cardiac function. Flies over-expressing 4E-BP showed improved cardiac performance in young individuals associated with incremented SERCA activity. Moreover, we demonstrate that SERCA interacts with translation initiation factors eIF4E-1, eIF4E-2 and eIF4E-4 in a yeast two-hybrid assay. The specific identification of eIF4E-4 in cardiac tissue leads us to propose that the interaction of elF4E-4 with SERCA may be the basis of the cardiac effects observed in 4E-BP over-expressing flies associated with incremented SERCA activity

Neprilysins regulate muscle contraction and heart function via cleavage of SERCA-inhibitory micropeptides

Muscle contraction depends on strictly controlled Ca2+ transients within myocytes. A major player maintaining these transients is the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase, SERCA. Activity of SERCA is regulated by binding of micropeptides and impaired expression or function of these peptides results in cardiomyopathy. To date, it is not known how homeostasis or turnover of the micropeptides is regulated. Herein, we find that the Drosophila endopeptidase Neprilysin 4 hydrolyzes SERCA-inhibitory Sarcolamban peptides in membranes of the sarcoplasmic reticulum, thereby ensuring proper regulation of SERCA. Cleavage is necessary and sufficient to maintain homeostasis and function of the micropeptides. Analyses on human Neprilysin, sarcolipin, and ventricular cardiomyocytes indicates that the regulatory mechanism is evolutionarily conserved. By identifying a neprilysin as essential regulator of SERCA activity and Ca2+ homeostasis in cardiomyocytes, these data contribute to a more comprehensive understanding of the complex mechanisms that control muscle contraction and heart function in health and disease