Neprilysin (NEP), a thermolysin-like zinc metalloendopeptidase, plays an important role in turning off peptide signalling events at the cell surface. It is involved in the metabolism of a number of regulatory peptides of the mammalian nervous, cardiovascular, inflammatory and immune systems. Examples include enkephalins, tachykinins, natriuretic and chemotactic peptides. NEP is an integral plasma membrane ectopeptidase of the M13 family of zinc peptidases. Other related mammalian NEP-like enzymes include the endothelin-converting enzymes (ECE-1 and ECE-2), KELL and PEX. A number of novel mammalian homologues of NEP have also recently been described. NEP family members are potential therapeutic targets, for example in cardiovascular and inflammatory disorders, and potent and selective inhibitors such as phosphoramidon have contributed to understanding enzyme function. Inhibitor design should be facilitated by the recent three-dimensional structural solution of the NEP-phosphoramidon complex. For several of the family members, however, a well-defined physiological function or substrate is lacking. Knowledge of the complete genomes of Caenorhabditis elegans and Drosophila melanogaster allows the full complement of NEP-like activities to be analysed in a single organism. These model organisms also provide convenient systems for examining cell-specific expression, developmental and functional roles of this peptidase family, and reveal the power of functional genomics.

Quiescence, or a sleep-like state, is a common and important feature of the daily lives of animals from both invertebrate and vertebrate taxa, suggesting that sleep appeared early in animal evolution. Recently, Drosophila melanogaster has been shown to be a relevant and powerful model for the genetic analysis of sleep behaviour. The sleep architecture of D. melanogaster is sexually dimorphic, with females sleeping much less than males during day-time, presumably because reproductive success requires greater foraging activity by the female as well as the search for egg-laying sites. However, this loss of sleep and increase in locomotor activity will heighten the risk for the female from environmental and predator hazards. In this study, we show that virgin females can minimize this risk by behaving like males, with an extended afternoon 'siesta'. Copulation results in the female losing 70 per cent of day-time sleep and becoming more active. This behaviour lasts for at least 8 days after copulation and is abolished if the mating males lack sex peptide (SP), normally present in the seminal fluid. Our results suggest that SP is the molecular switch that promotes wakefulness in the post-mated female, a change of behaviour compatible with increased foraging and egg-laying activity. The stress resulting from SP-dependent sleep deprivation might be an important contribution to the toxic side-effects of male accessory gland products that are known to reduce lifespan in post-mated females.

Peptides structurally related to mammalian tachykinins have recently been isolated from the brain and intestine of several insect species, where they are believed to function as both neuromodulators and hormones. Further evidence for the signaling role of insect tachykinin-related peptides was provided by the cloning and characterization of cDNAs for two tachykinin receptors from Drosophila melanogaster. However, no endogenous ligand has been isolated for the Drosophila tachykinin receptors to date. Analysis of the Drosophila genome allowed us to identify a putative tachykininrelated peptide prohormone (prepro-DTK) gene. A 1.5-kilobase pair cDNA amplified from a Drosophila head cDNA library contained an 870-base pair open reading frame, which encodes five novel Drosophila tachykininrelated peptides (called DTK peptides) with conserved C-terminal FXGXR-amide motifs common to other insect tachykinin-related peptides. The tachykinin-related peptide prohormone gene (Dtk) is both expressed and post-translationally processed in larval and adult midgut endocrine cells and in the central nervous system, with midgut expression starting at stage 17 of embryogenesis. The predicted Drosophila tachykinin peptides have potent stimulatory effects on the contractions of insect gut. These data provide additional evidence for the conservation of both the structure and function of the tachykinin peptides in the brain and gut during the course of evolution.Substance P was the first peptide signaling molecule to be identified by virtue of its effects upon blood pressure and smooth muscle contraction (1) and is the archetypal member of the tachykinin family of peptides. Vertebrate tachykinins represent a large family of peptides that elicit a wide range of both central and peripheral responses (2-5). Although these peptides are structurally diverse, all contain a conserved C-terminal FXGLM-amide motif. Like other biologically active peptides, substance P is derived from a larger prohormone polypeptide (preprotachykinin A (PPT-A) 1 ) that also allows the production of several other biologically active peptides (neurokinin A, neuropeptide K, and neuropeptide ␥) (6). Three different isoforms of preprotachykinin can be produced as a result of alternative splicing of the PPT-A mRNA, which, in conjunction with alternative post-translational processing of the prohormone, allows the production of these peptides in a tissue-specific manner (7-9). A fifth mammalian tachykinin, neurokinin B, is derived from a separate gene product, preprotachykinin B (10).The tachykinin family is not confined to vertebrates, and a large number of tachykinins have now been isolated from a variety of invertebrate species such as the cockroach Leucophaea maderae (11, 12), the mosquito Culex salinarius (13), and the echiuroid worm Urechis unicinctus (14). In contrast to the vertebrate tachykinins, almost all of the invertebrate tachykinins contain a conserved C-terminal FXGXR-amide motif and, for this reason, have been termed tachykinin-related peptides (TRPs). Not...

Peptide hormones are key messengers in the signaling network between the nervous system, endocrine glands, energy stores and the gastrointestinal tract that regulates feeding and metabolism. Studies on the Drosophila nervous system have uncovered parallels and homologies in homeostatic peptidergic signaling between fruit flies and vertebrates. Yet, the role of enteroendocrine peptides in the regulation of feeding and metabolism has not been explored, with research hampered by the unknown identity of peptides produced by the fly's intestinal tract. We performed a peptidomic LC/MS analysis of the fruit fly midgut containing the enteroendocrine cells. By MS/MS fragmentation, we found 24 peptides from 9 different preprohormones in midgut extracts, including MIP-4 and 2 forms of AST-C. DH(31), CCHamide1 and CCHamide2 are biochemically characterized for the first time. All enteroendocrine peptides represent brain-gut peptides, and apparently are processed by Drosophila prohormone convertase 2 (AMON) as suggested by impaired peptide detectability in amon mutants and localization of amon-driven GFP to enteroendocrine cells. Because of its genetic amenability and peptide diversity, Drosophila provides a good model system to study peptide signaling. The identification of enteroendocrine peptides in the fruit fly provides a platform to address functions of gut peptide hormones in the regulation of feeding and metabolism.

In polyandrous females, sperm storage permits competition between sperm of different mates, and in some species females influence the relative fertilization success of competing sperm in favor of a preferred mate [1, 2]. In female Drosophila melanogaster, sperm competition is strongly influenced by the timing of sperm ejection from the uterus [3, 4]. Understanding how female behavior influences sperm competition requires knowledge of the neuronal mechanisms controlling sperm retention and storage, which is currently lacking. Here, we show that D. melanogaster females eject male ejaculates from the uterus 1-6 hr after mating with a stereotypic behavior regulated by a brain signaling pathway composed of diuretic hormone 44 (Dh44), a neuropeptide related to vertebrate corticotropin-releasing factor (CRF), and its receptor, Dh44R1. Suppression of Dh44 signals in the brain expedites sperm ejection from the uterus, resulting in marked reduction of sperm in the storage organs and decreased fecundity, whereas enhancement of Dh44 signals delays sperm expulsion. The Dh44 function was mapped to six neurons located in the pars intercerebralis of the brain together with a small subset of Dh44R1 neurons that express the sex-specific transcription factor doublesex. This study identifies a neuronal pathway by which females can control sperm retention and storage and provides new insight into how the female might exercise post-copulatory sexual selection.

The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1-NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are "ciliopathies". Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.

Drosophila melanogaster express two distinct angiotensin-I-converting enzymes (ACEs) called Ance and Acer, which display a high level of primary structure similarity. We have expressed Acer in the yeast Pichia pastoris and purified the recombinant enzyme with a view to developing biochemical tools to distinguish between Acer and Ance. Purified Acer and Ance expressed in yeast were used to raise antiAcer Ig and anti-Ance Ig that specifically cross-reacted with the respective enzyme on immunoblotting, but did not act as specific inhibitors. Acer cleaves the C-terminal dipeptides from benzoylglycyl-histidylleucine and [Leu5]enkephalin, and Acer and Ance are both able to act as endopeptidases, releasing the C-terminal dipeptideamide from [Leu5]enkephalinamide. However, Acer hydrolyses this substrate at a slightly faster rate than [Leu5]enkephalin, whereas Ance hydrolyses the peptide with a free C-terminus with a k cat 15-fold higher than [Leu5]enkephalinamide. In addition, Acer did not cleave angiotensin I. In contrast, Ance hydrolysed 25% of this substrate at an 8-fold lower enzyme concentration. Furthermore, Acer did not hydrolyse the synthetic substrates Phe-Ser-Pro-Arg-Leu-Gly-Arg-Arg and Phe-Ser-Pro-ArgLeu-Gly-Lys-Arg, two partially processed putative locustamyotropin precursors, under conditions where Ance produced 82% substrate hydrolysis. Acer was inhibited by captopril, trandolaprilat and enalaprilat, with apparent K i values in the nanomolar range, whereas lisinopril and fosinoprilat were less potent. We show that the two Drosophila ACEs are alternatively expressed in stages P1 (white puparium)ϪP15 (eclosion) of pupal development; Ance is expressed predominantly during stages P4ϪP7, whereas the ACE activity expressed during stages P9ϪP12 is mainly due to Acer suggesting different roles for the two enzymes during pupal development.Keywords : angiotensin I-converting enzyme; Drosophila melanogaster; Pichia pastoris; angiotensin Iconverting enzyme substrates ; pupal development.Mammalian angiotensin I-converting enzyme (ACE, peptidyl dipeptidase A) is a zinc metallopeptidase, which plays a key role in blood pressure homeostasis both by cleaving the C-terminal His-Leu from angiotensin I to form the vasopressor peptide angiotensin II [1] and by inactivating the vasodilatory peptide bradykinin by the sequential removal of two C-terminal dipeptides [2,3]. ACE exists primarily as an ectoenzyme with a Cterminal transmembrane anchor, a small cytoplasmic domain and the bulk of the enzyme including the catalytic site(s) exCorrespondence to P. Corvol, INSERM Unité 36, Collège de France,

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