Genotoxic stress during DNA replication constitutes a serious threat to genome integrity and causes human diseases. Defects at different steps of DNA metabolism are known to induce replication ...stress, but the contribution of other aspects of cellular metabolism is less understood. We show that aminopeptidase P (APP1), a metalloprotease involved in the catabolism of peptides containing proline residues near their N-terminus, prevents replication-associated genome instability. Functional analysis of C. elegans mutants lacking APP-1 demonstrates that germ cells display replication defects including reduced proliferation, cell cycle arrest, and accumulation of mitotic DSBs. Despite these defects, app-1 mutants are competent in repairing DSBs induced by gamma irradiation, as well as SPO-11-dependent DSBs that initiate meiotic recombination. Moreover, in the absence of SPO-11, spontaneous DSBs arising in app-1 mutants are repaired as inter-homologue crossover events during meiosis, confirming that APP-1 is not required for homologous recombination. Thus, APP-1 prevents replication stress without having an apparent role in DSB repair. Depletion of APP1 (XPNPEP1) also causes DSB accumulation in mitotically-proliferating human cells, suggesting that APP1's role in genome stability is evolutionarily conserved. Our findings uncover an unexpected role for APP1 in genome stability, suggesting functional connections between aminopeptidase-mediated protein catabolism and DNA replication.
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.
Malaria poses a significant threat to approximately half of the world’s population with an annual death toll close to half a million. The emergence of resistance to front-line antimalarials in the ...most lethal human parasite species, Plasmodium falciparum (Pf), threatens progress made in malaria control. The prospect of losing the efficacy of antimalarial drugs is driving the search for small molecules with new modes of action. Asexual reproduction of the parasite is critically dependent on the recycling of amino acids through catabolism of hemoglobin (Hb), which makes metalloaminopeptidases (MAPs) attractive targets for the development of new drugs. The Pf genome encodes eight MAPs, some of which have been found to be essential for parasite survival. In this article, we discuss the biological structure and function of each MAP within the Pf genome, along with the drug discovery efforts that have been undertaken to identify novel antimalarial candidates of therapeutic value.
Sleep is a highly conserved state in animals, but its regulation and physiological function is poorly understood.
Drosophila melanogaster
is an excellent model for studying sleep regulation and has ...been used to investigate how sex and social interactions can influence wake-sleep profiles. Previously we have shown that copulation has a profound effect on day time activity and quiescence (siesta sleep) of individual post-mated females. Here we have the studied the effect of mating and the transfer of the 36 amino acid sex peptide in the seminal fluid on the behavior of mated female
Drosophila
populations, where there will be on-going social interactions. The locomotor activity and sleep patterns of virgin and post-mated female
D. melanogaster
from three laboratory strains (Oregon-R, Canton-S and Dahomey) were recorded in social groups of 20 individuals in a 12–12 h light–dark cycle. Virgin female populations from all three fly strains displayed consolidated periods of low activity in between two sharp peaks of activity, corresponding to lights-on and lights-off. Similar light-correlated peaks were recorded for the mated female populations, however, the low afternoon activity and siesta seen in virgin populations was abolished after mating in all three strains. In contrast, night activity appeared unaffected. This post-mating effect was sustained for several days and was dependent on the male SP acting as a pheromone. Evidence from mixed populations of virgin and mated females suggests that the siesta of non-mated females is not easily disturbed by the presence of highly active post-mated females.
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.
Peptides present in the seminal fluid of Drosophila melanogaster can function as antimicrobial agents, enzyme inhibitors and as pheromones that elicit physiological and behavioural responses in the ...post-mated female. Understanding the molecular interactions by which these peptides influence reproduction requires detailed knowledge of their molecular structures. However, this information is often lacking and cannot be gleaned from just gene sequences and standard proteomic data. We now report the native structures of four seminal fluid peptides (andropin, CG42782, Met75C and Acp54A1) from the ejaculatory duct of male D. melanogaster. The mature CG42782, Met75C and Acp54A1 peptides each have a cyclic structure formed by a disulfide bond, which will reduce conformational freedom and enhance metabolic stability. In addition, the presence of a penultimate Pro in CG42782 and Met75C will help prevent degradation by carboxypeptidases. Met75C has undergone more extensive post-translational modifications with the formation of an N-terminal pyroglutamyl residue and the attachment of a mucin-like O-glycan to the side chain of Thr4. Both of these modifications are expected to further enhance the stability of the secreted peptide. The glycan has a rare zwitterionic structure comprising an O-linked N-acetyl hexosamine, a hexose and, unusually, phosphoethanolamine. A survey of various genomes showed that andropin, CG42782, and Acp54A1 are relatively recent genes and are restricted to the melanogaster subgroup. Met75C, however, was also found in members of the obscura species groups and in Scaptodrosophila lebanonensis. Andropin is related to the cecropin gene family and probably arose by tandem gene duplication, whereas CG42782, Met75C and Acp54A1 possibly emerged de novo. We speculate that the post-translational modifications that we report for these gene products will be important not only for a biological function, but also for metabolic stability and might also facilitate transport across tissue barriers, such as the blood-brain barrier of the female insect.
Seminal fluid peptides of D. melanogaster function as antimicrobials, enzyme inhibitors and as pheromones, eliciting physiological and behavioural responses in the post-mated female. A fuller understanding of how these peptides influence reproduction requires knowledge not only of their primary structure, but also of their post-translational modification. However, this information is often lacking and difficult to glean from standard proteomic data. The reported modifications, including the unusual glycosylation, adds much to our knowledge of this important class of peptides in this model organism, par excellence.
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•The ejaculatory duct contributes peptides to the seminal fluid of D. melanogaster.•MALDI-TOF MS/MS identified the native forms of four peptides.•One peptide, Met75C, is a glycopeptide with a zwitterionic phosphoethanolamine.•All peptides are products of new taxon-restricted genes.
In Drosophila melanogaster mating triggers profound changes in the behaviour and reproductive physiology of the female. Many of these post-mating effects are elicited by sex peptide (SP), a 36-mer ...pheromone made in the male accessory gland and passed to the female in the seminal fluid. The peptide comprises several structurally and functionally distinct domains, one of which consists of five 4-hydroxyprolines and induces a female immune response. The SP gene predicts an isoleucine (Ile14) sandwiched between two of the hydroxyprolines of the mature secreted peptide, but the identity of this residue was not established by peptide sequencing and amino acid analysis, presumably because of modification of the side chain. Here we have used matrix-assisted laser desorption ionisation mass spectrometry together with Fourier-transform ion cyclotron resonance mass spectrometry to show that Ile14 is modified by oxidation of the side chain - a very unusual post-translational modification. Mass spectrometric analysis of glands from different geographical populations of male D. melanogaster show that SP with six hydroxylated side chains is the most common form of the peptide, but that a sub-strain of Canton-S flies held at Leeds only has two or three hydroxylated prolines and an unmodified Ile14. The D. melanogaster genome has remarkably 17 putative hydroxylase genes that are strongly and almost exclusively expressed in the male accessory gland, suggesting that the gland is a powerhouse of protein oxidation. Strain variation in the pattern of sex peptide hydroxylation might be explained by differences in the expression of individual hydroxylase genes.
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•Drosophila sex peptide of the male accessory gland controls female reproduction.•The 36-mer sex peptide is modified by oxidation of amino acid side chains.•Sex peptide has five functionally important hydroxyprolines and a rare hydroxyisoleucine.•Fully hydroxylated sex peptide is not found in all Drosophila strains.•A remarkable number of hydroxylase genes are expressed only in the male accessory gland.
Drosophila suzukii or spotted wing Drosophila is an economically important pest which can have a devastating impact on soft and stone fruit industries. Biological pesticides are being sought as ...alternatives to synthetic chemicals to control this invasive pest, but many are subject to degradation either in the environment or in the insect gut and as a result require protection. In this study we identified a sharp change in pH of the adult midgut from neutral to acidic (pH <3), which we then exploited to develop poly(2-vinylpyridine) (P2VP) microcapsules that respond to the change in midgut pH by dissolution and release of their cargo for uptake into the insect. First, we used labelled solid poly(methyl methacrylate) (PMMA) particles to show that microcapsules with a diameter less than 15 μm are readily ingested by the adult insect. To encapsulate water-soluble biological species in an aqueous continuous phase, a multiple emulsion template was used as a precursor for the synthesis of pH-responsive P2VP microcapsules with a fluorescent (FITC-dextran) cargo. The water-soluble agent was initially separated from the aqueous continuous phase by an oil barrier, which was subsequently polymerised. The P2VP microcapsules were stable at pH > 6, but underwent rapid dissolution at pH < 4.2. In vivo studies showed that the natural acidity of the midgut of D. suzukii also induced the breakdown of the responsive P2VP microcapsules to release FITC-dextran which was taken up into the body of the insect and accumulated in the renal tubules.
Human angiotensin I‐converting enzyme (ACE) has two isoforms, somatic ACE (sACE) and testis ACE (tACE). The functions of sACE are widespread, with its involvement in blood pressure regulation most ...extensively studied. sACE is composed of an N‐domain (nACE) and a C‐domain (cACE), both catalytically active but have significant structural differences, resulting in different substrate specificities. Even though ACE inhibitors are used clinically, they need much improvement because of serious side effects seen in patients (~ 25–30%) with long‐term treatment due to nonselective inhibition of nACE and cACE. Investigation into the distinguishing structural features of each domain is therefore of vital importance for the development of domain‐specific inhibitors with minimal side effects. Here, we report kinetic data and high‐resolution crystal structures of both nACE (1.75 Å) and cACE (1.85 Å) in complex with fosinoprilat, a clinically used inhibitor. These structures allowed detailed analysis of the molecular features conferring domain selectivity by fosinoprilat. Particularly, altered hydrophobic interactions were observed to be a contributing factor. These experimental data contribute to improved understanding of the structural features that dictate ACE inhibitor domain selectivity, allowing further progress towards designing novel 2nd‐generation domain‐specific potent ACE inhibitors suitable for clinical administration, with a variety of potential future therapeutic benefits.
Database
The atomic coordinates and structure factors for nACE‐fosinoprilat and cACE‐fosinoprilat structures have been deposited with codes 7Z6Z and 7Z70, respectively, in the RCSB Protein Data Bank, www.pdb.org.
Analysis of high‐resolution crystal structures of angiotensin I‐converting enzyme (ACE) N‐ and C‐domains in complex with fosinoprilat provide further insights into the requirements for high‐affinity domain‐specific inhibition that is required to reduce side effects of treatments that target ACE. In addition, the data highlights where modifications or redesign of fosinoprilat are needed to develop novel insecticides that act via ACE analogues.
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