Interaction of a Dimeric Single-Stranded DNA-Binding Protein (G5P) with DNA Hairpins. A Molecular Beacon Study

Gene-V protein (G5P/GVP) is a single-stranded (ss)DNA-binding protein (SBP) of bacteriophage f1 that is required for DNA synthesis and repair. In solution, it exists as a dimer that binds two antiparallel ssDNA strands with high affinity in a cooperative manner, forming a left-handed helical protein–DNA filament. Here, we report on fluorescence studies of the interaction of G5P with different DNA oligonucleotides having a hairpin structure (molecular beacon, MB) with a seven base-pair stem (dT24-stem7, dT18-stem7), as well as with DNA oligonucleotides (dT38, dT24) without a defined secondary structure. All oligonucleotides were end-labeled with a Cy3-fluorophore and a BHQ2-quencher. In the case of DNA oligonucleotides without a secondary structure, an almost complete quenching of their strong fluorescence (with about 5% residual intensity) was observed upon the binding of G5P. This implies an exact alignment of the ends of the DNA strand(s) in the saturated complex. The interaction of the DNA hairpins with G5P led to the unzipping of the base-paired stem, as revealed by fluorescence measurements, fluorescence microfluidic mixing experiments, and electrophoretic mobility shift assay data. Importantly, the disruption of ssDNA’s secondary structure agrees with the behavior of other single-stranded DNA-binding proteins (SBPs). In addition, substantial protein-induced fluorescence enhancement (PIFE) of the Cy3-fluorescence was observed

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

Identification of novel physiological processes regulated by Neprilysin activity in Drosophila melanogaster

Drosophila insulin like peptides (DILPs) and their human homolog insulin act as messengers to control many physiological processes in the body. Fields in which insulin signaling is crucial are e.g. growth, stress responses and aging. Consequently, many diseases are caused by disturbed insulin signaling, of which diabetes is the most prominent. During the last decades the functions of insulins and their signaling pathways have been studied in detail; what remains less well understood is how the production of insulin and insulin like peptides is regulated. The family of Neprilysins (Neps) belongs to the M13-zinc ion binding metallopeptidases. Neprilysins cleave peptides that regulate a wide range of cellular processes and are therefore linked to a variety of diseases like cancer, analgesia, hypertension or Alzheimer’s disease. In the fruit fly Drosophila melanogaster, five Neprilysins are expressed; but their in vivo substrates have not yet been identified. One of the Drosophila Neprilysins, Nep4, is expressed in the CNS, in muscle tissue, in cardiac tissue and in male reproductive organs. Nep4 is expressed in two isoforms, Nep4A and Nep4B. Isoform A is composed of a short intracellular domain, a transmembrane domain and a large extracellular domain containing the catalytically active center, whereas soluble Nep4B only consists of the extracellular domain. This thesis reveals that overexpression of catalytically active Nep4A in muscle tissue leads to animals with impaired insulin expression, decreased size and weight, affected feeding behavior and reduced locomotion speed. Further phenotypes are an impaired energy metabolism and larval lethality. Knockdown of the whole enzyme or knockout of its catalytic activity also interferes with feeding and locomotion speed and, in addition, causes pupal lethality. As an explanation for the phenotypes, Nep4 mediated hydrolysis of different short neuropeptide F (sNPF) species, which were identified as novel substrates of the peptidase, is proposed. sNPF is known to regulate insulin signaling and knockdown of sNPF phenocopies the Nep4 overexpression phenotypes, which suggests that Nep4 mediated hydrolysis of sNPF regulates insulin expression in the fly. Based on these results additional regulatory peptides were identified as novel Nep4 substrates. Among them are peptides that do not only regulate insulin signaling, but also feeding behavior (Hallier et al., 2016). These findings represent good evidence that muscle bound Nep4 is key to regulate homeostasis of distinct hemolymph circulating peptide hormones. Nep4 localizing to the surface of the central nervous system is likely necessary to ensure effective ligand clearance and thus proper regulation of corresponding peptide receptors

The bHLH Transcription Factor Hand Regulates the Expression of Genes Critical to Heart and Muscle Function in <i>Drosophila melanogaster</i>

<div><p>Hand proteins belong to the highly conserved family of basic Helix-Loop-Helix transcription factors and are critical to distinct developmental processes, including cardiogenesis and neurogenesis in vertebrates. In <i>Drosophila melanogaster</i> a single orthologous <i>hand</i> gene is expressed with absence of the respective protein causing semilethality during early larval instars. Surviving adult animals suffer from shortened lifespan associated with a disorganized myofibrillar structure being apparent in the dorsal vessel, the wing hearts and in midgut tissue. Based on these data, the major biological significance of Hand seems to be related to muscle development, maintenance or function; however, up to now the physiological basis for Hand functionality remains elusive. Thus, the identification of genes whose expression is, directly or indirectly, regulated by Hand has considerable relevance with respect to understanding its biological functionality in flies and vertebrates. Beneficially, <i>hand</i> mutants are viable and exhibit affected tissues, which renders <i>Drosophila</i> an ideal model to investigate up- or downregulated target genes by a comparative microarray approach focusing on the respective tissues from mutant specimens. Our present work reveals for the first time that <i>Drosophila</i> Hand regulates the expression of numerous genes of diverse physiological relevancy, including distinct factors required for proper muscle development and function such as Zasp52 or Msp-300. These results relate Hand activity to muscle integrity and functionality and may thus be highly beneficial to the evaluation of corresponding <i>hand</i> phenotypes.</p></div

Functional classification of genes exhibiting deviant expression levels in <i>hand</i>^<i>173</i> animals.

<p><i>Drosophila</i> Hand is apparently involved in regulating the expression of 545 genes with 385 genes being downregulated in <i>hand</i> mutants (<i>hand</i>^<i>173</i>) and 160 genes displaying an increased expression in the same line, compared to wild type. Functional classification of the corresponding protein products was done manually utilizing data from NCBI (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>) and Flybase (<a href="http://flybase.org/" target="_blank">http://flybase.org/</a>). Factors with yet unknown physiological functions are allocated to the category “assorted other”.</p

Relative expression of <i>Drosophila</i> homologs of selected vertebrate Hand target genes in <i>hand</i> mutant <i>Drosophila</i>, compared to wild type.

<p>Expression levels were assessed by Northern blot. Statistically relevant deviations are evident with respect to all genes tested. While <i>mef2</i> and <i>hedgehog</i> display an increased expression of 40.6% and 19.8% in <i>hand</i>^<i>173</i> animals, respectively, the expression of <i>cubitus interruptus</i> is decreased by 19.8% in the same line. Bars represent mean values ± SD of at least three independent experiments. Asterisks indicate statistical significance (Student´s <i>t</i>-test <i>P</i><0.05). Quantification of the relative band intensities in relation to the loading controls (ribosomal RNA, rRNA) was done by densitometric analysis.</p

Relative expression of selected genes in hand mutant animals, compared to wild type.

<p>Expression levels were assessed by Northern blot. Statistically relevant deviations are evident with respect to all genes tested. While <i>akirin</i> displays an expression that is reduced by 40.5% in the <i>hand</i> mutant (<i>hand</i>^<i>173</i>), the expression of <i>kugelei</i> is lowered by 57.5%, that of <i>msp-300</i> by 65% and that of <i>multiplexin</i> by 28.5%, respectively. With regard to <i>zasp52</i>, two major transcripts are detected by the applied riboprobes with the larger one (arrow) being downregulated by 44.8% in the <i>hand</i> mutant and the smaller one (arrowhead) being upregulated by 34.9% in the same line. Bars represent mean values <u>+</u> SD of at least three independent experiments. Asterisks indicate statistical significance (Student´s <i>t</i>-test <i>P</i><0.05). Quantification of the relative band intensities in relation to the loading controls (ribosomal RNA, rRNA) was done by densitometric analysis.</p