Drosophila insulin‐like peptide dilp1 increases lifespan and glucagon‐like Akh expression epistatic to dilp2

Insulin/IGF signaling (IIS) regulates essential processes including development, metabolism, and aging. The Drosophila genome encodes eight insulin/IGF‐like peptide (dilp) paralogs, including tandem‐encoded dilp1 and dilp2. Many reports show that longevity is increased by manipulations that decrease DILP2 levels. It has been shown that dilp1 is expressed primarily in pupal stages, but also during adult reproductive diapause. Here, we find that dilp1 is also highly expressed in adult dilp2 mutants under nondiapause conditions. The inverse expression of dilp1 and dilp2 suggests these genes interact to regulate aging. Here, we study dilp1 and dilp2 single and double mutants to describe epistatic and synergistic interactions affecting longevity, metabolism, and adipokinetic hormone (AKH), the functional homolog of glucagon. Mutants of dilp2 extend lifespan and increase Akh mRNA and protein in a dilp1‐dependent manner. Loss of dilp1 alone has no impact on these traits, whereas transgene expression of dilp1 increases lifespan in dilp1 ? dilp2 double mutants. On the other hand, dilp1 and dilp2 redundantly or synergistically interact to control circulating sugar, starvation resistance, and compensatory dilp5 expression. These interactions do not correlate with patterns for how dilp1 and dilp2 affect longevity and AKH. Thus, repression or loss of dilp2 slows aging because its depletion induces dilp1, which acts as a pro‐longevity factor. Likewise, dilp2 regulates Akh through epistatic interaction with dilp1. Akh and glycogen affect aging in Caenorhabditis elegans and Drosophila. Our data suggest that dilp2 modulates lifespan in part by regulating Akh, and by repressing dilp1, which acts as a pro‐longevity insulin‐like peptide.     

A noncoding RNA produced by arthropod-borne flaviviruses inhibits the cellular exoribonuclease XRN1 and alters host mRNA stability

All arthropod-borne flaviviruses generate a short noncoding RNA (sfRNA) from the viral 3′ untranslated region during infection due to stalling of the cellular 5′-to-3′ exonuclease XRN1. We show here that formation of sfRNA also inhibits XRN1 activity. Cells infected with Dengue or Kunjin viruses accumulate uncapped mRNAs, decay intermediates normally targeted by XRN1. XRN1 repression also resulted in the increased overall stability of cellular mRNAs in flavivirus-infected cells. Importantly, a mutant Kunjin virus that cannot form sfRNA but replicates to normal levels failed to affect host mRNA stability or XRN1 activity. Expression of sfRNA in the absence of viral infection demonstrated that sfRNA formation was directly responsible for the stabilization of cellular mRNAs. Finally, numerous cellular mRNAs were differentially expressed in an sfRNA-dependent fashion in a Kunjin virus infection. We conclude that flaviviruses incapacitate XRN1 during infection and dysregulate host mRNA stability as a result of sfRNA formation.     

Structural and biological properties of the Drosophila insulin-like peptide 5 show evolutionary conservation

We report the crystal structure of two variants of Drosophila melanogaster insulin-like peptide 5 (DILP5) at a resolution of 1.85 Å. DILP5 shares the basic fold of the insulin peptide family (T conformation) but with a disordered B-chain C terminus. DILP5 dimerizes in the crystal and in solution. The dimer interface is not similar to that observed in vertebrates, i.e. through an anti-parallel β-sheet involving the B-chain C termini but, in contrast, is formed through an anti-parallel β-sheet involving the B-chain N termini. DILP5 binds to and activates the human insulin receptor and lowers blood glucose in rats. It also lowers trehalose levels in Drosophila. Reciprocally, human insulin binds to the Drosophila insulin receptor and induces negative cooperativity as in the human receptor. DILP5 also binds to insect insulin-binding proteins. These results show high evolutionary conservation of the insulin receptor binding properties despite divergent insulin dimerization mechanisms.     

基于RNA-seq分析bdhA敲除对枯草芽孢杆菌产纳豆激酶和MK-7的影响

纳豆激酶(nattokinase, NK)具有多种生理功能,是治疗心血管疾病的理想药物。甲萘醌-7 (menaquinone-7, MK-7)是人体不可缺少的脂溶性维生素之一,可预防骨质疏松和帕金森等疾病。【目的】提高枯草芽孢杆菌中NK和MK-7共同生产的产量,揭示重组菌中共同生产NK和MK-7的机理,为MK-7和NK的生成提供新的代谢工程策略。【方法】以枯草芽孢杆菌为出发菌株,敲除2,3-丁二醇脱氢酶基因(bdhA),构建一株能增加NK和MK-7共同生产的枯草芽孢杆菌(Bacillus subtilis) 168-ΔbdhA。利用RNA-seq分析NK和MK-7合成途径关键酶编码基因的变化,总结NK和MK-7共同生产的机制。【结果】与原始菌株相比,Bacillus subtilis 168-ΔbdhA中2,3-丁二醇含量降低64.0%,为2.76 g/L。NK和MK-7的产量较原始菌株提高30.0%和60.0%。RNA-seq分析表明,中心碳代谢、氧化磷酸化和NK及MK-7合成等过程相关的基因表达存在差异。NK负调控因子codY下调2.19倍。在蛋白质分泌途径中,secA下调0.37倍,tatAD和tatC分别上调2.81倍和0.50倍。【结论】bdhA的敲除阻断了2,3-丁二醇的碳通量,促进甘油的吸收,碳通量更多地流向NK和MK-7的合成途径。负调控因子codY的下调促进NK转录,蛋白转运相关途径基因的上下调促进MK-7的胞外分泌,从而实现其产量的增加。     

Extraction and nutritional/hormonal regulation of tissue insulin-like growth factor 1 activity

Studies of insulin-like growth factor 1 (IGF-1) mRNA translation products suggest synthesis as a high Mr precursor, larger than circulating forms. To search for a precursor, we characterized IGF-1 immunoreactivity and IGF bioactivity in extracts from the liver and other body tissues. Sequential extraction with neutral followed by acid buffer was superior to extraction with acid/ethanol or acid alone in yield of immunoreactivity and specific activity. Extracts of normal rat liver exhibited both immuno- and bioactivity parallel to that of recombinant IGF-1 and serum IGFs over a 25-fold concentration range. Based on immunoreactivity, the liver of a 134-g rat appears to contain 1.2 micrograms of IGF-1 equivalents, 50% of the 2.45 micrograms in the circulation. Diaphragm, spleen, and kidney contained no significant IGF bioactivity and 8, 17, and 32% of the IGF-1 immunoreactivity of normal liver, respectively. Although serum IGFs were found at 7.5 kDa after size exclusion chromatography at pH 3, hepatic extracts contained a predominant peak of immuno- and bioactivity of apparent molecular mass of 30-35 kDa; both sizes were present in liver perfusates. Both immunoaffinity chromatography followed by Western blotting and IGF-binding protein affinity chromatography followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two predominant species, at 18-19 and 12 kDa. The 18-19-kDa species is consistent with the apparent size of the glycosylated propeptide encoded by IGF-1A mRNA, while the 12-kDa species may be nonglycosylated propeptide. Extract activity was pituitary-dependent; the livers of hypophysectomized rats contained 15.4 and 48.8% of normal immuno- and bioactivity, respectively. During fasting and refeeding of rats, fluctuations in hepatic extract IGF-1 immunoreactivity generally paralleled changes in serum IGF-1 (r = 0.93, p less than 0.001). These studies demonstrate that the liver contains a pituitary- and nutrition-dependent, high Mr form of IGF-1 with immunological and biological properties similar to circulating IGF-1. Processing of this 18-19-kDa molecule through a 12-kDa intermediate may contribute IGF-1 to the circulation.     

Neutrophil-activating peptide 2 and gro/melanoma growth-stimulatory activity interact with neutrophil-activating peptide 1/interleukin 8 receptors on human neutrophils

Neutrophil-activating peptide 1/interleukin 8 (NAP-1/IL-8), neutrophil-activating peptide 2 (NAP-2), and gro/melanoma growth-stimulatory activity (gro/MGSA) are potent inflammatory cytokines with homologous structure and similar neutrophil-activating properties. Receptors on human neutrophils that interact with these peptides were studied. Analysis of 125I-NAP-1/IL-8 binding at 0-4 degrees C revealed 64,500 +/- 14,000 receptors/cell with an apparent Kd of 0.18 +/- 0.07 nM (mean +/- S.D. of six independent experiments). Unlabeled NAP-1/IL-8, NAP-2, and gro/MGSA competed with 125I-NAP-1/IL-8 for binding to human neutrophils. Competition with increasing concentrations of unlabeled NAP-2 and gro/MGSA resolved two classes of NAP-1/IL-8 binding sites: about 70% of them bound NAP-2 and gro/MGSA with high affinity (Kd: 0.34 +/- 0.2 and 0.14 +/- 0.02), while 30% were of low affinity (Kd: 100 +/- 20 and 130 +/- 10 nM). Different binding sites, however, were not apparent upon competition with unlabeled NAP-1/IL-8, suggesting that both classes of receptors have similar affinities for NAP-1/IL-8. The existence of two receptors was also suggested by ligand cross-linking and cross-desensitization experiments. Two neutrophil membrane proteins with apparent Mr of 66,000-74,000 and 42,000-46,000 became cross-linked to 125I-NAP-1/IL-8, and the labeling was decreased when excess NAP-1/IL-8, NAP-2, or gro/MGSA was present. Stimulation of neutrophils with NAP-1/IL-8 resulted in desensitization toward a subsequent challenge with NAP-2 or gro/MGSA as shown by the rise in cytosolic free calcium. By contrast, following primary stimulation with NAP-2 or gro/MGSA, responses to NAP-1/IL-8 were only moderately attenuated, supporting the existence of NAP-1/IL-8 receptors which bind NAP-2 or gro/MGSA with low affinity. In conclusion, our results demonstrate that NAP-2 and gro/MGSA act upon human neutrophils by directly interacting with two classes of receptors for NAP-1/IL-8.     

Characterization of the XRN1 gene encoding a 5'-->3' exoribonuclease: sequence data and analysis of disparate protein and mRNA levels of gene-disrupted yeast cells.

Sequencing of the XRN1 gene of Saccharomyces cerevisiae, cloned in this laboratory as a gene encoding a 160-kDa 5'-->3' exoribonuclease (XRN1), shows that it is identical to a gene (DST2 or SEP1) encoding a DNA strand transferase and to genes involved in nuclear fusion, KEM1, and plasmid stability, RAR5. To better understand the various phenotypes associated with loss of XRN1 and the enzymatic activities associated with the protein, certain characteristics of our yeast cells lacking an active gene (xrn1) have been examined. Cells are larger (average volume is x 1.5-1.8) and have an increased doubling time (x1.9-2.1). The protein synthesis rate per cell is 80-90% that of wild-type (wt) cells, and the resultant cellular protein levels are higher. The rate of the 25S and 18S rRNA synthesis is approximately 45% that of wt cells and its cellular level is about 90% that of wt cells. Levels of protein bands resolved by one-dimensional PAGE show substantial differences. Synthesis rates observed for the same protein bands, as well as measurements of several specific mRNA levels by Northern analysis, suggest disparities in mRNA levels. Results show two to four times longer half-lives of specific short-lived mRNAs. The variations in levels of protein and RNA species found in the xrn1 cells may be the cause of some of the phenotypes found associated with gene loss.