Effects Of Neuronostatin On Gastrointestinal Motility And Depression And Semisynthesis Of Lipidated Protein LC3-Ⅱ

Based on bioinformatic analysis of evolutionarily conserved sequences in the pro-somatostatin mRNA, Samson WK and colleagues discovered a new peptide and named it neuronostatin in2008. Recent studies have demonstrated that neuronostatin is a brain/gut peptide with diverse physiological functions. It induces the expression of early response genes c-Fos in neuronal, anterior pituitary, gastrointestinal tissues. Neuronostatin inhibits food and water intake, depresses cardiac contractile function and increases mean arterial pressure. The post-translational modification of peptide has been linked to many biological functions and processes. C-terminal amidation is a common posttranslational modification in peptide hormones. Neuronostatin is a C-terminally amidated peptide. In the present study, neuronostatin and non-amidated neuronostatin were synthesized by manual solid-phase synthesis using standard Fmoc chemistry and the appropriate polystyrene resins resins:a Rink Amide-MBHA resin for the amidated, and the appropriate Wang linker resin for the non-amidated peptide. And their effects on pain, gastrointestinal motility and depression were accessed using animal model. Our results showed that i.c.v. administrated neuronostatin produced antinociceptive effect in acute pain tail immersion assay and induced hyperalgesia in tonic pain formalin test, and delayed gastric emptying and gastrointestinal transit in mice. The pain reponse and gastrointestinal inhibitory effect were mediated by the central melanocortin system and opioid system. I.c.v. administrated neuronostatin also induced depression-like behavior in the forced swim test of mice, and the effect was dependent upon the central melanocortin system and GABA receptor. However, we found that non-amidated neuronostatin had no effect on pain response, gastrointestinal motility and depression. In summary, neuronostatin may play a role in the regulation of pain response, gastrointestinal motility and depression, and C-terminal amidation modification of neuronostatin is essential to exert its activity.Autophagy is an evolutionally conserved self-eating process in eukaryotes, where excess or damaged organelles and both long-lived and aggregated proteins are cleaned. In mammalian autophagy, LC3is an ubiquitin-like conjugation protein and its target is phosphatidylethanolamine (PE). Newly synthesized proLC3is processed by a protease, Atg4B, to expose a C-terminal glycine and form LC3-I. LC3-I serves as a substrate in an ubiquitin-like conjugation reaction catalyzed by Atg7and Atg3and is conjugated to PE, and forms LC3-PE, also named it LC3-II. Atg4B releases lipidated LC3from the surface of closed autophagosomes. LC3-PE is upreulated when autophagy is induced by starvation or hormones, therefore, lipidated LC3has been used as a bona fide marker of autophagosome and progression of autophagy. Methods for generating post-translationally modified proteins are invaluable for studying the role of post-translational modification (PTM) in controlling protein function. However, recombinant production of post-translationally modified proteins is usually challenging in terms of homogeneity and output by means of molecular biological approaches. Liposomes containing LC3-PE have been generated either by reconstituting conjugation reaction in vitro with purified protein components or through chemical cross-linking of LC3with liposomes. However, preparation of lipidated LC3protein is not yet forthcoming. In the present study, LC3-PE was synthesized by the combination of expressed protein ligation (EPL) and organic synthesis. Ala114-Ser115was chosed as a possible ligation site, MBP-LC31-114thioester was prepared by intein strategy and lipidated peptide was synthesized by organic chemical techniques. LC3-PE was obtained after native chemical ligation and purification steps. The LC3-PE was completely cleaved by Atg4B, suggesting that semisynthetic LC3-PE protein is functionally active and can be further used for other studies on autophagy. Liposome assay showed semisynthetic LC3-PE induced membrane tethering and fusion. In conclusions, active LC3-PE is successful prepared by EPL, we envisage that this approach will will open a new avenue in the autophagy research field.