A Proteomic Study Of S-nitrosylation In The Rat Heart

Protein S-nitrosylation is a new type of protein post-translational modification, refers to the reversible attachment of nitric oxide(NO) moiety to specific cysteine residue(s) on selected proteins,producing labile S-nitrosothiol structure and functional alterations.S-nitrosylation is involved in the regulation of a diverse array of protein functions and plays a dominant role in many signaling pathways. Specifically in the heart,this modification appears to confer protective effects against myocardial ischemia/reperfusion injury(MI/RI).However,a systematic investigation of the overall protein targets of S-nitrosylation in the heart,or heart S-nitrosoproteome, is still lacking.What's more,some widely used cardiovascular drugs can increase the expression of NO,and whether the cardioprotective effect of these durgs are related with protein S-nitrosylation is still unknow.Thus,in order to prove the hypothesis mentioned above,relative studies are presented as below.The rat cardiac proteins were incubated in vitro with GSNO,to induce protein S-nitrosylation,and the resulting S-nitrosylated proteins were purified by the biotin switch method,followed by two-dimensional gel electrophoresis(2-DE) separation and matrix-assisted laser desorption ionization/time of flight tandem mass spectrometry(MALDI-TOF-MS/MS) identification.Candidate Westem blot analysis was also used to identify potential S-nitrosylated proteins.A total often proteins were unambiguously identified,among which AK1 was found as a novel target of S-nitrosylation.Further studies showed that AK1 activity in the rat heart extracts was significantly inhibited by GSNO but not oxidized glutathione(GSSG),and the inhibition was completely reversed by dithiothreitol(DTT) post-treatment, demonstrating that S-nitrosylation might serve as a new regulatory mechanism in controlling AK1 activity.The rat was treated with glyceryl trinitrate(GTN) to induce S-nitrosylation in vivo.The administration is 10mg·kg^-1.The biotin switch method was adopted to selectively purify the resulting S-nitrosylated proteins.Samples were then separated by 2-DE,and identified with MALDI-TOF-MS/MS.A total of seventeen proteins were unambiguously identified,among which Peroxiredoxin-1,Peroxiredoxin-3, Peroxiredoxin-6,aldolase A,Phosphoglycerate kinase 1,NDPK,transthyretin were found as novel targets of S-nitrosylation.These identified proteins are associated with the process of energy metabolism,redox regulatory and cardiac angina,signifing that S-nitrosylation might serve as a new regulatory mechanism of GTN playing a series of biological effects in vivo.To observe the effect of protein S-nitrosylation on Radix Ginseng and Radix Ophiopogonis extract(SMF)'s protecting MI/RI in rats.The serum levels of creatine kinase(CK),lactate dehydrogenase(LDH),and NO were determined to study the cardioprotective effects of SMF on the acute MI/RI rats.The change of the expression of endothelial nitric oxide synthase(eNOS) was detected by Western blot.The content of related S-nitrosylation proteins in myocardial tissue was measured by biotin switch method and DAN method.The result showed that SMF significantly decreased the serum levels of CK and LDH as well as increased the serum levels of NO and the expression of eNOS in myocardial tissue.The contents of S-nitrosylation proteins were significantly increased from 4.42±0.60 nmol·mg^-1to 8.78±1.37 nmol·mg^-1.The molecular weight of the majority S-nitrosylation proteins were in the range of 90-117kD.Thus increased expression of eNOS and NO induced by SMF may activate S-nitrosylation of many proteins in rat hearts.The change of the activities or functions of those proteins by S-nitrosylation may be an important mechanism for myocardial protective effects of SMF.Taken together,this study represents an initial attempt to characterize the S-nitrosoproteome in the rat heart,and successfully identified twenty-two cardiac proteins susceptible to S-nitrosylation and demonstrated that S-nitrosylation is a novel mechanism for regulating AK1 activity.What's more,protein S-nitrosylation may be an important mechanism for myocardial protective effects of SMF.