Study On Nitration Modification Induced By NOS1 In Melanoma Cells

Background and purposeNitric oxide(NO),catalyzed by three isoforms of nitric oxide synthase(NOS l,NOS2 and NOS3)to synthesize L-arginine,is a key signaling molecule and involves in various biological pathway.In tumor cells,NOS1 promotes tumor cell proliferation,glucose metabolism and immune escape.But the molecular mechanism of NOS 1 promoting tumor immune escape remains to be studied.NOSs regulates the structure,location,active sites and stability of target proteins by mediating two kinds of post-translational modifications(nitrosation and nitration).The NO group can form peroxynitrite with superoxide anion and modify the hydroxyl ortho-position on the specific tyrosine residue of the protein to form nitrotyrosine,which is called protein tyrosine nitration(PTN).The nitro group is similar to phosphoric acid in physical and chemical properties,and can competitively bind to the phosphoric acid binding site of protein to regulate the level of cellular phosphorylation.Interferon(IFN)binds to the receptors on the cell surface to induce the expression of interferon stimulating genes,exerting anti-proliferative,promoting apoptotic,immune regulated and anti-viral effects,which plays a critical role in tumor immunosurveillance and tumor therapy.IFNy stimulates the expression of NOS2 via IRF1.IFNs promotes the production of O2-,and regulates cellular glucose metabolism and cellular immune response.Whether PTN increases in tumor cells stimulated by IFN a and whether immune escape is regulated by PTN remains to be studied.In this study,liquid chromatography-tandem mass spectrometry(LC-MS/MS)was used to detect and analyze the nitration modification of proteins under the treatment of IFNa and the changes of PTN induced by NOS1 in melanoma cells.The purpose of this study is to provide protein nitrigenomics data for exploring the role and molecular targets of NOS1 in tumor immune escape.Results1.The result of CCK-8 assay showed that IFN α inhibited the proliferation of B16F10 cells in a concentration dependent manner.And the overexpression of NOS 1 decrease the sensitivity of cells to IFNα.The results of immunoprecipitation combined with liquid chromatography tandem mass spectrometry showed that 41 nitroproteins and 46 nitropeptides were detected in A375 cells overexpressing NOS1 treated with IFN α.A total of 16 nitroproteins and 18 nitrified peptides were detected in the control group.A total of 55 nitroproteins and 60 nitropeptides were detected in the two groups.GO and KEGG enrichment analysis showed that these nitrified proteins were mainly involved in regulating biological processes,such as protein conformation and localization,cytoskeleton tissue and intercellular adhesion.And molecular functions of them were mainly to bind to various substances(protein,ATP,calcium ion,etc.),and regulate the activity of kinase and NOSs.And they participate in some signal pathways,including antigen processing and presentation,endoplasmic reticulum protein processing and phagocytosis.A total of 20 nitroproteins and 25 nitropeptides were detected in the lung metastasis model of melanoma treated with IFNα.These nitroproteins are mainly involved in biological processes like RNA splicing,gluconeogenesis and glycolysis.The molecular functions of them are mainly protein kinase binding,cadherin binding and oxygen transporter binding.And the signal pathway they involved is splice bodies.2.The results of Western blot analysis showed that IFNa could not induce NOS2 expression and could not significantly increase the level of PTN.20ug/ml LPS induced the low expression of NOS2;the combination of IFN γ and 20ug/ml LPS induced the high expression of NOS2.The results of CCK-8 assay showed that low dose of LPS(10-50ug/ml)promoted proliferation in B16F10 cell.Our experiments also showed that the combination of IFN y and LPS inhibited the cellular viability,which could be reversed by 1400W.The result of Western blotananlysis showed that the combination of IFN y and LPS could significantly increase the level of protein nitrification in cells,and 1400W could reduce the effect of IFN γ and LPS.3.The results of ROS level assay showed that IFNa increased the generation of intracellular ROS.The results of Seahorse XF technology showed that IFNa increased ATP-related OCR and maximal OCR in A3 75 cells,suggesting that IFNa inhibits mitochondrial oxidative phosphorylation.13C-glutamine tracer combined with gas chromatography-mass spectrometry(GC-MS)showed that IFNα promoted TCA cycle intermediate synthesis,included Cit,Sum,α-kG,Fum and Mal.ConclusionThe protein nitration genomics study of melanoma cells and tissues stimulated by IFN α was established by mass spectrometry.A total of 55 human nitrified proteins were detected,of which 45 were newly discovered nitration target proteins.These nitroproteins are mainly involved in the regulation of antigen presentation,endoplasmic reticulum on protein processing,phagocytosis and other signal pathways.High concentration of IFN a slightly inhibited the proliferation of melanoma cells,and NO S1 increased the tolerance of melanoma cells to the inhibition effect of IFNα.This study provides protein nitrogenomics clues and theoretical basis for further exploring the role and molecular targets of NOS 1 in tumor immune escape.