Proteomic Analysis Of Sut Melanocytes In Response To XCT Deficiency

Objecti ve:This study is to explore the proteins related with xCT-deficiency in Sut melanocytes by proteinomics. The results would help to understand the mechanism of Sut melanocytes'growth inhibition caused by xCT-deficiency.Methods:1.Cells and extraction of total celluar proteins.Sut melanocytes and wild melanocytes were cultured respectively in DMEM/F12 medium contains 10% fetal calf serum and 100units/ml antibiotics in incubator with 5% CO2 at 37℃.Cells were collected at logarithmic growth phase, Sediments of Sut melanocytes and wild melanocytes were washed respectively and then centrifugated for extracting of total celluar proteins.2.2-dimensional gel electrophoresis (2-DE) of the total celluar proteins and tandem mass spectrometry of altered proteins2-DE was proformed on the total celluar proteins of Sut melanocytes and wild melanocytes respectively. Altered proteins were found by compared 2-DE map of Sut melanocytes with map of wild melanocytes by PDQuest software and then analyzed by tandem mass spectrometry.3. Database query and identification of the altered proteinsThe altered proteins are identified by searching SWISS-PROT database based on the information of tandem mass spectrometry.4.To confirm the mRNA level of Calmodulin, Annexin A3, Vimentin and S100-A4The total celluar RNA was extracted from Sut melanocytes and wild melanocytes and then reverse transcribed into cDNA. DNA of Calmodulin, Annexin A3, Vimentin and S100-A4 were amplified by RT-PCR. Agarose gel electrophoresis was proformed on the amplified DNA. The result was saved after the agarose gel with the amplified DN A was scanned, histogram was drawn based on the result of RT-PCR to show the mRNA altered degree of Sut melanocytes compared with wild melanocytes.Results:1. Identification of altered proteinsTwenty proteins were found altered significantly in xCT-deficient Sut melanocytes compared with wild melanocytes by 2-DE experiment. Ten of the altered proteins were up-regulated, while the other ten were down-regulated. These twenty proteins were identified by tandem mass spectrometry and database query. The ten up-regulated proteins were:Vimentin, Tubulin alpha-1b, Tubulin beta-5, Annexin A3, Calmodulin, protein S100-A4, unkown protein of 11kDa, protein S100-A6, Nucleoside diphosphate kinase B, S-formylglutathione. The ten down-regulated proteins were:Calumenin, protein N-Myc downstream regulated genel (NDRG1),Ornithine aminotransferase,protein disulfide isomerase A3(PDIA3), Dihydropyrimidinase-related protein2 (DPYSL2), ATP5A1, Histone H2B type 1-K (HISTLH2BK), unkown protein of 14kDa, unkown protein of gene sequence number of OTTMUSG00000007855 and unkown protein of 47kDa.2.The mRNA level of Calmodulin, Annexin A3, Vimentin and S100-A4.The mRNA level of Calmodulin, Annexin A3, Vimentin and S100-A4 were confirmed by RT-PCR experiment. The mRNA level of Calmodulin, Annexin A3, Vimentin and S100-A4 was found up-regulated significantly in Sut melanocytes compared with wild melanocytes.The mRNA altered degree was shown in histogram.Conclusions:1.Twenty proteins were found altered significantly in Sut melanocytes compared with wild melanocytes. Ten of them were up-regulated,the other ten were down-regulated.Three of up-regulated proteins were cell structure proteins(Vimentin, Tubulin alpha-1b, Tubulin beta-5), three up-regulated proteins were Ca2+-binding proteins(Calmodulin, Protein S100A-4,Protein S100A-6), three up-regulated proteins were enzymes( S-formylglutathione, Annexin A3). five down-regulated proteins were enzymes (NDRG1, PDIA3, ATP5A1, Ornithine aminotransferase, NME2), two down-regulated proteins were singal transduction factors (DPYSL2, HISTLH2BK3).2.The identified proteins play important roles in autophay, intracellular vesicle trafficking, MAP kinase signal pathway and DNA replication.Autophay and MAP kinase signal pathway may play vital role in Sut melanocytes'growth inhibition. Further study is required to elucidate how autophay and MAP kinase signal pathway effect on Sut melanocytes'growth inhibition.3.The mRNA level of Annexin A3, Calmodulin, Vimentin and S100-A4 in Sut melanocytes were up-regulated significantly compared with wild melanocytes, this result prove that Annexin A3, Calmodulin, Vimentin and S100-A4 protein expression degree were regulated by transcriptional level in xCT-deficent Sut melanocytes.