Effect Of TRAF6 On Proteome And Palmitoylated Proteome In Human Colon Cancer Cells

Colorectal cancer(CRC)is currently the third most common malignancy in Asia.Due to its high morbidity and mortality,CRC have became worldwide health problems.It is therefore particularly important to disclose the underlying molecular mechanisms of colon cancer.Tumor necrosis factor receptor-associated factor 6(TRAF6)is an adaptor protein and highly expressed in a variety of cancers.It is reported that TRAF6 plays an important role in tumorigenesis,invasion,and metastasis of tumor.But the role of TRAF6 in the human colon cancer has been rarely studied.AimsTo investigate the effect of TRAF6 on the proteome and palmitoylated proteome in human colon cancer cells using SILAC-based quantitative proteomics and explore the potential molecular mechanism underlying TRAF6-mediated regulation of tumorigenesis and development of human colon cancer at protein level and protein post-translational modification level.Methods1.Using q PCR and Western blot to verify the knockdown efficiency of TRAF6 in HCT116 cell line stably expressing TRAF6 sh RNA at m RNA and protein levels,respectively.2.Using SILAC-based quantitative proteomics to investigate the effect of TRAF6 on the whole proteome of human colon cancer cells.3.Appling bioinformatics software to analyze the biological processes,molecular component,biological functions and signal pathways involved by TRAF6-mediated differential proteins in human colon cancer cells.4.Using molecular biology techniques to verify the major signal pathways involved by TRAF6-mediated differential proteins in human colon cancer cells.5.Using SILAC-and click chemistry-based quantitative proteomics to develop a method for the quantitative analysis of palmitoylated proteome in wild-type HCT116 cells.6.Using the developed method above to analyze the palmitoylated proteome of TRAF6-regulated human colon cancer cells and to verify the palmitoylation sites.7.Using bioinformatics software to perform GO annotation analysis of palmitoylated proteins that are differentially expressed in TRAF6-mediated human colon cancer cells.Results1.Based on SILAC-based quantitative proteomics analysis of wild-type and TRAF6-knockdowned HCT116 cells,we found that TRAF6 is involved in the regulation of cholesterol biosynthesis pathway in human colon cancer cells.2.The knockdown of TRAF6 in human colon cancer cells promotes the expression of HMGCS1 and FDFT1,the key proteins in the cholesterol biosynthesis pathway,which not only raises the total cholesterol level in human colon cancer cells but also induces more cholesterol moving to cell membrane and the increase of cholesterol level in plasma membrane.3.The fact that down-regulation of TRAF6 increases the cholesterol level in human colon cancer cells was not regulated by the following mechanisms including LDLR-mediated uptake of exogenous cholesterol,SREBP2-mediated transcriptional regulation of key proteins in cholesterol biosynthesis pathway,ACSS2-mediated synthesis of acetyl-Co A,and ABCA1-mediated cholesterol efflux.4.Using SILAC-and click chemistry-based quantitative proteomics in combination with replacement of hydrophobic palmitoylate chain with NEM modification,we successfully performed the qualitative and quantitative analysis of palmitoylated proteome and identified their palmitoylation sites.5.Using the method developed above we investigate the effect of TRAF6 on palmitoylated proteome in human colon cancer cells.27 proteins was screened due to the change of their palmitoylation modification.These proteins are mostly located on the cell membrane and are mainly involved in cell-cell adhesion,m RNA stability regulation,positive regulation of m RNA catabolism,NIK/NF-?B signaling pathway,glucose metabolism and other biological processes.Commonly,these biological processes are associated with the development,invasion,and migration of human colon cancer.Conclusions1.TRAF6 knockdown raises the cholesterol level in human colon cells via mediating the cholesterol biosynthetic pathways,which might remodel the structure of lipid raft,thereby affect the localization and downstream signal transduction of colon cancer-associated proteins in lipd raft,leading to cell growth,proliferation,invasion,and migration of human colon cancer.2.TRAF6 participates in the biological processes associated with the development of colon cancer by mediating the palmitoylation modification of signal proteins,such as cell-cell adhesion,glucose metabolism and other biological processes.