Studies of post-translational modifications in differentially expressed proteins in the cell lines MELAN-A, TM1 E TM5. A murine model of melanoma progression

• Grant number: 05/04779-6
• Support Opportunities: Regular Research Grants
• Start date: May 01, 2006
• End date: November 30, 2008
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: José César Rosa
• Grantee: José César Rosa
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

The current proposal will investigate a murine melanoma progression model using a functional post-translational proteomics approach of three cell lines. Melan-a is a non-tumoral cell line arised from melanocytes; while Tm1 and Tm5 are two tumoral cell lines which present a vertical growth phenotype (VGP) and both of them are derived from Melan-a. Proteomics and SAGE analysis of these three cell lines were recently published by our group and the following results were obtained: 1) an accentuated decrease in the quantity of antioxidant enzymes concomitant with reduction of the efficiency of hydrogen peroxide degradation; 2) a reduction in the quantity of glutathione S-transferase in melanoma cells following the fall of glutathionylation in some yet non-identified proteins, and 3) decrease or loss of proteins, like cdkn2a (p16), galectin-3 and metallothionein that are proteins with CpG islets on their promoter regions. Thus, we propose to extent the study of this model to the following investigation: Characterization of post-translational modifications including glutathionylation, O-GlcNAc and phosphorylation by combination of 2D-gel electrophoresis, western-blotting and mass spectrometry for protein identification. All modifications are related to cellular stress provoked by ROS or external signal. Gluthationylation of proteins is a response to protect some active site from ROS whose function is based on cysteine residues. The dynamic modification of nuclear and cytoplasmatic proteins with O-linked beta-N-Acetylglucosamine (O-GlcNAc) is a regulatory post-translational modification that is rapidly responsive to morphogens, hormones, nutrients and cellular stress. O-GlcNAc and O-phosphorylation reciprocity has been documented by several group which have confirmed that a given Ser/Thr may exist in three states: glycosylated, phosphorylated or unmodified... (AU)