Quantitative chemical proteomics of dynamic palmitoylation in cells

细胞中动态棕榈酰化的定量化学蛋白质组学

• 批准号: 8335370
• 负责人: Brent Randall Martin
• 金额: $ 24.15万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-20 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8335370
• 关键词: Adenocarcinoma Cell; Affinity Chromatography; Azides; Biological Assay; Biotin; Cancer cell line; Cell Line; Cell physiology; Cells; Cellular Assay; Chemicals; Colon Adenocarcinoma; Complement; Coupled; Data; Detection; Development; Disseminated Malignant Neoplasm; Enzymes; Event; Family; Gel; Goals; Growth; HRAS gene; Human; Hydrolase; Individual; Label; Libraries; Light; Light Cell; Link; Lipids; Malignant - descriptor; Malignant Neoplasms; Mass Spectrum Analysis; Metabolic; Metastasis Suppression; Methods; Mind; Modification; Mus; Neoplasm Metastasis; Oncogenes; Palmitic Acylation Site; Participant; Pathology; Pathway interactions; Peptides; Play; Population; Post-Translational Protein Processing; Process; Protein Dynamics; Proteins; Proteome; Proteomics; Relative (related person); Research; Research Design; Resolution; Role; Series; Serine Hydrolase; Signal Transduction; Site; Staging; Stearic Acids; Testing; Transferase; Triazoles; Tumor Suppressor Proteins; Tumorigenicity; base; cancer cell; cancer proteomics; cell growth; comparative; complex biological systems; genetic regulatory protein; high throughput screening; inhibitor/antagonist; instrument; migration; novel; palmitoylation; small hairpin RNA; tool; trafficking; tumor progression; tumorigenesis

Protein palmitoylation is an essential post-translational modification necessary for trafficking and localization of numerous regulatory proteins that play key roles in cell growth and signaling. We have recently developed a chemo-proteomic method for metabolic incorporation and detection of palmitoylated proteins by multiple platforms, including fluorescent gel-based detection and mass spectrometry-based identification. This approach shows unprecedented sensitivity for profiling palmitoylated proteins in complex biological systems, leading to the identification of hundreds of palmitoylated proteins in cancer cells. These data indicate that palmitoylation is a widespread post-translational modification that influences the function of nearly all cellular pathways. In many cases, palmitoylation is thought to be dynamically regulated, although the mechanisms that control this lipid modification remain poorly characterized. In order to understand the processes regulating dynamic palmitoylation, we will develop a quantitative platform for global comparative proteomic analysis of palmitoylated proteins, including identification of exact sites of palmitoylation. We will use this platform to interrogate the population of palmitoylated proteins regulated by both palmitoyl transferases and thioesterases. Several oncogenes require palmitoylation to induce malignant transformation, suggesting protein palmitoyl thioesterases may repress aberrant growth signaling. By assaying de-palmitoylation of bio-orthogonally labeled substrates, we have identified a novel protein thioesterase, and plan to expand this assay to other uncharacterized hydrolases. We plan to further characterize the relationship between APT1 and cancer by proteomic identification of substrates coupled with cellular assays of transformation and tumorigenicity. Similarly, several DHHC palmitoyl acyl transferases (PATs) have been suggested to play important roles in cancer, yet deconvolution of their relative contributions to tumorigenesis has proven challenging. We propose to create the first activity-based proteomics probe for PATs and characterize their activity at different stages of cancer progression. We will also identify PAT substrates involved in suppressing metastasis. Currently, selective inhibitors of individual PAT enzymes are lacking. With this goal in mind, we will develop a general HTS assay for identifying PAT-specific inhibitors.

蛋白棕榈酰化是一种重要的翻译后修饰，是蛋白质运输和定位所必需的