Protein palmitoylation is an essential post-translational modification necessary for trafficking and localization ofnumerous regulatory proteins that play key roles in cell growth and signaling. We have recently developed achemo-proteomic method for metabolic incorporation and detection of palmitoylated proteins by multipleplatforms, including fluorescent gel-based detection and mass spectrometry-based identification. Thisapproach 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 thatpalmitoylation is a widespread post-translational modification that influences the function of nearly all cellularpathways. In many cases, palmitoylation is thought to be dynamically regulated, although the mechanisms thatcontrol this lipid modification remain poorly characterized. In order to understand the processes regulatingdynamic palmitoylation, we will develop a quantitative platform for global comparative proteomic analysis ofpalmitoylated proteins, including identification of exact sites of palmitoylation. We will use this platform tointerrogate the population of palmitoylated proteins regulated by both palmitoyl transferases and thioesterases.Several oncogenes require palmitoylation to induce malignant transformation, suggesting protein palmitoylthioesterases may repress aberrant growth signaling. By assaying de-palmitoylation of bio-orthogonally labeledsubstrates, we have identified a novel protein thioesterase, and plan to expand this assay to otheruncharacterized hydrolases. We plan to further characterize the relationship between APT1 and cancer byproteomic 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 incancer, yet deconvolution of their relative contributions to tumorigenesis has proven challenging. We proposeto create the first activity-based proteomics probe for PATs and characterize their activity at different stages ofcancer 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 generalHTS assay for identifying PAT-specific inhibitors.
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. 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. Unique chemical tools will be developed to profile the palmitoylation enzymes implicated in the development of metastatic cancer.
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