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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Transition Award (R00)
Project #
5R00CA151460-03
Application #
8335370
Study Section
Special Emphasis Panel (NSS)
Program Officer
Couch, Jennifer A
Project Start
2011-09-20
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
3
Fiscal Year
2012
Total Cost
$241,530
Indirect Cost
$72,357
Name
University of Michigan Ann Arbor
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Hernandez, Jeannie L; Davda, Dahvid; Majmudar, Jaimeen D et al. (2016) Correlated S-palmitoylation profiling of Snail-induced epithelial to mesenchymal transition. Mol Biosyst 12:1799-808
Majmudar, Jaimeen D; Konopko, Aaron M; Labby, Kristin J et al. (2016) Harnessing Redox Cross-Reactivity To Profile Distinct Cysteine Modifications. J Am Chem Soc 138:1852-9
Won, Sang Joon; Davda, Dahvid; Labby, Kristin J et al. (2016) Molecular Mechanism for Isoform-Selective Inhibition of Acyl Protein Thioesterases 1 and 2 (APT1 and APT2). ACS Chem Biol :
Xu, Hao; Majmudar, Jaimeen D; Davda, Dahvid et al. (2015) Substrate-Competitive Activity-Based Profiling of Ester Prodrug Activating Enzymes. Mol Pharm 12:3399-407
Foe, Ian T; Child, Matthew A; Majmudar, Jaimeen D et al. (2015) Global Analysis of Palmitoylated Proteins in Toxoplasma gondii. Cell Host Microbe 18:501-11
Majmudar, Jaimeen D; Martin, Brent R (2014) Strategies for profiling native S-nitrosylation. Biopolymers 101:173-9
Martin, Brent R (2014) The next frontier of post-translational modifications. Biopolymers 101:131-2
Davda, Dahvid; Martin, Brent R (2014) Acyl protein thioesterase inhibitors as probes of dynamic S-palmitoylation. Medchemcomm 5:268-276
Jia, Lixia; Chisari, Mariangela; Maktabi, Mohammad H et al. (2014) A mechanism regulating G protein-coupled receptor signaling that requires cycles of protein palmitoylation and depalmitoylation. J Biol Chem 289:6249-57
Davda, Dahvid; El Azzouny, Mahmoud A; Tom, Christopher T M B et al. (2013) Profiling targets of the irreversible palmitoylation inhibitor 2-bromopalmitate. ACS Chem Biol 8:1912-7

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