Date of Award
Reversible S-palmitoylation confers spatiotemporal control of protein function by modulating protein stability, trafficking and activity as well as protein-protein and membrane-protein associations. While it is evident that palmitoylation is regulated in vivo, mechanisms that mediate cellular stimuli-driven changes of the lipid modification are not understood. Furthermore, the requirement for substrate specificity among the highly redundant palmitoyl acyltransferases (PATs) remains unresolved. To study the regulation of PATs and palmitoylomes, I developed bioorthogonal chemical strategies for improved analysis of dynamic palmitoylation in mammalian cells. I showed that alkyne-functionalized fatty acids, in conjunction with azido-fluorophores, provide the most sensitive detection of acylated proteins following CuI-catalyzed azidealkyne cycloaddition. Linkage-specific hydrolysis, mutagenesis and inhibitor studies reveal that these alkynyl-fatty acids are incorporated into proteins by endogenous fattyacylation machinery via native linkages at specific amino acid residues. In addition, shorter and longer chain fatty acids label myristoylated and palmitoylated proteins respectively. Since myristoylation is co-translational and constitutive, I employed both palmitoylation and myristoylation chemical reporters with orthogonal fluorophores to simultaneously monitor palmitate and protein turnover. Dual pulse-chase analysis of Lck, a tyrosine kinase required for T-cell signaling, revealed accelerated palmitate cycling upon T-cell activation. Pharmacological perturbation of Lck palmitate turnover suggests yet uncharacterized serine hydrolases contribute to dynamic palmitoylation in cells. These significant improvements allow rapid and robust biochemical analysis of palmitoylated proteins without overexpression, facilitating the functional characterization of cellular factors and drugs that modulate protein palmitoylation. Taking advantage of the sensitive bioorthogonal detection of protein palmitoylation and the simple PAT network in the fission yeast Schizosaccharomyces pombe, I provided evidence for regulation of PATs and palmitoylomes in vivo at physiological enzyme and substrate concentrations. I showed that the Erf2-Erf4 PAT modulates sexual differentiation, and that upregulation of its expression is required to establish the meiotic palmitoylome. Importantly, I demonstrated that changes in Erf2- Erf4 levels within the physiological range control PAT specificity and result in the differential palmitoylation of its substrates in vegetative and meiotic cells. Underscoring the biological significance of controlling PAT levels, Erf2-Erf4 overproduction in proliferating cells alters the palmitoylome and the subcellular distribution of Rho3, a major meiotic target, stimulating sexual differentiation in the absence of normal physiological cues. From this study, I conclude that PAT substrate specificity depends on enzyme levels and propose the rheostatic control of PAT activity as a mechanism by which cells shape stimuli-induced palmitoylomes. Future questions stemming from this work are also discussed.
Zhang, Mingzi, "Develoment and Application of Chemical Strategies to Study Protein Fatty-Acylation in Eukaryotes" (2012). Student Theses and Dissertations. 157.