Optimizing protein S-acylation detection in autophagy

Abstract

S-acylation, sometimes referred to as S-palmitoylation or simply palmitoylation, is a posttranslational protein modification involving the covalent addition of a fatty acid to cysteine residues. Since the first characterization of protein palmitoylation over 40 years ago, this modification has been implicated in a variety of cellular processes, particularly membrane targeting, protein localization, and regulation of protein-protein interactions. These processes are essential in autophagy, a membrane-dependent cellular recycling and degradation mechanism to remove damaged organelles and toxic proteins. Due to its unique reversibility among lipid modifications as well its regulatory roles in membrane-dependent processes essential to autophagy, palmitoylation has recently began to be explored in the context of autophagy. Despite increasing evidence pointing to a regulatory role of palmitoylation in autophagy, methods for the detection of palmitoylated proteins specifically under autophagic conditions have not been a focus for development and optimization. The aim of this thesis project was to optimize detection methods of palmitoylated proteins during autophagy. In this regard, we combined existing techniques of chemical autophagy induction, metabolic labeling with bio-orthogonal fatty acid analogs that are detectable with click chemistry, as well as affinity purification for the detection and identification of palmitoylated proteins during autophagy. First, we tested several new commercial products and established a working protocol for the click chemistry assay. We then demonstrated that delivery of detectable alkynyl fatty acids into cells during metabolic labeling can be considerably improved with the use of delipidated media and saponification of fatty acids. Cellular incorporation of the 18-carbon alkynyl stearate, the most commonly used fatty acid analog, was shown to be improved the most through saponification and incubation with fatty acid free bovine serum albumin (BSA), leading to greater availability of the fatty acid label and significantly enhanced overall palmitoylation signal. In addition, saponification of fatty acids prior to addition to cell culture can protect cells from lipotoxicity and activation of stress pathways induced by the direct addition of fatty acids. Next, we tested two approaches to enrich and purify palmitoylated proteins following click chemistry linkage of an affinity probe. In addition, we determined optimal treatment times of autophagy induction using rapamycin to induce the most robust autophagic flux. Finally, we demonstrated low throughput confirmation and characterization of palmitoylation in two proteins of interest, the small VCP-interacting protein and the spike protein of the SARS-CoV2 virus, via immunoprecipitation and click chemistry detection. The established methods and findings of this study provide important foundations to identify palmitoylation targets and characterize pathways of palmitoylation regulation during autophagy, which ultimately will provide valuable insight on the molecular mechanisms of a wide range of diseases from cancer to neurodegeneration.