title: A fluorescence-based assay to monitor autopalmitoylation of zDHHC proteins applicable to high-throughput screening.
abstract: Palmitoylation, the posttranslational thioester-linked modification of a 16-carbon saturated fatty acid onto the cysteine residue of a protein, has garnered considerable attention due to its implication in a multitude of disease states. The signature DHHC motif (Asp-His-His-Cys) identifies a family of protein acyltransferases (PATs) that catalyze the S-palmitoylation of target proteins via a two-step mechanism. In the first step, autopalmitoylation, palmitate is transferred from palmitoyl-CoA to the PAT, creating a palmitoyl:PAT intermediate and releasing reduced CoA. The palmitoyl moiety is then transferred to a protein substrate in the second step of the reaction. We have developed an in vitro, single-well, fluorescence-based enzyme assay that monitors the first step of the PAT reaction by coupling the production of reduced CoA to the reduction of NAD(+) using the α-ketoglutarate dehydrogenase complex. This assay is suitable for determining PAT kinetic parameters, elucidating lipid donor specificity and measuring PAT inhibition by 2-bromopalmitate. Finally, it can be used for high-throughput screening (HTS) campaigns for modulators of protein palmitoylation.
authors.affiliation: Department of Molecular Medicine, University of South Florida, Tampa, FL 33612, USA.
authors.forename: Laura D
authors.initials: LD
authors.lastname: Hamel
authors.affiliation: Department of Molecular Medicine, University of South Florida, Tampa, FL 33612, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Deschenes
authors.affiliation: Department of Molecular Medicine, University of South Florida, Tampa, FL 33612, USA. Electronic address: dmitchel@health.usf.edu.
authors.forename: David A
authors.initials: DA
authors.lastname: Mitchell
mesh_terms: D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D020816:Amino Acid Motifs; D003065:Coenzyme A / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D057075:Enzyme Assays / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D007700:Kinetics; D050356:Lipid Metabolism; D054878:Lipoylation / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism; D010084:Oxidation-Reduction; D010168:Palmitates / Q000494:pharmacology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D003902:Detergents; D008565:Membrane Proteins; D010168:Palmitates; D029701:Saccharomyces cerevisiae Proteins; C022776:2-bromopalmitate; D000217:Acyltransferases; C403284:ERF2 protein, S cerevisiae; C092131:SHR5 protein, S cerevisiae; D003065:Coenzyme A
keywords: Autopalmitoylation; High-throughput screening; Posttranslational modification; Protein palmitoylation; zDHHC proteins