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 ?-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.

SUBMITTER: Hamel LD 

PROVIDER: S-EPMC6445550 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature