Abstract
A family of zDHHC protein acyltransferase (PAT) enzymes catalyze the S-palmitoylation of target proteins via a two-step mechanism. The first step involves transfer of palmitate from the palmitoyl-CoA donor to the active site cysteine of the zDHHC PAT enzyme, releasing reduced CoA (CoASH). In the second step, the palmitoyl–PAT intermediate thioester reacts with a cysteine side chain within the target substrate to produce the palmitoylated substrate product or, in the absence of a protein substrate, the palmitoyl–PAT intermediate thioester is hydrolyzed and releases palmitate. Formation and resolution of the palmitoyl–PAT intermediate complex (autopalmitoylation) is measured using a coupled enzyme system that monitors the production of CoASH via reduction of NAD+ by the α-ketoglutarate dehydrogenase complex. This assay can be used to isolate and characterize modulators of autopalmitoylation and is scalable to high-throughput screening (HTS). A second fluorescence-based assay is described that monitors the hydrolysis of the palmitoyl–PAT thioester linked intermediate by thin-layer chromatography using a palmitoyl-CoA analog, BODIPY®-C12:0-CoA, as a substrate. These two assays provide a methodology to quantify the first enzymatic step of the two-step zDHHC PAT reaction.
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References
Linder ME, Deschenes RJ (2007) Palmitoylation: policing protein stability and traffic. Nat Rev Mol Cell Biol 8:74–84
Mitchell DA, Vasudevan A, Linder ME, Deschenes RJ (2006) Protein palmitoylation by a family of DHHC protein S-acyltransferases. J Lipid Res 47:1118–1127
Lobo S, Greentree W, Linder M, Deschenes R (2002) Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae. J Biol Chem 277:41268–41273
Roth AF, Feng Y, Chen L, Davis NG (2002) The yeast DHHC cysteine-rich domain protein Akr1p is a palmitoyl transferase. J Cell Biol 159:23–28
Mitchell DA, Mitchell G, Ling Y, Budde C, Deschenes RJ (2010) Mutational analysis of Saccharomyces cerevisiae Erf2 reveals a two-step reaction mechanism for protein palmitoylation by DHHC enzymes. J Biol Chem 285:38104–38114
Jennings BC, Linder ME (2012) DHHC protein S-acyltransferases use similar ping-pong kinetic mechanisms but display different acyl-CoA specificities. J Biol Chem 287:7236–7245
Mitchell DA, Hamel LD, Ishizuka K, Mitchell G, Schaefer LM, Deschenes RJ (2012) The Erf4 subunit of the yeast Ras palmitoyl acyltransferase is required for stability of the Acyl-Erf2 intermediate and palmitoyl transfer to a Ras2 substrate. J Biol Chem 287:34337–34348
Mitchell DA, Hamel LD, Reddy KD, Farh L, Rettew LM, Sanchez PR, Deschenes RJ (2014) Mutations in the X-linked intellectual disability gene, zDHHC9, alter autopalmitoylation activity by distinct mechanisms. J Biol Chem 289:18582–18592
Hamel LD, Deschenes RJ, Mitchell DA (2014) A fluorescence-based assay to monitor autopalmitoylation of zDHHC proteins applicable to high throughput screening. Anal Biochem 460:1–8
Hamel LD, Lenhart BJ, MItchell DA, Santos RG, Giulianotti MA, Deschenes RJ (2016) Identification of protein palmitoylation inhibitors from a scaffold ranking library. Comb Chem High Throughput Screen 19:262–274
Massey V (1960) The composition of the ketoglutarate dehydrogenase complex. Biochim Biophys Acta 38:447–460
Storer AC, Cornish-Bowden A (1974) The kinetics of coupled enzyme reactions. Applications to the assay of glucokinase, with glucose 6-phosphate dehydrogenase as coupling enzyme. Biochem J 141:205–209
Garland PB, Shepherd D, Yates DW (1965) Steady-state concentrations of coenzyme A, acetyl-coenzyme A and long-chain fatty acyl-coenzyme A in rat-liver mitochondria oxidizing palmitate. Biochem J 97:587–594
Berthiaume L, Peseckis SM, Resh MD (1995) Synthesis and use of iodo-fatty acid analogs. Methods Enzymol 250:454–466
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Mitchell, D.A., Pendleton, L.C., Deschenes, R.J. (2019). In Vitro Assays to Monitor the Enzymatic Activities of zDHHC Protein Acyltransferases. In: Linder, M. (eds) Protein Lipidation. Methods in Molecular Biology, vol 2009. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9532-5_13
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DOI: https://doi.org/10.1007/978-1-4939-9532-5_13
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