A fluorescence-based assay to monitor autopalmitoylation of zDHHC proteins applicable to high-throughput screening.
A fluorescence-based assay to monitor autopalmitoylation of zDHHC proteins applicable to high-throughput screening.
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DOI：
10.1016/j.ab.2014.05.013
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发表时间：
2014-09-01
影响因子：
2.9
通讯作者：
Mitchell DA
中科院分区：
文献类型：
--
作者：
Hamel LD;Deschenes RJ;Mitchell DA
棕榈酰化是将16个碳饱和脂肪酸翻译后硫酯连接修饰到蛋白质的半胱氨酸残基上，由于其与多种疾病状态的关系而引起了相当大的关注。标志性DHHC基序（Asp-His-His-Cys）鉴定了通过两步机制催化靶蛋白的S-棕榈酰化的蛋白酰基转移酶（PAT）家族。在第一步中，自棕榈酰化，棕榈酸从棕榈酰辅酶A转移到PAT，产生棕榈酰：PAT中间体并释放还原的辅酶A。然后在反应的第二步中将棕榈酰基部分转移到蛋白质底物上。我们开发了一种体外、单孔、基于荧光的酶测定法，通过使用α-酮戊二酸脱氢酶复合物将还原CoA的产生与NAD+的还原偶联，监测PAT反应的第一步。该测定法适用于测定PAT动力学参数、阐明脂质供体特异性和测量2-溴棕榈酸酯对PAT的抑制。最后，它可用于蛋白质棕榈酰化调节剂的高通量筛选（HTS）活动。
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.
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影响因子：
4.8
作者：
Lobo, S;Greentree, WK;Deschenes, RJ
通讯作者：
Deschenes, RJ
影响因子：
3.2
作者：
ITO, H;FUKUDA, Y;KIMURA, A
通讯作者：
KIMURA, A
影响因子：
4
作者：
Davda D;El Azzouny MA;Tom CT;Hernandez JL;Majmudar JD;Kennedy RT;Martin BR
通讯作者：
Martin BR
影响因子：
5.2
作者：
Mansouri, MR;Marklund, L;Dahl, N
通讯作者：
Dahl, N
影响因子：
4.1
作者：
KERBEY, AL;RADCLIFFE, PM;RANDLE, PJ
通讯作者：
RANDLE, PJ