The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase Is Palmitoylated and Contains Tightly Bound Zinc
The Cysteine-rich Domain of the DHHC3 Palmitoyltransferase Is Palmitoylated and Contains Tightly Bound Zinc
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DOI：
10.1074/jbc.m115.691147
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发表时间：
2015-12-04
影响因子：
4.8
通讯作者：
Linder, Maurine E.
中科院分区：
文献类型：
--
作者：
Gottlieb, Colin D.;Zhang, Sheng;Linder, Maurine E.
DHHC棕榈酰基转移酶催化脂肪酸棕榈酸酯与细胞膜上的蛋白质加成。哺乳动物DHHC蛋白家族有23个成员，它们都含有一个保守的催化结构域，称为富含半胱氨酸的结构域(CRD)。DHHC蛋白通过两步催化机制转移棕榈酸酯，在这个过程中，酶首先用棕榈酸酯修饰自己，这一过程称为自酰化。然后，该酶将棕榈酸从自身转移到底物蛋白质上。棕榈酰化半胱氨酸在自酰化中间体中的数量和位置尚不清楚。在这项研究中，我们提出了利用质谱学的证据，证明DHHC3在DHHC基序的半胱氨酸上是棕榈酰化的。DHHC基序外高度保守的CRD半胱氨酸突变导致活性缺陷和有限的蛋白质降解所揭示的结构扰动。用螯合剂在体外处理DHHC3，复制了在保守的半胱氨酸突变体中观察到的特定结构扰动和活性缺陷，表明CRD中存在金属离子结合。使用荧光指示剂MAG-Fura-2，从DHHC3中释放的金属被鉴定为锌。锌结合的化学计量比为2摩尔锌/摩尔DHHC3蛋白。综上所述，我们的数据表明，CRD中的半胱氨酸残基与锌离子的配位是DHHC蛋白结构完整性所必需的。
DHHC palmitoyltransferases catalyze the addition of the fatty acid palmitate to proteins on the cytoplasmic leaflet of cell membranes. There are 23 members of the highly diverse mammalian DHHC protein family, all of which contain a conserved catalytic domain called the cysteine-rich domain (CRD). DHHC proteins transfer palmitate via a two-step catalytic mechanism in which the enzyme first modifies itself with palmitate in a process termed autoacylation. The enzyme then transfers palmitate from itself onto substrate proteins. The number and location of palmitoylated cysteines in the autoacylated intermediate is unknown. In this study, we present evidence using mass spectrometry that DHHC3 is palmitoylated at the cysteine in the DHHC motif. Mutation of highly conserved CRD cysteines outside the DHHC motif resulted in activity deficits and a structural perturbation revealed by limited proteolysis. Treatment of DHHC3 with chelating agents in vitro replicated both the specific structural perturbations and activity deficits observed in conserved cysteine mutants, suggesting metal ion-binding in the CRD. Using the fluorescent indicator mag-fura-2, the metal released from DHHC3 was identified as zinc. The stoichiometry of zinc binding was measured as 2 mol of zinc/mol of DHHC3 protein. Taken together, our data demonstrate that coordination of zinc ions by cysteine residues within the CRD is required for the structural integrity of DHHC proteins.