Neuronal palmitoyl acyl transferases exhibit distinct substrate specificity
Neuronal palmitoyl acyl transferases exhibit distinct substrate specificity
复制标题
DOI：
10.1096/fj.08-127399
复制
发表时间：
2009-08-01
期刊：
影响因子：
4.8
通讯作者：
El-Husseini, Alaa
中科院分区：
文献类型：
--
作者：
Huang, Kun;Sanders, Shaun;El-Husseini, Alaa
棕榈酰化是半胱氨酸残基与棕榈酸脂的一种翻译后修饰，近年来已成为调节蛋白质转运和功能的重要机制。随着23个DHHC哺乳动物棕榈酰转移酶(PATS)的鉴定，一个关键的问题是这些PATS的底物-酶专一性的性质。利用酰基-生物素交换棕榈酰化实验，我们比较了四个神经元PATT，即DHHC-3、DHHC-8、HIP14L(DHHC-13)和HIP14(DHHC-17)的底物特异性。在COS细胞中外源表达的酶和底物表明，HIP14L和HIP14调节亨廷顿蛋白棕榈酰化，DHHC-8调节paralemmin-1棕榈酰化，DHHC-3表现出最小的底物特异性。这些体外数据通过慢病毒siRNA介导的内源性HIP14和DHHC-3在培养的大鼠皮质神经元中的下调而得到验证。PATS需要棕榈酰化半胱氨酸的存在才能与其底物相互作用。为了进一步了解影响酶/底物专一性的因素，我们将HIP14 ankryin重复结构域融合到DHHC-3的N端，它不是Huntingtin的PAT。这一修饰使DHHC-3能够通过调节棕榈酰化和亨廷顿蛋白的运输而表现出与HIP14相似的行为。综上所述，这项研究表明，单个PAT具有特定的底物选择性，这是由酶的DHHC结构域之外的调节结构域决定的。-Huang，K.，Sanders，S.，Singaraja，R.，Orban，P.，CijSouw，T.，Arstikaitis，P.，Yanai，A.，Hayden，M.R.，El-Husseini，A.神经元棕榈酰酰基转移酶表现出明显的底物特异性。FASE B J.23,2605-2615(2009)
Palmitoylation, a post-translational modification of cysteine residues with the lipid palmitate, has recently emerged as an important mechanism for regulating protein trafficking and function. With the identification of 23 DHHC mammalian palmitoyl acyl transferases (PATs), a key question was the nature of substrate-enzyme specificity for these PATs. Using the acyl-biotin exchange palmitoylation assay, we compared the substrate specificity of four neuronal PATs, namely DHHC-3, DHHC-8, HIP14L (DHHC-13), and HIP14 (DHHC-17). Exogenous expression of enzymes and substrates in COS cells reveals that HIP14L and HIP14 modulate huntingtin palmitoylation, DHHC-8 modulates paralemmin-1 palmitoylation, and DHHC-3 shows the least substrate specificity. These in vitro data were validated by lentiviral siRNA- mediated knockdown of endogenous HIP14 and DHHC-3 in cultured rat cortical neurons. PATs require the presence of palmitoylated cysteines in order to interact with their substrates. To understand the elements that influence enzyme/substrate specificity further, we fused the HIP14 ankryin repeat domain to the N terminus of DHHC-3, which is not a PAT for huntingtin. This modification enabled DHHC-3 to behave similarly to HIP14 by modulating palmitoylation and trafficking of huntingtin. Taken together, this study indicates that individual PATs have specific substrate preference, determined by regulatory domains outside the DHHC domain of the enzymes.-Huang, K., Sanders, S., Singaraja, R., Orban, P., Cijsouw, T., Arstikaitis, P., Yanai, A., Hayden, M. R., El-Husseini, A. Neuronal palmitoyl acyl transferases exhibit distinct substrate specificity. FASEB J. 23, 2605-2615 ( 2009)