Identification of conserved regions and residues within Hedgehog acyltransferase critical for palmitoylation of Sonic Hedgehog.
Identification of conserved regions and residues within Hedgehog acyltransferase critical for palmitoylation of Sonic Hedgehog.
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DOI：
10.1371/journal.pone.0011195
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发表时间：
2010-06-23
期刊：
影响因子：
3.7
通讯作者：
Resh MD
中科院分区：
文献类型：
--
作者：
Buglino JA;Resh MD
Sonic Hedgehog (Shh) 是一种棕榈酰化蛋白，在哺乳动物发育和人类癌症中发挥着关键作用。 Shh 的棕榈酰化是有效的长程和短程 Shh 介导的信号传导所必需的。棕榈酸与 Shh 的结合是由 Hedgehog 酰基转移酶 (Hhat) 催化的，Hhat 是多通道膜蛋白膜结合 O-酰基转移酶 (MBOAT) 家族的成员。 MBOAT 蛋白的极度疏水性成分限制了其生化特征。除了两个保守残基的诱变外，尚未对 Hhat 进行结构功能分析，并且 Shh 棕榈酰化所需的蛋白质区域也是未知的。在这里，我们采用系统方法来鉴定 Hhat 中蛋白质稳定性和/或酶活性所需的残基。我们还确定了 Hhat 活性所需的第二个新颖的 MBOAT 同源区域（残基 196-234）。总共生成并分析了十个缺失突变体和十一个点突变体。 Hhat N 端和 C 端的截短产生稳定性降低的无活性蛋白质。四个在预测环区域内有缺失的 Hhat 突变体和五个点突变体保留了稳定性，但失去了棕榈酰化活性。我们纯化了两个点突变体 W378A 和 H379A，它们具有缺陷的 Hhat 活性。动力学分析揭示了 Shh 和/或棕榈酰 CoA 的表观 Km 和 Vmax 的变化，这些变化可能解释了在这些突变体中观察到的催化缺陷。这项研究精确定位了调节 Hhat 稳定性和催化作用的特定区域和多个残基。我们的研究结果应该适用于介导 Wnt 蛋白和 ghrelin 脂质修饰的其他 MBOAT 蛋白，并且应该作为了解棕榈酰酰基转移酶如何修饰分泌的形态发生素的模型。
Sonic hedgehog (Shh) is a palmitoylated protein that plays key roles in mammalian development and human cancers. Palmitoylation of Shh is required for effective long and short range Shh-mediated signaling. Attachment of palmitate to Shh is catalyzed by Hedgehog acyltransferase (Hhat), a member of the membrane bound O-acyl transferase (MBOAT) family of multipass membrane proteins. The extremely hydrophobic composition of MBOAT proteins has limited their biochemical characterization. Except for mutagenesis of two conserved residues, there has been no structure-function analysis of Hhat, and the regions of the protein required for Shh palmitoylation are unknown. Here we undertake a systematic approach to identify residues within Hhat that are required for protein stability and/or enzymatic activity. We also identify a second, novel MBOAT homology region (residues 196–234) that is required for Hhat activity. In total, ten deletion mutants and eleven point mutants were generated and analyzed. Truncations at the N- and C-termini of Hhat yielded inactive proteins with reduced stability. Four Hhat mutants with deletions within predicted loop regions and five point mutants retained stability but lost palmitoylation activity. We purified two point mutants, W378A and H379A, with defective Hhat activity. Kinetic analyses revealed alterations in apparent Km and Vmax for Shh and/or palmitoyl CoA, changes that likely explain the catalytic defects observed for these mutants. This study has pinpointed specific regions and multiple residues that regulate Hhat stability and catalysis. Our findings should be applicable to other MBOAT proteins that mediate lipid modification of Wnt proteins and ghrelin, and should serve as a model for understanding how secreted morphogens are modified by palmitoyl acyltransferases.