Identification and Characterization of an Extramitochondrial Human 3-Hydroxy-3-methylglutaryl-CoA Lyase
Identification and Characterization of an Extramitochondrial Human 3-Hydroxy-3-methylglutaryl-CoA Lyase
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DOI：
10.1074/jbc.m112.393231
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发表时间：
2012-09-28
影响因子：
4.8
通讯作者：
Miziorko, Henry M.
中科院分区：
文献类型：
--
作者：
Montgomery, Christa;Pei, Zhengtong;Miziorko, Henry M.
3-羟基-3-甲基戊二酰-CoA 裂解酶样蛋白 (HMGCLL1) 已在哺乳动物基因组保藏中注释为先前未鉴定的人类 HMG-CoA 裂解酶 (HMGCL)。为了测试该注释的有效性并评估该蛋白质的生理作用，构建了用于在大肠杆菌和巴斯德毕赤酵母中表达蛋白质的质粒。大肠杆菌中的蛋白质表达产生不溶性物质。相反，活性 HMGCLL1 在毕赤酵母中表达后可以恢复。针对蛋白质 N 末端发现的独特肽序列制备了抗体。在免疫检测实验中，抗体可区分 HMGCLL1 和线粒体 HMGCL。纯化的酶经过表征并证明能够将 HMG-CoA 裂解为乙酰乙酸和乙酰-CoA，其催化和亲和特性与人线粒体 HMGCL 相当。推导的 HMGCLL1 序列包含 N 端肉豆蔻酰化基序；通过构建 G2A HMGCLL1 消除了假定的修饰位点。尝试使用人 N-肉豆蔻酰转移酶和 [H-3] 肉豆蔻酰辅酶 A 修饰这两种蛋白质。野生型蛋白被明显修饰，而G2A蛋白没有被标记。 HMGCLL1 的肉豆蔻酰化影响其细胞定位。将适当的表达质粒转染至 COS1 细胞后，免疫荧光检测表明 G2A HMGCLL1 表现出弥散模式，表明其位于胞质中。相反，野生型 HMGCLL1 表现出点状以及核周免疫染色模式，表明肉豆蔻酰化依赖于非线粒体膜区室的关联。在使用 HMGCL 表达质粒的对照实验中，正如预期的那样，蛋白质定位于线粒体中。 COS1 细胞表达以及 U87 细胞内源性表达的可用结果表明 HMGCLL1 是一种线粒体外羟甲基戊二酰辅酶 A 裂解酶。
3-Hydroxy-3-methylglutaryl-CoA lyase-like protein (HMGCLL1) has been annotated in the Mammalian Genome Collection as a previously unidentified human HMG-CoA lyase (HMGCL). To test the validity of this annotation and evaluate the physiological role of the protein, plasmids were constructed for protein expression in Escherichia coli and Pichia pastoris. Protein expression in E. coli produced insoluble material. In contrast, active HMGCLL1 could be recovered upon expression in P. pastoris. Antibodies were prepared against a unique peptide sequence found in the N terminus of the protein. In immunodetection experiments, the antibodies discriminated between HMGCLL1 and mitochondrial HMGCL. Purified enzyme was characterized and demonstrated to cleave HMG-CoA to acetoacetate and acetyl-CoA with catalytic and affinity properties comparable with human mitochondrial HMGCL. The deduced HMGCLL1 sequence contains an N-terminal myristoylation motif; the putative modification site was eliminated by construction of a G2A HMGCLL1. Modification of both proteins was attempted using human N-myristoyltransferase and [H-3] myristoyl-CoA. Wild-type protein was clearly modified, whereas G2A protein was not labeled. Myristoylation of HMGCLL1 affects its cellular localization. Upon transfection of appropriate expression plasmids into COS1 cells, immunofluorescence detection indicates that G2A HMGCLL1 exhibits a diffuse pattern, suggesting a cytosolic location. In contrast, wildtype HMGCLL1 exhibits a punctate as well as a perinuclear immunostaining pattern, indicating myristoylation dependent association with nonmitochondrial membrane compartments. In control experiments with the HMGCL expression plasmid, protein is localized in the mitochondria, as anticipated. The available results for COS1 cell expression, as well as endogenous expression in U87 cells, indicate that HMGCLL1 is an extramitochondrial hydroxymethylglutaryl-CoA lyase.