Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes
Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes
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DOI：
10.1074/jbc.m407462200
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发表时间：
2004-10-01
影响因子：
4.8
通讯作者：
Brasaemle, DL
中科院分区：
文献类型：
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作者：
Subramanian, V;Rothenberg, A;Brasaemle, DL
周脂蛋白是覆盖脂肪细胞成熟脂滴表面的主要结构蛋白，在调节三酰甘油储存和水解方面发挥着重要作用。我们已经使用蛋白质组学分析，以确定CGI-58，α/β-水解酶折叠酶家族的成员，作为3 T3-L1脂肪细胞的脂滴的组成部分。CGI-58 mRNA在脂肪组织和睾丸（也表达周脂蛋白的组织）中高度表达，并且在肝脏、皮肤、肾脏和心脏中以较低水平表达。内源性CGI-58和异位CGI-58-GFP嵌合体在3 T3-L1前脂肪细胞中均表现出弥漫性细胞质定位，但在分化的3 T3-L1脂肪细胞中几乎仅定位于脂滴表面。使用显微镜在稳定表达突变形式的周脂蛋白的3 T3-L1细胞中研究内源性CGI-58的定位。CGI-58结合包被有周脂蛋白A或具有从氨基酸382至429的完整C-末端序列的周脂蛋白的突变形式的脂滴，但不结合包被有周脂蛋白B或缺乏该序列的突变的周脂蛋白A的脂滴。免疫沉淀研究证实了这些发现，但也显示周脂蛋白B和CGI-58的共沉淀。值得注意的是，通过用异丙肾上腺素和异丁基甲基黄嘌呤孵育3 T3-L1脂肪细胞来激活cAMP依赖性蛋白激酶，将CGI-58从脂滴表面分散到细胞质分布中。这种亚细胞定位的转变可以通过向培养基中加入心得安来逆转。因此，CGI-58以依赖于脂肪细胞的代谢状态和cAMP依赖性蛋白激酶的活性的方式与包被有周脂蛋白A的脂滴结合。
Perilipins, the major structural proteins coating the surfaces of mature lipid droplets of adipocytes, play an important role in the regulation of triacylglycerol storage and hydrolysis. We have used proteomic analysis to identify CGI-58, a member of the alpha/beta-hydrolase fold family of enzymes, as a component of lipid droplets of 3T3-L1 adipocytes. CGI-58 mRNA is highly expressed in adipose tissue and testes, tissues that also express perilipins, and at lower levels in liver, skin, kidney, and heart. Both endogenous CGI-58 and an ectopic CGI-58-GFP chimera show diffuse cytoplasmic localization in 3T3-L1 preadipocytes, but localize almost exclusively to the surfaces of lipid droplets in differentiated 3T3-L1 adipocytes. The localization of endogenous CGI-58 was investigated in 3T3-L1 cells stably expressing mutated forms of perilipin using microscopy. CGI-58 binds to lipid droplets coated with perilipin A or mutated forms of perilipin with an intact C-terminal sequence from amino acid 382 to 429, but not to lipid droplets coated with perilipin B or mutated perilipin A lacking this sequence. Immunoprecipitation studies confirmed these findings, but also showed co-precipitation of perilipin B and CGI-58. Remarkably, activation of cAMP-dependent protein kinase by the incubation of 3T3-L1 adipocytes with isoproterenol and isobutylmethylxanthine disperses CGI-58 from the surfaces of lipid droplets to a cytoplasmic distribution. This shift in subcellular localization can be reversed by the addition of propanolol to the culture medium. Thus, CGI-58 binds to perilipin A-coated lipid droplets in a manner that is dependent upon the metabolic status of the adipocyte and the activity of cAMP-dependent protein kinase.