Functional analysis of FSP27 protein regions for lipid droplet localization, caspase-dependent apoptosis, and dimerization with CIDEA
Functional analysis of FSP27 protein regions for lipid droplet localization, caspase-dependent apoptosis, and dimerization with CIDEA
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DOI：
10.1152/ajpendo.00188.2009
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发表时间：
2009-12-01
影响因子：
5.1
通讯作者：
Smas, Cynthia M.
中科院分区：
文献类型：
--
作者：
Liu, Kun;Zhou, Shengli;Smas, Cynthia M.
刘 K、周 S、金 J、蒂利森 K、Majors D、Rearick D、Lee JH、费尔南德斯-博亚纳帕利 RF、巴里克洛 K、休斯顿 MS、Smas CM。使用 CIDEA 对 FSP27 蛋白区域进行脂滴定位、半胱天冬酶依赖性细胞凋亡和二聚化的功能分析。 Am J Physiol Endocrinol Metab 297：E1395-E1413，2009。首次发表于 2009 年 10 月 20 日； doi:10.1152/ajpendo.00188.2009.-脂肪细胞特异性蛋白 FSP27，也称为 CIDEC，是三种细胞死亡诱导 DFF45 样效应蛋白 (CIDE) 之一。 CIDE 的第一个已知功能是在哺乳动物细胞中异位表达时促进细胞凋亡。最近在内源环境中的研究证明了 CIDE 在能量代谢中的关键作用。 FSP27 是一种脂滴相关蛋白，其异源表达增强了扩大脂滴的形成，并且是体内白色脂肪细胞典型的单眼脂滴所必需的。在这里，我们描述了 FSP27 的凋亡功能和脂滴定位之间的关系。我们证明，FSP27 的异位表达会在多种人类细胞系中诱导脂滴增大，这表明其机制涉及普遍存在的细胞机制，而不是脂肪细胞特异性的细胞机制。此外，通过外源油酸培养促进 HeLa 细胞中脂滴的形成抵消了 FSP27 介导的细胞凋亡。通过瞬时共转染和对稳定表达 FSP27 的 HeLa 细胞中的脂滴进行分析，我们发现 FSP27 不能保护脂滴免受 ATGL 脂肪酶的作用。使用 eGFP-FSP27 缺失构建体进行的结构域作图表明，FSP27 的脂滴定位需要其 CIDE C 结构域的氨基酸 174-192。我们展示的 FSP27 细胞凋亡机制涉及 caspase-9 和线粒体细胞色素 c，也需要这个 19 个氨基酸区域。相互作用测定确定了 FSP27 CIDE C 结构域与 CIDEA 的复合物，蛋白质印迹显示 FSP27 蛋白水平因 CIDEA 的共表达而降低。总体而言，我们的研究结果证明了 FSP27 CIDE C 结构域和/或其区域对于细胞凋亡、脂滴定位和 CIDEA 相互作用的功能。
Liu K, Zhou S, Kim J, Tillison K, Majors D, Rearick D, Lee JH, Fernandez-Boyanapalli RF, Barricklow K, Houston MS, Smas CM. Functional analysis of FSP27 protein regions for lipid droplet localization, caspase-dependent apoptosis, and dimerization with CIDEA. Am J Physiol Endocrinol Metab 297: E1395-E1413, 2009. First published October 20, 2009; doi:10.1152/ajpendo.00188.2009.-The adipocyte-specific protein FSP27, also known as CIDEC, is one of three cell death-inducing DFF45-like effector (CIDE) proteins. The first known function for CIDEs was promotion of apoptosis upon ectopic expression in mammalian cells. Recent studies in endogenous settings demonstrated key roles for CIDEs in energy metabolism. FSP27 is a lipid droplet-associated protein whose heterologous expression enhances formation of enlarged lipid droplets and is required for unilocular lipid droplets typical of white adipocytes in vivo. Here, we delineate relationships between apoptotic function and lipid droplet localization of FSP27. We demonstrate that ectopic expression of FSP27 induces enlarged lipid droplets in multiple human cell lines, which is indicative that its mechanism involves ubiquitously present, rather than adipocyte-specific, cellular machinery. Furthermore, promotion of lipid droplet formation in HeLa cells via culture in exogenous oleic acid offsets FSP27-mediated apoptosis. Using transient cotransfections and analysis of lipid droplets in HeLa cells stably expressing FSP27, we show that FSP27 does not protect lipid droplets from action of ATGL lipase. Domain mapping with eGFP-FSP27 deletion constructs indicates that lipid droplet localization of FSP27 requires amino acids 174-192 of its CIDE C domain. The apoptotic mechanism of FSP27, which we show involves caspase-9 and mitochondrial cytochrome c, also requires this 19-amino acid region. Interaction assays determine the FSP27 CIDE C domain complexes with CIDEA, and Western blot reveals that FSP27 protein levels are reduced by coexpression of CIDEA. Overall, our findings demonstrate the function of the FSP27 CIDE C domain and/or regions thereof for apoptosis, lipid droplet localization, and CIDEA interaction.