Novel lipid droplet-associated serine hydrolase regulates macrophage cholesterol mobilization.
Novel lipid droplet-associated serine hydrolase regulates macrophage cholesterol mobilization.
复制标题
DOI：
10.1161/atvbaha.113.302448
复制
发表时间：
2014-02
期刊：
影响因子：
--
通讯作者：
Paul A
中科院分区：
文献类型：
--
作者：
Goo YH;Son SH;Kreienberg PB;Paul A
载脂巨噬细胞或泡沫细胞的特征是大量胞质脂滴（LD）沉积，主要含有来自动脉壁清除的脂蛋白的胆固醇酯（CE）。从泡沫细胞流出的胆固醇被认为具有动脉粥样硬化保护作用。由于胆固醇以游离胆固醇（FC）的形式外排，LD中的CE蓄积可能会限制FC外排。我们的目标是确定蛋白质调节胆固醇运输通过LD。在RAW 264.7巨噬细胞LD组分的蛋白质组学分析中，我们鉴定了一种进化上保守的蛋白质，其具有典型的GXSXG脂肪酶催化基序和预测的α/β-水解酶折叠，RIKEN cDNA 1110057 K 04基因，我们将其命名为脂滴相关水解酶（LDAH）。通过免疫印迹和免疫细胞化学证实LDAH与LD的关联。LDAH用活性丝氨酸水解酶特异性探针标记。LDAH显示出相对较弱的体外CE水解酶活性。然而，完整细胞中的胆固醇测量支持LDAH在CE稳态中的重要作用，因为LDAH上调和下调分别降低和增加HEK 293细胞和RAW 264.7巨噬细胞中的细胞内胆固醇和CE。推定的亲核丝氨酸突变损害活性水解酶探针结合，在体外CE水解酶活性，和细胞中的胆固醇降低作用，而这种突变体仍然定位于LD。LDAH上调增加CE水解和巨噬细胞的胆固醇流出，有趣的是，LDAH在小鼠和人动脉粥样硬化病变内的巨噬细胞丰富区域中高度表达。这些数据确定了促进动脉粥样硬化病变胆固醇逆向转运的候选靶点。
Lipid-laden macrophages or foam cells are characterized by massive cytosolic lipid droplet (LD) deposition containing mostly cholesterol ester (CE) derived from the lipoproteins cleared from the arterial wall. Cholesterol efflux from foam cells is considered to be atheroprotective. Since cholesterol is effluxed as free cholesterol (FC), CE accumulation in LDs may limit FC efflux. Our objective was to identify proteins that regulate cholesterol trafficking through LDs. In a proteomic analysis of the LD fraction of RAW 264.7 macrophages we identified an evolutionarily conserved protein with a canonical GXSXG lipase catalytic motif and a predicted α/β-hydrolase fold, the RIKEN cDNA 1110057K04 gene, which we named lipid droplet-associated hydrolase (LDAH). LDAH association to LDs was confirmed by immunoblotting and immunocytochemistry. LDAH was labeled with a probe specific for active serine hydrolases. LDAH showed relatively weak in vitro CE hydrolase activity. However, cholesterol measurements in intact cells supported a significant role of LDAH in CE homeostasis, since LDAH upregulation and downregulation decreased and increased, respectively, intracellular cholesterol and CE in HEK293 cells and RAW 264.7 macrophages. Mutation of the putative nucleophilic serine impaired active hydrolase probe binding, in vitro CE hydrolase activity, and the cholesterol lowering effect in cells, while this mutant still localized to the LD. LDAH upregulation increased CE hydrolysis and cholesterol efflux from macrophages and, interestingly, LDAH is highly expressed in macrophage-rich areas within mouse and human atherosclerotic lesions. The data identify a candidate target to promote reverse cholesterol transport from atherosclerotic lesions.