Eichmann Thomas O, Grumet Lukas, Taschler Ulrike, Hartler Jürgen, Heier Christoph, Woblistin Aaron, Pajed Laura, Kollroser Manfred, Rechberger Gerald, Thallinger Gerhard G, Zechner Rudolf, Haemmerle Günter, Zimmermann Robert, Lass Achim
Institute of Molecular Biosciences, University of Graz, Graz, Austria.
Bioinformatics, Institute for Knowledge Discovery, Graz University of Technology, Graz, Austria.
J Lipid Res. 2015 Oct;56(10):1972-84. doi: 10.1194/jlr.M062372. Epub 2015 Aug 31.
Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/β-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs.
肝星状细胞（HSCs）中的脂滴（LDs）含有大量维生素A[以视黄酯（REs）的形式]以及其他中性脂质，如甘油三酯（TGs）。在维生素A供应不足时，RE储备会被调动起来，以确保身体的持续供应。迄今为止，对于负责水解HSCs中中性脂质酯，特别是REs的酶，人们了解甚少。在本研究中，我们旨在通过蛋白质组学方法，利用大鼠星状细胞系HSC-T6来鉴定与LD相关的中性脂质水解酶。首先，我们用视黄醇和脂肪酸处理细胞，以促进脂质合成和在LDs内的沉积。然后，分离出LDs并分析脂质组成和LD蛋白质组。在其他蛋白质中，我们发现了脂滴包被蛋白2、脂肪甘油三酯脂肪酶（ATGL）和比较基因识别-58（CGI-58），这些都是已知的和已确定的LD蛋白。对LD蛋白质组进行生物信息学搜索，寻找含α/β水解酶折叠结构的蛋白质，未发现尚未表征的中性脂质水解酶。在体外活性测定中，我们发现大鼠（r）ATGL在大鼠（r）CGI-58的共同激活下，能有效水解TGs和REs。这些发现表明，rATGL和rCGI-58是HSCs中驻留在LDs的蛋白质，并参与REs和TGs的动员。
