Gallegos Adalberto M, Atshaves Barbara P, Storey Stephen, Schoer Jonathan, Kier Ann B, Schroeder Friedhelm
Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 77843-4466, USA.
Chem Phys Lipids. 2002 Jun;116(1-2):19-38. doi: 10.1016/s0009-3084(02)00018-x.
Although the most exogenous lipids enter the cell via the LDL-receptor pathway, the mechanism(s) whereby lipids leave the lysosome for transport to intracellular sites are not clearly resolved. As shown herein, expression of sterol carrier protein-2 (SCP-2) in transfected L-cells altered lysosomal membrane lipid distribution, dynamics, and response to lipid transfer proteins. SCP-2 expression decreased the mass of cholesterol and lyso-bis-phosphatidic acid [LBPA], as well as the ratios of cholesterol/phospholipid and polyunsaturated/monounsaturated fatty acids esterified to lysosomal membrane phospholipids. Concomitantly, a fluorescent sterol transfer assay showed that SCP-2 expression decreased the initial rates of spontaneous and SCP-2-mediated sterol transfer 5.5- and 3.8-fold, respectively, from lysosomal membranes isolated from SCP-2 expressing cells as compared to controls. SCP-2, sphingomyelinase, low density lipoprotein, and high density lipoprotein directly enhanced the initial rates of sterol transfer from isolated lysosomal membranes by 50-, 12-, 4-, and 5-fold, respectively. In contrast, albumin and cholesterol esterase had no effect on lysosomal sterol transfer. Spontaneous sterol was very slow, t(1/2)>4 days, regardless of the source of the lysosomal membrane, while SCP-2 added in vitro induced formation of rapid and slowly transferable sterol pools in lysosomal membranes of control cells. In contrast, SCP-2 did not induce formation of a rapidly transferable sterol domain in lysosomal membranes isolated from SCP-2 expressing cells. These data suggest that SCP-2 expression selectively shifted the distribution of lipids (cholesterol, LBPA, esterified polyunsaturated fatty acids) away from lysosomal membranes. Furthermore, the cholesterol depleted lysosomal membrane isolated from SCP-2 expressing cells was resistant to additional direct action of SCP-2 to further enhance sterol transfer and induce rapidly transferable sterol pools in the lysosomal membrane.
尽管大多数外源性脂质通过低密度脂蛋白受体途径进入细胞，但脂质离开溶酶体并转运至细胞内位点的机制尚未完全明确。如本文所示，转染的L细胞中固醇载体蛋白2（SCP-2）的表达改变了溶酶体膜脂质分布、动力学以及对脂质转运蛋白的反应。SCP-2的表达降低了胆固醇和溶血双磷脂酸[LBPA]的含量，以及胆固醇/磷脂和与溶酶体膜磷脂酯化的多不饱和/单不饱和脂肪酸的比例。同时，荧光固醇转运试验表明，与对照相比，SCP-2的表达分别使从表达SCP-2的细胞中分离出的溶酶体膜的自发和SCP-2介导的固醇转运初始速率降低了5.5倍和3.8倍。SCP-2、鞘磷脂酶、低密度脂蛋白和高密度脂蛋白分别使从分离的溶酶体膜中固醇转运的初始速率直接提高了50倍、12倍、4倍和5倍。相比之下，白蛋白和胆固醇酯酶对溶酶体固醇转运没有影响。无论溶酶体膜的来源如何，自发的固醇转运都非常缓慢，t(1/2)>4天，而体外添加的SCP-2在对照细胞的溶酶体膜中诱导形成了快速和缓慢可转移的固醇池。相反, SCP-2并未在从表达SCP-2的细胞中分离出的溶酶体膜中诱导形成快速可转移的固醇结构域。这些数据表明，SCP-2的表达选择性地使脂质（胆固醇、LBPA、酯化多不饱和脂肪酸）的分布从溶酶体膜上转移。此外，从表达SCP-2的细胞中分离出的胆固醇耗尽的溶酶体膜对SCP-2的额外直接作用具有抗性，无法进一步增强固醇转运并在溶酶体膜中诱导形成快速可转移的固醇池。
