Pandak William M, Ren Shunlin, Marques Dalila, Hall Elizabeth, Redford Kaye, Mallonee Darrell, Bohdan Patricia, Heuman Douglas, Gil Gregorio, Hylemon Phillip
Department of Medicine, Veterans Affairs Medical Center, and Virginia Commonwealth University, Richmond 23298-0711, USA.
J Biol Chem. 2002 Dec 13;277(50):48158-64. doi: 10.1074/jbc.M205244200. Epub 2002 Oct 3.
Bile acid synthesis occurs mainly via two pathways: the "classic" pathway, initiated by microsomal cholesterol 7alpha-hydroxylase (CYP7A1), and an "alternative" (acidic) pathway, initiated by sterol 27-hydroxylase (CYP27). CYP27 is located in the inner mitochondrial membrane, where cholesterol content is very low. We hypothesized that cholesterol transport into mitochondria may be rate-limiting for bile acid synthesis via the "alternative" pathway. Overexpression of the gene encoding steroidogenic acute regulatory (StAR) protein, a known mitochondrial cholesterol transport protein, led to a 5-fold increase in bile acid synthesis. An increase in StAR protein coincided with an increase in bile acid synthesis. CYP27 overexpression increased bile acid synthesis by <2-fold. The rates of bile acid synthesis following a combination of StAR plus CYP27 overexpression were similar to those obtained with StAR alone. TLC analysis of (14)C-labeled bile acids synthesized in cells overexpressing StAR showed a 5-fold increase in muricholic acid; in chloroform-extractable products, a dramatic increase was seen in bile acid biosynthesis intermediates (27- and 7,27-hydroxycholesterol). High-performance liquid chromatography analysis showed that 27-hydroxycholesterol accumulated in the mitochondria of StAR-overexpressing cells only. These findings suggest that cholesterol delivery to the inner mitochondrial membrane is the predominant rate-determining step for bile acid synthesis via the alternative pathway.
“经典”途径，由微粒体胆固醇7α-羟化酶（CYP7A1）启动；以及“替代”（酸性）途径，由甾醇27-羟化酶（CYP27）启动。CYP27位于线粒体内膜，而线粒体内膜的胆固醇含量非常低。我们推测，胆固醇向线粒体的转运可能是通过“替代”途径进行胆汁酸合成的限速步骤。编码类固醇生成急性调节（StAR）蛋白（一种已知的线粒体胆固醇转运蛋白）的基因过表达，导致胆汁酸合成增加了5倍。StAR蛋白的增加与胆汁酸合成的增加同时出现。CYP27过表达使胆汁酸合成增加不到2倍。StAR与CYP27共同过表达后的胆汁酸合成速率与单独使用StAR时相似。对过表达StAR的细胞中合成的（14）C标记胆汁酸进行薄层层析分析显示，鼠胆酸增加了5倍；在氯仿可提取物中，胆汁酸生物合成中间体（27-羟胆固醇和7,27-二羟胆固醇）显著增加。高效液相色谱分析表明，仅在过表达StAR的细胞线粒体中积累了27-羟胆固醇。这些发现表明，胆固醇向线粒体内膜的输送是通过替代途径进行胆汁酸合成的主要限速步骤。
