Rone Malena B, Midzak Andrew S, Issop Leeyah, Rammouz Georges, Jagannathan Sathvika, Fan Jinjiang, Ye Xiaoying, Blonder Josip, Veenstra Timothy, Papadopoulos Vassilios
The Research Institute of the McGill University Health Centre and Departments of Medicine, Biochemistry and Pharmacology & Therapeutics, McGill University, Montreal, Quebec H3G 1A4, Canada.
Mol Endocrinol. 2012 Nov;26(11):1868-82. doi: 10.1210/me.2012-1159. Epub 2012 Sep 12.
Steroid hormones are critical for organismal development and health. The rate-limiting step in steroidogenesis is the transport of cholesterol from the outer mitochondrial membrane (OMM) to the cytochrome P450 enzyme CYP11A1 in the inner mitochondrial membrane (IMM). Cholesterol transfer occurs through a complex termed the "transduceosome," in which cytosolic steroidogenic acute regulatory protein interacts with OMM proteins translocator protein and voltage-dependent anion channel (VDAC) to assist with the transfer of cholesterol to OMM. It has been proposed that cholesterol transfer from OMM to IMM occurs at specialized contact sites bridging the two membranes composed of VDAC and IMM adenine nucleotide translocase (ANT). Blue native PAGE of Leydig cell mitochondria identified two protein complexes that were able to bind cholesterol at 66- and 800-kDa. Immunoblot and mass spectrometry analyses revealed that the 800-kDa complex contained the OMM translocator protein (18-kDa) and VDAC along with IMM CYP11A1, ATPase family AAA domain-containing protein 3A (ATAD3A), and optic atrophy type 1 proteins, but not ANT. Knockdown of ATAD3A, but not ANT or optic atrophy type 1, in Leydig cells resulted in a significant decrease in hormone-induced, but not 22R-hydroxycholesterol-supported, steroid production. Using a 22-phenoxazonoxy-5-cholene-3-beta-ol CYP11A1-specific probe, we further demonstrated that the 800-kDa complex offers the microenvironment needed for CYP11A1 activity. Addition of steroidogenic acute regulatory protein to the complex mobilized the cholesterol bound at the 800-kDa complex, leading to increased steroid formation. These results identify a bioactive, multimeric protein complex spanning the OMM and IMM unit that is responsible for the hormone-induced import, segregation, targeting, and metabolism of cholesterol.
类固醇激素对机体发育和健康至关重要。类固醇生成的限速步骤是胆固醇从线粒体外膜（OMM）转运至线粒体内膜（IMM）中的细胞色素P450酶CYP11A1。胆固醇转运通过一种名为“转导体”的复合物进行，其中胞质类固醇生成急性调节蛋白与OMM蛋白转位蛋白和电压依赖性阴离子通道（VDAC）相互作用，以协助胆固醇向OMM的转运。有人提出，胆固醇从OMM向IMM的转运发生在由VDAC和IMM腺嘌呤核苷酸转位酶（ANT）构成的连接两个膜的特殊接触位点。对睾丸间质细胞线粒体进行蓝色非变性聚丙烯酰胺凝胶电泳，鉴定出两种能够结合66 kDa和800 kDa胆固醇的蛋白质复合物。免疫印迹和质谱分析显示，800 kDa复合物包含OMM转位蛋白（18 kDa）、VDAC以及IMM CYP11A1、含ATP酶家族AAA结构域蛋白3A（ATAD3A）和视神经萎缩1型蛋白，但不包含ANT。在睾丸间质细胞中敲低ATAD3A而非ANT或视神经萎缩1型蛋白，导致激素诱导的而非22R-羟基胆固醇支持的类固醇生成显著减少。使用22-苯恶唑氧基-5-胆烯-3-β-醇CYP11A1特异性探针，我们进一步证明800 kDa复合物提供了CYP11A1活性所需的微环境。向复合物中添加类固醇生成急性调节蛋白可动员结合在800 kDa复合物上的胆固醇，导致类固醇形成增加。这些结果确定了一种跨越OMM和IMM单元的生物活性多聚体蛋白复合物，其负责激素诱导的胆固醇导入、分离、靶向和代谢。
