Cholesterol binding at the cholesterol recognition/ interaction amino acid consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV TAT-CRAC peptide.

在外周型苯并二氮杂受体的胆固醇识别 / 相互作用氨基酸共有 （ CRAC ） 处结合胆固醇 ， 并通过 HIV TAT - CRAC 肽抑制类固醇生成。

• 影响指数: 12.779
• 发表时间: Jan 2001 30
• 来源期刊: Proc Natl Acad Sci U S A
• DOI: 10.1073/pnas.031461598
• PMID: 11158628
• 文章类型: Journal Article
• 中科院分区: Q1
• 方向: 综合性期刊
• 作者列表: Li H,Yao Z,Degenhardt B,Teper G,Papadopoulos V

Mesh: Amino Acid Sequence; Animals; Binding Sites; Cell Line; Cholesterol / metabolism; Cloning, Molecular; Consensus Sequence; Gene Products, tat / chemistry metabolism; HIV / metabolism; Kinetics; Leydig Cells / physiology; Male; Mice; Mitochondria / metabolism; Molecular Sequence Data; Progesterone / metabolism; Promegestone / pharmacokinetics; Receptors, GABA-A / chemistry metabolism; Recombinant Proteins / chemistry metabolism; Tritium; tat Gene Products, Human Immunodeficiency Virus

Abstract

We previously defined a cholesterol recognition/interaction amino acid consensus (CRAC; ATVLNYYVWRDNS) in the carboxyl terminus of the peripheral-type benzodiazepine receptor (PBR), an outer mitochondrial membrane protein involved in the regulation of cholesterol transport into the mitochondria, the rate-determining step in steroid biosynthesis. We examined (i) the PBR-cholesterol interaction by UV crosslinking of the C17 side-chain containing progestin, promegestone, and (ii) the role of the CRAC domain of PBR in Leydig cell steroidogenesis by using a transducible peptide composed of the TAT domain of HIV and the CRAC domain of PBR. [(3)H]Promegestone photoincorporated into recombinant PBR, and this labeling was displaced by cholesterol. [(3)H]Promegestone also photoincorporated into the TAT-CRAC peptide. [(3)H]Promegestone crosslinking to TAT-CRAC could be displaced by cholesterol and promegestone, with IC50 values of 1 and 200 microM, respectively. TAT-CRAC efficiently transduced into MA-10 Leydig cells and inhibited the hCG- and cAMP-stimulated steroid production in a dose-dependent manner. TAT-CRAC did not affect the hCG-induced cAMP synthesis and the 22R-hydroxycholesterol-supported steroidogenesis. Mutated TAT-CRAC lost its ability to bind [(3)H]promegestone and to inhibit the hCG-stimulated steroidogenesis. These results show that TAT-CRAC binds cholesterol and competes for cholesterol interaction with endogenous PBR, suggesting that the cytosolic carboxyl-terminal domain of PBR is responsible for taking up and bringing steroidogenic cholesterol into the mitochondria.

摘要

我们先前在外周型苯二氮卓受体（PBR）的羧基末端定义了胆固醇识别/相互作用氨基酸共识（CRAC;ATVLNYYVWRDNS），一种线粒体外膜蛋白，参与调节胆固醇转运到线粒体中，类固醇生物合成中的速率决定步骤。我们检查了（i）通过含孕激素的C17侧链的UV交联的PBR-胆固醇相互作用，普美孕酮,和(ii)通过使用由HIV的TAT结构域和PBR的CRAC结构域组成的转导肽,PBR的CRAC结构域在Leydig细胞类固醇生成中的作用。[（3）H]Promegestone光掺入重组PBR，这个标签被胆固醇取代了。[(3)H]Promegestone也光掺入到TAT-CRAC肽中。[（3）H]与TAT-CRAC交联的Promegestone可被胆固醇和promegestone取代，IC50值为1和200μM，分别。TAT-CRAC有效地转导到MA-10睾丸间质细胞中，并以剂量依赖性方式抑制hCG和cAMP刺激的类固醇产生。TAT-CRAC不影响hCG诱导的cAMP合成和22R-羟基胆固醇支持的类固醇生成。突变的TAT-CRAC失去了结合[（3）H]孕酮和抑制hCG刺激的类固醇生成的能力。这些结果表明TAT-CRAC结合胆固醇并与内源性PBR竞争胆固醇相互作用，这表明PBR的胞质羧基末端结构域负责摄取并将类固醇胆固醇带入线粒体。