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.
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.
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DOI：
10.1073/pnas.98.3.1267
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发表时间：
2001-01
影响因子：
11.1
通讯作者：
Hua Li;Z. Yao;Babett Degenhardt;G. Teper;V. Papadopoulos
中科院分区：
文献类型：
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作者：
Hua Li;Z. Yao;Babett Degenhardt;G. Teper;V. Papadopoulos
我们之前在外周型苯二氮卓受体（PBR）的羧基端定义了胆固醇识别/相互作用氨基酸共识（CRAC； ATVLNYYVWRDNS）， PBR是一种线粒体外膜蛋白，参与胆固醇转运到线粒体的调节，这是类固醇生物合成的速率决定步骤。我们通过紫外线交联含有黄体酮、孕酮的C17侧链，研究了PBR与胆固醇的相互作用，以及（ii）利用由HIV的TAT结构域和PBR的CRAC结构域组成的可转导肽，研究了PBR的CRAC结构域在间质细胞甾体生成中的作用。[(3)H]将promegstone光掺入重组PBR中，并被胆固醇取代。[(3)H] promegstone也光结合到TAT-CRAC肽中。[3]与TAT-CRAC交联的黄体酮可被胆固醇和黄体酮置换，IC50值分别为1和200微米。TAT-CRAC有效地转导到MA-10间质细胞，并以剂量依赖的方式抑制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]孕激素和抑制hcg刺激的甾体生成的能力。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甾体源性胆固醇带入线粒体。
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.