ILEAL BILE ACID TRANSPORTER METABOLISM AND REGULATION

回肠顶端Na/胆汁酸共转运蛋白的鉴定和克隆 (ASBT)cDNA提供了有关其主要基因的新信息 结构，组织表达，调节，运输机制和作用 人类疾病。然而，人们对此知之甚少。 功能性ASBT，其质膜表达和周转的调节， 或胆汁酸跨细胞转运和外排。拟议的研究将 重点研究肠细胞胆汁酸摄取的分子机制。这个 这项工作的长远目标是了解其机制和调节 正常人群肠道胆汁酸转运与膳食脂肪代谢的关系 和疾病状态。将解决以下问题：(1)什么是 ASBT的一般结构和亚基化学计量？其中的步骤有哪些 ASBT的生物发生？与其一级结构不同的是，亚基 功能转运体复合体的化学计量和组装很差 明白了。初步研究表明，亚基内和可能的间隔区 亚基二硫键是ASBT折叠和 运输机复合体队形。在拟议的研究中，组装和亚单位 ASBT在转基因MDCK细胞和正常对照细胞中的化学计量比将被测定 大鼠胆管细胞。将确定相关联的亚基的身份 使用化学交联法、免疫共沉淀法和酵母菌的组合 双杂交筛选策略。(2)细胞质膜表达如何？ ASBT受到监管吗？ASBT的表达受底物、激素等调节 糖皮质激素和分泌素，以及cAMP。鉴于这一规定的一部分是 转录上，ASBT蛋白或血浆的翻译后调节 膜的活性似乎是一个重要的组成部分。在建议的 研究表明，ASBT的胞吐/内吞和降解的机制将是 下定决心。(3)肠道Oatp3在胆汁酸中的作用是什么？ 吸收？胆汁酸被主动的钠依赖转运吸收，并 被动的便利运输。推动者的候选运输者 转运蛋白Oatp3已被分离出来。Oatp3‘S的细胞表达及其作用 在肠道中胆汁酸的转运将被测定。

The identification and cloning of the ileal apical Na/bile acid cotransporter (ASBT) cDNA has provided new information regarding its primary structure, tissue expression, regulation, transport mechanism, and role in human disease. However, little is known about the assembly and structure of the functional ASBT, the regulation of its plasma membrane expression and turnover, or bile acid transcellular transport and efflux. The proposed research will focus on the molecular mechanism for enterocyte bile acid uptake. The long-range goal of this work is to understand the mechanism and regulation of intestinal bile acid transport as it relates to diet lipid metabolism in normal and disease states. The following questions will be addressed: (1) What are the general structure and subunit stoichiometry of the ASBT? What are the steps in the ASBT biogenesis? In contrast to its primary structure, the subunit stoichiometry and assembly of the function transporter complex are poorly understood. Preliminary studies suggest that intra-subunit and possibly inter subunit disulfide bonding are prerequisite steps for ASBT folding and transporter complex formation. In proposed studies, the assembly and subunit stoichiometry of the ASBT will be determined in transfect MDCK cells and normal rat cholangiocytes. The identity of associated subunits will be determined using combination of chemical cross-linking, coimmunoprecipitation, and a yeast two-hybrid screening strategy. (2) How is the plasma membrane expression of the ASBT regulated? ASBT expression is regulated by substrate, hormones such as glucocorticoids and secretin, and by cAMP. Whereas part of this regulation is transcriptionally, post-translational regulation of ASBT protein or plasma membrane activity appears to be an important component. In the proposed studies, the mechanism for ASBT exocytosis/endocytosis and degradation will be determined. (3) What is the role of the intestinal Oatp3 in bile acid absorption? Bile acids are absorbed by active sodium dependent transport and passive facilitative transport. A candidate transporter for the facilitative transporter Oatp3, has been isolated. The Oatp3's cellular expression and role in intestinal bile acid transport will be determined.