Functions of the Hepatic Gamma-Glutamyl Cycle

描述（由申请人提供）：还原性谷胱甘肽（GSH）在许多生化过程中起着关键作用，其体内平衡的紊乱与许多疾病的病因和进展有关。在所有哺乳动物细胞中，这种三肽转换的第一步是它穿过质膜进入细胞外空间；然而，介导谷胱甘肽外排的运输系统仍然不明确。肝脏是谷胱甘肽合成的主要部位，谷胱甘肽以高速率释放到血浆和胆汁中。谷胱甘肽转运到胆汁中作为胆汁分泌的驱动力，在药物、金属和其他内源性和外源性活性化合物的肝脏解毒中起重要作用。谷胱甘肽也通过窦状膜释放到血浆中，以输送到其他组织。我们最近的研究已经确定了运输蛋白Oatp和Mrp家族的一些成员在GSH从细胞输出中的关键作用。拟议研究的总体目标是表征谷胱甘肽运输的机制，并深入了解这些运输过程的生理意义。我们的具体目标是：(1)建立大鼠非洲蟾卵母细胞中表达的Oatp1对GSH转运的动力学和特异性，并检测人类主要的正弦型有机溶质转运蛋白OATP-C是否也介导GSH/有机溶质交换。(2)表征大鼠Mrp2转运谷胱甘肽的机制，并将其与Mrp2的酵母同源物Ycf1p转运谷胱甘肽的机制进行比较，验证巯基酸（n -乙酰半胱氨酸s缀合物）是Mrp2底物的假设。(3)检测凋亡细胞中观察到的谷胱甘肽外排是否由Oatp或Mrp蛋白介导，还是由其他谷胱甘肽输出机制介导；(4)通过克隆酿酒葡萄球菌的表达，鉴定出更多的GSH双向转运体。由于谷胱甘肽跨细胞膜运输是其稳态的关键步骤，并且与其生物学功能密切相关，因此拟议的研究应该为预防和治疗由氧化剂和反应性亲电化学物质引起的细胞和组织损伤提供关键信息。

DESCRIPTION (provided by applicant): Reduced glutathione (GSH) plays a critical role in a multitude of biochemical processes, and disturbances in its homeostasis are implicated in the etiology and progression of a number of diseases. The initial step in the turnover of this tripeptide in all mammalian cells is its transport across the plasma membrane into the extracellular space; however, the transport systems that mediate GSH efflux remain poorly defined. In the liver, a major site of GSH synthesis, GSH is released at high rates into both blood plasma and bile. GSH transport into bile functions as a driving force for bile secretion and plays an important role in hepatic detoxification of drugs, metals, and other reactive compounds of both endogenous and exogenous origin. GSH is also released across the sinusoidal membrane into blood plasma for delivery to other tissues. Our recent studies have identified a key role for some members of the Oatp and Mrp families of transport proteins in GSH export from cells. The overall goals of the proposed studies are to characterize the mechanisms of GSH transport, and to gain insight into the physiological significance of these transport processes. Our specific aims are: (1) Establish the kinetics and specificity of GSH transport on rat Oatp1 expressed in Xenopus laevis oocytes, and test whether human OATP-C, a major human sinusoidal organic solute transporter, also mediates GSH/organic solute exchange. (2) Characterize the mechanism of GSH transport by rat Mrp2, compare this mechanism with that for GSH transport by Ycf1p, the yeast orthologue of Mrp2, and test the hypothesis that mercapturic acids (N-acetylcysteine S-conjugates) are substrates for Mrp2. (3) Test whether the GSH efflux that is observed in cells undergoing apoptosis is mediated by the Oatp or Mrp proteins, or by other GSH export mechanisms; and (4) Identify additional bi-directional GSH transporters by expression cloning in S. cerevisiae. Because GSH transport across cell membranes is a key step in its homeostasis and is intimately linked to its biological functions, the proposed studies should provide information critical for the prevention and treatment of cell and tissue injury produced by oxidants and reactive electrophilic chemicals.