Developmental Pharmacology of Mitochondrial and Cytosolic GSTZ1

项目摘要。 这项研究的长期目标是了解谷胱甘肽转移酶Z1的性质 (GSTZ1)及其在人体内消除研究药物二氯乙酸(DCA)中的作用 寿命。这一点很重要，因为虽然线粒体丙酮酸脱氢酶激酶的抑制剂DCA， 作为代谢调节剂，在治疗乳酸中毒、某些实体肿瘤和肺部疾病方面有疗效。 动脉高血压，在药代动力学中表现出显著的年龄和遗传相关的个体变异性 与DCA的副作用有关。DCA代谢的第一步是脱氯为乙醛， 由GSTZ1催化。GSTZ1又称顺丁烯二酰乙酰乙酸异构酶，在人体内起着重要的生理作用 酪氨酸分解代谢途径内源性反应性代谢物马来酰乙酰乙酸酯的异构化作用 和顺丁烯二酸丙酮。GSTZ1主要在肝脏中表达，但也在肾脏、肠、脑和心脏中表达。它是 已知的唯一催化DCA脱卤化为其主要代谢物乙醛的酶。虽然 众所周知，DCA通过以下途径抑制自身代谢和内源底物的代谢 GSTZ1失活后DCA药代动力学个体差异显著的原因 人们对重复剂量的了解还只有一部分。大多数成年人清除重复剂量的DCA的速度比 在儿童和成人中，Clearance是GSTZ1单倍型依赖的。有初步证据表明 DCA术后DCA代谢的肝外部位比肝脏更重要 治疗，这是年龄相关的。最后，氯离子浓度影响酶的失活速度。 GSTZ1通过DCA以单倍型依赖的方式；线粒体中的[Cl-]远低于胞浆中的[Cl-] 被认为是线粒体基质酶更快失活的一个因素，可能是一种 在观察到的年龄相关差异中的因素，因为细胞内[Cl-]随着年龄的变化而变化。如何实现的机制 CL-保护GSTZ1免受DCA依赖的失活尚不清楚。 此应用程序旨在检查与年龄和遗传相关的表达和活性变化的原因 GSTZ1基因在人中的表达。提出了三个具体目标。第一个具体目标是研究microrna的作用。 (MIR)调节已证实的与年龄相关的肝脏GSTZ1表达的变化。这一目标将使用 储存不同年龄捐赠者的人类肝脏以识别MIR，并使用基于细胞的系统来验证参与情况。 第二个具体目的是研究DCA对GSTZ1失活的保护机制。 氯离子和某些其他阴离子的存在，以及单倍型的影响。表达的重组GSTZ1a和 1B将被结晶，并研究了DCA和阴离子的结合位置。第三个具体目标将使用青少年 和成年大鼠作为儿童和成年大鼠的模型，考察肝外组织相对于肝脏在 GSTZ1在不同剂量DCA作用下的表达和活性，以模拟临床应用。

Project Summary. The broad, long-term objectives of this research are to understand the properties of glutathione transferase Z1 (GSTZ1) and its role in the elimination of the investigational drug dichloroacetic acid (DCA) across the human lifespan. This is important because while DCA, an inhibitor of mitochondrial pyruvate dehydrogenase kinase, has therapeutic benefits as a metabolic regulator in treating lactic acidosis, certain solid tumors and pulmonary arterial hypertension, it exhibits marked age- and genetics- related individual variability in pharmacokinetics that are linked to DCA's side effects. The first step in DCA metabolism is dechlorination to glyoxylate, catalyzed by GSTZ1. Also known as maleylacetoacetate isomerase, GSTZ1 plays an important physiological role in isomerizing endogenous reactive metabolites of the tyrosine catabolism pathway, maleylacetoacetate and maleylacetone. GSTZ1 is expressed chiefly in the liver, but also in kidney, intestine, brain and heart. It is the only enzyme known to catalyze the dehalogenation of DCA to its primary metabolite, glyoxylate. Although it is well recognized that DCA inhibits its own metabolism as well as that of its endogenous substrates, through inactivation of GSTZ1, the reasons for the marked individual variability in pharmacokinetics of DCA after repeated doses are only partially understood. Most adults clear repeated doses of DCA more slowly than children, and clearance is GSTZ1 haplotype dependent in children and adults. There is preliminary evidence that extrahepatic sites of DCA metabolism become of greater importance relative to liver following DCA treatment, and that this is age-dependent. Finally, chloride (Cl-) concentration affects the rate of inactivation of GSTZ1 by DCA in a haplotype-dependent manner; the much lower [Cl-] in mitochondria compared with cytosol is thought to be a factor in the more rapid inactivation of the mitochondrial matrix enzyme, and could be a factor in the observed age-dependent differences, as intracellular [Cl-] varies with age. The mechanism of how Cl- protects GSTZ1 from DCA-dependent inactivation is not known. This application seeks to examine reasons for age- and genetics- related changes in expression and activity of GSTZ1 in people. Three specific aims are proposed. The first specific aim will study the role of microRNA (miR) in regulating the documented age-related changes in hepatic GSTZ1 expression. This aim will use banked human liver of different aged donors to identify miRs, and cell-based systems to verify involvement. The second specific aim will investigate the mechanism of protection of GSTZ1 from inactivation by DCA in the presence of Cl- and certain other anions, and the influence of haplotype. Expressed recombinant GSTZ1A and 1B will be crystallized and the binding sites of DCA and anions studied. The third specific aim will use juvenile and adult rats as models of children and adults to examine the roles of extrahepatic tissues relative to liver in the expression and activity of GSTZ1 following multiple DCA doses, to mimic the clinical use.