Zinc Regulation & Toxicity in Cellular Energy Metabolism

描述（由申请人提供）：最近，越来越多的报道试图将细胞内游离Zn++的变化与病理过程联系起来，特别是在神经系统中。 细胞内Zn++增加与线粒体功能障碍、线粒体防御丧失、ROS产生增加和细胞死亡相关。 我们已经证明：1）升高的Zn++干扰线粒体能量利用（呼吸），部分原因是抑制了在碳水化合物代谢中起作用的酶，特别是α-酮戊二酸脱氢酶。 2)Zn++降低线粒体膜电位并诱导线粒体渗透孔开放，从而释放Ca 2+并抑制ATP生成。 3)Zn++还抑制吡啶核苷酸依赖性巯基还原酶的活性，这对维持线粒体氧化还原防御至关重要。 4)Zn++激活线粒体酶硫辛酰胺脱氢酶的NADH氧化酶活性。 这加速了超氧化物（O2）和过氧化氢（H2 O2）的产生。 5)Zn++诱导线粒体细胞色素c的释放，细胞色素c是细胞凋亡的胞质传播者。 我们的假设是：线粒体和金属硫蛋白形成一个生理反馈回路，利用Zn 2+作为信号来调节ATP（ROS）和还原巯基之间的平衡。 升高的细胞内Zn++（被转运到线粒体中并且）通过抑制参与碳水化合物代谢和电子转运的酶直接降低细胞能量利用，并且通过干扰线粒体氧化还原平衡间接降低细胞能量利用。 细胞溶质Zn 2+的过量（病理）水平降低线粒体保护并使线粒体完整性（膜渗透性）不稳定。 Zn++还诱导线粒体释放ROS和细胞色素c。 具体而言，我们将：1）表征由Zn++刺激的LADH的NADH-脱氢酶活性的反应产物的光谱。 2)表征Zn++在酶和线粒体水平上对巯基氧化还原活性的抑制作用。 3)建立大鼠肝线粒体中外源性添加Zn++后线粒体事件的顺序。 4)目的2和3中在肝线粒体中发现和表征的氧化还原平衡和其他Zn++诱导的变化将在大鼠脑线粒体中得到证实。

DESCRIPTION (provided by applicant): Recently a growing number of reports have attempted to link changes intracellular free Zn++ to pathological processes, particularly in the nervous system. Increased intracellular Zn++ is correlated with mitochondria dysfunction, loss of mitochondria defenses, increased production of ROS and cellular death. We have demonstrating that 1) Elevated Zn++ interferes with mitochondria energy utilization (respiration), due in part to inhibition of enzymes that play a role in carbohydrate metabolism, in particular alpha-ketoglutarate dehydrogenase. 2) Zn++ lowers mitochondria membrane potential and induces the opening of the mitochondria permeability pore, which releases Ca2+ and inhibits ATP generation. 3) Zn++ also inhibits the activity of pyridine nucleotide-dependent thiol reductases, which are critical to maintenance of mitochondria redox defenses. 4) Zn++ activates the NADH-oxidase activity of the mitochondria enzyme lipoamide dehydrogenase. This accelerates generation of superoxide (O2) and hydrogen peroxide (H2O2). 5) Zn++ induces release of mitochondria cytochrome c, which is a cytosolic propagator of apoptosis. Our hypotheses are that: Mitochondria and metallothioneins form a physiological feedback loop that utilizes Zn2+ as signal to regulate the balance of production between ATP, (ROS) and reduced thiols. Elevated intracellular Zn++ (is transported into mitochondria and) reduces cellular energy utilization directly by inhibiting enzymes involved in carbohydrate metabolism and electron transport and indirectly by interfering with mitochondrial redox balance. Excess (pathological) levels of cytosolic Zn2+ reduce mitochondria protection and destabilize mitochondria integrity (membrane permeability). Zn++ also induces mitochondria release of ROS and cytochrome c. Specifically, we will: 1) Characterize the spectrum of reactive products of the NADH-dehydrogenase activity of LADH that are stimulated by Zn++. 2) Characterize the inhibitory effect of Zn++ on thiol-redox activity at both the enzyme and mitochondrial level. 3) Establish the order of mitochondria events following exogenous addition of Zn++ in rat liver mitochondria. 4) Redox balance and other Zn++ -induced changes discovered and characterized in liver mitochondria in Aims 2 and 3 will be confirmed in rat brain mitochondria.