REGULATION OF PYRUVATE DEHYDROGENASE PHOSPHATASE

丙酮酸脱氢酶(PDH)复合体催化丙酮酸的不可逆脱羧化反应。在该反应中产生的乙酰辅酶A的命运取决于特定组织的代谢环境。一般来说，对能量有很高需求的氧气充足的组织(脑、心脏、骨骼肌)使用乙酰辅酶A作为克雷布斯循环的燃料。相比之下，脂肪生成组织(脂肪、肝脏、乳腺)主要使用乙酰辅酶A作为脂肪生成的前体，即将多余的碳水化合物转化为甘油三酯。因此，PDH活性的调节必须足够复杂，以满足酶的分解代谢和合成代谢功能。这一建议的主要工作假设是，这一任务是通过最近发现的丙酮酸脱氢酶磷酸酶(PDH磷酸酶)的组织特异性表达来完成的。PDH磷酸酶是一种专一的线粒体蛋白磷酸酶，它能使失活的磷酸二氢酶去磷酸化并重新激活。磷酸酶被认为介导了各种营养和激素刺激对PDH活性的影响。因此，我们认为分解代谢和合成代谢(造脂)组织表达不同的磷酸酶同工酶，进而对营养和激素刺激做出不同的反应，从而定义PDH复合体的组织特异性反应。这项提案中概述的研究计划旨在检验这一假设。为此，我们计划研究PDH磷酸酶同工酶的酶学和调控特性。我们还建议进一步研究同工酶的组织分布。最后，我们将评估饥饿和糖尿病对两种同工酶表达模式的影响，这些情况已知与PDH磷酸酶活性下调有关。这项建议的具体目的是：(1)确定丙酮酸脱氢酶磷酸酶催化的分子基础和严格的底物特异性；(2)进一步表征负责调节磷酸酶同工酶催化活性的分子因素；(3)评估每种同工酶对丙酮酸脱氢酶复合体活性的组织特异性调节的贡献；以及(4)阐明在饥饿和糖尿病中观察到的磷酸酶活性稳定变化的分子机制。这些目标的实现将为PDH反应在调节一般碳水化合物和脂肪代谢中的作用提供新的线索。从长远来看，它可能会导致高度特异的药物的开发，这些药物将减少与糖尿病、缺血和脓毒症等疾病相关的并发症。

Pyruvate dehydrogenase (PDH) complex catalyzes the irreversible decarboxylation of pyruvic acid. The fate of acetyl-CoA produced in this reaction depends upon the metabolic context of a particular tissue. In general, well-oxygenated tissues with high demand for energy (brain, heart, skeletal muscle) use acetyl-CoA as a fuel for the Krebs cycle. In contrast, the lipogenic tissues (fat, liver, mammary gland) primarily use acetyl-CoA as a precursor for lipogenesis, i.e. to convert excess carbohydrate into triglycerides. Therefore, the regulation of PDH activity must be sophisticated enough to fulfill both catabolic and anabolic functions of the enzyme. The major working hypothesis of this proposal is that this task is accomplished through tissue-specific expression of recently discovered isozymes of pyruvate dehydrogenase phosphatase (PDH phosphatase). PDH phosphatase is a dedicated mitochondrial protein phosphatase that dephosphorylates and re-activates catalytically inactive phospho-PDH. Phosphatase is believed to mediate the effects of a variety of nutritional and hormonal stimuli on PDH activity. Thus, we suggest that catabolic and anabolic (lipogenic) tissues express different isozymes of phosphatase, which in turn, differently respond to nutritional and hormonal stimulation, by this means defining the tissue-specific responses of PDH complex. The research plan outlined in this proposal is designed to test this hypothesis. Toward this goal, we plan to study the enzymatic and regulatory properties of isozymes of PDH phosphatase. We also propose to further characterize the tissue distribution of isozymes. Finally, we will evaluate the effects of starvation and diabetes, the conditions known to be associated with down-regulation of PDH phosphatase enzymatic activity, on the expression patterns of the two isozymes. The detailed specific aims for this proposal are to: (1) determine the molecular basis for the catalysis and for the strict substrate specificity of pyruvate dehydrogenase phosphatase; (2) further characterize the molecular factors responsible for the regulation of catalytic activity of the isozymes of phosphatase; (3) evaluate the contribution of each isozyme to the tissue specific regulation of the activity of pyruvate dehydrogenase complex; and (4) elucidate the molecular mechanisms responsible for the stable changes in phosphatase activity observed in starvation and diabetes. The accomplishment of these objectives will shed new light on the role of PDH reaction in the regulation of general carbohydrate and lipid metabolism. In the long run, it may lead to the development of highly specific drugs that will alleviate complications associated with diseases like diabetes, ischemia and sepsis.