V-ATPase H+ PUMP REGULATION IN FUEL ENERGY SELECTION

描述(申请人提供)：V-ATPase是保守的质子泵，对pH动态平衡很重要。V-ATPase位于溶酶体、空泡和内体的膜上，维持蛋白质分类和降解所需的酸性管腔pH，以及病毒和细菌毒素进入宿主细胞所需的酸性管腔pH。专门用于活跃质子分泌的细胞，如肾单位的1-嵌入细胞，在质膜上表达V-ATPase，在那里他们微调全身的酸碱平衡。我们的目标是展示仔细调节V-ATPase功能和组装的基本机制，以便深入了解V-ATPase如何帮助控制管腔、胞液和细胞外的pH。V-ATPase是一种动态结构，可以可逆地分解以控制pH。在酵母和肾脏细胞中，活性和V-ATPase组装与糖酵解有关，但涉及的机制尚不清楚。利用酵母模型系统，我们发现了V-ATPase和葡萄糖-脂肪酸循环之间的一种新的联系，表明葡萄糖和脂肪代谢通过影响V-ATPase来重塑pH动态平衡。我们的假设是，当代谢在葡萄糖和脂肪酸之间切换时，V-ATPase组装被调节为维持细胞pH动态平衡的一种手段。我们认为，由V-ATPase泵和糖酵解酶组成的复合体在功能和结构上耦合了pH动态平衡和能量代谢；这种大分子结构的代谢控制调节了V-ATPase的组装。三个特定的目标将检验这一模型：目标1将剖析将V-ATPase连接到糖酵解途径的代谢信号；目标2将阐明糖酵解酶调节重塑V-ATPase组装和活性的机制；以及目标3将确定葡萄糖-脂肪酸循环的激活如何与V-ATPase相互作用。为了达到所提出的目标，本研究将对缺乏糖酵解、脂肪酸2-氧化和葡萄糖-脂肪酸循环等关键步骤的VMA突变体和代谢突变体的V-ATPase组装和拆解进行研究。我们将在细胞内代谢中间产物的水平和V-ATPase和糖酵解酶之间的结合动力学之间建立类似的关系，使我们能够理解V-ATPase如何帮助细胞适应代谢变化。由于全面的代谢研究和V-ATPase泵的可逆分解调节的复杂性，酿酒酵母是从遗传和生化水平上解决这一机制的优秀系统。通过展示V-ATPase的贡献，将出现对调整燃料能量选择的机制的新见解。由于代谢的改变，癌细胞使用V-ATPase来调节pH；因此，关于V-ATPase在癌症中维持pH动态平衡的机制的新知识可能会被揭示。由于葡萄糖-脂肪酸循环的病理生理学导致包括糖尿病和慢性肾脏疾病在内的代谢紊乱，我们的研究也将有助于他们的理解。

DESCRIPTION (provided by applicant): V-ATPases are conserved proton pumps important to pH homeostasis. Located at the membrane of lysosomes, vacuoles and endosomes, V-ATPases sustain the acidic luminal pH needed for protein sorting and degradation; and for entry of viruses and bacterial toxins into host cells. Cells specialized for active proton secretion such as the 1-intercalated cells of the kidney nephron, express V-ATPases at the plasma membrane where they fine-tune the systemic acid-base balance. It is our goal to demonstrate the fundamental mechanisms that carefully regulate V-ATPase function and assembly in order to gain insight into how V- ATPases assist in controlling luminal, cytosolic, and extracellular pH. V-ATPases are dynamic structures that reversibly disassemble to control pH. In yeast and kidney cells, activity and V-ATPase assembly are coupled with glycolysis, but the mechanisms involved are unclear. Using yeast model systems, we have shown a novel link between V-ATPases and the glucose-fatty acid cycle, suggesting that glucose and lipid metabolism remodel pH homeostasis via effects on V-ATPases. It is our hypothesis that V-ATPase assembly is regulated as a means of maintaining cellular pH homeostasis when metabolism switches between glucose and fatty acids. We propose that a complex consisting of the V-ATPase pump and glycolytic enzymes functionally and structurally couples pH homeostasis and energy metabolism; and that metabolic control of this macromolecular structure regulates V-ATPase assembly. Three specific aims will test this model: Aim 1 will dissect the metabolic signals that link V-ATPase to the glycolytic pathway; Aim 2 will elucidate the mechanisms by which regulation of glycolytic enzymes remodels V-ATPase assembly and activity; and Aim 3 will establish how activation of the glucose-fatty acid cycle cross-talks to V-ATPases. In order to accomplish the aims proposed, this study will measure V-ATPase assembly and disassembly in vma mutants and metabolic mutants deficient in key steps of glycolysis, 2- oxidation of fatty acids, and the glucose-fatty acid cycle. Parallels will be established between intracellular levels of metabolic intermediates and dynamics of binding between V-ATPase and glycolytic enzymes enabling us to understand how V-ATPases assist cells in adjusting to metabolic changes. Because of the complexity involved in both comprehensive metabolic studies and regulation of V-ATPase pumps by reversible disassembly, S. cerevisiae is an outstanding system to address this mechanism at both the genetic and biochemical levels. By showing the contribution of V-ATPases, new insights into the mechanisms that tune fuel energy selection will emerge. Cancer cells use V-ATPases to regulate pH as a result of changes in metabolism; thus new knowledge on the mechanisms by which V-ATPases maintain pH homeostasis in cancer may be revealed. As pathophysiology of the glucose-fatty acid cycle results in metabolic disorders including diabetes and chronic kidney disease, our studies will also contribute towards their understanding.