Structure, Mechanism and Regulation of the V-ATPases

超越空间 提供了 本建议的长远目标是确定 液泡（H+）-ATP酶（或V-ATP酶）。V-ATP酶是ATP依赖性质子泵家族， 在细胞内室的酸化和质子穿过血浆的运输中起作用 某些细胞的膜。细胞内区室的酸化对于以下过程是重要的， 受体介导的内吞作用、细胞内运输、病毒和毒素进入、蛋白质加工和 降解和小分子的耦合运输。肾组织中V-ATP酶的功能 酸化、骨吸收、pH稳态和肿瘤转移。 V-ATP酶由负责ATP水解的外周V1结构域和整合的 VO结构域负责质子运输。尽管V-ATP酶通过旋转机构操作， 这一机制的细节仍不清楚。为了进一步阐明V- ATP酶复合物，并深入了解其功能，将采用各种方法，包括 突变分析、半胱氨酸介导的交联和电子显微镜。标记的表位的纯化 还将进行酵母中表达的复合物以确定结晶试验的可行性。 其中要解决的问题是V1和被动的ATP酶活性的机制 通过VO的质子转运在这些结构域解离后沉默（重要的体内调节活性）。 机制）。VOsubunits之间的螺旋接触对质子传输的重要性将阐明使用 二硫键介导的交联和对转运和抑制剂结合至关重要的残基 诱变细胞内环境和V-ATP酶的几个关键结构域的作用， 调节体内解离将被探索，包括亚基A的非同源区域和N- 亚基A末端亲水结构域。一种新的筛选将被用来鉴定在这种情况下被阻断的突变体。 过程这些研究将为进一步了解V- ATP酶由于它们在胆固醇稳态、骨吸收、病毒和 毒素进入，肾酸化和肿瘤转移，所获得的见解直接相关， 人类疾病如动脉粥样硬化、骨质疏松症、流感、炭疽、肾病和癌症。

EXCEEDTHE SPACE PROVIDED. The long term objectives of this proposal are to determine the structure, mechanism and regulation of the vacuolar (H+)-ATPases (or V-ATPases). The V-ATPases are a family of ATP dependent proton pumps that function both in acidification of intracellular compartments and in proton transport across the plasma membrane of certain cells. Acidification of intracellular compartments is important for such processes as receptor-mediated endocytosis, intracellular trafficking, viral and toxin entry, protein processing and degradation and coupled transport of small molecules. Plasma membrane V-ATPases function in renal acidification, bone resorption, pH homeostasis and tumor metastasis. The V-ATPases are composed of a peripheral V1 domain responsible for ATP hydrolysis and an integral VO domain responsible for proton transport. Although the V-ATPases operate by a rotary mechanism, the details of this mechanism remain uncertain. To further elucidate the arrangement of subunits within the V- ATPase complex and to gain insight into their function, a variety of approaches will be employed, including mutational analysis, cysteine-mediated crosslinking and electron microscopy. Purification of epitope tagged complexes expressed in yeast to determine the feasibility of crystallization trials will also be performed. Among the questions to be addressed are the mechanisms by which the ATPase activity of V1 and passive proton transport by VOare silenced upon dissociation of these domains (an important in vivo regulatory mechanism). Helical contacts between VOsubunits important for proton transport will be elucidated using disulfide-mediated crosslinking and residues critical for transport and inhibitor binding identified by mutagenesis. The role of the intracellular environment and of several critical domains of the V-ATPase in regulating in vivo dissociation will be explored, including the non-homologous region of subunit A and the N- terminal hydrophilic domain of subunit a. A novel screen will be employed to identify mutants blocked in this process. These studies will provide further insight into the structure, mechanism and regulation of the V- ATPases. Because of their role in such processes as cholesterol homeostasis, bone resorption, viral and toxin entry, renal acidification and tumor metastasis, the insights gained are directly relevant to such diverse human diseases as atherosclerosis, osteoporosis, influenza, anthrax, renal disease and cancer.