Mechanisms of Receptor Regulated Na+-H+ Exchange

描述（由申请人提供）：细胞内pH值（pHi）的动态变化调节了一系列正常和病理细胞过程。pHi升高促进细胞增殖、分化和迁移，pHi降低诱导细胞凋亡。失调的pHi被认为有助于癌症进展、糖尿病和心脏和脑缺血后的组织损伤。这项资助的长期目标是确定pHi动力学的调节和功能。过去四个资助周期的重点是如何通过质膜Na-H交换剂NHE1调节H+通量并驱动pHi动力学。我们还显示了nhe1依赖性pHi增加对细胞增殖、细胞周期进展和细胞迁移的功能意义。尽管pHi依赖的细胞功能具有广泛的意义，但我们对pHi变化如何影响蛋白质和大分子组装的理解有限。为了解决这一限制，我们最近开始研究pH传感器的结构和功能，或对pH的微小生理变化敏感的具有活性或结合亲和力的蛋白质。目前的应用应用了我们在前四个资助周期中共同学到的知识，以确定pH传感器调节癌细胞中异常的基本细胞过程的设计原则和功能。无论组织起源或遗传背景如何，pHi升高是大多数癌症的标志。这可能反映了代谢适应、增殖增加和转移对高pHi的依赖。我们将验证控制代谢和细胞周期进程的pH传感器在NHE1和pHi如何直接癌症中失调的细胞功能中发挥关键作用的假设。我们的研究采用了一种创新的综合方法，将蛋白质结构、蛋白质生物化学和细胞生理学联系起来。在Aim 1中，我们将确定NHE1如何调节控制代谢的激酶。基于我们最近的结构和生化发现，我们将测试关于磷酸肌苷3-激酶如何对pH敏感的预测，即其p85调节亚基是pH传感器。我们还将确定NHE1对磷酸果糖激酶1 （PFK1）的调控，PFK1是糖酵解中的第一个限速酶，它直接结合NHE1的c端细胞质结构域，具有非常ph敏感的活性。在Aim 2中，我们将通过关注我们之前的研究结果来确定NHE1如何促进细胞增殖，即NHE1的H+外排乘以G2/M。基于新的计算和生化数据，我们将测试Wee1作为假定的pH传感器的预测，以及细胞周期蛋白B1表达在缺乏NHE1活性的细胞中如何减弱。在Aim 3中，我们将使用我们生成的果蝇模型来确定NHE活性和失调的pHi在肿瘤发生中的作用。我们将测试Dnhe2缺失如何对转化细胞而非正常细胞造成合成致死的预测，并测试在Aim 1和Aim 2中产生的突变pH传感器对表型的拯救。我们还将探讨Dnhe2过表达是否与癌基因激活或肿瘤抑制因子缺失协同诱导转移性癌症，并将使用修饰子筛选来鉴定Dnhe2过表达表型的介质。

DESCRIPTION (provided by applicant): Dynamic changes in intracellular pH (pHi) regulate a range of normal and pathological cell processes. Increased pHi promotes cell proliferation, differentiation, and migration, and decreased pHi induces apoptosis. Dysregulated pHi is thought to contribute to cancer progression, diabetes, and tissue damage after cardiac and cerebral ischemia. The long-term goals of this grant are to determine the regulation and function of pHi dynamics. The past four funding cycles focused on how H+ fluxes by the plasma membrane Na-H exchanger NHE1 are regulated and drive pHi dynamics. We also showed the functional significance of NHE1-dependent increases in pHi for cell proliferation, cell cycle progression, and cell migration. Despite the broad significance of pHi-dependent cell functions, we have limited understanding of how changes in pHi affect proteins and macromolecular assemblies. To address this limitation we recently began studying the structure and function of pH sensors, or proteins with activities or binding affinities that are sensitive to small physiological changes in pH. The current application applies what we collectively learned in the previous four funding cycles to determine the design principles and function of pH sensors regulating basic cell processes that are aberrant in cancer cells. Increased pHi is a hallmark of most cancers, regardless of the tissue origin or genetic background. This likely reflects a dependence on higher pHi for metabolic adaptation, increased proliferation, and metastasis. We will test the hypothesis that pH sensors controlling metabolism and cell cycle progression play critical roles in how NHE1 and pHi direct cell functions that are dysregulated in cancer. Our studies use an innovative comprehensive approach that bridges protein structure, protein biochemistry, and cell physiology. In Aim 1 we will determine how kinases controlling metabolism are regulated by NHE1. We will test predictions on how phosphoinositide 3-kinase is pH sensitive, based on our recent structural and biochemical findings that its p85 regulatory subunit is a pH sensor. We also will determine NHE1 regulation of phosphofructokinase 1 (PFK1), the first rate-limiting enzyme in glycolysis, which directly binds the C-terminal cytoplasmic domain of NHE1 and has extremely pH-sensitive activity. In Aim 2 we will determine how NHE1 promotes cell proliferation by focusing on our previous findings that H+ efflux by NHE1 times G2/M. We will test predictions on Wee1 as a putative pH sensor based on our new computational and biochemical data and on how cyclin B1 expression is attenuated in cells lacking NHE1 activity. In Aim 3 we will determine the role of NHE activity and dysregulated pHi in tumorigenesis using Drosophila models we generated. We will test predictions on how loss of Dnhe2 may be a synthetic lethal for transformed but not normal cells, and test rescue of phenotypes with mutant pH sensors generated in Aim 1 and 2. We also will ask whether over expression of Dnhe2 cooperates with oncogene activation or tumor suppressor deletion to induce metastatic cancer, and will use modifier screens to identify mediators of the Dnhe2 over expression phenotype.