Mechanisms of Receptor Regulated Na+-H+ Exchange

描述(由申请人提供)：细胞内pH(Phi)的动态变化调节一系列正常和病理的细胞过程。Phi升高促进细胞的增殖、分化和迁移，phi降低则诱导细胞凋亡。PHI调节失调被认为是心脏和脑缺血后癌症进展、糖尿病和组织损伤的原因之一。这笔赠款的长期目标是确定PHI动态的调节和功能。在过去的四个资金周期中，重点关注质膜Na-H交换器NHE1的H+通量是如何被调节并驱动PHI动态的。我们还显示了NHE1依赖的PHI增加对细胞增殖、细胞周期进展和细胞迁移的功能意义。尽管依赖于phi的细胞功能具有广泛的意义，但我们对phi的变化如何影响蛋白质和大分子组装的了解有限。为了解决这一局限性，我们最近开始研究pH传感器的结构和功能，即对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东西，以确定调节癌细胞中异常的基本细胞过程的pH传感器的设计原则和功能。PHI升高是大多数癌症的标志，无论其组织来源或遗传背景如何。这可能反映了对高PHI代谢适应、增殖增加和转移的依赖。我们将测试这一假设，即控制新陈代谢和细胞周期进展的pH传感器在NHE1和Phi如何指导癌症中失调的细胞功能方面发挥关键作用。我们的研究使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。在目标1中，我们将确定NHE1如何调节控制新陈代谢的激酶。我们将基于我们最近的结构和生物化学发现，即其P85调节亚基是一个pH传感器，来测试关于磷脂酰肌醇3-激酶对pH敏感的预测。我们还将确定NHE1对磷酸果糖激酶1(PFK1)的调节，PFK1是糖酵解中的第一个限速酶，直接结合NHE1的C末端细胞质结构域，具有极强的pH敏感活性。在目标2中，我们将通过专注于我们之前的发现来确定NHE1是如何促进细胞增殖的，即H+外流是G2/M的两倍。我们将基于我们新的计算和生化数据以及在缺乏NHE1活性的细胞中如何减弱细胞周期蛋白B1的表达来测试Wee1作为假定的pH传感器的预测。在目标3中，我们将使用我们建立的果蝇模型来确定NHE活性和调节失调的PHI在肿瘤发生中的作用。我们将测试Dnhe2的丢失如何对转化的但不是正常的细胞造成合成致命的预测，并使用在Aim 1和2中产生的突变的pH传感器来测试表型的挽救。我们还将询问Dnhe2的过度表达是否与癌基因激活或肿瘤抑制基因缺失协同作用来诱导转移癌，并将使用修饰筛选来识别Dnhe2过度表达的表型的介体。

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.