Identification of regulatory domains that mediate the membrane-binding of BAX

描述（由申请人提供）：BAX是BCL-2家族的一员，可以通过使线粒体和内质网释放凋亡因子来促进细胞死亡。许多不同的蛋白质已经被确定可以调节BAX的活性，但是使BAX易位到膜的构象变化仍然未知。因此，在知识基础上存在一个根本性的空白——没有统一的机制来协调BAX如何与许多不同的调节因子相互作用，控制其与膜的结合能力。为了解决这个问题，提出的研究的长期目标是阐明BAX与膜相互作用的手段，并了解这种相互作用如何调节蛋白质的功能。本应用程序的目的是确定一个突出的疏水沟槽的占用如何调节BAX与膜结合的能力。核心假设是，BAX的c端19螺旋可以在疏水沟槽内以正向或反向两种不同的方向结合，并且结合的方向调节沟槽的稳定性，从而调节BAX与膜的结合能力，这一过程由钠氢交换器（NHE）介导的瞬时凋亡碱化增强。这项研究的基本原理是，了解19螺旋占据疏水沟如何介导BAX的膜易位，可能会导致开发一种有效的方法，用于在疾病状态下对蛋白质进行药理学操作。拟议的研究与NIH的使命有关，该使命涉及发展基础知识，以减轻人类疾病的负担。在强有力的初步数据支持下，中心假设将通过以下三个具体目标进行验证：(1)确定调节疏水槽内BAX c端19螺旋结合的分子相互作用；(2)确定疏水沟槽的占用如何调节BAX的膜结合；(3)确定细胞内碱化如何增强BAX与膜的结合。为了实现第一个目标，疏水沟槽中一个关键位点的诱变将用于检查与19螺旋的分子相互作用。为了实现第二个目标，BAX的二聚化将与新的跨膜结构域的暴露一起进行评估。为了实现第三个目标，脂质在膜上的扩散将在NHE缺陷细胞的背景下进行检查。该研究设计具有创新性，因为它涉及跨学科的方法，将生物物理测量与功能分析结合起来，以确定BAX如何转运到膜上。提出的研究意义重大，因为通过证明疏水沟槽的稳定性控制跨膜结构域的可及性，我们将揭示沟槽以前未知的功能。这一发现将推进该领域的工作，导致开发新的策略来治疗人类疾病中的BAX蛋白失调。提出的研究将通过揭示使BAX过渡到膜的关键构象变化的机制来填补知识库中的现有空白。从拟议的研究中获得的健康益处将包括在疾病状态下治疗性地操纵BAX活性的潜力：关闭BAX的凋亡活性以防止导致神经退行性变的神经元或导致心脏损伤的心肌细胞的死亡，或打开BAX的凋亡活性以使癌细胞对化疗敏感。拟议研究的结果将提高对BCL-2家族蛋白如何与膜相互作用的基本理解，这是一项在改善人类健康方面具有广泛应用的一般性发现。

DESCRIPTION (provided by applicant): BAX is a member of the BCL-2 family that can promote cell death by making possible the release of apoptotic factors from mitochondria and the endoplasmic reticulum. Many different proteins have been identified that regulate the activity of BAX, but the conformational changes that enable the translocation of BAX to membranes remain unknown. Hence a fundamental gap in the knowledge base exists - there is no unifying mechanism that reconciles how the interaction of BAX with many distinct regulatory factors controls its ability to bind to membranes. To address this, the long-term goals of the proposed studies are to elucidate the means by which BAX interacts with membranes and understand how this interaction modulates the protein's function. The objective of this application is to determine how occupancy of a prominent hydrophobic groove regulates the capacity of BAX to associate with membranes. The central hypothesis is that the C-terminal 19 helix of BAX can bind in two different orientations, forward or reverse, within the hydrophobic groove, and that the orientation of binding regulates stability of the groove and thereby the ability of BAX to bind to membranes, a process that is enhanced by a transient apoptotic alkalinization mediated by the sodium hydrogen exchanger (NHE). The rationale for the proposed research is that understanding how occupancy of the hydrophobic groove by the 19 helix mediates the membrane translocation of BAX can potentially lead to the development of an effective approach for the pharmacological manipulation of the protein in disease states. The proposed research is relevant to that part of NIH's mission that involves developing fundamental knowledge with the goal of reducing the burden of human disease. Supported by strong preliminary data, the central hypothesis will be tested by pursuing the following three specific aims: (1) Identify the molecular interactions that regulate the binding of the C-terminal 19 helix of BAX within the hydrophobic groove; (2) Determine how occupancy of the hydrophobic groove modulates the membrane association of BAX; and (3) Establish how intracellular alkalinization enhances the binding of BAX to membranes. To achieve the first aim, mutagenesis of a critical site in the hydrophobic groove will be used to examine molecular interactions with the 19 helix. To achieve the second aim, dimerization of BAX will be assessed in conjunction with the exposure of novel transmembrane domains. To achieve the third aim, lipid diffusion in membranes will be examined in the context of NHE defective cells. The research design is innovative because it involves an interdisciplinary approach, uniting biophysical measurements with functional assays to determine how BAX translocates to membranes. The proposed research is significant because, by demonstrating that the stability of the hydrophobic groove controls the accessibility of transmembrane domains, we will have revealed a previously unknown function for the groove. This finding will advance work in the field, leading to the development of novel strategies for the therapeutic manipulation of BAX in human diseases for which dysregulation of this protein figures prominently. The proposed research will fill an existing gap in the knowledge base by revealing the mechanism underlying key conformational changes that enable BAX to transition to membranes. The health benefits derived from the proposed studies will include the potential to therapeutically manipulate the activity of BAX in disease states: turning off the apoptotic activity of BAX to prevent the death of neurons that cause neurodegeneration or cardiomyocytes that cause heart damage or turning on the apoptotic activity of BAX to sensitize cancerous cells to chemotherapy. Results from the proposed studies will improve the fundamental understanding of how proteins of the BCL-2 family interact with membranes, a general finding with broad application in the improvement of the health of human beings.