Investigation of Electrolyte Homeostasis via Quantitative Proteomics

描述(由申请人提供)：电解质平衡对细胞水平的生命至关重要，因为细胞必须对渗透挑战做出反应，以抵御可能导致破裂的细胞水分和离子含量的变化。在更大的范围内，人类通过维持肾脏中钠的重新吸收和排泄的适当平衡来调节血压。高血压是影响6000多万美国人的一个主要健康问题，它是电解质平衡失调的结果。因此，了解电解质动态平衡的调控机制对人类健康具有重要意义。越来越清楚的是，一组核心的调节蛋白可以感知和维持电解质的稳态。我们对如何在分子水平上实现这一点缺乏了解。电解质稳态的分子机制对人类的健康和疾病状态都是至关重要的，因此必须了解才能释放它们的治疗潜力。我们的目标是了解蛋白质和信号机制的网络，主要是调节磷酸化事件，它们连接着细胞体积控制和血压稳态的机制。红细胞作为一个模型系统具有巨大的潜力，可以用来理解对电解质稳态至关重要的基本元素。我们将使用定量蛋白质组学的方法来研究调节红细胞电解质通量的信号蛋白网络。我们将重点研究K-Cl共转运蛋白作为电解质通量的代表性直接介体，以及Wnk1和Wnk4作为离子通量的关键信号成分。这些研究将为WNK功能的上游调控和下游信号事件提供新的见解，这些信号事件通过识别关键的调节磷酸化位点来控制电解质动态平衡。为了将这些观察结果与体内环境联系起来，我们将在小鼠红细胞中使用SILAC技术来量化对特定生理扰动做出反应的关键调节磷酸化位点。这项研究的目的是为协调电解质稳态的机制提供一个基本的理解。此外，我们正在寻找血压控制和细胞体积调节的机制联系，以寻找新的靶点来治疗高血压和镰状细胞性贫血等疾病。

DESCRIPTION (provided by applicant): Electrolyte homeostasis is essential for life at the cellular level, in that, cells must respond to osmotic challenge to fend off changes in cellular water and ion content that could lead to rupture. On a larger scale, humans regulate blood pressure by maintaining an appropriate balance of sodium reabsorption and excretion in the kidney. Hypertension, a major health problem affecting more than 60 million Americans, is a result of a dysfunction in electrolyte homeostasis. Therefore, understanding mechanisms that control electrolyte homeostasis is important for human health. It is becoming increasingly clear that a core set of regulatory proteins senses and maintains electrolyte homeostasis. Our knowledge is lacking in how this is achieved at the molecular level. The molecular mechanisms of electrolyte homeostasis are of critical importance for both healthy and disease states in humans and thus must be understood in order unlock their therapeutic potential. We aim to understand the network of proteins and signaling mechanisms, mainly regulatory phosphorylation events, which connect mechanisms of cell volume control and blood pressure homeostasis. The red blood cell holds great potential as a model system to understand the fundamental elements critical for electrolyte homeostasis. We will use a quantitative proteomic approach to study networks of signaling proteins that regulate electrolyte flux in red blood cells. We will focus our studies on the K-Cl cotransporters as a representative direct mediator of electrolyte flux and the kinases Wnk1 and Wnk4 as critical signaling components of ion flux. These studies will provide new insight into the upstream regulation of Wnk function and downstream signaling events that control electrolyte homeostasis by identifying critical regulatory phosphorylation sites. To link these observations to the in vivo setting, we will use SILAC technology in the mouse red blood cell to quantify critical regulatory phosphorylation sites that respond to specific physiologic perturbation. The purpose of this study is to provide a fundamental understanding of the mechanisms that coordinate electrolyte homeostasis. Furthermore, we are seeking mechanistic links in blood pressure control and cell volume regulation in order to find new target points to treat diseases such as hypertension and sickle cell anemia.