Regulation of Erythrocyte Volume Homeostasis

描述(由申请人提供)：维持水和溶质的平衡对红细胞的存活至关重要。原发性红细胞水合障碍是一组遗传性疾病，范围从脱水到过度水合的细胞。根据容量平衡的紊乱程度，可能会导致溶血性贫血。当细胞水合作用的紊乱与另一种情况有关时，就会发生继发性红细胞水合障碍，例如，通常伴随镰状细胞病或贝塔血红蛋白病的脱水。在继发性疾病中，红细胞水合作用的改变可能是疾病病理的主要因素。PIEZO1最近被确定为参与哺乳动物机械感觉和拉伸激活阳离子通道激活的长期追逐的蛋白质。我们发现PIEZO1基因突变导致遗传性干细胞症(HX)，这是一种以红细胞脱水为特征的溶血性贫血，表明PIEZO1在细胞体积稳态中发挥重要作用。PIEZO1是红细胞中未知的拉伸诱导阳离子通路的候选者，这些通路在红细胞老化、疟疾侵袭和循环应激中发挥关键作用。PIEZO1也是镰刀的一个很好的候选者，镰刀是镰刀红细胞中一种未知的阳离子渗透途径，在脱水级联反应的启动对镰刀细胞的病理生物学具有重要意义。尽管PIEZO1很重要，但我们还不知道它的表达、调节、结构或功能的机制，以及它在调节红系细胞体积稳态中的作用。拟议的研究结合了最先进的细胞、遗传、蛋白质组和生理学技术，以创新的多学科方式表征PIEZO1的表达、结构和功能。研究包括对PIEZO1膜蛋白表达、转运和电生理学的功能性、基于细胞的分析，在一种新的、体内稳定转染、单拷贝、可诱导的PIEZO1表达CLL模型中。将创建新的Piezo1转基因小鼠模型，包括HX小鼠模型，并对其进行表征。最后，基于MRM的定量蛋白质组学研究和最先进的机械转导生理学技术将在各种细胞条件下应用于HX患者的红细胞。PIEZO1广泛存在于淋巴细胞、血管内皮细胞、肾脏和神经细胞等多种细胞中，表明PIEZO1在多种细胞中发挥重要作用。因此，对红系细胞的研究可能会产生适用于许多关键细胞过程或人类疾病的机制或生物学原理。

DESCRIPTION (provided by applicant): Maintenance of water and solute homeostasis is critical to survival of the erythrocyte. Primary disorders of erythrocyte hydration are a group of inherited disorders ranging from dehydrated to overhydrated cells. Depending on the degree of perturbation of volume homeostasis, hemolytic anemia may result. Secondary disorders of erythrocyte hydration occur when perturbation in cell hydration is associated with another condition, for instance the dehydration that commonly accompanies sickle cell disease or beta hemoglobinopathies. In secondary disorders, altered erythrocyte hydration may be a major contributor to disease pathology. PIEZO1 has recently been identified as the long sought after protein involved in mammalian mechanosensation and stretch-activated cation channel activation. We have discovered mutations in PIEZO1 that lead to hereditary xerocytosis (HX), a hemolytic anemia characterized by primary erythrocyte dehydration, indicating PIEZO1 plays an important role in cellular volume homeostasis. PIEZO1 is a candidate for unidentified stretch-induced cation pathways in the erythrocyte that play critical roles in erythrocyte aging, malaria invasion, and circulatory sheer stress. PIEZO1 is also an excellent candidate for Psickle, an unidentified cation permeability pathway in sickle erythrocytes at the initiation of the dehydratio cascade of fundamental importance to sickle cell pathobiology. Despite its importance, we have no knowledge of the mechanisms controlling PIEZO1 expression, regulation, structure or function and its role in regulation of volume homeostasis in erythroid cells. The proposed studies combine state of the art cellular, genetic, proteomic, and physiologic technologies to characterize the expression, structure and function of PIEZO1, in an innovative, multidisciplinary manner. Studies include functional, cell-based assays of PIEZO1 membrane protein expression, trafficking, and electrophysiology in a novel, in vivo stably-transfected, single-copy, inducible cll model of PIEZO1 expression. New genetically modified murine models of Piezo1, including a murine model of HX, will be created and characterized. Finally, quantitative MRM-based proteomic studies and state-of-the-art mechanotransduction physiologic techniques will be applied to erythrocytes from HX patients under a variety of cellular conditions. PIEZO1 is found in many cell types including lymphocytes, endothelial, kidney, and neural cells, indicating it likey mediates important functions in a wide variety of cells. Thus studies in erythroid cells may yield mechanistic or biological principles generalizable to many critical cellular processes or human diseases.