白念珠菌质膜蛋白Rch1对细胞质内钙离子稳态和耐药调控功能的机理研究

In budding yeast, calcium homeostasis is tightly regulated to maintain an optimal concentration of calcium ions in the cytosol through a calcium channel consisted of Cch1 and Mid1 that exists in the plasma membrane, the vacuolar calcium pump Pmc1 and Ca2+/H+ exchanger Vcx1, and the ER/Golgi calcium pump Pmr1. We have recently found that the Candida albicans plasma membrane protein Rch1 is a novel regulator of cytosolic calcium homeostasis. However, the interactions between RCH1 and CCH1, MID1, PMC1 or PMR1 as well as how Rch1 regulates cytosolic calcium homeostasis remain unknown. In this project, we will take genetic, molecular and biochemical approaches to study the genetic interactions of RCH1 with other calcium regulator genes. We will try to identity the interacting proteins of Rch1 in cells through Mass Spectrum and biochemical approaches in order to elucidate the mechanisms by which Rch1 regulates calcium homeostasis and drug tolerance in C. albicans. Meanwhile, we will examine the role of RCH1 in the pathogenesis of C. albicans in an immunocompromised mouse model. In addition, we will study the cellular functions and regulation of ScRch1 in budding yeast, which will provide clues for the functions and regulatory mechanisms of CaRch1.

酿酒酵母胞质内的钙离子稳态是通过质膜上的钙通道(Cch1和Mid1组成)，高尔基和内质网膜上的钙泵Pmr1以及液泡膜上的钙泵Pmc1和钙/质子交换蛋白Vcx1来调控的。我们最近从白念珠菌细胞质膜上发现了一个钙离子稳态的新调控因子Rch1， 但是它和已知的调控因子之间的关系以及它是如何调控胞质内钙离子稳态的仍然未知。本项目拟通过遗传学，分子生物学和生物化学等技术研究RCH1与其它钙离子调控基因之间在钙离子稳态和耐药方面的相互作用。通过质谱和生化方法鉴定Rch1蛋白在细胞内的互作蛋白，以阐明其调控作用的机理。 同时，通过小鼠模型研究RCH1在白念珠菌致病过程中的作用。此外，通过系统研究模型菌-酿酒酵母中Rch1同源蛋白的细胞功能和表达调控，为深入研究白念珠菌Rch1的功能和调控机理提供线索。

钙离子稳态和钙离子/钙调磷酸酯酶信号途径在真核细胞中高度保守。和最简单的模式真核生物(酿酒酵母菌)一样，人体病原真菌白念珠菌的细胞中存在各种钙通道、钙泵和钙交换器以及完整的钙离子/钙调磷酸酯酶信号途径成员，它们在维持白念珠菌胞内钙离子稳态，以及应答外界环境压力、耐药性、形态发生和致病性等方面有着至关重要的作用。对白念珠菌钙离子稳态和钙离子/钙调磷酸酯酶信号途径调控机理的认知，有助于我们了解其致病过程和耐药机理，同时可以为发现和开发新的抗真菌药物提供。本研究我们在白念珠菌和酿酒酵母菌中发现和鉴定了一个新型的细胞质膜上钙离子内流的负向调控因子Rch1。酿酒酵母菌ScRCH1是白念珠菌CaRCH1的同功基因。CaRch1在白念珠菌细胞质膜上持续表达，不受钙信号途径的调控，但是ScRch1基因的转录表达本身却受到钙离子信号途径的正向调控。这可能与酿酒酵母和白念珠菌细胞所处的环境有关。酿酒酵母平时不接触高钙环境，当它应对高钙胁迫时ScRch1被诱导表达可以起到一个反馈调控的作用。相对而言，白念珠菌一直需要持续面对人体内部的高钙环境，CaRch1可以起到一个持续性降低外界（人体内）钙离子流入白念珠菌细胞内的作用。然而，Rch1如何调控钙离子的内流仍然未知，需要进一步研究来阐明其作用机理。