Molecular study of calcium channels in fungal pathogens.

描述（由申请人提供）：为了在宿主细胞内增殖并随后促进疾病，真菌病原体需要活性钙-钙调蛋白依赖性信号级联。决定钙反应如何在真菌病原体中启动和传播的分子机制在很大程度上仍然未知。一个可能的工作模型将说明，钙调磷酸酶被激活的细胞溶质钙水平和钙调素的增加，以响应特定于宿主环境的信号。活化的钙调磷酸酶随后会使真菌致病所需的特定蛋白质去磷酸化。我们认为致病真菌细胞中的钙通道通过响应特定于宿主环境的特定刺激（即碱性pH、5%CO2、铁水平等）来启动钙信号传导。信号特异性通过钙通道与关键信号蛋白的结合来实现，所述关键信号蛋白可以通过钙调蛋白与钙通道的C-末端的相互作用来募集。拟议研究的总体目标是表征致病真菌使用钙通道将宿主特异性信号耦合到宿主环境定植所需的钙/钙调素介导的信号级联的细胞和分子机制。为了阐明钙通道功能和调节的分子机制，将在模拟宿主环境的条件下使用常规膜片钳技术进行结构-功能研究。将在隐球菌脑膜炎动物模型中测试用于结构-功能研究的钙通道突变体的毒力。将在缺乏关键信号分子的细胞中检查通道激活和调节，以确定这些信号蛋白是否调节通道功能作为赋予信号特异性的手段。对钙通道功能和调节的详细研究不仅对于清楚地理解致病真菌-宿主关系中的信号-响应耦合的机制是必要的，而且对于可能发挥作用以防止宿主中真菌增殖的小分子的潜在开发也是必要的。 例如，通道孔的闭塞，或通道电压敏感性的变化或防止调节蛋白与通道相互作用可以代表可行的手段，通过该手段小分子可以起到扰乱通道活性、抑制宿主内的真菌细胞增殖并最终预防疾病的作用。

DESCRIPTION (provided by applicant): In order to proliferate within host cells and subsequently promote disease, fungal pathogens require an active calcium-calmodulin-dependent signaling cascade. The molecular mechanisms that determine how the calcium response is initiated and propagated in fungal pathogens remain largely unknown. A possible working model would state that calcineurin is activated by an increase in cytosolic calcium levels and calmodulin in response to signals specific to the host environment. Activated calcineurin would then subsequently dephosphorylate specific proteins required for fungal pathogenesis. We propose that calcium channels in pathogenic fungal cells initiate calcium signaling by responding to particular stimuli that are specific to the host environment (i.e. alkaline pH, 5% CO2, iron levels etc). Signal-specificity is achieved by the association of the calcium channel with key signaling proteins that could be recruited by calmodulin's interaction with the C-terminus of the calcium channel. The overall aim of the proposed research is to characterize the cellular and molecular mechanism by which pathogenic fungi use calcium channels to couple host-specific signals to a calcium/calmodulin-mediated signaling cascade that is required for colonization of the host environment. In order to elucidate the molecular mechanism of calcium channel function and regulation, structure-function studies using conventional patch clamp techniques in conditions that mimic the host environment will be performed. The calcium channel mutants generated for the structure-function studies, will be tested for virulence in an animal model of cryptococcal meningitis. Channel activation and regulation will be examined in cells that lack key signaling molecules in order to determine whether these signaling proteins regulate channel function as a means to impart signal specificity. A detailed study of calcium channel function and regulation is imperative not only for a clear understanding of the mechanism(s) underlying the signal-response coupling in the pathogenic fungal-host relationship but also for the potential development of small molecules that could function to prevent fungal proliferation in the host. For example, occlusion of the channel pore, or a change in channel voltage-sensitivity or the prevention of regulatory proteins from interacting with the channel could represent viable means by which small molecules may function to perturb channel activity, inhibit fungal cell proliferation within the host and ultimately prevent disease.