Molecular study of calcium channels in fungal pathogens.

描述(申请人提供)：为了在宿主细胞内增殖并随后促进疾病，真菌病原体需要活跃的钙调蛋白依赖的信号级联反应。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不清楚。一种可能的工作模型表明，钙调神经磷酸酶是由胞浆钙水平的增加和钙调蛋白对宿主环境特有的信号做出反应而激活的。激活的钙调神经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对宿主环境特有的刺激(如碱性pH、5%CO2、铁水平等)做出反应来启动钙信号。信号特异性是通过钙通道与关键信号蛋白的关联实现的，钙调素与钙通道的C末端相互作用可以招募关键信号蛋白。这项研究的总体目标是描述病原真菌利用钙通道将宿主特异性信号耦合到钙/钙调蛋白介导的信号级联的细胞和分子机制，而钙/钙调蛋白介导的信号级联是宿主环境定居所必需的。为了阐明钙通道功能和调控的分子机制，将在模拟宿主环境的条件下使用传统的膜片钳技术进行结构-功能研究。为结构功能研究产生的钙通道突变体将在隐球菌性脑膜炎的动物模型中进行毒力测试。将在缺乏关键信号分子的细胞中检查通道的激活和调节，以确定这些信号蛋白是否调节通道功能，作为传递信号特异性的一种手段。深入研究钙通道的功能和调控，不仅是为了清楚地了解致病真菌-宿主关系中信号-反应耦合的机制(S)，而且也是为了潜在地开发能够防止宿主真菌增殖的小分子。例如，阻断通道孔，或改变通道电压敏感性，或阻止调节蛋白与通道相互作用，可能代表了一种可行的方法，通过这些方法，小分子可以发挥作用，扰乱通道活动，抑制宿主内真菌细胞的增殖，并最终预防疾病。

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.