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，真菌病原体需要一个活跃的钙-钙调素依赖性信号级联。决定真菌病原体中钙反应如何启动和传播的分子机制在很大程度上仍然未知。一种可能的工作模型表明，钙调磷酸酶是由细胞质钙水平和钙调蛋白的增加激活的，这是对宿主环境特异性信号的响应。活化的钙调磷酸酶随后会使真菌发病所需的特定蛋白去磷酸化。我们提出病原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。信号特异性是通过钙通道与关键信号蛋白的结合来实现的，这些关键信号蛋白可以通过钙调蛋白与钙通道c端的相互作用来募集。本研究的总体目的是表征病原真菌利用钙通道将宿主特异性信号偶联到钙/钙调素介导的信号级联的细胞和分子机制，这是宿主环境定植所必需的。为了阐明钙通道功能和调控的分子机制，我们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-功能研究而产生的钙通道突变体将在隐球菌脑膜炎的动物模型中进行毒力测试。通道激活和调节将在缺乏关键信号分子的细胞中进行检查，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。对钙通道功能和调控的详细研究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，也是为了潜在地开发可以阻止真菌在宿主中增殖的小分子。例如，通道孔的闭塞，或通道电压敏感性的改变，或防止调节蛋白与通道相互作用，都可能是小分子干扰通道活性、抑制宿主内真菌细胞增殖并最终预防疾病的可行手段。

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.