Molecular study of calcium channels in fungal pathogens.

真菌病原体中钙通道的分子研究。

• 批准号: 6941780
• 负责人: ANGIE GELLI
• 金额: $ 33.4万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6941780
• 关键词: Cryptococcus neoformans; acidity /alkalinity; biological signal transduction; calcium channel; calmodulin; cell membrane; drug resistance; fungal genetics; fungal proteins; gene environment interaction; genetic regulation; host organism interaction; intermolecular interaction; laboratory mouse; meningitis; molecular pathology; mycosis; osmotic pressure; protein localization; protein structure function; temperature; virulence; voltage /patch clamp

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.

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的钙-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仍然很大程度上未知。一种可能的工作模型表明，钙调神经磷酸酶是通过胞质钙水平和钙调蛋白的增加而激活的，以响应宿主环境特有的信号。激活的钙调神经磷酸酶随后会使真菌发病机制所需的特定蛋白质去磷酸化。我们认为病原真菌细胞中的钙通道通过响应宿主环境特有的特定刺激（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，这些蛋白可以通过钙调蛋白与钙通道 C 末端的相互作用来招募。拟议研究的总体目标是表征病原真菌利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/钙调蛋白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传统膜片钳技术进行结构功能研究。用于结构功能研究的钙通道突变体将在隐球菌脑膜炎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手段。对钙通道功能和调节的详细研究不仅对于清楚地了解病原真菌-宿主关系中信号-反应耦合的机制至关重要，而且对于开发可预防真菌感染的小分子的潜力也至关重要。真菌在宿主体内增殖。 例如，通道孔的堵塞、通道电压敏感性的改变或防止调节蛋白与通道相互作用可能是小分子扰乱通道活性、抑制宿主内真菌细胞增殖的可行方法。并最终预防疾病。