Identification of calcineurin-binding proteins in A. fumigatus septum formation

描述（由申请方提供）：由真菌烟曲霉引起的侵袭性曲霉病（IA）已超过侵袭性念珠菌病，成为免疫功能低下患者最常见的真菌性死亡原因。美国传染病学会强调了A。烟曲霉是仅有的六种病原体之一，迫切需要实质性的治疗突破。尽管发病率和死亡率不断增加，但A.烟曲霉的发病机制还不清楚。我们的长期目标是了解A.烟曲霉生长和疾病。隔膜是许多真菌生长的基本生物结构，也是理想的治疗靶点。a.烟曲霉菌丝产生分隔隔膜以推进和侵入宿主组织。我们已经证明，钙调磷酸酶稳定地定位于A。烟曲霉隔膜和钙调磷酸酶的遗传或药理学抑制阻断菌丝生长并破坏隔膜。尽管其在生长中的关键作用，没有研究集中在菌丝隔膜，以了解发病机制，和钙调神经磷酸酶介导的控制隔膜的形成是不明白的。我们的中心假设是钙调神经磷酸酶与其他蛋白质在A.烟曲霉菌隔膜调节隔膜的形成和菌丝的延伸。本申请的目的是定义那些在菌丝隔膜处与钙调磷酸酶结合的蛋白质。我们的方法将分为两个具体目标：1）鉴定A.通过钙调磷酸酶A和B下拉测定，然后进行LC/MS/MS分析。钙调神经磷酸酶A和B高度集中在隔膜处，这表明它们之间存在强烈的相互作用，这种作用不同于钙调神经磷酸酶作为磷酸酶的已知功能。我们将确定什么钙调磷酸酶结合在隔膜开始了解生长调节的机制。2)假定的钙调神经磷酸酶结合蛋白的中隔定位和钙调神经磷酸酶依赖性。我们将优先考虑和定位这些新发现的野生型A蛋白。fumigatus来验证它们的细胞位置和功能。为了表征所鉴定的钙调神经磷酸酶结合间隔蛋白的钙调神经磷酸酶依赖性，我们将利用我们独特的一组钙调神经磷酸酶缺失菌株。这些结果将有一个 对理解潜在的生长机制产生了重大影响，重点是钙调神经磷酸酶直接调节隔膜形成以协调菌丝生长和疾病的新理论。我们的方法，以确定蛋白质相互作用的钙调磷酸酶在隔膜将开始解开更大的分子基础，A。烟曲霉致病性这一策略将使我们朝着我们的长期目标迈进，即通过鉴定可利用的真菌特异性靶标来阻断隔膜形成和生长，从而对IA死亡率产生实质性影响，从而开发新型IA治疗剂。

DESCRIPTION (provided by applicant): Invasive aspergillosis (IA) caused by the fungus Aspergillus fumigatus has surpassed invasive candidiasis as the most frequent fungal cause of death in immunocompromised patients. The Infectious Diseases Society of America highlighted A. fumigatus as one of only six pathogens for which a substantive treatment breakthrough is urgently needed. Despite an increasing incidence of disease and mortality, the basic determinants underlying A. fumigatus pathogenesis are not well understood. Our long-term goal is to understand the molecular control of A. fumigatus growth and disease. The septum is a fundamental biologic structure important for the growth of many fungi and an ideal therapeutic target. A. fumigatus hyphae produce compartmentalizing septa to advance and invade host tissue. We have shown that calcineurin stably localizes to the A. fumigatus septum and genetic or pharmacologic inhibition of calcineurin blocks hyphal growth and disrupts septation. Despite its pivotal role in growth, no studies have focused on the hyphal septum to understand pathogenesis, and calcineurin-mediated control of septum formation is not understood. Our central hypothesis is that calcineurin complexes with other proteins at the A. fumigatus septum to regulate septum formation and hyphal extension. The objective of this application is to define those proteins that bind to calcineurin at the hyphal septum. Our approach will be divided into two specific aims: 1) Identify calcineurin- binding proteins in A. fumigatus through calcineurin A and B pulldown assays followed by LC/MS/MS analysis. Calcineurin A and B are highly concentrated at the septum, arguing for a strong interaction, a role different than calcineurin's known function as a phosphatase. We will identify what calcineurin binds at the septum to begin to understand the mechanism of growth regulation. 2) Validate septal localization and calcineurin-dependency of putative calcineurin-binding protein. We will prioritize and localize those newly identified proteins in wild-type A. fumigatus to verify their cellular location and function. To characterize the calcineurin-dependency of the identified calcineurin-binding septal proteins, we will utilize our unique set of calcineurin deletion strains. These results will have a significant impact in understanding underlying growth mechanisms, focusing on the novel theory that calcineurin directly regulates septum formation to orchestrate hyphal growth and disease. Our approach to define the proteins interacting with calcineurin at the septum will begin to unravel the larger molecular basis of A. fumigatus pathogenesis. This strategy will move us toward our long- term goal of developing novel IA therapeutics by identifying exploitable fungal-specific targets to block septum formation and growth, leading to a substantial impact on IA mortality.