Cell Wall Integrity Signaling in Yeast

描述（由申请人提供）：由于免疫功能低下患者人数的迅速增加，对安全有效的抗真菌药物的需求日益增长。由于人类细胞不具备构建细胞壁所需的机制，真菌病原体的细胞壁构建过程为新疗法提供了一个有吸引力的靶点。该项目的长期目标是了解酵母细胞在生长和形态发生过程中如何保持其细胞壁的结构完整性。这些研究可能会揭示合适的分子靶点，用于开发对真菌细胞具有选择性毒性的抗真菌药物。酵母细胞检测和响应细胞壁压力的主要机制是由两个细胞表面传感器家族介导的信号通路，一个小的GTPase (Rho1)，一个蛋白激酶C （Pkc1）和一个MAP激酶级联，尽管其他途径也有助于细胞壁的结构完整性。该项目的具体目的是：1)确定Pkc1是否通过在纺锤体形成过程中调节Mps1蛋白激酶来促进G2/M转变。已经积累了大量证据支持Pkc1在有丝分裂中的作用。我们的数据暗示Pkc1参与Mps1有丝分裂检查点激酶的调控。我们将验证Mps1是Pkc1靶点的假设，并探索这种磷酸化对有丝分裂的影响。2)确定Mpk1在细胞壁应激条件下是否作为转录因子。我们有令人兴奋的证据表明，Mpk1 MAP激酶可以以一种独立于其蛋白激酶活性的方式调节SBF转录因子。我们建议测试Mpk1在DMA上与SBF形成三元复合物的不同寻常的概念，并探索Mpk1驱动转录的机制。3)确定Wsc1传感器是否通过β -1,3-葡聚糖结合域被招募到极化生长位点。Wsc1的胞外区域具有一个富含cys的结构域，我们提出该结构域与葡聚糖链结合，并负责将Wsc1定位为葡聚糖合成酶。我们将通过生物化学和分子遗传学方法的结合来测试这个模型。4)了解PH结构域蛋白Ask10和Ypr115w如何促进细胞壁完整性的维持。我们已经确定了两个多余的蛋白质，其功能丧失导致细胞裂解。我们提出了一种多方面的方法来确定它们在维持细胞壁完整性中的作用。

DESCRIPTION (provided by applicant): There is a growing need for safe and effective antifungal agents that stems from the rapidly increasing population of immunecompromised patients. Because human cells do not possess the machinery needed to construct cell walls, the process of wall construction in fungal pathogens provides an attractive target for novel therapeutics. The long-term objective of this project is to understand how yeast cells maintain the structural integrity of their cell walls during growth and morphogenesis. These studies are likely to reveal suitable molecular targets for the development of antifungal agents that display selective toxicity against fungal cells. The principal mechanism by which yeast cells detect and respond to wall stress is a signaling pathway mediated by two families of cell surface sensors, a small GTPase (Rho1), protein kinase C (Pkc1), and a MAP kinase cascade, although additional pathways also contribute to the structural integrity of the wall. The specific aims of this project are 1) To determine if Pkc1 contributes to the G2/M transition by regulating the Mps1 protein kinase during spindle formation. Considerable evidence has accumulated to support a role for Pkc1 in mitosis. Our data implicates Pkc1 in the regulation of the Mps1 mitotic checkpoint kinase. We will test the hypothesis that Mps1 is a Pkc1 target and explore the mitotic effects of this phosphorylation. 2) To determine if Mpk1 acts as a transcription factor under conditions of cell wall stress. We have exciting evidence revealing that the Mpk1 MAP kinase can regulate the SBF transcription factor in a manner that is independent of its protein kinase activity. We propose to test the unusual notion that Mpk1 forms a ternary complex with SBF on the DMA and to explore the mechanism by which Mpk1 drives transcription. 3) To determine if the Wsc1 sensor is recruited to the site of polarized growth through a beta-1,3- glucan-binding domain. The extracellular region of Wsc1 possesses a cys-rich domain that we propose binds to glucan chains and is responsible for localization of Wsc1 to the glucan synthase. We will test this model by a combination of biochemical and molecular genetic approaches. 4) To understand how the PH domain proteins, Ask10 and Ypr115w, contribute to the maintenance of cell wall integrity. We have identified two redundant proteins whose loss of function results in cell lysis. We propose a multifaceted approach to establishing their role in the maintenance of cell wall integrity.