Cell Wall Integrity Signaling in Yeast

描述（由申请人提供）：由于免疫受损患者人群的迅速增加，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，因此真菌病原体中的细胞壁构建过程为新型疗法提供了有吸引力的靶点。该项目的长期目标是了解酵母细胞在生长过程中和面对渗透压时如何保持其细胞壁的结构完整性。这些研究可能揭示合适的分子靶点，用于开发对真菌细胞显示选择性毒性的抗真菌剂。酵母细胞检测和响应细胞壁应激的主要机制是通过细胞壁完整性（CWI）信号传导途径，其将在细胞表面产生的应激信号传递到激活MAP激酶级联的GTdR开关。然而，有一个额外的途径，有助于响应低渗休克的细胞壁的结构完整性。该途径通过Fps 1甘油通道释放细胞内甘油而降低膨压。本研究的具体目的是：1）确定Mpk 1细胞壁应激MAP激酶与Paf 1-RNA聚合酶转录延伸复合物（PafC）结合时的功能。最近的发现表明，Mpk 1通过非催化机制激活其转录程序的一个子集，该机制涉及其与其转录靶点的启动子和编码区的关联。新的数据表明，Mpk 1从启动子移动到PafC。实验中描述了解剖的功能MPK 1的背景下，这个复杂的。2)建立CWI信号与DNA损伤检查点信号之间关系的本质。Mpk 1在DNA损伤剂的作用下被激活，但其细胞壁转录程序在这些条件下不被激活。据推测，由DNA损伤检查点激酶引起的Mpk 1上的新型丝氨酸磷酸化将其从细胞壁靶点重定向到与DNA损伤反应相关的其他功能。提出实验来验证这一假设，并确定MPK 1在这种反应中的作用。3)建立Fps 1甘油通道的一对新调节剂Rgc 1和Rgc 2激活Fps 1的机制，并确定这些蛋白质上响应各种应激信号的调节磷酸化位点。初步数据表明，Rgc 1/2作为多个蛋白激酶的调节节点。实验提出，以确定RGC 1/2的上游和下游的通路组件。4)确定CWI通路的Pkc 1蛋白激酶是否通过RSC染色质重塑复合物促进G2/M转换。最近发现Pkc 1和RSC复合物的Rsc 1亚基之间的物理相互作用表明Pkc 1有助于G2/M转换的机制。提出实验来测试这种可能性。 公共卫生相关性：由于免疫功能低下患者的迅速增加，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，因此真菌病原体中的细胞壁构建过程为新型疗法提供了有吸引力的靶点。拟议的研究可能会揭示合适的分子靶点，用于开发对真菌细胞显示选择性毒性的抗真菌剂。

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 in the face of osmotic stress. 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 cell wall stress is through the Cell Wall Integrity (CWI) signaling pathway, which transmits stress signals generated at the cell surface to a GTPase switch that activates a MAP kinase cascade. However, there is an additional pathway that contributes to the structural integrity of the cell wall in response to hypo-osmotic shock. This pathway culminates in the reduction of turgor pressure by release of intracellular glycerol through the Fps1 glycerol channel. The specific aims of this project are 1) To determine the function of the Mpk1 cell wall stress MAP kinase when bound to the Paf1-RNA polymerase transcription elongation complex (PafC). Recent discoveries have revealed that Mpk1 activates a subset of its transcriptional program through a non-catalytic mechanism that involves its association with the promoters and coding regions of its transcriptional targets. New data suggests that Mpk1 moves from the promoter to the PafC. Experiments are described to dissect the function of Mpk1 within the context of this complex. 2) To establish the nature of the relationship between CWI signaling and DNA damage checkpoint signaling. Mpk1 is activated in reponse to DNA damaging agents, but its cell wall transcriptional program is not activated under these conditions. It is hypothesized that novel serine phosphorylations on Mpk1 provoked by DNA damage checkpoint kinases redirect it from cell wall targets to other functions relevant to the DNA damage response. Experiments are proposed to test this hypothesis and to identify the role of Mpk1 in this response. 3) To establish the mechanism by which a pair of novel regulators of the Fps1 glycerol channel, Rgc1 and Rgc2, activate Fps1 and to identify regulatory phosphorylation sites on these proteins in response to various stress signals. Preliminary data suggest that Rgc1/2 serve as regulatory nodes for multiple protein kinases. Experiments are proposed to identify pathway components both upstream and downstream of Rgc1/2. 4) To determine if the Pkc1 protein kinase of the CWI pathway contributes to the G2/M transition through the RSC chromatin remodeling complex. A recently-discovered physical interaction between Pkc1 and the Rsc1 subunit of the RSC complex suggests a mechanism by which Pkc1 contributes to the G2/M transition. Experiments are proposed to test this possibility. PUBLIC HEALTH RELEVANCE: There is a growing need for safe and effective antifungal agents that stems from the rapidly increasing population of immune-compromised 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. Proposed studies are likely to reveal suitable molecular targets for the development of antifungal agents that display selective toxicity against fungal cells.