Diverse Roles for RIC8 During G Protein Signaling in Fungi

描述(申请人提供)：杂三聚体(a？？)G蛋白是调控真核生物环境感知、生长发育的信号转导通路的重要组成部分。G蛋白偶联受体(GPCRs)是G亚基的鸟嘌呤核苷酸交换因子(GEF)。最近，通过非GPCRGEF促进GDP/GTP交换，如RIC8蛋白，已成为动物GA调节的重要机制。在线虫和果蝇中，RIC8是细胞不对称分裂所必需的。在果蝇中，ric8的缺失会导致G蛋白水平的降低，这表明除了全球环境基金的活性外，还有一种伴侣功能。RIC8不存在于植物、原生生物和酿酒酵母中。 RIC8在动物体内的功能与模式真核丝状真菌粗糙脉孢菌有显著的相似之处。缺失ric8基因会导致脉孢子虫生长、无性和有性发育的严重缺陷，类似于观察到的缺失GA基因gna-1和gna-3的突变体的情况。RIC8菌株的G蛋白水平显著降低，GNA-1和GNA-3的成分激活等位基因挽救了RIC8突变株的许多缺陷，初步结果支持RIC8与GNA-1和GNA-3之间的物理相互作用。此外，丝状真菌是动物细胞不对称分裂的优秀模型，而脉孢子菌是含有RIC8同系物的最具特征的微生物系统。基于这些观察，我们假设RIC8通过作为GNA-1和GNA-3的环境基金和/或作为保护G蛋白异源三聚体不被蛋白酶体降解的蛋白伴侣来调节脉孢子菌的细胞分裂。具体目的是：1)确定导致缺乏G蛋白菌株的G蛋白水平低的机制(S)。二聚体或RIC8。G蛋白亚基在RIC8和G？突变体将在影响空泡蛋白水解酶或蛋白酶体的蛋白分解活性的试剂存在的情况下确定。G蛋白水平将在缺乏E3连接酶复合体成分的突变体中进行测量。2)研究RIC8和G蛋白之间的相互作用。缺失ric8和G？？的突变体将对基因进行分析。RIC8和G蛋白之间的物理相互作用将使用免疫共沉淀进行探索。将对RIC8进行镓全球环境基金活性测试。3)探讨RIC8与GA蛋白协同作用调节细胞分裂和不对称分裂的可能性。肌动蛋白、微管蛋白和GFP标记的RIC8、Rho-4、GA和G？蛋白质将在不同的突变菌株背景下确定。RIC8将接受全球环境基金针对RHO-4的活性测试。4)利用遗传筛选识别新的通路组件和旁路机制。影响动物ric8功能的基因突变将在ric8背景下进行效果测试。一种新的单核苷酸多态切割扩增多态序列方法(SNP-CAPS)将用于克隆分离于GNA-1、GNA-3和RIC8遗传背景中的抑制子。来自基因敲除项目的突变体将被筛选出RIC8和G蛋白突变体的共同缺陷。 公共卫生相关性：这些研究将揭示粗糙脉孢菌中新的G蛋白信号机制，这些机制与动物相同，但在酿酒酵母中没有发现。所获得的信息将适用于针对人类细胞中RIC8-G蛋白通路的新药的开发。此外，由于RIC8在丝状真菌中保守，而G蛋白是这类生物毒力所必需的，这项研究还将揭示可用于治疗人类感染的抗真菌药物的新靶点。

DESCRIPTION (provided by applicant): Heterotrimeric (a??) G proteins are essential components of signal transduction pathways that regulate environmental sensing, growth and development in eukaryotes. G protein coupled receptors (GPCRs) have been characterized as guanine nucleotide exchange factors (GEFs) for Ga subunits. Recently, facilitated GDP/GTP exchange by non-GPCR GEFs, such as the RIC8 protein, has emerged as an important mechanism for Ga regulation in animals. RIC8 is required for asymmetric cell division in both Caenorhabditis elegans and Drosophila. Loss of ric8 leads to reduced levels of G proteins in Drosophila, suggesting a chaperone function in addition to GEF activity. RIC8 is absent from plants, protists and the yeast Saccharomyces cerevisiae. There are striking similarities between the functions of RIC8 in animals and the model eukaryotic filamentous fungus Neurospora crassa. Deletion of ric8 leads to severe defects in growth and asexual and sexual development in Neurospora, similar to those observed for a mutant lacking the Ga genes gna-1 and gna-3. ?ric8 strains have greatly reduced levels of G proteins, constitutively-activated alleles of gna-1 and gna- 3 rescue many defects of ?ric8 mutants and preliminary results support a physical interaction between RIC8 and GNA-1 and GNA-3. Furthermore, filamentous fungi are excellent models for asymmetric cell division in animals and Neurospora is the best-characterized microbial system that contains a RIC8 homologue. Based on these observations, we hypothesize that RIC8 modulates cell division in Neurospora by acting as a GEF for GNA-1 and GNA-3 and/or by serving as a protein chaperone that protects the G protein heterotrimer from degradation by the proteasome. The Specific Aims are: 1) Determine the mechanism(s) that lead to low levels of G proteins in strains lacking the G?? dimer or RIC8. The stability of G protein subunits in ?ric8 and G?? mutants will be determined in the presence of agents that influence the proteolytic activity of vacuolar proteases or the proteasome. G protein levels will be measured in mutants lacking components of E3 ligase complexes. 2) Characterize interactions between RIC8 and G proteins. Mutants lacking ric8 and G?? genes will be analyzed. Physical interactions between RIC8 and G proteins will be probed using co-immunoprecipitation. RIC8 will be tested for Ga GEF activity. 3) Explore the possibility that RIC8 acts in concert with Ga proteins to regulate septation and asymmetrical cell division. The localization of actin, tubulin and GFP-tagged RIC8, RHO-4, Ga and G? proteins will be determined in various mutant strain backgrounds. RIC8 will be tested for GEF activity toward RHO-4. 4) Identify novel pathway components and bypass mechanisms using genetic screens. Mutations in genes that influence ric8 function in animals will be tested for effects in the ?ric8 background. A new Single Nucleotide Polymorphism Cleaved Amplified Polymorphic Sequence-based method (SNP-CAPS) will be used to clone suppressors isolated in the ?gna-1 ?gna-3 and ?ric8 genetic backgrounds. Mutants from the knockout project will be screened for defects common to ?ric8 and G protein mutants. PUBLIC HEALTH RELEVANCE: These studies will reveal novel G protein signaling mechanisms in Neurospora crassa that are shared with animals, but are not found in the yeast Saccharomyces cerevisiae. The information obtained will be applicable to development of new drugs that target RIC8-G protein pathways in human cells. Moreover, because RIC8 is conserved in filamentous fungi and G proteins are required for virulence in this class of organisms, the studies will also reveal new targets for antifungal agents that can be used to treat infections in humans.