Diverse Roles for RIC8 During G Protein Signaling in Fungi

性状（由申请人提供）：异源三聚体（a？？）G蛋白是调节真核生物环境感知、生长和发育的信号转导途径的重要组成部分。G蛋白偶联受体（GPCRs）是G亚基的鸟嘌呤核苷酸交换因子（GEFs）。最近，促进GDP/GTP交换的非GPCR GEF，如RIC 8蛋白，已成为一个重要的机制，Ga调节动物。RIC 8是秀丽隐杆线虫和果蝇细胞不对称分裂所必需的。ric 8基因缺失导致果蝇G蛋白水平降低，提示除了GEF活性外，还有分子伴侣功能。RIC 8在植物、原生生物和酿酒酵母中不存在。 RIC 8在动物中的功能与模式真核丝状真菌粗糙脉孢菌（Neurospora crassa）的功能有惊人的相似之处。缺失ric 8导致严重的缺陷，在生长和无性和有性发育的脉孢菌，类似于观察到的突变体缺乏Ga基因gna-1和gna-3。? ric 8菌株的G蛋白水平大大降低，组成型激活的gna-1和gna- 3等位基因拯救了许多缺陷？ric 8突变体和初步结果支持RIC 8与GNA-1和GNA-3之间的物理相互作用。此外，丝状真菌是动物中不对称细胞分裂的极好模型，脉孢菌是含有RIC 8同源物的最佳表征的微生物系统。基于这些观察结果，我们假设RIC 8通过充当GNA-1和GNA-3的GEF和/或通过充当保护G蛋白异源三聚体免受蛋白酶体降解的蛋白伴侣来调节脉孢菌中的细胞分裂。具体目的是：1）确定导致缺乏G蛋白的菌株中G蛋白水平低的机制。二聚体或RIC 8。G蛋白亚基在细胞内的稳定性？Ric 8和G？突变体将在影响液泡蛋白酶或蛋白酶体的蛋白水解活性的试剂存在下测定。将在缺乏E3连接酶复合物组分的突变体中测量G蛋白水平。2)描述RIC 8和G蛋白之间的相互作用。缺失ric 8和G？基因将被分析。RIC 8和G蛋白之间的物理相互作用将使用免疫共沉淀进行探测。将对RIC 8进行Ga GEF活动测试。3)探索RIC 8与Ga蛋白协同作用调节分隔和不对称细胞分裂的可能性。肌动蛋白，微管蛋白和GFP标记的RIC 8，RHO-4，Ga和G？将在各种突变株背景中测定蛋白质。将对RIC 8进行测试，以了解全球环境基金在RHO-4方面的活动。4)使用基因筛选识别新的通路成分和旁路机制。在动物中影响ric 8功能的基因突变将被测试在？ric 8背景。一个新的单核苷酸多态性切割扩增多态性序列为基础的方法（SNP-CAPS）将被用来克隆抑制分离在？gna-1？gna-3和？ric 8基因背景。基因敲除项目中的突变体将被筛选出常见的缺陷？ric 8和G蛋白突变体。 公共卫生相关性：这些研究将揭示粗糙脉孢菌中与动物共享的新G蛋白信号传导机制，但在酵母酿酒酵母中没有发现。所获得的信息将适用于开发靶向人类细胞中RIC 8-G蛋白通路的新药。此外，由于RIC 8在丝状真菌中是保守的，而G蛋白是这类生物体毒力所必需的，因此这些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.