Diverse Roles for RIC8 During G Protein Signaling in Fungi

RIC8 在真菌 G 蛋白信号转导过程中的多种作用

• 批准号: 8231349
• 负责人: KATHERINE A BORKOVICH
• 金额: $ 29.86万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8231349
• 关键词: Actins; Alleles; Animal Model; Animals; Antifungal Agents; Base Sequence; Binding; Biological Assay; Bypass; Caenorhabditis elegans; Cell division; Cells; Cellular Morphology; Cleaved cell; Co-Immunoprecipitations; Complex; Cycloheximide; Cytoskeleton; Defect; Development; Drosophila genus; Drug Delivery Systems; Embryo; Employee Strikes; Eukaryota; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genes; Genetic; Genetic Epistasis; Genetic Screening; Growth; Growth and Development function; Guanine Nucleotide Exchange Factors; Guanine Nucleotides; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Homologous Gene; Human; Hydrolysis; Immunofluorescence Microscopy; Infection; Knock-out; Lead; Measures; Methods; Microtubules; Modeling; Molds; Molecular Chaperones; Molecular Genetics; Mutation; Neurospora; Neurospora crassa; Organism; Pathway interactions; Peptide Hydrolases; Phenotype; Physiologic pulse; Plants; Protein Subunits; Proteins; Proteolysis; Regulation; Role; Saccharomyces cerevisiae; Sexual Development; Signal Transduction; Signal Transduction Pathway; Single Nucleotide Polymorphism; System; Testing; Time; Translations; Tubulin; Vesicle; Virulence; Yeasts; asexual; base; cell type; dimer; fungus; microbial; multicatalytic endopeptidase complex; mutant; neuroblast; novel; presynaptic; public health relevance; research study; ubiquitin-protein ligase; zygote

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.

描述（由申请人提供）：异三聚体（A ??）G蛋白是信号转导途径的重要组成部分，可调节真核生物的环境感应，生长和发展。 G蛋白偶联受体（GPCR）的特征是GA亚基的鸟嘌呤核苷酸交换因子（GEFS）。最近，非GPCR GEF（例如RIC8蛋白）促进了GDP/GTP交换，已成为动物中GA调节的重要机制。 RIC8是秀丽隐杆线虫和果蝇的不对称细胞分裂所必需的。 RIC8的损失导致果蝇中G蛋白水平降低，表明除GEF活性外，伴侣功能。 RIC8不含植物，原生和酿酒酵母的酵母菌。 RIC8在动物中的功能与模型真核丝状真菌神经孢子Crassa之间存在惊人的相似性。 RIC8的缺失导致神经孢菌的生长和无性发育的严重缺陷，类似于缺乏GA基因GNAES GNA-1和GNA-3的突变体观察到的。 ？RIC8菌株的G蛋白水平大大降低，GNA-1和GNA-3的组成激活等位基因营救了？RIC8突变体的许多缺陷，初步结果支持RIC8和GNA-1和GNA-1和GNA-3之间的物理相互作用。此外，丝状真菌是动物不对称细胞分裂的绝佳模型，而神经孢子是包含RIC8同源物的最佳特征微生物系统。基于这些观察结果，我们假设RIC8通过充当GNA-1和GNA-3和/或通过用作保护G蛋白质异三聚体免受蛋白酶降解的蛋白伴侣的GEF来调节神经孢子中的细胞分裂。具体目的是：1）确定导致缺乏G的菌株中G蛋白水平较低的机制？二聚体或RIC8。 G ric8和g中G蛋白亚基的稳定性？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药物的存在下确定。 G蛋白水平将在缺乏E3连接酶复合物成分的突变体中进行测量。 2）表征RIC8和G蛋白之间的相互作用。缺乏RIC8和G的突变体？基因将进行分析。 RIC8和G蛋白之间的物理相互作用将使用共免疫沉淀进行探测。 RIC8将测试GA GEF活性。 3）探索RIC8与GA蛋白协同作用以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RIC8，RHO-4，GA和G的定位？蛋白质将在各种突变应变背景中确定。 RIC8将测试用于RHO-4的GEF活性。 4）使用遗传筛选确定新的途径成分和旁路机制。影响动物中RIC8功能的基因突变将在“ RIC8背景”中测试作用。一种新的单核苷酸多态性裂解基于扩增的多态性序列方法（SNP-CAP）将用于克隆在？gna-1？gna-3和？ric8遗传背景中分离出来的抑制器。敲除项目的突变体将被筛选，以获取与ric8和G蛋白突变体共同的缺陷。 公共卫生相关性：这些研究将揭示与动物共享的Neurospora Crassa中新型G蛋白信号传导机制，但在酿酒酵母中未发现。获得的信息将适用于针对人类细胞中RIC8-G蛋白途径的新药物的开发。此外，由于RIC8在丝状真菌中是保守的，并且在这类生物体中毒力需要G蛋白，因此这些研究还将揭示可用于治疗人类感染的抗真菌剂的新靶标。

项目成果

RIC8 is a guanine-nucleotide exchange factor for Galpha subunits that regulates growth and development in Neurospora crassa.

RIC8 是 Galpha 亚基的鸟嘌呤核苷酸交换因子，可调节粗糙脉孢菌的生长和发育。

• DOI: 10.1534/genetics.111.129270
• 发表时间: 2011
• 期刊: Genetics
• 影响因子: 3.3
• 作者: Wright,SaraJ;Inchausti,Regina;Eaton,CarlaJ;Krystofova,Svetlana;Borkovich,KatherineA
• 通讯作者: Borkovich,KatherineA

Analysis of mitogen-activated protein kinase phosphorylation in response to stimulation of histidine kinase signaling pathways in Neurospora.

分析脉孢菌中组氨酸激酶信号通路刺激后丝裂原激活蛋白激酶的磷酸化。

• DOI: 10.1016/s0076-6879(10)71017-x
• 发表时间: 2010
• 期刊: Methods in enzymology
• 作者: Jones,CarolA;Borkovich,KatherineA
• 通讯作者: Borkovich,KatherineA