PHOSPHOPROTEOME ANALYSIS OF SELF-FUSION REGULATION IN NEUROSPORA CRASSA

粗糙脉孢菌自融合调控的磷酸蛋白质组分析

• 批准号: 8170717
• 负责人: N Louise GLASS
• 金额: $ 3.21万
• 依托单位: BATTELLE PACIFIC NORTHWEST LABORATORIES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170717
• 关键词: Alleles; Animal Model; Animals; Ascomycota; Biogenesis; Biological; Cell fusion; Cells; Chemotactic Factors; Collaborations; Communication; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Defect; Development; Drug Metabolic Detoxication; Eukaryota; Fertilization; Funding; Grant; Growth; Homologous Gene; Institution; Liver; Mammals; Mitogen-Activated Protein Kinases; Molds; Molecular; Molecular Biology; Muscle; Neoplasm Metastasis; Neurospora crassa; Orthologous Gene; Partner in relationship; Pathogenesis; Pathway interactions; Phosphorylation; Phosphotransferases; Physiological; Placenta; Plants; Process; Proteins; Regulation; Research; Research Personnel; Resources; Role; Self Perception; Source; System; Tissues; United States National Institutes of Health; analog; bone; comparative; fungus; mutant; pathogen

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cell fusion is an essential process in eukaryotes. It occurs during fertilization and the biogenesis of tissues including muscle, bone and placenta. Defects in fusion also have consequences for liver detoxification and cancer metastasis. In fungi, fusion between mating cells has been extensively studied, while self-fusion between genetically identical cells remains largely uncharacterized. The filamentous fungus Neurospora crassa is a model organism for molecular biology, and is now used to study self-fusion. In N. crassa, self-fusion occurs between genetically identical cells that are in the same developmental state, the mechanisms of which are unknown in any system. Recent data has shown that N. crassa cells alternate between two different physiological states in order to facilitate communication. The conserved MAP kinase MAK2 is essential for self-fusion in N. crassa and has a direct role in chemotropic attraction between fusing cells. In addition to cell fusion, mak-2 mutants show growth defects, highlighting the importance of this pathway in filamentous fungi. Furthermore, orthologs of MAK2 are important regulators of plant and animal pathogenesis caused by a variety of ascomycete species. The molecular details of self-fusion are still unknown in N. crassa, including the identity of the chemoattractant molecule and any targets of the kinase pathway. We constructed allele MAK2Q100G, whose kinase activity can be specifically inhibited through the addition of the ATP analog 1NM-PP1. Using this allele, we showed that MAK2 kinase activity is required for MAK2 complex dynamics in the partner cell, and also for the dynamic activity of SO, a protein of unknown molecular function. The proposed collaboration aims to identify targets of MAK2, which will not only reveal proteins involved in self-fusion, but also some required for vegetative growth. Homolog comparisons will reveal targets of the orthologous kinases in related pathogenic species. Specific aims include: (1) Comparative phosphoproteome analysis of kinase active/inactive MAK2Q100G cells. (2) Identify specific phosphorylation motifs on MAK2 targets. (3) Biological characterization of the roles of identified MAK2 targets. This collaboration has the potential to significantly increase the knowledge of self-fusion mechanisms in filamentous fungi, and can be easily expanded to studies of fusion in other multicellular eukaryotes, potentially including mammals. The data obtained from this study will also have other implications, since MAK2 homologs are essential for the invasion of several pathogens.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 细胞融合是真核生物的一个重要过程。它发生在受精和肌肉、骨骼和胎盘等组织的生物发生过程中。融合缺陷也会导致肝脏解毒和癌症转移。在真菌中，交配细胞之间的融合已被广泛研究，而遗传相同的细胞之间的自我融合在很大程度上仍未确定。丝状真菌粗糙脉孢霉是一种分子生物学的模式生物，目前被用于研究自融合。在粗粒新月球藻中，自我融合发生在处于相同发育状态的遗传相同的细胞之间，其机制在任何系统中都是未知的。最近的数据表明，粗毛拟青霉细胞在两种不同的生理状态之间交替，以促进交流。保守的MAP激酶MAK2对于粗毛拟青霉的自融合是必不可少的，并且在融合细胞之间的趋化吸引中起着直接作用。除了细胞融合，mak-2突变体还显示出生长缺陷，突显了这一途径在丝状真菌中的重要性。此外，MAK2的同源基因是由多种子囊菌引起的动植物致病的重要调节因子。 粗粒新月形藻自我融合的分子细节尚不清楚，包括趋化分子的身份和激酶途径的任何靶标。我们构建了等位基因MAK2Q100G，其激酶活性可以通过添加ATP类似物1 nm-PP1而被特异性地抑制。利用这个等位基因，我们证明了MAK2激酶活性是伴侣细胞中MAK2复合体动力学所必需的，也是分子功能未知的蛋白质SO的动态活性所必需的。这项拟议的合作旨在确定MAK2的靶点，这不仅将揭示参与自我融合的蛋白质，还将揭示一些营养生长所需的蛋白质。同源比较将揭示相关致病物种中同源激酶的靶标。 具体目标包括： (1)蛋白激酶激活/失活MAK2Q100G细胞的比较蛋白组学分析。 (2)鉴定MAK2靶点上的特异性磷酸化基序。 (3)识别的MAK2靶标作用的生物学特性。 这种合作有可能显著增加对丝状真菌自我融合机制的了解，并可以很容易地扩展到其他多细胞真核生物的融合研究，潜在地包括哺乳动物。从这项研究中获得的数据还将具有其他意义，因为MAK2同源基因对于几种病原体的入侵是必不可少的。