Glycoregulation of Skp1 in the cytoplasm and nucleus

Skp1 在细胞质和细胞核中的糖调节

基本信息

批准号：
7744704
负责人：
CHRISTOPHER M. WEST
金额：
$ 26.48万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-01-01 至 2012-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7744704
关键词：
Address Affect Ally Amoeba genus Animal Model Animals Antibodies Binding Biochemical Biochemical Genetics Bioinformatics Biological Candidate Disease Gene Cell Nucleus Cell surface Cells Chimera organism Competence Complement Complex Cyclic AMP-Dependent Protein Kinases Cytoplasm Cytoplasmic Protein Development Dictyostelium Dictyostelium discoideum Entamoeba Enzyme Gene Enzymes Eukaryota Eukaryotic Cell Evolution Figs - dietary Future Galactose Genes Genetic Genome Glycopeptides Grant Haploidy Human Hydroxylation Hydroxyproline In Vitro Investigation Knock-out Knowledge Life Life Style Link Medical Modeling Modification Molecular Monitor Mutate Mutation Organelles Organism Orthologous Gene Parasites Pathway interactions Peptides Peripheral Phosphorylation Phytophthora Plants Polysaccharides Population Positioning Attribute Post-Translational Protein Processing Procollagen-Proline Dioxygenase Proline Protein Glycosylation Proteins Proteome Quality Control Regulation Relative (related person)Role Signal Pathway Signal Transduction Testing Toxoplasma Toxoplasma gondii Toxoplasmosis Trisaccharides United States National Institutes of Health Variant Work base cell type chemical synthesis enzyme activity extracellular genetic analysis genetic manipulation glycosylation glycosyltransferase in vivo interest mutant novel overexpression p19(SKP1) Protein pathogen positional cloning public health relevance response social sugar ubiquitin-protein ligase

项目摘要

DESCRIPTION (provided by applicant): Evolution has enlisted a large variety of posttranslational modifications to provide temporal, spatial and functional regulation of the protein machinery of the cell. This project focuses on a specific example of a type that has seemingly been borrowed from the secretory pathway of eukaryotic cells, glycosylation, but might actually have first evolved in the cytoplasm of bacterial cells. We propose that complex cytoplasmic glycosylation exerts unique glycoregulatory functions in eukaryotes, and is subject to distinct controls relative to `conventional' protein glycosylation in the secretory pathway. The initial organism of analysis is the social amoeba Dictyostelium, and the target of the pathway studied here is Skp1, an adaptor of the SCF class of E3 ubiquitin ligases whose targets are frequently activated by phosphorylation and for which there may be a need for an independent mode of covalent regulation. Most remarkable is that this modification involves six enzymatic steps resulting in the assembly of a pentasaccharide attached to a highly conserved residue of proline. This modification, with a structural richness rivaling that of a peptide, is hypothesized to target only Skp1 and modulate its regulation of a critical developmental transition (culmination). Genetic manipulation of prolyl hydroxylase expression controls the O2-requirement for development suggesting a normal role for this enzyme in O2-regulation. Recent analysis of the effects of disrupting enzyme genes required for the sequential hydroxyproline-dependent glycosylation of Skp1 gives evidence for additional levels of hierarchical regulation of O2-dependent development, which is to be characterized in this project. Our recent discovery of the last enzyme (AgtA) needed to construct the pentasaccharide has positioned us finally to address these ideas genetically and biochemically. At the outset, we will in aim 1 define the linkages of the two 1-linked galactose sugars whose additions appear to be catalyzed by AgtA, which will enable chemical synthesis of the glycan for the later aims. Aim 2 will examine the basis for apparent AgtA processivity in adding the two sugars, and how Skp1 and the catalytic and 2-propeller-like domains of AgtA mutually regulate each other's activity, hypothesized to be associated with quality control. Aim 3 will employ reverse genetic and epistatic analysis of the glycosylation genes to test whether hierarchical regulation is linear or involves parallel signaling pathways. In addition, new antibodies will be developed to monitor progressive variations in Skp1 glycosylation in the cells which signal development. Finally, to identify the functionally most important features of the modification pathway, aim 4 will carry out tests for the evolutionary conservation of Skp1 glycoregulation in the apicomplexan Toxoplasma gondii, the agent for human toxoplasmosis. The knowledge gained is expected to generate new ideas of how the proteome is regulated in select protists in response to external signals such as O2 and internal signals such as sugar metabolites. PUBLIC HEALTH RELEVANCE The study utilizes the NIH model organism Dictyostelium discoideum, a social amoeba allied genomically with the human parasite Entamoeba. Dictyostelium, which offers special advantages for molecular, biochemical and cell biological studies owing to its free-living, wall-less lifestyle and haploid genome, has proven to be a useful model for select pathways of protein glycosylation of various other types of protists. One example is the cytoplasmic glycosylation pathway examined in this investigation. Bioinformatics and early biochemical studies indicate that the main part of the pathway is present in the large Phytophthora group of plant pathogens, the agent for human toxoplasmosis Toxoplasma gondii, and other protists. Since T. gondii is an intracellular pathogen, Dictyostelium offers an attractive surrogate host for the biochemical and reverse genetic analysis of the Toxoplasma genes that are candidates for the Skp1 modification pathway in this organism. This will serve as a prelude for future direct studies on the function of the pathway for O2-regulation of T. gondii, which is widely disseminated latently in the human population and for which, if it is re-activated, pharmacological therapies are extremely limited.

描述（由申请人提供）：进化已吸引了多种翻译后修饰，以提供细胞蛋白质机械的时间，空间和功能调节。该项目着重于似乎是从真核细胞（糖基化）分泌途径中借来的一种类型的特定示例，但实际上可能首先在细菌细胞的细胞质中演变。我们提出，复杂的细胞质糖基化在真核生物中发挥独特的糖调节功能，并且相对于分泌途径中的“常规”蛋白糖基化，也要受到不同的控制。分析的最初有机体是社会变形虫柱状固有的，此处研究的途径的靶标是SKP1，SKP1是E3泛素连接酶SCF类的适配器，其靶标经常被磷酸化激活，并且可能需要独立的共价调节模式。最引人注目的是，这种修饰涉及六个酶促的步骤，导致组装连接到高度保守的脯氨酸残基上的五糖。假设这种修饰具有与肽的结构丰富度相媲美的修饰，以仅针对SKP1并调节其对关键发育过渡（Culmination）的调节。丙酰羟化酶表达的遗传操纵控制着开发的O2征值，这表明该酶在O2-调节中的正常作用。最近对破坏SKP1顺序羟基依赖性糖基所需的破坏酶基因的作用的分析为O2依赖性发育的其他层次结构调节提供了证据，这在该项目中应表征。我们最近对构建五糖的最后一个酶（AGTA）的发现最终使我们定位了这些思想，从遗传和生化上解决了这些思想。首先，我们将在AIM 1中定义两种1连锁半乳糖糖的连锁，它们的添加似乎是由AGTA催化的，这将使聚糖化学合成以后的目标。 AIM 2将检查添加两种糖的明显AGTA加工率的基础，以及SKP1以及AGTA的催化和2型螺旋桨样结构域如何相互调节彼此的活性，假设与质量控制有关。 AIM 3将对糖基化基因进行反向遗传和上皮分析，以测试层次调节是线性还是涉及并行信号通路。此外，将开发新的抗体，以监测信号发育的细胞中SKP1糖基化的逐步变化。最后，为了确定修饰途径的功能上最重要的特征，AIM 4将进行测试，以进化在Apicomplexan毒素弓形虫中的SKP1糖调节（人类弓形虫病的药物）。预计所获得的知识将产生有关如何在某些原生物中调节蛋白质组的新想法，该蛋白质组应响应外部信号（例如O2和内部信号），例如糖代谢物。公共卫生相关性这项研究利用了NIH模型生物Dictyostelium Discoideum，这是一种与人类寄生虫Entamoeba的社会变形虫结盟。事实证明，由于其自由生活，无壁的生活方式和单倍体基因组，dictyostelium为分子，生化和细胞生物学研究提供了特殊优势，已被证明是对其他各种类型生物的蛋白质糖基化的精选途径的有用模型。一个例子是在本研究中检查的细胞质糖基化途径。生物信息学和早期生物化学研究表明，该途径的主要部分存在于大型植物病原体，人类弓形虫病弓形虫弓形虫的药物和其他生物学家中。由于T. gondii是一种细胞内病原体，因此Dictyostelium为弓形虫基因的生化和反向遗传分析提供了一种有吸引力的替代宿主，这些基因是该生物体中SKP1修饰途径的候选者。这将是对未来直接研究T. gondii的途径功能的直接研究的前奏，该途径在人群中被广泛传播，如果重新激活该途径，药理学疗法非常有限。