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：
7997231
负责人：
CHRISTOPHER M. WEST
金额：
$ 37.89万
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/7997231
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 Future Galactose Genes Genetic Genome Glycopeptides Grant Haploidy Health 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 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）：进化已经涉及多种翻译后修饰，以提供细胞蛋白质机制的时间、空间和功能调节。该项目重点关注一种类型的具体例子，这种类型似乎是从真核细胞的分泌途径——糖基化中借用的，但实际上可能首先是在细菌细胞的细胞质中进化出来的。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”蛋白质糖基化受到不同的控制。分析的初始生物体是社会阿米巴盘基网柄菌，这里研究的途径的靶标是 Skp1，它是 E3 泛素连接酶 SCF 类的适配器，其靶标经常被磷酸化激活，因此可能需要一个独立的共价调节方式。最值得注意的是，这种修饰涉及六个酶促步骤，导致五糖组装到高度保守的脯氨酸残基上。这种修饰的结构丰富度可与肽相媲美，假设仅针对 Skp1 并调节其对关键发育转变（顶峰）的调节。脯氨酰羟化酶表达的基因操作控制发育的 O2 需求，表明该酶在 O2 调节中发挥正常作用。最近对 Skp1 的顺序羟脯氨酸依赖性糖基化所需的破坏酶基因的影响的分析为 O2 依赖性发育的层次调节的额外水平提供了证据，这将在本项目中进行表征。我们最近发现了构建五糖所需的最后一种酶（AgtA），这使我们最终能够从遗传学和生物化学角度解决这些问题。首先，我们将在目标 1 中定义两个 1-连接半乳糖的连接，这两个半乳糖的添加似乎是由 AgtA 催化的，这将为后续目标实现聚糖的化学合成。目标 2 将研究添加两种糖时 AgtA 持续加工能力的基础，以及 Skp1 和 AgtA 的催化域和 2-螺旋桨状域如何相互调节彼此的活性，假设与质量控制相关。目标 3 将采用糖基化基因的反向遗传和上位分析来测试分级调控是否是线性的或涉及平行的信号通路。此外，还将开发新的抗体来监测细胞中发出发育信号的 Skp1 糖基化的渐进变化。最后，为了确定修饰途径在功能上最重要的特征，目标 4 将在人类弓形体病的病原体弓形虫 apicomplexan Toxoplasma gondii 中进行 Skp1 糖调节的进化保守性测试。所获得的知识预计将产生关于如何在特定原生生物中响应外部信号（例如 O2）和内部信号（例如糖代谢物）来调节蛋白质组的新想法。公共健康相关性该研究利用了 NIH 模式生物盘基网柄菌，这是一种与人类寄生虫内阿米巴基因组相关的社会阿米巴原虫。盘基网柄菌因其自由生活、无壁生活方式和单倍体基因组而为分子、生化和细胞生物学研究提供了特殊优势，已被证明是各种其他类型原生生物的蛋白质糖基化选择途径的有用模型。一个例子是本研究中检查的细胞质糖基化途径。生物信息学和早期生化研究表明，该途径的主要部分存在于大型植物病原体疫霉属、人类弓形虫病弓形虫和其他原生生物中。由于弓形虫是一种细胞内病原体，盘基网柄菌为弓形虫基因的生化和反向遗传分析提供了一个有吸引力的替代宿主，弓形虫基因是该生物体中 Skp1 修饰途径的候选者。这将为未来直接研究弓形虫 O2 调节途径的功能奠定序幕，弓形虫在人群中潜在地广泛传播，如果重新激活，药物治疗极其有限。