Glycoregulation of Skp1 in the cytoplasm and nucleus

Skp1 在细胞质和细胞核中的糖调节

• 批准号: 8065710
• 负责人: CHRISTOPHER M. WEST
• 金额: $ 9.84万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 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/8065710
• 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; response; social; sugar; ubiquitin-protein ligase

SUMMARY 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 ¿-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 ¿-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.

总结 进化已经招募了大量的翻译后修饰，以提供时间，空间和生物学特性。 细胞蛋白质机器的功能调节。这个项目的重点是一个具体的例子， 糖基化似乎是从真核细胞的分泌途径中借来的，但 可能最早是在细菌细胞的细胞质中进化出来的。我们认为，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，并受到不同的控制 相对于分泌途径中的“常规”蛋白质糖基化。最初的分析有机体是 社会阿米巴Dictyosteoba，这里研究的通路的靶点是SCF的适配器Skp1 一类E3泛素连接酶，其靶标经常被磷酸化激活， 可能需要一种独立的共价调控模式。最值得注意的是， 涉及六个酶促步骤，导致五糖连接到高度保守的 脯氨酸残基。这种修饰具有与肽相匹敌的结构丰富性，被假设为 仅靶向Skp1并调节其对关键发育转变（顶点）的调节。遗传 脯氨酰羟化酶表达的操纵控制了发育对O2的需求，这表明 这种酶在O2调节中的正常作用。酶基因干扰效应的最新分析 Skp1的顺序羟脯氨酸依赖性糖基化所需的蛋白质提供了额外的证据， O2依赖性发展的分级调节水平，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）， 最后，从遗传学和生物化学的角度来阐述这些想法。首先，我们将在目标1中定义 两个半乳糖的连接，其加成似乎是由AgtA催化的， 使得能够化学合成聚糖以用于以后的目的。目标2将检查表观AgtA的基础 添加两种糖的持续合成能力，以及Skp1和AgtA的催化和螺旋桨样结构域如何 相互调节彼此的活动，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性分析，以测试分级调节是否是 线性或涉及平行信号传导途径。此外，还将开发新的抗体， 细胞中Skp1糖基化的进行性变化，这是发育的信号。最后，为了确定 功能上最重要的修饰途径的特点，目的4将进行测试， Skp1糖调节在顶复门弓形虫中的进化保守性， 人类弓形体病所获得的知识有望产生关于蛋白质组是如何 在选择原生生物中响应于外部信号如O2和内部信号如糖 代谢物。