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.

概括 进化已经利用了大量的翻译后修饰来提供时间、空间和 细胞蛋白质机器的功能调节。该项目重点关注一个具体示例 似乎是从真核细胞的分泌途径糖基化中借用的类型，但是 实际上可能首先在细菌细胞的细胞质中进化。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，并受到不同的控制 相对于分泌途径中的“传统”蛋白质糖基化。分析的初始有机体是 社会性变形虫盘基网柄菌，这里研究的途径的目标是 Skp1，SCF 的适配器 一类 E3 泛素连接酶，其靶点经常被磷酸化激活，并且有 可能需要一种独立的共价调节模式。最引人注目的是这个修改 涉及六个酶促步骤，导致组装连接到高度保守的五糖 脯氨酸残基。这种修饰的结构丰富度可与肽相媲美，推测是 仅针对 Skp1 并调节其对关键发育转变（顶峰）的调节。遗传 操纵脯氨酰羟化酶表达可控制发育的 O2 需求，这表明 该酶在 O2 调节中的正常作用。破坏酶基因影响的最新分析 Skp1 的连续羟脯氨酸依赖性糖基化所需的提供了额外的证据 依赖 O2 的发展的分级调节水平，这是本项目的特征。 我们最近发现了构建五糖所需的最后一种酶 (AgtA)，这为我们奠定了基础 最后从遗传学和生物化学角度解决这些想法。首先，我们将在目标 1 中定义 两个 ¿-连接的半乳​​糖的连接，其添加似乎是由 AgtA 催化的，这将 为后续目标实现聚糖的化学合成。目标 2 将检查表面 AgtA 的基础 添加两种糖的持续过程，以及 Skp1 和 AgtA 的催化和 ¿-螺旋桨状结构域如何 相互调节彼此的活动，假设与质量控制有关。目标 3 将雇用 对糖基化基因进行反向遗传和上位分析，以测试分级调控是否有效 线性或涉及平行信号传导途径。此外，还将开发新的抗体来监测 细胞中 Skp1 糖基化的渐进变化预示着发育。最后，要识别 修饰途径功能上最重要的特征，目标 4 将进行测试 顶复门弓形虫中 Skp1 糖调节的进化保守性 人类弓形体病。所获得的知识有望产生关于蛋白质组如何运作的新想法 在特定的原生生物中响应外部信号（例如 O2）和内部信号（例如糖）而受到调节 代谢物。