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：
8197789
负责人：
CHRISTOPHER M. WEST
金额：
$ 34.56万
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 2013-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8197789
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 Human Hydroxylation Hydroxyproline In Vitro Investigation Knock-out Knowledge Life Life Style Link 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

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、空间和该项目重点关注细胞蛋白质机制的功能调节。似乎是从真核细胞的分泌途径糖基化中借用的类型，但是我们认为复杂的细胞质实际上可能首先在细菌细胞的细胞质中进化。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”蛋白质糖基化分析的初始生物体是。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，最值得注意的是这种修饰。涉及六个酶促步骤，导致组装连接到高度保守的五糖这种修饰具有可与肽相媲美的结构丰富性。仅针对 Skp1 并调节其对关键发育转变（基因顶峰）的调节。操纵脯氨酰羟化酶表达可控制发育的 O2 需求，这表明这种酶在 O2 调节中的正常作用最近对破坏酶基因的影响进行了分析。 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基因和生物化学角度解决这些想法首先，我们将在目标 1 中定义两者的联系 ¿ - 连接的半乳糖，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 和催化作用如何？ -AgtA 的螺旋桨状结构域相互监管彼此的活动，追求与质量控制相关。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 糖基化的渐进变化是发育的信号。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虫病有望产生关于蛋白质组的新想法。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。