New Methods to Access GPI-Anchored Proteins and Study GPI-Anchored Proteomics

获取 GPI 锚定蛋白和研究 GPI 锚定蛋白质组学的新方法

基本信息

批准号：
9027236
负责人：
Zhongwu Guo
金额：
$ 8.15万
依托单位：
WAYNE STATE UNIVERSITY
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：
2009-09-30 至 2017-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9027236
关键词：
Address Affinity Affinity Chromatography Anabolism Bacterial Toxins Binding Biocompatible Materials Biological Biological Process Biotin Catalytic Domain Cell membrane Cell surface Cells Chemicals Chemistry Cleaved cell Complex Coupled Coupling Development Disease Endoplasmic Reticulum Enzymes Eukaryotic Cell Event GPI Membrane Anchors Glycolipids Glycopeptides Glycoproteins Glycosylphosphatidylinositols Goals HSV glycoprotein C Health Human In Vitro Investigation Label Lead Ligation Link Literature Membrane Membrane Glycoproteins Membrane Proteins Methods Molecular Nature Pathologic Processes Pathway interactions Peptides Pharmaceutical Preparations Phosphatidylinositols Phospholipase C Play Post-Translational Protein Processing Property Protein Binding Protein Biosynthesis Protein C Proteins Proteomics Recombinant Proteins Recombinants Research Research Project Grants Role Sepharose Signal Transduction Structure-Activity Relationship Techniques Tissues Trypanosoma brucei brucei analog base biophysical analysis chemical synthesis design disease diagnosis extracellular genetically modified cells innovation link protein metabolic engineering molecular marker novel novel diagnostics novel strategies novel therapeutics research study sortase synthetic peptide tool transamidases

项目摘要

DESCRIPTION (provided by applicant): New Methods to Access GPI-Anchored Proteins and Study GPI-Anchored Proteomics Glycosylphosphatidylinositol (GPI) attachment to the protein and glycoprotein C-terminus is an important and ubiquitous posttranslational modification in eukaryotic species, which helps anchor proteins and glycoproteins to the extracellular membrane. GPI-anchored proteins and glycoproteins play a pivotal role in various biological and pathological processes. However, currently, detailed studies on these molecules and their functions are limited, mainly because of the difficulty to access them in pure form and sufficient quantity and the lack of proper tools to analyze these diverse, complex, and amphipathic molecules. Therefore, it is highly desirable to have strategies that can facilitate access to and investigation of GPI-anchored proteins and glycoproteins. The ultimate goals of this research project are to develop strategies that enable access to homogeneous and structurally defined natural GPI-anchored proteins and glycoproteins and strategies that enable rapid, effective isolation and analysis of GPI-anchored proteins and glycoproteins. Accordingly, this proposal has three specific aims. Aim 1 is to prepare both the recombinant catalytic subunit GPI8 of GPI transamidase (GPI-T), the natural enzyme used by eukaryotic cells to attach GPIs to proteins, and membrane-associated intact GPI-T derived from the cell endoplasmic reticulum (ER) and uses them to create a potentially general method for enzymatic synthesis of natural GPI-anchored proteins and glycoproteins. Aim 2 is to develop a practical strategy for the study of GPI- anchored proteins expressed by cells via metabolic engineering of GPI-anchored protein biosynthetic pathways, namely, to give cells or isolated ERs a tagged synthetic GPI analog that can be used by GPI-T to add to proteins bound for GPI attachment. This will result in the specific labeling of GPI-anchored proteins to enable their rapid isolation and then MS-based proteomics analysis. Aim 3 is to develop a practical strategy for the study of cell surface GPI-anchored proteomics by using CAPM factor, a bacterial toxin that has a high-affinity binding to GPI anchors, to facilitate the isolation of GPI-linked proteins and glycoproteins released from cells upon treatment with phosphatidylinositol-specific phospholipase C enzyme and subsequent GPI-anchored proteomics analysis. Both the strategy for natural GPI-anchored protein and glycoprotein synthesis and the two strategies for GPI- anchored proteomics study are original and innovative, because currently there is no method for the synthesis of truly natural GPI-anchored proteins/glycoproteins and no proper method for systematic study of GPI-anchored proteomics. The proposed research will have a broad and significant impact. A practical synthetic method for GPI-anchored proteins and glycoproteins will allow access to these important molecules and their functionalized analogs in pure and defined forms for various biological and biophysical studies. Strategies allowing systematic study of GPI-anchored proteomics will help reveal the relationships between GPI-anchored proteins and diseases, as well as other important information, and help identify new protein markers. The results will be widely useful for the development of new diagnostic and therapeutic strategies with modulated activity, targeting ability, etc.

描述（申请人提供）：访问GPI锚定蛋白质并研究GPI生成的蛋白质组学糖基磷脂酰肌醇（GPI）附着在蛋白质和糖蛋白C-末端是一种重要的，无处不在的蛋白质蛋白质中的蛋白质，可帮助锚定蛋白质和GGLYC促成gymyc的蛋白质。 GPI锚定的蛋白质和糖蛋白在各种生物学和病理过程中起关键作用。但是，目前，对这些分子及其功能的详细研究是有限的，这主要是因为很难以纯形式和足够数量访问它们，并且缺乏分析这些多样，复杂和两亲性分子的适当工具。因此，非常希望拥有可以促进GPI锚定蛋白和糖蛋白的策略。该研究项目的最终目标是制定策略，使能够获得均质和结构定义的天然GPI锚定蛋白质以及糖蛋白以及策略，从而可以快速，有效地隔离和分析GPI锚定的蛋白质和糖蛋白。因此，该提案具有三个具体目标。目的1是准备GPI经济酶（GPI-T）的重组催化亚基GPI8，真核细胞使用的天然酶将GPI连接到蛋白质上，以及与膜相关的完整GPI-GPI-T，从而从细胞端质（ER）中产生了启发性的综合方法，以启用了enz的综合方法，以使其具有启发性的综合法（ER）。和糖蛋白。目的2是制定一种实用策略，以研究由细胞通过GPI锚定蛋白生物合成途径的代谢工程表达的GPI-锚定蛋白，即，可以使细胞或隔离的ERS与GPI-T可以使用标记为标记的合成GPI模拟，以添加到GPI附着的蛋白质中添加到蛋白质中。这将导致GPI锚定蛋白的特定标记，以使其快速分离，然后可以基于MS的蛋白质组学分析。目的3是通过使用CAPM因子（一种与GPI锚固型具有高亲和力结合的细菌毒素来研究细胞表面GPI锚定蛋白质组学的实用策略，以促进GPI链接的蛋白质和糖蛋白分离，并在用磷酸化磷酸化磷酸化磷酸化磷脂中从细胞中释放出来，并促进细胞从细胞中释放出来。分析。天然GPI锚定蛋白和糖蛋白合成的策略以及GPI-锚定蛋白质组学研究的两种策略都是原始且创新的，因为目前尚无合成真正天然GPI锚定的蛋白/糖蛋白/糖蛋白的方法，也没有用于对GPI-Cashered蛋白质组学进行系统研究的正确方法。拟议的研究将产生广泛而重大的影响。 GPI锚定蛋白和糖蛋白的实际合成方法将允许以纯和定义的形式访问这些重要的分子及其功能化的类似物，以进行各种生物学和生物物理研究。允许对GPI锚定蛋白质组学进行系统研究的策略将有助于揭示GPI锚定蛋白质和疾病之间的关系以及其他重要信息，并有助于识别新的蛋白质标记物。该结果将广泛用于开发具有调制活动，靶向能力等的新诊断和治疗策略。