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:8628408
- 负责人:
- 金额:$ 29.16万
- 依托单位:
- 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:
- 关键词:AddressAffinityAffinity ChromatographyAnabolismBacterial ToxinsBindingBiocompatible MaterialsBiologicalBiological ProcessBiotinCatalytic DomainCell membraneCell surfaceCellsChemicalsChemistryCleaved cellComplexCoupledCouplingDevelopmentDiseaseEndoplasmic ReticulumEnzymesEukaryotic CellEventGPI Membrane AnchorsGlycolipidsGlycopeptidesGlycoproteinsGlycosylphosphatidylinositolsGoalsHSV glycoprotein CHumanIn VitroInvestigationLabelLeadLigationLinkLiteratureMembraneMembrane GlycoproteinsMembrane ProteinsMethodsMolecularNaturePathologic ProcessesPathway interactionsPeptidesPharmaceutical PreparationsPhosphatidylinositolsPhospholipase CPlayPost-Translational Protein ProcessingPropertyProtein BindingProtein BiosynthesisProtein CProteinsProteomicsRecombinant ProteinsRecombinantsResearchResearch Project GrantsRoleSepharoseSignal TransductionStructure-Activity RelationshipTechniquesTissuesTrypanosoma brucei bruceianalogbasechemical synthesisdesigndisease diagnosisextracellulargenetically modified cellsinnovationlink proteinmetabolic engineeringmolecular markernovelnovel diagnosticsnovel strategiesnovel therapeuticspublic health relevanceresearch studysortasesynthetic peptidetooltransamidases
项目摘要
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 use 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.
获取 GPI 锚定蛋白和研究 GPI 锚定蛋白质组学的新方法
糖基磷脂酰肌醇 (GPI) 连接到蛋白质和糖蛋白 C 末端是一个重要且
真核物种中普遍存在的翻译后修饰,有助于将蛋白质和糖蛋白锚定
细胞外膜。 GPI 锚定蛋白和糖蛋白在多种生物和分子生物学中发挥着关键作用。
病理过程。然而,目前对这些分子及其功能的详细研究还很有限,
主要是因为难以以纯粹的形式和足够的数量获取它们,并且缺乏适当的工具来获取它们
分析这些多样化、复杂的两性分子。因此,非常需要有策略
可以促进 GPI 锚定蛋白和糖蛋白的获取和研究。
该研究项目的最终目标是制定策略,使人们能够获得同质和
结构明确的天然 GPI 锚定蛋白和糖蛋白以及能够快速、有效地
GPI 锚定蛋白和糖蛋白的分离和分析。据此,本提案提出了三项具体内容:
目标。目的 1 制备 GPI 转酰胺酶 (GPI-T) 的重组催化亚基 GPI8、天然
真核细胞用来将 GPI 附着到蛋白质上的酶,以及源自膜的完整 GPI-T
细胞内质网 (ER) 并使用它们创建潜在的酶合成通用方法
天然 GPI 锚定蛋白和糖蛋白。目标 2 是为 GPI 研究制定实用策略
通过 GPI 锚定蛋白生物合成途径的代谢工程由细胞表达的锚定蛋白,
即,为细胞或分离的 ER 提供标记的合成 GPI 类似物,GPI-T 可以使用该类似物添加到蛋白质中
绑定 GPI 附件。这将导致 GPI 锚定蛋白的特异性标记,使其能够快速
分离,然后基于 MS 的蛋白质组学分析。目标 3 是制定细胞研究的实用策略
使用 CAPM 因子(一种与 GPI 具有高亲和力结合的细菌毒素)进行表面 GPI 锚定的蛋白质组学
锚,以促进 GPI 连接蛋白和糖蛋白在处理后从细胞中释放的分离
磷脂酰肌醇特异性磷脂酶 C 酶和随后的 GPI 锚定蛋白质组学分析。
天然 GPI 锚定蛋白和糖蛋白合成的策略以及 GPI- 的两种策略
锚定蛋白质组学研究具有原创性和创新性,因为目前还没有合成方法
真正天然的 GPI 锚定蛋白/糖蛋白,并且没有适当的方法来系统研究 GPI 锚定蛋白
蛋白质组学。拟议的研究将产生广泛而重大的影响。一种实用的合成方法
GPI 锚定蛋白和糖蛋白将允许访问这些重要的分子及其功能化
用于各种生物学和生物物理学研究的纯和确定形式的类似物。允许系统化的策略
GPI锚定蛋白质组学研究将有助于揭示GPI锚定蛋白质与疾病之间的关系,
以及其他重要信息,并帮助识别新的蛋白质标记。研究结果将广泛用于
开发具有调节活性、靶向能力等的新诊断和治疗策略。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Zhongwu Guo其他文献
Zhongwu Guo的其他文献
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{{ truncateString('Zhongwu Guo', 18)}}的其他基金
Brain glycosphingolipids and Alzheimer's disease
脑鞘糖脂与阿尔茨海默病
- 批准号:
10738379 - 财政年份:2023
- 资助金额:
$ 29.16万 - 项目类别:
Synthetic and Biological Studies of GPI Conjugates and GPI Anchorage to Cell Membranes
GPI 缀合物和 GPI 细胞膜锚定的合成和生物学研究
- 批准号:
9902533 - 财政年份:2019
- 资助金额:
$ 29.16万 - 项目类别:
Synthetic and Biological Studies of GPI Conjugates and GPI Anchorage to Cell Membranes
GPI 缀合物和 GPI 细胞膜锚定的合成和生物学研究
- 批准号:
10371134 - 财政年份:2019
- 资助金额:
$ 29.16万 - 项目类别:
Synthetic and Biological Studies of GPI Conjugates and GPI Anchorage to Cell Membranes
GPI 缀合物和 GPI 细胞膜锚定的合成和生物学研究
- 批准号:
10584557 - 财政年份:2019
- 资助金额:
$ 29.16万 - 项目类别:
Novel Approaches to Access GPIs and GPI-Anchored Proteins for the Study of GPI An
获取 GPI 和 GPI 锚定蛋白用于 GPI An 研究的新方法
- 批准号:
8324038 - 财政年份:2009
- 资助金额:
$ 29.16万 - 项目类别:
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
- 批准号:
8989113 - 财政年份:2009
- 资助金额:
$ 29.16万 - 项目类别:
Novel Approaches to Access GPIs and GPI-Anchored Proteins for the Study of GPI An
获取 GPI 和 GPI 锚定蛋白用于 GPI An 研究的新方法
- 批准号:
7938107 - 财政年份:2009
- 资助金额:
$ 29.16万 - 项目类别:
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
- 批准号:
9027236 - 财政年份:2009
- 资助金额:
$ 29.16万 - 项目类别:
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