AGROBACTERIUM VIRD2 INTERACTION WITH HOST CYCLOPHILINS

农杆菌 VIRD2 与宿主亲环蛋白的相互作用

基本信息

批准号：
6178841
负责人：
DEREK W WOOD
金额：
$ 3.92万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-08-01 至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/6178841
关键词：
Agrobacterium tumefaciens Arabidopsis affinity chromatography antisense nucleic acid bacteria infection mechanism bacterial proteins cyclosporines genetic library host organism interaction immunoprecipitation intracellular transport peptidylprolyl isomerase polymerase chain reaction yeast two hybrid system

项目摘要

The long term objective of this proposal is to clarify the cellular role of cyclophilins. Cyclophilins are present in a wide range of cell types suggesting that they play an essential role in cell biology. Cyclophilins have recently been implicated in a number of processes including apoptosis, signal transduction, protein stabilization (chaperones), immunosuppression and pathogenesis of a variety of organisms (i.e. Leishmania and HIV-1). The specific goals of this research are to: 1) verify the interaction between the VirD2 protein of Agobacterium tumefaciens and two cyclophilins of Arabidopsis thaliana, 2) demonstrate that this interaction is important in tumorigenesis, 3) examine the interaction of these cyclophilins with VirD2 and other host proteins in an effort to define the cellular role of host cyclophilins and 4) investigate the involvement of cyclophilins in Agrobacterium-mediated transformation of yeast. Affinity chromatography and in vivo across- linking analysis will confirm the specific interactions between VirD2 and cyclophilins previously defined using yeast two-hybrid analysis. Cyclosporin A inhibition antisense RNA analysis will be used to examine the importance of this interaction in tumorigenesis initiated by agrobacterium. Site and region specific mutagenesis will be used in concert with forward and reverse yeast two-hybrid analysis to identify VirD2 residues essential to this interaction and to construct suppressor mutations in cyclophilins will be identified in an effort to elucidate host transport or signal transduction pathways linked to cyclophilins. Further work will utilize yeast as a tractable genetic model in which to examine the effect of cyclophilin disruptions on transformations.

该提案的长期目标是阐明细胞的作用亲环蛋白。亲环蛋白存在于多种细胞类型中表明它们在细胞生物学中发挥着重要作用。亲环蛋白最近涉及许多进程，包括细胞凋亡、信号转导、蛋白质稳定（伴侣）、多种生物体的免疫抑制和发病机制（即利什曼原虫和 HIV-1）。本研究的具体目标是：1）验证农杆菌VirD2蛋白之间的相互作用根瘤菌和拟南芥的两种亲环蛋白，2) 证明这种相互作用在肿瘤发生中很重要，3）检查这些亲环蛋白与 VirD2 和其他宿主蛋白的相互作用努力定义宿主亲环蛋白的细胞作用和 4) 研究亲环蛋白在农杆菌介导中的参与酵母的转化。亲和层析和体内交叉链接分析将确认 VirD2 和先前使用酵母双杂交分析定义了亲环蛋白。环孢素 A 抑制反义 RNA 分析将用于检查这种相互作用在肿瘤发生中的重要性农杆菌。位点和区域特异性诱变将用于与正向和反向酵母双杂交分析相配合来识别 VirD2 残基对于这种相互作用和构建抑制子至关重要将鉴定亲环蛋白的突变，以努力阐明与亲环蛋白相关的宿主运输或信号转导途径。进一步的工作将利用酵母作为易于处理的遗传模型检查亲环蛋白破坏对转化的影响。