Structure/Function of PKR and eIF-2a using vaccinia virus K3L Gene Product

使用痘苗病毒 K3L 基因产物的 PKR 和 eIF-2a 的结构/功能

• 批准号: 9317264
• 负责人: Rosemary Jagus
• 金额: $ 25.05万
• 依托单位: University of Maryland Biotechnology Institute
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-06-15 至 1997-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9317264&HistoricalAwards=false
• 关键词: Structure; Function; PKR; eIF; 2a

Jagus 9305314 The objective of this proposal is to elucidate the mechanism of action the dsRNA-dependent elF-2cx specific protein kinase, PKR, at the molecular structural level, utilizing a vaccinia virus gene product, pK3, that functions as a pseudosubstrate. PKR, a potent inhibitor of protein synthesis, is an enzyme that serves a regulatory role in host defense against viral infection, cell proliferation, cell differentiation, and the inflammatory response. The specific aims are 1) to identify the substrate binding domain(s) of PKR using pK3, the vaccinia virus homologue of elF-2cx that binds stably to PKR; 2) to confirm the results of the invitro studies in vivo, using wild-type and variant forms of PKR transiently expressed in COS-1 cells, and 3) to extrapolate the conclusions of elF-2cx itself. The methods to be employed are recombinant DNA techniques, polymerase chain reaction, site-specific mutagenesis, deoxynucleotide sequencing, in vitro transcription and translation, protein kinase assays, gel electrophoresis, immunoblotting, immunoprecipitation, and expression of recombinant proteins in bacteria and mammalian cells. PKR plays a key role in the inhibition of viral growth by interferon, in the maintenance of low proliferation rates, the initiation of differentiation, and the production of cytokines and cellular adhesion molecules. Despite the importance of this enzymes and wealth of information recently uncovered on its regulatory domains, little is currently known about how it interacts with its only known cellular substrate, elF02cx. This proposal represents a strategy to identify substrate binding domains in PKR using a vaccinia virus homologue to elF-cx that binds stably to PKR. The ability of pK3 to form tight associations with PKR, as well as its homology to elF-2cx, forms the basis of a simple binding assay to investigate functional domains of PKR and pK3, and by extrapolation of elF-2cx. %%% The synthesis of proteins is accomplished by a c omplex machinery involving hundreds of proteins and is regulated by the protein synthesis factor, elF-2. elF-2 exists in two forms, an active and an inactive form. The change from an active to an inactive form of elF-2 is accomplished by a modifying protein called PKR. To increase our understanding of the mechanism of action of PKR at a molecular level, we will identify the site within the molecule that interacts with elF-2. The gene for PKR will be mutated and the variant PKR's will be expressed in bacteria and purified. The activities of the variant PKR's will be compared for their ability to bind to a competitor for elF-2, pK3, a protein produced in vaccinia virus infected cells. The results from these studies using isolated components will be confirmed by expressing the mutant PKR's in a model cell system and monitoring their ability to modify elF-2. PKR plays a key role in many cellular processes such as the inhibition of viral growth by interferon, the initiation of cell differentiation, and the production of molecules that promote cell- cell interaction. In addition, PKR seems to function as a tumor suppressor gene. ***

本提案的目的是利用牛痘病毒基因产物pK3作为假底物，在分子结构水平上阐明dsrna依赖性elF-2cx特异性蛋白激酶PKR的作用机制。PKR是一种有效的蛋白质合成抑制剂，是一种在宿主防御病毒感染、细胞增殖、细胞分化和炎症反应中起调节作用的酶。具体目的是：1)利用pK3鉴定PKR的底物结合域(s)， pK3是elF-2cx的牛痘病毒同源物，能稳定地与PKR结合；2)利用在COS-1细胞中瞬时表达的PKR的野生型和变异形式，在体内证实体外研究的结果。3)推断elF-2cx本身的结论。所采用的方法包括重组DNA技术、聚合酶链反应、位点特异性诱变、脱氧核苷酸测序、体外转录和翻译、蛋白激酶测定、凝胶电泳、免疫印迹、免疫沉淀以及重组蛋白在细菌和哺乳动物细胞中的表达。PKR在干扰素抑制病毒生长、维持低增殖率、启动分化、产生细胞因子和细胞粘附分子等方面发挥关键作用。尽管这种酶很重要，而且最近发现了大量关于其调控结构域的信息，但目前对它如何与唯一已知的细胞底物elF02cx相互作用知之甚少。这一建议代表了一种利用与elF-cx稳定结合的牛痘病毒同源物来鉴定PKR中底物结合域的策略。pK3与PKR紧密结合的能力，以及它与elF-2cx的同源性，构成了一个简单的结合试验的基础，以研究PKR和pK3的功能域，并通过外推elF-2cx。蛋白质的合成是由涉及数百种蛋白质的复杂机制完成的，并由蛋白质合成因子elF-2调节。elF-2以两种形式存在，一种是活性形式，另一种是非活性形式。elF-2从活性到非活性的转变是由一种叫做PKR的修饰蛋白完成的。为了增加我们在分子水平上对PKR作用机制的理解，我们将确定分子内与elF-2相互作用的位点。PKR基因将发生突变，变异的PKR将在细菌中表达并纯化。变体PKR的活性将被比较其与elF-2的竞争对手pK3的结合能力，pK3是牛痘病毒感染细胞中产生的一种蛋白质。这些使用分离成分的研究结果将通过在模型细胞系统中表达突变PKR并监测其修饰elF-2的能力来证实。PKR在许多细胞过程中发挥关键作用，如干扰素抑制病毒生长，细胞分化的开始，以及促进细胞-细胞相互作用的分子的产生。此外，PKR似乎作为肿瘤抑制基因发挥作用。＊＊＊