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

该建议的目的是利用作为假底物的牛痘病毒基因产物pK 3，在分子结构水平上阐明dsRNA依赖性eIF-2cx特异性蛋白激酶PKR的作用机制。 PKR是一种蛋白质合成的有效抑制剂，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）使用pK 3（eIF-2cx的稳定结合PKR的牛痘病毒同源物）鉴定PKR的底物结合结构域; 2）使用在COS-1细胞中瞬时表达的PKR的野生型和变体形式，确认体内体外研究的结果，和3）外推eIF-2cx本身的结论。 所采用的方法是重组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、维持低增殖速率、启动分化以及产生细胞因子和细胞粘附分子中起关键作用。 尽管这种酶的重要性和最近发现的关于其调节结构域的丰富信息，但目前对其如何与其唯一已知的细胞底物eIF 02 cx相互作用知之甚少。 该提议代表了使用稳定结合PKR的eIF-cx的牛痘病毒同源物来鉴定PKR中的底物结合结构域的策略。 pK 3与PKR形成紧密结合的能力，以及其与eIF-2cx的同源性，形成了研究PKR和pK 3的功能结构域的简单结合测定的基础，并通过外推eIF-2cx。 蛋白质的合成是通过涉及数百种蛋白质的复杂机制来完成的，并且由蛋白质合成因子eIF-2调节。 eIF-2以两种形式存在，活性和非活性形式。 elF-2从活性形式到非活性形式的变化是通过称为PKR的修饰蛋白完成的。 为了增加我们对PKR在分子水平上的作用机制的理解，我们将鉴定分子内与eIF-2相互作用的位点。 PKR的基因将被突变，并且变体PKR将在细菌中表达并纯化。 将比较变体PKR的活性与eIF-2的竞争者pK 3（在牛痘病毒感染的细胞中产生的蛋白质）结合的能力。 通过在模型细胞系统中表达突变体PKR并监测其修饰eIF-2的能力，将证实使用分离组分的这些研究的结果。 PKR在许多细胞过程中起关键作用，例如干扰素抑制病毒生长、细胞分化的起始和促进细胞-细胞相互作用的分子的产生。 此外，PKR似乎是一种肿瘤抑制基因。 ***

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. ***