PROTEIN INTERACTIONS AND REPRESSION FUNCTION OF THE E1A

E1A 的蛋白质相互作用和抑制功能

• 批准号: 3198310
• 负责人: Elizabeth Moran
• 金额: $ 25.15万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-09 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3198310
• 关键词: Adenoviridae; DNA binding protein; gene expression; genetic mapping; genetic transcription; laboratory mouse; laboratory rabbit; laboratory rat; membrane proteins; molecular cloning; neoplastic transformation; oncogenes; phosphoproteins; posttranslational modifications; protein structure function; retinoblastoma; transcription factor; transforming virus; virus protein

The long term objective of this proposal is to understand the biochemical steps which transform a normal resting cell to one with malignant characteristics. This proposal focuses on the mechanism by which the Adenovirus E1A oncogene induces proliferation of quiescent primary cells. Recent evidence indicates that this process is related to E1A binding of an unidentified 300kDa cellular product. The specific aims of this project are to characterize the 300kDa product by (1) purifying and raising antibodies to it; (2) cloning and overexpressing it; (3) characterizing its posttranslational modifications, and (4) determining its intrinsic biological activities. The identification and characterization of p300 will focus largely on obtaining specific antibodies that recognize this product. When antibodies or some sequence are obtained, cDNA libraries will be screened to identify a p300 clone. The isolation and expression of a p300 clone will permit more extensive characterization including in vitro mutagenesis to study its biological activity. The biochemical and biological properties of the 300kDa protein will be characterized with particular emphasis on properties related to cell growth control and regulation of gene expression. The 300kDa product is a phosphoprotein which appears to undergo specific hyperphosphorylation during mitosis (preliminary results). The phosphorylation patterns of interphase and mitotic p300 will be mapped using techniques such as phosphopeptide analysis. Ultimately, phosphorylation events will be correlated with biological activity. The overall goal of the present proposal is to determine what biological activity of p300 makes it an apparent target for E1A cell growth regulating activity. Obtaining purified p300 will allow us to test its intrinsic biological activity. We will determine, for example, whether it is a DNA binding protein. We will determine the subcellular localization of p300, and the level of protein expression in various normal and aberrantly regulated cells. Possible interactions between p300 and other cellular products will be probed to identify cellular factors that may influence p300 function. Recent discoveries (reviewed in Green, 1989) have emphasized the relationship between E1A transforming functions and human cancer. The transforming mechanisms of adenoviruses are closely related to those of the human papillomaviruses, implicated in human malignancies. E1A association with the retinoblastoma protein provides further evidence that study of the E1A-associated 300-kilodalton product will promote our understanding of human tumorigenesis.

该提案的长期目标是了解生化 将正常静息细胞转变为恶性细胞的步骤 特征。 该提案的重点是 腺病毒 E1A 癌基因诱导静止原代细胞增殖。 最近的证据表明，这个过程与 E1A 结合有关 未鉴定的 300kDa 细胞产物。 该项目的具体目标 通过 (1) 纯化和提高来表征 300kDa 产品 其抗体； (2) 克隆并过表达； (3) 表征其 翻译后修饰，以及（4）确定其内在 生物活性。 p300 的鉴定和表征将主要集中在 获得识别该产品的特异性抗体。 当抗体 或者获得某些序列，将筛选cDNA文库以鉴定 p300 克隆。 p300 克隆的分离和表达将允许 更广泛的表征，包括体外诱变来研究其 生物活性。 的生化和生物学特性 300kDa 蛋白质的特征将特别强调特性 与细胞生长控制和基因表达调控有关。 这 300kDa 产物是一种磷蛋白，似乎经历了特定的 有丝分裂期间的过度磷酸化（初步结果）。 这 间期和有丝分裂 p300 的磷酸化模式将被绘制 使用磷酸肽分析等技术。 最终， 磷酸化事件将与生物活性相关。 这 本提案的总体目标是确定哪些生物 p300 的活性使其成为 E1A 细胞生长调节的明显靶标 活动。 获得纯化的 p300 将使我们能够测试其内在 生物活性。 例如，我们将确定它是否是DNA 结合蛋白。 我们将确定 p300 的亚细胞定位， 以及各种正常和异常的蛋白质表达水平 调节细胞。 p300 和其他细胞之间可能存在的相互作用 将探测产品以确定可能影响的细胞因素 p300 功能。 最近的发现（Green, 1989 中的评论）强调了 E1A转化功能与人类癌症之间的关系。 这 腺病毒的转化机制与腺病毒的转化机制密切相关。 人类乳头瘤病毒，与人类恶性肿瘤有关。 E1A协会 与视网膜母细胞瘤蛋白的研究提供了进一步的证据 与 E1A 相关的 300 千道尔顿产品将促进我们对 人类肿瘤发生。