STRUCTURE FUNCTION RELATIONSHIP OF TUMOR SUPRESSORS

描述：（来自申请人摘要的逐字逐句） 项目是了解新的结构-功能关系 发现了肿瘤抑制因子，特别是p53-Rb通路中的抑制因子。的 最后一个授予期的重点是p16-INK 4A。有三大目标 在下一个授予期。具体目标1是扩展以前的研究， INK 4蛋白与细胞周期蛋白依赖性 激酶4（CDK 4）或CDK 6在两个方向：（a）定点诱变将 用于探测许多表面的结构和功能作用， 在不同的INK 4蛋白中具有相反电荷的残基。(b)基因 改组将继续用于获得新的INK 4变体， 特异性，以及最有趣的新构建体的特性 性质将在蛋白质水平上表征。在这些 研究中，将开发两种额外的测定方法，以补充 目前用途：是一种荧光结合分析，用于定量 INK 4-CDK解离常数的测定和INK 4的体内测定 癌细胞系中的蛋白质。具体目标2是扩展INK 4的研究 蛋白质与其与人HTLV-1 Tax蛋白的相互作用。申请人 计划确定税收三角洲109的结构，并使用现场指导 诱变以鉴定来自Tax和p16两者的关键残基，所述关键残基涉及 相互作用，并将p16-Tax相互作用与p16-CDK 4（或CDK 6）进行比较 交互.具体目标3是将研究扩展到其他新蛋白质 与p53-Rb通路有关。这两种蛋白质是人类的 p14 ARF是一种与p16基因相同的肿瘤抑制基因，但在p16基因中表达。 交替阅读框架和人类TRIP-Br 1，一个新发现的 已发现参与转录因子的新家族， E2 F-1的转录机制。该方法涉及以下方面的结合： 遗传、生物化学和生物物理技术。

DESCRIPTION: (Verbatim from the Applicant's Abstract) The objective of this project is to understand the structure-function relationship of newly discovered tumor suppressors, particularly those in the p53-Rb pathway. The focus of the last granting period was on p16-INK4A. There are three major goals for the next granting period. Specific Aim 1 is to extend previous studies on the specificity of the interactions between INK4 protein and cyclin-dependent kinase 4 (CDK4) or CDK6 in two directions: (a) Site-directed mutagenesis will be used to probe the structural and functional roles of a number of surface residues that have contrasting charges among different INK4 proteins. (b) Gene shuffling will continue to be used to obtain new INK4 variants with altered specificity, and the properties of new constructs with most interesting properties will be characterized at the protein level. As part of these studies, two additional assay methods will be developed to complement the inhibitory assay currently in use: a fluorescent binding assay for quantitative determination of INK4-CDK dissociation constants, and an in vivo assay for INK4 proteins in cancer cell lines. Specific Aim 2 is to extend the studies of INK4 proteins to their interactions with human HTLV-1 Tax protein. The applicant plans to determine the structures of Tax delta109 and use site-directed mutagenesis to identify key residues from both Tax and p16 that are involved in the interactions, and compare p16-Tax interactions to p16-CDK4 (or CDK6) interactions. Specific Aim 3 is to extend the studies to other new proteins relevant to the p53-Rb pathway. The two proteins to be pursued are human p14ARF, a tumor suppressor expressed from the same gene locus as p16 but in an alternate reading frame, and human TRIP-Br1, a member of a newly discovered novel family of transcription factors that has been found to be involved in the transcriptional machinery of E2F-1. The approach involves a combination of genetic, biochemical and biophysical techniques.