STRUCTURE FUNCTION RELATIONSHIP OF TUMOR SUPPRESSORS

该项目的目标是了解结构-功能 近年来发现的抑癌基因p16与肿瘤细胞凋亡的关系， 相对p15。它们是人类细胞周期蛋白依赖性 激酶4（Cdk 4）及其近缘变体cdk 6。该方法涉及一个 遗传、生物化学和生物物理技术的组合。 的 具体目标1是通过以下方法确定p16和p15的三级结构： NMR结合15 N/13 C同位素标记。具体目标2 通过位点特异性鉴定p16中与CDK 4结合关键残基 诱变，从肿瘤细胞中存在的p16突变体开始。的 将分析显示没有整体构象变化的突变体， 基于它们对CDK 4的抑制和结合的活性。 的 结果，沿着由NMR确定的三级结构，将允许 可能的CDK 4结合基序的“工作模型”的形成， 每个肿瘤抑制因子。工作模型将在 通过突变体的合理设计的特异性目的3。 目标是：提供 用于合理设计新cdk 4抑制剂的信息 抗癌药物（这将是第二个项目期的目标）， 并确定p16和p15是否具有相同的CDK 4结合位点。的 具体目标4是将p16和/或p15的特异性从cdk 4改变为cdk 4。 通过体外分子进化技术结合 酵母双杂交系统和噬菌体展示技术作为筛选 方法. 特异性改变的蛋白质的三级结构 并与天然p16和p15的那些进行比较; 这些资料将有助于了解cdk的性质。 肿瘤抑制剂的特异性。第5章寻找新的 来自p16或p15的表位文库的CDK 4的肽抑制剂和 随机组合文库，使用两步筛选过程： 杂交系统或噬菌体展示，以及与p16或p15竞争， 抑制CDK 4活性。 新的抑制剂和它们的肽- 模拟物或非肽类似物（稍后设计）是潜在的 抗癌药 具体目标6是寻找肽模拟物， CDK 4能够结合来自表位的p16或p15， 图书馆. 除了基于结合的筛选方法之外， 通过NMR的结构分析将进一步用于确定 肽结合位点与特异性推导的CDK 4结合位点一致。 目标1-3。 目的是为抑制子提供一个结构模型 cdk 4复合体，这将是有用的，在破译“失踪” 肿瘤抑制因子的疾病相关突变体中的“功能性”， 药物设计的重要信息。

The objective of this project is to understand the structure-function relationship of the recently discovered tumor suppressor p16 and its close relative p15. They are specific inhibitors of human cyclin-dependent kinase 4 (Cdk4) and its close variant cdk6. The approach involves a combination of genetic, biochemical, and biophysical techniques. The Specific Aim 1 is to determine the tertiary structures of p16 and p15 by NMR in combination with 15N/13C isotope labeling. The Specific Aim 2 s to identify key residues in p16 for their binding to cdk4 by site-specific mutagenesis, starting with the p16 mutants existing in tumor cells. The mutants showing no global conformational change will be analyzed for activity on the basis of their inhibition of, and binding to cdk4. The results, along with the tertiary structures determined by NMR, will allow formulation of a "working model" for the possible cdk4 binding motif for each tumor suppressor. The working models will be further refined in Specific Aim 3 by rational design of mutants. The goals are: to provide information for rational design of new cdk4 inhibitors as potential anticancer drugs (which will be the goal of the second project period), and to address whether p16 and p15 have identical cdk4 binding sites. The Specific Aim 4 is to change the specificity of p16 and/or p15 from cdk4 to cdk1 by an in vitro molecular evolution technique in combination with the yeast two-hybrid system and a phage-display assay as the screening methods. The tertiary structures of the proteins with altered specificity will be determined and compared with those of the native p16 and p15; the information will be useful in understanding the nature of the cdk specificity of tumor suppressors. The Specific Aim 5 is to search for new peptide inhibitors of cdk4 from epitope libraries of p16 or p15 and a random combinatorial library, using a two-step screening process: the two- hybrid system or phage-display, and competition with p16 or p15 for inhibition of the cdk4 activity. The new inhibitors and their peptide- mimetic or non-peptide analogs (to be designed later) are potential anticancer drugs. The Specific Aim 6 is to search for a peptide mimic of cdk4 capable of binding to p16 or p15 from epitope and combinatorial libraries. In addition to the screening methods based on binding, structural analysis by NMR will be further used to determine whether the peptide binding site agrees with the cdk4 binding site deduced in specific aims 1-3. The goal is to provide a structural model for the suppressor cdk4 complex, which will be useful in deciphering the "missing functionality" in the disease-related mutants of tumor suppressors, an important information for drug design.