CHARACTERIZATION OF NEW HUMAN P53 DISSOCIATORS

新人类 P53 解离器的表征

• 批准号: 6378026
• 负责人: RAINER K BRACHMANN
• 金额: $ 27.72万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6378026
• 关键词: Saccharomyces cerevisiae; apoptosis; binding proteins; cell growth regulation; cell line; complementary DNA; gene expression; genetic library; glutathione transferase; human papillomavirus; luciferin monooxygenase; microorganism culture; molecular cloning; p53 gene /protein; plasmids; protein protein interaction; reporter genes; virus protein; yeast two hybrid system

The human tumor suppressor protein and transcription factor p53 integrates stress signals, such as DNA damage, hypoxia and ribonucleotide depletion, and induces either cell cycle arrest or apoptosis. Approximately 50 percent of all human cancers carry p53 gene mutations. Another 5 to 10 percent inactivate the p53 protein directly by overexpression of high-risk human papilloma virus (HPV) E6 or MDM2, a physiological negative regulator of p53. Considering the plethora of upstream signals, all using different mechanisms of p53 activation, and of p53 downstream effector genes, it is very likely that the remaining human cancers also directly inactivate p53 protein by overexpressing currently unknown negative regulators of p53. We devised a genetic assay for p53 in S. cerevisiae, called the "p53 dissociator assay", to select for such, and other, proteins important for p53 biology in highly efficient screens. We showed that SV40 large T antigen, a known negative regulator, scores in this assay, and we recreated the complex interplay of p53, E6-associated protein (E6-AP) and high-risk HPV E6 resulting in p53 degradation. Three p53 dissociator screens yielded MDM2 and high-risk HPV E6, two established negative regulators, 53BP1, a coactivator of p53, and 23 candidate proteins without current connections to p53 biology. Besides negative and positive regulators of p53, we may also have isolated proteins negatively regulated by p53 in human cells, as suggested by their biology. All three classes of proteins represent important drug targets for improved cancer therapies: inhibition of negative regulators will unleash p53 and induce apoptosis in cancer cells, studies of p53 enhancers will elucidate new mechanisms of further potentiating p53 activity and proteins negatively regulated by p53 will provide new directions to interfere pharmacologically with unchecked proliferation. We propose to study the set of candidate proteins using secondary yeast and mammalian assays, followed by assays tailored to the classes that confirmed p53 dissociators likely belong to. Novel negative regulators of p53 will be pursued further by checking for their overexpression in cancer cell lines with wild-type p53 genes and by setting up reverse two-hybrid assays to screen for drugs able to abrogate their effect on p53. Several specific ways of direct p53 protein inactivation exist, but their exact mechanisms have not been elucidated: cytoplasmic sequestration, p53 degradation involving the human protein E6-AP and nuclear sequestration in teratocarcinoma cell lines. To identify the proteins underlying these mechanisms, we will perform additional targeted D53 dissociator screens in the presence of E6-A P and using specific cDNA libraries.

人类肿瘤抑制蛋白和转录因子p53整合了应力信号，例如DNA损伤，缺氧和核糖核苷酸耗竭，并诱导细胞周期停滞或凋亡。 所有人类癌症中约有50％携带p53基因突变。 另外5％至10％的人直接通过高危人乳头瘤病毒（HPV）E6或MDM2（p53的生理阴性调节剂）过度表达p53蛋白。 考虑到大量上游信号，都使用p53激活的不同机制以及p53下游效应子基因的基因，其余的人类癌症也很可能还通过过度表达p53的p53蛋白而直接使当前未知的负调控剂过度表达p53的蛋白质。 我们在酿酒酵母中设计了一种p53的遗传测定，称为“ p53分离剂测定”，以选择对p53生物学重要的蛋白质和其他高效筛选中重要的蛋白质。 我们表明，该测定法中的SV40大T抗原（一种已知的负调节剂）得分，我们重新创建了p53，与E6相关蛋白（E6-AP）和高危HPV E6的复杂相互作用，从而导致p53降解。 三个p53分解器筛选产生了MDM2和高风险HPV E6，两个已建立的负调节剂，53BP1，一个p53的共激活剂和23个没有当前与P53生物学连接的候选蛋白。 除了p53的负调节剂和阳性调节剂外，我们的生物学建议还可能具有人类细胞中p53负调控的孤立蛋白。所有三类蛋白质都代表了改善癌症治疗的重要药物靶标：抑制负调节剂将释放p53并诱导癌细胞中的凋亡，p53增强剂的研究将阐明进一步增强p53活性的新机制，并由p53负调节p53的所有新机制，将在p53中构成新的方向，从而使新的方向呈插入式繁殖。 我们建议使用二级酵母和哺乳动物测定法研究一组候选蛋白质，然后是针对确认p53解散剂可能属于的类别量身定制的测定方法。 p53的新型负调节剂将通过检查其在具有野生型p53基因的癌细胞细胞系中的过表达，并设置反面两种杂交测定法以筛选能够消除其对p53作用的药物。存在几种特定的直接p53蛋白质灭活方式，但尚未阐明它们的确切机制：细胞质隔离，涉及人蛋白E6-AP的p53降解和在Teratocarcinoma瘤细胞系中的核隔离。 为了识别这些机制的蛋白质，我们将在存在E6-A P并使用特定的cDNA文库的情况下执行其他有针对性的D53分离剂筛选。