MECHANISM OF P53 SILENCING BY ADENOVIRUS E1B 55K PROTEIN

腺病毒 E1B 55K 蛋白沉默 P53 的机制

• 批准号: 2008589
• 负责人: ARNOLD J BERK
• 金额: $ 18.21万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-02-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2008589
• 关键词: Adenoviridae; DNA binding protein; DNA directed RNA polymerase; DNA footprinting; affinity chromatography; chemical association; gel electrophoresis; gel mobility shift assay; gene induction /repression; genetic promoter element; genetic transcription; intermolecular interaction; molecular site; mutant; oncogenes; protein purification; protein reconstitution; transcription factor; tumor suppressor proteins; virus protein; western blottings

P 53 is a sequence-specific DNA-binding transcription factor which functions to arrest the cell cycle and induce apoptosis in response to DNA damage. Both p 53 alleles are frequently mutated in metastatic human cancers indicating that p53 is a tumor suppressor. This is probably because inactivation of p53 allows the accumulation of mutations and gene amplifications which activate oncogenes resulting in tumor progression. Adenovirus E1B 55K protein binds to p53 and represses p53 transactivation through specific protein-protein interactions, activities which are critical for the oncogenic transforming activity of 55K in conjunction with adenovirus E1A. We propose to study the molecular mechanisms of both p 53 activation and E1B 55K transcriptional silencing through in vitro studies with purified general transcription factors. The polypeptides with which p53 interacts and the mechanism by which these interactions stimulate transcription will be determined. The surface of the TATA-box binding protein (TCP) to which p 53 binds will be determined by analyzing p53 binding to a large collection of mutant TBPs with alterations in specific surface residues. Interactions with purified TFIID-TAFs will be analyzed by protein blotting and chemical cross-linking. Functional targets will be identified by determining which purified factors can overcome transcriptional squelching by high concentrations of p53. Agarose gel electrophoresis of large DNA-protein complexes and footprinting will be used to analyze the influence of p53 on initiation complex assembly and on disassociation of the initiation complex following initiation. Since recent work has shown that 55K contains a repression domain, a LexA-55K fusion protein will be expressed and purified, and its ability to repress transcription will be investigated. If basal transcription in inhibited by 55K, the general transcription factors inhibited by 55K will be identified and specific interactions defined. If activated transcription is specifically inhibited, coactivators inhibited by 55K will be identified and the mechanisms of inhibition studied. Understanding the mechanism of transcriptional activation by p53 and its inhibition by adenovirus E1B 55K may allow the design of therapies which reactivate p53 in human tumors, leading to cell-cycle arrest and apoptosis of tumor cells.

p53是一种序列特异性dna结合转录因子