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Regulation of p53 Transcription by Viral Oncoproteins & Covalent Modifications

病毒癌蛋白对 p53 转录的调节

基本信息

批准号：
8514765
负责人：
CHENG-MING CHIANG
金额：
$ 7.5万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-09-13 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8514765
关键词：
Acetylation Adenovirus E1B 19K Protein Adenoviruses Apoptosis Binding Biochemical Biological Assay Cell Cycle Arrest Cell Cycle Regulation Cells Cellular Stress Chemicals Chromatin Chromatin Modeling Chromatin Structure Code Complex DNA DNA Tumor Viruses Degradation Pathway EP300 gene Event Excision Fractionation Gene Activation Gene Targeting Genes Genetic Transcription Genome Growth HPV-High Risk Histone H2A Histones Human Human Papillomavirus ISWI In Vitro Knock-out Laboratories Lesion Low risk HPV MG132 Malignant Neoplasms Mediating Modification Molecular Molecular Chaperones Monitor Oncogene Proteins Pathway interactions Play Polyomaviruses Large T Proteins Promoter Regions Proteasome Inhibitor Protein p53 Proteins Recombinants Recruitment Activity Regulation Reporter Genes Reporting Repression Research Personnel Reverse Transcriptase Polymerase Chain Reaction Risk Role Simian virus 40 Small Interfering RNA Stress Testing Transactivation Transfection Tumor Suppressor Proteins Ubiquitin Viral activating transcription factor cofactor drug development gene repression high risk histone acetyltransferase histone deacetylase 6 human UBE3A protein in vivo inhibitor/antagonist multicatalytic endopeptidase complex mutant novel nucleosome assembly protein I pathogen programs protein degradation protein function reconstitution repaired research study transcription factor ubiquitin-protein ligase ultraviolet irradiation

项目摘要

DESCRIPTION (provided by applicant): p53 is a tumor suppressor protein that functions as a cellular genome guardian. Under normal growth conditions, p53 is kept at low levels due to a fast protein turnover rate. When cells are stressed, p53 becomes stabilized and leads to cell cycle arrest. This transient block allows cells to overcome the stress and efficiently repair DMA lesions, if necessary. When the damage is too severe to be restored, p53 induces apoptosis. The dual function of p53 lies in its ability to act as a sequence-specific transcription factor that activates transcription of gene products involved in cell cycle control and apoptosis. Not surprisingly, p53 is a frequent target for DNA tumor virus-encoded oncoproteins that are able to functionally inactivate p53 and block the activation of p53 target gene transcription. For over a decade, this inhibition of p53 transactivation has been attributed to the effect of these viral oncoproteins on p53 protein stability. Recently, we have uncovered a new pathway for human papillomavirus (HPV)-encoded E6 oncoprotein to inactivate the function of chromatin-bound p53 by inhibiting acetylation of p53 and nucleosomal core histones on the p53-targeted human p21 gene. To understand the repression mechanisms employed by HPV E6 and other DNA tumor virus-encoded oncoproteins, we propose two aims. 1) To identify cellular proteins involved in E6-mediated repression of p53 target gene transcription. We found that both p53 and histone acetyltransferase (HAT) p300 are essential for E6-mediated repression of p21 gene transcription. Since p300 autoacetylation is not inhibited by E6, we hypothesize that acetylated p300 may provide a protein code for recruitment of other cellular proteins to further modify the function of p300 and p53, resulting in a condensed chromatin structure on p53 target genes. This hypothesis will be tested by performing in vitro chromatin transcription, HAT assays, DNA/chromatin-binding assays as well as in vivo ChIP, RT-PCR, siRNA and reporter gene assays to follow the recruitment of transcription factors and cofactors to p53-regulated genes. Moreover, we will identify additional cellular proteins involved in this repression pathway by isolating and characterizing E6 cellular complexes. An unbiased biochemical fractionation will also be conducted to identify cellular factors involved in E6 repressor function. 2) To define the repression mechanism used bv other DNA tumor virus-encoded oncoproteins. We will examine whether SV40 and polyomavirus large T-antigens and adenovirus E1B-55K and E1B-19K proteins also employ a similar but non-identical mechanism to repress p53 target gene transcription. Collectively, these studies will establish a general principle of this novel repression mechanism employed by DNA tumor virus- encoded oncoproteins, independently of the proteasome-mediated degradation pathway, and provide a new direction for the development of drug inhibitors to block the propagation of these human pathogens.

描述（申请人提供）：p53是一种肿瘤抑制蛋白，起着细胞基因组守护者的作用。在正常生长条件下，由于蛋白质周转率快，p53保持在较低水平。当细胞受到压力时，p53变得稳定，导致细胞周期停滞。如果有必要，这种短暂的阻滞允许细胞克服压力并有效地修复DMA病变。当损伤严重到无法修复时，p53诱导细胞凋亡。p53的双重功能在于它能够作为一个序列特异性转录因子，激活参与细胞周期控制和凋亡的基因产物的转录。毫不奇怪，p53是DNA肿瘤病毒编码的癌蛋白的常见靶标，这些癌蛋白能够在功能上灭活p53并阻断p53靶基因转录的激活。十多年来，这种对p53反活化的抑制被归因于这些病毒癌蛋白对p53蛋白稳定性的影响。最近，我们发现了人类乳头瘤病毒（HPV）编码的E6癌蛋白通过抑制p53的乙酰化和核小体核心组蛋白在p53靶向的人类p21基因上灭活染色质结合p53的功能的新途径。为了了解HPV E6和其他DNA肿瘤病毒编码的癌蛋白的抑制机制，我们提出了两个目标。1)鉴定参与e6介导的p53靶基因转录抑制的细胞蛋白。我们发现p53和组蛋白乙酰转移酶（HAT） p300对于e6介导的p21基因转录抑制至关重要。由于p300的自乙酰化不受E6的抑制，我们假设乙酰化的p300可能为募集其他细胞蛋白提供了一个蛋白质代码，从而进一步修饰p300和p53的功能，导致p53靶基因上的染色质结构浓缩。这一假设将通过进行体外染色质转录、HAT测定、DNA/染色质结合测定以及体内ChIP、RT-PCR、siRNA和报告基因测定来验证，以跟踪转录因子和辅助因子对p53调节基因的募集。此外，我们将通过分离和表征E6细胞复合物来鉴定参与该抑制途径的其他细胞蛋白。一个无偏倚的生化分离也将进行，以确定细胞因子参与E6阻遏因子的功能。2)明确其他DNA肿瘤病毒编码癌蛋白的抑制机制。我们将研究SV40和多瘤病毒大t抗原以及腺病毒E1B-55K和E1B-19K蛋白是否也采用类似但不相同的机制来抑制p53靶基因转录。总之，这些研究将建立DNA肿瘤病毒编码的癌蛋白独立于蛋白酶体介导的降解途径所采用的这种新型抑制机制的一般原理，并为开发阻断这些人类病原体传播的药物抑制剂提供新的方向。