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：
7322019
负责人：
CHENG-MING CHIANG
金额：
$ 29.83万
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 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7322019
关键词：
Acetylation Adenovirus E1B 19K Protein Adenoviruses Apoptosis Binding Biochemical Biological Assay CDKN1A gene Cell Cycle Arrest Cell Cycle Regulation Cells Chemicals Chromatin Chromatin Modeling Chromatin Structure Class Code Complex Condition 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 Activities Human Papillomavirus ISWI In Vitro Knock-out Laboratories Lesion Low risk HPV MG132 Malignant Neoplasms Mediating Modification Molecular Molecular Chaperones Monitor Oncogene Proteins Other Finding Papillomavirus Pathway interactions Play Polyomaviruses Large T Proteins Promoter Regions Proteasome Inhibitor Protein p53 Proteins Rate Recombinants Recruitment Activity Regulation Reporter Genes Reporting Repression Research Personnel Reverse Transcriptase Polymerase Chain Reaction Risk Role Simian virus 40 Small Interfering RNA Stress TP53 gene Testing Transactivation Transfection Tumor Suppressor Proteins Ubiquitin Viral activating transcription factor cofactor drug development gene repression histone acetyltransferase histone deacetylase 6 human UBE3A protein in vivo inhibitor/antagonist multicatalytic endopeptidase complex mutant novel nucleosome assembly protein I oncoprotein p21 pathogen programs protein degradation protein function reconstitution repaired research study transcription factor ubiquitin 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（由申请人提供）：p53是一种肿瘤抑制蛋白，用作细胞基因组监护人。在正常的生长条件下，由于快速蛋白质周转率，p53保持较低水平。当细胞受到压力时，p53稳定并导致细胞周期停滞。这种瞬时块使细胞在必要时可以克服压力并有效地修复DMA病变。当损害太严重以至于无法恢复时，p53会诱导凋亡。 p53的双重功能在于它充当序列特异性转录因子的能力，该转录因子激活了参与细胞周期控制和凋亡的基因产物的转录。毫不奇怪，p53是能够在功能上失活p53并阻止p53靶基因转录的激活的DNA肿瘤病毒代码蛋白的常见靶标。十多年来，这种对p53反式激活的抑制一直归因于这些病毒癌蛋白对p53蛋白稳定性的影响。最近，我们发现了人乳头瘤病毒（HPV）编码的E6癌蛋白的新途径，从而通过抑制p53和核小体核心组蛋白的乙酰化对P53靶向的人P21基因的乙酰化和核小体核心组蛋白的乙酰化来灭活p53的功能。为了了解HPV E6和其他DNA肿瘤病毒所使用的抑制机制，我们提出了两个目标。 1）确定参与E6介导的p53靶基因转录抑制的细胞蛋白。我们发现p53和组蛋白乙酰基转移酶（HAT）P300对于E6介导的P21基因转录的抑制至关重要。由于E6不抑制p300自乙酰化，因此我们假设乙酰化的p300可以提供蛋白质代码，用于募集其他细胞蛋白，以进一步改变p300和p53的功能，从而导致p53靶基因上的凝聚蛋白结构。该假设将通过进行体外染色质转录，HAT分析，DNA/染色质结合测定以及体内芯片，RT-PCR，siRNA和报告基因测定法进行检验，以遵循转录因子和辅助因子和辅助因子的募集到P53受调控的基因。此外，我们将通过隔离和表征E6细胞复合物来鉴定在该抑制途径中涉及的其他细胞蛋白。还将进行无偏的生化分馏，以识别E6阻遏功能中涉及的细胞因素。 2）定义使用BV其他DNA肿瘤病毒编码的抑制机制。我们将检查SV40和多瘤病毒大型T-抗原和腺病毒E1B-55K和E1B-19K蛋白是否也采用了类似但非相同的机制来抑制p53靶基因转录。总的来说，这些研究将建立这种新型抑制机制的一般原理，该机制是由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，并为开发药物抑制剂的开发提供了新的方向，以阻止这些人类病原体的传播。