REDOX REGULATION OF TRANSCRIPTION FACTORS

转录因子的氧化还原调节

• 批准号: 6344112
• 负责人: JAMIL A MOMAND
• 金额: $ 7.33万
• 依托单位: CALIFORNIA STATE UNIVERSITY LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-06-14 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6344112
• 关键词: DNA binding protein; MCF7 cell; cysteine; gel mobility shift assay; gene environment interaction; gene induction /repression; mass spectrometry; oxidation reduction reaction; p53 gene /protein; protein structure function; site directed mutagenesis; tissue /cell culture; transcription factor; transfection

The p53 tumor suppressor gene is a frequently inactivated gene in human cancer cells. In response to cell stressors p53 induces cell cycle arrest or programmed cell death. We have discovered that a small thiol-containing agent (pyrrolidine dithiocarbamate) inhibits some aspects of p53 activity in cultured cells and increase our understanding of the mechanisms by which p53 activity is modulated as such mechanisms may alter its ability to respond to genotoxic agents and perform its tumor suppressor activities. Dithiocarbamates are commonly used as herbicides, fungicides, and insecticides world-wide. Environmental toxins that act on p53 in a fashion similar to pyrrolidine dithiocarbamate may contribute to some types of diseases. We developed a novel technique to measure protein cysteine oxidation reactions in cultured cells. Evidence from our laboratory and others suggest that the redox state of p53 modulates its ability to specifically bind its DNA promoter targets, activate transcription of its effector genes, and suppress cell cycle progression. The hypothesis to be tested are: 1) p53 protein undergoes cysteine residue, oxidation after cellular treatment with some oxidizing reagents; 2) p53 oxidation alters its ability to bind its consensus sequence with DNA promoters and insertion/deletion mismatch DNA sequences. The specific aims are: 1. Identify the sites of cysteine residue oxidation on p53 by site-directed mutagenesis and mass spectroscopy. 2. Determine if Ref-1 is required for maintaining a low p53 redox state in the presence of dithiocarbamate-based pesticides and hydrogen peroxide. 3. Determine the redox potential of p53 as a function of oxidation, binding to p53 consensus sequence DNA and binding to insertion/deletion mismatch DNA. Successful completion of this project will uncover new insight into this novel p53 modulation mechanism.

p53肿瘤抑制基因是人类癌细胞中经常失活的基因。响应细胞应激源p53诱导细胞周期停滞或编程细胞死亡。我们发现，含硫代硫醇的小剂（吡咯烷二硫代氨酸）抑制培养细胞中p53活性的某些方面，并增加了我们对p53活性受到调节的机制的理解，因为这种机制可能会改变其对遗传毒性和性能的能力，并改变其遗传毒性的能力，并它的肿瘤抑制活性。二硫代氨基植物通常用作全球范围内的除草剂，杀真菌剂和杀虫剂。以类似于吡咯烷二硫代氨酸类似的方式对p53作用的环境毒素可能会导致某些类型的疾病。我们开发了一种新型技术来测量培养细胞中蛋白质半胱氨酸氧化反应。我们实验室和其他实验室的证据表明，p53的氧化还原状态调节其特异性结合其DNA启动子靶标，激活其效应基因的转录并抑制细胞周期进程的能力。要检验的假设是：1）p53蛋白经历半胱氨酸残基，用一些氧化试剂在细胞处理后氧化； 2）p53氧化改变了其与DNA启动子和插入/缺失不匹配DNA序列结合其共识序列的能力。具体目的是：1。通过定点诱变和质谱法确定p53上半胱氨酸残基氧化的位点。 2。确定在存在基于二硫代氨基氨基盐的农药和过氧化氢的情况下维持低p53氧化还原状态的REF-1。 3。确定p53作为氧化，与p53共有序列DNA的结合并与插入/缺失不匹配DNA结合的氧化还原电位。该项目的成功完成将发现对这种新型P53调制机制的新见解。