DEPENDENCE OF P53 ON THIOL MAINTENANCE PROTEINS

P53 对硫醇维持蛋白的依赖性

• 批准号: 6377389
• 负责人: GARY F MERRILL
• 金额: $ 13.61万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6377389
• 关键词: DNA binding protein; NAD(P)H oxidoreductase; SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; alkylation; autoradiography; chimeric proteins; cysteine; disulfide bond; enzyme activity; flavoproteins; gel mobility shift assay; gene expression; glutathione; immunoprecipitation; oxidation; p53 gene /protein; peptide chemical synthesis; polymerase chain reaction; reporter genes; site directed mutagenesis; thiols; thioredoxin; western blottings

Stimulation of transcription by the human tumor suppressor protein p53 is compromised in yeast lacking the enzyme thioredoxin reductase (Trr1). The result suggests that p53 is prone to oxidative inactivation. This hypothesis and the mechanism of inactivation will be investigated using genetic tools unique to the yeast system. Unlike deletion of the TRR1 gene, deletion of both yeast thioredoxin genes (TRX1 and 2) does not affect p53 activity. Before concluding that Trr1 affects p53 independently of thioredoxin, the possibility that loss of p53 activity is due to accumulation of oxidized thioredoxin will be investigated by determining whether deleting TRX1 and 2 suppresses the effect of deleting TRR1 (Aim 1). Deletion of TRR1 results in accumulation of oxidized glutathione. To test whether reduced p53 activity in deltatrr1 yeast is an indirect consequence of increased glutathione oxidation, the effect of deleting and overexpressing the glutathione reductase gene GLR1 on p53 activity and glutathione redox state will be determined (Aim 2). If deleting GLR1 results in glutathione oxidation but does not affect p53 activity, and if high copy GLR1 restores glutathione to the reduced state in deltatrr1 yeast but does not restore p53 activity, it would indicate that the thioredoxin system and not the glutathione system is critical for p53 activity. A LexA/full length p53 fusion protein stimulates a Lex operator/LacZ reporter gene in wildtype but not deltatrr1 yeast. A fusion protein lacking the last 256 p53 residues is active in both yeast. To identify the domain that causes p53 to be Trr1- dependent, a finer set of C-terminally-truncated LexA/p53 fusion proteins will be analyzed (Aim 3). To identify cysteines that cause p53 to be Trr-dependent, the effect of Cys-to-Ser mutations on p53 activity in wildtype and deltatrr1 yeast will be determined (Aim 4). To complement the genetic approaches, biochemical evidence of p53 oxidation will be sought (Aim 5). Immunoblots done in the presence or absence of reducing agent will investigate whether p53 is disulfide-bonded to other proteins in deltatrr1 yeast. Differential alkylation assays will investigate whether p53 has fewer free thiols in deltatrr1 yeast. Band shift assays will determine whether p53 modifications that result in Trr1-independent transcriptional activity in vivo result in dithiothreitol-independent DNA binding activity in vitro. Understanding the basis for Trr1-dependent p53 activity in yeast will provide a framework for testing whether p53 is subject to oxidative inactivation in higher eucaryotes, and may provide a molecular explanation for the effects of antioxidants and hypoxia on tumor incidence and progression. Equally important, the experiments test a general approach for deriving oxidation-resistant proteins using yeast.

在缺乏硫氧还蛋白还原酶 (Trr1) 的酵母中，人类肿瘤抑制蛋白 p53 对转录的刺激会受到损害。 结果表明p53容易氧化失活。 将使用酵母系统特有的遗传工具来研究这一假设和失活机制。 与删除 TRR1 基因不同，删除两个酵母硫氧还蛋白基因（TRX1 和 2）不会影响 p53 活性。 在得出 Trr1 独立于硫氧还蛋白影响 p53 的结论之前，将通过确定删除 TRX1 和 2 是否会抑制删除 TRR1 的效果来研究 p53 活性丧失是由于氧化硫氧还蛋白积累所致的可能性（目标 1）。 TRR1 的缺失会导致氧化型谷胱甘肽的积累。 为了测试 deltatrr1 酵母中 p53 活性降低是否是谷胱甘肽氧化增加的间接结果，将确定删除和过表达谷胱甘肽还原酶基因 GLR1 对 p53 活性和谷胱甘肽氧化还原状态的影响（目标 2）。 如果删除 GLR1 导致谷胱甘肽氧化但不影响 p53 活性，并且如果高拷贝 GLR1 将 deltatrr1 酵母中的谷胱甘肽恢复为还原态但不恢复 p53 活性，则表明硫氧还蛋白系统而不是谷胱甘肽系统对p53 活性。 LexA/全长 p53 融合蛋白可刺激野生型酵母中的 Lex 操纵基因/LacZ 报告基因，但不会刺激 deltatrr1 酵母。缺少最后 256 个 p53 残基的融合蛋白在两种酵母中均具有活性。 为了识别导致 p53 成为 Trr1 依赖性的结构域，将分析一组更精细的 C 末端截短的 LexA/p53 融合蛋白（目标 3）。 为了鉴定导致 p53 成为 Trr 依赖性的半胱氨酸，将确定 Cys 到 Ser 突变对野生型和 deltatrr1 酵母中 p53 活性的影响（目标 4）。 为了补充遗传方法，将寻找 p53 氧化的生化证据（目标 5）。在存在或不存在还原剂的情况下进行的免疫印迹将研究 p53 是否与 deltatrr1 酵母中的其他蛋白质形成二硫键。 差异烷基化测定将研究 deltatrr1 酵母中 p53 是否具有较少的游离硫醇。带位移分析将确定导致体内 Trr1 独立转录活性的 p53 修饰是否会导致体外二硫苏糖醇独立 DNA 结合活性。 了解酵母中 Trr1 依赖性 p53 活性的基础将为测试 p53 在高等真核生物中是否受到氧化失活提供框架，并可能为抗氧化剂和缺氧对肿瘤发生和进展的影响提供分子解释。同样重要的是，这些实验测试了使用酵母产生抗氧化蛋白质的通用方法。

项目成果

Protein electrophoretic mobility shift assay to monitor redox state of thioredoxin in cells.

蛋白质电泳迁移率变动测定法监测细胞中硫氧还蛋白的氧化还原状态。

• DOI: 10.1016/s0076-6879(02)47031-0
• 发表时间: 2002
• 期刊: Methods in enzymology
• 作者: Bersani,NeilA;Merwin,JasonR;Lopez,NathanI;Pearson,GeorgeD;Merrill,GaryF
• 通讯作者: Merrill,GaryF

Reporter gene transactivation by human p53 is inhibited in thioredoxin reductase null yeast by a mechanism associated with thioredoxin oxidation and independent of changes in the redox state of glutathione.

在硫氧还蛋白还原酶无效酵母中，人 p53 的报告基因反式激活受到与硫氧还蛋白氧化相关的机制的抑制，且与谷胱甘肽氧化还原状态的变化无关。

• DOI: 10.1093/carcin/23.10.1609
• 发表时间: 2002
• 期刊: Carcinogenesis
• 影响因子: 4.7
• 作者: Merwin,JR;Mustacich,DJ;Muller,EGD;Pearson,GD;Merrill,GF
• 通讯作者: Merrill,GF

Effect of thioredoxin deletion and p53 cysteine replacement on human p53 activity in wild-type and thioredoxin reductase null yeast.

硫氧还蛋白缺失和 p53 半胱氨酸替换对野生型和硫氧还蛋白还原酶无效酵母中人 p53 活性的影响。

• DOI: 10.1021/bi900757q
• 发表时间: 2009
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Stoner,ChristopherS;Pearson,GeorgeD;Koç,Ahmet;Merwin,JasonR;Lopez,NathanI;Merrill,GaryF
• 通讯作者: Merrill,GaryF