Mechanism of Redox Regulation by Ape1/Ref-1

Ape1/Ref-1 的氧化还原调节机制

• 批准号: 7424924
• 负责人: Millie M Georgiadis
• 金额: $ 23.32万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7424924
• 关键词: Affect; Affinity; Base Excision Repairs; Binding; Biological Assay; Cancer Etiology; Cancer cell line; Chemicals; Conflict (Psychology); Cysteine; DNA Damage; Data; Deuterium; Development; E 3330; Enzymes; Excision; Future; Goals; Hydrogen; In Vitro; Literature; Malignant Neoplasms; NF-kappa B; Numbers; Oxidation-Reduction; Play; Propionic Acids; Propionic acid; Quinones; Reaction; Regulation; Research Personnel; Role; Signal Transduction; Site; Site-Directed Mutagenesis; Structure; TP53 gene; Therapeutic; Thinking; Transcription Factor AP-1; X-Ray Crystallography; base; benzoquinone; cancer cell; crosslink; density; endonuclease; neoplastic cell; novel; programs; repair enzyme; repaired; research study; response; transcription factor

DESCRIPTION (provided by applicant): Apurinic/apyrimidinic endonuclease (Ape1), an essential Base Excision Repair (BER) enzyme, has been shown to have elevated or altered levels of expression in a number of cancers.(1-8) In addition to its DMA repair activity, Ape1 has a redox function that serves to reduce a number of transcription factors including AP-1 (Fos/Jun), HIF-1a, NF-kB, PAX, HLF, p53, and others (reviewed in 1). Despite the discovery of Apel's redox activity more than 10 years ago, there is currently no detailed mechanism to describe how Ape1 reduces these transcription factors and conflicting data in the literature regarding the role of cysteine 65, thought to be critical for the redox function. We propose to revisit the mechanism of Apel's redox activity and its role in cancer etiology with the specific goal of exploring this unique and as yet unexplored activity as a target for the development of cancer therapeutics in the future. Our ability to detail the mechanism of Apel's redox activity and its role in cancer cells will be aided greatly by the use of 3-[5-(2,3-dimethoxy-6-methyl-1,4- benzoquinoyl)]-2-nonyl-2-propionic acid, (E3330), a quinone derivative, that has been shown (1) to bind specifically to Ape1 in a direct binding assay with high affinity and (2) to inhibit the redox activity of Ape1 both in vitro and in cancer cell lines. As detailed in Specific Aims 1 and 2, we have proposed to use X-ray crystallography, site-directed mutagenesis, chemical cross-linking, and hydrogen/deuterium exchange experiments in order to identify the residues that are required for Apel's redox activity and sites of interactions between Ape1 and the transcription factors that it reduces. In Specific Aim 3, we propose to inhibit the redox activity of Ape1 with E3330 and thereby determine the role of Apel's redox activity on downstream targets in normal and cancer cell lines. Relevance: Apurinic/apyrimidinic endonuclease (Ape1) has been shown to have elevated or altered levels of expression in a number of cancers. This multifunctional enzyme is an attractive target for the development of chemotherapeutics. We propose here to elucidate the mechanism of the redox function of Ape1 and explore its role in tumor cell response following treatment with DNA damaging agents.

描述（由申请人提供）：脱嘌呤/脱嘧啶核酸内切酶 (Ape1) 是一种重要的碱基切除修复 (BER) 酶，已显示在多种癌症中表达水平升高或改变。(1-8) 除了 DMA 修复活性外，Ape1 还具有氧化还原功能，可减少多种转录因子，包括 AP-1 (Fos/Jun)、HIF-1a、 NF-kB、PAX、HLF、p53 等（已在 1 中进行了综述）。尽管十多年前就发现了 Apel 的氧化还原活性，但目前还没有详细的机制来描述 Ape1 如何减少这些转录因子，并且文献中关于半胱氨酸 65 的作用的数据相互矛盾，半胱氨酸 65 被认为对氧化还原功能至关重要。我们建议重新审视 Apel 氧化还原活性的机制及其在癌症病因学中的作用，具体目标是探索这种独特且尚未探索的活性，作为未来癌症治疗开发的目标。使用 3-[5-(2,3-二甲氧基-6-甲基-1,4-苯并喹酰基)]-2-壬基-2-丙酸 (E3330)（一种醌衍生物）将极大地帮助我们详述 Apel 氧化还原活性的机制及其在癌细胞中的作用，该衍生物已被证明 (1) 在直接结合测定中以高亲和力与 Ape1 特异性结合，并且 (2)在体外和癌细胞系中抑制Ape1的氧化还原活性。正如具体目标 1 和 2 中详述的，我们建议使用 X 射线晶体学、定点诱变、化学交联和氢/氘交换实验来鉴定 Apel 氧化还原活性所需的残基以及 Ape1 与其还原的转录因子之间的相互作用位点。在具体目标 3 中，我们建议用 E3330 抑制 Ape1 的氧化还原活性，从而确定 Apel 的氧化还原活性对正常细胞系和癌细胞系下游靶标的作用。相关性：脱嘌呤/脱嘧啶核酸内切酶 (Ape1) 已被证明在多种癌症中表达水平升高或改变。这种多功能酶是化疗药物开发的一个有吸引力的靶标。我们在此建议阐明 Ape1 氧化还原功能的机制，并探讨其在 DNA 损伤剂治疗后肿瘤细胞反应中的作用。