Mechanism of Redox Regulation by Ape1/Ref-1

描述（由申请人提供）：脱嘌呤/脱嘧啶核酸内切酶（Ape 1）是一种必需的碱基切除修复（BER）酶，已显示在许多癌症中具有升高或改变的表达水平。(1-8)除了DNA修复活性外，Ape 1还具有氧化还原功能，可减少许多转录因子，包括AP-1（Fos/Jun），HIF-1a，NF-kB，PAX，HLF，p53等（综述见1）。尽管Apel的氧化还原活性在10多年前就被发现了，但目前还没有详细的机制来描述Ape 1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被认为是氧化还原功能的关键。我们建议重新审视Apel的氧化还原活性的机制及其在癌症病因学中的作用，其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发展的目标。我们能够详细说明Apel的氧化还原活性的机制及其在癌细胞中的作用，这将极大地有助于使用3-[5-（2-甲氧基苯基）-2-甲基-N-（2-甲氧基苯基）-2-甲基-（2-甲氧基苯基）-2-（2-甲氧基苯基）-（2，3-二甲氧基-6-甲基-1，4-苯醌基）]-2-壬基-2-丙酸，（E3330），醌衍生物，其已显示（1）在直接结合测定中以高亲和力特异性结合Ape 1和（2）在体外和癌细胞系中抑制Ape 1的氧化还原活性。如具体目标1和2所述，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，以确定Apel氧化还原活性所需的残基以及Ape 1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，我们提出用E3330抑制Ape 1的氧化还原活性，从而确定Apel的氧化还原活性对正常和癌细胞系中下游靶点的作用。相关性：脱嘌呤/脱嘧啶核酸内切酶（Ape 1）已被证明在许多癌症中表达水平升高或改变。这种多功能酶是开发化学治疗剂的有吸引力的靶标。我们拟阐明Ape 1的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。

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.