Mechanism of Redox Regulation by Ape1/Ref-1

描述（由申请人提供）：无嘌呤/无嘧啶核酸内切酶（Ape1）是一种必需的碱基切除修复（BER）酶，已被证明在许多癌症中表达水平升高或改变。（1-8）除了其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（见1）。尽管早在10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是如何减少这些转录因子的，而且关于半胱氨酸65的作用，文献中的数据相互矛盾，而半胱氨酸65被认为是氧化还原功能的关键。我们建议重新研究Apel氧化还原活性的机制及其在癌症病因学中的作用，以探索这种独特的、尚未被探索的活性，作为未来癌症治疗学发展的目标。我们详细了解Apel氧化还原活性的机制及其在癌细胞中的作用的能力将极大地帮助使用3-[5-（2,3-二甲氧基-6-甲基-1,4-苯并喹啉基）]-2-壬基-2-丙酸，（E3330），一种醌衍生物，已被证明：(1)在直接结合试验中以高亲和力特异性结合Ape1，(2)在体外和癌细胞系中抑制Ape1的氧化还原活性。如具体目标1和2中所述，我们建议使用x射线晶体学、定点诱变、化学交联和氢/氘交换实验来确定Apel氧化还原活性所需的残基以及Ape1与其减少的转录因子之间的相互作用位点。在Specific Aim 3中，我们提出用E3330抑制Ape1的氧化还原活性，从而确定Apel氧化还原活性对正常和癌细胞系下游靶点的作用。相关性：无嘌呤/无嘧啶内切酶（Ape1）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疗药物的一个有吸引力的靶标。我们建议阐明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。

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.