Predicting Novel Arsenic Targets in DNA Repair Pathways

描述（由申请人提供）：有令人信服的证据表明，DNA 修复的抑制会导致砷的致癌和共致癌作用。据报道，基于对锌指功能的干扰，两种 DNA 修复蛋白（PARP-1 和 XPA）可作为砷的直接靶标。迄今为止，DNA 修复途径中砷靶标的识别仅基于经验证据，尚不清楚是否存在其他直接且高度敏感的 DNA 修复靶标。我们最近的工作表明，亚砷酸盐与含有 3 或 4 个半胱氨酸残基的锌指肽优先相互作用，并且从 C3H1（例如 PARP-1）和 C4（例如 XPA）中暴露的细胞中分离出的特定 DNA 修复蛋白中亚砷酸盐依赖性锌释放，但不包括 C2H2 锌指蛋白。这些发现为锌指蛋白基于半胱氨​​酸残基数量的靶点选择性提供了证据。该项目的目标是实施迭代生物信息学/实验方法来识别、测试和完善DNA修复途径中高亲和力砷靶标的选择，以深入了解砷协同致癌和DNA修复抑制的机制。这项工作将产生关于已识别目标的相对敏感性、半胱氨酸残基的数量和构型在控制观察到的敏感性中的重要性以及不同的锌指二级结构（例如无名指、高音谱号、锌带）在确定砷攻击脆弱性中的作用的关键信息。使用生物信息学方法的初步结果确定了新的候选 DNA 修复靶点，其中包含不同于 PARP-1 或 XPA 的锌指结构和活性，表明砷在 DNA 修复抑制中可能具有新的作用。根据我们已发表的初步研究结果，我们假设可以开发并应用耦合生物信息学/实验方法来预测基于锌指配置的 DNA 修复中的高亲和力砷靶标。为了检验这个假设，我们将： 1) 使用无偏锌指基序模式识别算法识别 DNA 修复中假定的砷靶标，与来自自动在线数据库搜索的结构生物信息学数据和文献注释相关联，并通过系统发育和通路分析进一步分类。 DNA 修复途径被用作验证集，因为它代表了对砷癌症生物学和流行病学具有直接意义的成熟且生物学相关的焦点。 2) 使用生化和细胞生物学方法测试亚砷酸盐与预测目标的相互作用，以验证潜在目标，建立对砷的相对敏感性，并为生物信息学方法的迭代细化提供结构特征信息。拟议研究的结果预计将通过以下方式推进该领域的发展：1）扩大我们对砷对锌指 DNA 修复蛋白破坏范围的理解，2）识别新颖且敏感的靶标，3）确定特定的锌指结构是否代表优先靶标。这些结果将为有关癌症和其他砷相关疾病中其他潜在砷靶点的可检验假设提供信息。 公共健康相关性：考虑到公众广泛接触市政和私人供水中的砷，人们对砷浓度达到或接近 EPA 最大污染物水平的观察感到兴趣和担忧，这会大大增强其他 DNA 损伤剂的致癌潜力并抑制 DNA 修复。因此，当个体通过职业、环境或生活方式接触其他致癌物时，砷可能会导致癌症风险升高。该项目首次通过计算方式预测高度敏感的砷目标，以更好地了解砷对 DNA 修复的影响，并为扭转或预防砷暴露对人类健康造成的不利影响提供策略。

DESCRIPTION (provided by applicant): There is compelling evidence that inhibition of DNA repair contributes to the carcinogenic and co-carcinogenic actions of arsenic. Two DNA repair proteins (PARP-1 and XPA) have been reported as direct arsenic targets based on interference with zinc finger function. To date, identification of arsenic targets in DNA repair pathways has been based solely on empirical evidence and it is unknown whether there are additional direct and highly sensitive DNA repair targets. Our recent work demonstrates preferential interaction of arsenite with zinc finger peptides containing 3 or 4 cysteine residues and arsenite-dependent zinc release from specific DNA repair proteins isolated from exposed cells in C3H1 (e.g. PARP-1) and C4 (e.g. XPA), but not C2H2, zinc finger proteins. These findings provide evidence for target selectivity of zinc finger proteins based on the number of cysteine residues. The objective of this project is to implement an iterative bioinformatic/ experimental approach to identify, test and refine the selection of high-affinity arsenic targets in the DNA repair pathway, in order to gain insights into mechanisms of arsenic co-carcinogenicity and DNA repair inhibition. This work will yield critical information on the relative sensitivities of identified targets, the importanceof number and configuration of cysteine residues in governing observed sensitivities, and the role of distinct zinc finger secondary structures (e.g. ring finger, treble clef, zinc ribbon) in determining vulnerability to arsenic attack. Preliminary results using a bioinformatic approach identified novel candidate DNA repair targets containing zinc finger structures and activities distinct from PARP-1 or XPA, suggesting possible new actions of arsenic in DNA repair inhibition. Based on our published and preliminary findings, we hypothesize that a coupled bioinformatic/ experimental approach can be developed and applied to predict high affinity arsenic targets in DNA repair, based on zinc finger configuration. To test this hypothesis we will: 1) Identify putative arsenic targets in DNA repair using an unbiased zinc finger motif pattern recognition algorithm, correlated with structural bioinformatic data and literature annotations from automated online database searches, and further classified through phylogenetic and pathway analyses. The DNA repair pathway is used as the validation set since it represents a well-established and biologically-relevant focus of direct significance to arsenic cancer biology and epidemiology. 2) Test arsenite interaction with predicted targets using biochemical and cell biology approaches to validate potential targets, establish relative sensitivities to arsenic, and provide information on structural characteristics for iterative refinement of the bioinformatics approach. The outcomes from the proposed studies are expected to advance the field by 1) expanding our understanding of the scope of zinc finger DNA repair protein disruption by arsenic, 2) identifying novel and sensitive targets, and 3) establishing whether specific zinc finger structures represent preferential targets. These results will inform testable hypotheses regarding additional potential arsenic targets in cancer and other arsenic-associated diseases. PUBLIC HEALTH RELEVANCE: Given the widespread public exposure to arsenic in municipal and private water supplies, there is interest and concern in observations that arsenic concentrations at or near the EPA maximum contaminant level greatly enhance the carcinogenic potential of other DNA damaging agents and inhibit DNA repair. Thus, arsenic may contribute to elevated cancer risk when individuals are exposed to other carcinogens through occupational, environmental or lifestyle exposures. This project represents the first effort to computationally predict highly sensitive arsenic targets to better understand the impact of arsenic on DNA repair and inform strategies to reverse or prevent the adverse health effects of arsenic exposure in humans.