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，抑制DNA修复有助于砷的致癌和共同致癌作用。两种DNA修复蛋白(PARP-1和XPA)已被报道为干扰锌指功能的直接砷靶点。到目前为止，识别DNA修复途径中的砷靶标完全是基于经验证据，尚不清楚是否存在额外的直接和高度敏感的DNA修复靶标。我们最近的工作证明了亚砷酸盐与含有3或4个半胱氨酸残基的锌指多肽的优先相互作用，以及从暴露于C3H1(例如PARP-1)和C4(例如XPA)的细胞中分离的特定DNA修复蛋白对亚砷酸盐依赖的锌释放，但不包括C2H2和锌指蛋白。这些发现为基于半胱氨酸残基数量的锌指蛋白的靶向选择性提供了证据。本项目的目的是实施一种迭代的生物信息学/实验方法来识别、测试和改进DNA修复途径中高亲和力砷靶标的选择，以深入了解砷的共同致癌和DNA修复抑制的机制。这项工作将提供关于以下方面的关键信息：已识别靶标的相对敏感性、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(如无名指、高音环、锌带)在确定砷攻击易感性方面的作用。利用生物信息学方法的初步结果发现了新的候选DNA修复靶点，其中包含与PARP-1或XPA不同的锌指结构和活性，这表明砷可能在DNA修复抑制中发挥新的作用。基于我们已发表的和初步的发现，我们假设一种生物信息学/实验耦合的方法可以开发并应用于基于锌指构型的DNA修复中的高亲和力砷靶标的预测。为了检验这一假设，我们将： 1)使用无偏锌指模体模式识别算法识别DNA修复中可能的砷靶标，与来自自动化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，并通过系统发育和途径分析进一步分类。DNA修复途径被用作验证集，因为它代表了一个对砷癌生物学和流行病学具有直接意义的成熟的和生物相关的焦点。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，确定对砷的相对敏感性，并为生物信息学方法的迭代完善提供结构特征信息。这些拟议研究的结果有望通过1)扩大我们对砷破坏锌指DNA修复蛋白质的范围的理解，2)确定新的和敏感的靶点，以及3)确定特定的锌指结构是否代表优先靶点，从而推动该领域的发展。这些结果将为关于癌症和其他砷相关疾病的其他潜在砷靶点的可测试假设提供信息。 与公共健康相关：鉴于市政和私人供水中的砷在公众中的广泛暴露，人们对观察到的砷浓度达到或接近EPA最大污染物水平会极大地提高其他DNA损伤剂的致癌潜力并抑制DNA修复感到兴趣和担忧。因此，当个人通过职业、环境或生活方式暴露于其他致癌物时，砷可能会增加患癌症的风险。该项目是通过计算预测高度敏感的砷目标的第一次努力，以更好地了解砷对DNA修复的影响，并为扭转或防止人体接触砷对健康的不利影响提供战略信息。

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.