Thermodynamics of Coupled Binding of Zn(II) and DNA to a Zinc Finger Tumor Suppre

描述(申请人提供)：这项研究的长期目标是通过表征ZFP转录因子与锌金属离子和DNA相互作用的基本热力学，改进人工锌指蛋白(ZFP)转录因子疗法的合理设计。ZFP是人类基因组中最大的一类金属蛋白，通过结合锌金属离子和DNA或RNA来调节基因转录，从而调节蛋白质的表达。由于ZFP转录因子可以潜在地设计为激活或抑制基因组中的任何基因，这些蛋白质是药物设计的极佳靶点。事实上，人工ZFP的合理设计和组合选择正开始产生潜在的候选药物，其中一种正在进行糖尿病神经病变和Lou Gehrig病的临床试验。此外，最近在哺乳动物细胞中转导ZFP蛋白的成功表明它们可能适合于基因治疗应用。阻碍人工ZFP设计进展的一个问题是ZFP中锌金属离子结合、DNA结合和蛋白质折叠的耦合能量学。基于我们使用简单设计的多肽成功地分离了锌金属离子结合和蛋白质折叠的能量学，我们处于一个独特的位置来解偶联锌金属离子和DNA在天然ZFP中的结合，Wilms肿瘤抑制因子。我们将集中精力描述ZFP与锌金属离子和DNA结合的基本热力学。我们的方法是测量锌(II)和DNA与ZFP结合的平衡热力学。基于目前对锌(II)和DNA与ZFP的偶联结合的了解，我们确定了在所要求的资助期内需要完成的两个具体目标。具体目的1：研究DNA存在下锌指蛋白与锌(II)的亲和力。具体目的2：脱脂锌指蛋白折叠的研究。 公共卫生相关性：这项研究提供了对锌(II)和DNA与锌指蛋白结合的相互关系的详细了解，将有助于更好地理解一般金属调控过程，特别是人类癌症中的基因激活。这些结果将有助于肿瘤抑制药物的合理设计和改进对癌症基因激活过程的预测。因此，这项工作的成功完成有望给医疗保健和公众的普遍福利带来好处。

DESCRIPTION (provided by applicant): The long-term goal of this research is to improve the rational design of artificial zinc finger protein (ZFP) transcription factor therapeutics by characterizing the fundamental thermodynamics of ZFP transcription factor interactions with zinc metal-ions and DNA. ZFPs, the largest single class of metalloproteins in the human genome, regulate gene transcription and, therefore, protein expression, by binding both zinc metal-ions and DNA or RNA. Since ZFP transcription factors can be potentially designed to activate or repress any gene in the genome, these proteins are excellent targets for drug design. Indeed the rational design and combinatorial selection of artificial ZFPs is beginning to yield potential drug candidates, one of which is in clinical trials for diabetic neuropathy and Lou Gehrig's disease. Furthermore, recent success in ZFP protein transduction in mammalian cells indicates they may be suitable for gene therapy applications. One issue that slows the progress of artificial ZFP design is coupled energetics of zinc metal-ion binding, DNA-binding, and protein folding in ZFPs. Based on our success in parsing apart the energetics of zinc metal-ion binding and protein folding using simple designed peptides, we are in a unique position to decouple the binding of zinc metal-ions and DNA in a natural ZFP, the Wilms tumor suppressor. We will focus our research efforts in delineating the fundamental thermodynamics of ZFP binding to zinc metal-ions and DNA. Our approach is to measure the equilibrium thermodynamics of Zn(II) and DNA binding to ZFPs. Based on the current lack of understanding of the coupled binding of Zn(II) and DNA to ZFPs, we have identified two Specific Aims to be completed during the requested funding period. Specific Aim 1: Study of zinc finger protein Zn(II) affinity in the presence of DNA. Specific Aim 2: Study of apo- zinc finger protein folding. PUBLIC HEALTH RELEVANCE: The detailed understanding of the interrelationship of Zn(II) and DNA binding to zinc finger proteins provided by this study will lead to a greater understanding of general metalloregulatory processes, and specifically gene activation in human cancer. These results will facilitate both the rational design of tumor suppressor drugs and improved prediction of the process of cancer gene activation. Therefore, benefits to healthcare and the general welfare of the public are anticipated from the successful completion of this work.