Biochemical and Molecular Genetic Studies of the HMT-1-Dependent Heavy Metal Detoxification Pathway in C. Elegans

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。虽然金属污染的环境日益受到关注，但所有生物的保护性适应和机制仍有待了解。有效开发重金属污染环境生物修复策略的能力取决于对这些细胞解毒和耐受机制的详细了解。因此，秀丽隐杆线虫（Caenorhabditis elegans）中的半分子atp结合盒（ABC）转运体HMT-1是重金属解毒的迫切需要，并且在包括莱因哈特衣藻（Chlamydomonas reinhardtii）、pombe裂糖菌（Schizosaccharomyces pombe）、果蝇和人类在内的许多其他物种中具有同源物。HMT-1依赖通路的功能以及其他ABC转运蛋白相对于HMT-1的作用尚不清楚。本项目以秀丽隐杆线虫作为一个简单且定义良好的模型系统，利用生化和遗传方法研究hmt -1依赖性解毒途径。目的1将重点关注复合物中与HMT-1功能相关的蛋白质的鉴定和定位。在目标2中进行的平行系统遗传分析将确定重金属耐受性所需的ABC转运蛋白，并评估它们与HMT-1的功能关系。考虑到秀丽隐杆线虫ABC转运基因家族的庞大和多样性，这项工作有望对更广泛的秀丽隐杆线虫群落产生相当大的兴趣，并进一步了解这些基因的功能以及与其他应激反应和发育途径的潜在参与。在提供有关hmt -1依赖性通路的第一个基本信息时，该项目将识别和分类ABC转运蛋白的功能作用，识别新的通路，并将功能归因于以前未确定参与金属解毒的已知通路，从而将有助于理解金属耐受性的细胞资源的长期目标。更广泛的影响。该项目的研究部分与学生、博士后研究员和公众关于重金属及其毒性以及环境和农业生物技术方面更广泛问题的教育相结合。首先，一系列讨论重金属及其毒性的讲座被整合到两个高级讲座课程中。第二，研究的目的是培训和指导实验室的本科生、研究生和博士后，并特别努力招收妇女和少数民族。将现代基因组学和生物化学方法结合起来研究环境问题将为下一代科学家提供宝贵的经验。第三，该项目旨在让纽约州的农民和园丁了解重金属及其毒性，以及安全土壤管理对减少重金属进入食物链的重要性。这项外联活动将包括演讲和编写用户友好的土壤测试口译指导文件，这些文件将在一个网络模块中进行总结。这一举措具有令人兴奋的广泛应用潜力，超出了纽约州公民的范围，并可能为科学专业知识和研究与公共可用信息的有效接口提供范例。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5). While metal polluted environments are a growing concern, much remains to be learned about the protective adaptations and mechanisms in all organisms. The ability to effectively develop bioremediation strategies for heavy metal-polluted environments is contingent on gaining a detailed understanding of these cellular detoxification and tolerance mechanisms. In this regard, HMT-1, a half molecule ATP-binding cassette (ABC) transporter in the nematode worm Caenorhabditis elegans is acutely required for heavy metal detoxification and has homologs in many other species including Chlamydomonas reinhardtii, Schizosaccharomyces pombe, Drosophila and humans. How the HMT-1-dependent pathway functions and the roles of other ABC transporters relative to HMT-1 are unknown. This project uses C. elegans as a simple and well-defined model system to study the HMT-1-dependent detoxification pathway using biochemical and genetic approaches. Objective 1 will focus on the identification and localization of proteins that functionally associate with HMT-1 in a complex. Parallel systematic genetic assays pursued in Objective 2 will identify ABC transporters that are required for heavy metal tolerance, and assess their functional relationships to HMT-1. Given the large and diverse nature of the C. elegans ABC transporter gene family, this work promises to be of considerable interest to the broader C. elegans community and beyond to learn the function of these genes and potential involvement with other stress responses and developmental pathways. In providing the first fundamental information about the HMT-1-dependent pathway, this project will identify and classify functional roles of the ABC transporters, identify new pathways, and ascribe functions to known pathways that have not previously been determined to be involved in metal detoxification, and thus will contribute to the long-term goal of understanding of cellular resources for metal tolerance. Broader Impacts. The research component of this project is integrated with the education of students, postdoctoral fellows and the general public about heavy metals and their toxicity as well as broader issues in environmental and agricultural biotechnology. First, a series of lectures discussing heavy metals and their toxicity is integrated into two upper-level lecture courses. Second, research is directed towards the training and mentoring of undergraduate, graduate students and postdoctoral fellows in the laboratory with special efforts to recruit women and minorities. The incorporation of modern genomic and biochemical approaches to study environmental issues will provide valuable experience for the next generation of scientists. Third, the project is designed to reach New York State (NYS) farmers and gardeners to increase their knowledge about heavy metals and their toxicity, and the importance of safe soil management for decreasing heavy metal entrance into the food chain. This outreach activity will consist of lecture presentations, and the development of user-friendly soil-test interpretation guidance documents, which will be summarized in a web module. This initiative has exciting potential for broad usage, going beyond the citizens of NYS, and may provide a paradigm for effective interfacing of scientific expertise and research with publicly-available information.