Elucidating the Molecular Basis of Cellular Metal Stress by using Mass Spectrometry-Based Proteomic Methods

项目摘要/摘要 金属离子是细胞功能所必需的营养物质，但如果调控不当，也可能是有毒的。我们的免疫系统 利用这种二分法，通过部署机制来阻止病原体的营养金属以及 用毒性水平让它们不堪重负，尤其是铜和锌。细胞水平上的金属失衡也 与神经退行性疾病有关，并正在作为可能的抗癌策略进行研究。 但是，细胞金属应力的目标和机制到底是什么？这里提出的研究探索了 通过寻求识别异常金属相互作用的蛋白质靶点来解决细胞金属应激的问题 通过蛋白质组测量蛋白质稳定性的全球变化。拟议的工作建立在初步和 这个合作团队最近发表的结果显示了脉冲蛋白水解团的效用 Pi Fitzgerald实验室共同开发的鉴定大肠杆菌中铜的蛋白质靶标的光谱方法， 建立这些研究金属-蛋白质相互作用的蛋白质组学方法。总的目标是 目前的应用是识别当细胞经历应激诱导时受到功能影响的蛋白质 通过暴露于过量的锌和铜。这一目标将通过使用质量的强大组合来实现 基于光谱的蛋白质组学方法解决四个特定目标：1)确定蛋白质的全球图谱 稳定性作为细胞金属超载和金属缺乏的函数在细菌、真菌和 人类癌细胞；2)建立将Aim 1蛋白的差异稳定性与功能联系起来的机制基础；3) 确定蛋白质组中蛋白质对铜和锌结合引起的错误折叠的相对敏感性；以及 4)为了解蛋白质对金属诱导的错误折叠的相对敏感性奠定了生物物理基础。 了解当暴露于正常或异常水平时，蛋白质组的稳定性是如何受到影响的 铜和锌对理解金属的毒性和细胞利用的机制具有重要意义 在与金属相关的压力面前，保持金属的动态平衡。通过研究微生物、真菌、 和人类癌细胞，这些研究的结果将促进我们对这些生物如何 在蛋白质组水平上对宿主免疫系统施加的不断变化的金属环境做出反应。这些 研究将提供信息并影响针对微生物病原体和 并与申请人的长期目标保持一致，即开发化学工具来操纵生物 潜在治疗益处的金属离子的位置、形态和反应性。

PROJECT SUMMARY/ABSTRACT Metal ions are required nutrients for cellular function, but can also be toxic if misregulated. Our immune system leverages this dichotomy by deploying mechanisms both to withhold nutrient metals from pathogens as well as overwhelm them with toxic levels, particularly of Cu and Zn. Metal imbalances at the cellular level have also been implicated in neurodegenerative diseases, and are being investigated as possible anticancer strategies. But what exactly are the targets and mechanisms of cellular metal stress? The research proposed here explores this question of cellular metal stress by seeking to identify protein targets of aberrant metal interactions by measuring global changes in protein stability across the proteome. The proposed work builds on preliminary and recently published results from this collaborative team showing the utility of a pulse proteolysis mass spectrometry method developed in co-PI Fitzgerald’s laboratory to identify protein targets of Cu in E. coli, establishing these proteomic methodologies for the study of metal-protein interactions. The overall objective of the current application is to identify proteins that are functionally affected when cells experience stress induced by exposure to excess levels of Zn and Cu. This objective will be met by using a powerful combination of mass spectrometry-based proteomic methods to address four specific aims: 1) Determine global profiles of protein stability changes as a function of cellular metal overload and metal deficiency across bacterial, fungal, and human cancer cells; 2) Establish a mechanistic basis linking differential stability of Aim 1 proteins to function; 3) Identify the relative sensitivity of proteins across the proteome to misfolding induced by Cu and Zn binding; and 4) Establish a biophysical basis for understanding the relative sensitivity of proteins to metal-induced misfolding. Understanding how protein stability is impacted across the proteome upon exposure to normal or aberrant levels of Cu and Zn has important implications for understanding metal-induced toxicity and mechanisms cells use to maintain metal homeostasis in the face of metal-associated stress. By studying proteomes from microbial, fungal, and human cancer cells, the outcomes of these studies will advance our understanding of how these organisms respond at the proteome level to changing metal environments imposed by the host immune system. These studies will inform and impact the development of pharmacological agents against microbial pathogens and cancer cells, and align with the applicant’s long-term goals to develop chemical tools to manipulate biological metal ion location, speciation, and reactivity for potential therapeutic benefit.