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

使用基于质谱的蛋白质组学方法阐明细胞金属应力的分子基础

• 批准号: 10467488
• 负责人: Michael C Fitzgerald
• 金额: $ 43.97万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-05 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10467488
• 关键词: Address; Affect; Binding; Biological; Biological Assay; Biophysics; Cancer cell line; Candida albicans; Cell Line; Cell Survival; Cell physiology; Cells; Chemicals; Collaborations; Copper; Data; Development; Environment; Escherichia coli; Escherichia coli Proteins; Event; Exposure to; Foundations; Functional disorder; Global Change; Goals; Growth; Homeostasis; Human; Immune system; In Vitro; Ionophores; Ions; Laboratories; Lead; Link; Location; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Metabolism; Metal exposure; Metalloproteins; Metals; Methodological Studies; Methods; Molecular; Monitor; Neurodegenerative Disorders; Nutrient; Organism; Outcome; Outcome Study; Pathway interactions; Pharmacology; Physiologic pulse; Precipitation; Predisposition; Proteins; Proteolysis; Proteome; Proteomics; Proxy; Publishing; Research; Signal Transduction; Stress; Techniques; Testing; Therapeutic; Time; Toxic effect; Trace Elements; Translations; Work; anti-cancer; antimicrobial; base; biophysical properties; cancer cell; cell type; cofactor; design; experience; microbial; pathogen; pathogenic microbe; prostate cancer cell; protein folding; protein function; protein misfolding; tool; trafficking

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.

项目总结/摘要 金属离子是细胞功能所需的营养素，但如果调控不当也可能有毒。我们的免疫系统 利用这一二分法，通过部署机制，既阻止营养金属从病原体， 使它们不堪重负，尤其是铜和锌的毒性水平。细胞水平的金属失衡也 与神经退行性疾病有关，并且正在研究作为可能的抗癌策略。 但细胞金属应激的靶点和机制究竟是什么？这里提出的研究探索 这个问题的细胞金属压力，寻求确定蛋白质目标的异常金属相互作用， 测量整个蛋白质组中蛋白质稳定性的整体变化。拟议的工作建立在初步和 该合作团队最近发表的结果显示了脉冲蛋白水解质量的实用性， 光谱分析方法在共同PI菲茨杰拉德的实验室开发，以确定蛋白质目标的铜在大肠杆菌。大肠杆菌， 建立了研究金属-蛋白质相互作用的蛋白质组学方法。的总体目标 当前的应用是鉴定当细胞经历诱导的应激时功能上受影响的蛋白质 通过暴露于过量的锌和铜。这一目标将通过使用强大的质量组合来实现 基于光谱学蛋白质组学方法解决四个具体目标：1）确定蛋白质的全局谱 稳定性变化作为细胞金属超载和金属缺乏的功能，在细菌，真菌， 人癌细胞; 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） 确定整个蛋白质组中蛋白质对Cu和Zn结合诱导的错误折叠的相对敏感性;以及 4)建立一个生物物理学基础，以了解蛋白质对金属诱导的错误折叠的相对敏感性。 了解暴露于正常或异常水平时蛋白质稳定性如何在蛋白质组中受到影响 铜和锌的结合对于理解金属诱导的毒性和细胞用于 在面对与金属相关的压力时保持金属体内平衡。通过研究来自微生物、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体如何 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 研究将为开发抗微生物病原体的药物提供信息并产生影响， 癌细胞，并与申请人开发化学工具来操纵生物学的长期目标保持一致 金属离子的位置、形态和反应性，以获得潜在的治疗益处。