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.

项目摘要/摘要 金属离子是细胞功能所需的营养素，但如果错过，也可能是有毒的。我们的免疫系统 通过部署机制来保留病原体以及 以有毒水平的水平，尤其是Cu和Zn淹没它们。细胞水平的金属失衡也有 在神经退行性疾病中隐含，并正在研究作为可能的抗癌策略。 但是，细胞金属应激的目标和机制到底是什么？这里提出的研究探讨了 通过寻求通过识别通过鉴定异常金属相互作用的蛋白质靶标的细胞金属应激问题 测量整个蛋白质蛋白质稳定性的全球变化。拟议的工作以初步为基础 最近发布了这个合作团队的结果，显示了脉冲蛋白水解质量的实用性 在Co-Pi Fitzgerald的实验室中开发的光谱法，以鉴定大肠杆菌中Cu的蛋白质靶标， 建立这些蛋白质组学方法来研究金属蛋白质相互作用。总体目标 当前的应用是识别当细胞经历应力诱导时功能影响的蛋白质 通过暴露于锌和铜的过量水平。通过使用强大的质量组合来实现这一目标 基于光谱法的蛋白质组学方法来解决四个特定目的：1）确定蛋白质的全局特征 稳定性随细菌，真菌和细菌的金属过载和金属缺乏症的作用而变化 人类癌细胞； 2）建立一个机械基础，将目标1蛋白的差异稳定性联系起来； 3） 确定蛋白质组跨蛋白质对Cu和Zn结合引起的错误折叠的相对灵敏度；和 4）建立一个生物物理基础，以理解蛋白质对金属诱导的错误折叠的相对敏感性。 了解蛋白质稳定性如何在暴露于正常水平或异常的情况下在蛋白质上产生影响 Cu和Zn的of对于理解金属诱导的毒性和机制的重要意义 面对金属相关的压力，保持金属稳态。通过研究微生物，真菌的蛋白质组织， 和人类癌细胞，这些研究的结果将提高我们对这些生物如何的理解 在蛋白质组水平上响应宿主免疫系统施加的不断变化的金属环境。这些 研究将告知和影响药物针对微生物病原体的开发和 癌细胞，并与申请人的长期目标保持一致，以开发化学工具来操纵生物学 金属离子位置，规格和反应性，以实现潜在的治疗益处。