PROTEOMIC PROFILING OF CANDIDA ALBICANS RESPONSES TO NITROSATIVE AND OXIDATIVE

白色念珠菌对亚硝化和氧化反应的蛋白质组学分析

• 批准号: 8359570
• 负责人: DAVID J SINGEL
• 金额: $ 19.82万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8359570
• 关键词: Address; Biochemical; Biochemical Pathway; Candida albicans; Cells; Chemical Agents; Chemicals; Chemistry; Color; Data; Detection; Disulfides; Drug Metabolic Detoxication; Funding; Generations; Goals; Grant; Health; Host Defense; Human; Immune response; Immunocompromised Host; Individual; Invaded; Label; Measurement; Measures; Methods; Modeling; Modification; Molecular; National Center for Research Resources; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Pharmaceutical Preparations; Post-Translational Protein Processing; Principal Investigator; Proteins; Proteome; Proteomics; Relative (related person); Research; Research Infrastructure; Resistance; Resources; Risk; Role; S-Nitrosothiols; Signal Transduction; Source; Stress; Sulfenic Acids; Sulfhydryl Compounds; Systems Biology; United States National Institutes of Health; Validation; base; cell growth; computerized data processing; cost; design; macrophage; neutrophil; nitrosative stress; pathogen; programs; response; sensor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Candida albicans is a prevalent human pathogen, which presents a serious health risk in immunocompromised individuals. The first line of host defense against C. albicans involves innate immune responses that challenge the invading pathogen with macrophage- and neutrophil-generated nitrosative and oxidative stress. The counter-measures that pathogens muster against this stress are not well understood, but appear to include roles for proteins involved in enzymatic detoxification of stress-inducing chemical agents, and in signaling cascades that regulate of cell growth. Very little is known, however, at the molecular level, of the responses of C. albicans to oxidative and nitrosative stress. The broad goal of this research is to develop enhanced differential proteomics methods and to apply them to advance our understanding of the molecular bases of C. albicans defenses against host nitrosative and oxidative challenges. Elucidation of the primary biochemical steps in defense pathways will establish new targets for the design of more effective and specific drugs against C. albicans. This research will focus on early responses that are likely to involve reversible modification of reactive protein thiols that yield S-nitrosothiols (RSNO), S- sulfenic acids (RSOH) and transient disulfices. The fundamental questions that we will address are: (1) what components of the C. albicans proteome are modified by different types and levels of oxidative and nitrosative stress; (2) which of these modifications involve redox signaling processes in which protein sensors detect and transduce the stress; and (3) what biochemical pathways are modulated by stress-responsive protein modifications? Differential thiol redox profiling (dThiRP) - featuring a new generation of "Zdyes", that promise unsurpassed threshold sensitivities and the capacity for multi-color differential profiling - will be used to address these questions. Specifically, the research program entails: (1) validation of thiol "switch" detection chemistry with the Zdye labels; (2) global proteomic measurements of changes in protein levels, S-nitrosothiols, sulfenic acids, and disulfides in C. albicans cells subject to exogenous chemical, and macrophage challenge; (3) measurements of the relative resistance to chemical and macrophage challenge of homozygous and heterozygous KO strains, suggested by modeling of the wild-type proteomic data.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的首席研究员可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 白色念珠菌是一种常见的人类病原体，对免疫功能低下的个体构成严重的健康风险。宿主针对白色念珠菌的第一道防线涉及先天免疫反应，利用巨噬细胞和中性粒细胞产生的亚硝化和氧化应激来挑战入侵的病原体。病原体针对这种应激而采取的对策尚不清楚，但似乎包括参与应激诱导化学试剂的酶促解毒以及调节细胞生长的信号级联的蛋白质的作用。然而，在分子水平上，人们对白色念珠菌对氧化和亚硝化应激的反应知之甚少。这项研究的总体目标是开发增强的差异蛋白质组学方法，并应用它们来加深我们对白色念珠菌防御宿主亚硝化和氧化挑战的分子基础的理解。阐明防御途径中的主要生化步骤将为设计针对白色念珠菌更有效和更特异性的药物建立新的目标。 这项研究将重点关注可能涉及反应性蛋白硫醇可逆修饰的早期反应，这些反应产生 S-亚硝基硫醇 (RSNO)、S- 磺酸 (RSOH) 和瞬时二硫化物。我们要解决的基本问题是：（1）白色念珠菌蛋白质组的哪些成分会被不同类型和水平的氧化和亚硝化应激改变； (2) 其中哪些修饰涉及氧化还原信号传导过程，其中蛋白质传感器检测并转导应激； (3) 应激反应蛋白修饰调节哪些生化途径？ 差异硫醇氧化还原分析 (dThiRP) - 采用新一代“Zdyes”，具有无与伦比的阈值灵敏度和多色差异分析能力 - 将用于解决这些问题。具体来说，该研究计划包括：(1) 使用 Zdye 标记验证硫醇“开关”检测化学； (2) 对受到外源化学物质和巨噬细胞攻击的白色念珠菌细胞中蛋白质水平、S-亚硝基硫醇、次磺酸和二硫化物的变化进行整体​​蛋白质组学测量； (3)通过野生型蛋白质组数据建模建议测量纯合和杂合KO菌株对化学和巨噬细胞攻击的相对抗性。