Multiscale chemical approaches to map oxidative stress

绘制氧化应激图谱的多尺度化学方法

基本信息

批准号：
8751150
负责人：
Brent Randall Martin
金额：
$ 222.21万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-30 至 2019-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8751150
关键词：
Aging Alkylation Antioxidants Award Biochemical Biotin Buffers Cells Chemicals Cysteine Detection Development Diabetes Mellitus Environment Enzymes Fluorescence Fluorescent Probes Genome Health Heart Diseases Human Life Link Magnetic Resonance Imaging Malignant Neoplasms Maps Mass Spectrum Analysis Methodology Methods Mitochondria Modification Molecular Chaperones Nerve Degeneration Neurodegenerative Disorders Oxidation-Reduction Oxidative Stress Parkinson Disease Physiological Post-Translational Protein Processing Proteins Proteome Reactive Oxygen Species Reagent Research Respiration Role SKIL gene Series Signal Transduction Sulfhydryl Compounds Sulfinic Acids Validation analytical method base cellular imaging in vivo in vivo imaging innovation novel oxidation oxidative damage public health relevance ratiometric repaired research study small molecule tool

项目摘要

DESCRIPTION (provided by applicant): Radical species are an unavoidable consequence of respiration and the environment, and are tightly buffered by small molecule antioxidants and redo detoxifying enzymes. Oxidative stress emerges when an imbalance develops between the levels of reactive oxygen species and the cell's ability to readily eliminate the reactive intermediates or to repair the resulting damage. Aberrant oxidative signaling is perhaps one of the most important factors contributing to aging, neurodegeneration, heart disease, diabetes, and cancer. In order to induce a phenotypic change, oxidative stress must induce biochemical alterations to the genome, proteome and/or metabolome. Crystallographic analysis revealed that DJ-1 harbors a stabile sulfinic acid, and this oxidative modification is required for the suppression of mitochondrial oxidative stress. We have now shown that this sulfinic acid can react with nitrosothiols to form a thiosulfonate linkage, which can then be reduced by cellular thiols. Amazingly, sulfinates react with nitrosothiols faster than thiolates in standard physiological buffers. This provides a potential mechanism for DJ-1 function, which will be further explored with this award. We extended this approach to develop biotin-linked sulfinates for the direct detection and enrichment of endogenous nitrosylated proteins. In preliminary experiments, this method led to the identification of >1500 endogenous nitrosylated proteins, and establishes a robust new platform to functionally interrogate the dynamics of S-nitrosylation. In addition, we describe a new methodology for the selective enrichment of sulfinic acids based on orthogonal alkylation reagents, and propose to identify novel functional sulfinates in the proteome. Finally, we present a new class of ratiometric fluorescent probes for live-cell imaging and 19F-NMR of protein sulfenylation in vivo. Despite the central role of oxidative stress in human health, our ability to study the precise mechanisms of such modifications is hampered by a lack of selective chemical and analytical methods. In this proposal, we present a series of innovative chemical approaches to study oxidative damage across experimental scales, from live-cell imaging to in vivo imaging, in addition to proteome-wide annotation of oxidative post-translational modifications. Furthermore, we present a likely mechanism for the Parkinson's disease-linked redox chaperone DJ-1, and present new mechanism-based probes to functionally annotate and profile S-nitrosylation (R-SNO), S-sulfenylation (R-SOH), and S-sulfinylation (R-SO2H).

描述（由申请人提供）：自由基物种是呼吸和环境的不可避免的结果，并且被小分子抗氧化剂和重做排毒酶紧密缓冲。当活性氧的水平与细胞容易消除反应性中间体或修复产生的损伤的能力之间发生不平衡时，就会出现氧化应激。异常的氧化信号传导可能是导致衰老，神经退行性，心脏病，糖尿病和癌症的最重要因素之一。为了诱导表型变化，氧化应激必须诱导基因组，蛋白质组和/或代谢组的生化改变。晶体学分析表明，DJ-1具有稳定的硫酸酸，并且这种氧化性修饰是抑制线粒体氧化应激所必需的。现在，我们已经表明，这种硫酸可以与硝基硫醇反应形成硫代磺酸盐链接，然后可以通过细胞硫醇减少。令人惊讶的是，在标准生理缓冲液中，硫酸盐与硝基硫醇的反应快于硫醇酸盐。这为DJ-1功能提供了潜在的机制，该奖项将进一步探讨。我们扩展了这种开发生物素连接的硫酸盐的方法，以直接检测和富集内源性硝基基化蛋白。在初步实验中，这种方法导致识别> 1500个内源性硝基乙酰化蛋白，并建立了一个强大的新平台，以在功能上询问S-硝基化的动力学。此外，我们描述了一种基于正交烷基化试剂选择性富集硫酸酸的新方法，并建议鉴定蛋白质组中新型的功能性硫酸盐。最后，我们提出了一类新的比率荧光探针，用于活体内的活细胞成像和19F-NMR。尽管氧化应激在人类健康中的核心作用，但我们研究这种修饰的精确机制的能力受到缺乏选择性化学和分析方法的阻碍。在此提案中，除了对氧化后翻译后修饰的氧化注释外，我们提出了一系列创新的化学方法，以研究从活细胞成像到体内成像的实验量表的氧化损伤。此外，我们提出了帕金森氏病与疾病相关的氧化还原伴侣DJ-1的可能机制，并提出了基于机制的新探针，以在功能上注释和剖面S-硝基化（R-SNO）（R-SNO），S-磺烯基化（R-SOH）（R-SOH）和S-硫固醇（R-SO2H）（R-SO2H）。