Mechanism-based probes to image protein sulfenylation in live cells and in vivo

基于机制的活细胞和体内蛋白质磺酰化成像探针

基本信息

批准号：
8922802
负责人：
Christina Aiyami Tom
金额：
$ 2.78万
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：
2014-09-01 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8922802
关键词：
Affinity Chromatography Alkynes Animal Model Area Azides B-Lymphocytes Basic Science Biochemical Biochemistry Biological Biological Assay Biological Models Biomedical Research Biotin Buffers Carbon Cell Nucleus Cell model Cells Chemicals Cysteine Detection Development Diagnostic Disease Electronics Environment Enzymes Faculty Fluorescence Fluorescence Microscopy Fluorescent Probes Fluorine Generations Glutathione Goals Growth Factor Health Hydrogen Peroxide Image Ketones Label Lead Left Libraries Life Link Magnetic Resonance Imaging Mammalian Cell Masks Measures Mentors Methodology Methods Modification Monitor NADPH Oxidase Organic Synthesis Organism Outcome Oxidants Oxidation-Reduction Oxidative Stress Peroxides Physiological Play Positioning Attribute Post-Translational Protein Processing Protein Dynamics Protein phosphatase Proteins Proteomics Protons Reaction Regulation Reporting Research Role Signal Pathway Signal Transduction Spectrum Analysis Sulfenic Acids Sulfhydryl Compounds Sulfinic Acids System Techniques Testing Tissues Training Universities Yeasts analog base carbene cell fixing cellular imaging cysteinesulfenic acid design dimedone enolate fluorescence imaging fluorophore functional group human disease improved in vivo in vivo imaging novel novel diagnostics novel strategies oxidation ratiometric sample fixation skills training small molecule spatiotemporal thioether tool

项目摘要

DESCRIPTION (provided by applicant): Protein sulfenylation describes the reversible post-translational modification of cysteine by cellular oxidants, like hydrogen peroxide. Growth factor stimulation induces a burst of hydrogen peroxide, which transiently oxidizes the nucleophilic cysteine of protein phosphatases and other proximal redox active thiols. Cysteine oxidation begins with the reaction of the cysteine thiolate with hydrogen peroxide, which forms a highly unstable sulfenic acid intermediate. Further oxidation by peroxide leads to formation of sulfinic acids, which are generally irreversible modifications. The small molecule dimedone (5,5-dimethylcyclohexane-1,3-dione) acts as a covalent trap, reacting with sulfenic acids to form a stable and irreversible thioether linkage. Several groups have recently reported the development of functionalized analogues of dimedone (azide, alkyne, biotin, etc.) for affinity purification and proteomic annotation of dimedone-reactive oxidized proteins. Despite this recent progress, there are no probes for live-cell or in vivo imaging of sulfenylation. Our long-term goal is to develop new methods to visualize the role of protein sulfenylation in vivo, and apply these tools to characterize the functional significance of this modification in disease. In this application, I propose to develop mechanism- based chemical probes to analyze the spatiotemporal dynamics in cells and in vivo. These methods will introduce new methods for visualizing protein sulfenylation in live cells and tissues using advanced ratiometric fluorescent imaging and emerging MRI methods. These approaches will be validated using model organisms predicted to display altered redox regulation, providing a path for extended studies in mammalian systems. Finally, developing these methods will provide a unique training environment bridging organic synthesis, biochemistry, and spectroscopy. This broad training approach, supervised by a committee of junior and senior faculty mentors, will provide me with the skills and training to pursue my long-term goal advancing to an academic position at a diverse, research university.

描述（由申请人提供）：蛋白质磺酰化描述了细胞氧化剂（例如过氧化氢）对半胱氨酸的可逆翻译后修饰。生长因子刺激诱导过氧化氢爆发，短暂氧化蛋白磷酸酶和其他近端氧化还原活性硫醇的亲核半胱氨酸。半胱氨酸氧化始于半胱氨酸硫醇盐与过氧化氢的反应，形成高度不稳定的次磺酸中间体。过氧化物的进一步氧化导致亚磺酸的形成，这通常是不可逆的修饰。小分子双甲酮（5,5-二甲基环己烷-1,3-二酮）充当共价陷阱，与次磺酸反应形成稳定且不可逆的硫醚键。几个小组最近报道了双甲酮功能化类似物（叠氮化物、炔烃、生物素等）的开发，用于双甲酮反应性氧化蛋白的亲和纯化和蛋白质组注释。尽管最近取得了这些进展，但还没有用于磺酰化活细胞或体内成像的探针。我们的长期目标是开发新方法来可视化体内蛋白质磺酰化的作用，并应用这些工具来表征这种修饰在疾病中的功能意义。在此应用中，我建议开发基于机制的化学探针来分析细胞和体内的时空动态。这些方法将引入使用先进的比例荧光成像和新兴 MRI 方法可视化活细胞和组织中蛋白质磺酰化的新方法。这些方法将使用预测显示氧化还原调节改变的模型生物体进行验证，为哺乳动物系统的扩展研究提供途径。最后，开发这些方法将提供一个连接有机合成、生物化学和光谱学的独特培训环境。这种广泛的培训方法由初级和高级教师导师委员会监督，将为我提供技能和培训，以实现我的长期目标，在多元化的研究型大学取得学术职位。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Free Radical Initiated Peptide Sequencing for Direct Site Localization of Sulfation and Phosphorylation with Negative Ion Mode Mass Spectrometry.

使用负离子模式质谱法进行自由基引发肽测序，用于硫酸化和磷酸化的直接位点定位。

DOI：
发表时间：
2018-08-21
期刊：
影响因子：
7.4
作者：
Borotto, Nicholas B;Ileka, Kevin M;Tom, Christina A T M B;Martin, Brent R;Håkansson, Kristina
通讯作者：
Håkansson, Kristina

Chemoselective ratiometric imaging of protein S-sulfenylation.

蛋白质 S-磺酰化的化学选择性比例成像。

DOI：
10.1039/c7cc02285a
发表时间：
2017-06-29
期刊：
Chemical communications
影响因子：
4.9
作者：
Tom CTMB;Crellin JE;Motiwala HF;Stone MB;Davda D;Walker W;Kuo YH;Hernandez JL;Labby KJ;Gomez-Rodriguez L;Jenkins PM;Veatch SL;Martin BR
通讯作者：
Martin BR