Project 4: Protein adduction by electrophilic products of lipid oxidation

项目4：脂质氧化亲电子产物的蛋白质加合

基本信息

批准号：
7540268
负责人：
Daniel C Liebler
金额：
$ 29.47万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-09-12 至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7540268
关键词：
Affinity Affinity Labels Alkylation Amino Acid Sequence Apolipoprotein A-I Archives Avidin Biological Biological Assay Biological Markers Cell model Cell physiology Cells Cellular Stress Response Chemistry Collaborations Condition Coupled Data Databases Disease Environmental Exposure Epitopes Equipment and supply inventories Evaluation Fatty Acids Funding Gene Expression Graph High Density Lipoproteins Human In Vitro Information Networks Label Link Lipid Peroxidation Lipids Maps Mass Spectrum Analysis Measurement Measures Mediating Methods Monitor Oxidative Stress Pathway interactions Penetration Peptides Phospholipids Plasma Plasma Cells Plasma Proteins Predisposition Protein Analysis Protein Chemistry Proteins Proteome Proteomics Reaction Reagent Regulation Resistance Signal Induction Signal Pathway Signal Transduction Site Specificity Specimen Spectrometry Stress System Techniques Testing Thinking Tissues Work adduct affinity labeling base cellular targeting in vivo new technology novel oxidation protein protein interaction response sensor theories transcription factor

项目摘要

During the initial funding period of this PPG, we and our colleagues have implemented mass spectrometry (MS)-based proteomics technologies and novel affinity labeling and capture techniques to enable the selective capture and analysis of proteins labeled by specific lipid oxidation products. Our overall objective in this competing renewal application is to define the scope of protein damage by lipid electrophiles in cells and plasma and to apply sensitive MS and immunoaffinity methods to study protein adducts as modulators of biological responses to oxidative stress and as biomarkers for oxidative stress. A major thrust of the proposed work is an evaluation of the target selectivity and biological effects of lipid electrophiles in both their "free" and phospholipid-esterified forms. These studies will collectively advance our understanding of how protein damage by lipid electrophiles transduces environmentally-induced oxidatve stress into discrete signaling effects and will identify new candidate biomarkers for oxidative damage in plasma and tissues. The specific aims of Project 4 are: 1) To identify the protein targets of lipid electrophiles in cell models. These studies will characterize protein adduction by lipid electrophiles using Click chemistry-based methods to capture and analyze protein and peptide adducts. 2) To identify potential damage-susceptible cellular systems and candidate electrophile sensors by mapping electrophile-adducted proteins to cellular processes, canonical pathways, and protein-protein interaction networks archived in a local database. 3) To identify protein targets and corresponding adduction sites on proteins that regulate responses to oxidative stress in cells. This work will be done in collaboration with Project 3 to test the hypothesis that alkylation of specific proteins by lipid electrophiles triggers signaling and transcription factor regulation changes associated with stress. 4) To evaluate apolipoprotein A1 (ApoA1) adducts as biomarkers of oxidative stress 'n vivo. We will evaluate the hierarchical reactivity of ApoA1 nucleophiles with lipid electrophiles from electrophile probes and endogenous HDL lipids in plasma oxidations in vitro. Multiple reaction monitoring (MRM)-based LC-MS-MS quantitative assays will be developed for specific adducts, which will be evaluated as biomarker candidates in vivo in human specimens in collaboration with Project 2.

在此PPG的最初资助期间，我们和我们的同事实施了质谱法（MS）基于基于的蛋白质组学技术和新颖的亲和力标记和捕获技术以实现选择性捕获和分析由特定脂质氧化产物标记的蛋白质。我们的整体目标在这种竞争的更新应用中，是定义细胞中脂质电力的蛋白质损伤范围和血浆，并应用敏感的MS和免疫亲和力方法来研究蛋白质加合物作为调节剂对氧化应激的生物反应和作为氧化应激的生物标志物的反应。是拟议的工作是评估脂质电子的靶标选择性和生物学作用它们的“自由”和磷脂层的形式。这些研究将共同提高我们对脂质电力损伤如何将环境诱导的氧化应激转导为离散信号传导效应，并将确定新的候选生物标志物，以实现等离子体和组织中的氧化损伤。项目4的具体目的是：1）鉴定细胞模型中脂质电力的蛋白质靶标。这些研究将使用单击基于化学的方法来表征脂质电力的蛋白质收益捕获和分析蛋白质和肽加合物。 2）确定潜在的受损敏感的细胞系统和候选电力传感器通过将亲电的蛋白映射到细胞在本地数据库中存档的过程，规范途径和蛋白质 - 蛋白质相互作用网络。 3）到鉴定蛋白质靶标和相应的内收位点，这些蛋白质调节对氧化反应的反应细胞中的压力。这项工作将与项目3合作进行，以检验以下假设。脂质电力的特定蛋白质触发信号传导和转录因子调节变化与压力有关。 4）评估载脂蛋白A1（APOA1）加合物作为氧化应激的生物标志物 'n Vivo。我们将评估apoA1亲核试剂与脂质电子的分层反应性在体外血浆氧化中的亲电探针和内源性HDL脂质。多反应监测（MRM）基于LC-MS-MS-MS定量测定将针对特定加合物开发，将对此进行评估作为与项目2合作的人类标本中的生物标志物候选人。