ASK signalosomes and environmental sensing

ASK 信号体和环境传感

• 批准号: 8695895
• 负责人: Daniel C Liebler
• 金额: $ 35.29万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8695895
• 关键词: Address; Apoptosis; Biological Assay; Cell Death; Cell model; Chemicals; Chemistry; Complex; Cysteine; Disease; Energy Transfer; Environment; Environmental Exposure; Enzymes; Equipment and supply inventories; Goals; Homologous Protein; Human; Hydrogen Peroxide; Immunoprecipitation; Insulin Resistance; Kinetics; Link; Lipids; MAP Kinase Kinase Kinase; MAP3K5 gene; MAPK14 gene; Maps; Mass Spectrum Analysis; Mediating; Metabolic syndrome; Methodology; Methods; Mitogen-Activated Protein Kinases; Modeling; Modification; Monitor; Multiprotein Complexes; Neurodegenerative Disorders; Non-Insulin-Dependent Diabetes Mellitus; Oxidants; Oxidation-Reduction; Oxidative Stress; Peptides; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Process; Proteins; Proteomics; Reaction; Regulation; Relative (related person); Reporter; Risk; Role; Shotguns; Signal Induction; Signal Transduction; Site; Stimulus; Stress; Sulfhydryl Compounds; System; Testing; Thioredoxin; Transducers; Translations; Ubiquitination; Work; adduct; base; cysteinesulfenic acid; environmental agent; environmental chemical; environmental stressor; hazard; molecular marker; oxidation; prevent; protein misfolding; prototype; public health relevance; response; screening; sensor

DESCRIPTION (provided by applicant): The mitogen-activated protein kinase (MAPK) signaling cascade is activated by oxidants and reactive lipid electrophiles generated by diverse environmental exposures. MAPK signaling is a key driver of environmentally-induced disease processes. The principal upstream trigger for MAPK activation is the MAP kinase kinase kinase (MAP3K) enzyme apoptosis signal-regulating kinase 1 (ASK1), which serves as a key integrating sensor/transducer for MAPK signaling induced by environmental stressors. ASK1 activation requires the assembly of a multiprotein ASK "signalosome", which can be regulated in part through ASK1 complexation with reduced thioredoxin 1 (TRX1) and other proteins, including the homologous kinases ASK2 and ASK3. This project addresses the central question of how oxidative stress activates the ASK signalosome. We hypothesize that oxidants and lipid electrophiles oxidize and covalently modify ASK proteins or their interacting partners to destabilize ASK inhibitory complexes and enable assembly of an active ASK signalosome. We will apply quantitative mass spectrometry (MS)-based analyses in human cell models to test this hypothesis through the following specific aims: 1) Define the composition of pre- and post-activation ASK signalosomes. ASK complexes will be analyzed by capture of TAP-tagged ASK1/2/3 and protein partners in 3 human cell models. We will screen a panel of lipid electrophiles and H2O2 for activation of MAPK signaling and then inventory pre- and post-activation ASK signalosomes by shotgun proteomics. We will configure a multiplexed panel of parallel reaction monitoring (PRM) mass spectrometry assays to quantify the components of pre- and post-activation ASK signalosomes. 2) Identify oxidative modifications, electrophile adducts and ubiquitination signatures associated with ASK activation and regulation. We will identify protein components of ASK signalosomes that are targets for electrophile adduction and cysteine thiol oxidation and then map sites of modification by MS/MS. We will develop PRM assays for modified peptides to enable quantitation of phosphorylations, ubiquitination tags, cysteine redox changes, and electrophile adducts. Alkynyl-electrophile probes will allow covalent capture of adducts and cysteine sulfenic acids using Click chemistry methods. 3) Characterize the dynamics of ASK signalosome activation by oxidants and lipid electrophiles. These studies will quantify ASK signalosomes at the level of protein composition, adduction and oxidation as signalosome activation evolves. These studies will establish an "all components" methodology for multiprotein complex dynamics that will have broad applicability to functional multiprotein systems. 4) Develop prototype reporter systems for monitoring ASK activation in cell models. We will co-express fluorescent reporter-tagged ASK and interacting proteins to establish F¿rster resonance energy transfer (FRET) assays for perturbation of key interactions in the ASK signalosome. These models will enable translation of this work into highly specific and molecularly informative standardized screening methods for ASK and MAPK activation by chemicals.

描述（由申请方提供）：丝裂原活化蛋白激酶（MAPK）信号级联由不同环境暴露产生的氧化剂和反应性脂质亲电体激活。MAPK信号转导是环境诱导的疾病过程的关键驱动因素。MAPK激活的主要上游触发因子是MAP激酶（MAP 3 K）酶凋亡信号调节激酶1（ASK 1），其作为环境应激物诱导的MAPK信号传导的关键整合传感器/换能器。ASK 1激活需要组装多蛋白ASK“信号体”，其可以部分地通过ASK 1与还原型硫氧还蛋白1（TRX 1）和其他蛋白质（包括同源激酶ASK 2和ASK 3）的复合来调节。该项目解决了氧化应激如何激活ASK信号体的核心问题。我们假设氧化剂和脂质亲电体氧化并共价修饰ASK蛋白或其相互作用伴侣，以使ASK抑制复合物不稳定，并使活性ASK信号体组装成为可能。我们将在人类细胞模型中应用基于定量质谱（MS）的分析，通过以下具体目标来测试这一假设：1）定义激活前和激活后ASK信号体的组成。将通过捕获3种人类细胞模型中TAP标记的ASK 1/2/3和蛋白质伴侣来分析ASK复合物。我们将筛选一组脂质亲电体和过氧化氢激活MAPK信号，然后通过鸟枪蛋白质组学清点激活前后的ASK信号体。我们将配置平行反应监测（PRM）质谱分析的多路复用面板，以量化激活前和激活后ASK信号体的组分。2)识别与ASK激活和调节相关的氧化修饰、亲电加合物和泛素化标记。我们将确定ASK信号体的蛋白质成分，这些信号体是亲电体加合和半胱氨酸巯基氧化的靶点，然后通过MS/MS绘制修饰位点。我们将开发PRM测定法，用于修饰肽，以定量磷酸化，泛素化标签，半胱氨酸氧化还原变化和亲电体加合物。炔基亲电探针将允许使用点击化学方法共价捕获加合物和半胱氨酸次磺酸。3)表征氧化剂和亲脂电子体激活ASK信号体的动力学。这些研究将在蛋白质组成、内收和氧化的水平上量化ASK信号体，因为信号体活化不断发展。这些研究将建立一个“所有组件”的多蛋白质复合物动力学的方法，将有广泛的适用性功能多蛋白质系统。4)开发用于监测细胞模型中ASK激活的原型报告系统。我们将共同表达荧光标记的ASK和相互作用的蛋白质，以建立弗斯特共振能量转移（FRET）分析的ASK信号体中的关键相互作用的扰动。这些模型将使这项工作转化为高度特异性和分子信息的标准化筛选方法的ASK和MAPK激活的化学品。