ASK signalosomes and environmental sensing

描述（由申请人提供）：有丝分裂原活化蛋白激酶（MAPK）信号级联被不同环境暴露产生的氧化剂和反应性脂质亲电试剂激活。MAPK信号是环境诱导疾病过程的关键驱动因素。MAPK激活的主要上游触发器是MAP3K激酶（MAP3K）酶凋亡信号调节激酶1 (ASK1)，它是环境应激源诱导的MAPK信号传导的关键集成传感器/传感器。ASK1的激活需要多蛋白ASK“信号体”的组装，这可以部分通过ASK1与还原硫氧还蛋白1 （TRX1）和其他蛋白质（包括同源激酶ASK2和ASK3）的络合来调节。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试剂氧化并共价修饰ASK蛋白或其相互作用的伙伴，以破坏ASK抑制复合物的稳定，并使活性ASK信号体的组装成为可能。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验证这一假设：1)定义激活前和激活后ASK信号体的组成。通过在3种人类细胞模型中捕获tap标记的ASK1/2/3和蛋白伴侣来分析ASK复合物。我们将筛选一组脂质亲电试剂和H2O2来激活MAPK信号，然后通过散弹枪蛋白质组学对激活前和激活后的ASK信号体进行清查。我们将配置一个平行反应监测（PRM）质谱分析的多路复用面板来量化激活前和激活后的ASK信号体的成分。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特征。我们将鉴定ASK信号小体中亲电内合和半胱氨酸硫醇氧化的靶蛋白组分，然后通过质谱联用技术绘制修饰位点。我们将开发修饰肽的PRM分析，以定量磷酸化、泛素化标记、半胱氨酸氧化还原变化和亲电加合物。烷基亲电探针将允许共价捕获加合物和半胱氨酸磺酸使用点击化学方法。3)表征氧化剂和脂质亲电试剂激活ASK信号体的动力学。随着信号体激活的发展，这些研究将在蛋白质组成、内聚和氧化水平上量化ASK信号体。这些研究将为多蛋白复合物动力学建立一种“全组分”方法，该方法将广泛适用于功能性多蛋白系统。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报告标记的ASK和相互作用蛋白，建立f¿rster共振能量转移（FRET）检测，以检测ASK信号体中关键相互作用的扰动。这些模型将使这项工作转化为化学物质激活ASK和MAPK的高度特异性和分子信息性的标准化筛选方法。

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.