ASK signalosomes and environmental sensing

描述(由申请人提供)：丝裂原活化蛋白激酶(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体激活。MAPK信号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器是MAP3K(MAP3K)酶凋亡信号调节激酶1(ASK1)，它是环境应激诱导的MAPK信号转导的关键整合传感器。ASK1的激活需要一个多蛋白ASK“信号小体”的组装，这可以通过ASK1与还原的硫氧还蛋白1(TRX1)和其他蛋白质，包括同源激酶ASK2和ASK3的络合来部分调节。这个项目解决了氧化应激如何激活ASK信号体这一中心问题。我们假设，氧化剂和脂类亲电体氧化并共价修饰ASK蛋白或它们的相互作用伙伴，以破坏ASK抑制复合体的稳定，并使活性ASK信号体能够组装。我们将在人类细胞模型中应用基于定量质谱仪(MS)的分析来验证这一假设，具体目的如下：1)确定激活前和激活后的ASK信号体的组成。ASK复合体将通过捕获TAP标记的ASK1/2/3和3个人类细胞模型中的蛋白质伙伴来分析。我们将筛选一组脂质亲电体和过氧化氢来激活MAPK信号，然后用鸟枪式蛋白质组学方法清点激活前和激活后的ASK信号体。我们将配置一个平行反应监测(PRM)质谱分析的多路面板，以定量激活前和激活后ASK信号体的成分。2)确定与ASK激活和调节相关的氧化修饰、亲电加合物和泛素化信号。我们将确定ASK信号小体的蛋白质组分，这些蛋白质是凝胶加合和半胱氨酸硫醇氧化的目标，然后用MS/MS绘制修饰位点的图谱。我们将开发修饰多肽的PRM分析，以实现对磷酸化、泛素化标记、半胱氨酸氧化还原变化和电泳性加合物的定量。使用Click化学方法，炔基亲电探针将允许共价捕获加合物和半胱氨酸磺酸。3)表征氧化剂和脂类亲电体激活ASK信号体的动力学。这些研究将在蛋白质组成、内收和氧化水平上量化ASK信号小体，随着信号小体激活的进化。这些研究将建立一种多蛋白质复合体动力学的“全组分”方法学，该方法学将广泛适用于功能性多蛋白质系统。4)开发用于监测细胞模型中ASK激活的原型报告系统。我们将共表达荧光报告标记的ASK和相互作用的蛋白质，以建立Fürster共振能量转移(FRET)分析，以干扰ASK信号体中的关键相互作用。这些模型将使这项工作能够转化为高度特异和分子信息丰富的标准化筛选方法，用于化学物质激活ASK和MAPK。

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.