Biochemistry connecting glutathione and isocyanate asthma

描述（由申请人提供）：异氰酸酯是制造聚氨酯必不可少的交联化学品，是世界范围内职业性哮喘的主要原因。然而，它们致病的机制仍不清楚。我们推测，异氰酸酯哮喘的发病机制取决于异氰酸酯与多种/不同的自身蛋白/肽的动态生化反应，其代谢途径此前未被发现与异氰酸酯致敏性有关。我们的具体假设是，吸入的异氰酸酯首先优先与自肽谷胱甘肽（GSH）的游离硫醇发生反应，这在气道液中高浓度（100-1000 μ M）时是不存在的。然而，巯基异氰酸酯-谷胱甘肽反应产物在生理pH值下是“准稳定的”，如果不能迅速代谢和排泄，可以将异氰酸酯基团“转移”到另一种自身蛋白/肽，特别是白蛋白，这是体内最丰富的蛋白质之一。已知异氰酸酯结合白蛋白引起抗原改变，引发适应性免疫反应（特异性IgE， t细胞反应），已知可促进气道炎症和哮喘。与先前提出的异氰酸酯哮喘机制相比，当前假说的新颖性在于，异氰酸酯抗原（异氰酸酯-白蛋白偶联物）通过谷胱甘肽介导的转甲氧基化形成，而不是直接与异氰酸酯发生反应。如果正确，这一模式将解释“异氰酸酯”与典型“环境”哮喘的许多独特特征，并为疾病的发病机制、危险因素和诊断指标提供重要的新见解。在这项拨款申请中，我们提出了未发表的初步数据，有力地支持了我们的新假设，并概述了一项研究计划，以更好地了解gsh -异氰酸酯反应性及其与职业暴露致病反应的关系。鉴于异氰酸酯的毒性，研究设计包括体外实验，然后是异氰酸酯哮喘动物模型的体内实验，以一种不可能在人类受试者中进行的方式直接验证我们的假设。这项雄心勃勃的提案将通过申请人所在机构的独特基础设施（W.M. Keck生物技术资源实验室）和正在进行的临床异氰酸酯哮喘研究的合作来促进。该资助的实验数据有望确定异氰酸酯哮喘发病机制和生物标志物的关键步骤，并以此为基础制定更好的疾病监测和诊断策略。拟议研究的成功完成也将为进一步的临床研究提供令人信服的证据，将这些初步发现转化为预防疾病的实际方法。两个具体目标是Aim 1。表征谷胱甘肽（GSH）与异氰酸酯在体外生理条件下的反应产物及其氨基酰基化能力。目标2。在动物模型中确定谷胱甘肽-异氰酸酯相互作用在哮喘发展中的作用。

DESCRIPTION (provided by applicant): Isocyanate, the essential cross-linking chemical for making polyurethane, are a major cause of occupational asthma world-wide. However, the mechanisms by which they cause disease remain unclear. We theorize that isocyanate asthma pathogenesis depends upon dynamic biochemical reactivity of isocyanate with multiple/different self-proteins/peptides, through metabolic pathways previously unappreciated for their connection to isocyanate allergenicity. Our specific hypothesis is that inhaled isocyanate reacts first, and preferentially, with the free thiol of the self-peptide, glutathione (GSH), which is preent at high concentrations (100-1000 ¿M) in the airway fluid. However, thiol-linked isocyanate-GSH reaction products are "quasi-stable" at physiologic pH, and if not rapidly metabolized and excreted, can "transfer" the isocyanate group to another self-protein/peptide, especially albumin, one of the most abundant proteins in the body. Isocyanate conjugation of albumin is known to cause antigenic changes that trigger adaptive immunologic responses (specific IgE, T-cell responses) known to promote airway inflammation and asthma. The novelty of the present hypothesis vs. previously proposed mechanisms of isocyanate asthma is the formation of immunogenically distinct isocyanate antigens (isocyanate-albumin conjugates) via GSH-mediated transcarbomylation, rather than direct reactivity with isocyanate. If correct, this paradigm would explain many of the unique features of "isocyanate" vs. typical "environmental" asthma, and provide critical new insights into pathogenesis, risk factors, and diagnostic indicators of disease. In this grant application, we present unpublished Preliminary data that strongly support our novel hypotheses, and outline a Research Plan to better understand GSH-isocyanate reactivity and its connection with pathogenic responses to occupational exposure. Given the toxicity of isocyanates, the study design involves in vitro experiments followed by in vivo experiments in an animal model of isocyanate asthma, to directly test our hypotheses in a manner not possible in human subjects. The highly ambitious proposal will be facilitated through unique infrastructure (W.M. Keck Biotechnology Resource Laboratory) at the applicant's institution and collaboration with ongoing clinical isocyanate asthma research. The experimental data from the grant is expected to define key steps in isocyanate asthma pathogenesis, and biomarkers, upon which to base better strategies for disease surveillance and diagnosis. Successful completion of the proposed studies should also provide compelling evidence for further clinical studies to translate these initial findings into practical methods of disease prevention. The two specific aims are to Aim 1. Characterize the reaction products between glutathione (GSH) and isocyanate, and their carbamoylating capacity, under physiologic conditions in vitro. Aim 2. Determine the role of GSH-isocyanate interactions in the development of asthma in vivo in an animal model.