Biochemistry connecting glutathione and isocyanate asthma

描述（由申请人提供）：异氰酸酯是制造聚氨酯的基本交联化学品，是全球职业性哮喘的主要原因。然而，它们引起疾病的机制仍不清楚。 我们推测异氰酸酯哮喘的发病机理取决于异氰酸酯与多种/不同的自身蛋白质/肽的动态生化反应性，通过以前未被认识到的与异氰酸酯过敏性相关的代谢途径。我们的具体假设是，吸入的异氰酸酯首先反应，并优先与自身肽的游离巯基，谷胱甘肽（GSH），这是在气道液中的高浓度（100-1000 μ M）。然而，巯基连接的异氰酸酯-GSH反应产物在生理pH下是“准稳定的”，并且如果不被快速代谢和排泄，则可以将异氰酸酯基团“转移”到另一自身蛋白质/肽，特别是白蛋白，其是体内最丰富的蛋白质之一。已知白蛋白的异氰酸酯缀合引起抗原变化，其触发已知促进气道炎症和哮喘的适应性免疫应答（特异性IgE、T细胞应答）。 与先前提出的异氰酸酯哮喘机制相比，本假设的新奇在于通过GSH介导的转羧化作用形成免疫原性不同的异氰酸酯抗原（异氰酸酯-白蛋白缀合物），而不是与异氰酸酯直接反应。如果正确的话，这种范式将解释“异氰酸酯”与典型的“环境”哮喘的许多独特特征，并为疾病的发病机制，风险因素和诊断指标提供重要的新见解。在这项资助申请中，我们提出了未发表的初步数据，这些数据强烈支持我们的新假设，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联系。考虑到异氰酸酯的毒性，研究设计包括体外实验，然后在异氰酸酯哮喘动物模型中进行体内实验，以直接测试我们的假设，这在人类受试者中是不可能的。这项雄心勃勃的计划将通过独特的基础设施（W.M.凯克生物技术资源实验室）在申请人的机构和正在进行的临床异氰酸酯哮喘研究的合作。来自该基金的实验数据预计将确定异氰酸酯哮喘发病机制的关键步骤和生物标志物，并以此为基础制定更好的疾病监测和诊断策略。成功完成拟议的研究还应为进一步的临床研究提供令人信服的证据，以将这些初步发现转化为预防疾病的实际方法。这两个具体目标是目标1。在体外生理条件下，对谷胱甘肽（GSH）与异氰酸酯的反应产物及其氨甲酰化能力进行了表征。目标2.在动物模型中确定GSH-异氰酸酯相互作用在体内哮喘发展中的作用。

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.