Trichothecene Toxicity and the Ribotoxic Stress Response

描述(申请人提供)：三氯乙烯是一组倍半萜类真菌毒素，在世界各地普遍被视为食品污染物，在病因学上与人类和动物的疾病有关，免疫系统是主要目标。在实验动物中，这些真菌毒素和其他与真核细胞核糖体高亲和力的天然毒素(例如。蓖麻毒素、志贺毒素、茴香霉素)可诱导淋巴组织中致炎基因的表达和细胞凋亡。由于它们可能用于化学恐怖主义，三氯乙烯和其他核毒素现在被列入疾控中心精选毒剂名单。虽然已知核糖体导向的药物通过激活丝裂原激活的蛋白激酶而发挥毒性作用，但这种“核毒应激反应”(RSR)的潜在机制在很大程度上仍不清楚。因此，我们对核毒素调节基因表达和细胞凋亡的信号转导机制的了解存在着一个关键的空白。这项建议的目的是检验指导性假说，即核糖体在蛋白激酶介导的对三茂铁和其他核毒剂的应激反应的启动和整合中发挥核心作用。这一假说是基于在巨噬细胞中观察到的：1)脱氧雪腐镰刀菌烯醇(DON)，一种常见的食源性毛霉烯类，介导18S和28S核糖体(R)RNA的裂解，2)双链RNA激活的蛋白激酶(PKR)，一种核糖体相关的丝氨酸-苏氨酸激酶，是DON诱导的蛋白激酶激活所必需的，以及3)DON诱导几种蛋白激酶动员到核糖体，在那里它们被磷酸化。为了验证我们的假设，我们提出了三个具体目标。在目标1中，我们将描述DON诱导的rRNA相对于靶的切割，巨噬细胞中的动力学和机制。在目标2中，我们将使用巨噬细胞和无细胞模型来表征PKR作为DON诱导的rRNA损伤的早期传感器的作用。在目标3中，我们将跟踪DON诱导的巨噬细胞中核糖体相关蛋白相对于组成、动力学和激酶活性的变化。通过这些研究，我们希望了解核糖体如何介导多种细胞内信号级联反应的诱导和整合，这些信号级联反应导致单核吞噬细胞中基因表达的改变和对核毒素的反应。预期成果包括：1)提高对三环烯和其他核毒素扰乱免疫的分子基础的了解，2)加强评估和管理与接触三环烯和其他核毒素有关的风险的能力，3)基于机制的战略，预防和/或治疗因自然污染或化学恐怖主义而暴露于三环烯和核毒性化学品的人。总体而言，这些结果将对公众健康产生积极影响，为针对这一重要类别的毒素提出合理建议提供科学基础，并在发生暴露时采取适当的补救行动。公共卫生相关性：我们建议了解一类有效的生物毒素如何干扰免疫系统所必需的细胞的功能和生存。这项研究将增强我们评估和管理与接触这些毒素相关的风险的能力，并产生基于机制的战略，以预防和/或治疗因无意中的食物污染或蓄意的化学恐怖主义而暴露于这些毒剂的人。总体而言，这些结果将为产生关于这些重要毒素的合理建议和发生暴露时的适当补救行动提供科学基础，从而对公众健康产生积极影响。

DESCRIPTION (provided by applicant): The trichothecenes, a group of sesquiterpenoid mycotoxins commonly encountered as food contaminants worldwide, have been etiologically linked to human and animal illnesses with the immune system being a major target. In experimental animals, these mycotoxins and other natural toxins that bind with high affinity to eukaryotic ribosomes (eg. ricin, Shiga toxin, anisomycin) induce proinflammatory gene expression and apoptosis in lymphoid tissues. Because of their potential for use in chemical terrorism, trichothecenes and other ribotoxins are now included on the CDC Select Agents list. Although ribosome-directed agents are known to exert toxicity by activating mitogen-activated protein kinases, the underlying mechanisms for this "ribotoxic stress response" (RSR) remain largely undefined. Thus, a critical gap exists in our knowledge of the signal transduction mechanisms by which ribotoxins modulate gene expression and apoptosis. The objective of this proposal is to test the guiding hypothesis that the ribosome plays a central role in the initiation and integration of protein kinase-mediated stress responses to trichothecenes and other ribotoxic agents. This hypothesis is based on observations in the macrophage that: 1)deoxynivalenol (DON), a common foodborne trichothecene, mediates cleavage of 18S and 28S ribosomal (r)RNA, 2) double-stranded RNA-activated protein kinase (PKR), a ribosome-associated serine-threonine kinase, is essential for DON-induced protein kinase activation and 3) DON induces mobilization of several protein kinases to the ribosome whereupon they are phosphorylated. To test our hypothesis, three Specific Aims are proposed. In Aim 1, we will characterize DON-induced rRNA cleavage relative to targets, kinetics and mechanisms in the macrophage. In Aim 2, we will use both macrophage and cell-free models to characterize the role of PKR as an early sensor of DON-induced rRNA damage. In Aim 3, we will track DON-induced changes in ribosome-associated proteins in the macrophage relative to composition, kinetics and kinase activites. From these studies, we expect to understand how the ribosome mediates the induction and integration of multiple intracellular signaling cascades that drive altered gene expression and apoptosis in mononuclear phagocytes in response to ribotoxic agents. Anticipated outcomes include: 1) improved understanding of the molecular basis by which trichothecenes and other ribotoxins disrupt immunity, 2) enhanced capacity to assess and manage risks associated with exposure to trichothecenes and other ribotoxins, and 3) mechanism-based strategies for preventing and/or treating persons exposed to trichothecenes and ribotoxic chemicals via natural contamination or chemical terrorism. Collectively, these outcomes will positively impact public health by providing a scientific basis for generating sound recommendations relative to this important class of toxins and appropriate remedial actions should exposure occur. PUBLIC HEALTH RELEVANCE: We propose to learn how a class of potent biological toxins interferes with the function and survival of cells that are essential to the immune system. This research will enhance our capacity to assess and manage risks associated with exposure to these toxins as well as yield mechanism-based strategies for preventing and/or treating persons exposed to these agents via inadvertent food contamination or deliberate chemical terrorism. Collectively, these outcomes will positively impact public health by providing a scientific basis for generating sound recommendations relative to these important toxins and appropriate remedial actions should exposures occur.