Trichothecene Toxicity and the Ribotoxic Stress Response

描述（由申请人提供）：毛霉烯是一组倍半萜类真菌毒素，通常在世界范围内作为食品污染物遇到，与人类和动物疾病有病原学联系，免疫系统是主要目标。在实验动物中，这些真菌毒素和其他天然毒素与真核核糖体高亲和力结合。蓖麻毒素、志贺毒素、大霉素)诱导淋巴组织促炎基因表达和细胞凋亡。由于它们在化学恐怖主义中的潜在用途，毛霉烯和其他核素毒素现在被列入疾病控制与预防中心的选定药剂名单。虽然已知核糖体导向的药物通过激活有丝分裂原激活的蛋白激酶来发挥毒性，但这种“核糖体毒性应激反应”（RSR）的潜在机制仍未明确。因此，我们对核糖素毒素调节基因表达和细胞凋亡的信号转导机制的认识存在一个关键的空白。本研究的目的是验证核糖体在蛋白激酶介导的对毛孢烯和其他核糖体毒性药物的应激反应的启动和整合中起核心作用的指导假设。这一假设是基于对巨噬细胞的观察：1)脱氧镰刀菌醇（DON），一种常见的食源性毛菌烯，介导18S和28S核糖体（r)RNA的裂解；2)双链RNA激活蛋白激酶（PKR），一种与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；3）DON诱导几种蛋白激酶动员到核糖体，然后它们被磷酸化。为了验证我们的假设，提出了三个具体目标。在Aim 1中，我们将描述巨噬细胞中don诱导的rRNA切割的相关靶点、动力学和机制。在Aim 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Aim 3中，我们将追踪巨噬细胞中don诱导的核糖体相关蛋白的变化，这些变化与组成、动力学和激酶活性有关。从这些研究中，我们期望了解核糖体如何介导诱导和整合多种细胞内信号级联反应，这些信号级联反应驱动单核吞噬细胞对核糖体毒性药物的基因表达改变和细胞凋亡。预期的结果包括：1)增进对毛霉烯和其他核素毒素破坏免疫的分子基础的了解；2)加强评估和管理与接触毛霉烯和其他核素毒素有关的风险的能力；3)基于机制的战略，以预防和/或治疗通过自然污染或化学恐怖主义接触毛霉烯和核素毒性化学品的人。总的来说，这些结果将对公共卫生产生积极影响，为就这类重要毒素提出合理建议以及在发生接触时采取适当补救行动提供科学依据。公共卫生相关性：我们建议了解一类强效生物毒素如何干扰对免疫系统至关重要的细胞的功能和存活。这项研究将提高我们评估和管理与接触这些毒素有关的风险的能力，并产生基于机制的策略，以预防和/或治疗通过无意的食品污染或故意的化学恐怖主义接触这些物质的人。总的来说，这些结果将对公共卫生产生积极影响，为就这些重要毒素提出合理建议以及在发生接触时采取适当补救行动提供科学依据。

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.