Mechanism of cytotoxicity of ricin

蓖麻毒素的细胞毒性机制

基本信息

批准号：
7630083
负责人：
NILGUN E TUMER
金额：
$ 3.64万
依托单位：
RUTGERS, THE STATE UNIV OF N.J.
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-03-15 至 2012-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7630083
关键词：
Address Adenine Aerosols Antidotes Apoptosis Apoptotic Binding Biochemical Biological Models Biological Warfare Bioterrorism Breathing Categories Cell Death Cells Cessation of life Cytosol Defect Depurination Development Endocytosis Endoplasmic Reticulum Enterotoxins Genes Genome Genomics Induction of Apoptosis Intoxication Lectin Lung MAPK14 gene MAPK8 gene Mammalian Cell Measures Medical Membrane Messenger RNA Molecular Mutagenesis N glycosidase National Institute of Allergy and Infectious Disease Plants Plasmids Process Protein Biosynthesis Protein Synthesis Inhibition Proteins RNA Splicing Resistance Respiratory System Ribosomal RNA Ribosomes Ricin Ricinus communis Risk Role Signal Pathway Signal Transduction Small Interfering RNA System Techniques Terrorism Toxin Translations Vaccines Yeasts base biodefense cell injury cell killing cytotoxicity design instrument mutant novel therapeutics prevent prototype response ribosomal A-protein

项目摘要

Ribosome inactivating proteins (RIPs) have been used as instruments of biological warfare and terrorism. Ricin is a heterodimeric plant toxin that consists of A and B-chains and the prototype of type II RIPs. Its B- chain is a lectin that enables cell binding. After endocytosis, the A-chain translocates through the endoplasmic reticulum membrane to reach the cytosol where its N-glycosidase activity removes a specific adenine from the highly conserved, sarcin/ricin loop (SRL) in the large rRNA. Ricin has been classified as Category B priority for biodefense. Very little is known about the membrane translocation and ribosome interactions of ricin and the molecular mechanism by which it causes apoptosis in mammalian cells. We have established yeast as a biologically relevant model system to study the activity of RTA, and isolated mutant forms of RTA, which do not kill cells. Translation inhibition by ribosome depurination has been hypothesized to be responsible for the cytotoxicity of ricin. However, our preliminary analysis of the nontoxic RTA mutants indicates that ribosome depurination is necessary, but not sufficient for cytotoxicity. We further showed that wild type RTA inhibits induction of the unfolded protein response (UPR) in yeast and in mammalian cells and induces activation of JNK, p38 and ERK signaling pathways in mammalian cells. This project aims to use yeast and mammalian cells as complementary systems to understand the molecular basis for ricin intoxication. Specific Aims: 1. Using the nontoxic RTA mutants, determine if ribosome binding and depurination are required for cytotoxicity in yeast and induction of apoptosis in mammalian cells. 2. Characterize the interaction between RTA and ribosomal protein POand determine if binding to POis essential for ribosome depurination by RTA. 3. Determine if RTA causes cell death by inhibiting the development of UPR in yeast and in mammalian cells. 4. Identify the cellular genes necessary for cytotoxicity of RTA in yeast. Ricin is not only a bioterrorism threat, but inhibits translation by a similar mechanism as the bacterial enterotoxins. Therefore, the studies outlined in this application will have important implications for the design of protection strategies against AB-toxins that are classified as high-risk candidates for bioterrorism.

核糖体失活蛋白（RIP）已被用作生物战和恐怖主义的工具。蓖麻毒素是一种异二聚体植物毒素，由 A 链和 B 链组成，是 II 型 RIP 的原型。它的B- 链是一种能够实现细胞结合的凝集素。内吞作用后，A链通过内质网膜到达细胞质，其 N-糖苷酶活性去除特定的来自大 rRNA 中高度保守的肌氨酸/蓖麻毒素环 (SRL) 的腺嘌呤。蓖麻毒素被分类为生物防御 B 类优先。关于膜易位和核糖体知之甚少蓖麻毒素的相互作用及其引起哺乳动物细胞凋亡的分子机制。我们建立了酵母作为生物学相关的模型系统来研究 RTA 的活性，并分离出 RTA 的突变形式，不会杀死细胞。核糖体脱嘌呤的翻译抑制作用推测与蓖麻毒素的细胞毒性有关。然而，我们对无毒的初步分析 RTA 突变体表明核糖体脱嘌呤是必要的，但不足以产生细胞毒性。我们进一步表明野生型 RTA 抑制酵母和酵母中未折叠蛋白反应 (UPR) 的诱导哺乳动物细胞并诱导哺乳动物细胞中 JNK、p38 和 ERK 信号通路的激活。这项目旨在利用酵母和哺乳动物细胞作为互补系统来理解分子为蓖麻毒素中毒的基础。具体目标： 1. 使用无毒的 RTA 突变体，确定核糖体是否结合和脱嘌呤是酵母细胞毒性和哺乳动物细胞凋亡诱导所必需的。 2. 表征 RTA 与核糖体蛋白 PO 之间的相互作用，并确定是否与 PO 结合对于 RTA 核糖体脱嘌呤至关重要。 3. 确定 RTA 是否通过抑制 UPR 在酵母和哺乳动物细胞中的发展。 4. 确定必要的细胞基因 RTA 在酵母中的细胞毒性。蓖麻毒素不仅是一种生物恐怖主义威胁，而且通过类似的物质抑制翻译作用机制为细菌肠毒素。因此，本申请中概述的研究将具有对于设计针对高风险 AB 毒素的保护策略具有重要意义生物恐怖主义的候选者。