What makes ricin toxic

蓖麻毒素为何有毒

• 批准号: 8702992
• 负责人: NILGUN E TUMER
• 金额: $ 38.7万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-15 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8702992
• 关键词: Active Sites; Adenine; Affect; Affinity; Antidotes; Binding; Bioterrorism; C-terminal; Categories; Cells; Complex; Cytosol; Depurination; Development; Dislocations; Dissociation; Docking; Endoplasmic Reticulum; Escherichia coli O157; Eukaryota; Event; Gene Deletion; Generations; Genes; Genetic Models; Genetic Screening; Glycine decarboxylase; Goals; Health Priorities; Human; Immunotoxins; Integration Host Factors; Intoxication; Knowledge; Lead; Libraries; Mammalian Cell; Measures; Mediating; Methods; Morbidity - disease rate; Myelin P2 Protein; Pathway interactions; Peptides; Plants; Poison; Protein Biosynthesis; Proteins; Public Health; Research; Ribosomal Proteins; Ribosomal RNA; Ribosome Inactivation; Ribosomes; Ricin; Ricin A Chain; Role; Shiga Toxin; Speed; Structure; Surface; System; Testing; Therapeutic; Toxic effect; Toxin; United States Food and Drug Administration; Vaccines; Work; Yeasts; base; cancer cell; cell killing; cytotoxicity; density; design; genome wide association study; glycosylation; in vivo; inhibitor/antagonist; insight; killings; mortality; mutant; new therapeutic target; novel; response; screening; therapeutic target; tool; weapons

DESCRIPTION (provided by applicant): The plant toxin ricin is one of the most toxic substances known and can cause severe morbidity and mortality. It is a category B select agent. There are no specific protective measures or therapeutics effective against ricin intoxication and there is an urgent unmet need for therapy. Therefore, understanding how ricin kills cells and developing antidotes to protect exposed people remain top health priorities. Ricin inhibits protein synthesis by removing a specific adenine from the highly conserved ?-sarcin/ricin loop (SRL) in the large rRNA. The toxicity of ricin is thought to be due to irreversibe inactivation of ribosomes and subsequent translational arrest. Our work challenged this paradigm by demonstrating that ribosome depurination does not directly correlate with the cytotoxicity of RTA in yeast and in mammalian cells. We showed that RTA binds to the ribosomal stalk to depurinate ribosomes with an exceptionally high rate of association and dissociation, allowing it to depurinate the SRL at a much higher rate on intact ribosomes than on the naked 28S rRNA. Our preliminary results in human cells demonstrated that the human ribosomal stalk is also critical for the depurination activity of RTA. We present new preliminary evidence that the ribosome binding surface of RTA, which is distinct from the active site, is required for full toxicity. We showed that RTA inhibits the unfolded protein response (UPR) in yeast and in mammalian cells and inhibition of the UPR contributes to cytotoxicity of ricin. Our genome-wide screen in yeast identified novel host factors that mediate the toxicity of RTA. We obtained recent evidence that N-glycosylation is important for dislocation of RTA from the ER to the cytosol and identified a host factor critical for N- glycosylation. We will test the hypothesis that the high speed with which RTA binds the ribosome together with its interaction with the host factors that facilitate translocation contribute to the cytotoxicity of ricin. We will carry out structure function analysis to identify residues that are critical for ribosome binding and examine the depurination activity and cytotoxicity of these mutants. We will determine if depletion of stal proteins in human cells will affect depurination activity and cytotoxicity of RTA. We will screen a high density peptide array library to identify peptide inhibitors of the ribosome docking event. We will determine how genes identified from the genetic screen in yeast mediate RTA toxicity in mammalian cells to identify potential therapeutic targets. These discoveries will impact our understanding ricin toxicity and will be critical for development of countermeasures with post-exposure potential.

描述(申请人提供)：植物毒素蓖麻毒素是已知的毒性最强的物质之一，可导致严重的发病率和死亡率。它是B类选择代理。目前还没有针对蓖麻毒素中毒的具体保护措施或有效的治疗方法，而且对治疗的迫切需求尚未得到满足。因此，了解蓖麻毒素是如何杀死细胞的，并开发解毒剂来保护接触过蓖麻毒素的人，仍然是健康的首要任务。蓖麻毒素通过从大rRNA中高度保守的？-sarcin/ricin环(SRL)中移除特定的腺嘌呤来抑制蛋白质的合成。蓖麻毒素的毒性被认为是由于核糖体不可逆转地失活和随后的翻译停滞。我们的工作挑战了这一范式，证明了核糖体脱嘌呤与RTA在酵母和哺乳动物细胞中的细胞毒性没有直接关系。我们发现RTA与核糖体的柄结合，以异常高的结合和解离率去纯化核糖体，使其在完整的核糖体上比在裸露的28S rRNA上以更高的速率去纯化SRL。我们在人类细胞中的初步结果表明，人的核糖体茎对RTA的去嘌呤活性也是至关重要的。我们提出了新的初步证据，即RTA的核糖体结合表面不同于活性部位，是完全毒性所必需的。我们发现RTA抑制酵母和哺乳动物细胞中的未折叠蛋白反应(UPR)，抑制UPR有助于蓖麻毒素的细胞毒性。我们在酵母全基因组筛查中发现了介导RTA毒性的新宿主因子。我们最近获得的证据表明，N-糖基化对RTA从内质网到胞浆的错位非常重要，并确定了一个对N-糖基化至关重要的宿主因子。我们将检验这一假设 RTA与核糖体的快速结合及其与促进转位的宿主因子的相互作用导致了蓖麻毒素的细胞毒性。我们将进行结构功能分析，以确定对核糖体结合至关重要的残基，并检查 这些突变体的脱嘌活性和细胞毒性。我们将确定人类细胞中Stal蛋白的耗尽是否会影响RTA的脱嘌活性和细胞毒性。我们将放映一部 高密度多肽阵列文库用于鉴定核糖体对接事件的多肽抑制物。我们 将确定从酵母的遗传筛选中识别的基因如何在哺乳动物细胞中介导RTA毒性，以确定潜在的治疗靶点。这些发现将影响我们对蓖麻毒素毒性的理解，并将对开发具有暴露后潜力的对策至关重要。