What makes ricin toxic

蓖麻毒素为何有毒

基本信息

批准号：
8891184
负责人：
NILGUN E TUMER
金额：
$ 40.46万
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 2017-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8891184
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 analysis glycosylation in vivo inhibitor/antagonist insight killings mortality mutant new therapeutic target novel response screening therapeutic target tool weapons

项目摘要

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 α-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.

说明（由申请人提供）：植物毒素蓖麻毒素是已知的毒性最强的物质之一，可导致严重的发病和死亡。它是 B 类选择剂，目前没有有效对抗蓖麻毒素中毒的具体保护措施或治疗方法。因此，了解蓖麻毒素如何杀死细胞并开发解毒剂来保护暴露的人仍然是首要健康问题，蓖麻毒素通过从高度保守的腺嘌呤中去除特定的腺嘌呤来抑制蛋白质合成。大 rRNA 中的 α-sarcin/ricin 环 (SRL)。蓖麻毒素的毒性被认为是由于核糖体的不可逆失活和随后的翻译停滞而引起的，我们的工作通过证明核糖体脱嘌呤与细胞毒性不直接相关来挑战这一范式。我们发现，RTA 与核糖体柄结合，以极高的结合率和解离率使核糖体脱嘌呤，从而允许它在完整核糖体上对 SRL 的脱嘌呤率比在裸露的 28S rRNA 上的脱嘌呤率高得多。我们在人类细胞中的初步结果表明，人类核糖体柄对于 RTA 的脱嘌呤活性也至关重要。 RTA 的结合表面与活性位点不同，是完全毒性所必需的。我们表明，RTA 抑制酵母和哺乳动物细胞中的未折叠蛋白反应 (UPR)，并抑制 UPR。我们在酵母中进行全基因组筛选，发现了介导 RTA 毒性的新宿主因子。我们获得了最近的证据，表明 N-糖基化对于 RTA 从 ER 到细胞质的移位非常重要，并确定了一个关键的宿主因子。我们将测试 RTA 与核糖体结合的高速及其与促进易位的宿主因子的相互作用有助于蓖麻毒素的细胞毒性的假设。我们将进行功能分析，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呤活性和细胞毒性。我们将确定人类细胞中 sta 蛋白的消耗是否会影响 RTA 的脱嘌呤活性和细胞毒性。我们将确定从酵母基因筛选中鉴定出的基因如何介导哺乳动物细胞中的 RTA 毒性，从而确定潜在的治疗靶点。蓖麻毒素的毒性，对于制定具有暴露后潜力的对策至关重要。