What makes ricin toxic

蓖麻毒素为何有毒

• 批准号: 8532806
• 负责人: NILGUN E TUMER
• 金额: $ 44.12万
• 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/8532806
• 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（由申请人提供）：蓖麻毒素是已知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类精选代理。目前还没有针对蓖麻毒素中毒的有效保护措施或治疗方法，迫切需要治疗。因此，了解蓖麻毒素如何杀死细胞并开发解毒剂以保护接触者仍然是卫生的首要任务。蓖麻毒素通过去除高度保守的？大rRNA中的-sarcin/ricin loop （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阻滞。我们的工作挑战了这一范式，证明核糖体去嘌呤化与酵母和哺乳动物细胞中RTA的细胞毒性没有直接关系。我们发现RTA与核糖体柄结合，以极高的结合和解离率去嘌呤核糖体，使其在完整核糖体上的去嘌呤SRL比在裸28S rRNA上的去嘌呤率要高得多。我们在人类细胞中的初步结果表明，人类核糖体柄对RTA的去嘌呤活性也至关重要。我们提出了新的初步证据，表明RTA的核糖体结合表面与活性部位不同，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），并且UPR的抑制有助于蓖麻毒素的细胞毒性。我们在酵母中进行了全基因组筛选，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-糖基化对于RTA从内质网到细胞质的错位很重要，并确定了一个对N-糖基化至关重要的宿主因子。我们将检验这个假设