What makes ricin toxic

描述（由申请人提供）：植物毒素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。它是一种B类精选药剂。没有有效对抗蓖麻毒素中毒的具体保护措施或治疗方法，并且存在迫切的未满足的治疗需求。因此，了解蓖麻毒素如何杀死细胞并开发解毒剂以保护暴露人群仍然是首要的健康优先事项。蓖麻毒素通过从高度保守的？大rRNA中的八叠球菌素/蓖麻毒素环（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。我们的工作通过证明核糖体脱嘌呤与RTA在酵母和哺乳动物细胞中的细胞毒性没有直接关系来挑战这种范式。我们发现，RTA结合到核糖体柄脱嘌呤核糖体的缔合和解离的异常高的速率，使其脱嘌呤的SRL在一个更高的速率在完整的核糖体比裸露的28 S rRNA。我们在人类细胞中的初步结果表明，人类核糖体茎也是RTA的脱嘌呤活性的关键。我们提出了新的初步证据表明，RTA的核糖体结合表面，这是不同的活性位点，需要充分的毒性。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），并且抑制UPR有助于蓖麻毒素的细胞毒性。我们在酵母中的全基因组筛选鉴定了介导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ER到胞质溶胶的移位是重要的，并鉴定了对N-糖基化至关重要的宿主因子。我们将检验这个假设 RTA与核糖体结合的高速以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。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 这些突变体的脱嘌呤活性和细胞毒性。我们将确定是否在人类细胞中的stal蛋白的消耗将影响RTA的脱嘌呤活性和细胞毒性。我们将筛选一个 高密度肽阵列文库以鉴定核糖体对接事件的肽抑制剂。我们 将确定从酵母遗传筛选中鉴定的基因如何介导哺乳动物细胞中的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，并对开发具有暴露后潜力的对策至关重要。

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 ?-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.