Mechanism of cytotoxicity of ricin

描述(申请人提供)：核糖体失活蛋白(RIPs)已被用作生物战和恐怖主义的工具。蓖麻毒素是一种由A链和B链组成的异二聚体植物毒素，是II型RIPs的原型。它的B链是一种凝集素，可以使细胞结合。内吞作用后，A链通过内质网膜转移到胞浆，在胞浆中，其N-糖苷酶活性从大rRNA中高度保守的sarcin/ricin环(SRL)中移除特定的腺嘌呤。蓖麻毒素已被列为生物防御的B类优先事项。关于蓖麻毒素的膜转位和核糖体相互作用，以及它引起哺乳动物细胞凋亡的分子机制，人们知之甚少。我们已经建立了酵母作为生物相关的模型系统来研究RTA的活性，并分离出不会杀死细胞的RTA的突变形式。核糖体脱嘌呤对翻译的抑制被认为是导致蓖麻毒素细胞毒性的原因。然而，我们对无毒的RTA突变体的初步分析表明，核糖体脱嘌呤不足以产生细胞毒性。该项目旨在利用酵母和哺乳动物细胞作为互补系统来了解蓖麻毒素中毒的分子基础。具体目的：1.利用无毒的RTA突变体，确定酵母细胞毒性和诱导哺乳动物细胞凋亡是否需要核糖体结合和去嘌呤。2.鉴定RTA与核糖体蛋白PO的相互作用，并确定与PO的结合是否是RTA对核糖体去嘌呤所必需的。3.确定RTA在酵母中的细胞毒性所必需的细胞基因，并确定RTA是否通过影响未折叠蛋白反应(UPR)的诱导而导致细胞死亡。蓖麻毒素不仅是一种生物恐怖主义威胁，而且通过与细菌肠毒素相似的机制抑制翻译。因此，本申请中概述的研究将对设计针对被归类为生物恐怖主义高危候选者的AB毒素的保护策略具有重要意义。

DESCRIPTION (provided by applicant): 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 not sufficient for cytotoxicity. 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 PO and determine if binding to PO is essential for ribosome depurination by RTA. 3. Identify the cellular genes necessary for cytotoxicity of RTA in yeast and determine if RTA causes cell death by affecting induction of the unfolded protein response (UPR). 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.