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.
|Li, Xiao-Ping; Kahn, Jennifer N; Tumer, Nilgun E (2018) Peptide Mimics of the Ribosomal P Stalk Inhibit the Activity of Ricin A Chain by Preventing Ribosome Binding. Toxins (Basel) 10:|
|Zhou, Yijun; Li, Xiao-Ping; Kahn, Jennifer N et al. (2018) Functional Assays for Measuring the Catalytic Activity of Ribosome Inactivating Proteins. Toxins (Basel) 10:|
|Li, Xiao-Ping; Tumer, Nilgun E (2017) Differences in Ribosome Binding and Sarcin/Ricin Loop Depurination by Shiga and Ricin Holotoxins. Toxins (Basel) 9:|
|Zhou, Yijun; Li, Xiao-Ping; Chen, Brian Y et al. (2017) Ricin uses arginine 235 as an anchor residue to bind to P-proteins of the ribosomal stalk. Sci Rep 7:42912|
|Basu, Debaleena; Li, Xiao-Ping; Kahn, Jennifer N et al. (2016) The A1 Subunit of Shiga Toxin 2 Has Higher Affinity for Ribosomes and Higher Catalytic Activity than the A1 Subunit of Shiga Toxin 1. Infect Immun 84:149-61|
|Basu, Debaleena; Kahn, Jennifer N; Li, Xiao-Ping et al. (2016) Conserved Arginines at the P-Protein Stalk Binding Site and the Active Site Are Critical for Ribosome Interactions of Shiga Toxins but Do Not Contribute to Differences in the Affinity of the A1 Subunits for the Ribosome. Infect Immun 84:3290-3301|
|Jetzt, Amanda E; Li, Xiao-Ping; Tumer, Nilgun E et al. (2016) Toxicity of ricin A chain is reduced in mammalian cells by inhibiting its interaction with the ribosome. Toxicol Appl Pharmacol 310:120-128|
|Basu, Debaleena; Tumer, Nilgun E (2015) Do the A subunits contribute to the differences in the toxicity of Shiga toxin 1 and Shiga toxin 2? Toxins (Basel) 7:1467-85|
|Grela, Przemys?aw; Li, Xiao-Ping; Tchórzewski, Marek et al. (2014) Functional divergence between the two P1-P2 stalk dimers on the ribosome in their interaction with ricin A chain. Biochem J 460:59-67|
|Yan, Qing; Li, Xiao-Ping; Tumer, Nilgun E (2014) Wild type RTA and less toxic variants have distinct requirements for Png1 for their depurination activity and toxicity in Saccharomyces cerevisiae. PLoS One 9:e113719|
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