title: Development of a quantitative RT-PCR assay to examine the kinetics of ribosome depurination by ribosome inactivating proteins using Saccharomyces cerevisiae as a model.
abstract: Ricin produced by the castor bean plant and Shiga toxins produced by pathogenic Escherichia coli (STEC) and Shigella dysenteriae are type II ribosome inactivating proteins (RIPs), containing an enzymatically active A subunit that inhibits protein synthesis by removing an adenine from the α-sarcin/ricin loop (SRL) of the 28S rRNA. There are currently no known antidotes to Shiga toxin or ricin, and the ability to screen large chemical libraries for inhibitors has been hindered by lack of quantitative assays for catalytic activity that can be adapted to a high throughput format. Here, we describe the development of a robust and quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay that can directly measure the toxins' catalytic activity on ribosomes and can be used to examine the kinetics of depurination in vivo. The qRT-PCR assay exhibited a much wider dynamic range than the previously used primer extension assay (500-fold vs. 16-fold) and increased sensitivity (60 pM vs. 0.57 nM). Using this assay, a 400-fold increase in ribosome depurination was observed in yeast expressing ricin A chain (RTA) relative to uninduced cells. Pteroic acid, a known inhibitor of enzymatic activity, inhibited ribosome depurination by RTA and Shiga toxin 2 with an IC(50) of ∼ 100 μM, while inhibitors of ricin transport failed to inhibit catalytic activity. These results demonstrate that the qRT-PCR assay would enable refined kinetic studies with RIPs and could be a powerful screening tool to identify inhibitors of catalytic activity.
authors.affiliation: Department of Plant Biology and Pathology, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, New Jersey 08901-8520, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Pierce
authors.forename: Jennifer Nielsen
authors.initials: JN
authors.lastname: Kahn
authors.forename: Jiachi
authors.initials: J
authors.lastname: Chiou
authors.forename: Nilgun E
authors.initials: NE
authors.lastname: Tumer
mesh_terms: D001681:Biological Assay; D002619:Chemical Warfare Agents / Q000032:analysis; D008954:Models, Biological; D011622:Pterins / Q000494:pharmacology; D011687:Purines / Q000378:metabolism*; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D054788:Ribosome Inactivating Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D012276:Ricin / Q000032:analysis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D022621:Shiga Toxin / Q000032:analysis* / Q000235:genetics
chemical_list: D002619:Chemical Warfare Agents; D011622:Pterins; D011687:Purines; D012331:RNA, Fungal; D012333:RNA, Messenger; D022621:Shiga Toxin; C005584:pteroic acid; D012276:Ricin; D054788:Ribosome Inactivating Proteins