SummaryRibosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga-like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the a-sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic ribosomes. Ricin depurinates the eukaryotic ribosomes more efficiently than the prokaryotic ribosomes, while PAP can depurinate both types of ribosomes. Accumulating evidence suggests that different docking sites on the ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the a-sarcin/ ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild-type ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the DP1 and DP2 mutants in vivo and these mutants were more resistant to the cytotoxicity of RTA than the wild-type cells. We further show that while RTA, Stx1 and Stx2 have similar requirements for ribosome depurination, PAP has different requirements, providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their ribosome specificity.
Ricin is a ribosome inactivating protein that catalytically removes a universally conserved adenine from the α-sarcin/ricin loop (SRL) of the 28S rRNA. We recently showed that ricin A chain (RTA) interacts with the P1 and P2 proteins of the ribosomal stalk to depurinate the SRL in yeast. Here we examined the interaction of RTA with wild type and mutant yeast ribosomes deleted in the stalk proteins by surface plasmon resonance. The interaction between RTA and wild type ribosomes did not follow a single step binding model, but was best characterized by two distinct types of interactions. The AB1 interaction had very fast association and dissociation rates, was saturable and required an intact stalk, while the AB2 interaction had slower association and dissociation rates, was not saturable and did not require the stalk. RTA interacted with the mutant ribosomes by a single type of interaction, which was similar to the AB2 interaction with the wild type ribosomes. Both interactions were dominated by electrostatic interactions and the AB1 interaction was stronger than the AB2 interaction. Based on these results we propose a two-step interaction model. The slow and ribosomal stalk nonspecific AB2 interactions concentrate the RTA molecules on the surface of the ribosome. The AB2 interactions facilitate the diffusion of RTA towards the stalk and promote the faster, more specific AB1 interactions with the ribosomal stalk. The electrostatic AB1 and AB2 interactions work together allowing RTA to depurinate the SRL at a much higher rate on the intact ribosomes than on the naked 28S rRNA.Ricin is a ribosome inactivating protein (RIP) isolated from the castor bean plant, Ricinus communis that consists of a Ricin Toxin A chain (RTA) and a galactose-binding B chain (RTB). RTA and RTB are linked by a disulfide bond between the Cys 259 near the C-terminus of RTA and the Cys 4 of RTB (1). The holotoxin is not active on ribosomes (2-6). The enzymatic activity of ricin is due to RTA, which is an N-glycosidase that specifically cleaves the Nglycosidic bond at A4324 of the 28S rRNA of rat ribosomes (7) and inhibits the elongation factor dependent ribosomal functions (8-10). RTA can also cleave the A2660 in naked 23S rRNA from E. coli but not the ribosomes from E. coli (11)(12)(13)(14). Both A4324 of rat 28S rRNA and A2660 of E. coli 23S rRNA are located at a universally conserved rRNA stem loop called the α-sarcin/ricin loop (SRL) named after the toxins (9). The SRL is the site where elongation factors interact with the ribosome and is involved in peptide translocation during protein synthesis (15). RTA depurinates the 28S rRNA on rat ribosomes about 5000 times faster than the naked rat 28S rRNA (7), indicating that the conformation of the rRNA on intact ribosomes and the ribosomal proteins play an important role in ribosome depurination by ricin.The α-sarcin/ricin loop (SRL) is located on the 60S subunit in close proximity to the ribosomal stalk, which is a lateral flexible structure on the large subunit. The stalk region was ...
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 a-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 mM, 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.
We report the discovery of a promising NDM-1 inhibitor, ebselen, through a cell-based screening approach. Enzymatic kinetic study and ESI-MS analysis suggested that ebselen could bind to NDM-1 by forming a S-Se bond with the Cys(221) residue at the active site, thereby exhibiting a new inhibition mechanism with broad spectrum inhibitory potential.
The coronavirus outbreak that commenced at the end of 2019 has led to a dramatic increase in the demand for face masks. In countries that are experiencing a shortage of face masks as a result of panic buying or inadequate supply, reusable fabric masks have become a popular option, because they are often considered more cost-effective and environmentally friendly than disposable medical masks. Nevertheless, there remains a significant variation in the quality and performance of existing face masks; not all are simultaneously able to provide protection against the extremely contagious virus and be comfortable to wear. This study aims to examine the influential factors that affect the comfort of reusable face masks, but not to assess the antimicrobial or antiviral potential. Seven types of masks were selected in this study and subjected to air and water vapor permeability testing, thermal conductivity testing and a wear trial. The results indicate that washable face masks made of thin layers of knitted fabric with low density and a permeable filter are more breathable. Additionally, masks that contain sufficient highly thermally conductive materials and have good water vapor permeability are often more comfortable to wear as they can transfer heat and moisture from the body quickly, and thus do not easily dampen and deteriorate.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.