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Ricin A-chain ( RTA) depurinates the sarcin-ricin loop of 28S ribosomal RNA and inhibits protein synthesis in mammalian cells. In yeast, the ribosomal stalk facilitates the interaction of RTA with the ribosome and subsequent depurination. Despite homology between the stalk structures from yeast and humans, there are notable differences. The human ribosomal stalk contains two identical heterodimers of P1 and P2 bound to P0, whereas the yeast stalk consists of two different heterodimers, P1α- P2β and P2α- P1β, bound to P0. RTA exhibits higher activity towards mammalian ribosomes than towards ribosomes from other organisms, suggesting that the mode of interaction with ribosomes may vary. Here, we examined whether the human ribosomal stalk proteins facilitate the interaction of RTA with human ribosomes and subsequent depurination of the sarcin-ricin loop. Using small interfering RNA-mediated knockdown of P1/ P2 expression in human cells, we demonstrated that the depurination activity of RTA is lower when P1 and P2 levels are reduced. Biacore analysis showed that ribosomes from P1/ P2-depleted cells have a reduced ability to bind RTA, which correlates with reduced depurination activity both in vitro and inside cells. RTA interacts directly with recombinant human P1- P2 dimer, further demonstrating the importance of human P1 and P2 in enabling RTA to bind and depurinate human ribosomes. Structured digital abstract P2, P1 and RTA physically interact by surface plasmon resonance (View interaction), P2, P1 and RTA physically interact by anti bait coimmunoprecipitation (View interaction) [ABSTRACT FROM AUTHOR]

Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga-like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the α-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 α-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 ΔP1 and ΔP2 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. [ABSTRACT FROM AUTHOR]