Ribosome-inactivating proteins: potent poisons and molecular tools

Abstract

Ribosome-inactivating proteins (RIPs) were first isolated over a century ago and have been shown to be catalytic toxins that irreversibly inactivate protein synthesis. Elucidation of atomic structures and molecular mechanism has revealed these proteins to be a diverse group subdivided into two classes. RIPs have been shown to exhibit RNA N-glycosidase activity and depurinate the 28S rRNA of the eukaryotic 60S ribosomal subunit. In this review, we compare archetypal RIP family members with other potent toxins that abolish protein synthesis: the fungal ribotoxins which directly cleave the 28S rRNA and the newly discovered Burkholderia lethal factor 1 (BLF1). BLF1 presents additional challenges to the current classification system since, like the ribotoxins, it does not possess RNA N-glycosidase activity but does irreversibly inactivate ribosomes. We further discuss whether the RIP classification should be broadened to include toxins achieving irreversible ribosome inactivation with similar turnovers to RIPs, but through different enzymatic mechanisms.

Keywords: BLF1; BPSL1549; Shiga toxins; abrin; analytical and therapeutic applications; biological weapon; eukaryotic protein synthesis inhibition; ricin; saporin; α-sarcin.

Figure 1. Mechanisms of cellular entry and actions of RIPs. Shiga toxins are delivered into the cell after the B-chain binds globotriaosylceramide (Gb3) to stimulate clathrin-dependent/independent endocytosis. Upon reaching the Golgi the A-chain is cleaved by the protease Furin. Ricin is delivered into the cell after the binding of the lectin-like B chain to glycoproteins/glycolipids and clathrin-dependent/independent endocytosis. After retrograde translocation from the Golgi to the ER the A- and B-chains of ricin and Shiga toxins are separated. Saporin is delivered into the cytoplasm via a clathrin-dependent mechanism of uptake aided by saponins. Shiga toxins, ricin and saporin all inhibit translation using RNA N-glycosidase to depurinate 28S rRNA at the same adenine residue (A₄₃₂₄). Alpha-sarcin enters the cell via pinocytosis and cleaves the phosphodiester bond between G₄₃₂₅ and A₄₃₂₆. BLF1 is delivered into the cytoplasm via an intra-cellular pathogenic bacterium and deamidates the translation initiation factor eIF4A inactivating its RNA-helicase activity leading to inactivation of initiating ribosomes.

Figure 2. Ribbon representations of the X-ray crystallography structures of type 1/2 RIPs and BLF1. Ricin, saporin, and Shiga toxin Stx2 have N-glycosidase activity that cleaves the 28S rRNA while BLF1 catalyzes the deamidation of the eIF4A initiation translation factor. (A) Structure of Ricin A-chain in complex with the cyclic tetranucleotide inhibitor; PDB ID code 3HIO. (B) Structure of saporin in complex with the cyclic tetranucleotide inhibitor; PDB ID code 3HIW. (C) Structure of Shiga-like Stx2 A-chain from Escherichia coli in complex with adenine; PDB ID code 2GA4. Red-labeled residues Tyr-80, Tyr-123, and Arg-180 in the active site of ricin A-chain are equivalent in saporin and Stx2 (Tyr-73, Tyr-123, Arg-177 and Tyr-77, Tyr-114, Arg-170, respectively).^,- The catalytic water molecule and crystallized inhibitors within the active sites are not represented for improving clarity. In contrast, Glu-177 of Stx2 is not conserved with the Glu-177/174 in the active sites of ricin and saporin respectively. Glutamate residues are labeled in blue. (D) Structure of BLF1, PDB ID code 3TU8. The primary sequence Leu-91 Ser-92 Gly-93 is (cyan) is conserved in the carboxyl-terminal domain of CNF1 along with Cys-94. Residues forming the catalytic triad of BLF1 (Thr-88, Cys-94, and His-106) are shown in red. Both the tertiary structure and catalytic site of BLF1 are different from type 1 and 2 RIPs represented in (A–C).