Ricin A-Chain Activity on Stem−Loop and Unstructured DNA Substrates

Amukele, Tim K.; Roday, Setu; Schramm, Vern L.

doi:10.1021/bi0474362

Cited by 28 publications

(27 citation statements)

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“…Truncated oligonucleotide constructs of the ribosomal sarcinricin loop are RTA and SAP substrates (13)(14)(15)(16). In addition to hairpin stem-loop structures, RTA hydrolyzes adenine from cyclic GAGA loops that possess a 5Ј-to 3Ј-covalently closed synthetic linker (17,18) (Fig.…”

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confidence: 99%

Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins

Sturm

Almo

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Ricin A-chain (RTA) and saporin-L1 (SAP) catalyze adenosine depurination of 28S rRNA to inhibit protein synthesis and cause cell death. We present the crystal structures of RTA and SAP in complex with transition state analogue inhibitors. These tight-binding inhibitors mimic the sarcin-ricin recognition loop of 28S rRNA and the dissociative ribocation transition state established for RTA catalysis. RTA and SAP share unique purine-binding geometry with quadruple -stacking interactions between adjacent adenine and guanine bases and 2 conserved tyrosines. An arginine at one end of the -stack provides cationic polarization and enhanced leaving group ability to the susceptible adenine. Common features of these ribosome-inactivating proteins include adenine leaving group activation, a remarkable lack of ribocation stabilization, and conserved glutamates as general bases for activation of the H2O nucleophile. Catalytic forces originate primarily from leaving group activation evident in both RTA and SAP in complex with transition state analogues.is the catalytic subunit of ricin, a Centers for Disease Control and Prevention category B bioterrorism agent derived from Ricinus communis seeds. RTA catalyzes the depurination of an invariant adenosine residue, A 4234, within the GA 4234 GA tetraloop motif of the highly conserved sarcin-ricin loop of eukaryotic 28S rRNA (1). Clinical trials have exploited the toxicity of RTA in RTA-antibody constructs to kill leukemia and lymphoma cells (e.g., RTA conjugated to anti-CD22) (2-5). Side effects limit the utility of RTA immunotoxins (6, 7). Targeted inhibitors against ribosome-inactivating proteins (RIPs) could improve immunotoxin cancer therapies by rescuing normal cells following toxin treatment.Saporin-L1 (SAP), a homologue of RTA from Saponaria officinalis (soapwort) leaves, exhibits N-glycohydrolase activities on 80S ribosomes, poly(A) RNA, and other cellular . SAP releases multiple adenines from ribosomes, whereas RTA shows exquisite specificity.Truncated oligonucleotide constructs of the ribosomal sarcinricin loop are RTA and SAP substrates (13-16). In addition to hairpin stem-loop structures, RTA hydrolyzes adenine from cyclic GAGA loops that possess a 5Ј-to 3Ј-covalently closed synthetic linker (17, 18) (Fig. 1).Transition state analysis of RTA-mediated depurinations established that hydrolysis of adenine involves a ribocation intermediate, followed by attack of an activated water. Adenine activation is a major driving force for RTA catalysis (19,20). Efficient catalysis by RTA requires the invariant Glu-177 and Arg-180 residues (21-25). RTA and other RIPs have evolved to become near-perfect catalysts for mammalian ribosomes (21,(26)(27)(28). Here, we use transition state analogues to establish the catalytic site features contributing to this remarkable catalytic activity.RTA transition state structures have guided the design and synthesis of potent RIP inhibitors (17, 29). Inhibitors for RTA and SAP include 1Ј-aza-sugars with a nonhydrolyzable 9-deazaadenine to mimic the...

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confidence: 99%

Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins

Sturm

Almo

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…RTA depurinates an adenine from stem-loop RNA and RNA/DNA hybrid substrates at pH 4.0, but not at pH 7 8, 15, 22. Small RNA stem-loops were assayed discontinuously at pH 4.0 and the reaction stopped by the addition of 2× coupling buffer to provide a neutral pH.…”

Section: Resultsmentioning

confidence: 99%

“…RTA catalysis of stem-loop substrates was not detectable above pH 6.5 with either A-10 or A-14 2-dA stem-loop oligonucleotides (up to 100 µM) with 2 µM RTA for 10 minutes. The pH 4.0 catalytic optimum of RTA on 6 to 18-mer stem-loop DNA and RNA 28S SRL mimic oligonucleotides is known to result from the protonation of two ionizable residues on substrate and/or RTA 8, 15, 22…”

Section: Resultsmentioning

confidence: 99%

Detecting Ricin: Sensitive Luminescent Assay for Ricin A-Chain Ribosome Depurination Kinetics

2009

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Ricin is a family member of the lethal ribosome-inactivating proteins (RIP) found in plants. Ricin toxin A-chain (RTA) from castor beans catalyzes the hydrolytic depurination of a single base from a GAGA tetraloop of eukaryotic ribosomal RNA to release a single adenine from the sarcin-ricin loop (SRL). Protein synthesis is inhibited by loss of elongation factor binding resulting in cell death. We report a sensitive coupled assay for the measurement of adenine released from ribosomes or small stem-loop RNAs by RTA catalysis. Adenine phosphoribosyl transferase (APRTase) and pyruvate orthophosphate dikinase (PPDK) convert adenine to ATP for quantitation by firefly luciferase. The resulting AMP is cycled to ATP to give sustained luminescence proportional to adenine concentration. Sub-picomole adenine quantitation permits the action of RTA on eukaryotic ribosomes to be followed in continuous, high-throughput assays. Facile analysis of RIP catalytic activity will have applications in plant toxin detection, inhibitor screens, mechanistic analysis of depurinating agents on oligonucleotides and intact ribosomes, and in cancer immunochemotherapy. Kinetic analysis of the catalytic action of RTA on rabbit reticulocyte 80S ribosomes establishes a catalytic efficiency of 2.6 × 10 8 M −1 s −1 , a diffusion limited reaction indicating catalytic perfection even with large reactants.

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“…The team further demonstrated the prophylactic protective effects of Retro-2 against lethal intranasal ricin challenge, but similar prophylactic effects in mice was not observed with Retro-1 (Stechmann et al 2010). Inhibitors based on the oligonucleotide stem-loop RNA inhibitors of ricin A-chain were also developed, but this drug development approach faces additional obstacles with respect to bioavailability and hydrolysis by circulating nucleases (Amukele et al 2005). At present, the detailed mechanism through which Retro-1-and Retro-2-mediated inhibition of retrograde trafficking of ricin and shiga-like toxin remains to be established; early evidence suggested targeting and relocation of syntaxin 5 activity by these novel small molecules (Stechmann et al 2010).…”

Section: Targeted Therapy and Intervention Points In Ricin-mediated Cmentioning

confidence: 99%

Biological Toxins and Bioterrorism

Gopalakrishnakone¹,

Balali-Mood²,

Llewellyn³

et al. 2015

Toxinology

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In recent years, the field of toxinology has expanded substantially. On the one hand it studies venomous animals, plants and micro organisms in detail to understand their mode of action on targets. While on the other, it explores the biochemical composition, genomics and proteomics of toxins and venoms to understand their three interaction with life forms (especially humans), development of antidotes and exploring their pharmacological potential. Therefore, toxinology has deep linkages with biochemistry, molecular biology, anatomy and pharmacology. In addition, there is a fast-developing applied subfield, clinical toxinology, which deals with understanding and managing medical effects of toxins on human body. Given the huge impact of toxin-based deaths globally, and the potential of venom in generation of drugs for so-far incurable diseases (for example, diabetes, chronic pain), the continued research and growth of the field is imminent. This has led to the growth of research in the area and the consequent scholarly output by way of publications in journals and books. Despite this ever-growing body of literature within biomedical sciences, there is still no all-inclusive reference work available that collects all of the important biochemical, biomedical and clinical insights relating to toxinology. Composed of 11 volumes, Toxinology provides comprehensive and authoritative coverage of the main areas in toxinology, from fundamental concepts to new developments and applications in the field. Each volume comprises a focused and carefully chosen collection of contributions from leading names in the subject.

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Ricin A-Chain Activity on Stem−Loop and Unstructured DNA Substrates

Cited by 28 publications

References 40 publications

Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins

Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins

Detecting Ricin: Sensitive Luminescent Assay for Ricin A-Chain Ribosome Depurination Kinetics

Biological Toxins and Bioterrorism

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