Fluorescence studies on the interaction of adenine with ricin A‐chain

Honjo, Eijiro; Tsukamoto, Takuji; Funatsu, Gunki

doi:10.1016/0014-5793(92)80630-y

Cited by 17 publications

(16 citation statements)

References 23 publications

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“…Because of this finding, we attempted to detect (data not shown) the binding of [3H]-AZT to RIC holotoxin, and the binding of nonradioactive AZT to RIC A chain, using a tryptophan fluorescence assay. In both cases we failed to detect AZT binding to RIC or RIC A chain; although in the latter case, we did confirm a n earlier report (Watanabe et al, 1992) that adenine binding to RIC A chain could be detected by measuring changes in tryptophan fluorescence. A variety of agents can perturb intracellular trafficking in cultured eukaryotic cells.…”

Section: Effects Of Various Agents On the Azt Inhibition Of Ric And Psupporting

confidence: 60%

Characterization of 3′‐azido‐3′‐deoxythymidine inhibition of ricin and pseudomonas exotoxin A toxicity in CHO and vero cells

Wellner¹,

Pless²,

Thompson³

1994

Journal Cellular Physiology

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Ricin (RIC), modeccin (MOD), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) are protein toxins that enter cells by receptor-mediated endocytosis. After intracellular transport and membrane translocation to the cytosol, these toxins inhibit protein synthesis by enzymatically removing a specific adenine residue from ribosomal RNA (RIC, MOD), or by ADP-ribosylation of elongation factor-2 (PE, DT). Recently, Thompson and Pace (1992) reported that AZT (3'-azido-3'-deoxythymidine) inhibited RIC toxicity in Vero cells, and this inhibition was not due to a block of RIC enzymatic activity. This paper extends these findings and examines the effects of AZT treatment on the toxicities of other protein toxins in Chinese hamster ovary (CHO) and Vero cell lines. AZT treatment did not significantly alter the toxicity of DT or MOD in either cell line, but it markedly reduced RIC and PE toxicity in both cell lines. The ID50 values (concentration of toxin required to inhibit protein synthesis by 50%) for RIC and PE in CHO cells increased approximately 6.5- and 12.5-fold, respectively; while in Vero cells the ID50 values increased ca. 8.5- and 4.5-fold, respectively. Results of further studies revealed differences in the mechanisms by which AZT inhibited RIC and PE toxicity. Results of cell-free translation indicated that, unlike its effects on RIC, AZT blocked the ability of PE to perform its enzymatic activity. As AZT did not block RIC enzymatic activity, we examined the effects of AZT on earlier steps in the RIC intoxication process. AZT treatment did not inhibit cell-surface binding or internalization of [125I]-RIC. Results of kinetic studies showed that when AZT was incubated with cells at the time of RIC exposure, it caused no major change in the lag phase, during which RIC reaches the site of translocation. However, it clearly reduced the subsequent first-order reduction in the rate of protein synthesis, suggesting an effect on translocation. Monensin (an ionophore that perturbs intracellular trafficking and increases the toxicities of RIC and PE) reduced AZT protection against both toxins. Nocodazole and colchicine (agents that disrupt microtubules and some routes of intracellular trafficking) reduced the ability of AZT to inhibit RIC, but not PE, toxicity. In summary, our results suggest that (1) AZT acts within the cytosol to inhibit (directly or indirectly) the enzymatic action of PE, and (2) the AZT inhibition of RIC cytotoxicity does not involve perturbations of RIC cell-surface binding, internalization, or enzymatic activity but might result from an alteration in RIC translocation.

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Section: Effects Of Various Agents On the Azt Inhibition Of Ric And Psupporting

confidence: 60%

Characterization of 3′‐azido‐3′‐deoxythymidine inhibition of ricin and pseudomonas exotoxin A toxicity in CHO and vero cells

Wellner¹,

Pless²,

Thompson³

1994

Journal Cellular Physiology

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“…The activity of RA does not survive deletion, even one at a time, of three residues (Trp-211, Gly-212, and Arg-213) in helix H (Table I); however, Arg-213 can be chemically modified with phenylglyoxal, and presumably inactivated, without loss of RA activity (40). The invariant Trp-211 is thought to participate in the binding of the adenosine in the RNA substrate (39).…”

Section: Discussionmentioning

confidence: 99%

Systematic Deletion Analysis of Ricin A-Chain Function

Munishkin

Wool

1995

Journal of Biological Chemistry

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The A-chain of ricin is a cytotoxic RNA N-glycosidase that inactivates ribosomes by depurination of the adenosine at position 4324 in 28 S rRNA. Of the 267 amino acids in the protein, 222 could be deleted, in one or another of 74 mutants, without the loss of the capacity to catalyze hydrolysis of a single specific nucleotide in rRNA (Morris, K. N., and Wool, I. G. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4869 -4873). The 45 amino acids that could not be omitted when the deletions were in sets of 20, 5, or 2 residues have now been deleted one at a time; 9 of these deletion mutants retained activity. A RNP-like structural motif in ricin A-chain that may mediate binding to ribosomal RNA has been identified.Ricin is a cytotoxic RNA N-glycosidase that is synthesized in the castor bean Ricinus communis. Proricin is a polypeptide of approximately 65 kDa which is processed by removal of 12 amino acids to form an A-chain of 267, and a B-chain of 262, residues linked by a disulfide bond (1). The toxic ricin A-chain (RA) 1 inhibits protein synthesis by inactivating ribosomes; the inhibition is the result of the hydrolysis of the bond between the base and the ribose of the adenosine at position 4324 (A4324) in 28 S rRNA (2, 3). RA is extraordinarily toxic: a single molecule will inactivate 1500 ribosomes min Ϫ1, indeed, a single molecule resident in a cell is sufficient to kill it (1); finally, this one covalent modification accounts entirely for the cytotoxicity.A value of an analysis of the mechanism of action of antibiotics and of ribotoxins is that it directs attention to components of the ribosome where efforts to comprehend functional correlates of structure are likely to be rewarded. That this one RA-catalyzed depurination inactivates the ribosome implies that this region of 28 S rRNA is crucial for the function of the particle and there is evidence that the ricin domain is involved in elongation factor-1 dependent binding of aminoacyl-tRNA to the ribosomal A-site and elongation factor-2 catalyzed GTP hydrolysis and translocation of peptidyl-tRNA to the P-site (4).A substantial effort has been made to relate the structure of RA to its mechanism of action spurred in part by the use of the protein in the construction of immunotoxins for cancer therapy (5). The amino acid sequences of ricin and of several homologous proteins have been determined (6 -15) and an atomic structure of ricin has been obtained from x-ray diffraction of crystals (16 -18). In the three-dimensional structure there is a prominent cleft that was proposed to be the active site of RA before the mechanism of action of the toxin had been defined, indeed, before the substrate had been identified (16). Strong support for the suggestion has come from the observation that most of the amino acids that are invariant in related plant and bacterial toxins are clustered at the bottom of the putative active site cleft (18); from site-directed mutagenesis (19 -24); and from an analysis by systematic deletion of amino acids (25).Three series of amino acid deletion...

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“…Since adenine is the product of the N-glycosidase activity of RIPs, we reasoned that it would bind directly to the active site of StxA1 (32). However, attempts to demonstrate direct binding of adenine to the A1 polypeptide by equilibrium dialysis were unsuccessful.…”

Section: Discussionmentioning

confidence: 99%

Investigation of ribosome binding by the Shiga toxin A1 subunit, using competition and site-directed mutagenesis

Skinner

Jackson

1997

J Bacteriol

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The enzymatic subunit of Shiga toxin (StxA1) is a member of the ribosome-inactivating protein (RIP) family, which includes the ricin A chain as well as other examples of plant toxins. StxA1 catalytically depurinates a well-conserved GAGA tetra-loop of 28S rRNA which lies in the acceptor site of eukaryotic ribosomes. The specific activities of native StxA1, as well as mutated forms of the enzyme with substitutions in catalytic site residues, were measured by an in vitro translation assay. Electroporation was developed as an alternative method for the delivery of purified A1 polypeptides into Vero cells. Site-directed mutagenesis coupled with N-bromosuccinimide modification indicated that the sole tryptophan residue of StxA1 is required for binding it to the 28S rRNA backbone. Northern analysis established that the catalytic site substitutions reduced enzymatic activity by specifically interfering with the capacity of StxA1 to depurinate 28S rRNA. Ribosomes were protected from StxA1 by molar excesses of tRNA and free adenine, indicating that RIPs have the capacity to enter the acceptor site groove prior to binding and depurinating the GAGA tetra-loop.Shiga toxin (STX) is a multimeric molecule consisting of a single 32-kDa enzymatic A subunit (StxA) noncovalently associated with a ring of five 7.7-kDa B subunits (StxB) (17). The StxB subunits mediate the binding of toxin to its receptor on eukaryotic membranes, and the entire STX molecule enters cells via endocytosis from coated pits (28). StxA is processed in the trans-Golgi network by the cellular protease furin (11) to release StxA1, which possesses the enzymatic activity, and StxA2, which acts as a bridge between StxA1 and the B pentamer. StxA1 is an N-glycosidase which catalytically removes a specific adenine from a well-conserved tetra-loop of 28S rRNA and thus inhibits protein synthesis by preventing the binding of aminoacyl tRNAs to the ribosome and halting protein elongation (7). StxA1 is a member of the ribosome-inactivating protein (RIP) family of enzymes, which are a large group of protein synthesis inhibitors primarily found in the leaves, seeds, and roots of dicotyledonous plants (5,7,30). The only RIPs identified so far which are not produced by plants are the members of the STX family of toxins.The catalytic sites of StxA1 have been identified by sequence comparison of RIP family members and site-specific mutagenesis. Amino acid substitutions in three regions of StxA1 which are highly conserved among the RIPs identified tyrosine 77, glutamic acid 167, arginine 170, and tryptophan 203 as important for catalytic activity (2,16,33). Corresponding mutations in another RIP, the A chain of the plant lectin ricin (RTA), corroborated the findings with StxA1 (24, 29).The X-ray crystal structures of StxA1 and RTA have been solved at 2.5 Å (10, 20). Comparison of these structures revealed an active site cleft which contains all of the catalytic site residues previously identified by site-specific mutagenesis. Xray crystallographic analysis of StxA1 and RTA als...

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Fluorescence studies on the interaction of adenine with ricin A‐chain

Cited by 17 publications

References 23 publications

Characterization of 3′‐azido‐3′‐deoxythymidine inhibition of ricin and pseudomonas exotoxin A toxicity in CHO and vero cells

Characterization of 3′‐azido‐3′‐deoxythymidine inhibition of ricin and pseudomonas exotoxin A toxicity in CHO and vero cells

Systematic Deletion Analysis of Ricin A-Chain Function

Investigation of ribosome binding by the Shiga toxin A1 subunit, using competition and site-directed mutagenesis

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