Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.9 (ribonuclease)
 6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pseudomonas exotoxin A (PE) is a protein toxin composed of three structural domains. Functional analysis of PE has revealed that domain I is the cell-binding domain and that domain III functions in ADP ribosylation. Domain II was originally designated as the translocation domain, mediating the transfer of domain III to the cytosol, because mutations in this domain result in toxin molecules with normal cell-binding and ADP-ribosylation activities but which are not cytotoxic. However, the results do not rule out the possibility that regions of PE outside of domain II also participate in the translocation process. To investigate this problem, we have now constructed a toxin in which domain III of PE is replaced with barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens. This chimeric toxin, termed PE1-412-Bar, is cytotoxic to a murine fibroblast cell line and to a murine hybridoma resistant to the ADP-ribosylation activity of PE. A mutant form of PE1-412-Bar with an inactivating mutation in domain II at position 276 was significantly less toxic. Because the cytotoxic effect of PE1-412-Bar was due to the ribonuclease-activity of barnase molecules which had been translocated to the cytosol, we conclude that domain II of PE is not only essential but also probably sufficient to carry out the translocation process.
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PMID:Translocation mediated by domain II of Pseudomonas exotoxin A: transport of barnase into the cytosol. 156 15

Pseudomonas exotoxin A (PE) is a protein toxin composed of three structural domains which are responsible for cell binding (domain Ia, amino acids 1-252), translocation into the cytosol (domain II, amino acids 253-364) and ADP-ribosylation activity (domain III, amino acids 405-613). We have previously described (Prior, T. I., FitzGerald, D. J., and Pastan, I. (1992) Biochem. 31, 3555-3559) a molecule composed of amino acids 1-412 of PE and the extracellular ribonuclease of Bacillus amyloliquefaciens, barnase (Bar), and shown that this protein (PE1-412-Bar) is toxic to cells due to its ribonuclease activity, which had been delivered to the cytosol. We have now used this model to evaluate the role played by the carboxyl end of domain II (amino acids 347-364), domain Ib, and the amino end of domain III (amino acids 405-412) in the translocation event. Toxins completely lacking domain III, termed PE1-380-Bar, or both domains Ib and III, termed PE1-364-Bar, were equally cytotoxic to a murine fibroblast cell line (L929) as was PE1-412-Bar. Extending the deletion to include part of the E-helix and all of the F-helix of domain II (amino acids 347-364) resulted in a toxin (PE1-346-Bar) that was 10-fold less toxic. Previously tested on only murine cell lines, we demonstrate that barnase toxins are cytotoxic also to a variety of human cell lines. Cytotoxicity was assessed by measuring inhibition of DNA synthesis. Surprisingly, PE1-380-Bar is not lethal when injected into mice, either intraperitoneally or intravenously, at 9 nmol, which is 2200-fold more than the amount required for killing by PE (4 pmol). In cell culture these barnase-containing toxins are 100-fold less toxic to murine fibroblast cells than PE. Barnase toxin has a greater survival time in the blood of mice than PE, with a half-life of 102 min. We conclude that domain II is sufficient to transport proteins into the cytosol. Further, since domain Ia can be replaced with other cell targeting moieties, we propose that barnase-toxins should be evaluated for utility in targeted cancer therapy.
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PMID:Studies on the activity of barnase toxins in vitro and in vivo. 874 87