Onconase: an unusually stable protein.
Several members of the RNase A superfamily are endowed with antitumor activity, showing selective cytotoxicity toward tumor cell lines. One of these is onconase, the smallest member of the superfamily, which at present is undergoing phase-III clinical trials as an antitumor drug. Our investigation focused on other interesting features of the enzyme, such as its unusually high denaturation temperature, its low catalytic activity, and its renal toxicity as a drug. We used differential scanning calorimetry, circular dichroism, fluorescence measurements, and limited proteolysis to investigate the molecular determinants of the stability of onconase and of a mutant, (M23L)-ONC, which is catalytically more active than the wild-type enzyme, and fully active as an antitumor agent. The determination of the main thermodynamic parameters of the protein led to the conclusion that onconase is an unusually stable protein. This was confirmed by its resistance to proteolysis. On the basis of this analysis and on a comparative analysis of the (M23L)-ONC variant of the protein, which is less stable and more sensitive to proteolysis, a model was constructed in line with available data. This model supports a satisfactory hypothesis of the molecular basis of onconase stability and low-catalytic activity.
Notomista
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Dipartimento di Chimica Organica e Biologica, Università di Napoli Federico II, Via Mezzocannone 16, 80134 Naples, Italy.
Catanzano
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Graziano
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Dal Piaz
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Barone
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D'Alessio
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Di Donato
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D019943:Amino Acid Substitution; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D004527:Egg Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D011894:Rana pipiens; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D004527:Egg Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012260:Ribonucleases; D002918:Chymotrypsin; D010434:Pepsin A; C058703:ranpirnase