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Onconase: an unusually stable protein
E Notomista1, F Catanzano, G Graziano
1Dipartimento di Chimica Organica e Biologica, Università di Napoli Federico II, Via Mezzocannone 16, 80134 Naples, Italy.
Abstract:
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.
Insights
Onconase, a small antitumor enzyme, exhibits remarkable stability and low catalytic activity. This study explores the molecular basis for its stability and low activity, crucial for its drug development.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- The RNase A superfamily contains enzymes with antitumor properties.
- Onconase, a small RNase A member, shows selective cytotoxicity and is in phase-III clinical trials.
- Onconase possesses unusual stability, low catalytic activity, and potential renal toxicity.
Purpose of the Study:
- To investigate the molecular determinants of onconase stability and low catalytic activity.
- To compare the stability and proteolysis sensitivity of onconase and its catalytically active mutant (M23L)-ONC.
- To develop a model explaining onconase's stability and low activity.
Main Methods:
- Differential scanning calorimetry
- Circular dichroism
- Fluorescence spectroscopy
- Limited proteolysis
- Thermodynamic parameter determination
Main Results:
- Onconase is an unusually stable protein, confirmed by its resistance to proteolysis.
- (M23L)-ONC mutant is less stable and more sensitive to proteolysis than wild-type onconase.
- Thermodynamic analysis revealed key parameters contributing to onconase's stability.
Conclusions:
- Onconase's high stability is a key characteristic, potentially linked to its low catalytic activity.
- A model was constructed explaining the molecular basis of onconase stability and low activity.
- Understanding these properties is vital for onconase's therapeutic application.