Onconase:  An Unusually Stable Protein

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.

The Importance of Dynamic Effects on the Enzyme Activity

“…Our present and previous (17,45) results clearly indicate two distinctive features of Onconase with respect to other ribonucleases: very small flexibility and very high thermal stability, both unusual properties for a mesophilic enzyme. The two properties are mainly governed by different structural features.…”

“…Crystallization and Data Collection-Mutants of Onconase were expressed in Escherichia coli and purified as described previously (17)(18)(19). Crystallization was achieved by the sitting-drop vapor-diffusion technique at 277 and 293 K for (C87S,des103-104)-ONC and M23L-ONC, respectively.…”

“…Although ONC is a frog protein, it is immunologically tolerated by humans. It only causes a reversible renal toxicity (16), probably linked to the enzyme stability, which is unusually high for a protein isolated from a mesophilic source (17). ONC has a denaturation temperature (T d ) close to 90°C at pH 6.0 and is particularly resistant to proteolysis (17).…”

“…These mutants were proved to have a reduced thermal stability and/or increased catalytic activity (17)(18)(19). In particular, the mutant in which methionine 23 has been replaced by leucine (M23L-ONC) is 5-fold more active than the native enzyme and fully toxic toward tumor cells (17 (19), whereas their catalytic action and antitumor activity are practically unaffected.…”

Cytotoxic Ribonucleases and RNA Interference (RNAi)

“…The best characterized is Onconase (Onc), a basic and remarkably stable ribonuclease of approximately 12,000 MW . [5][6][7][8][9][10] Onc is cytostatic and cytotoxic to tumor cell lines of different lineage, [11][12][13][14][15] inhibits tumor growth in mice, 16,17 and under the trade name of ONCONASE® its antitumor properties are being currently evaluated in Phase III clinical trials. 18 The mechanism by which Onc exerts cytostatic and cytotoxic activity is not well understood.…”