Onconase dimerization through 3D domain swapping: structural investigations and increase in the apoptotic effect in
Andrea Fagagnini1, Andrea Pica2, Sabrina Fasoli1
1Dipartimento di Neuroscienze, Biomedicina e del Movimento, Sezione di Chimica Biologica, Università degli Studi di Verona, Strada Le Grazie, 8, Verona I-37134, Italy.
Abstract:
Onconase® (ONC), a protein extracted from the oocytes of the Rana pipiens frog, is a monomeric member of the secretory 'pancreatic-type' RNase superfamily. Interestingly, ONC is the only monomeric ribonuclease endowed with a high cytotoxic activity. In contrast with other monomeric RNases, ONC displays a high cytotoxic activity. In this work, we found that ONC spontaneously forms dimeric traces and that the dimer amount increases about four times after lyophilization from acetic acid solutions. Differently from RNase A (bovine pancreatic ribonuclease) and the bovine seminal ribonuclease, which produce N- and C-terminal domain-swapped conformers, ONC forms only one dimer, here named ONC-D. Cross-linking with divinylsulfone reveals that this dimer forms through the three-dimensional domain swapping of its N-termini, being the C-terminus blocked by a disulfide bond. Also, a homology model is proposed for ONC-D, starting from the well-known structure of RNase A N-swapped dimer and taking into account the results obtained from spectroscopic and stability analyses. Finally, we show that ONC is more cytotoxic and exerts a higher apoptotic effect in its dimeric rather than in its monomeric form, either when administered alone or when accompanied by the chemotherapeutic drug gemcitabine. These results suggest new promising implications in cancer treatment.
Insights
Onconase (ONC), a cytotoxic frog protein, forms a unique dimer (ONC-D) through N-terminal domain swapping. This ONC-D form exhibits enhanced anti-cancer effects, suggesting new therapeutic potential.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Onconase (ONC) is a unique monomeric ribonuclease from Rana pipiens oocytes with high cytotoxic activity.
- Unlike other RNases, ONC's cytotoxic potential is not fully understood.
- Investigating ONC's structural dynamics and their impact on activity is crucial.
Purpose of the Study:
- To investigate the dimerization of Onconase (ONC).
- To characterize the structure and formation mechanism of the ONC dimer (ONC-D).
- To evaluate the cytotoxic and apoptotic effects of ONC monomer versus dimer.
Main Methods:
- Spontaneous dimerization analysis of ONC.
- Lyophilization from acetic acid solutions to induce dimerization.
- Cross-linking with divinylsulfone to identify dimer formation sites.
- Homology modeling of ONC-D based on RNase A dimer structure.
- Spectroscopic and stability analyses.
- Cytotoxicity and apoptosis assays of monomeric and dimeric ONC.
Main Results:
- ONC spontaneously forms dimers, with increased amounts after lyophilization.
- ONC forms a unique N-terminal domain-swapped dimer (ONC-D), distinct from other RNases.
- ONC-D exhibits significantly higher cytotoxic and apoptotic activity compared to monomeric ONC.
- The dimeric form enhances the efficacy of gemcitabine chemotherapy.
Conclusions:
- ONC dimerization via N-terminal domain swapping creates a distinct structural conformer, ONC-D.
- The dimeric form of ONC possesses superior cytotoxic and apoptotic properties.
- ONC-D represents a promising candidate for novel cancer therapeutic strategies, potentially in combination with chemotherapy.
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