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Ribonuclease A variants with potent cytotoxic activity
P A Leland1, L W Schultz, B M Kim
1Departments of Biochemistry and Chemistry, University of Wisconsin, Madison, WI 53706, USA.
Summary
Cytotoxic ribonucleases kill cancer cells by degrading RNA after evading ribonuclease inhibitor (RI). Low affinity for RI enhances cytotoxic potency, paving the way for novel pancreatic ribonuclease-based chemotherapy.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Bovine pancreatic ribonuclease A (RNase A) superfamily members can be potent cytotoxins.
- Cytotoxic ribonucleases enter the cytosol to degrade cellular RNA, leading to cell death.
- Ribonuclease inhibitor (RI) is a cytosolic protein that binds RNase A superfamily members with varying affinities.
Purpose of the Study:
- To investigate the role of ribonuclease affinity for RI in determining cytotoxic potency.
- To engineer RNase A variants with altered RI binding and assess their cytotoxic activity.
Main Methods:
- Site-directed mutagenesis was used to replace specific RNase A residues (Asp-38, Gly-88, Ala-109) with arginine.
- The affinity of wild-type RNase A and its variants for RI was measured.
- Cytotoxicity of the engineered ribonucleases against a transformed cell line was evaluated.
Main Results:
- RNase A binds RI with high affinity and is not cytotoxic.
- Onconase, a cytotoxic homolog, binds RI with low affinity.
- The G88R RNase A variant showed retained catalytic activity in the presence of RI and was cytotoxic.
- The G88D RNase A variant also exhibited decreased RI affinity and cytotoxic activity.
- Cytotoxic potency correlated with the ability to evade RI.
Conclusions:
- Ribonucleases that maintain catalytic activity despite RI binding are cytotoxic.
- The affinity of a ribonuclease for RI is a critical determinant of its cytotoxic potency.
- These findings support the development of pancreatic ribonucleases as a new class of chemotherapeutic agents.