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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.
Abstract:
Select members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. These cytotoxic ribonucleases enter the cytosol, where they degrade cellular RNA and cause cell death. Ribonuclease inhibitor (RI), a cytosolic protein, binds to members of the RNase A superfamily with inhibition constants that span 10 orders of magnitude. Here, we show that the affinity of a ribonuclease for RI plays an integral role in defining the potency of a cytotoxic ribonuclease. RNase A is not cytotoxic and binds RI with high affinity. Onconase, a cytotoxic RNase A homolog, binds RI with low affinity. To disrupt the RI-RNase A interaction, three RNase A residues (Asp-38, Gly-88, and Ala-109) that form multiple contacts with RI were replaced with arginine. Replacing Asp-38 and Ala-109 with an arginine residue has no effect on the RI-RNase interaction. In addition, these variants are not cytotoxic. In contrast, replacing Gly-88 with an arginine residue yields a ribonuclease (G88R RNase A) that retains catalytic activity in the presence of RI and is cytotoxic to a transformed cell line. Replacing Gly-88 with aspartate also yields a ribonuclease (G88D RNase A) with a decreased affinity for RI and cytotoxic activity. The cytotoxic potency of onconase, G88R RNase A, and G88D RNase A correlate with RI evasion. We conclude that ribonucleases that retain catalytic activity in the presence of RI are cytotoxins. This finding portends the development of a class of chemotherapeutic agents based on pancreatic ribonucleases.
Insights
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.