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A ribonuclease A variant with low catalytic activity but high cytotoxicity
L E Bretscher1, R L Abel, R T Raines
1Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Abstract:
Onconase, a homolog of ribonuclease A (RNase A) with low ribonucleolytic activity, is cytotoxic and has efficacy as a cancer chemotherapeutic. Here variants of RNase A were used to probe the interplay between ribonucleolytic activity and evasion of the cytosolic ribonuclease inhibitor protein (RI) in the cytotoxicity of ribonucleases. K41R/G88R RNase A is a less active catalyst than G88R RNase A but, surprisingly, is more cytotoxic. Like Onconase, the K41R/G88R variant has a low affinity for RI, which apparently compensates for its low ribonucleolytic activity. In contrast, K41A/G88R RNase A, which has the same affinity for RI as does the K41R/G88R variant, is not cytotoxic. The nontoxic K41A/G88R variant is a much less active catalyst than is the toxic K41R/G88R variant. These data indicate that maintaining sufficient ribonucleolytic activity in the presence of RI is a requirement for a homolog or variant of RNase A to be cytotoxic. This principle can guide the design of new chemotherapeutics based on homologs and variants of RNase A.
Insights
Cancer chemotherapeutics based on ribonuclease A (RNase A) variants require both sufficient ribonucleolytic activity and evasion of the ribonuclease inhibitor protein (RI) to be cytotoxic. This finding guides the design of novel RNase A-based cancer drugs.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Onconase, a ribonuclease A (RNase A) homolog, exhibits cytotoxicity and anticancer efficacy despite low ribonucleolytic activity.
- The cytosolic ribonuclease inhibitor protein (RI) modulates the activity of RNase A variants.
Purpose of the Study:
- To investigate the relationship between ribonucleolytic activity, RI evasion, and cytotoxicity in RNase A variants.
- To establish design principles for novel RNase A-based chemotherapeutics.
Main Methods:
- Creation and characterization of specific RNase A variants (K41R/G88R, G88R, K41A/G88R).
- Assays to determine ribonucleolytic activity and affinity for RI.
- Cytotoxicity assessments of the variants.
Main Results:
- K41R/G88R RNase A, despite lower catalytic activity than G88R RNase A, demonstrated higher cytotoxicity.
- K41R/G88R and K41A/G88R variants showed low affinity for RI, similar to Onconase.
- K41A/G88R RNase A was not cytotoxic, contrasting with the toxic K41R/G88R variant, despite similar RI affinity.
- The non-toxic K41A/G88R variant exhibited significantly lower catalytic activity than the toxic K41R/G88R variant.
Conclusions:
- Sufficient ribonucleolytic activity, even in the presence of RI, is crucial for RNase A homologs/variants to be cytotoxic.
- This principle is key for developing new chemotherapeutics derived from RNase A and its variants.