Onconase is an amphibian member of the pancreatic ribonuclease family of enzymes that is in clinical trials for the treatment of cancer. Onconase, which has an abundance of lysine residues, is internalized by cancer cells through endocytosis in a mechanism similar to that of cell-penetrating peptides. Here, we compare the effect of lysine versus arginine residues on the biochemical attributes necessary for Onconase to elicit its cytotoxic activity. In the variant R-Onconase, ten of the twelve lysine residues in Onconase are replaced with arginine, leaving only the two active-site lysines intact. Cytometric assays quantifying internalization showed a 3-fold increase in the internalization of R-Onconase compared with Onconase. R-Onconase also showed greater affinity for heparin and a 2-fold increase in ribonucleolytic activity. Nonetheless, arginine substitution endowed only a slight increase in toxicity towards human cancer cells. Analysis of denaturation induced with guanidine–HCl showed that R-Onconase has less conformational stability than does the wild-type enzyme; moreover, R-Onconase is more susceptible to proteolytic degradation. These data indicate that arginine residues are more effective than lysine in eliciting cellular internalization, but can compromise other aspects of protein structure and function.
Pancreatic-type ribonucleases show clinical promise as chemotherapeutic agents, but are limited in efficacy by the inefficiency of their uptake by human cells. Cellular uptake can be increased by the addition of positive charges to the surface of ribonucleases, either by site-directed mutagenesis or by chemical modification. This observation has led to the hypothesis that ribonuclease-uptake by cells depends on electrostatics. Here, we use a combination of experimental and computational methods to ascertain the contribution of electrostatics to the cellular uptake of ribonucleases. We focus on three homologous ribonucleases: Onconase (frog), ribonuclease A (cow), and ribonuclease 1 (human). Our results support the hypothesis that electrostatics are necessary for the cellular uptake of Onconase. In contrast, specific interactions with cell-surface components likely contribute more to the cellular uptake of ribonuclease A and ribonuclease 1 than do electrostatics. These findings provide insight for the design of new cytotoxic ribonucleases.
Revealing the thermodynamic driving force of protein/DNA interactions is crucial to the understanding of factors that dictate the properties and function of protein-DNA complexes. For the binding of DNA to DNA-wrapping proteins, such as the integration host factor (IHF), Record and co-workers have proposed that the disruption of a large number of pre-existing salt-bridges is coupled with the binding process (J. Mol. Biol., 310, 2001, 379). To test this proposal, we have carried out explicit solvent MD simulations (multiple ~ 25–50 ns trajectories for each salt concentration) to examine the behavior of charged residues in IHF, especially concerning their ability to form salt bridges under different salt concentrations. Of the 17 cationic residues noted by Record and co-workers, most are engaged in salt-bridge interactions for a significant portion of the trajectories, especially in the absence of salt. This observation suggests that, from a structural point of view, their proposal is plausible. However, the complex behaviors of charged residues observed in the MD simulations also suggest that the unusual thermodynamic characteristics for IHF-DNA binding likely arise from the interplay between complex dynamics of charged residues both in and beyond the DNA binding site. Moreover, a comparison of MD simulations at different salt concentrations suggests that the large dependence of the IHF-DNA binding enthalpy on salt concentration may not be due to a significant decrease in the number of stable salt bridges in apo IHF at high salt concentration. In addition to the Hofmeister effects quantified in more recent studies of IHF-DNA binding, we recommend consideration of variation of the enthalphy change of salt bridge disruption at different salt concentrations. Finally, the current simulation study explicitly highlights that the electrostatic properties of DNA-binding proteins can be rather different in the apo and DNA bound states, which has important implication to the design of robust methods for predicting DNA binding sites in proteins.
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