| The mammalian ribonuclease inhibitor (RI) is a 50-kDa cytosolic protein that binds to members of the bovine pancreatic ribonuclease (RNase A) superfamily with inhibition constants that span 10 orders of magnitude. Thus, RI plays an integral role in defining the biological activities of RNase A and its homologs. This dissertation describes the role of RI in the biological activities of pancreatic-type ribonucleases.; Variants of RNase A that evade RI can be toxic to tumor cells. The conformation stability of a ribonuclease also contributes to the cytotoxicity of a ribonuclease. I created A4C/K41R/G88R/V 118C RNase A to reduce RI affinity (K41R/G88R) and increase the conformational stability (A4C/V 118C). The variant was highly resistant to RI binding, but suffered a significant decrease in catalytic activity. The (kcat/Km) cyto parameter, which reports on the ability of a ribonuclease to exert its ribonucleolytic activity in the presence of cytosolic RI, predicted that the toxicity of A4C/K41R/G88R/V118C RNase A would not exceed its predecessors, as was observed, validating the utility of this parameter.; The complex formed by ANG and RI is amongst the tightest known in biology (Kd ≈ 1 fM). ANG exerts its biological activity in the nucleus, whereas RI is present in the cytosol. I constructed G85R/G86R ANG, which possessed 106-fold weaker affinity for RI but retained its catalytic activity. G85R/G86R ANG maintained its ability to translocate to the nucleus of HUVE cells and stimulated their migration at lower protein concentrations than did wild-type ANG. In addition, blood vessel growth stimulated by G85R/G86R ANG in rabbit cornea was more robust than with ANG. Thus, RI serves to regulate ANG-induced neovascularization.; Finally, I examined the effect of RI silencing on ribonuclease toxicity in human tumor cells using RNAi. Expression of an shRNA targeted to the RI gene resulted in a significant decrease in cytosolic RI levels. RI levels in some but not all tumor cell lines limited the cytotoxicity of ribonucleases. I conclude that salient features of the mechanism of ribonuclease cytotoxicity remain to be discovered. |
