Interaction of human pancreatic ribonuclease with human ribonuclease inhibitor - Generation of inhibitor-resistant cytotoxic variants

Mammalian ribonucleases interact very strongly with the intracellular ribon uclease inhibitor (RI), Eukaryotic cells exposed to mammalian ribonucleases are protected from their cytotoxic action by the intracellular inhibition of ribonucleases by RI. Human pancreatic ribonuclease (HPR) is structurally and functionally very similar to bovine RNase A and interacts with human R I with a high affinity. In the current study, we have investigated the invo lvement of Lys-7, Gln-11, Asn-71, Asn-88, Gly-89, Ser-90, and Glu-111 in HP R in its interaction with human ribonuclease inhibitor. These contact resid ues were mutated either individually or in combination to generate mutants K7A, Q11A, N71A, E111A, N88R, G89R, S90R, K7A/E111A, Q11A/E111A, N71A/E111A , K7A/ N71A/E111A, Q11A/N71A/E111A, and K7A/Q11A/N71A/ E111A. Out of these, eight mutants, K7A, Q11A, N71A, S90R, E111A, Q11A/E111A, N71A/E111A, and K 7A/N71A/ E111A, showed an ability to evade RI more than the wild type HPR, with the triple mutant K7A/N71A/E111A having the maximum RI resistance. As a result, these variants exhibited higher cytotoxic activity than wild type HPR, The mutation of Gly-89 in HPR produced no change in the sensitivity o f HPR for RI, whereas it has been reported that mutating the equivalent res idue Gly-88 in RNase A yielded a variant with increased RI resistance and c ytotoxicity, Hence, despite its considerable homology with RNase A, HPR sho ws differences in its interaction with RI. We demonstrate that interaction between human pancreatic ribonuclease and RI can be disrupted by mutating r esidues that are involved in HPR-RI binding. The inhibitor-resistant cytoto xic HPR mutants should be useful in developing therapeutic molecules.