Intraspecies Regulation of Ribonucleolytic Activity

Abstract

The evolutionary rate of proteins involved in obligate protein-protein interactions is slower and the degree of co-evolution higher than that for non-obligate protein-protein interactions. The coevolution of the proteins involved in certain non-obligate interactions is, however, essential to cell survival. To gain insight into the co-evolution of one such non-obligate protein pair, the cytosolic ribonuclease inhibitor (RI) proteins and secretory pancreatic-type ribonucleases from cow (Bos taurus) and human (Homo sapiens) were produced in Escherichia coli and purified, and their physicochemical properties were analyzed. The two intraspecies complexes were found to be extremely tight (bovine K d = 0.69 fM; human K d = 0.34 fM). Human RI binds to its cognate ribonuclease (RNase 1) with 100-fold greater affinity than to the bovine homologue (RNase A). In contrast, bovine RI binds to RNase 1 and RNase A with nearly equal affinity. This broader specificity is consistent with there being more pancreatic-type ribonucleases in cows (20) than humans (13). Human RI (32 cysteine residues) also has 4-fold less resistance to oxidation by hydrogen peroxide than does bovine RI (29 cysteine residues). This decreased oxidative stability of human RI, which is caused largely by Cys74, implies a larger role for human RI as an antioxidant. The conformational and oxidative stabilities of both RIs increase upon complex formation with ribonucleases. Thus, RI has evolved to maintain its inhibition of invading ribonucleases, even when confronted with extreme environmental stress. That role appears to take precedence over its role in mediating oxidative damage.The discovery of extensive protein-protein interaction networks has informed investigations of protein evolution (1-3). For example, the rate of evolution within a protein-protein interaction network, has been found to depend on the number of binding partners, the concentration of each protein, and the evolutionary age of the proteins (4-10). These relationships extend to subclasses of interactions, as obligate protein-protein interactions, which are defined as interactions necessary for the stability of an individual protein (11), evolve at a slower rate and show a greater degree of co-evolution than do non-obligate interactions, which are interactions between proteins that can remain stable independently (7). Nonetheless, the co-evolution of certain non-obligate protein-protein interactions, such as particular receptor-ligand and enzyme-inhibitor interactions, can be essential for cell survival (11). To understand the co-evolution of such a non-obligate pair, we have investigated the complex formed between the cytosolic ribonuclease inhibitor protein (RI 1 ) and secretory pancreatictype ribonucleases, as failure to inhibit the enzymatic activity of an invading ribonuclease can lead to cell death (12)(13)(14)(15) RIs (25,26), which binds to some members of the ribonuclease A superfamily with affinities in the femtomolar range (27,28). RI is able to exert this affinity using...

Bovine Brain Ribonuclease Is the Functional Homolog of Human Ribonuclease 1

“…Like human RNase 1, BRB was found to be less thermostable than either RNase A or BSR. BRB was found to bind tightly to its endogenous inhibitor, bovine RI, similar to other homologous ribonucleases (21,31,46,47).…”

“…d Value is for the complex with bovine ribonuclease inhibitor, determined as described (31,35,36). e Value was determined with circular dichroism spectroscopy (75).…”

“…Ribonucleases were purified as inclusion bodies, and variants containing a free cysteine residue were labeled with either 2Ј,7Ј-diethylfluorescein (29) or BODIPY FL (Molecular Probes) as described (30). Bovine RI was purified via RNase A affinity chromatography as described (31). Dimeric BSR was isolated as a monomer and allowed to dimerize upon refolding as described (32).…”

“…Inhibitor Dissociation Rate-The equilibrium dissociation rates of the RI⅐ribonuclease complexes were determined as described (31,35,36). Briefly, RI and 2Ј,7Ј-diethylfluoresceinlabeled ribonucleases were mixed in equimolar ratios, and the resulting solution was incubated at 25°C for 5 min.…”

Ribonucleases, Nucleases and Antiangiogenins in Antiproliferative Activities

“…Overall, the hRIRNase1 (human RI) complex has both more intermolecular hydrogen bonds than does the pRI (pig RI) complex and more between residues of opposite charge. Moreover those hydrogen bonds that are conserved are shorter in the hRIRNase 1 complex [87]. The antitumor activity of various animal ribonucleases has been demonstrated both in vitro and in vivo [2][3][4][5][6]88].…”