Select members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. These cytotoxic ribonucleases enter the cytosol, where they degrade cellular RNA and cause cell death. Ribonuclease inhibitor (RI), a cytosolic protein, binds to members of the RNase A superfamily with inhibition constants that span 10 orders of magnitude. Here, we show that the affinity of a ribonuclease for RI plays an integral role in defining the potency of a cytotoxic ribonuclease. RNase A is not cytotoxic and binds RI with high affinity. Onconase, a cytotoxic RNase A homolog, binds RI with low affinity. To disrupt the RI-RNase A interaction, three RNase A residues (Asp-38, Gly-88, and Ala-109) that form multiple contacts with RI were replaced with arginine. Replacing Asp-38 and Ala-109 with an arginine residue has no effect on the RI-RNase interaction. In addition, these variants are not cytotoxic. In contrast, replacing Gly-88 with an arginine residue yields a ribonuclease (G88R RNase A) that retains catalytic activity in the presence of RI and is cytotoxic to a transformed cell line. Replacing Gly-88 with aspartate also yields a ribonuclease (G88D RNase A) with a decreased affinity for RI and cytotoxic activity. The cytotoxic potency of onconase, G88R RNase A, and G88D RNase A correlate with RI evasion. We conclude that ribonucleases that retain catalytic activity in the presence of RI are cytotoxins. This finding portends the development of a class of chemotherapeutic agents based on pancreatic ribonucleases.

Thiol:disulfide oxidoreductases have a CXXC motif within their active sites. To initiate the reduction of a substrate disulfide bond, the thiolate form of the N-terminal cysteine residue (CXXC) of this motif performs a nucleophilic attack. Escherichia coli thioredoxin [Trx (CGPC)] is the best characterized thiol:disulfide oxidoreductase. Previous determinations of the active-site pKa values of Trx have led to conflicting interpretations. Here, 13C-NMR spectroscopy, site-specific isotopic labeling, and site-directed mutagenesis were used to demonstrate that analysis of the titration behavior of wild-type Trx requires the invocation of microscopic pKa values for two interacting active-site residues: Asp26 (7.5 and 9.2) and Cys32 (CXXC; 7.5 and 9.2). By contrast, in two Trx variants, D26N Trx and D26L Trx, Cys32 exhibits a pKa near 7.5 and has a well-defined, single-pKa titration curve. Similarly, in oxidized wild-type Trx, Asp26 has a pKa near 7.5. In CVWC and CWGC Trx, Cys32 exhibits a single pKa near 6.2. In all five enzymes studied here, there is no evidence for a Cys35 (CXXC) pKa of < 11. This study demonstrates that a comprehensive approach must be used to unravel complex titration behavior of the functional groups in a protein.

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Onconase 0 (ONC) is a homolog of ribonuclease A (RNase A) that has unusually high conformational stability and is toxic to human cancer cells in vitro and in vivo. ONC and its amphibian homologs have a C-terminal disulfide bond, which is absent in RNase A. Replacing this cystine with a pair of alanine residues greatly decreases the conformational stability of ONC. In addition, the C87A/C104A variant is 10-fold less toxic to human leukemia cells. These data indicate that the synapomorphic disulfide bond of ONC is an important determinant of its cytotoxicity. ß 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Human angiogenin (ANG) is a homologue of bovine pancreatic ribonuclease (RNase A) that induces neovascularization. ANG is the only human angiogenic factor that possesses ribonucleolytic activity.To stimulate blood-vessel growth, ANG must be transported to the nucleus and must retain its catalytic activity. Like other mammalian homologues of RNase A, ANG forms a femtomolar complex with the cytosolic ribonuclease inhibitor protein (RI). To determine whether RI affects ANG-induced angiogenesis, we created G85R/G86R ANG, which possesses 10 6 -fold lower affinity for RI but retains wild-type ribonucleolytic activity. The neovascularization of rabbit corneas by G85R/G86R ANG was more pronounced and more rapid than by wild-type ANG. These findings provide the first direct evidence that RI serves to regulate the biological activity of ANG in vivo.Angiogenin (ANG) is a potent inducer of blood vessel growth (1) and has been implicated in the establishment, growth, and metastasis of tumors (2,3). A homologue of bovine pancreatic ribonuclease (RNase A (4-6); EC 3.1.27.5), ANG is the only human angiogenic factor that displays ribonucleolytic activity. ANG was first isolated from the conditioned medium of human adenocarcinoma cells (1), and is present in normal human plasma (7) as well as numerous other tissues and organs (8). After receptor-mediated endocytosis (9), a nuclear localization sequence (NLS) directs ANG to the nucleus (10). The receptor-binding, nuclear localization, and ribonucleolytic activity of ANG are all required for angiogenic activity (9-11). In endothelial and smooth muscle cells, ANG induces a wide range of cellular responses, including transcriptional activation (12), differentiation (13), cell migration and invasion (14), and tube formation (13).The ribonuclease inhibitor (RI (15)), a cytosolic protein found in all mammalian tissues analyzed to date, binds to mammalian ribonucleases with extraordinary affinity. The RI·ANG complex ( Figure 1A) is among the tightest of known protein-protein interactions with K d = † This work was supported in part by Grants CA073808 (NIH) and M10749000231-08N4900-23110 (Korea Science and Engineering Foundation A known role for RI is to protect cellular RNA from invading ribonucleases (19,20). ANG, however, possesses <1% of the ribonucleolytic activity of its homologues with cytotoxic activity (21). Moreover, the IC 50 values for cytotoxic ribonucleases are ≥10 2 -fold greater than the concentration of ANG required to induce endothelial cell proliferation in vitro. Thus, a major role for RI as an antagonist of the cytotoxic activity of ANG is unlikely.Does RI play a role in ANG-induced angiogenesis? The exogenous addition of extracellular RI is known to antagonize angiogenesis (22,23). That experiment, however, puts RI in a nonnative location. We sought to determine whether endogenous, intracellular RI regulates ANG-induced neovascularization. We reasoned that we could do so by using a variant of ANG that evades RI.To disrupt its interaction with RI, we introduce...