Leland P A, Schultz L W, Kim B M, Raines R T
Departments of Biochemistry and Chemistry, University of Wisconsin, Madison, WI 53706, USA.
Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10407-12. doi: 10.1073/pnas.95.18.10407.
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.
牛胰核糖核酸酶A（RNase A）超家族的某些成员是强效细胞毒素。这些具有细胞毒性的核糖核酸酶进入细胞质，在那里它们降解细胞RNA并导致细胞死亡。核糖核酸酶抑制剂（RI）是一种细胞质蛋白，它与RNase A超家族的成员结合，抑制常数跨度达10个数量级。在此，我们表明核糖核酸酶对RI的亲和力在定义细胞毒性核糖核酸酶的效力中起着不可或缺的作用。RNase A没有细胞毒性，并且与RI高亲和力结合。癌酶是一种具有细胞毒性的RNase A同源物，与RI低亲和力结合。为了破坏RI-RNase A的相互作用，将与RI形成多个接触点的三个RNase A残基（Asp-38、Gly-88和Ala-109）替换为精氨酸。用精氨酸残基替换Asp-38和Ala-109对RI-RNase相互作用没有影响。此外，这些变体没有细胞毒性。相反，用精氨酸残基替换Gly-88产生一种核糖核酸酶（G88R RNase A），它在RI存在下保留催化活性，并且对转化细胞系具有细胞毒性。用天冬氨酸替换Gly-88也产生一种对RI亲和力降低且具有细胞毒性活性的核糖核酸酶（G88D RNase A）。癌酶、G88R RNase A和G88D RNase A的细胞毒性效力与逃避RI相关。我们得出结论，在RI存在下保留催化活性的核糖核酸酶是细胞毒素。这一发现预示着一类基于胰腺核糖核酸酶的化疗药物的开发。
