KFERQ Sequence in Ribonuclease A-mediated Cytotoxicity

Marcia C Haigis,Erin L Kurten,Richele L Abel,Ronald T Raines

doi:10.1074/jbc.m112227200

Marcia C Haigis, Erin L Kurten + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m112227200

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Abstract

Onconase(ONC) is an amphibian ribonuclease that is in clinical trials as a cancer chemotherapeutic agent. ONC is a homolog of ribonuclease A (RNase A). RNase A can be made toxic to cancer cells by replacing Gly(88) with an arginine residue, thereby enabling the enzyme to evade the endogenous cytosolic ribonuclease inhibitor protein (RI). Unlike ONC, RNase A contains a KFERQ sequence (residues 7-11), which signals for lysosomal degradation. Here, substitution of Arg(10) of the KFERQ sequence has no effect on either the cytotoxicity of G88R RNase A or its affinity for RI. In contrast, K7A/G88R RNase A is nearly 10-fold more cytotoxic than G88R RNase A and has more than 10-fold less affinity for RI. Up-regulation of the KFERQ-mediated lysosomal degradation pathway has no effect on the cytotoxicity of these ribonucleases. Thus, KFERQ-mediated degradation does not limit the cytotoxicity of RNase A variants. Moreover, only two amino acid substitutions (K7A and G88R) are shown to endow RNase A with cytotoxic activity that is nearly equal to that of ONC.

Highlights

Onconase® (ONC)1 is a homolog of ribonuclease A (RNase A; EC 3.1.27.5) from the Northern leopard frog, Rana pipiens [1, 2]
Using other RNase A variants with substitutions in the KFERQ sequence that do not disrupt ribonuclease inhibitor protein (RI) binding, we find that targeted lysosomal degradation via the KFERQ sequence does not modulate ribonuclease toxicity
Design of Ribonuclease A Variants—RNase A variants were designed with the primary goal of discerning a role for the KFERQ sequence in cytotoxicity

Summary

EXPERIMENTAL PROCEDURES

Materials—K-562 cells, which derive from a continuous human chronic myelogenous leukemia line, were from the American Type Culture Collection (Manassas, VA). Fluorescence Assay of Ribonuclease Inhibitor Binding—The value of Kd for the complex between porcine RI and RNase A variants was determined by using a competitive binding assay. Cuvettes of PBS containing fluoresceinϳG88R RNase A (50 nM), an unlabeled RNase A variant (1 nM–2 ␮M), and dithiothreitol (1 mM) were incubated at room temperature (23 Ϯ 2 °C). 0.6-ml siliconized microcentrifuge tubes of 0.10 M MES-NaOH buffer (pH 6.0) containing NaCl (0.10 M), dithiothreitol (1 mM), yeast rRNA (4 ␮g), and a ribonuclease (10 ng) were mixed with RI (0, 10, 20, or 40 units, where 1 unit is the amount required to inhibit the activity of 5 ng of RNase A by 50%). Assay of Cytotoxicity—The effect of RNase A, its variants, and ONC on cell proliferation was determined by measuring the incorporation of [methyl-3H]thymidine into cellular DNA.

RNase A

RESULTS

DISCUSSION