KFERQ Sequence in Ribonuclease A-mediated Cytotoxicity

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.

The ribonuclease/angiogenin inhibitor is also present in mitochondria and nuclei

“…It has been proposed that the role of RI in the cell is that of a ''sentry'', to protect its cytosolic RNA from extracellular RNases that may enter that compartment [5]. This hypothesis is based on the following evidence: RI resistant RNases, such as bovine seminal RNase [6] and onconase [7], are cytotoxic; noncytotoxic RNases, strongly inhibited by RI, become cytotoxic when they are engineered into relatively RI-resistant RNases [8]; cells manipulated to increase the RI levels become more resistant to RNase cytotoxicity [5,8,9].…”

X-ray Structure of Two Crystalline Forms of aStreptomycete Ribonuclease with Cytotoxic Activity

“…Data represent the average of quadruplicate samples within an individual assay. The IC 50 value of each cytotoxic ribonuclease was calculated by using the equation: S ϭ 100 ϫ IC 50 /(IC 50 ϩ [ribonuclease]) where S is the percent of total DNA synthesis after the incubation period (24). 34 is from a molecule related by symmetry.…”

“…The cytosol of mammalian cells contains a ribonuclease inhibitor (RI) 1 protein that binds tightly to mammalian secretory ribonucleases (19,20). The ability of secretory ribonucleases from human (21), cow (22)(23)(24), bull (25)(26)(27), and frog (28) to evade RI endows them with toxicity for mammalian cells. These secretory ribonucleases from vertebrates are not homologous to microbial ribonucleases.…”

Secretory ribonucleases are internalized by a dynamin-independent endocytic pathway

“…A preincubation of metabolic inhibitors protects K-562 cells from the toxicity of ONC and K7A/G88R RNase A (Fig. 3A), which is the most toxic known variant of RNase A (Haigis et al, 2002). In addition, metabolic inhibitors block the internalization of RNase A (Fig.…”

“…Briefly, cells (95 µl of a solution of 5×10 4 cells/ml) were incubated with PBS containing a known concentration of a ribonuclease (5 µl) in a 96-well plate for 20 hours at 37°C in a humidified incubator containing CO2 (g; 5% v/v). Data on cell proliferation were obtained and analyzed as described previously (Haigis et al, 2002).…”