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* This work was supported in part by National Institutes of Health Grant CA73808. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Supported by a Pfizer Summer Undergraduate Research Fellowship, Wisconsin/Hilldale Undergraduate/Faculty Research Award, and Trewartha Undergraduate Honors Research Grant. ¶ Supported by Molecular Biophysics Training Grant T32 GM08293 from the National Institutes of Health.
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 Gly88 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 Arg10 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.
Several studies have focused on understanding the contribution of ribonucleolytic activity and affinity for RI to cytotoxicity. RNase A itself does not have marked antitumor activity, but variants of RNase A are toxic to cancer cells. For example, substituting the glycine residue at position 88 with arginine decreases the affinity for RI and endows RNase A with cytotoxic activity (
). K41R/G88R RNase A has enhanced toxicity to K-562 cells as compared with G88R RNase A.
Some endosomal pathways end in the lysosomal degradation of proteins. The KFERQ pentapeptide sequence targets cytosolic proteins for lysosomal degradation via an alternative pathway (for reviews, see Refs.
) found that microinjected RNase A associates with lysosomes upon cellular fractionation. Subsequent studies found that the KFERQ pentapeptide, which comprises residues 7–11 of RNase A, regulates lysosomal degradation (
). ONC does not contain a KFERQ sequence (Fig. 2). The significance of KFERQ-targeted lysosomal decay in ribonuclease-mediated cytotoxicity is unknown. This sequence, along with RI, could serve to protect cells against an invading ribonuclease.
Here, we determine the effect of the KFERQ sequence on G88R RNase A-mediated cytotoxicity. Replacing Lys7 of the KFERQ sequence with an alanine residue has little effect on the conformational stability or catalytic activity of G88R RNase A. K7A/G88R RNase A does, however, have a marked decrease in affinity for RI compared with G88R RNase A and is the most cytotoxic variant of RNase A reported to date. Using other RNase A variants with substitutions in the KFERQ sequence that do not disrupt RI binding, we find that targeted lysosomal degradation via the KFERQ sequence does not modulate ribonuclease toxicity. Moreover, the toxicity of ribonucleases is not diminished in serum-deprived cells, which have enhanced KFERQ-mediated lysosomal degradation (
K-562 cells, which derive from a continuous human chronic myelogenous leukemia line, were from the American Type Culture Collection (Manassas, VA). Cell culture medium and supplements were from Invitrogen.
) were prepared as described. Enzymes used for DNA manipulation were from Promega (Madison, WI) or New England Biolabs (Beverly, MA).
[methyl-3H]Thymidine was from PerkinElmer Life Sciences (Boston, MA). 6-Carboxyfluorescein∼dArU(dA)2∼6-carboxytetramethylrhodamine (6-FAM∼dArU(dA)2∼6-TAMRA) was from Integrated DNA Technologies (Coralville, IA). Yeast rRNA (16 S and 23 S) was from Roche Molecular Biochemicals. All other chemicals were of commercial reagent grade or better and were used without further purification.
Ultraviolet and visible absorption was measured with a Cary model 50 spectrophotometer from Varian (Sugar Land, TX). Fluorescence was measured with a QuantaMaster1 photon-counting spectrofluorometer from Photon Technology International (South Brunswick, NJ), using fluorescence-grade quartz or glass cuvettes (1.0-cm path length, 3.0-ml volume) from Starna Cells (Atascadero, CA). Radioactivity was measured with a Beckman model LS 3801 liquid scintillation counter from Beckman Instruments (Fullerton, CA).
). The K7A/G88R and R10A/G88R substitutions within RNase A were created by ligating DNA fragments using the ApaI and MunI restriction sites. The K7A/K41R/G88R RNase A variant was created by ligating the DNA fragments using the ApaI and ClaI restriction sites.
Wild-type RNase A and its variants were purified by using methods described previously (
), but with the following modifications. Refolding solutions for RNase A variants with the G88R substitution contained 0.50 marginine, instead of 0.10 m NaCl. Protein concentrations were determined by UV spectroscopy using ε = 0.72 ml mg−1 cm−1 at 277.5 nm for RNase A (
). All ribonucleases used in biological assays were dialyzed exhaustively versus phosphate-buffered saline (PBS), which contained in 1.00 liter: KCl (0.20 g), KH2PO4 (0.20 g), NaCl (8.0 g), and Na2HPO4 (2.16 g).
Assay of Conformational Stability
The conformational stability of RNase A variants was determined by monitoring the absorbance at 287 nm (A287) with increasing temperature (
). The temperature of solution of protein (0.3 mg/ml) in PBS was increased from 25 to 75 °C in 1 °C increments. The A287 was recorded after a 7-min equilibration at each temperature. The value of Tm is the temperature at the midpoint of the thermal denaturation. Data were collected and analyzed with the program THERMAL (Varian Analytical Instruments; Walnut Creek, CA).
Assay of Catalytic Activity
Ribonucleolytic activity was measured by using a fluorogenic substrate (
). Assays were performed at 23 °C in 2.00 ml of 0.10 m MES-NaOH buffer (pH 6.0) containing NaCl (0.10 m). Solutions contained 6-FAM∼dArU(dA)2∼6-TAMRA (50 nm) and enzyme (1.0–5.0 pm). Fluorescence was monitored by using 493 and 515 nm for the excitation and emission wavelengths, respectively. Kinetic parameters were determined by a linear least-squares regression analysis of the initial velocity using Equation 1 (
where V/K is the first-order rate constant, ΔF/Δt is the slope from the linear regression, Fmax is the final fluorescence intensity after the reaction has reached completion, and F0 is the initial fluorescence intensity before enzyme is added. The value of kcat/Km was calculated by dividing V/K by the enzyme concentration.
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. It has been shown that the fluorescence of fluorescein-labeled A19C/G88R RNase A (fluorescein∼G88R RNase A) decreases by ∼15% upon binding to RI.
R. A. Abel, M. C. Haigis, C. Park, and R. T. Raines (2002), submitted manuscript.
Thus, fluorescence spectroscopy can be used to evaluate the ability of an unlabeled ribonuclease to compete with fluorescein∼G88R RNase A for binding to RI. Specifically, cuvettes of PBS containing fluorescein∼G88R RNase A (50 nm), an unlabeled RNase A variant (1 nm–2 μm), and dithiothreitol (1 mm) were incubated at room temperature (23 ± 2 °C). After 15 min, the initial fluorescence intensity was measured with 490 and 511 nm for the excitation and emission wavelengths, respectively. Next, RI was added with stirring (to 50 nm), and the average fluorescence intensity was measured after an additional incubation of 4 min. The maximum fluorescence decrease upon RI binding was measured with samples that lacked unlabeled ribonuclease. The concentration of the RI·fluorescein∼G88R RNase A complex was determined by comparing the fluorescence of a sample with the fluorescence decrease observed when all of the fluorescein∼G88R RNase A was bound by RI. The Kd value was determined as described.2
Gel Assay of Ribonuclease Inhibitor Binding
The effect of RI binding on catalytic activity was monitored directly, but qualitatively, by an agarose gel-based assay as described previously (
). Briefly, 0.6-ml siliconized microcentrifuge tubes of 0.10m MES-NaOH buffer (pH 6.0) containing NaCl (0.10m), dithiothreitol (1 mm), yeast rRNA (4 μg), 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%). After a 15-min incubation at 37 °C, 10 mm Tris-HCl buffer (pH 7.5) containing EDTA (50 mm), glycerol (30%, w/v), xylene cyanol FF (0.25%, w/v), and bromphenol blue (0.25%, w/v) was added. Samples were analyzed by electrophoresis through an agarose gel (1%, w/v) containing ethidium bromide (0.4 μg/ml). Control samples were incubated in the absence of a ribonuclease or RI (or both).
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. K-562 cells were grown in RPMI 1640 medium. Unless indicated otherwise, all culture medium contained fetal bovine serum (10%, v/v), penicillin (100 units/ml), and streptomycin (100 μg/ml). Cells were cultured at 37 °C in a humidified incubator containing CO2 (g; 5%, v/v). All toxicity studies were performed using asynchronous log-phase cultures. For toxicity assays, cells (95 μl of a solution of 5 × 104 cells/μl) were incubated with a 5-μl solution of a ribonuclease or PBS in the wells of a 96-well plate. Cells were incubated for 44 h at 37 °C in a humidified incubator containing CO2 (g; 5%, v/v). Next, the proliferation of cells was monitored with a 4-h pulse of [methyl-3H]thymidine (0.4 μCi/well). Cells were harvested onto glass fiber filters using a PHD Cell Harvester (Cambridge Technology; Watertown, MA). Filters were washed with water and dried with methanol, and their 3H content was measured with liquid scintillation counting.
Serum-deprived cells have enhanced KFERQ-mediated lysosomal degradation of RNase A (
). To discern the effect of RNase A, its variants, and ONC on the proliferation of cells with enhanced KFERQ-mediated degradation, K-562 cells were grown in RPMI medium without fetal bovine serum for 18 h prior to the addition of a ribonuclease. Ribonuclease-mediated cytotoxicity was measured as described above.
Cytotoxicity data were analyzed with the programs SIGMAPLOT (SPSS Science; Chicago, IL) and DELTAGRAPH (DeltaPoint; Monterey, CA). Each data point represents the mean (± S.E.) of at least three experiments, each performed in triplicate. The IC50 value of each variant was calculated by using Equation 2,
where S is the percent of total DNA synthesis after the incubation period (48 h).
Design of Ribonuclease A Variants
RNase A variants were designed with the primary goal of discerning a role for the KFERQ sequence (residues 7–11) in cytotoxicity. Because RNase A itself is not cytotoxic, the cytotoxic G88R RNase A variant was used as a basis for this work. In addition, amino acid substitutions were combined with the secondary goal of producing variants that have high ribonucleolytic activity, low affinity for RI, and thus (presumably) high cytotoxicity.
Lys7 and Arg10
Lys7 and Arg10 comprise the enzymic P2 subsite, which interacts with a phosphoryl group of an RNA substrate (
) found that replacing either Lys7 or Arg10 alone with a glutamine residue has only a minor effect on catalysis of RNA cleavage, but that replacing both Lys7 and Arg10 decreases catalytic activity by 60-fold. Likewise, replacing both Lys7 and Arg10 with alanine residues results in a kcat/Km value that is 60-fold lower than that of wild-type RNase A (
) found that replacing Lys7 with an S-methyl cysteine residue resulted in a >50-fold decrease in affinity for RI. This result is consistent with the structure of the RI·RNase A complex in which the side-chain nitrogen of Lys7 donates a hydrogen bond to the C-terminal carboxyl group of RI (Fig. 1) (
Accordingly, we replaced Lys7 and Arg10 of RNase A independently with an alanine residue. The resulting K7A and R10A variants are designed to disrupt the KFERQ sequence, without decreasing ribonucleolytic activity. By systematically incorporating these changes in a cytotoxic variant, G88R RNase A, we were able to investigate the contribution of Lys7 and Arg10to cytotoxicity.
Phe8, Glu9, and Gln11
Phe8, Glu9, and Gln11 are important to the structure and function of RNase A. Replacing Gln11 with an alanine, glutamine, or histidine residue enables the enzyme to bind a substrate in a nonproductive manner (
) with other residues decreases its conformational stability or catalytic activity (or both). Because these three residues of the KFERQ sequence play roles other than in lysosomal degradation, we left them intact.
The side chain of Lys41 of RNase A donates a hydrogen bond to the transition state during catalysis of RNA cleavage (
). Changing Lys41to an arginine residue results in a 102-fold decrease in catalytic activity. Although K41R/G88R RNase A has low catalytic activity, it binds RI with less affinity than does G88R RNase A. Moreover, K41R/G88R RNase A is more cytotoxic than G88R RNase A (
). Hence, we used the K7A/K41R/G88R RNase A variant to explore the additivity of single substitutions that disrupt RI binding, as well as the relationship between catalytic activity, RI affinity, and cytotoxicity.
The conformational stability of the RNase A variants was measured to ensure that the proteins were folded properly during all assays. Values of Tm for RNase A, its variants, and ONC are listed in Table I. The Tm values of both wild-type RNase A and G88R RNase A in PBS were determined to be 63 °C, respectively; this value is similar to those reported previously (
). K7A RNase A, K7A/G88R RNase A, R10A RNase A, and R10A/G88R RNase A were found to have Tm values of 63, 62, 60, and 62 °C, respectively. K41R/G88R RNase A and K7A/K41R/G88R RNase A were both found to have a Tm value of 63 °C. Hence, all RNase A variants were essentially completely folded during assays at 37 °C or room temperature. The Tm value of ONC in PBS was reported previously to be 90 °C (
). Ribonucleolytic activity was measured by using a fluorogenic substrate, 6-FAM∼dArU(dA)2∼6-TAMRA, which exhibits a nearly 200-fold increase in fluorescence upon cleavage of the P–O5′ bond on the 3′ side of the single ribonucleotide-embedded residue (
). Values of kcat/Km for RNase A, its variants, and ONC are listed in Table I. The values of kcat/Km for RNase A and ONC were 4.3 × 107 and 3.5 × 102m−1 s−1, respectively, which is in good agreement with those reported previously (
). The values of kcat/Km for K7A RNase A and R10A RNase A were 9.2 × 106 and 8.2 × 106m−1 s−1, respectively, which are lower by 4–5-fold than those of the wild-type enzyme. These values are not reduced by the G88R substitution; the kcat/Km values for K7A/G88R RNase A and R10A/G88R RNase A were 8.8 × 106 and 1.2 × 107m−1s−1, respectively. As expected, the K41R variants have greatly diminished catalytic activity. The kcat/Km value of 4.1 × 105m−1 s−1 for K41R/G88R RNase A is in good agreement with the activity reported previously (
), 0.57 nm,2 and 0.55 nm,2 respectively. Here, the Kd values for the complex between RI and other RNase A variants were measured by using a competition assay (Table I). The K7A substitution disturbs the interaction of RI and RNase A, giving Kd values of 0.07 and 7.2 nm for K7A RNase A and K7A/G88R RNase A, respectively. In contrast, the R10A substitution has no significant effect on the affinity for RI, as the value of Kd for the RI·R10A/G88R RNase A complex is indistinguishable from that of the RI·G88R RNase A complex. The Kd value for the RI·K41R/G88R RNase A complex is 2.9 nm, which is comparable with the value of Ki = 3.0 nm reported previously (
). Addition of the K41R substitution to K7A/G88R RNase A decreases the affinity for RI by 5-fold, giving a Kd value of 47 nm for the triple variant. These Kd values were used to calculate the change in the free energy of interaction for RI and the RNase A variants. These values of ΔΔG are listed in Table I.
An agarose gel-based RI-evasion assay was used to verify that affinity for RI correlates with inhibition of catalytic activity. In this assay, ribonucleases were incubated with increasing amounts of RI in the presence of yeast rRNA as substrate. The samples were subjected to electrophoresis through an agarose gel, and degraded RNA was observed by its faster mobility and less efficient staining compared with control samples without ribonuclease. We find that K7A/G88R RNase A degrades yeast rRNA more thoroughly in the presence of RI than does either G88R RNase A or K7A RNase A (Fig. 3).
The toxicity of each ribonuclease was measured with the K-562 human leukemia cell line. The resulting IC50 values are listed in Table I. ONC, G88R RNase A, and K41R/G88R RNase A had IC50 values similar to those reported previously (
). Like wild-type RNase A, K7A RNase A and R10A RNase A were not cytotoxic, even at protein concentrations of 25 μm. The cytotoxicity of R10A/G88R RNase A did not differ from that of G88R RNase A. In contrast, the IC50 value of K7A/G88R RNase A was 7-fold lower than that of G88R RNase A (Fig. 4A). The IC50value of K7A/G88R RNase A was within 2-fold of that of ONC. Unexpectedly, the triple variant, K7A/K41R/G88R RNase A had an IC50 value that was similar to that of G88R RNase A, and 2- and 12-fold greater than that of K41R/G88R RNase A and K7A/G88R RNase A, respectively.
To test further whether KFERQ-mediated degradation contributes to differences between the cytotoxicity of ONC and RNase A variants, assays were performed with serum-deprived K-562 cells. The IC50 values of ONC, K7A/G88R RNase A, and G88R RNase A did not change significantly in serum-deprived cells as compared with control cells cultured with fetal bovine serum (Fig. 4B; Table II).
Table IIToxicity of ribonuclease A, its variants, and Onconase® for serum-deprived cells
). The ribonucleolytic activity within the cytosol is regulated by two factors: (i) the concentration of enzyme within the cytosol, and (ii) how much of that enzyme is bound by RI. The cytosolic concentration of a ribonuclease is dependent on the balance between its import and degradation. The ability of a ribonuclease to reach the cytosol is known to limit its toxicity. For example, even wild-type RNase A is toxic to cells when injected directly into the cytosol (
M. C. Haigis and R. T. Raines, unpublished results.
The contribution of protein degradation to ribonuclease cytotoxicity has been less studied, but its importance can be inferred. For example, ONC is more cytotoxic in the presence of protease inhibitors (
The sequence-specific, lysosome-targeted degradation of cytosolic proteins can also lower the cellular concentration of a protein. RNase A, unlike ONC, contains a KFERQ sequence (Fig. 2). This sequence is required for the targeted lysosomal degradation of cytosolic RNase A (
), the biochemical nature of the side chains must be conserved. Thus, replacing residues of the KFERQ sequence could enhance the cytotoxicity of an RNase A variant.
Arg10 of RNase A is located in the KFERQ sequence, but does not form any interaction with RI. Hence, we used the R10A variant to isolate the consequence of lysosomal degradation from RI evasion. R10A RNase A is not toxic to cells. In addition, R10A/G88R RNase A has an IC50 value similar to that of G88R RNase A. We also investigated the toxicity of a G88R RNase A variant with Gln11 replaced by a histidine or alanine residue. The cytotoxicity of the Q11H/G88R and Q11A/G88R variants does not differ from that of G88R RNase A (data not shown). Hence, disrupting the KFERQ sequence has no effect on ribonuclease-mediated cytotoxicity.
We measured the cytotoxicity of ribonucleases in cells with up-regulated lysosomal degradation. This experiment was based on the hypothesis that if KFERQ-mediated degradation limits the concentration of cytosolic ribonuclease, then enhancing this pathway would result in decreased toxicity. Cells cultured in the absence of serum show enhanced degradation of cytosolic RNase A (
). The data demonstrate that toxic variants of RNase A do not have lowered potency in serum-deprived cells (Fig. 4B and Table II). These results indicate that the KFERQ-mediated degradation of cytosolic ribonucleases does not limit their potency.
RI binds to members of the RNase A superfamily in a 1:1 stoichiometry (
), which corresponds to Ser460 of human RI. The distance between the side chain nitrogen of Lys7 and the side-chain oxygen of Ser456 is 3.1 Å. The side-chain nitrogen is 3.5 and 4.1 Å away from the two oxygens of the C-terminal carboxyl group of RI. Replacing Lys7 with an alanine residue removes any hydrogen bonds and favorable Coulombic interactions with Ser456 of RI. The value of Kd for the RI·K7A RNase A complex is 70 pm (Table I). The cooresponding value of Kd for the double variant, K7A/G88R RNase A, is 7.2 nm. Moreover, K7A/G88R RNase A is endowed with enhanced cytotoxicity.
Surprisingly, we find that the interactions of Lys7, Lys41, and Gly88 with RI are not additive. Single substitutions at Lys7 or Gly88 result in decreases of binding free energy of 4.1 or 5.3 kcal/mol, respectively (Table I). The double variants, K7A/G88R RNase A and K41R/G88R RNase A, have lost 6.9 and 6.3 kcal/mol of binding free energy, respectively. Yet, the triple variant, K7A/K41R/G88R RNase A, has lost only 8.0 kcal/mol of binding free energy. If the interactions had been additive, then the effect of single substitutions would contribute fully to the loss of binding free energy. Such conservation in binding free energy loss would suggest rigidity between the interface of the complex. For example, the effect of single substitutions at Lys
). In contrast, our data show that the interface between RI and RNase A is not rigid. Rather, compensatory changes occur upon perturbation of key contacts. A similar conclusion was reached by Shapiro et al. (
) who measured the affinity of RI variants for wild-type RNase A. Thus, the dynamic nature of the RI-RNase A interface must be addressed when engineering new ribonuclease variants to evade RI.
In conclusion, we have shown that the KFERQ sequence does not contribute to a decrease in ribonuclease-mediated cytotoxicity. We find that Lys7 of RNase A contributes to a key interaction that tethers the N terminus of RNase A with the C terminus of RI. The K7A/G88R RNase A variant has >105-fold lower affinity for RI than does wild-type RNase A. The ribonucleolytic activity of K7A/G88R RNase A is, however, within 10-fold of that of the wild-type enzyme. Together, its high ribonucleolytic activity and low affinity for RI make K7A/G88R RNase A the most cytotoxic known variant of RNase A, with an IC50 value within 2-fold of that of ONC. Finally, we have found that the RI-RNase A interface is dynamic; disruption of one contact can alter other contacts.
We thank K. J. Woycechowsky, K. A. Dickson, and Dr. B. G. Miller for contributive discussions.