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. 2007 Sep 11;46(36):10308-16.
doi: 10.1021/bi700857u. Epub 2007 Aug 18.

Cytotoxic ribonucleases: the dichotomy of Coulombic forces

R Jeremy Johnson  1 , Tzu-Yuan Chao, Luke D Lavis, Ronald T Raines
Affiliations

Cytotoxic ribonucleases: the dichotomy of Coulombic forces

R Jeremy Johnson et al. Biochemistry. .

Abstract

Cells tightly regulate their contents. Still, nonspecific Coulombic interactions between cationic molecules and anionic membrane components can lead to adventitious endocytosis. Here, we characterize this process in a natural system. To do so, we create variants of human pancreatic ribonuclease (RNase 1) that differ in net molecular charge. By conjugating a small-molecule latent fluorophore to these variants and using flow cytometry, we are able to determine the kinetic mechanism for RNase 1 internalization into live human cells. We find that internalization increases with solution concentration and is not saturable. Internalization also increases with time to a steady-state level, which varies linearly with molecular charge. In contrast, the rate constant for internalization (t1/2 = 2 h) is independent of charge. We conclude that internalization involves an extracellular equilibrium complex between the cationic proteins and abundant anionic cell-surface molecules, followed by rate-limiting internalization. The enhanced internalization of more cationic variants of RNase 1 is, however, countered by their increased affinity for the cytosolic ribonuclease inhibitor protein, which is anionic. Thus, Coulombic forces mediate extracellular and intracellular equilibria in a dichotomous manner that both endangers cells and defends them from the potentially lethal enzymatic activity of ribonucleases.

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Figures

Figure 1
Figure 1
Design of ribonuclease variants and latent fluorophore. (A) Ribbon diagram of RNase 1 (PDB entry 1Z7X, chain Z (40)). Residues substituted herein are depicted in red. The image was created with the program PyMOL (DeLano Scientific, South San Francisco, CA). (B) Structure of latent fluorophore before and after activation by an intracellular esterase. (C,D) Confocal microscopy images of unwashed K-562 cells incubated at 37 °C for 30 min with fluorescein-labeled RNase 1 (10 μM; C) or latent fluorophore-labeled RNase 1 (10 μM; D). Nuclei were stained by adding Hoechst 33342 (2 μg/mL) during the final 5 min of incubation. Insets: bright-field images. Scale bar: 10 μm.
Figure 2
Figure 2
Cytotoxicity of ribonuclease variants. Effect of ribonucleases on the proliferation of K-562 cells was determined by monitoring the incorporation of [methyl-3H]thymidine into cellular DNA in the presence of ribonucleases. Data points are mean values (±SE) from ≥3 experiments, each carried out in triplicate, and were fitted to eq 3. Variants in order of increasing cytotoxicity: D38R/R39D/N67R/G88R RNase A (◇); DRRDD RNase 1 (◆); DRRRD RNase 1 (▼); DDADD RNase 1 (•); LRRDD RNase 1 (▲); LRRRD RNase 1 (■); and wild-type RNase 1 (○).
Figure 3
Figure 3
Kinetics of ribonuclease internalization. (A) Initial velocity of cellular internalization versus time (≤2 h). Internalization was determined by incubating latent fluorophore-labeled RNase 1 (○) with K-562 cells at 37 °C. Incubations were quenched at known times by immersing the K-562 cells in ice-cold PBS and storing them on ice before quantitation by flow cytometry. Data points are mean values (±SE) from ≥3 cell populations. (B) Confocal microscopy images of unwashed K-562 cells incubated at 37 °C for varying time periods (0–6 h) with latent fluorophore-labeled RNase 1 (10 μM). Nuclei were stained by adding Hoechst 33342 (2 μg/mL) during the final 5 min of incubation. Scale bar: 10 μm.
Figure 4
Figure 4
Properties of RNase 1, its variants, and RNase A: wild-type RNase 1 (○), DDADD RNase 1 (•), DRRDD RNase 1 (◆), LRRDD RNase 1 (▲), LLALL RNase 1 (□), DRRRD RNase 1 (▼), LRRRD RNase 1 (■), and wild-type RNase A (◇). (A) Plot of internalization of a ribonuclease into K-562 cells versus time. Internalization was measured by using flow cytometry and following the fluorescence manifested by activation of a latent fluorophore attached to the ribonuclease (10 μM) after incubation for the specified times points. Data points are mean values (±SE) for 20,000 cells from ≥3 cell populations, and were fitted to eq 4. (B) Plot of internalization of a ribonuclease into K-562 cells versus its net charge. Internalization was monitored as in panel A, except that all incubations were for 30 min at 37 °C. (C) Plot of internalization of a ribonuclease into K-562 cells versus its concentration. Internalization was followed as in panel A, except for the variable concentration of ribonuclease (0.1, 1.0, or 10 μM). (D) Semilog plot of affinity of a ribonuclease for RI versus its net charge. Data points are mean values (±SE). Variants of RNase 1 from ref are indicated by an asterisk and their amino acids at positions 39, 67, 88, 89, and 91, with “–” indicating the wild-type residue.
Figure 5
Figure 5
Coulombic effects on ribonuclease-mediated cytotoxicity. Cationic and anionic molecules are depicted in blue and red, respectively. (A) Ribonucleases form an extracellular equilibrium complex with abundant anionic cell-surface molecules, such as heparan sulfate. Bound ribonucleases are internalized into endosomes with rate constant kI. (B) Internalized ribonucleases translocate to the cytosol by an unknown mechanism. (C) In the cytosol, ribonucleases form an intracellular equilibrium complex with RI. (D) Ribonucleases that evade RI degrade cellular RNA, leading to apoptosis.
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