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. 2011 Oct 4;50(39):8374-82.
doi: 10.1021/bi2009079. Epub 2011 Sep 7.

Mechanism of Ribonuclease A Endocytosis: Analogies to Cell-Penetrating Peptides

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Free PMC article

Mechanism of Ribonuclease A Endocytosis: Analogies to Cell-Penetrating Peptides

Tzu-Yuan Chao et al. Biochemistry. .
Free PMC article

Abstract

Pancreatic-type ribonucleases can exert toxic activity by catalyzing the degradation of cellular RNA. Their ability to enter cells is essential for their cytotoxicity. Here, we determine the mechanism by which bovine pancreatic ribonuclease (RNase A) enters human cells. Inhibiting clathrin-dependent endocytosis with dynasore or chlorpromazine decreases RNase A-uptake by ~70%. Limited colocalization between RNase A and transferrin indicates that RNase A is not routed through recycling endosomes. Instead, vesicular staining of RNase A overlaps substantially with that of nona-arginine and the cationic peptide corresponding to residues 47-57 of the HIV-1 TAT protein. At low concentrations (<5 μM), internalization of RNase A and these cell-penetrating peptides (CPPs) is inhibited by chlorpromazine as well as the macropinocytosis inhibitors cytochalasin D and 5-(N-ethyl-N-isopropyl)amiloride to a similar extent, indicative of common endocytic mechanism. At high concentrations, CPPs adopt a nonendocytic mechanism of cellular entry that is not shared by RNase A. Collectively, these data suggest that RNase A is internalized via a multipathway mechanism that involves both clathrin-coated vesicles and macropinosomes. The parallel between the uptake of RNase A and CPPs validates reference to RNase A as a "cell-penetrating protein".

Figures

Figure 1
Figure 1
Effect of endocytic inhibitors on RNase A-uptake. HeLa cells at 37 °C were pretreated with endocytic inhibitors for 30 min, and then incubated with fluorogenic RNase A for 1 h in the presence of the inhibitors. (A) Confocal microscopy. (i) Untreated cells; (ii) dynasore at 80 μM; (iii) CPZ at 10 μg/mL; (iv) nystatin at 25 μg/mL; (v) MβCD at 5 mM; (vi) CD at 5 μg/mL; (vii) EIPA at 10 μM; (viii) wortmannin at 50 nM. Scale bars: 10 μm. (B) Flow cytometry. Data are mean values (±SE) of total fluorescence for 20,000 washed cells from ≥6 cell populations.
Figure 2
Figure 2
Colocalization of RNase A with endocytic markers. Confocal microscopy of HeLa cells incubated with fluorogenic RNase A (27) (10 μM) and (i) Alexa Flour 594–transferrin (1 μM), (ii) Alexa Flour 594–CTB (1 μg/mL), (iii) 10 μM TAMRA–R9 (10 μM), or (iv) TAMRA–TAT (10 μM) for 1 h at 37 °C, and washed. Incubations with CPPs followed a 15-min pretreatment with RNase A. Scale bars: 10 μm.
Figure 3
Figure 3
Concentration-dependence of RNase A- and CPP-uptake. (A) Confocal microscopy. HeLa cells were incubated with fluorescein–RNase A (i and iv), FAM–R9 (ii and v), and FAM–TAT (iii and vi) for 1 h at 37 °C, and washed. Scale bars: 10 μm. (B) Flow cytometry. Data points are mean values (±SE) of total fluorescence for 20,000 washed cells from 3 cell populations.
Figure 4
Figure 4
Comparative effect of endocytic inhibitors on RNase A- and CPP-uptake HeLa cells at 37 °C were pretreated with CD, CPZ, or EIPA for 30 min, and then incubated with fluorogenic RNase A, FAM–R9, or FAM–TAT for 1 h in the presence of the endocytic inhibitors. Data are mean values (±SE) of total fluorescence for 20,000 washed cells from ≥6 cell populations.
Figure 5
Figure 5
Portals of entry into a mammalian cell. RNase A and CPPs enter cells in a similar manner as discerned, in part, by the blocking of portals with the indicated drugs. Image was adapted from ref. .
Figure 6
Figure 6
Cationic residues of RNase A. Lysine residues (10) are light blue; arginine residues (4) are dark blue. A large cationic patch consists of Lys1, Lys7, Arg10, Lys37, Arg39, and Lys41. A small cationic patch consists of Lys66, Arg85, Lys98, and Lys104.

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