title: Cytotoxic ribonucleases: the dichotomy of Coulombic forces.
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.
authors: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706-1544, USA.
authors: R Jeremy
authors: RJ
authors: Johnson
authors: Tzu-Yuan
authors: TY
authors: Chao
authors: Luke D
authors: LD
authors: Lavis
authors: Ronald T
authors: RT
authors: Raines
mesh_terms: D001696:Biomechanical Phenomena; D016923:Cell Death; D004705:Endocytosis; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D020014:K562 Cells; D007700:Kinetics; D050505:Mutant Proteins / Q000737:chemistry; D017433:Protein Structure, Secondary; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D050505:Mutant Proteins; D012259:Ribonuclease, Pancreatic
references: Biochemistry. 2001 Jun 26;40(25):7518-24
references: 11412105
references: J Biol Chem. 2003 Aug 15;278(33):31192-201
references: 12783857
references: Chem Rev. 1998 May 7;98(3):1045-1066
references: 11848924
references: Electrophoresis. 1997 Jun;18(7):1109-19
references: 9237565
references: Electrophoresis. 1999 Feb;20(2):362-71
references: 10197444
references: Prog Nucleic Acid Res Mol Biol. 2005;80:349-74
references: 16164979
references: Cell. 2006 Mar 10;124(5):897-900
references: 16530038
references: Methods Enzymol. 1983;91:49-60
references: 6855597
references: J Biol Chem. 1991 Nov 5;266(31):21208-14
references: 1939163
references: J Control Release. 2005 Jan 20;102(1):247-53
references: 15653149
references: J Biol Chem. 2005 Apr 15;280(15):15300-6
references: 15687490
references: Biotechnol Lett. 2006 Oct;28(20):1615-22
references: 16902846
references: Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10407-12
references: 9724716
references: FEBS Lett. 2003 Apr 10;540(1-3):15-20
references: 12681476
references: Biochim Biophys Acta. 2006 Aug;1761(8):812-26
references: 16928468
references: Anal Biochem. 2002 Jul 1;306(1):100-7
references: 12069420
references: Biochemistry. 2004 Mar 9;43(9):2438-44
references: 14992581
references: Chem Biol. 2001 May;8(5):405-13
references: 11358688
references: Biochim Biophys Acta. 2001 Dec 1;1515(2):101-9
references: 11718666
references: Adv Drug Deliv Rev. 2005 Feb 28;57(4):529-45
references: 15722162
references: Nucleic Acids Res. 1999 Sep 15;27(18):3696-701
references: 10471739
references: Mol Cell. 2002 Jan;9(1):145-54
references: 11804593
references: J Biol Chem. 2003 Jun 6;278(23):20934-8
references: 12649287
references: J Biochem. 2002 Aug;132(2):223-8
references: 12153719
references: J Biol Chem. 1997 Apr 11;272(15):9818-24
references: 9092516
references: J Control Release. 2007 Feb 12;117(2):148-62
references: 17196289
references: J Cell Sci. 2003 Jan 15;116(Pt 2):313-24
references: 12482917
references: ACS Chem Biol. 2006 May 23;1(4):252-60
references: 17163679
references: Curr Pharm Des. 2005;11(28):3613-28
references: 16305498
references: Biochem J. 1995 Jun 1;308 ( Pt 2):547-50
references: 7772040
references: Biol Pharm Bull. 2002 Jun;25(6):722-7
references: 12081136
references: Biochemistry. 2004 Mar 2;43(8):2167-77
references: 14979713
references: Nucleic Acids Res. 2003 Feb 1;31(3):1024-32
references: 12560499
references: Arch Biochem Biophys. 2003 Jul 15;415(2):172-7
references: 12831839
references: Expert Opin Biol Ther. 2006 Apr;6(4):391-9
references: 16548765
references: Comp Biochem Physiol C Toxicol Pharmacol. 2001 Jul;129(3):175-91
references: 11461835
references: J Am Chem Soc. 2005 Feb 16;127(6):1652-3
references: 15700995
references: FEBS J. 2006 Aug;273(16):3687-97
references: 16911519
references: J Mol Biol. 2005 Nov 18;354(1):41-54
references: 16188273
references: J Biol Chem. 2000 Apr 7;275(14):9893-6
references: 10744660
references: Mol Biosyst. 2005 Oct;1(4):294-302
references: 16880994
references: Biochem Biophys Res Commun. 2004 Feb 6;314(2):550-4
references: 14733942
references: ACS Chem Biol. 2007 Mar 20;2(3):167-70
references: 17319644
references: Protein Sci. 2002 Oct;11(10):2522-5
references: 12237473
references: J Biol Chem. 2002 Mar 29;277(13):11576-81
references: 11801605
references: Protein Eng Des Sel. 2007 Oct;20(10):505-9
references: 17954521
references: J Mol Biol. 2007 Apr 27;368(2):434-49
references: 17350650
references: Annu Rev Biophys Biomol Struct. 2005;34:119-51
references: 15869386
references: J Biol Chem. 2001 Nov 16;276(46):43095-102
references: 11555655
references: J Biol Chem. 2000 Jun 9;275(23):17463-7
references: 10747991
references: Cell Mol Life Sci. 2006 Aug;63(16):1819-22
references: 16909213
references: Nature. 2003 Mar 6;422(6927):37-44
references: 12621426