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 steadystate level, which varies linearly with molecular charge. In contrast, the rate constant for internalization (t 1/2 = 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.Cells control their intracellular environment through careful gating of the influx of extracellular material (1,2). To distinguish between molecules to be internalized from those to be excluded, cells use specific interactions with cell-surface proteins, lipids, and carbohydrates. Nonspecific interactions mediated by Coulombic forces can also lead to internalization, often in an unregulated manner (1-5). 1 Such nonspecific interactions can be modulated by increasing, decreasing, or masking the cationic charge on the biomolecule (3,5).Two classes of molecules that exploit high cationicity to effect nonspecific adsorption to the cell surface and internalization are cell-penetrating peptides (CPPs 2 ) and ribonucleases (7-10). CPPs have received considerable attention due to their ability to transport otherwise membrane-impermeable cargo into cells (9,11,12). The detailed mechanism of CPP internalization is unclear, but is known to involve multiple steps. Those steps include binding † This work was supported by Grant CA73808 (NIH). R.J.J. and L.D.L. were supported by Biotechnology Training grant 08349 (NIH).L.D.L. was also supported by an ACS Division of Organic Chemistry Fellowship, sponsored by the Genentech Foundation. The Biophysics Instrumentation Facility was established with grants BIR-9512577 (NSF) and RR13790 (NIH).*To whom correspondence should be addressed: Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706-1544. Telephone: 608-262-8588. Fax: 608-262-3453. raines@biochem.wisc.edu.. 1 We prefer to use the term Coulombic rather than electrostatic to describe the for...

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