title: Cell-surface thiols affect cell entry of disulfide-conjugated peptides.
abstract: Cell-penetrating peptides (CPPs) can cross the cell membrane and are widely used to deliver bioactive cargoes inside cells. The cargo and the CPP are often conjugated through a disulfide bridge with the common acceptation that this linker is stable in the extracellular biological medium and should not perturb the internalization process. However, with the use of thiol-specific reagents combined with mass spectrometry (as a quantitative method to measure intracellular concentrations of peptides) and confocal microscopy (as a qualitative method to visualize internalized peptides) analyses, we could show that, depending on the peptide sequence, thiol/disulfide exchange reactions could happen at the cell surface. These exchange reactions lead to the reduction of disulfide conjugates. In addition, it was observed that not only disulfide- but also thiol-containing peptides could cross-react with cell-surface thiols. The peptides cross-linked by thiol-containing membrane proteins were either trapped in the membrane or further internalized. Therefore, a new route of cellular uptake was unveiled that is not restricted to CPPs: a protein kinase C peptide inhibitor that is not cell permeant could cross cell membranes when an activated cysteine (with a 3-nitro-2-pyridinesulfenyl moiety) was introduced in its sequence.
authors.affiliation: UPMC Université Paris 06, UMR 7613, Synthesis, Structure, and Function of Bioactive Molecules, Paris, France.
authors.forename: Soline
authors.initials: S
authors.lastname: Aubry
authors.forename: Fabienne
authors.initials: F
authors.lastname: Burlina
authors.forename: Edmond
authors.initials: E
authors.lastname: Dupont
authors.forename: Diane
authors.initials: D
authors.lastname: Delaroche
authors.forename: Alain
authors.initials: A
authors.lastname: Joliot
authors.forename: Solange
authors.initials: S
authors.lastname: Lavielle
authors.forename: Gérard
authors.initials: G
authors.lastname: Chassaing
authors.forename: Sandrine
authors.initials: S
authors.lastname: Sagan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002463:Cell Membrane Permeability; D006224:Cricetinae; D003412:Cricetulus; D004220:Disulfides / Q000737:chemistry / Q000493:pharmacokinetics; D004337:Drug Carriers; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D010455:Peptides / Q000138:chemical synthesis / Q000493:pharmacokinetics*; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004220:Disulfides; D004337:Drug Carriers; D010455:Peptides; D013438:Sulfhydryl Compounds