Cell-surface thiols affect cell entry of disulfide-conjugated peptides

FASEB J. 2009 Sep;23(9):2956-67. doi: 10.1096/fj.08-127563. Epub 2009 Apr 27.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • CHO Cells
  • Cell Membrane / chemistry
  • Cell Membrane / metabolism*
  • Cell Membrane Permeability*
  • Cricetinae
  • Cricetulus
  • Disulfides / chemistry
  • Disulfides / pharmacokinetics
  • Drug Carriers
  • Mass Spectrometry
  • Microscopy, Confocal
  • Peptides / chemical synthesis
  • Peptides / pharmacokinetics*
  • Sulfhydryl Compounds / chemistry
  • Sulfhydryl Compounds / metabolism*


  • Disulfides
  • Drug Carriers
  • Peptides
  • Sulfhydryl Compounds