title: Assessing delivery of lipopeptides into the cytoplasm of intact cells by a functional assay based on PKC inhibition. I. The Jurkat model.
abstract: We have developed a functional assay to verify the delivery into the cytoplasm of a 14-amino acid hydrophilic peptide, modified by the incorporation of a palmitoyl-lysine residue into the N- or C-terminal end. This assay is based on the use of a pseudo-substrate sequence for the protein kinase C (PKC) isoenzymes of alpha and beta for the quantification of PKC-mediated tumor necrosis factor (TNF) secretion after T-cell activation by phorbol ester and anti-CD3 MAb. This cellular assay is simple, and it allows for a rapid and comparative study of several peptides. The lipidic analogues of the pseudo-substrate peptide were able to inhibit TNF secretion in intact-activated Jurkat cells, with an EC50 in the 40-60 microM range, whereas the unmodified peptide was not active. Two control lipopeptides were also inactive, demonstrating that the palmitoyl-lysine group had no effect by itself. This study confirms that the modification of a relatively long peptide by the insertion of a palmitoyl-lysine into the N- or C-terminal end is sufficient to allow entry into intact cells.
authors.affiliation: URA CNRS 1309, Faculté de Pharmacie, Pasteur Institute, Lille, France.
authors.forename: E
authors.initials: E
authors.lastname: Loing
authors.forename: A
authors.initials: A
authors.lastname: Delanoye
authors.forename: C
authors.initials: C
authors.lastname: Sergheraert
authors.forename: A
authors.initials: A
authors.lastname: Tartar
authors.forename: H
authors.initials: H
authors.lastname: Gras-Masse
mesh_terms: D000595:Amino Acid Sequence; D001681:Biological Assay / Q000379:methods; D001692:Biological Transport; D002463:Cell Membrane Permeability; D003593:Cytoplasm / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008074:Lipoproteins / Q000737:chemistry / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D008074:Lipoproteins; C104968:N(epsilon)-palmitoyllysine; D010446:Peptide Fragments; D014409:Tumor Necrosis Factor-alpha; C104969:protein kinase C (15-28); D011493:Protein Kinase C; D008239:Lysine