Inhibition of NF-kappa B activation by peptides targeting NF-kappa B essential modulator (nemo) oligomerization.
NF-kappa B essential modulator/IKK-gamma (NEMO/IKK-gamma) plays a key role in the activation of the NF-kappa B pathway in response to proinflammatory stimuli. Previous studies suggested that the signal-dependent activation of the IKK complex involves the trimerization of NEMO. The minimal oligomerization domain of this protein consists of two coiled-coil subdomains named Coiled-coil 2 (CC2) and leucine zipper (LZ) (Agou, F., Traincard, F., Vinolo, E., Courtois, G., Yamaoka, S., Israel, A., and Veron, M. (2004) J. Biol. Chem. 279, 27861-27869). To search for drugs inhibiting NF-kappa B activation, we have rationally designed cell-permeable peptides corresponding to the CC2 and LZ subdomains that mimic the contact areas between NEMO subunits. The peptides were tagged with the Antennapedia/Penetratin motif and delivered to cells prior to stimulation with lipopolysaccharide. Peptide transduction was monitored by fluorescence-activated cell sorter, and their effect on lipopolysaccharide-induced NF-kappa B activation was quantified using an NF-kappa B-dependent beta-galactosidase assay in stably transfected pre-B 70Z/3 lymphocytes. We show that the peptides corresponding to the LZ and CC2 subdomains inhibit NF-kappa B activation with an IC(50) in the mum range. Control peptides, including mutated CC2 and LZ peptides and a heterologous coiled-coil peptide, had no inhibitory effect. The designed peptides are able to induce cell death in human retinoblastoma Y79 cells exhibiting constitutive NF-kappa B activity. Our results provide the "proof of concept" for a new and promising strategy for the inhibition of NF-kappa B pathway activation through targeting the oligomerization state of the NEMO protein.
Agou
Fabrice
F
Unité de Régulation Enzymatique des Activités Cellulaires, CNRS URA 2185, 75724 Paris, France. fagou@pasteur.fr
Courtois
Gilles
G
Chiaravalli
Jeanne
J
Baleux
Françoise
F
Coïc
Yves-Marie
YM
Traincard
François
F
Israël
Alain
A
Véron
Michel
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D015195:Drug Design; D056827:Endosomal Sorting Complexes Required for Transport; D005434:Flow Cytometry; D006801:Humans; D051550:I-kappa B Kinase; D007376:Interleukin-2 / Q000235:genetics; D019169:Jurkat Cells; D016350:Leucine Zippers / Q000235:genetics / Q000502:physiology; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry; D011993:Recombinant Fusion Proteins; D012175:Retinoblastoma; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics; D000099177:Tsg101 Protein
D004247:DNA; D004268:DNA-Binding Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D051550:I-kappa B Kinase; D007376:Interleukin-2; D008070:Lipopolysaccharides; D016328:NF-kappa B; D010455:Peptides; D017346:Protein Serine-Threonine Kinases; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D001616:beta-Galactosidase; D000099177:Tsg101 Protein; C496559:CHUK protein, human; C496564:Chuk protein, mouse; C496560:IKBKB protein, human; C496565:IKBKE protein, human; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse