Novel insights into the cellular mechanisms of the anti-inflammatory effects of NF-kappaB essential modulator binding domain peptides.
The classical nuclear factor kappaB (NF-kappaB) signaling pathway is under the control of the IkappaB kinase (IKK) complex, which consists of IKK-1, IKK-2, and NF-kappaB essential modulator (NEMO). This complex is responsible for the regulation of cell proliferation, survival, and differentiation. Dysregulation of this pathway is associated with several human diseases, and as such, its inhibition offers an exciting opportunity for therapeutic intervention. NEMO binding domain (NBD) peptides inhibit the binding of recombinant NEMO to IKK-2 in vitro. However, direct evidence of disruption of this binding by NBD peptides in biological systems has not been provided. Using a cell system, we expanded on previous observations to show that NBD peptides inhibit inflammation-induced but not basal cytokine production. We report that these peptides cause the release of IKK-2 from an IKK complex and disrupt NEMO-IKK-2 interactions in cells. We demonstrate that by interfering with NEMO-IKK-2 interactions, NBD peptides inhibit IKK-2 phosphorylation, without affecting signaling intermediates upstream of the IKK complex of the NF-kappaB pathway. Furthermore, in a cell-free system of IKK complex activation by TRAF6 (TNF receptor-associated factor 6), we show that these peptides inhibit the ability of this complex to phosphorylate downstream substrates, such as p65 and inhibitor of kappaB alpha (IkappaB alpha). Thus, consistent with the notion that NEMO regulates IKK-2 catalytic activity by serving as a scaffold, appropriately positioning IKK-2 for activation by upstream kinase(s), our findings provide novel insights into the molecular mechanisms by which NBD peptides exert their anti-inflammatory effects in cells.
Baima
Eric T
ET
Department of Inflammation, Pfizer Inc., Chesterfield, Missouri 63017, USA.
Guzova
Julia A
JA
Mathialagan
Sumathy
S
Nagiec
Eva E
EE
Hardy
Medora M
MM
Song
Lily R
LR
Bonar
Sheri L
SL
Weinberg
Robin A
RA
Selness
Shaun R
SR
Woodard
Scott S
SS
Chrencik
Jill
J
Hood
William F
WF
Schindler
John F
JF
Kishore
Nandini
N
Mbalaviele
Gabriel
G
D000893:Anti-Inflammatory Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D006801:Humans; D051550:I-kappa B Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D046912:Multiprotein Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism; D051996:Transcription Factor RelA / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000893:Anti-Inflammatory Agents; D016207:Cytokines; C491731:IKBKG protein, human; D046912:Multiprotein Complexes; D010455:Peptides; C498143:RELA protein, human; D048029:TNF Receptor-Associated Factor 6; D051996:Transcription Factor RelA; C496559:CHUK protein, human; D051550:I-kappa B Kinase