Identification of a p65 peptide that selectively inhibits NF-kappa B activation induced by various inflammatory stimuli and its role in down-regulation of NF-kappaB-mediated gene expression and up-regulation of apoptosis.
Because of the critical role of the nuclear transcription factor NF-kappaB in inflammation, viral replication, carcinogenesis, antiapoptosis, invasion, and metastasis, specific inhibitors of this nuclear factor are being sought and tested as treatments. NF-kappaB activation is known to require p65 phosphorylation at serine residues 276, 529, and 536 before it undergoes nuclear translocation. Small protein domains, termed protein transduction domains (PTDs), which are able to penetrate cell membranes can be used to transport other proteins across the cell membrane. We have identified two peptides from the p65 subunit of NF-kappaB (P1 and P6 were from amino acid residues 271-282 and 525-537, respectively) that, when linked with a PTD derived from the third helix sequence of antennapedia, inhibited tumor necrosis factor (TNF)-induced NF-kappaB activation in vivo. Linkage to the PTD was not, however, required to suppress the binding of the p50-p65-heterodimer to the DNA in vitro. PTD-p65-P1 had no effect on TNF-induced AP-1 activation. PTD-p65-P1 suppressed NF-kappaB activation induced by lipopolysaccharide, interleukin-1, okadaic acid, phorbol 12-myristate 13-acetate, H(2)O(2), and cigarette smoke condensate as well as that induced by TNF. PTD-p65-P1 had no effect on TNF-induced inhibitory subunit of NF-kappaB(IkappaBalpha) phosphorylation, IkappaBalpha degradation, or IkappaBalpha kinase activation, but it blocked TNF-induced p65 phosphorylation and nuclear translocation. NF-kappaB-regulated reporter gene expression induced by TNF, TNF receptor 1, TNF receptor-associated death domain, TNF receptor-associated factor-2, NF-kappaB-inducing kinase, IkappaBalpha kinase, and p65 was also suppressed by these peptides. Suppression of NF-kappaB by PTD-p65-P1 enhanced the apoptosis induced by TNF and chemotherapeutic agents. Overall, our results demonstrate the identification of a p65 peptide that can selectively inhibit NF-kappaB activation induced by various inflammatory stimuli, down-regulate NF-kappaB-mediated gene expression, and up-regulate apoptosis.
Takada
Yasunari
Y
Cytokine Research Laboratory, Department of Bioimmunotherapy, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and Imgenex, San Diego, California 92121, USA.
Singh
Sujay
S
Aggarwal
Bharat B
BB
D021581:Active Transport, Cell Nucleus; D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D017209:Apoptosis; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D004396:Coloring Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D007375:Interleukin-1 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D008969:Molecular Sequence Data; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D019319:Okadaic Acid / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D051996:Transcription Factor RelA / Q000737:chemistry; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation
D004396:Coloring Agents; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D007375:Interleukin-1; D008070:Lipopolysaccharides; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D010446:Peptide Fragments; D010455:Peptides; C579474:RelA protein (525-536); D013778:Tetrazolium Salts; D013844:Thiazoles; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D019319:Okadaic Acid; D006861:Hydrogen Peroxide; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; D000469:Alkaline Phosphatase; C022616:thiazolyl blue; D013755:Tetradecanoylphorbol Acetate