Identification of a p65 Peptide That Selectively Inhibits NF-κ B Activation Induced by Various Inflammatory Stimuli and Its Role in Down-regulation of NF-κB-mediated Gene Expression and Up-regulation of Apoptosis

Abstract

Because of the critical role of the nuclear transcription factor NF-B in inflammation, viral replication, carcinogenesis, antiapoptosis, invasion, and metastasis, specific inhibitors of this nuclear factor are being sought and tested as treatments. NF-B 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-B (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-B 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-B 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-B (IB␣) phosphorylation, IB␣ degradation, or IB␣ kinase activation, but it blocked TNF-induced p65 phosphorylation and nuclear translocation. NF-B-regulated reporter gene expression induced by TNF, TNF receptor 1, TNF receptor-associated death domain, TNF receptor-associated factor-2, NF-B-inducing kinase, IB␣ kinase, and p65 was also suppressed by these peptides. Suppression of NF-B 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-B activation induced by various inflammatory stimuli, down-regulate NF-B-mediated gene expression, and up-regulate apoptosis.NF-B represents a group of five proteins, namely c-Rel, RelA (p65), RelB, NF-B1 (p50 and p105), and NF-B2 (p52) (1). Ways to modulate NF-B expression therapeutically have focused on its active-inactive state transition mechanisms. NF-B is regulated by a family of inhibitors, called IB 1 (2). In an inactive state, NF-B is present in the cytoplasm as a heterotrimer consisting of p50, p65, and IB␣ subunits. In response to an activation signal, the IB␣ subunit is phosphorylated at serine residues 32 and 36, ubiquitinated at lysine residues 21 and 22, and degraded through the proteosomal pathway, thus exposing the nuclear localization signals on the p50-p65 heterodimer. The p65 is then phosphorylated, leading to the nuclear translocation and binding to a specific sequence in DNA, which in turn results in transcriptions of various genes including cyclin D1, cyclooxygenase (COX)-2, and matrix metalloproteinase (MMP)-9.NF-B has been shown to regulate the expression of a number of genes whose products are involved in inflammation, viral replication, carcinogenesis, ant...

Suppression of NF-κB and NF-κB-regulated gene expression by sulforaphane and PEITC through IκBα, IKK pathway in human prostate cancer PC-3 cells

“…Thus, the inhibition of phosphorylated p65 in the nucleus was mainly due to the decrease of nuclear translocation of p65 by treatment with SFN and PEITC in PC-3 C4 cells, and not by the direct inhibition of phosphorylation elicited by SFN and PEITC. Phosphorylation of p65 at serine 529 is known to be required for NF-kB transcriptional activation (Takada et al, 2004b). Since SFN and PEITC could inhibit the basal and UVCinduced NF-kB transcriptional activity, our results suggest that inhibition on NF-kB transcriptional activation by SFN and PEITC is possibly due to the inhibition of nuclear translocation of p65 and consequently phosphorylation of p65 in PC-3 C4 cells.…”

“…Recent studies have shown that the p65 subunit is directly phosphorylated by an upstream kinase before activating downstream genes (Catley et al, 2004;Takada et al, 2004b). Based on those results, we tested whether SFN and PEITC could also inhibit the nuclear translocation and phosphorylation Effects of SFN and PEITC on IKKa and IKKb activity.…”

“…Recent studies revealed that the protein-tyrosine kinase Syk could phosphorylate IkBa through tyrosine and activate NF-kB (Fan et al, 2003;Takada et al, 2003). It is still controversial which kinase is responsible for the phosphorylation of p65, but the phosphorylation of p65 at serine 529 is known to be required for NF-kB transcriptional activation (Takada et al, 2004b). NF-kB has been reported to play an important role in several signal transduction pathways involved in various cancers as well as in chronic inflammatory diseases (Rayet and Gelinas, 1999;Bremner and Heinrich, 2002;Aggarwal et al, 2004).…”

“…The most common nuclear factor kappa B (NF-kB) is a heterodimer composed of p65 and p50 (Greten and Karin, 2004;Suh and Rabson, 2004). NF-kB is activated by a wide variety of stimuli such as tumor necrosis factor (TNF)-alpha, interleukin-1 (IL-1), LPS, UV light, and oxidative stress (Jeong et al, 2004;Takada et al, 2004b). In most cell types, NF-kB is present in the cytosol in an inactive form and is associated with its inhibitor proteins, called inhibitor of NF-kB (IkBs).…”

Interaction of TRAF6 with MAST205 Regulates NF-κB Activation and MAST205 Stability

“…(17) have been used to deliver peptides and small proteins to cells by an energy-and receptor-independent mechanism with the aim of interfering in signaling processes. Examples include protein transduction domain peptides directed against STAT3 (18), extracellular signal-regulated kinase (19), and NF-B (20). To analyze the importance of the N terminus of MAST205, we synthesized protein transduction domain MAST205 peptides targeting a high scoring PEST sequence, thought to control proteasomal degradation (21) (residues 218 -233; peptide 1) and a potential TRAF2 binding epitope (residues 188 -201; peptide 2).…”