Is a phenylalanine-glucosamine derivative able to affect the main responsible for IκB protein phosphorylation?

Nuclear factor κB (NFkB) is an ubiquitous transcription factor well known for its role in the innate immune response. In fact, it is involved as activator of inflammatory mediators such as cytokines. It consists of subunit dimers including p65, RelB, c-Rel, p50 and p52. Under normal conditions, they are bound in the cytosol by an inhibitory protein known as inhibitor of NFkB (IkB). The responsible for IkB phosphorylation is IkB kinase (IKK) complex, consisting of two catalytic subunits (IKKα and IKKβ) and a regulatory one. When activated, IKK phosphorylates IkB, thus tagging it for proteasomal degradation and freeing the NFkB subunit dimers which translocate to the nucleus and bind to specific DNA sequences within the promoter region of vast array of genes. Previous in vivo and in vitro studies in our laboratory demonstrate that Glucosamine (GlcN) and its N-acetyl phenylalanine derivative (NAPA) are able to counteract effects induced by inflammatory cytokines, particularly modulating some genes, under the control of NFkB, upregulated by TNF-α. So, the aim of this study is to better understand whether these two molecules may affect the activity of IKKα and IKKβ through some specific interactions. HTB-94, treated respectively with TNF-α and single molecules, are grown, lysated, incubated with anti-IKKα antibody and eventually treated with magnetic beads. This assay is performed to immunoprecipitate the activated IKK complex. Subsequently, an in vitro kinase assay is performed using a recombinant protein as substrate while in presence and absence of both molecules, to analyze the immunoprecipitated complex (IP-IKK). An immunocytochemistry is performed to visualize if IKKα re-localization is affected by GlcN and NAPA. Owing to immunoprecipitated complex formation, molecules can directly interact with kinases because they don’t need to cross cell membrane. In presence of GlcN, IP-IKK can phosphorylate the recombinant substrate, whileas in presence of NAPA 0.5mM the phosphorylation is inhibited. Further studies are performed evaluating IKK inhibition activity by GlcN and NAPA on recombinant protein using recombinant IKKα and IKKβ molecules. As previously observed, NAPA strongly inhibits IKKα activity on itself and on the recombinant substrate, but no effect is observed on IKKβ. On the contrary, GlcN cannot inhibit either IKKα or IKKβ at any concentrations used. In immunocytochemistry IKKα is mainly cytoplasmic in untreated cells, but it accumulates in nucleus under TNF-α stimulus. Pre-treatment with both molecules shows a mainly cytoplasmic IKKα localization. In conclusion, it is observed that both molecules affect IKKα localization, but only NAPA is an effective IkB phosphorylation inhibitor explaining how both molecules modulate expression level gene under NFkB control. Scotto d'Abusco A; Politi A; Giordano C; Scandurra R¸ Arthritis Research & Therapy 2010; doi: 10.1186/ar2920.