There has recently been identified a strong connection between infection, chronic inflammation and oncogenesis. Tissue damage resulting from leukocyte infiltration is the hallmark of many acute and chronic inflammatory diseases. NF-kappaB transcription factors are activated by removal of their cellular inhibitors, the IkappaBs, and differentially activate the proinflammatory gene program. IkappaB phosphorylation by the IkappaB kinase (IKK) is the key regulatory event in this process. IKK exists as a large molecular weight protein complex and is activated by a wide variety of extracellular stimuli. Only three IKK subunits have been identified unambiguously, the related kinases (IKKalpha and IKKbeta), and a structural component (IKKgamma). Virtually nothing is known about, structure or subunits stoichiometry. We hypothesize that an undescribed IKK kinase(s) (IKK-K) exists that specifically links the IKKs with upstream signaling molecules. Our Preliminary Results from in vitro kinase assays indicate IKKgamma is the target of the IKK-K and that phosphorylation of IKKgamma leads to its partial removal from IKK allowing of IKK. We hypothesize that stimulus-induced activated IKK-K phosphorylates a protein of the IKKgamma triggering a conformational change in IKKgamma causing either its partial removal from the IKK complex an activation of IKK or alternatively, an efficient interaction with an IKK inhibitory molecule and then phosphorylated IKKgamma and the inhibitor leave the complex resulting in activation of IKK. To test this hypothesis, in Aim 1 we will biochemically and biophysically characterize the modifications on IKK- associated and freed IKKgamma in resting and stimulus- induced cells using immunoaffinity purification and phospgopeptide mapping/sequencing techniques.
In Aim 2, we will use both traditional and immuno-affinity, chromatography to purify IKK-Ks which associate and phpsphorylate IKKgamma following a TNFalpha stimulus. To determine if there are different types of IKK complexes in cells, in Aim 3 we will characterize a number of IKK subunits immunoaffinity reagents and also develop functional monoclonal antibodies (single chain antibodies (scFvs) that can be used to identify and select subunits-specific IKK complexes. These will also be expressed intracellularly to determine the physiological function of a particular type of IKK complex in response to TNF or IL-1 activation. Uncovering how the IKK complex is regulated in response to environmental cues and defining the structural basis for this will allow the future development of pharmaceuticals to specifically target IKK activation and NF-kappaB target gene transcription. These pharmaceuticals will be of great medical benefit in combating diseases of a acute and chronic inflammation, which can lead to cancer.
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