Identification of New IκBα Complexes by an Iterative Experimental and Mathematical Modeling Approach

The transcription factor nuclear factor kappa-B (NFκB) is a key regulator of pro-inflammatory and pro-proliferative processes. Accordingly, uncontrolled NFκB activity may contribute to the development of severe diseases when the regulatory system is impaired. Since NFκB can be triggered by a huge variety of inflammatory, pro-and anti-apoptotic stimuli, its activation underlies a complex and tightly regulated signaling network that also includes multi-layered negative feedback mechanisms. Detailed understanding of this complex signaling network is mandatory to identify sensitive parameters that may serve as targets for therapeutic interventions. While many details about canonical and non-canonical NFκB activation have been investigated, less is known about cellular IκBα pools that may tune the cellular NFκB levels. IκBα has so far exclusively been described to exist in two different forms within the cell: stably bound to NFκB or, very transiently, as unbound protein. We created a detailed mathematical model to quantitatively capture and analyze the time-resolved network behavior. By iterative refinement with numerous biological experiments, we yielded a highly identifiable model with superior predictive power which led to the hypothesis of an NFκB-lacking IκBα complex that contains stabilizing IKK subunits. We provide evidence that other but canonical pathways exist that may affect the cellular IκBα status. This additional IκBα:IKKγ complex revealed may serve as storage for the inhibitor to antagonize undesired NFκB activation under physiological and pathophysiological conditions.Author Summary: In unstimulated cells, the transcription factor NFκB resides in the cytosol bound to its inhibitor IκBα. Canonical activation of NFκB by numerous stimuli leads to proteasomal depletion of IκBα, thereby liberating NFκB to translocate into the nucleus to induce transcription of genes leading to proliferation, angiogenesis, metastasis, or chronic inflammation. Consequently, only transient activity needs to be warranted by immediate NFκB-dependent induction of negative regulatory mechanisms, including up-regulation of its inhibitor IκBα. Resynthesized IκBα consequently terminates NFκB activity by binding to its nuclear localization sequence. However, under physiological or pathophysiological conditions, random NFκB activation may occur, which needs to be avoided in order to guarantee proper cellular function. Using detailed dynamical modeling, we have now identified an additional IκBα containing complex to exist in un-stimulated cells which lacks NFκB but includes IKKγ (IκBα:IKKγ complex). This additional IκBα is not depleted from cells in the canonical fashion and may therefore serve as a cellular backup to avoid random NFκB activation.