Structural plasticity of NF-kappaB essential modulator demonstrates the active regulatory roles of scaffold proteins
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Citation
Abstract
Within the chaos of intracellular signaling pathways, scaffold proteins serve as the control boards to organize the comings and goings of a variety of signals and proteins. There are over 300 known scaffold proteins, which act as switchboards that lessen this chaos of the cellular “soup” and enable proper cellular responses. While the terms scaffold, adaptor, docking, and anchoring protein have sometimes been used interchangeably, this dissertation focuses on scaffolds as a distinct class of proteins that are central to enhancing signaling cascades, have multiple interaction domains to facilitate higher order complex formation, and are highly conserved. In addition, scaffold proteins are not simply inert or passive platforms. Rather, there are multiple examples of scaffolds that, while catalytically inactive, are dynamic in their mechanism of action. Studying these dynamic roles is critical to our understanding of signaling cascades and how signaling-related disease states occur. NF-κB Essential Modulator (aka NEMO or IKK gamma) is a scaffold protein that has a pivotal role in the NF-κB signaling pathway. In this dissertation, NEMO is examined both for its changes in activity upon mutation and the mechanical/structural effects of mutations within a highly conserved central region termed the intervening domain (IVD). The IVD of NEMO is essential for proper function of the protein and for the coordination of phosphorylation of the inhibitor IκBα in the activation of canonical NF-κB pathway. The proper structural organization of NEMO is required for cytokine-induced activation of IκB kinase (IKK), and the impact that the IVD has on this structural organization is demonstrated. Mutations within the conserved IVD core are detrimental to the activation of the canonical NF-κB pathway and reduce the ability of NEMO to form ubiquitin-induced liquid-liquid phase separated droplets in vitro. The effects of the IVD mutations also correlate with a reduced ability of NEMO to form signal-induced puncta in vivo. Thermal and chemical denaturation studies of truncated NEMO variants indicate that the IVD affects the stability of the full-length NEMO molecule, due to conflicting structural demands of this region on upstream and downstream domains. This conformational strain in the IVD mediates allosteric communication between N- and C-terminal regions of NEMO. Overall, these findings support the hypothesis that the IVD of NEMO participates in signal-induced activation of the IKK/NF-κB pathway by acting as a mediator of conformational change in NEMO. These findings demonstrate the importance of conformational change for scaffold protein function and provide new information about how clinical mutations in scaffolds can affect function and can serve as targets for therapeutic intervention.
Description
2024
License
Attribution 4.0 International