A conserved core region of the scaffold NEMO is essential for signal-induced conformational change and liquid-liquid phase separation.

Scaffold proteins help mediate interactions between protein partners, often to optimize intracellular signaling. Herein, we use comparative, biochemical, biophysical, molecular, and cellular approaches to investigate how the scaffold protein NEMO contributes to signaling in the NF-κB pathway. Comparison of NEMO and the related protein optineurin from a variety of evolutionarily distant organisms revealed that a central region of NEMO, called the Intervening Domain (IVD), is conserved between NEMO and optineurin.

A Conserved Core Region of the Scaffold NEMO is Essential for Signal-induced Conformational Change and Liquid-liquid Phase Separation.

Scaffold proteins help mediate interactions between protein partners, often to optimize intracellular signaling. Herein, we use comparative, biochemical, biophysical, molecular, and cellular approaches to investigate how the scaffold protein NEMO contributes to signaling in the NF-κB pathway. Comparison of NEMO and the related protein optineurin from a variety of evolutionarily distant organisms revealed that a central region of NEMO, called the Intervening Domain (IVD), is conserved between NEMO and optineurin.

Scaffold proteins as dynamic integrators of biological processes.

Scaffold proteins act as molecular hubs for the docking of multiple proteins to organize efficient functional units for signaling cascades. Over 300 human proteins have been characterized as scaffolds, acting in a variety of signaling pathways. While the term scaffold implies a static, supportive platform, it is now clear that scaffolds are not simply inert docking stations but can undergo conformational changes that affect their dependent signaling pathways.

Comparison of NF-κB from the protists Capsaspora owczarzaki and Acanthoeca spectabilis reveals extensive evolutionary diversification of this transcription factor.

We provide a functional characterization of transcription factor NF-κB in protists and provide information about the evolution and diversification of this biologically important protein. We characterized NF-κB in two protists using phylogenetic, cellular, and biochemical techniques. NF-κB of the holozoan Capsaspora owczarzaki (Co) has an N-terminal DNA-binding domain and a C-terminal Ankyrin repeat (ANK) domain, and its DNA-binding specificity is more similar to metazoan NF-κB proteins than to Rel proteins.

CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein.

The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter D) is largely responsible for NEMO transcription in liver cells.