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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. Compar...

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Autores principales: DiRusso, Christopher J., DeMaria, Anthony M., Wong, Judy, Wang, Wei, Jordanides, Jack J., Whitty, Adrian, Allen, Karen N., Gilmore, Thomas D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10694592/
https://www.ncbi.nlm.nih.gov/pubmed/37890781
http://dx.doi.org/10.1016/j.jbc.2023.105396
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author DiRusso, Christopher J.
DeMaria, Anthony M.
Wong, Judy
Wang, Wei
Jordanides, Jack J.
Whitty, Adrian
Allen, Karen N.
Gilmore, Thomas D.
author_facet DiRusso, Christopher J.
DeMaria, Anthony M.
Wong, Judy
Wang, Wei
Jordanides, Jack J.
Whitty, Adrian
Allen, Karen N.
Gilmore, Thomas D.
author_sort DiRusso, Christopher J.
collection PubMed
description 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. Previous studies have shown that this central core region of the IVD is required for cytokine-induced activation of IκB kinase (IKK). We show that the analogous region of optineurin can functionally replace the core region of the NEMO IVD. We also show that an intact IVD is required for the formation of disulfide-bonded dimers of NEMO. Moreover, inactivating mutations in this core region abrogate the ability of NEMO to form ubiquitin-induced liquid-liquid phase separation droplets in vitro and signal-induced puncta in vivo. Thermal and chemical denaturation studies of truncated NEMO variants indicate that the IVD, while not intrinsically destabilizing, can reduce the stability of surrounding regions of NEMO due to the conflicting structural demands imparted on this region by flanking upstream and downstream domains. This conformational strain in the IVD mediates allosteric communication between the N- and C-terminal regions of NEMO. Overall, these results support a model in which the IVD of NEMO participates in signal-induced activation of the IKK/NF-κB pathway by acting as a mediator of conformational changes in NEMO.
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spelling pubmed-106945922023-12-05 A conserved core region of the scaffold NEMO is essential for signal-induced conformational change and liquid-liquid phase separation DiRusso, Christopher J. DeMaria, Anthony M. Wong, Judy Wang, Wei Jordanides, Jack J. Whitty, Adrian Allen, Karen N. Gilmore, Thomas D. J Biol Chem Research Article 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. Previous studies have shown that this central core region of the IVD is required for cytokine-induced activation of IκB kinase (IKK). We show that the analogous region of optineurin can functionally replace the core region of the NEMO IVD. We also show that an intact IVD is required for the formation of disulfide-bonded dimers of NEMO. Moreover, inactivating mutations in this core region abrogate the ability of NEMO to form ubiquitin-induced liquid-liquid phase separation droplets in vitro and signal-induced puncta in vivo. Thermal and chemical denaturation studies of truncated NEMO variants indicate that the IVD, while not intrinsically destabilizing, can reduce the stability of surrounding regions of NEMO due to the conflicting structural demands imparted on this region by flanking upstream and downstream domains. This conformational strain in the IVD mediates allosteric communication between the N- and C-terminal regions of NEMO. Overall, these results support a model in which the IVD of NEMO participates in signal-induced activation of the IKK/NF-κB pathway by acting as a mediator of conformational changes in NEMO. American Society for Biochemistry and Molecular Biology 2023-10-27 /pmc/articles/PMC10694592/ /pubmed/37890781 http://dx.doi.org/10.1016/j.jbc.2023.105396 Text en © 2023 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Article
DiRusso, Christopher J.
DeMaria, Anthony M.
Wong, Judy
Wang, Wei
Jordanides, Jack J.
Whitty, Adrian
Allen, Karen N.
Gilmore, Thomas D.
A conserved core region of the scaffold NEMO is essential for signal-induced conformational change and liquid-liquid phase separation
title A conserved core region of the scaffold NEMO is essential for signal-induced conformational change and liquid-liquid phase separation
title_full A conserved core region of the scaffold NEMO is essential for signal-induced conformational change and liquid-liquid phase separation
title_fullStr A conserved core region of the scaffold NEMO is essential for signal-induced conformational change and liquid-liquid phase separation
title_full_unstemmed A conserved core region of the scaffold NEMO is essential for signal-induced conformational change and liquid-liquid phase separation
title_short A conserved core region of the scaffold NEMO is essential for signal-induced conformational change and liquid-liquid phase separation
title_sort conserved core region of the scaffold nemo is essential for signal-induced conformational change and liquid-liquid phase separation
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10694592/
https://www.ncbi.nlm.nih.gov/pubmed/37890781
http://dx.doi.org/10.1016/j.jbc.2023.105396
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