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Consequences of fuzziness in the NF?B/I?B? interaction.

ABSTRACT: This chapter provides a short review of various biophysical experiments that have been applied to the inhibitor of kappa B, I?B? and its binding partner, nuclear factor kappa B, or NF?B. The picture that emerges from amide hydrogen/deuterium exchange, NMR and binding kinetics experiments is one in which parts of both proteins are "fuzzy" in the free-state and some parts remain "fuzzy" in the NF?B-I?B? complex. The NF?B family of transcription factors responds to inflammatory cytokines with rapid transcriptional activation, in which NF?B enters the nucleus and binds DNA. Just as rapidly as transcription is activated, it is subsequently repressed by newly synthesized I?B??that also enters the nucleus and removes NF?B from the DNA. Because I?B??is an ankyrin repeat protein, it's "fuzziness" can be controlled by mutagenesis to stabilized the folded state. Experimental comparison with such stabilized mutants helps provide evidence that much of the system control depends on the "fuzziness" of I?B?.

SUBMITTER: Komives EA

PROVIDER: S-EPMC3603378 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Consequences of fuzziness in the NFκB/IκBα interaction.

Komives Elizabeth A EA

Advances in experimental medicine and biology 20120101

This chapter provides a short review of various biophysical experiments that have been applied to the inhibitor of kappa B, IκBα and its binding partner, nuclear factor kappa B, or NFκB. The picture that emerges from amide hydrogen/deuterium exchange, NMR and binding kinetics experiments is one in which parts of both proteins are "fuzzy" in the free-state and some parts remain "fuzzy" in the NFκB-IκBα complex. The NFκB family of transcription factors responds to inflammatory cytokines with rapid ...[more]

PMID: 22399319

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Project description:We have previously documented that in liver cells, the multifunctional protein scaffold p62/SQSTM1 is closely associated with IκBα, an inhibitor of the transcriptional activator NF-κB. Such an intimate p62-IκBα association we now document leads to a marked 18-fold proteolytic IκBα-stabilization, enabling its nuclear entry and termination of the NF-κB-activation cycle. In p62-/--cells, such termination is abrogated resulting in the nuclear persistence and prolonged activation of NF-κB following inflammatory stimuli. Utilizing various approaches both classic (structural deletion, site-directed mutagenesis) as well as novel (in-cell chemical crosslinking), coupled with proteomic analyses, we have defined the precise structural hotspots of p62-IκBα association. Accordingly, we have identified such IκBα hotspots to reside around N-terminal (K38, K47, and K67) and C-terminal (K238/C239) residues in its fifth ankyrin repeat domain. These sites interact with two hotspots in p62: One in its PB-1 subdomain around K13, and the other comprised of a positively charged patch (R183/R186/K187/K189) between its ZZ- and TB-subdomains. APEX proximity analyses upon IκBα-cotransfection of cells with and without p62 have enabled the characterization of the p62 influence on IκBα-protein-protein interactions. Interestingly, consistent with p62's capacity to proteolytically stabilize IκBα, its presence greatly impaired IκBα's interactions with various 20S/26S proteasomal subunits. Furthermore, consistent with p62 interaction with IκBα on an interface opposite to that of its NF-κB-interacting interface, p62 failed to significantly affect IκBα-NF-κB interactions. These collective findings together with the known dynamic p62 nucleocytoplasmic shuttling leads us to speculate that it may be involved in "piggy-back" nuclear transport of IκBα following its NF-κB-elicited transcriptional activation and de novo synthesis, required for termination of the NF-κB-activation cycle. Consequently, mice carrying a liver-specific deletion of p62-residues 68 to 252 reveal age-dependent-enhanced liver inflammation. Our findings reveal yet another mode of p62-mediated pathophysiologically relevant regulation of NF-κB.

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Consequences of fuzziness in the NF?B/I?B? interaction.

Publications

Consequences of fuzziness in the NFκB/IκBα interaction.

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