Link to bioRxiv paper:
http://biorxiv.org/cgi/content/short/2022.10.13.512146v1?rss=1

Authors: Liu, Y., Trnka, M. J., He, L., Burlingame, A., Correia, M. A.

Abstract:
We have previously documented that in liver cells, the multifunctional protein scaffold p62/SQSTM1 is closely associated with I{kappa}B, an inhibitor of the transcriptional activator NF-{kappa}B. Such an intimate p62-I{kappa}Bassociation we now document leads to a marked 18-fold proteolytic I{kappa}B-stabilization, enabling its nuclear entry and termination of the NF-{kappa}B-activation cycle. In p62-/--cells, such termination is abrogated resulting in the nuclear persistence and prolonged activation of NF-{kappa}B 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-I{kappa}B association. Accordingly, we have identified such I{kappa}B hotspots to reside around N-terminal (K38, K47 and K67) and C-terminal (K238/C239) residues in its 5th 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) in the intervening region between its ZZ- and TB-subdomains. APEX proximity analyses upon I{kappa}B co-transfection of cells with and without p62 have enabled the characterization of the p62 influence on I{kappa}B-protein-protein interactions. Interestingly, consistent with p62 capacity to proteolytically stabilize I{kappa}B, its presence greatly impaired I{kappa}B interactions with various 20S/26S proteasomal subunits. Furthermore, consistent with p62-interaction with I{kappa}B on an interface opposite to that of its NF-{kappa}B-interacting interface, p62 failed to significantly affect I{kappa}B-NF-{kappa}B 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 I{kappa}B following its NF-{kappa}B-elicited transcriptional activation and de novo synthesis, required for the termination of the NF-{kappa}B-activation cycle. Consequently, mice carrying a liver specific deletion of p62-residues 68-252 harboring its positively charged patch, reveal age-dependent enhanced liver inflammation. Our findings reveal yet another mode of p62-mediated pathophysiologically relevant regulation of NF-{kappa}B.

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