In-Cell Chemical Crosslinking Identifies Hotspots for SQSTM-1/p62-IKB Interaction That Underscore a Critical Role of p62 in Limiting NF-KB Activation Through IKB Stabilization

We have previously documented that in liver cells, multifunctional protein scaffold p62/SQSTM1 is closely associated with IxBa, an inhibitor of the transcriptional activator NF-xB. Such an intimate p62-IxBa association we now document leads to a marked 18-fold proteolytic IxBa-stabilization, enabling its nuclear entry and termi-nation of the NF-xB-activation cycle. In p62-/--cells, such termination is abrogated resulting in the nuclear persis-tence and prolonged activation of NF-xB following flammatory stimuli. Utilizing various approaches classic (structural deletion, site-directed mutagenesis) well as novel (in-cell chemical crosslinking), coupled with proteomic analyses, we have defined the precise struc-tural hotspots of p62-IxBa association. Accordingly, have identified such IxBa hotspots to reside around 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 sub domain around K13, and the other comprised of a posi-tively charged patch (R183/R186/K187/K189) between ZZ-and TB-subdomains. APEX proximity analyses upon IxBa-cotransfection of cells with and without p62 enabled the characterization of the p62 influence on IxBa- protein-protein interactions. Interestingly, consistent with p62's capacity to proteolytically stabilize IxBa, its pres-ence greatly impaired IxBa ' s interactions with various 20S/26S proteasomal subunits. Furthermore, consistent with p62 interaction with IxBa on an interface opposite that of its NF-xB-interacting interface, p62 failed significantly affect IxBa-NF-xB interactions. These lective findings together with the known dynamic nucleocytoplasmic shuttling leads us to speculate that may be involved in "piggy-back" nuclear transport of following its NF-xB-elicited transcriptional activation de novo synthesis, required for termination of the NF-xB- 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 pathophysio- logically relevant regulation of NF-xB.