ABSTRACT
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 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 IκBα co-transfection 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 the termination of the NF-κB-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-κB.
Highlights
p62 binds to and stabilizes IκBα by preventing its proteolytic degradation
In-cell chemical crosslinking/LC-MS/MS identified the inter-crosslinked sites
Hotspots of p62-IκBα association are defined
APEX proximity labeling revealed p62 impaired IκBα-interaction with proteasome
p62 chaperones newly synthesized IκBα to terminate NF-κB activation.
In Brief The transcriptional activator NF-κB inhibitor, IκBα is proteolytically unstable when uncomplexed. How newly synthesized IκBα escapes degradation to terminate nuclear NF-κB-activation is unknown. Using in-cell chemical crosslinking and proximity labeling MS analyses, we uncovered a novel association of p62 with IκBα via well-defined structural hotspots, which impairs its interaction with the 26S/20S proteasome, extending its life-span and enabling termination of NF-κB-activation. Mice carrying liver-specific genetic deletion of p62-IκBα hotspot exhibit enhanced liver inflammation upon aging, validating this novel p62 role.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Conflict of interest: The authors have no conflict of interest to declare.
ABBREVIATIONS
- ABC
- ammonium bicarbonate
- AGC
- automatic gain control
- AR
- Ankyrin repeat
- ARD
- AR-domain
- APEX
- a 27 kDa engineered monomeric peroxidase (APEX2)
- BCA
- bicinchoninic acid
- BFDR
- Bayesian False Discovery Rate
- CE
- cytoplasmic Extract
- CSL
- Cell Lysis
- CLMS
- chemical crosslinking mass spectrometry
- Co-IP
- co-immunoprecipitation
- CHX
- cycloheximide
- DMEM
- Dulbecco’s Modified Eagle high glucose medium
- DSS
- disuccinimidyl suberate
- EThcD
- electron transfer/high-energy collision dissociation
- FBS
- fetal bovine serum
- FDR
- false discovery rates;
- HA
- hemagglutinin
- HA-IκBα
- HA-tagged IκBα
- HCD
- High collision dissociation
- iBMK
- immortalized baby mouse kidney cells
- IB
- Immunoblotting
- IL-1β
- interleukin-1β
- IDR-1
- innate defense regulator
- IR
- intervening region
- Lys-C
- lysylendopeptidase C
- MEFs
- mouse embryo fibroblasts
- MEM
- minimal Eagle’s medium
- NCE
- normalized collision energy
- NE
- nuclear extract
- NIS
- nuclear import sequences
- NLS
- nuclear localization signal
- NPC
- nuclear pore complex
- NSAF
- normalized spectral abundance factor
- Nup153
- nucleoporin 153
- p62 flp/flp
- p62-floxed mouse
- p62-Myc
- Myc-tagged p62
- p62mut
- p62 genetic mutant mouse
- PB-1
- Phox and Bem1p-domain
- P-p65
- phosphorylated p65
- RanBP2
- a SUMO E3-ligase/Nup358
- Ran-GDP
- an abundant GTPase involved in nuclear import
- SA
- streptavidin
- SOD1
- Cu-Zn superoxide dismutase
- SIAB [succinimidyl (4- iodoacetyl)aminobenzoate); SQSTM-1
- Sequestosome 1
- TNFα
- tumor necrosis factor α
- TB
- TRAF6-binding
- Ub
- ubiquitin
- UPD
- Ub-dependent 26S proteasomal degradation
- ZnPP
- Znprotoporphyrin IX
- ZZ
- Zn-finger binding motifs