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

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 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.

SUBMITTER: Liu Y

PROVIDER: S-EPMC9947424 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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

Liu Yi Y Trnka Michael J MJ He Liang L Burlingame A L AL Correia Maria Almira MA

Molecular & cellular proteomics : MCP 20230110 2

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<sup>-/-</sup>-cells, such termination is abrogated resulting in the nuclear persistence and prolonged activation of NF-κB ...[more]

PMID: 36634736

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Project description:BackgroundRegulator of cullins 1 (ROC1) is an important catalytic subunit of cullin-RING E3 ligase. Nuclear factor-kappa B (NF-κB) signaling is closely related to tumor invasion and metastasis. Earlier, we reported that ROC1 was associated with a poor prognosis in patients with bladder cancer (BCa). However, it is unclear whether ROC1 is involved in the NF-κB signaling associated with malignant BCa progression.MethodsThe expression of ROC1 and p65 in bladder cancer and paracancerous tissues were detected by immunohistochemistry (IHC). Pearson correlation was used to assess correlation between ROC1 and p65 protein expressions. The wound-healing and transwell assays were used to monitor cell invasion and migration. The effect of ROC1 on the expression of key proteins in the NF-κB signaling was determined by immunofluorescence and western blot (WB). Cycloheximide (CHX), MG132 and immunoprecipitation assays were used to evaluate the effect of ROC1 on the ubiquitination of phosphorylated inhibitor of kappa B alpha (p-IκBα). A lung metastasis mouse model was generated to detect the role of ROC1 in tumor metastasis.ResultsWe found that ROC1 was up-regulated in BCa tissues and cell lines, and high ROC1 levels were positively correlated with higher tumour grade, lymph node metastasis, distant metastasis and poor prognosis. Linear-regression analysis showed significant a Pearson correlation between ROC1 and nuclear p65 expression in BCa tissue microarray (TMA) samples. Functional studies demonstrated that ROC1 promoted BCa cell invasion and migration. In vitro and in vivo experiments showed that ROC1 activated NF-κB signaling by enhancing the ubiquitination of p-IκBα, which caused p65 nuclear translocation and promoted the transcription of some metastasis-related target genes, such as urokinase-type plasminogen activator receptor (uPAR), intracellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), and matrix metalloproteinase 9 (MMP9), resulting in promoting BCa metastasis.ConclusionROC1 plays an important role in the progression of BCa and serves as a potential diagnostic and therapeutic target for patients with BCa.

Dataset Information

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

Publications

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

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