A Novel Mechanism for NF-κB-Activation via IκB-Aggregation: Implications for Hepatic Mallory-denk-body Induced Inflammation

Background & Aims Mallory-Denk-bodies (MDBs) are hepatic protein aggregates associated with inflammation both clinically and in MDB-inducing models. Similar protein aggregation in neurodegenerative diseases also triggers inflammation and NF-κB activation. However, the precise mechanism that links protein aggregation to NFκB-activation and inflammatory response remains unclear. Methods Herein, we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin, protoporphyrin IX (PPIX), or ZnPPIX) elicited an IκBα-loss with consequent NF-κB activation. We characterized the underlying mechanism in detail using hepatocytes from various knockout mice and MEF cell lines and multiple approaches including immunoblotting, EMSA, RT-PCR, confocal immunofluorescence microscopy, affinity immunoprecipitation, and protein solubility assays. Additionally, we performed rigorous proteomic analyses to identify the proteins aggregating upon PPIX treatment and/or co-aggregating with IκBα. Results Four known mechanisms of IκBα-loss were probed and excluded. Immunofluorescence analyses of ZnPPIX-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this IκBα-loss was due to its sequestration along with IκBβ into insoluble aggregates. Through proteomic analyses we identified 47 aggregation-prone proteins that co-aggregate with IκBα through direct interaction or proximity. Of these ZnPPIX-aggregation targets, the nucleoporins Nup153 and Nup358/RanBP2 were identified through RNA-interference, as likely mediators of IκBα-nuclear import. Conclusion We discovered a novel mechanism of inflammatory NF-κB activation through IκB-sequestration into insoluble aggregates along with interacting aggregation-prone proteins. This mechanism may account for the protein aggregate-induced inflammation observed in MDB-associated liver diseases, thereby identifying novel targets for therapeutic intervention. Because of inherent commonalities this MDB cell model is a bona fide protoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria. Lay Summary Mallory-Denk-bodies (MDBs) are hepatic protein aggregates commonly featured in many liver diseases. MDB-presence is associated with the induction of inflammatory responses both clinically and in all MDB-inducing models. Similar protein aggregation in neurodegenerative diseases is also known to trigger inflammation and NFκB pathway activation via an as yet to be characterized non-canonical mechanism. Herein using a MDB-inducing cell model, we uncovered a novel mechanism for NFκB activation via cytosolic IκB-sequestration into insoluble aggregates. Furthermore, using a proteomic approach, we identified 47 aggregation-prone proteins that interact and co-aggregate with IκBα. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thereby identifying novel targets for therapeutic intervention.


INTRODUCTION
13 for identification of IκBα-interacting proteins in both soluble and insoluble fractions of ZnPP-286 treated and untreated cells (Fig. S5). The overlapping proteome identified via both co-IP and 287 BAR approaches revealed a very high-confidence IκBα-interactome. More importantly, of the 288 previously identified 47 IκBα-interacting proteins in common with the ZnPP-aggregate proteome, 289 37 were also detected via BAR to be interacting with IκBα in ZnPP-triggered protein aggregates 290 ( Fig 5B), indicating that they were indeed co-aggregating. HepG2 ZnPP-aggregate proteome. We therefore verified Nup153 presence through IB analyses 296 of Triton-and urea-solubilized cellular aggregates upon a 2 h-ZnPP-treatment of HEK293 and 297 HepG2 cells. Indeed, whereas native Nup153 exhibited its intrinsic 153 kDa-mobility in 298 untreated cells, upon ZnPP-treatment it was found as HMM-protein aggregates in Triton-and 299 urea extracts, along with endogenous IκBα (Fig. 6A). Furthermore, co-immunoprecipitation (Co-300 IP) using HEK293T cells revealed a significant fraction of endogenous Nup153 interacted with 301 IκBα both in cytoplasmic and nuclear extracts of GFP-IκBα-transfected cells, but not in mock-or 302 C1-GFP-transfected cells (Fig. 6B). A similar interaction was also evident among the 303 endogenous counterparts under basal conditions. However, this Nup153-IκBα-interaction was 304 greatly enhanced upon TNFα-treatment at times (1 and 1.5 h) when rapid translocation of de 305 novo synthesized IκBα across the nuclear pore would be expected to trigger its post-induction 306 repression of NF-κB-activation (Fig. 6C). Furthermore, ZnPP-treatment of TNFα-pretreated cells 307 led to accelerated IκBα sequestration, whose timing revealed that ZnPP may preferentially 308 target de novo synthesized IκBα as it increasingly interacts with cytoplasmic Nup153 during its 309 14 nuclear import (Fig. 6C-D). Consistent with this, our CMIF of HepG2 cells revealed that under 310 basal conditions, both NF-κB (p65-Rel A subunit) and IκBα are localized in the cytoplasm (Fig.  311   6E). But upon TNFα-treatment, IκBα UPD results in NF-κB nuclear translocation within 0.5 h. 312 Subsequently, upon transcriptional activation of the NF-κB-responsive IκBα-gene, newly 313 synthesized IκBα enters the nucleus resulting in NF-κB dissociation and nuclear export, all 314 within 1 h of TNFα-treatment. By contrast, upon ZnPP-treatment, in spite of this robust IκBα-315 restoration at 1 h after TNFα-treatment, it is apparently functionally defective as a nuclear NF-316 κB-repressor, as NF-κB persisted in the nucleus (Fig. 6E). This stalled IκBα appears 317 prominently both in the cytoplasm as well as clustered around the outer nuclear envelope rim 318 ( Fig. 6E). Additional support for this likelihood is provided by our Nup153 and RanBP2 siRNA-319 knockdown analyses that revealed the marked attenuation of nuclear IκBα-import upon TNFα-320 activation, without affecting its ZnPP-sequestration (Figs. 7 & S6). 321 322

Why is IκBα vulnerable to ZnPP-elicited physical sequestration from the cytoplasm? 347
Recent evidence increasingly supports a sequestration and co-aggregation model of 348 pathogenesis [41]. In a snowballing effect, aggregation-prone disease proteins "hijack" their 349 interacting partners with vital functions, sequestering them into cytotoxic insoluble inclusions 350 [41]. The protein scaffold p62 is one such IκBα-co-aggregating protein. However, scrutiny of 351 ZnPP-treated p62 -/hepatocytes and MEF cells reveals that it is not essential for ZnPP-elicited 352 IκBα-sequestration. This is consistent with the report that in p62 -/mouse liver, p62 is not 353 required for MDB formation, just for their maturation and stability [42]. These aggregation-prone 354 proteins share some common physicochemical properties: Preexistent proteins are relatively 355 large in size, enriched in domains with high intrinsic disorder or unstructured regions, and exhibit 356 low average hydrophobicity [43]; newly synthesized proteins on the other hand, are more 357 16 vulnerable due to prolonged exposure of their relatively hydrophobic domains either during or 358 soon after synthesis during their subsequent folding, assembly, or transport [43]. 359 None of the 47 proteins that interacted with IκBα and co-aggregated upon ZnPP-360 treatment is a well-established stable IκBα interactor. Although most of the known stable IκBα 361 interactors were repeatedly found in our co-IP analyses (Fig 4C), they were never detected in 362 the ZnPP-aggregate proteome. This suggests that these 47 proteins may be transient 363 IκBα interactors that are difficult to capture without highly sensitive and selective approaches. 364 Our analyses of the physicochemical properties of these 47 proteins indicated that they ranged 365 widely in size, with ~90% exhibiting relatively low hydrophobicity, and ~80% predicted to contain 366 a long (>30 residues) disordered segment (Table S1). These dual features of low hydrophobicity 367 coupled with high intrinsic disorder could synergistically contribute to their ZnPP-elicited co-368 aggregation upon transient interaction, and may explain why ZnPP initially targets preexistent 369 IκBα species exhibiting both features (Table S1). The structurally related IκBβ is similarly 370 vulnerable to ZnPP-elicited sequestration thus accounting for the remarkably profound NF-κB-371 activation. Whether this vulnerability stems from their common structural ankyrin-repeat domain 372 feature remains to be determined. Free IκBα species is apparently unstable and requires NF-κB 373 binding for folding to a stable conformation [44]. After TNFα stimulation, newly synthesized 374 IκBα would be unstable and thus greatly susceptible to ZnPP-elicited aggregation without 375 nuclear import in order to bind NF-κB and properly fold, thus resulting in the sustained ZnPP-376 triggered NF-κB-activation. 377 Although NF-κB and IκBα form a stable complex, they are not static cytoplasmic 378 residents but shuttle continuously between the nucleus and cytoplasm [45]. Interestingly, p65 379 and IκBα translocate into the nucleus via different pathways. p65 enters the nucleus via the 380 classical NLS-and importin (α3/α4)-dependent machinery [46], whereas IκBα and several other 381 ankyrin repeat domain-containing proteins (ARPs) enter the nucleus via a NLS-and importin-382 independent machinery [47]. This machinery depends on a direct interaction between the ARPs, 383 RanGDP as well as mobile nucleoporins such as Nup153 and RanBP2 for entry across the NPC 384 into the nucleus. Indeed, plausibly relevant to this IκBα-nucleocytoplasmic shuttling process, our 385 proteomic analyses of the 47 common IκBα-interacting and ZnPP-co-aggregating proteins 386 underscored significant enrichment of mobile NPC nucleoporins. Of these, our heat-map 387 analyses singled out Nup153 as both the top-ranked IκBα-interactant and ZnPP-aggregation 388 target (Fig. 5B). By contrast, importins α3 and α4 relevant to p65 nuclear import were not found 389 in the ZnPP-aggregate proteome, which may explain why p65 is not co-aggregated during its 390 nuclear import. 391 392

Potential role of Nup153 in IκBα-mediated NF-κB-repression:
The nucleoporin Nup153 393 primarily exists N-terminally anchored to the nuclear pore basket while its disordered and 394 flexible FG-rich C-terminus extends into the cytoplasm [48]. Another Nup153 pool apparently 395 exists that shuttles between the cytoplasmic and NPC faces [49], and interacts directly with 396 cargos (i.e. Stat1, Smad2, and PU.1) for their nuclear import via a transporter-independent 397 machinery [48]. Nup153, in contrast to other FG-rich nucleoporins i.e. Nup98, Nup62, Nup214, 398 also strongly binds ARs of iASPP and ASPP2, two representative substrates of the NLS-399 independent ARP-RanGDP nuclear import pathway [47]. Structural/biochemical analyses 400 indicate that Nup153 binds RanGDP at a higher affinity than RanGTP [50]. This collective 401 evidence suggests a critical role of Nup153 in facilitating ARP-RanGDP nuclear import. Our 402 findings that IκBα co-immunoprecipitated not only with Nup153 in the nuclear extracts (NER), 403 but also more appreciably with that in the cytoplasmic extracts (CER), even though the basal 404 cytoplasmic Nup153-content is much lower than its nuclear content (compare inputs, Fig. 6B), 405 are consistent with a minor, albeit highly dynamic, cytoplasmic Nup153 pool . This pool directly  406   interacts with IκBα and may be responsible for the continuous IκBα-nucleocytoplasmic traffic at  407 steady state, as indeed verified by our Nup153 knockdown analyses (Fig. 7). The concurrent 408 ZnPP-elicited aggregation of Nup153 and IκBα suggests that the basal nuclear import of 409 IκBα as well as that of newly synthesized IκBα after TNFα-stimulation is disrupted, leading to 410 NF-κB nuclear persistence and prolonged response. 411 Another noteworthy proteomic finding is our quite prominent detection of the SUMO E3-412 ligase RanBP2 in this 47-protein cohort. RanBP2, an outer NPC component, fans its FG-rich 413 filaments into the cytoplasm [51]. The proteomic detection of RanBP2, but no other cytoplasmic 414 NPC nucleoporin i.e. Nup214 in this common 47-protein cohort is functionally intriguing because 415 besides Nup153, RanBP2 is also involved in receptor-independent nuclear import through direct 416 interactions with its specific cytoplasmic cargos [51]. This is intriguing given that IκBα 417 SUMOylation is reportedly involved in its nuclear entry and subsequent post-induction NF-κB-418 repression [52]. Because RanBP2 also strongly binds RanGDP, conceivably a RanBP2-419 RanGDP-Nup153-IκBα-SUMO complex is involved in IκBα-nuclear import, a possibility 420 consistent with our RanBP2 knockdown analyses (Fig. 7). The inviability of actively dividing 421 HEK293T cells precluded our simultaneous knockdown of both nucleoporins to mimic their 422 concurrent ZnPP-elicited sequestration. The failure of Nup153 or RanBP2 knockdown to affect 423 ZnPP-elicited IκBα-sequestration suggests that the newly synthesized IκBα is itself vulnerable, 424 particularly in the absence of both Nup153 and RanBP2 to chaperone its nuclear entry. 425 We find it quite instructive that many of these nucleocytoplasmic trafficking proteins as The precise mechanism of PPIX-induced protein aggregation remains to be elucidated. 453 Porphyrins trigger protein cross-linking through a secondary reaction between the 454 photooxidative products of histidine, tyrosine and/or tryptophan and free NH 2 -groups of amino 455 acids [58]. Given the light-sheltered in vivo environment of the liver, such a photooxidative 456 protein crosslinking seems unlikely in PPIX-mediated hepatic MDB formation, although PPIX 457 can trigger protein aggregation and cell toxicity in the dark, albeit at a slower rate [ Samples subject to MS proteomics analyses were processed using standard in-gel digestion 488 and the resulting peptide mixture was desalted using C18-Ziptips (Millipore) and then injected 489 into an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher). Raw data were processed 490      Results:

NMPP-elicited IκBα-loss is independent of HRI eIF2α-kinase activation:
The concurrence of IκBα loss and NF-κB activation and its attenuation by hemin-treatment led us to consider whether NMPP-elicited heme depletion was responsible for these findings. Conceivably, heme depletion could activate the hepatic heme-sensor HRI eIF2α kinase resulting in the translational suppression of IκBα and consequent NF-κB-activation. Similar IκBα translational suppression and consequent NF-κB-activation has been documented upon specific activation of the other cellular eIF2α kinases [1,2]. Indeed, in parallel with IκBα-loss (Fig S2A), NMPP treatment increased the relative ratio of phosphorylated eIF2α (eIF2αP) over basal eIF2α levels, indicating HRI activation [3]. However, NMPP-treatment of cultured hepatocytes from cultured hepatocytes from HRI WT (HRI +/+ ) and knockout (KO; HRI -/-) mice elicited a comparable IκBα, excluding causal HRI activation (Fig. 1D).

ZnPP-elicited IκBα-loss is independent of autophagy, calpain-mediated proteolysis and reactive oxygen species (ROS):
Given the reported IκBα-degradation via ALD [4] and the known p62-role in this process [5], we conclusively excluded ALD in this process by documenting that knockout of the essential autophagic gene ATG5 failed to mitigate ZnPPelicited IκBα loss (Fig. S2B). Because of possible calpain-mediated IκBα-degradation [6,7], we also excluded its involvement by documenting that knockout of the calpain-degradation pathway (capn4 KO MEF cells) failed to abrogate ZnPP-elicited IκBα loss (Fig. S2C). These findings coupled with those with ALD and calpain inhibitors (Fig. 2D) revealed that neither ALD nor calpain degradation played any role in this IκBα loss. Furthermore, through the use of various ROS-quenchers (Fig. S3), we excluded any possible PPIX-photoactivation and consequent ROS-mediated oxidative stress in this IκBα-loss [8].

Discussion:
The precise mechanism of the ZnPP-elicited IκB-protein sequestration is currently unknown, but some plausible mechanisms are presented. The fact that newly synthesized IκBα is particularly vulnerable to such ZnPP-sequestration implicates the involvement of its intrinsic disordered domains at a stage when it has not yet reached its mature folded state.
In search of clues on potential ZnPP-mechanisms of protein sequestration, we sought the advice of various internationally recognized expert heme/porphyrin chemists and biochemists and porphyria experts on the possible mechanistic causes. Not a single investigator was aware that ZnPP caused protein aggregation, much less IκBα-sequestration. ZnPP (along with SnPP), is actually believed to be safe and is recommended for the treatment of neonatal jaundice due to its effective inhibition of microsomal heme oxygenase (HO1), the key rate-limiting enzyme in the conversion of heme to biliverdin [9]. However, in vivo, overproduction of protoporphyrin IX (PPIX) due to defects in ferrochelatase (congenital erythropoietic protoporphyria) or PPIXoverproduction in X-linked protoporphyria, as well as iron-deficiency or lead-poisoning induced anemias (that can exhaust iron-stores) leads to ZnPP-generation [10][11][12]. Our findings suggest that under these conditions, these patients may easily succumb to ZnPP-elicited IκBαsequestration and consequently unabated hepatic NF-κB-elicited activation of cytokines and chemokines. We believe, these findings would for the first-time alert physicians of this pathological potential of ZnPP and are clinically relevant not just in MDB-inducing diseases but also in clinical protoporphyrias and iron-deficiency/lead-induced anemias.

Contact for reagent and resource sharing
Information and requests for resources and reagents should be directed to M. A. Correia (almira.correia@ucsf.edu).

Experimental model details
Animal studies: C57BL/6 wild type male mice (8-12-week old) purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were fed a standard chow-diet and maintained under a normal diurnal light cycle. All animal experiments were carried out strictly by protocols specifically approved by the UCSF/Institutional Animal Care and Use Committee (IACUC) and its care and use of laboratory animal guidelines. For the ZnPP-treatment, ZnPP was dissolved in sterile DMSO at 100 mM, and then diluted 1:10 (v:v) with sterile 7.5% BSA to a final concentration of 10 mM. Mice were weighed and then injected daily with either ZnPP (50 μmol/kg, i.p.), or vehicle (controls) (10% DMSO in 7.5% BSA) for 7 days. Liver samples were collected and frozen at -80°C until RNA isolation.

Cell culture:
All cells were grown at 37°C with 5% CO 2 in a humidified incubator. C57BL/6 wild type male mice (8-12-week old) purchased from the Jackson Laboratory (Bar Harbor, ME) were used for primary hepatocyte preparation. Hepatocytes were isolated by in situ collagenase perfusion and purified by Percoll-gradient centrifugation by the UCSF Liver Center Cell Biology Core, as described previously [13]. Fresh primary mouse hepatocytes were cultured on Type I collagencoated 60 mm Permanox plates (Thermo Scientific, Grand Island, NY) in William's E Medium supplemented with 2 mM L-glutamine, insulin-transferrin-selenium, 0.1% bovine albumin Fraction V, Penicillin-Streptomycin and 0.1 μM dexamethasone. Cells were allowed to attach for 4 to 6 h and then overlaid with Matrigel. From the 2nd day after plating, the medium was replaced daily, and cells were further cultured for 4-5 days with daily light microscopic examination for any signs of cell death and/or cytotoxicity. On day 5, some cells were treated with 30 μM NMPP (dissolved in DMSO), 10 μM PPIX (dissolved in DMSO with sonication) or 10 μM zinc (II)-PPIX (ZnPP, dissolved in DMSO and complexed with bovine serum albumin (BSA) at a molar ratio of 4:1 to keep it solubilized in the medium) for various times as indicated (Results). In some cases, hepatocytes were pretreated with various inhibitors as indicated (Results) for 1 h before treatment with NMPP, PPIX or ZnPP.
HepG2 and HeLa cells were cultured in minimal Eagle's medium (MEM) containing 10% v/v fetal bovine serum (FBS) and supplemented with nonessential amino acids and 1 mM sodium pyruvate. HEK293T and MEF cells were cultured with Dulbecco's Modified Eagle high glucose medium (DMEM) containing 10% v/v FBS. For transfection experiments, cells were seeded onto 6-well plates, and when cells were 60% confluent, each cell well was transfected with 3 μg plasmid DNA complexed with TurboFect transfection reagent for HEK293T cells and X-tremeGENE HP transfection reagent for HepG2 cells according to the manufacturers' ! 4! instructions. At 40-72 h after transfection, cells were either treated as indicated or directly harvested for assays.

Method details
Hepatic PPIX content: PPIX content was determined using the intrinsic PPIX fluorescence as described [14]. Briefly, 50 μl of cell lysates were first extracted with 400 μl of EtOAc-HAc (3:1) and then re-extracted with another 400 μl of EtOAc-HAc. The extracts were pooled and reextracted with 400 μl of 3 M HCl. After centrifugation, the aqueous phase was recovered for fluorescent determination in a SpectrumMax M5 plate reader at an excitation 405 nm. The intensity at emission 610 nm was quantified relative to a standard curve prepared with known concentrations of pure PPIX. Co-Immunoprecipitation (Co-IP) analyses: Whole-cell extracts were prepared as described above. Cell lysates (1 mg) were then incubated with indicated antibodies (2 µg) or control IgGs at 4°C overnight. Antibody-antigen complexes were then captured by protein G Dynabeads at room temperature for 1 h, and then eluted by heating at 95°C for 10 min in 2X SDS-loading buffer. Eluates were subjected to IB analyses as described above.
Sequential solvent extraction of cell lysates: Cells were harvested in cell lysis buffer as described above and the cell lysates were cleared by centrifugation at 14,000g. The pellet was then solubilized in RIPA buffer supplemented with 0.1% SDS, 10% glycerol and protease/phosphatase inhibitor cocktail with sonication followed by centrifugation at the highest speed for 10 min. The resulting pellet was then solubilized with sonication in urea/CHAPS buffer containing 8 M urea, 2 M thiourea, 4% CHAPS, 20 mM Tris-base, and 30 mM DTT and supplemented with protease/phosphatase inhibitor cocktail. High salt buffer (HSB)-extraction ( Fig. 4C) was carried out as described previously [15]. Briefly, cells were first harvested in cell lysis buffer containing 1% Triton as described above, the resulting pellet was then suspended in a cell lysis buffer supplemented with 1.5 M KCl (HSB) with sonication. Upon sedimentation, the resulting pellet was solubilized in Laemmli buffer containing 4% SDS or in urea/CHAPS buffer as described above.
Confocal Immunofluorescence microscopy (CIFM): Cells were grown on collagen-coated glass coverslips and treated as indicated. Cells were fixed with 4% formaldehyde for 20 min at room temperature followed by methanol at -20°C for 1 min. After that, cells were rinsed with PBS and blocked for 1 h with 10% normal goat serum in PBS /0.1% Tween at room temperature, and then stained with indicated primary antibodies at 4°C overnight. Cells were then washed in PBS 0.1% Tween three times and then stained with secondary antibodies for 1 h at room temperature. Cells were further washed three times in PBS 0.1% Tween and then mounted using ProLong Diamond Antifade Mountant with DAPI nuclear stain (Molecular Probes, Grand Island, NY). The following secondary antibodies were applied: Goat anti-rabbit IgG Alexa Fluor 488 (Invitrogen, Grand Island, NY), anti-mouse IgG Alexa Fluor 647 (Cell Signaling Technology, Danvers, MA). These particular fluor dyes were selected to circumvent any specific interference from ZnPP intrinsic fluorescence. Images were taken with a Nikon Yokogawa CSU-22 Spinning Disk Confocal Microscope using a Plan Apo VC 100x/1.4 oil lens or on a Nikon high-speed wide-field Andor Borealis CSU-W1 spinning disk confocal microscope using a Plan Apo VC 60x/1.4 oil lens. Images were processed using ImageJ software. For quantification, at least 600 cells at each condition were evaluated. Statistical significance was tested using two-sided unpaired Student's t-test.

Immunoaffinity purification (IAP):
We employed high-affinity alpaca Nanobody crosslinked beads (GFP-Trap) for IAP, which not only enabled a high-level enrichment of target proteins but ! 6! also eliminated IgG contaminants that confound downstream LC-MS/MS analyses. N-terminally mEmerald (GFP)-tagged IκBα (GFP-IκBα) was transiently transfected into HEK293T cells, with mEmerald-transfected cells as background control (C1-GFP). Two wells of HEK293T cells grown on 6-well plates were pooled and lysed in 1 ml lysis buffer supplemented with 10% glycerol, protease/phosphatase inhibitor cocktail and 20 mM N-ethylmaleimide (NEM). Centrifugation-cleared cell lysates were incubated with 50 μl GFP-trap agarose beads at 4°C overnight. Subsequently, GFP-trap beads were collected by centrifugation at 3,000g for 30s and then washed 5 times with cell lysis buffer. Co-immunoprecipitated proteins were eluted by incubating beads with 2X Laemmli buffer at 70°C for 15 min. Eluates were then subjected to SDS-PAGE and stained with Coomassie Blue to visualize the bands for subsequent in-gel digestion.
Biotinylation by antibody-recognition (BAR): Traditional approaches for in vivo proteinprotein interactions such as co-IP are based on affinity capture of stable protein complexes that will be disrupted under harsh denaturing conditions. By contrast, biotinylation proximity labeling circumvents this limitation by introducing an enzyme to the target protein that can generate distance-constrained reactive biotin molecules to covalently link neighboring proteins, providing a permanent tag that survives purification under harsh conditions for downstream identification [16]. We chose BAR/APEX over BioID (the two popular methods for in vivo biotinylation proximity) because the BAR approach [17] is much more robust as it enables quicker capture of interacting proteins (as the peroxidase mediated biotinylation takes only a few minutes) without the more protracted BioID methodology (requiring 18-24 h tagging time), which could overlook short-term/transient interactants. BAR analyses were performed according to [17]. Briefly, two wells of HEK293T cells grown on collagen-coated 6-well plates were first treated as indicated and then fixed with 4% formaldehyde for 10 min at room temperature and permeabilized for 7 min with 0.5% Triton-X in PBS. After rinsing with PBS, cells were incubated with 0.5% H 2 O 2 for 10 min. After rinsing with PBS, cells were then blocked for 1 h with 10% normal goat serum in PBS/0.1% Tween, and then stained with IκBα antibody (mouse monoclonal, 44D4) at a 1:100 dilution in 1% normal goat serum in PBS/0.1% Tween at 4°C overnight. Negative control staining with no antibody was also performed. Cells were then washed in PBS 0.1% Tween for over 1 h with at least 5 buffer changes and then stained with secondary poly-HRP-conjugated goat anti-mouse IgGs (from Biotin XX Tyramide SuperBoost™ Kit) at a 1:1000 v:v dilution in 1% normal goat serum in PBS/0.1% Tween for 1 h. Cells were further washed for over 2 h in PBS 0.1% Tween with at least 5 buffer changes. After that, cells were labeled with biotin using Biotin XX Tyramide SuperBoost™ Kit following product instructions. Briefly, cells were pre-incubated with 300 ml of reaction buffer containing biotin-XX-tyramide (Biotin-Phenol) for 15 min; H 2 O 2 was then added to obtain a final concentration of 0.5 mM and incubated for another 5 min. Negative controls without H 2 O 2 were also included. The labeling reaction was then stopped by quickly exchanging the reaction solution with 1 ml of 500mM sodium ascorbate. Cells were washed 3 times with PBS and then lysed in RIPA buffer supplemented with 2% SDS and boiled for 1 h to reverse formaldehyde crosslinking. The cell lysates were cleared by centrifugation at 14,000g, the supernatants were used as whole cell lysates (WCL). The resulting pellet was further solubilized in urea/CHAPS buffer as described above. Aliquots from both fractions were used for streptavidin-HRP blot (Invitrogen, Grand Island, NY, #S911), to characterize the specificity and efficiency of BAR. The remaining WCL and urea/CHAPS fractions were separately diluted 1:2 (v;v) in RIPA buffer for streptavidin (SA) pull-down. SA pull-downs were performed according to [18]. Briefly, 50 μl of streptavidin magnetic beads were added to the lysates to incubate at 4°C overnight on a rotator. Beads were then washed twice with RIPA buffer, once with 1 M KCl, once with 2 M urea in 20 mM Tris base, and lastly, twice with RIPA buffer. Biotinylated proteins were then eluted by incubating beads in 40 μl of 2X Laemmli buffer ! 9! * The GRAVY number of a protein is a measure of its hydrophobicity or hydrophilicity. The hydropathy values range from -2 to +2 for most proteins, with the positively rated proteins being more hydrophobic. Less hydrophobic proteins are labeled red. ** Proteins are listed with SLIDER score, the higher the score the more likely a protein has a long (>= 30 AAs) disordered segment. Scores above 0.538 indicates that a given protein has a long disorder segment (labeled red).   Fig 1). A. IB analyses of eIF2α, eIF2αP and IκBα in lysates from mouse hepatocytes treated with NMPP or NMPP plus hemin for the indicated times. The temporal profiles of the ratio of eIF2αP to total eIF2α as well as IκBα content were determined (Mean ± SD, n=3). B. ATG5 wild-type (ATG5WT) and knockout (ATG5KO) MEF cells were treated with 10 μM ZnPP for the indicated time. Cell lysates were used for IB analysis of p62, IκBα, and LC3, with actin as the loading control. C. MEF cells wild-type calpain (capn4WT), capn4 knockout (capn4KO) and capn4KO-rescue (wherein capn4 knockout cells were rescued by transfection of a Capn4 lentiviral vector) were treated with ZnPP for the indicated time. Cell lysates were used for IB analysis of p62 and IκBα with actin as the loading control.  Fig 1). A. Primary mouse hepatocytes were left untreated, or pretreated for 1 h with 50mM mannitol, or 100µM BHT, or 50µM lycopene (Lyp) , or BHT coupled with BSA to help keep it solubilized (BHT-BSA), or lycopene coupled with BSA (Lyp-BSA), and then treated with vehicle control (C), 10 μM hemin (H), 30 μM NMPP (N), or 10 μM PPIX (PP), or 10 μM ZnPP (ZnPP) for 4 h as indicated. Cell lysates were used for IB analyses of p62 and IκBα. B. HEK293T cells were cotransfected with pcDNA6-p62-myc or pCMV4-3HA-IκBα. 48 h after transfection, cells were pretreated with various concentrations of cystamine as indicated and then treated with 10 μM ZnPP for 2 h. Cell lysates were used for IB analyses of p62 and IκBα.