Biology subjects

Correia, M. A.

Publications and source records attributed to Correia, M. A..

7 recordsLinked to original sources

bioRxiv2025

Hepatic cytochrome P450 endoplasmic reticulum-associated degradation (ERAD): Topological determinants and cellular partnerships that dictate the preferential P450 proteolytic sorting into macroautophagy rather than UPS.

Many N-terminally endoplasmic reticulum (ER)-anchored cytochrome P450 proteins (P450s) turn over proteolytically via ER-associated degradation (ERAD), others via ER-to-lysosomal-associated degradation (ERLAD), and yet others via both pathways. What precisely dictates their differential proteolytic turnover is unknown. Herein, we employed rabbit liver CYPs 1A1 and 1A2, which reportedly reside in liquid-disorded (ld)- and detergent-resistant, liquid-ordered (lo)-ER-microdomains, respectively, governed by their specific N-terminal (NT) signal-anchor (SA) subdomains. We now report that this precise SA-dependent ER-topology not only determines the proclivity of CYP1A1 towards ERAD and CYP1A2 towards ERLAD, but also their differential lifespans. We further document that the detergent-resistant lo-ER-membranes (DRMs) are morphologically quite similar to mitochondria-associated ER-membranes (MAMs), documented cellular platforms for autophagic-initiation complexes. DRMs and MAMs, composed of saturated fatty-acids, glycosphingolipids and cholesterol, harbor many common morphological markers including the ER-specific prohibitin, erlin-1. Herein employing SURF, a split fluorogenic bifunctional complementation assay, we show that intracellularly, erlin-1 and CYP1A2 interact closely via their ER NT-SAs. siRNA-knockdown (KD) of erlin-1 in HepG2-cells, not only relocated CYP1A2 from DRMs to non-DRMs, but also impaired its ERLAD, resulting in insoluble cellular CYP1A2 aggregates. Upon erlin-1 KD, CYP1A2 ERLAD could be rescued by co-expression of a siRNA-resistant intact erlin-1 or just its NT-1-30 residue SA-domain. Our findings are the first to reveal that the CYP1A2 lifespan and preferential proclivity towards ERLAD is determined by its close association with erlin-1 within DRMs/MAMs. As proof of concept, we document that the ERLAD-proclivity of CYP2B1 is also similarly dependent upon erlin-1-DRM-association. SIGNIFICANCE STATEMENTThe endoplasmic reticulum (ER)-anchored cytochromes P450 (P450s) incur ER-associated degradation (ERAD) and/or ER-to-lysosomal-associated degradation (ERLAD). What determines their preferential proteolytic turnover is unknown. Here, employing P450s CYP1A1 and CYP1A2 that reside in their N-terminal signal anchor-determined liquid-disordered (ld)- and detergent-resistant, liquid-ordered (lo)-ER-microdomains, respectively, we documented that in HepG2-cells these ER-microdomains determine the CYP1A proteolytic preferences for ERAD vs ERLAD as well as their lifespans. More importantly, we discovered that its intimate interaction with the ER-specific prohibitin erlin-1 colocalized in these lo-ER-microdomains, specifically dictates CYP1A2s ERLAD-preference. Accordingly, siRNA-elicited erlin-1-knockdown disrupted CYP1A2-ERLAD, which was rescued upon coexpression of either a siRNA-resistant erlin-1 or just its N-terminal 1-30 residues. As proof of concept, we document similar characteristics for CYP2B1, another ERLAD-targeted P450.

cell biology↗

bioRxiv2024

Liver CYP4A autophagic-lysosomal degradation (ALD): A major role for the autophagic receptor SQSTM1/p62 through an uncommon target interaction site

The hepatic P450 hemoproteins CYPs 4A are typical N-terminally anchored Type I endoplasmic reticulum (ER)-proteins, that are inducible by hypolipidemic drugs and other "peroxisome proliferators". They are engaged in the {omega}-/{omega}-1-oxidation of various fatty acids including arachidonic acid, prostaglandins and leukotrienes and in the biotransformation of some therapeutic drugs. Herein we report that of the mammalian liver CYPs 4A, human CYP4A11 and mouse Cyp4a12a are preferential targets of the ER-lysosome-associated degradation (ERLAD). Consequently, these proteins are stabilized both as 1%Triton X100-soluble and -insoluble species in mouse hepatocytes and HepG2-cells deficient in the autophagic initiation ATG5-gene. Although these proteins exhibit surface LC3-interacting regions (LIRs) that would target them directly to the autophagosome, they nevertheless interact intimately with the autophagic receptor SQSTM1/p62. Through structural deletion analyses and site-directed mutagenesis, we have identified the Cyp4A-interacting p62 subdomain to lie between residues 170 and 233, which include its Traf6-binding and LIM-binding subdomains. Mice carrying a liver-specific genetic deletion of p62 residues 69-251 (p62Mut) that includes the CYP4A-interacting subdomain also exhibit Cyp4a-protein stabilization both as Triton X100-soluble and -insoluble species. Consistently, p62Mut mouse liver microsomes exhibit enhanced {omega}- and {omega}-1-hydroxylation of arachidonic acid to its physiologically active metabolites 19- and 20-HETEs relative to the corresponding wild-type mouse liver microsomes. Collectively, our findings suggest that any disruption of CYP4A ERLAD results in functionally active P450 protein and consequent production of proinflammatory metabolites on one hand, and insoluble aggregates on the other, which may contribute to pathological aggregates i.e. Mallory-Denk bodies/inclusions, hallmarks of many liver diseases.

cell biology↗

bioRxiv2022

In-cell chemical crosslinking identifies hotspots for p62-IκBα interaction that underscore a critical role of p62 in limiting NF-κB activation through IκBα-stabilization

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}B association 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 p62s capacity to proteolytically stabilize I{kappa}B, its presence greatly impaired I{kappa}Bs 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/512146v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@a2cce1org.highwire.dtl.DTLVardef@38da91org.highwire.dtl.DTLVardef@95847aorg.highwire.dtl.DTLVardef@2c829b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIp62 binds to and stabilizes I{kappa}B by preventing its proteolytic degradation C_LIO_LIIn-cell chemical crosslinking/LC-MS/MS identified the inter-crosslinked sites C_LIO_LIHotspots of p62-I{kappa}B association are defined C_LIO_LIAPEX proximity labeling revealed p62 impaired I{kappa}B-interaction with proteasome C_LIO_LIp62 chaperones newly synthesized I{kappa}B to terminate NF-{kappa}B activation. C_LI In BriefThe transcriptional activator NF-{kappa}B inhibitor, I{kappa}B is proteolytically unstable when uncomplexed. How newly synthesized I{kappa}B escapes degradation to terminate nuclear NF-{kappa}B-activation is unknown. Using in-cell chemical crosslinking and proximity labeling MS analyses, we uncovered a novel association of p62 with I{kappa}B via well-defined structural hotspots, which impairs its interaction with the 26S/20S proteasome, extending its life-span and enabling termination of NF-{kappa}B-activation. Mice carrying liver-specific genetic deletion of p62-I{kappa}B hotspot exhibit enhanced liver inflammation upon aging, validating this novel p62 role.

cell biology↗

bioRxiv2019

Human hepatic tryptophan 2,3-dioxygenase ubiquitin-dependent protein degradation: The critical role of its exosite as the molecular lynchpin of its substrate-mediated protein stabilization

Hepatic tryptophan 2,3-dioxygenase (TDO) is a cytoplasmic homotetrameric hemoprotein and the rate-limiting enzyme in the irreversible degradation of the essential amino acid L-tryptophan (L-Trp) to N-formylkynurenine, thus controlling the flux of L-Trp into its serotonergic and kynureninic/NAD pathways. TDO has long been recognized to be substrate-inducible via protein stabilization, but the molecular mechanism of this stabilization has remained elusive. Recent elucidation of human TDO (hTDO) crystal structure has identified a high-affinity (Kd {approx} 0.5 M) Trp-binding exosite in each of its 4 monomeric subunits. Mutation of the Glu105, Trp208 and Arg211 comprising this exosite not only abolished the high-affinity L-Trp binding, but also accelerated the ubiquitin-dependent proteasomal degradation of hTDO. We have further characterized this hTDO degradation by documenting that its ubiquitination by gp78/AMFR and CHIP E2/E3 ligase complexes occurs on external Lys-residues within or vicinal to acidic Asp/Glu and phosphorylated pSer/pThr (DEpSpT)-clusters. Furthermore, we have identified the unstructured hTDO N- and C-termini as imparting relatively high proteolytic instability, as their deletion ({Delta}NC) markedly prolonged hTDO t1/2. Additionally, although previous studies reported that upon hepatic heme-depletion, the heme-free apoTDO turns over with a t1/2 {approx} 2.2 h relative to the t1/2 of 7.7 h of holoTDO, mutating the axial heme-ligating His328 to Ala has the opposite effect of prolonging hTDO t1/2. Most importantly, introducing the exosite mutation into the {Delta}NC-deleted or H328A-mutant completely abolished their prolonged half-lives irrespective of L-Trp presence or absence, thereby revealing that the exosite is the molecular lynchpin that defines L-Trp-mediated TDO induction via protein stabilization.

biochemistry↗

bioRxiv2019

Mitochondrial dysfunction is signaled to the integrated stress response by OMA1, DELE1 and HRI

In mammalian cells, mitochondrial dysfunction triggers the integrated stress response (ISR), in which eIF2 phosphorylation upregulates the transcription factor ATF4. However, how mitochondrial stress is relayed to the ISR is unknown. We found that HRI is the eIF2 kinase necessary and sufficient for this relay. Using an unbiased CRISPRi screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease, and DELE1, a little-characterized protein we found to be associated with the inner mitochondrial membrane. Mitochondrial stress stimulates the OMA1-dependent cleavage of DELE1, leading to its accumulation in the cytosol, where it interacts with HRI and activates its eIF2 kinase activity. Blockade of the OMA1-DELE1-HRI pathway is beneficial during some, but not all types of mitochondrial stress, and leads to an alternative response that induces specific molecular chaperones. Therefore, this pathway is a potential therapeutic target enabling fine-tuning of the ISR for beneficial outcomes in diseases involving mitochondrial dysfunction.

cell biology↗

bioRxiv2019

Induction via Functional Protein Stabilization of Hepatic Cytochromes P450 upon gp78/ Autocrine Motility Factor Receptor (AMFR) Ubiquitin E3-ligase Genetic Ablation in Mice: Therapeutic and Toxicological Relevance.

3.The hepatic endoplasmic reticulum (ER)-anchored monotopic proteins, cytochromes P450 (P450s) are enzymes that metabolize endobiotics (physiologically active steroids and fatty acids) as well as xenobiotics including therapeutic/chemotherapeutic drugs, nutrients, carcinogens and toxins. Alterations of hepatic P450 content through synthesis, inactivation or proteolytic turnover influence their metabolic function. P450 proteolytic turnover occurs via ER-associated degradation (ERAD) involving ubiquitin (Ub)-dependent proteasomal degradation (UPD) as a major pathway. UPD critically involves P450 protein ubiquitination by E2/E3 Ub-ligase complexes. We have previously identified the ER-polytopic gp78/AMFR (autocrine motility factor receptor) as a relevant E3 in CYP3A4, CYP3A23 and CYP2E1 UPD. We now document that liver-conditional genetic ablation of gp78/AMFR in mice disrupts P450 ERAD, resulting in significant stabilization of Cyp2a5 and Cyps 2c, in addition to that of Cyps 3a and Cyp2e1. More importantly, we establish that such stabilization is of the functionally active P450 proteins, leading to corresponding significant enhancement of their drug metabolizing capacities. Our findings with clinically relevant therapeutic drugs (nicotine, coumarin, chlorzoxazone, and acetaminophen) and the prodrug (tamoxifen) as P450 substrates, reveal that P450 ERAD disruption could influence therapeutic drug response and/or toxicity, warranting serious consideration as a potential source of clinically significant drug-drug interactions (DDIs). Because gp78/AMFR is not only an E3 Ub-ligase, but also a cell-surface prometastatic oncogene that is upregulated in various malignant cancers, our finding that hepatic gp78/AMFR-knockout can enhance P450-dependent bioactivation of relevant cancer chemotherapeutic prodrugs is of therapeutic relevance and noteworthy in prospective drug design and development.\n\n4. Significance StatementThe cell surface and ER transmembrane protein gp78/AMFR, a receptor for the prometastatic autocrine motility factor (AMF), as well as an E3 ubiquitin-ligase involved in the ERAD of not only the tumor metastatic suppressor KAI1, but also of hepatic cytochromes P450, is upregulated in various human cancers, enhancing their invasiveness, metastatic potential and poor prognosis. Liver specific gp78/AMFR genetic ablation results in functional protein stabilization of several hepatic P450s and consequently enhanced drug and prodrug metabolism, a feature that could be therapeutically exploited in the bioactivation of chemotherapeutic prodrugs, through design and development of novel short-term gp78/AMFR chemical inhibitors.

pharmacology and toxicology↗

bioRxiv2019

A NOVEL MECHANISM FOR NF-κB-ACTIVATION VIA IκB-AGGREGATION: IMPLICATIONS FOR HEPATIC MALLORY-DENK-BODY INDUCED INFLAMMATION

Background & AimsMallory-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-{kappa}B activation. However, the precise mechanism that links protein aggregation to NF{kappa}B-activation and inflammatory response remains unclear.\n\nMethodsHerein, we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin, protoporphyrin IX (PPIX), or ZnPPIX) elicited an I{kappa}B-loss with consequent NF-{kappa}B 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 I{kappa}B.\n\nResultsFour known mechanisms of I{kappa}B-loss were probed and excluded. Immunofluorescence analyses of ZnPPIX-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this I{kappa}B-loss was due to its sequestration along with I{kappa}B{beta} into insoluble aggregates. Through proteomic analyses we identified 47 aggregation-prone proteins that co-aggregate with I{kappa}B 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 I{kappa}B-nuclear import.\n\nConclusionWe discovered a novel mechanism of inflammatory NF-{kappa}B activation through I{kappa}B-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.\n\nLay SummaryMallory-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 NF{kappa}B 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 NF{kappa}B activation via cytosolic I{kappa}B-sequestration into insoluble aggregates. Furthermore, using a proteomic approach, we identified 47 aggregation-prone proteins that interact and co-aggregate with I{kappa}B. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thereby identifying novel targets for therapeutic intervention.

cell biology↗