Showing papers on "Aryl hydrocarbon receptor nuclear translocator published in 2023"

Direct interaction between mortalin and HIF-1α at the mitochondria inhibits apoptosis by blocking recruitment of Bax.

Abstract: Hypoxia Inducible Factor 1, a heterodimer of alpha (HIF-1α) and beta (HIF-1β or ARNT) subunits, is a major regulator of the transcriptional response to hypoxia. However, HIF-1α, the oxygen-regulated subunit, also exerts non-transcriptional functions through interaction with proteins other than ARNT. We have previously shown that the subcellular localization and protein interactions of HIF-1α are controlled by ERK-mediated phosphorylation at Ser641/643. When HIF-1α is modified at these sites, it is nuclear, binds to ARNT, interacts with NPM1, and activates transcription of hypoxia-target genes. On the other hand, unmodified HIF-1α is bound by CRM1, exits the nucleus and, via its association with mortalin, is targeted to the mitochondria to form an anti-apoptotic complex. To further characterize the latter function, recombinant fragments of HIF-1α and mortalin were used for in vitro binding assays and immunoprecipitation experiments to map the respective binding sites and show that their interaction is direct and functional. We could also show that embelin, a natural product and known inhibitor of the mortalin-p53 interaction, also disrupts the mortalin-HIF-1α association and, furthermore, removes unmodified HIF-1α from mitochondria. Mitochondrial dissociation of HIF-1α, either by embelin or overexpression of a HIF-1α peptide harboring the mortalin binding site, under stress conditions leads to mitochondrial localization of the pro-apoptotic protein Bax and induction of apoptosis. We suggest that when ERK activity is low under hypoxia, binding of HIF-1α to mortalin inhibits mitochondrial recruitment of Bax and protects cells from apoptotic cell death.

Molecular dynamics simulations depict structural motions of the whole human aryl hydrocarbon receptor influencing its binding of ligands and HSP90.

Abstract: The aryl hydrocarbon receptor (AhR) has broad biological functions when its ligands activate it; the non-binding interactions with AhR have not been fully elucidated due to the absence of a complete tridimensional (3D) structure. Therefore, utilization of the whole 3D structure from Homo sapiens AhR by in silico studies will allow us to better study and analyze the binding mode of its full and partial agonists, and antagonists, as well as its interaction with the HSP90 chaperone. The 3D AhR structure was obtained from I-TASSER and subjected to molecular dynamics (MD) simulations to obtain different structural conformations and determine the most populated AhR conformer by clustering analyses. The AhR-3D structures selected from MD simulations and those from clustering analyses were used to achieve docking studies with some of its ligands and protein-protein docking with HSP90. Once the AhR-3D structure was built, its Ramachandran maps and energy showed a well-qualified 3D model. MD simulations showed that the per-Arnt-Sim homology (PAS) PAS A, PAS B, and Q domains underwent conformational changes, identifying the conformation when agonists were binding also, and HSP90 was binding near the PAS A, PAS B, and Q domains. However, when antagonists are binding, HSP90 does not bind near the PAS A, PAS B, and Q domains. These studies show that the complex agonist-AhR-HSP90 can be formed, but this complex is not formed when an antagonist is binding. Knowing the conformations when the ligands bind to AHR and the behavior of HSP90 allows for an understanding of its activity.Communicated by Ramaswamy H. Sarma.

Multi-level interaction between HIF and AHR transcriptional pathways in kidney carcinoma

Abstract: This pan-genomic study of crosstalk between HIF and AHR transcription factors reveals both antagonistic and cooperative interactions and highlights their relevance to kidney cancer and its treatment. Hypoxia-inducible factor (HIF) and aryl hydrocarbon receptor (AHR) are members of the bHLH-PAS family of transcription factors that underpin cellular responses to oxygen and to endogenous and exogenous ligands, respectively, and have central roles in the pathogenesis of renal cancer. Composed of heterodimers, they share a common HIF-1β/ARNT subunit and similar DNA-binding motifs, raising the possibility of crosstalk between the two transcriptional pathways. Here, we identify both general and locus-specific mechanisms of interaction between HIF and AHR that act both antagonistically and cooperatively. Specifically, we observe competition for the common HIF-1β/ARNT subunit, in cis synergy for chromatin binding, and overlap in their transcriptional targets. Recently, both HIF and AHR inhibitors have been developed for the treatment of solid tumours. However, inhibition of one pathway may promote the oncogenic effects of the other. Therefore, our work raises important questions as to whether combination therapy targeting both of these pro-tumourigenic pathways might show greater efficacy than targeting each system independently.

Monoterpenoid aryl hydrocarbon receptor allosteric antagonists protect against ultraviolet skin damage in female mice

Abstract: Abstract The human aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is a pivotal regulator of human physiology and pathophysiology. Allosteric inhibition of AhR was previously thought to be untenable. Here, we identify carvones as noncompetitive, insurmountable antagonists of AhR and characterize the structural and functional consequences of their binding. Carvones do not displace radiolabeled ligands from binding to AhR but instead bind allosterically within the bHLH/PAS-A region of AhR. Carvones do not influence the translocation of ligand-activated AhR into the nucleus but inhibit the heterodimerization of AhR with its canonical partner ARNT and subsequent binding of AhR to the promoter of CYP1A1 . As a proof of concept, we demonstrate physiologically relevant Ahr-antagonism by carvones in vivo in female mice. These substances establish the molecular basis for selective targeting of AhR regardless of the type of ligand(s) present and provide opportunities for the treatment of disease processes modified by AhR.

TCDD-Induced Allosteric Perturbation of the AhR:ARNT Binding to DNA

Abstract: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the biological and toxicological effects of structurally diverse chemicals, including halogenated aromatic hydrocarbons. In this work, we investigate the effects of the binding of the AhR prototypical ligand, TCDD, on the stability of the AhR:ARNT complex, as well as the mechanisms by which ligand-induced perturbations propagate to the DNA recognition site responsible for gene transcription. To this aim, a reliable structural model of the overall quaternary structure of the AhR:ARNT:DRE complex is proposed, based on homology modelling. The model shows very good agreement with a previous one and is supported by experimental evidence. Moreover, molecular dynamics simulations are performed to compare the dynamic behaviour of the AhR:ARNT heterodimer in the presence or absence of the TCDD. Analysis of the simulations, performed by an unsupervised machine learning method, shows that TCDD binding to the AhR PASB domain influences the stability of several inter-domain interactions, in particular at the PASA-PASB interface. The inter-domain communication network suggests a mechanism by which TCDD binding allosterically stabilizes the interactions at the DNA recognition site. These findings may have implications for the comprehension of the different toxic outcomes of AhR ligands and drug design.

Ligand‐dependent protein interactions of the juvenile hormone receptor captured in real time

Abstract: Juvenile hormone (JH) signalling provides vital regulatory functions during insect development via transcriptional regulation of genes critical for the progression of metamorphosis and oogenesis. Despite the importance of JH signalling, the underlying molecular mechanisms remain largely unknown. Our current understanding of the pathway depends on static end‐point information and suffers from the lack of time‐resolved data. Here, we have addressed the dynamic aspect of JH signalling by monitoring in real time the interactions of insect JH receptor proteins. Use of two tags that reconstitute a functional luciferase when in proximity enabled us to follow the rapid assembly of a JH receptor heterodimer from basic helix–loop–helix/Per‐Arnt‐SIM (bHLH‐PAS) proteins, methoprene‐tolerant (Met) and taiman (Tai), upon specific JH binding to Met. On a similar timescale (minutes), the dissociation of Met‐Met complexes occurred, again strictly dependent on Met interaction with specific agonist ligands. To resolve questions regarding the regulatory role of the chaperone Hsp90/83 in the JHR complex formation, we used the same technique to demonstrate that the Met‐Hsp83 complex persisted in the agonist absence but readily dissociated upon specific binding of JH to Met. Preincubation with the Hsp90 inhibitor geldanamycin showed that the chaperone interaction protected Met from degradation and was critical for Met to produce the active signalling dimer with Tai. Thus, the JH receptor functions appear to be governed by principles similar to those regulating the aryl hydrocarbon receptor, the closest vertebrate homologue of the arthropod JH receptor.

A Novel HIF-2α/ARNT Signaling Pathway Protects Against Microvascular Dysfunction and heart failure After Myocardial Infarction

Abstract: Rationale Cardiac microvascular leakage and inflammation are triggered during myocardial infarction (MI) and contribute to heart failure. Hypoxia-inducible factor 2α (Hif2α) is highly expressed in endothelial cells (ECs) and rapidly activated by myocardial ischemia, but whether it has a role in endothelial barrier function during MI is unclear. Objective To test our hypothesis that the expression of Hif2α and its binding partner aryl hydrocarbon nuclear translocator (ARNT) in ECs regulate cardiac microvascular permeability in infarcted hearts. Methods and Results Experiments were conducted with mice carrying an inducible EC-specific Hif2α-knockout (ecHif2α–/–) mutation, with mouse cardiac microvascular endothelial cells (CMVECs) isolated from the hearts of ecHif2α–/– mice after the mutation was induced, and with human CMVECs and umbilical-vein endothelial cells transfected with ecHif2α siRNA. After MI induction, echocardiographic assessments of cardiac function were significantly lower, while measures of cardiac microvascular leakage (Evans blue assay), plasma IL6 levels, and cardiac neutrophil accumulation and fibrosis (histology) were significantly greater, in ecHif2α–/– mice than in control mice, and RNA-sequencing analysis of heart tissues from both groups indicated that the expression of genes involved in vascular permeability and collagen synthesis was enriched in ecHif2α–/– hearts. In cultured ECs, ecHif2α deficiency was associated with declines in endothelial barrier function (electrical cell impedance assay) and the reduced abundance of tight-junction proteins, as well as an increase in the expression of inflammatory markers, all of which were largely reversed by the overexpression of ARNT. We also found that ARNT, but not Hif2α, binds directly to the IL6 promoter and suppresses IL6 expression. Conclusions EC-specific deficiencies in Hif2α expression significantly increase cardiac microvascular permeability, promote inflammation, and reduce cardiac function in infarcted mouse hearts, and ARNT overexpression can reverse the upregulation of inflammatory genes and restore endothelial-barrier function in Hif2α-deficient ECs. GRAPHIC ABSTRACT A graphic abstract is available for this article. Diagram of the role of HF2a/ARNT in the cardiac MI.

Discovery of Norisoboldine Analogue III11 as a Novel and Potent Aryl Hydrocarbon Receptor Agonist for the Treatment of Ulcerative Colitis.

Abstract: The aryl hydrocarbon receptor (AhR) is a transcript factor, belonging to the basic helix-loop-helix-Per-ARNT-SIM family, is closely associated with health and diseases. Targeting AhR is an emerging therapeutic strategy for various diseases. Norisoboldine (NOR), which is the main alkaloid of Linderae Radix, has been known to activate AhR. Unfortunately, the oral bioavailability (F) of NOR is only 2.49%. To improve the chemical efficacy and bioavailability, we designed and synthesized NOR analogues. Using various in vitro assays, 2-methoxy-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-9-ol (III11) was discovered as a potent AhR agonist. Compound III11 enhanced the expression of AhR downstream target genes, triggered AhR nuclear translocation, and promoted differentiation of regulatory T cells. More importantly, III11 exhibited good bioavailability (F = 87.40%) and remarkable therapeutic effects in a mouse model of ulcerative colitis at a dosage of 10 mg/kg. These findings may serve as a reference for the design of novel AhR agonists against immune and inflammatory diseases.

Aryl Hydrocarbon Receptor Agonism Antagonizes the Hypoxia-driven Inflammation in Cystic Fibrosis

Abstract: Hypoxia contributes to the exaggerated yet ineffective airway inflammation that fails to oppose infections in cystic fibrosis (CF). However, the potential for impairment of essential immune functions by HIF-1α (hypoxia-inducible factor 1α) inhibition demands a better comprehension of downstream hypoxia-dependent pathways that are amenable for manipulation. We assessed here whether hypoxia may interfere with the activity of AhR (aryl hydrocarbon receptor), a versatile environmental sensor highly expressed in the lungs, where it plays a homeostatic role. We used murine models of Aspergillus fumigatus infection in vivo and human cells in vitro to define the functional role of AhR in CF, evaluate the impact of hypoxia on AhR expression and activity, and assess whether AhR agonism may antagonize hypoxia-driven inflammation. We demonstrated that there is an important interferential cross-talk between the AhR and HIF-1α signaling pathways in murine and human CF, in that HIF-1α induction squelched the normal AhR response through an impaired formation of the AhR:ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF-1β heterodimer. However, functional studies and analysis of the AhR genetic variability in patients with CF proved that AhR agonism could prevent hypoxia-driven inflammation, restore immune homeostasis, and improve lung function. This study emphasizes the contribution of environmental factors, such as infections, in CF disease progression and suggests the exploitation of hypoxia:xenobiotic receptor cross-talk for antiinflammatory therapy in CF.

AhR and HIF-1α Signaling Pathways in Benign Meningioma under Hypoxia

Abstract: The role of hypoxia in benign meningiomas is less clear than that in the malignant meningiomas. Hypoxia-induced transcription factor 1 subunit alpha (HIF-1α) and its downstream signaling pathways play a central role in the mechanism of hypoxia. HIF-1α forms a complex with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein and can compete for ARNT with aryl hydrocarbon receptor (AhR). In this work, the status of HIF-1α- and AhR-dependent signaling pathways was investigated in World Health Organization (WHO) grade 1 meningioma and patient-derived tumor primary cell culture under hypoxic conditions. mRNA levels of HIF-1α, AhR, and of their target genes as well as of ARNT and nuclear receptor coactivator NCOA2 were determined in tumor tissues from patients in whom the tumor was promptly removed either with or without prior endovascular embolization. Using the patient-derived nonembolized tumor primary cell culture, the effects of a hypoxia mimetic cobalt chloride (CoCl2) and an activator of the AhR signaling pathway benzo(α)pyrene (B[a]P) on mRNA levels of HIF-1α, AhR, and their target genes were investigated. Our findings show active functioning of AhR signaling in meningioma tissue of patients with tumor embolization and crosstalk between HIF-1α and AhR signaling in meningeal cells under hypoxia.

Bioluminescence imaging of Cyp1a1-luciferase reporter mice demonstrates prolonged activation of the aryl hydrocarbon receptor in the lung

Abstract: Aryl hydrocarbon receptor (AHR) signalling integrates biological processes that sense and respond to environmental, dietary, and metabolic challenges to ensure tissue homeostasis. AHR is a transcription factor that is inactive in the cytosol but upon encounter with ligand translocates to the nucleus and drives the expression of AHR targets, including genes of the cytochrome P4501 family of enzymes such as Cyp1a1. To dynamically visualise AHR activity in vivo, we generated reporter mice in which firefly luciferase (Fluc) was non-disruptively targeted into the endogenous Cyp1a1 locus. Exposure of these animals to FICZ, 3-MC or to dietary I3C induced strong bioluminescence signal and Cyp1a1 expression in many organs including liver, lung and intestine. Longitudinal studies revealed that AHR activity was surprisingly long-lived in the lung, with sustained Cyp1a1 expression evident in discrete populations of cells including columnar epithelia around bronchioles. Our data link diet to lung physiology and also reveal the power of bespoke Cyp1a1-Fluc reporters to longitudinally monitor AHR activity in vivo.

#6635 arnt and alk3 mediate reno protection after ischemic preconditioning

Abstract: Ischemia and following hypoxia are a common cause of acute kidney injury (AKI). Strategies for prevention and treatment of AKI are limited. In several animal models, ischemic preconditioning (IPC) has been established as an effective intervention to prevent ischemic organ injury. An IPC protocol of ischemia and reperfusion mediates organ protection for a few hours. This first phase of organ protection is followed by a second, protective phase occurring after 24–48 h. The second phase, named second window of protection (SWOP), persists for 3–4 d. Mechanisms and transcriptional mediators during SWOP are mostly unknown. The aim of the underlying study was to investigate effectors and target genes during SWOP and to identify a potential pharmacological therapeutic target. 6–8-week-old C57BL/6 mice were undergoing an IPC intervention; five minutes of renal sinistra artery ischemia was followed by five minutes of reperfusion. This cycle was performed five times. Mice were sacrificed 2, 4, 8, 12, 25, 48, 72 h after IPC. Two additional groups of mice were pharmacologically preconditioned for 48 h with low dose FK506 and small molecule GPI1046. Analysis of murine kidneys were performed by SDS-Page and Western blot. The first phase after IPC (2–4 h) was characterized by an increasing expression of hypoxia inducible factor-1α (HIF1α); 48 h after intervention a significant increase of expression of aryl hydrocarbon receptor nuclear translocator (ARNT) and consecutive activin receptor-like kinase 3 (ALK3) was observed. Comparable induction of ARNT and ALK3 expression was obtained by pharmacological preconditioning with FK506 or GPI1046. Affected by hypoxia, HIF1α activates transcriptional upregulation of ARNT 48 h after IPC. The increasing expression of ARNT, follows an increased ARNT homodimerization. ARNT homodimer binds to the palindromic E-box 5′-CACGTG-3′ of the proximal ALK3 promoter. By this it induces the expression of the renoprotective ALK3 during SWOP. The organ-protective effects of constitutively expressed ARNT during SWOP can be mimicked pharmacologically by administration of FK506 or GPI1046 and may be used as a future therapeutic target.

Bioluminescence imaging of Cyp1a1-luciferase reporter mice demonstrates prolonged activation of the aryl hydrocarbon receptor in the lung

Abstract: Abstract Aryl hydrocarbon receptor (AHR) signalling integrates biological processes that sense and respond to environmental, dietary, and metabolic challenges to ensure tissue homeostasis. AHR is a transcription factor that is inactive in the cytosol but upon encounter with ligand translocates to the nucleus and drives the expression of AHR targets, including genes of the cytochrome P4501 family of enzymes such as Cyp1a1 . To dynamically visualise AHR activity in vivo, we generated reporter mice in which firefly luciferase ( Fluc ) was non-disruptively targeted into the endogenous Cyp1a1 locus. Exposure of these animals to FICZ, 3-MC or to dietary I3C induced strong bioluminescence signal and Cyp1a1 expression in many organs including liver, lung and intestine. Longitudinal studies revealed that AHR activity was surprisingly long-lived in the lung, with sustained Cyp1a1 expression evident in discrete populations of cells including columnar epithelia around bronchioles. Our data link diet to lung physiology and also reveal the power of bespoke Cyp1a1-Fluc reporters to longitudinally monitor AHR activity in vivo.

Molecular Docking of Natural Compounds for Potential Inhibition of AhR

Abstract: The aryl hydrocarbon receptor (AhR) is a highly conserved environmental sensor, historically known for mediating the toxicity of xenobiotics. It is involved in numerous cellular processes such as differentiation, proliferation, immunity, inflammation, homeostasis, and metabolism. It exerts a central role in several conditions such as cancer, inflammation, and aging, acting as a transcription factor belonging to the basic helix–loop–helix/Per-ARNT-Sim (bHLH-PAS) protein family. A key step in the canonical AhR activation is AhR-ARNT heterodimerization followed by the binding to the xenobiotic-responsive elements (XREs). The present work aims to investigate the potential AhR inhibitory activity of selected natural compounds. Due to the absence of a complete structure of human AhRs, a model consisting of the bHLH, the PAS A, and the PAS B domains was constructed. Blind and focused docking simulations revealed the presence of further binding pockets, different from the canonical one presented in the PAS B domain, which could be important for AhR inhibition due to the possibility to impede AhR:ARNT heterodimerization, either preventing conformational changes or masking crucial sites necessary for protein–protein interaction. Two of the compounds retrieved from the docking simulations, i.e., β-carotene and ellagic acid, confirmed their capacity of inhibiting benzo[a]pyrene (BaP)-induced AhR activation in in vitro tests on the human hepatoma cell line HepG2, validating the efficacy of the computational approach.

Discovery of a highly potent NPAS3 heterodimer inhibitor by covalently modifying ARNT.

Abstract: Neuronal PAS domain protein 3 (NPAS3), a basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) family member, is a pivotal transcription factor in neuronal regeneration, development, and related diseases, regulating the expression of downstream genes. Despite several modulators of certain bHLH-PAS family proteins being identified, the NPAS3-targeted compound has yet to be reported. Herein, we discovered a hit compound BI-78D3 that directly blocks the NPAS3-ARNT heterodimer formation by covalently binding to the aryl hydrocarbon receptor nuclear translocator (ARNT) subunit. Further optimization based on the hit scaffold yielded a highly potent Compound 6 with a biochemical EC50 value of 282 ± 61 nM and uncovered the 5-nitrothiazole-2-sulfydryl as a cysteine-targeting covalent warhead. Compound 6 effectively down-regulated NPAS3's transcriptional function by disrupting the interface of NPAS3-ARNT complexes at cellular level. In conclusion, our study identifies the 5-nitrothiazole-2-sulfydryl as a cysteine-modified warhead and provides a strategy that blocks the NPAS3-ARNT heterodimerization by covalently conjugating ARNT Cys336 residue. Compound 6 may serve as a promising chemical probe for exploring NPAS3-related physiological functions.

Aryl hydrocarbon receptor nuclear translocator limits the recruitment and function of regulatory neutrophils against colorectal cancer by regulating the gut microbiota

Abstract: Although the role and mechanism of neutrophils in tumors have been widely studied, the precise effects of aryl hydrocarbon receptor nuclear translocator (ARNT) on neutrophils remain unclear. In this study, we investigated the roles of ARNT in the function of CD11b+Gr1+ neutrophils in colitis-associated colorectal cancer.Wild-type (WT), ARNT myeloid-specific deficient mice and a colitis-associated colorectal cancer mouse model were used in this study. The level and functions of CD11b+Gr1+ cells were evaluated by flow cytometry and confocal microscopy.We found that ARNT deficiency drives neutrophils recruitment, neutrophil extracellular trap (NET) development, inflammatory cytokine secretion and suppressive activities when cells enter the periphery from bone marrow upon colorectal tumorigenesis. ARNT deficiency displays similar effects to aryl hydrocarbon receptor (AHR) deficiency in neutrophils. CXCR2 is required for NET development, cytokine production and recruitment of neutrophils but not the suppressive activities induced by Arnt-/- in colorectal cancer. The gut microbiota is essential for functional alterations in Arnt-/- neutrophils to promote colorectal cancer growth. The colorectal cancer effects of Arnt-/- neutrophils were significantly restored by mouse cohousing or antibiotic treatment. Intragastric administration of the feces of Arnt-/- mice phenocopied their colorectal cancer effects.Our results defined a new role for the transcription factor ARNT in regulating neutrophils recruitment and function and the gut microbiota with implications for the future combination of gut microbiota and immunotherapy approaches in colorectal cancer.

Η επίδραση της υποξίας στους μηχανισμούς επιβίωσης και απόπτωσης των καρκινικών κυττάρων

Abstract: Ως υποξία ορίζεται η ελάττωση των διαθέσιμων επιπέδων οξυγόνου που παρατηρείται σε κύτταρα ή ιστούς και είναι χαρακτηριστικό πολλών φυσιολογικών (π.χ. μεγάλο υψόμετρο και έντονη μυϊκή άσκηση) και παθολογικών καταστάσεων (π.χ. ισχαιμία, καρκίνος). Ο HIF-1 αποτελεί μεταξύ άλλων, την κύρια πρωτεΐνη, που ρυθμίζει την απόκριση των κυττάρων στην υποξία, κυρίως μέσω της δράσης του ως μεταγραφικός παράγοντας. Επιπλέον, η δράση του έχει συσχετιστεί με προώθηση της επιβίωσης και αναστολή της απόπτωσης σε διάφορους καρκίνους. Ο ΗΙF-1 είναι ετεροδιμερές αποτελούμενος από μία υπομονάδα που εκφράζεται συνεχώς, την HIF-1β ή ARNT, και από την υπομονάδα HIF-1α, η έκφραση της οποίας ρυθμίζεται οξυγονο-εξαρτώμενα μέσω μετα-μεταφραστικής τροποποίησης (υδροξυλίωσης) και αποικοδόμησης. Ωστόσο ο HIF-1α επιδρά σε διάφορες κυτταρικές λειτουργίες και μέσω μηχανισμών ανεξάρτητων από τη μεταγραφική του δράση, αλληλεπιδρώντας με άλλες πρωτεΐνες εκτός του ARNT. Από προηγούμενες δημοσιεύσεις του εργαστηρίου Βιοχημείας, δείχθηκε ότι η υποκυτταρική κατανομή και οι αλληλεπιδράσεις του HIF-1α με άλλες πρωτεΐνες, ρυθμίζονται μέσω φωσφορυλίωσης από τις κινάσες ERK1/2 στα κατάλοιπα Ser641/Ser643. Όταν ο HIF-1α τροποποιείται σε αυτές τις θέσεις, συγκεντρώνεται στο εσωτερικό του πυρήνα, όπου αλληλεπιδρά με την νουκλεοφοσμίνη (NPM1) και ενεργοποιεί τη μεταγραφή των γονιδίων-στόχων του. Η απώλεια της φωσφορυλίωσης στις θέσεις 641 και 643, έχει ως αποτέλεσμα την αλληλεπίδραση του HIF-1α με την εξπορτίνη CRM1 και την εξαγωγή του από τον πυρήνα και στο κυτταρόπλασμα, όπου αλληλεπιδρά με τη μορταλίνη, η οποία διαμεσολαβεί τη στόχευση του στα μιτοχόνδρια και την συγκρότηση ενός συμπλόκου που αναστέλλει την έναρξη της απόπτωσης.Κύριο θέμα της παρούσας διατριβής αποτέλεσε ο χαρακτηρισμός της αλληλεπίδρασης μεταξύ HIF-1α και μορταλίνης. Για το σκοπό αυτό πραγματοποιήθηκαν, τόσο πειράματα in vitro δέσμευσης, χρησιμοποιώντας ανασυνδυασμένα τμήματα των δύο πρωτεϊνών, όσο και ανοσοκατακρήμνισης, ώστε να χαρτογραφηθούν οι αντίστοιχες θέσεις δέσμευσης μεταξύ τους και να δειχθεί ότι η αλληλεπίδραση είναι άμεση και λειτουργική. Συγκεκριμένα αποδείχθηκε ότι η καρβοξυτελική επικράτεια της μορταλίνης δεσμεύεται άμεσα και εξειδικευμένα με τον μη φωσφορυλιωμένο HIF-1α. Επίσης, δείχθηκε ότι ένα φυσικό παράγωγο, που ονομάζεται εμπελίνη και είναι γνωστός αναστολέας της αλληλεπίδρασης μεταξύ της μορταλίνης και της p53, μπορεί να αναστείλει την αλληλεπίδραση μορταλίνης/HIF-1α και να οδηγήσει σε απομάκρυνση του μη φωσφορυλιωμένου HIF-1α από τα μιτοχόνδρια. Η επαγόμενη από την εμπελίνη, απομάκρυνση του μη φωσφορυλιωμένου HIF-1α από την εξωτερική μιτοχονδριακή μεμβράνη οδηγεί στη μετακίνηση της προ-αποπτωτικής πρωτεΐνης Bax (B-cell lymphoma 2-associated X protein) στα μιτοχόνδρια, κάτω από συνθήκες στρες. Συνοψίζοντας, τα αποτελέσματα αποδεικνύουν ότι σε συνθήκες υποξίας και απενεργοποίησης των ERK1/2, η άμεση και ειδική δέσμευση μεταξύ της μορταλίνης και του HIF-1α, παρεμποδίζει τη στόχευση της Bax στα μιτοχόνδρια και προστατεύει τα κύτταρα από κυτταρικό θάνατο μέσω απόπτωσης.

Cap ‘n’ Collar C and Aryl Hydrocarbon Receptor Nuclear Translocator Facilitate the Expression of Glutathione S-Transferases Conferring Adaptation to Tannic Acid and Quercetin in Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae)

Abstract: Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae) is a notorious pest of poplar. Coevolution with poplars rich in plant secondary metabolites prompts M. troglodyta to expand effective detoxification mechanisms against toxic plant secondary metabolites. Although glutathione S-transferases (GSTs) play an important role in xenobiotic detoxification in M. troglodyta, it is unclear how GSTs act in response to toxic secondary metabolites in poplar. In this study, five GST gene core promoters were accurately identified by a 5’ loss luciferase reporter assay, and the core promoters were significantly induced by two plant secondary metabolites in vitro. Two transcription factors, cap ‘n’ collar C (CncC) and aryl hydrocarbon receptor nuclear translocator (ARNT), were cloned in M. troglodyta. MtCncC and MtARNT clustered well with other insect CncCs and ARNTs, respectively. In addition, MtCncC and MtARNT could bind the MtGSTt1 promoter and strongly improve transcriptional activity, respectively. However, MtCncC and MtARNT had no regulatory function on the MtGSTz1 promoter. Our findings revealed the molecular mechanisms of the transcription factors MtCncC and MtARNT in regulating the GST genes of M. troglodyta. These results provide useful information for the control of M. troglodyta.