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1.
Comput Biol Med ; 149: 106006, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36027865

RESUMO

Hypoxia-inducible factor 1 (HIF-1), a transcriptional activator that mediates cellular responses to hypoxic stress, is essential for tumor progression. It is a heterodimer comprising HIF1α and HIF1ß, with multiple interfaces among their PAS-A, PAS-B, and bHLH domains. HIF1ß is also known as aryl hydrocarbon receptor nuclear translocator (ARNT). Casein kinase 1δ-dependent phosphorylation of the solvent-front residue S247 on the HIF1α PAS-B domain interrupts HIF1α-ARNT complex formation and reduces HIF-1 transcription activity. However, S247 is involved in neither HIF1α-ARNT complex formation nor stabilization of the relative orientation between the HIF1α PAS-A and PAS-B domains. To uncover the underlying allosteric mechanism, we conducted Gaussian accelerated molecular dynamics simulations and identified two distinct conformations of the pS247-carrying HIF1α PAS-B domain: H291-in and H291-out. The H291-in structure can associate with the HIF1α PAS-A domain and form a V-shaped pouch to accommodate the ARNT PAS-A domain, but it cannot associate with the ARNT PAS-B domain. By contrast, the H291-out structure can bind to the ARNT PAS-B domain, but its association with the HIF1α PAS-A domain leads to an unsuitable relative orientation to accommodate the ARNT PAS-A domain. Both conformations were also collected in parallel simulations of the unphosphorylated PAS-B domain. Both structures manage to associate with the ARNT PAS-B and HIF1α PAS-A domains; thus, they are adequate for HIF1α-ARNT complex formation. The domain-domain contact pattern in a phosphorylated variant is shuffled by an order-to-disorder structural switch, triggered by the newly formed K251-pS247 interaction.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Subunidade alfa do Fator 1 Induzível por Hipóxia , Translocador Nuclear Receptor Aril Hidrocarboneto/química , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Caseína Quinases/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fosforilação , Solventes
2.
Viruses ; 14(7)2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35891329

RESUMO

Increasing evidence suggests that the polymerase acidic (PA) protein of influenza A viruses plays an important role in viral replication and pathogenicity. However, information regarding the interaction(s) of host factors with PA is scarce. By using a yeast two-hybrid screen, we identified a novel host factor, aryl hydrocarbon receptor nuclear translocator (ARNT), that interacts with the PA protein of the H5N1 virus. The interaction between PA and human ARNT was confirmed by co-immunoprecipitation and immunofluorescence microscopy. Moreover, overexpression of ARNT downregulated the polymerase activity and inhibited virus propagation, whereas knockdown of ARNT significantly increased the polymerase activity and virus replication. Mechanistically, overexpression of ARNT resulted in the accumulation of PA protein in the nucleus and inhibited both the replication and transcription of the viral genome. Interaction domain mapping revealed that the bHLH/PAS domain of ARNT mainly interacted with the C-terminal domain of PA. Together, our results demonstrate that ARNT inhibits the replication of the H5N1 virus and could be a target for the development of therapeutic strategies against H5N1 influenza viruses.


Assuntos
Virus da Influenza A Subtipo H5N1 , Vírus da Influenza A , Influenza Humana , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Humanos , RNA Polimerase Dependente de RNA/metabolismo , Replicação Viral/genética
3.
Biochem J ; 479(13): 1441-1454, 2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35730699

RESUMO

Single-minded 2 (SIM2) is a neuron-enriched basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH/PAS) transcription factor essential for mammalian survival. SIM2 is located within the Down syndrome critical region (DSCR) of chromosome 21, and manipulation in mouse models suggests Sim2 may play a role in brain development and function. During the screening of a clinical exome sequencing database, nine SIM2 non-synonymous mutations were found which were subsequently investigated for impaired function using cell-based reporter gene assays. Many of these human variants attenuated abilities to activate transcription and were further characterized to determine the mechanisms underpinning their deficiencies. These included impaired partner protein dimerization, reduced DNA binding, and reduced expression and nuclear localization. This study highlighted several SIM2 variants found in patients with disabilities and validated a candidate set as potentially contributing to pathology.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Síndrome de Down , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Núcleo Celular/metabolismo , Síndrome de Down/metabolismo , Humanos , Mamíferos/metabolismo , Camundongos , Fenótipo , Receptores de Hidrocarboneto Arílico/metabolismo , Fatores de Transcrição/metabolismo
4.
Am J Physiol Cell Physiol ; 323(2): C322-C332, 2022 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-35704696

RESUMO

The increasing amount of data points to the circadian timing system as an essential part of processes regulating androgen homeostasis. However, the relationship between stress response, timekeeping-, and steroidogenesis-related systems is unexplored. Here, the stress-response of the testosterone-producing rat Leydig cells depending on the time of stressful events was studied. The study analyzes the effects of 3-h immobilization (IMO) applied at different periods during the day. The IMO performed once [1 time immobilization stress (1×IMO)] or repeated in 10 consecutive days [10 time repeated immobilization stress (10×IMO)]. Both types of IMO increased corticosterone and decreased testosterone blood level. However, the effect of 10×IMO occurring in the active phase on blood testosterone was less pronounced. This is related to different sensitivity to IMO-events depending on the diurnal time. Most steroidogenesis-related genes [gene encoding luteinizing hormone/choriogonadotropin receptor (Lhcgr), gene encoding cytochrome P450, family 11, subfamily a, polypeptide 1 (Cyp11a1), gene encoding hydroxy-δ-5-steroid dehydrogenase, 3 ß- and steroid δ-isomerase 1 (Hsd3b1/2), and gene encoding cytochrome P450, family 17, subfamily a, polypeptide 1 (Cyp17a1)] were downregulated in the inactive phase but unchanged or even upregulated in the active phase of the day. Both types of IMO stimulated the expression of clock elements gene encoding aryl hydrocarbon receptor nuclear translocator-like (Bmal1)/aryl hydrocarbon receptor nuclear translocator-like (BMAL1), gene encoding period circadian regulator 1 (Per1)/period circadian regulator 1 (PER1) regardless of the day's stage and reduced gene encoding nuclear receptor subfamily 1, group D, member 1 (Rev-erba) in the inactive phase. The principal component analysis (PCA) confirmed a major shift, for both IMO-types, in the transcription of the genes across the passive/active stage. Further, 10×IMO changed a diurnal pattern of the glucocorticoid receptor [gene encoding nuclear receptor subfamily 3, group C, member 1 (Nr3c1)/nuclear receptor subfamily 3, group C, member 1 (GR)] expression, whereas the observed time-dependent IMO-response of the Leydig cells correlated with different corticosterone engagements. Altogether, the Leydig cell's stress response depends on the daytime of the stressful event, emphasizing the importance of the circadian system in supporting androgen homeostasis and male fertility.


Assuntos
Androgênios , Células Intersticiais do Testículo , Fatores de Transcrição ARNTL/metabolismo , Androgênios/metabolismo , Androgênios/farmacologia , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/farmacologia , Corticosterona/farmacologia , Células Intersticiais do Testículo/metabolismo , Masculino , Ratos , Ratos Wistar , Testosterona/farmacologia
5.
Zhongguo Zhong Yao Za Zhi ; 47(10): 2698-2704, 2022 May.
Artigo em Chinês | MEDLINE | ID: mdl-35718489

RESUMO

This study aimed to explore the effect of artesunate(ARS) on bone destruction in rheumatoid arthritis(RA) based on the aryl hydrocarbon receptor(AhR)/AhR nucleart ranslocator(ARNT)/NAD(P)H quinone dehydrogenase 1(NQO1) signaling pathway. Macrophage-colony stimulating factor(M-CSF) and receptor activator of nuclear factor-κB(RANKL) were used to induce the differentiation of primary bone marrow-derived mouse macrophages into osteoclasts. After intervention with ARS(0.2, 0.4, and 0.8 µmol·L~(-1)), the formation and differentiation of osteoclasts were observed by tartrate-resistant acid phosphatase(TRAP) and F-actin staining. The protein expression levels of AhR and NQO1 were detected by Western blot, and their distribution in osteoclasts was observed by immunofluorescence localization. Simultaneously, the collagen induced arthritis(CIA) rat model was established using type Ⅱ bovine collagen emulsion and then treated with ARS(7.5, 15, and 30 mg·kg~(-1)) by gavage for 30 days. Following the observation of spinal cord and bone destruction in CIA rats by Masson staining, the expression of AhR and ARNT in rat knee joint tissue was measured by immunohistochemistry and the NQO1 protein expression in the knee joint tissue by Western blot. The results showed that a large number of TRAP-positive cells were present in RANKL-induced rats. Compared with the RANKL-induced group, ARS(0.2, 0.4, and 0.8 µmol·L~(-1)) inhibited the number of TRAP-positive cells in a dose-dependent manner. F-actin staining results showed that the inhibition of F-actin formation was enhanced with the increase in ARS dose. As revealed by Western blot and immunofluorescence assay, ARS significantly promoted the expression of AhR and its transfer to the nucleus, thereby activating the protein expression of downstream ARNT and antioxidant enzyme NQO1. At the same time, the CIA rat model was successfully established. Masson staining revealed serious joint destruction in the model group, manifested by the failed staining of surface cartilage, disordered arrangement of collagen fibers, and unclear boundaries of cartilage and bone. The positive drug and ARS at different doses all improved cartilage and bone destruction to varying degrees, with the best efficacy detected in the high-dose ARS group. According to immunohistochemistry, ARS promoted AhR and ARNT protein expression in knee cartilage and bone of CIA rats and also NQO1 protein expression in rat knee and ankle joint tissues. In conclusion, ARS inhibited osteoclast differentiation by activating the AhR/ARNT/NQO1 signaling pathway, thus alleviating RA.


Assuntos
Artrite Experimental , Artrite Reumatoide , Actinas/metabolismo , Animais , Artesunato/farmacologia , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/farmacologia , Bovinos , Colágeno Tipo II/metabolismo , Camundongos , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Osteoclastos , Ratos , Transdução de Sinais
6.
Biochem Soc Trans ; 50(3): 1227-1243, 2022 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-35695677

RESUMO

Proteins that contain basic helix-loop-helix (bHLH) and Per-Arnt-Sim motifs (PAS) function as transcription factors. bHLH-PAS proteins exhibit essential and diverse functions throughout the body, from cell specification and differentiation in embryonic development to the proper function of organs like the brain and liver in adulthood. bHLH-PAS proteins are divided into two classes, which form heterodimers to regulate transcription. Class I bHLH-PAS proteins are typically activated in response to specific stimuli, while class II proteins are expressed more ubiquitously. Here, we discuss the general structure and functions of bHLH-PAS proteins throughout the animal kingdom, including family members that do not fit neatly into the class I-class II organization. We review heterodimerization between class I and class II bHLH-PAS proteins, binding partner selectivity and functional redundancy. Finally, we discuss the evolution of bHLH-PAS proteins, and why a class I protein essential for cardiovascular development in vertebrates like chicken and fish is absent from mammals.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/química , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Dimerização , Mamíferos/metabolismo , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/metabolismo
7.
Mol Cell Biol ; 42(5): e0050321, 2022 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-35404107

RESUMO

Aberrant alternative splicing (AS) of pre-mRNAs promotes the development and proliferation of cancerous cells. Accordingly, we had previously observed higher levels of the aryl hydrocarbon receptor nuclear translocator (ARNT) spliced variant isoform 1 in human lymphoid malignancies compared to that in normal lymphoid cells, which is a consequence of increased inclusion of alternative exon 5. ARNT is a transcription factor that has been implicated in the survival of various cancers. Notably, we found that ARNT isoform 1 promoted the growth and survival of lymphoid malignancies, but the regulatory mechanism controlling ARNT AS is unclear. Here, we report cis- and trans-regulatory elements which are important for the inclusion of ARNT exon 5. Specifically, we identified recognition motifs for the RNA-binding protein RBFOX2, which are required for RBFOX2-mediated exon 5 inclusion. RBFOX2 upregulation was observed in lymphoid malignancies, correlating with the observed increase in ARNT exon 5 inclusion. Moreover, suppression of RBFOX2 significantly reduced ARNT isoform 1 levels and cell growth. These observations reveal RBFOX2 as a critical regulator of ARNT AS in lymphoid malignancies and suggest that blocking the ARNT-specific RBFOX2 motifs to decrease ARNT isoform 1 levels is a viable option for targeting the growth of lymphoid malignancies.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Neoplasias , Processamento Alternativo/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Éxons/genética , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas Repressoras/metabolismo
8.
BMC Pharmacol Toxicol ; 23(1): 26, 2022 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-35473600

RESUMO

BACKGROUND: The main causes of lung cancer are smoking, environmental pollution and genetic susceptibility. It is an indisputable fact that PAHs are related to lung cancer, and benzo(a) pyrene is a representative of PAHs. The purpose of the current investigation was to investigate the interaction between AhR and HIF-1 signaling pathways in A549 cells, which provide some experimental basis for scientists to find drugs that block AhR and HIF-1 signaling pathway to prevent and treat cancer. METHODS: This project adopts the CYP1A1 signaling pathways and the expression of CYP1B1 is expressed as a measure of AhR strength index. The expression of VEGF and CAIX volume as a measure of the strength of the signal path HIF-1 indicators. Through the construction of plasmid vector, fluorescence resonance energy transfer, real-time quantitative PCR, western blotting and immunoprecipitation, the interaction between AhR signaling pathway and HIF-1 signaling pathway was observed. RESULTS: BaP can enhance the binding ability of HIF-1α protein to HIF-1ß/ARNT in a dose-dependent manner without CoCl2. However, the binding ability of AhR protein to HIF-1ß/ARNT is inhibited by HIF-1α signaling pathway in a dose-dependent manner with CoCl2. CONCLUSION: It is shown that activation of the AhR signaling pathway does not inhibit the HIF-1α signaling pathway, but activation of the HIF-1α signaling pathway inhibits the AhR signaling pathway.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Neoplasias Pulmonares , Receptores de Hidrocarboneto Arílico , Células A549 , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais
9.
Proc Natl Acad Sci U S A ; 119(12): e2114336119, 2022 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-35290121

RESUMO

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor present in immune cells as a long and short isoform, referred to as isoforms 1 and 3, respectively. However, investigation into potential ARNT isoform­specific immune functions is lacking despite the well-established heterodimerization requirement of ARNT with, and for the activity of, the aryl hydrocarbon receptor (AhR), a critical mediator of immune homeostasis. Here, using global and targeted transcriptomics analyses, we show that the relative ARNT isoform 1:3 ratio in human T cell lymphoma cells dictates the amplitude and direction of AhR target gene regulation. Specifically, shifting the ARNT isoform 1:3 ratio lower by suppressing isoform 1 enhances, or higher by suppressing isoform 3 abrogates, AhR responsiveness to ligand activation through preprograming a cellular genetic background that directs explicit gene expression patterns. Moreover, the fluctuations in gene expression patterns that accompany a decrease or increase in the ARNT isoform 1:3 ratio are associated with inflammation or immunosuppression, respectively. Molecular studies identified the unique casein kinase 2 (CK2) phosphorylation site within isoform 1 as an essential parameter to the mechanism of ARNT isoform­specific regulation of AhR signaling. Notably, CK2-mediated phosphorylation of ARNT isoform 1 is dependent on ligand-induced AhR nuclear translocation and is required for optimal AhR target gene regulation. These observations reveal ARNT as a central modulator of AhR activity predicated on the status of the ARNT isoform ratio and suggest that ARNT-based therapies are a viable option for tuning the immune system to target immune disorders.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Neoplasias , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Humanos , Ligantes , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Linfócitos T/metabolismo
10.
Sci Rep ; 12(1): 3045, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35197517

RESUMO

Transport and Golgi Organization protein 2 Homolog (TANGO2)-related disease is an autosomal recessive disorder caused by mutations in the TANGO2 gene. Symptoms typically manifest in early childhood and include developmental delay, stress-induced episodic rhabdomyolysis, and cardiac arrhythmias, along with severe metabolic crises including hypoglycemia, lactic acidosis, and hyperammonemia. Severity varies among and within families. Previous studies have reported contradictory evidence of mitochondrial dysfunction. Since the clinical symptoms and metabolic abnormalities are suggestive of a broad dysfunction of mitochondrial energy metabolism, we undertook a broad examination of mitochondrial bioenergetics in TANGO2 deficient patients utilizing skin fibroblasts derived from three patients exhibiting TANGO2-related disease. Functional studies revealed that TANGO2 protein was present in mitochondrial extracts of control cells but not patient cells. Superoxide production was increased in patient cells, while oxygen consumption rate, particularly under stress, along with relative ATP levels and ß-oxidation of oleate were reduced. Our findings suggest that mitochondrial function should be assessed and monitored in all patients with TANGO2 mutation as targeted treatment of the energy dysfunction could improve outcome in this condition.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/deficiência , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Adolescente , Adulto , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Células Cultivadas , Criança , Ácidos Graxos/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo
11.
Front Endocrinol (Lausanne) ; 12: 748216, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34858331

RESUMO

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) and excessive accumulation of dysfunctional PVAT are hallmarks of pathogenesis after angioplasty. Recent genome-wide association studies reveal that single-nucleotide polymorphism (SNP) in MIA3 is associated with atherosclerosis-relevant VSMC phenotypes. However, the role of MIA3 in the vascular remodeling response to injury remains unknown. Here, we found that expression of MIA3 is increased in proliferative VSMCs and knockdown of MIA3 reduces VSMCs proliferation, migration, and inflammation, whereas MIA3 overexpression promoted VSMC migration and proliferation. Moreover, knockdown of MIA3 ameliorates femoral artery wire injury-induced neointimal hyperplasia and increases brown-like perivascular adipocytes. Collectively, the data suggest that MIA3 deficiency prevents neointimal formation by decreasing VSMC proliferation, migration, and inflammation and maintaining BAT-like perivascular adipocytes in PVAT during injury-induced vascular remodeling, which provide a potential therapeutic target for preventing neointimal hyperplasia in proliferative vascular diseases.


Assuntos
Tecido Adiposo Marrom/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima/metabolismo , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Humanos , Camundongos , Neointima/genética , Polimorfismo de Nucleotídeo Único
12.
Sci Rep ; 11(1): 24075, 2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34912030

RESUMO

Aryl hydrocarbon receptor nuclear translocator (ARNT) mediates anti-fibrotic activity in kidney and liver through induction of ALK3-receptor expression and subsequently increased Smad1/5/8 signaling. While expression of ARNT can be pharmacologically induced by sub-immunosuppressive doses of FK506 or by GPI1046, its anti-fibrotic activity is only realized when ARNT-ARNT homodimers form, as opposed to formation of ARNT-AHR or ARNT-HIF1α heterodimers. Mechanisms underlying ARNTs dimerization decision to specifically form ARNT-ARNT homodimers and possible cues to specifically induce ARNT homodimerization have been previously unknown. Here, we demonstrate that phosphorylation of the Ser77 residue is critical for ARNT-ARNT homodimer formation and stabilization. We further demonstrate that inhibition of PP2A phosphatase activity by LB100 enhances ARNT-ARNT homodimers both in vivo and in vitro (mouse tubular epithelial cells and human embryonic kidney cells). In murine models of kidney fibrosis, and also of liver fibrosis, combinations of FK506 or GPI1046 (to induce ARNT expression) with LB100 (to enhance ARNT homodimerization) elicit additive anti-fibrotic activities. Our study provides additional evidence for the anti-fibrotic activity of ARNT-ARNT homodimers and reveals Ser77 phosphorylation as a novel pharmacological target to realize the therapeutic potential of increased ARNT transactivation activity.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Inibidores Enzimáticos/farmacologia , Multimerização Proteica , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/química , Biomarcadores , Modelos Animais de Doenças , Suscetibilidade a Doenças , Fibrose , Nefropatias/etiologia , Nefropatias/metabolismo , Nefropatias/patologia , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , Fosforilação/efeitos dos fármacos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais
13.
Proc Natl Acad Sci U S A ; 118(47)2021 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-34799448

RESUMO

Circadian transcriptional timekeepers in pacemaker neurons drive profound daily rhythms in sleep and wake. Here we reveal a molecular pathway that links core transcriptional oscillators to neuronal and behavioral rhythms. Using two independent genetic screens, we identified mutants of Transport and Golgi organization 10 (Tango10) with poor behavioral rhythmicity. Tango10 expression in pacemaker neurons expressing the neuropeptide PIGMENT-DISPERSING FACTOR (PDF) is required for robust rhythms. Loss of Tango10 results in elevated PDF accumulation in nerve terminals even in mutants lacking a functional core clock. TANGO10 protein itself is rhythmically expressed in PDF terminals. Mass spectrometry of TANGO10 complexes reveals interactions with the E3 ubiquitin ligase CULLIN 3 (CUL3). CUL3 depletion phenocopies Tango10 mutant effects on PDF even in the absence of the core clock gene timeless Patch clamp electrophysiology in Tango10 mutant neurons demonstrates elevated spontaneous firing potentially due to reduced voltage-gated Shaker-like potassium currents. We propose that Tango10/Cul3 transduces molecular oscillations from the core clock to neuropeptide release important for behavioral rhythms.


Assuntos
Relógios Circadianos/fisiologia , Proteínas de Drosophila/metabolismo , Neuropeptídeos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Proteínas Culina/genética , Proteínas Culina/metabolismo , Drosophila , Proteínas de Drosophila/genética , Neurônios/metabolismo , Neuropeptídeos/genética , Proteômica , Sono
14.
Mol Hum Reprod ; 27(12)2021 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-34665260

RESUMO

Placental hypoxia and increased levels of maternal blood anti-angiogenic protein, soluble fms-like tyrosine kinase-1 (sFLT1), are associated with the pathogenesis of pre-eclampsia. We have demonstrated that hypoxia-inducible factor (HIF)-2α mediates the upregulation of the hypoxia-induced FLT1 gene in trophoblasts and their cell lines. Here, we investigated the involvement of HIF-1ß, which acts as a dimerization partner for HIF-α, in the upregulation of the FLT1 gene via hypoxia. We confirmed the interactions between HIF-1ß and HIF-2α in the nuclei of BeWo, JAR and JEG-3 cells under hypoxia via co-immunoprecipitation. We found that hypoxia-induced upregulation of the FLT1 gene in BeWo cells and secretion of sFLT1 in human primary trophoblasts were significantly reduced by siRNAs targeting HIF-1ß. Moreover, the upregulation of the FLT1 gene in BeWo cells induced by dimethyloxaloylglycine (DMOG) was also inhibited by silencing either HIF-2α or HIF-1ß mRNA. It was recently shown that DNA demethylation increases both basal and hypoxia-induced expression levels of the FLT1 gene in three trophoblast-derived cell lines. In the demethylated BeWo cells, siRNAs targeting HIF-2α and HIF-1ß suppressed the further increase in the expression levels of the FLT1 gene due to hypoxia or treatment with DMOG. However, luciferase reporter assays and bisulfite sequencing revealed that a hypoxia response element (-966 to -962) of the FLT1 gene is not involved in hypoxia or DMOG-induced upregulation of the FLT1 gene. These findings suggest that HIF-1ß is essential for the elevated production of sFLT1 in the hypoxic trophoblasts and that the HIF-2α/HIF-1ß complex may be a crucial therapeutic target for pre-eclampsia.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Trofoblastos/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Aminoácidos Dicarboxílicos/farmacologia , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Hipóxia Celular , Linhagem Celular Tumoral , Metilação de DNA , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Gravidez , Trofoblastos/efeitos dos fármacos , Regulação para Cima , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética
15.
Cell Rep ; 36(11): 109707, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34525362

RESUMO

Secretory cargos are collected at endoplasmic reticulum (ER) exit sites (ERES) before transport to the Golgi apparatus. Decades of research have provided many details of the molecular events underlying ER-Golgi exchanges. Essential questions, however, remain about the organization of the ER-Golgi interface in cells and the type of membrane structures mediating traffic from ERES. To investigate these, we use transgenic tagging in Drosophila flies, 3D-structured illumination microscopy (SIM), and focused ion beam scanning electron microscopy (FIB-SEM) to characterize ERES-Golgi units in collagen-producing fat body, imaginal discs, and imaginal discs overexpressing ERES determinant Tango1. Facing ERES, we find a pre-cis-Golgi region, equivalent to the vertebrate ER-Golgi intermediate compartment (ERGIC), involved in both anterograde and retrograde transport. This pre-cis-Golgi is continuous with the rest of the Golgi, not a separate compartment or collection of large carriers, for which we find no evidence. We observe, however, many vesicles, as well as pearled tubules connecting ERES and Golgi.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Drosophila/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Fator 1 de Ribosilação do ADP/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Transporte Biológico , Proteínas de Drosophila/metabolismo , Retículo Endoplasmático/química , Complexo de Golgi/química , Proteínas da Matriz do Complexo de Golgi/metabolismo , Microscopia Eletrônica de Varredura , Proteínas Monoméricas de Ligação ao GTP/metabolismo
16.
Artigo em Inglês | MEDLINE | ID: mdl-34496301

RESUMO

The white shrimp Litopenaeus vannamei is exposed to hypoxic conditions in natural habitats and in shrimp farms. Hypoxia can retard growth, development and affect survival in shrimp. The hypoxia-inducible factor 1 (HIF-1) regulates many genes involved in glucose metabolism, antioxidant proteins, including metallothionein (MT) and apoptosis. In previous studies we found that the L. vannamei MT gene expression changed during hypoxia, and MT silencing altered cell apoptosis; in this study we investigated whether the silencing of HIF-1 affected MT expression and apoptosis. Double-stranded RNA (dsRNA) was used to silence HIF-1α and HIF-1ß under normoxia, hypoxia, and hypoxia plus reoxygenation. Expression of HIF-1α, HIF-1ß and MT, and apoptosis in hemocytes or caspase-3 expression in gills, were measured at 0, 3, 24 and 48 h of hypoxia and hypoxia followed by 1 h of reoxygenation. The results showed that hemocytes HIF-1α expression was induced during hypoxia and reoxygenation at 3 h, while HIF-1ß decreased at 24 and 48 h. In normoxia, HIF-1 silencing in hemocytes increased apoptosis at 3 h and decreased at 48 h; while in gills, caspase-3 increased at 3, 24 and 48 h. In hypoxia, HIF-1 silencing decreased apoptosis in hemocytes at 3 h, but caspase-3 increased in gills. During reoxygenation, apoptosis in hemocytes and caspase-3 in gills increased. During normoxia in hemocytes, silencing of HIF-1 decreased MT expression, but in gills, MT increased. During hypoxia and reoxygenation, silencing induced MT in hemocytes and gills. These results indicate HIF-1 differential participation in MT expression regulation and apoptosis during different oxygen conditions.


Assuntos
Apoptose , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Proteínas de Peixes/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Metalotioneína/metabolismo , Oxigênio/metabolismo , Penaeidae/metabolismo , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Brânquias/metabolismo , Brânquias/patologia , Hemócitos/metabolismo , Hemócitos/patologia , Hipóxia/genética , Hipóxia/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Metalotioneína/genética , Penaeidae/genética , Espécies Reativas de Oxigênio/metabolismo
17.
Sci Rep ; 11(1): 18194, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521881

RESUMO

The human aryl hydrocarbon receptor (AHR) is predominantly located in the cytoplasm, while activation depends on its nuclear translocation. Binding to endogenous or xenobiotic ligands terminates the basal nucleo-cytoplasmic shuttling and stabilizes an exclusive nuclear population. The precise mechanisms that facilitate such stable nuclear accumulation remain to be clarified as essential step in the activation cascade. In this study, we have tested whether the sustained nuclear compartmentalization of ligand-bound or basal AHR might further require heterodimerization with the AHR-nuclear translocator (ARNT) and binding to the cognate XRE-motif. Mutagenesis of the DNA-binding motif or of selected individual residues in the ARNT-binding motif did not lead to any variation in AHR's nucleo-cytoplasmic distribution. In response to ligands, all mutants were retained in the nucleus demonstrating that the stable compartmentalization of activated AHR in the nucleus is neither dependent on interactions with DNA, nor ARNT. Knocking down the ARNT gene using small interfering RNA confirmed that ARNT does not play any role in the intracellular trafficking of AHR.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Núcleo Celular/metabolismo , DNA/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Transporte Ativo do Núcleo Celular , Translocador Nuclear Receptor Aril Hidrocarboneto/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Sítios de Ligação , Células Hep G2 , Humanos , Células MCF-7 , Ligação Proteica , Multimerização Proteica , Receptores de Hidrocarboneto Arílico/química
18.
J Cell Sci ; 134(17)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34350936

RESUMO

Complex machinery is required to drive secretory cargo export from the endoplasmic reticulum (ER), which is an essential process in eukaryotic cells. In vertebrates, the MIA3 gene encodes two major forms of transport and Golgi organization protein 1 (TANGO1S and TANGO1L), which have previously been implicated in selective trafficking of procollagen. Using genome engineering of human cells, light microscopy, secretion assays, genomics and proteomics, we show that disruption of the longer form, TANGO1L, results in relatively minor defects in secretory pathway organization and function, including having limited impacts on procollagen secretion. In contrast, loss of both long and short forms results in major defects in cell organization and secretion. These include a failure to maintain the localization of ERGIC53 (also known as LMAN1) and SURF4 to the ER-Golgi intermediate compartment and dramatic changes to the ultrastructure of the ER-Golgi interface. Disruption of TANGO1 causes significant changes in early secretory pathway gene and protein expression, and impairs secretion not only of large proteins, but of all types of secretory cargo, including small soluble proteins. Our data support a general role for MIA3/TANGO1 in maintaining secretory pathway structure and function in vertebrate cells.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Via Secretória , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Transporte Proteico
19.
J Mol Neurosci ; 71(9): 1752-1760, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34173192

RESUMO

To study the potential role of brain-derived neurotrophic factor (BDNF) methylation in heroin addiction, we first detected the methylation level of seven CpG islands that included 106 CpG sites in the promoter regions of BDNF from 120 people addicted to heroin and 113 controls. Methylation quantitative trait locus (mQTL) analysis was then employed to determine the association between the single-nucleotide polymorphism rs6265, a well-known locus shown to be correlated with heroin addiction, and the methylation levels of these CpG sites. Finally, we used the JASPAR database to predict whether transcription factors could bind to these CpG sites. We found that the methylation levels of CpG islands 6 and 7 and the methylation levels of BDNF_45 and BDNF_80 were significantly higher in the heroin addiction group than in the control group. We also found that rs6265 was an mQTL and was associated with the methylation level of BDNF_58. Using the JASPAR database, we found that ALX homeobox 3 (ALX3), achaete-scute family bHLH transcription factor 1 (ASCL1) and aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) could bind to CpG island 6, and ALX3 could bind to CpG island 7. In summary, we showed that increased DNA methylation in the promoter regions of the BDNF gene was associated with heroin addiction in Han Chinese.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Metilação de DNA , Dependência de Heroína/genética , Adulto , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Ilhas de CpG , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas
20.
Infect Immun ; 89(8): e0012421, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34031127

RESUMO

Vascular remodeling is a phenomenon seen in the cutaneous lesions formed during infection with Leishmania parasites. Within the lesion, Leishmania major infection leads to the infiltration of inflammatory cells, including macrophages, and is associated with hypoxic conditions and lymphangiogenesis in the local site. This low-oxygen environment is concomitant with the expression of hypoxic inducible factors (HIFs), which initiate the expression of vascular endothelial growth factor-A (VEGF-A) in macrophages during the infection. Here, we found that macrophage hypoxia is elevated in the skin, and the HIF target Vegfa is preferentially expressed at the site of infection. Further, transcripts indicative of both HIF-1α and HIF-2α activation were increased at the site of infection. Given that HIF mediates VEGF-A and that VEGF-A/VEGFR-2 signaling induces lymphangiogenesis, we wanted to investigate the link between myeloid HIF activation and lymphangiogenesis during L. major infection. We show that myeloid aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF/VEGF-A signaling promotes lymphangiogenesis (the generation of newly formed vessels within the local lymphatic network), which helps resolve the lesion by draining away inflammatory cells and fluid. Concomitant with impaired lymphangiogenesis, we find the deletion of myeloid ARNT/HIF signaling leads to an exacerbated inflammatory response associated with a heightened CD4+ Th1 immune response following L. major infection. Altogether, our data suggest that VEGF-A-mediated lymphangiogenesis occurs through myeloid ARNT/HIF activation following Leishmania major infection and this process is critical in limiting immunopathology.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Leishmania major/fisiologia , Leishmaniose Cutânea/etiologia , Leishmaniose Cutânea/metabolismo , Linfangiogênese/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Transdução de Sinais , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Biomarcadores , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Suscetibilidade a Doenças/imunologia , Interações Hospedeiro-Patógeno/imunologia , Leishmaniose Cutânea/patologia
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