RESUMO
Cargo translocation across membranes is a crucial aspect of secretion. In conventional secretion signal peptide-equipped proteins enter the endoplasmic reticulum (ER), whereas a subset of cargo lacking signal peptides translocate into the ER-Golgi intermediate compartment (ERGIC) in a process called unconventional protein secretion (UcPS). The regulatory events at the ERGIC in UcPS are unclear. Here we reveal the involvement of ERGIC-localized small GTPases, Rab1 (Rab1A and Rab1B) and Rab2A, in regulating UcPS cargo transport via TMED10 on the ERGIC. Rab1 enhances TMED10 translocator activity, promoting cargo translocation into the ERGIC, whereas Rab2A, in collaboration with KIF5B, regulates ERGIC compartmentalization, establishing a UcPS-specific compartment. This study highlights the pivotal role of ERGIC-localized Rabs in governing cargo translocation and specifying the ERGIC's function in UcPS.
Assuntos
Retículo Endoplasmático , Complexo de Golgi , Transporte Proteico , Retículo Endoplasmático/metabolismo , Humanos , Complexo de Golgi/metabolismo , Células HeLa , Cinesinas/metabolismo , Cinesinas/genética , Células HEK293 , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Animais , Proteínas rab1 de Ligação ao GTP/metabolismo , Proteínas rab1 de Ligação ao GTP/genéticaRESUMO
Non-alcoholic steatohepatitis (NASH) is a severe type of the non-alcoholic fatty liver disease (NAFLD). NASH is a growing global health concern due to its increasing morbidity, lack of well-defined biomarkers and lack of clinically effective treatments. Using metabolomic analysis, the most significantly changed active lipid sphingosine d18:1 [So(d18:1)] is selected from NASH patients. So(d18:1) inhibits macrophage HIF-2α as a direct inhibitor and promotes the inflammatory factors secretion. Male macrophage-specific HIF-2α knockout and overexpression mice verified the protective effect of HIF-2α on NASH progression. Importantly, the HIF-2α stabilizer FG-4592 alleviates liver inflammation and fibrosis in NASH, which indicated that macrophage HIF-2α is a potential drug target for NASH treatment. Overall, this study confirms that So(d18:1) promotes NASH and clarifies that So(d18:1) inhibits the transcriptional activity of HIF-2α in liver macrophages by suppressing the interaction of HIF-2α with ARNT, suggesting that macrophage HIF-2α may be a potential target for the treatment of NASH.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Macrófagos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica , Esfingosina , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Masculino , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Humanos , Camundongos , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Fígado/metabolismo , Fígado/patologia , Fígado/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Cirrose Hepática/genética , Modelos Animais de DoençasRESUMO
Transcriptional response to changes in oxygen concentration is mainly controlled by hypoxia-inducible transcription factors (HIFs). Besides regulation of hypoxia-responsible gene expression, HIF-3α has recently been shown to be involved in lung development and in the metabolic process of fat tissue. However, the precise mechanism for such properties of HIF-3α is still largely unknown. To this end, we generated HIF3A gene-disrupted mice by means of genome editing technology to explore the pleiotropic role of HIF-3α in development and physiology. We obtained adult mice carrying homozygous HIF3A gene mutations with comparable body weight and height to wild-type mice. However, the number of litters and ratio of homozygous mutation carriers born from the mating between homozygous mutant mice was lower than expected due to sporadic deaths on postnatal day 1. HIF3A gene-disrupted mice exhibited abnormal configuration of the lung such as a reduced number of alveoli and thickened alveolar walls. Transcriptome analysis showed, as well as genes associated with lung development, an upregulation of stearoyl-Coenzyme A desaturase 1, a pivotal enzyme for fatty acid metabolism. Analysis of fatty acid composition in the lung employing gas chromatography indicated an elevation in palmitoleic acid and a reduction in oleic acid, suggesting an imbalance in distribution of fatty acid, a constituent of lung surfactant. Accordingly, administration of glucocorticoid injections during pregnancy resulted in a restoration of normal alveolar counts and a decrease in neonatal mortality. In conclusion, these observations provide novel insights into a pivotal role of HIF-3α in the preservation of critically important structure and function of alveoli beyond the regulation of hypoxia-mediated gene expression.
Assuntos
Proteínas Reguladoras de Apoptose , Alvéolos Pulmonares , Proteínas Repressoras , Animais , Feminino , Masculino , Camundongos , Animais Recém-Nascidos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Ácidos Graxos/metabolismo , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismoRESUMO
Glioblastoma (GBM) is the most aggressive and lethal brain tumor in adults. This study aimed to investigate the functional significance of aryl hydrocarbon receptor nuclear translocator (ARNT) in the pathogenesis of GBM. Analysis of public datasets revealed ARNT is upregulated in GBM tissues compared to lower grade gliomas or normal brain tissues. Higher ARNT expression correlated with the mesenchymal subtype and poorer survival in GBM patients. Silencing ARNT using lentiviral shRNAs attenuated the proliferative, invasive, and stem-like capabilities of GBM cell lines, while ARNT overexpression enhanced these malignant phenotypes. Single-cell RNA sequencing uncovered that ARNT is highly expressed in a stem-like subpopulation and is involved in regulating glycolysis, hypoxia response, and stress pathways. Mechanistic studies found ARNT activates p38 mitogen-activated protein kinase (MAPK) signaling to promote chemoresistance in GBM cells. Disrupting the ARNT/p38α protein interaction via the ARNT PAS-A domain restored temozolomide sensitivity. Overall, this study demonstrates ARNT functions as an oncogenic driver in GBM pathogenesis and represents a promising therapeutic target.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Resistencia a Medicamentos Antineoplásicos , Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/genética , Camundongos , Regulação Neoplásica da Expressão Gênica , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Camundongos Nus , Transdução de Sinais/efeitos dos fármacosRESUMO
Aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (ARNT) mediate the responses of adaptive metabolism to various xenobiotics. Here, we found that BoAhR and BoARNT are highly expressed in the midgut of Bradysia odoriphaga larvae. The expression of BoAhR and BoARNT was significantly increased after exposure to imidacloprid and phoxim. The knockdown of BoAhR and BoARNT significantly decreased the expression of CYP6SX1 and CYP3828A1 as well as P450 enzyme activity and caused a significant increase in the sensitivity of larvae to imidacloprid and phoxim. Exposure to ß-naphthoflavone (BNF) significantly increased the expression of BoAhR, BoARNT, CYP6SX1, and CYP3828A1 as well as P450 activity and decreased larval sensitivity to imidacloprid and phoxim. Furthermore, CYP6SX1 and CYP3828A1 were significantly induced by imidacloprid and phoxim, and the silencing of these two genes significantly reduced larval tolerance to imidacloprid and phoxim. Taken together, the BoAhR/BoARNT pathway plays key roles in larval tolerance to imidacloprid and phoxim by regulating the expression of CYP6SX1 and CYP3828A1.
Assuntos
Proteínas de Insetos , Inseticidas , Larva , Neonicotinoides , Nitrocompostos , Receptores de Hidrocarboneto Arílico , Animais , Inseticidas/farmacologia , Larva/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/efeitos dos fármacos , Nitrocompostos/farmacologia , Nitrocompostos/metabolismo , Neonicotinoides/farmacologia , Neonicotinoides/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Dípteros/metabolismo , Dípteros/genética , Dípteros/efeitos dos fármacos , Dípteros/crescimento & desenvolvimento , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Inativação Metabólica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Single, high doses of TCDD in rats are known to cause wasting, a progressive loss of 30 to 50% body weight and death within several weeks. To identify pathway perturbations at or near doses causing wasting, we examined differentially gene expression (DGE) and pathway enrichment in centrilobular (CL) and periportal (PP) regions of female rat livers following 6 dose levels of TCDD - 0, 3, 22, 100, 300, and 1000 ng/kg/day, 5 days/week for 4 weeks. At the higher doses, rats lost weight, had increased liver/body weight ratios and nearly complete cessation of liver cell proliferation, signs consistent with wasting. DGE curves were left shifted for the CL versus the PP regions. Canonical Phase I and Phase II genes were maximally increased at lower doses and remained elevated at all doses. At lower doses, ≤ 22 ng/kg/day in the CL and ≤ 100 ng/kg/day, upregulated genes showed transcription factor (TF) enrichment for AHR and ARNT. At the mid- and high-dose doses, there was a large number of downregulated genes and pathway enrichment for DEGs which showed downregulation of many cellular metabolism processes including those for steroids, fatty acid metabolism, pyruvate metabolism and citric acid cycle. There was significant TF enrichment of the hi-dose downregulated genes for RXR, ESR1, LXR, PPARalpha. At the highest dose, there was also pathway enrichment with upregulated genes for extracellular matrix organization, collagen formation, hemostasis and innate immune system. TCDD demonstrates most of its effects through binding the aryl hydrocarbon receptor (AHR) while the downregulation of metabolism genes at higher TCDD doses is known to be independent of AHR binding to DREs. Based on our results with DEG, we provide a hypothesis for wasting in which high doses of TCDD shift circadian processes away from the resting state, leading to greatly reduced synthesis of steroids and complex lipids needed for cell growth, and producing gene expression signals consistent with an epithelial-to-mesenchymal transition in hepatocytes.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Fígado , Dibenzodioxinas Policloradas , Receptores de Hidrocarboneto Arílico , Animais , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Feminino , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Dibenzodioxinas Policloradas/toxicidade , Ratos , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Transcriptoma/efeitos dos fármacos , Perfilação da Expressão Gênica/métodos , Ratos Sprague-Dawley , Relação Dose-Resposta a DrogaRESUMO
The p24 family of proteins have been regarded as cargo receptors for endoplasmic reticulum (ER) to Golgi transport; however, their precise functions have yet to be revealed. In this issue, Pastor-Pareja and colleagues (https://doi.org/10.1083/jcb.202309045) show that the interaction of these proteins with Tango1 is critical for their localization at the ER exit site (ERES) and efficient transport of secretory proteins in Drosophila.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Drosophila , Retículo Endoplasmático , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Drosophila/citologia , Drosophila/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Transporte Proteico/fisiologia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates biological signals to control various complicated cellular functions. It plays a crucial role in environmental sensing and xenobiotic metabolism. Dysregulation of AhR is associated with health concerns, including cancer and immune system disorders. Upon binding to AhR ligands, AhR, along with heat shock protein 90 and other partner proteins undergoes a transformation in the nucleus, heterodimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT), and mediates numerous biological functions by inducing the transcription of various AhR-responsive genes. In this manuscript, the 3-dimensional structure of the entire human AhR is obtained using an artificial intelligence tool, and molecular dynamics (MD) simulations are performed to study different structural conformations. These conformations provide insights into the protein's function and movement in response to ligand binding. Understanding the dynamic behavior of AhR will contribute to the development of targeted therapies for associated health conditions. Therefore, we employ well-tempered metadynamics (WTE-metaD) simulations to explore the conformational landscape of AhR and obtain a better understanding of its functional behavior. Our computational results are in excellent agreement with previous experimental findings, revealing the closed and open states of helix α1 in the basic helix-loop-helix (bHLH domain) in the cytoplasm at the atomic level. We also predict the inactive form of AhR and identify Arginine 42 as a key residue that regulates switching between closed and open conformations in existing AhR modulators.
Assuntos
Inteligência Artificial , Receptores de Hidrocarboneto Arílico , Humanos , Receptores de Hidrocarboneto Arílico/metabolismo , Ligantes , Translocador Nuclear Receptor Aril Hidrocarboneto/química , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismoRESUMO
The eukaryotic p24 family, consisting of α-, ß-, γ- and δ-p24 subfamilies, has long been known to be involved in regulating secretion. Despite increasing interest in these proteins, fundamental questions remain about their role. Here, we systematically investigated Drosophila p24 proteins. We discovered that members of all four p24 subfamilies are required for general secretion and that their localizations between ER exit site (ERES) and Golgi are interdependent in an αâßδâγ sequence. We also found that localization of p24 proteins and ERES determinant Tango1 requires interaction through their respective GOLD and SH3 lumenal domains, with Tango1 loss sending p24 proteins to the plasma membrane and vice versa. Finally, we show that p24 loss expands the COPII zone at ERES and increases the number of ER-Golgi vesicles, supporting a restrictive role of p24 proteins on vesicle budding for efficient transport. Our results reveal Tango1-p24 interplay as central to the generation of a stable ER-Golgi interface.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Proteínas de Drosophila , Retículo Endoplasmático , Complexo de Golgi , Proteínas de Membrana Transportadoras , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Membrana Celular , Drosophila melanogaster , Proteínas de Drosophila/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Domínios de Homologia de src , Proteínas de Membrana Transportadoras/metabolismoRESUMO
Breast cancer is one of the main causes of malignancy-related deaths globally and has a significant impact on women's quality of life. Despite significant therapeutic advances, there is a medical need for targeted therapies in breast cancer. Aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor mediates responses to environment stimuli, is emerging as a unique pleiotropic target. Herein, a combined molecular simulation and in vitro investigations identified 3-(3-fluorophenyl)-1H-pyrazolo[3,4-b]pyridine (3FPP) as a novel AhR ligand in T47D and MDA-MB-231 breast cancer cells. Its agonistic effects induced formation of the AhR-AhR nuclear translocator (Arnt) heterodimer and prompted its binding to the penta-nucleotide sequence, called xenobiotic-responsive element (XRE) motif. Moreover, 3FPP augmented the promoter-driven luciferase activities and expression of AhR-regulated genes encoding cytochrome P450 1A1 (CYP1A1) and microRNA (miR)-212/132 cluster. It reduced cell viability, migration, and invasion of both cell lines through AhR signaling. These anticancer properties were concomitant with reduced levels of B-cell lymphoma 2 (BCL-2), SRY-related HMG-box4 (SOX4), snail family zinc finger 2 (SNAI2), and cadherin 2 (CDH2). In vivo, 3FPP suppressed tumor growth and activated AhR signaling in an orthotopic mouse model. In conclusion, our results introduce the fused pyrazolopyridine 3FPP as a novel AhR agonist with AhR-specific anti-breast cancer potential in vitro and in vivo.
Assuntos
Neoplasias da Mama , Pirazóis , Receptores de Hidrocarboneto Arílico , Animais , Camundongos , Feminino , Humanos , Receptores de Hidrocarboneto Arílico/metabolismo , Ligantes , Qualidade de Vida , Citocromo P-450 CYP1A1/metabolismo , Neoplasias da Mama/genética , Piridinas/farmacologia , Piridinas/uso terapêutico , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Fatores de Transcrição SOXC/metabolismoAssuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Optogenética , Proteínas Circadianas Period , Transdução de Sinais , Peptídeos , Preparações Farmacêuticas , Domínios Proteicos , Proteínas Circadianas Period/química , Translocador Nuclear Receptor Aril Hidrocarboneto/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Humanos , AnimaisRESUMO
Tight junctions are involved in skin barrier functions. In this study, the expression of CLDN1, CLDN4, and OCLN was found to decrease in skin lesions of atopic dermatitis by bioinformatics analysis. Immunohistochemistry staining in skin specimens from 12 patients with atopic dermatitis and 12 healthy controls also showed decreased CLDN1, CLDN4, and OCLN expression in atopic dermatitis lesions. In vitro studies showed that IL-4 and IL-13 downregulated CLDN1, CLDN4, and OCLN expression in HaCaT cells as well as CLDN4 and OCLN expression in human primary keratinocytes. This effect, which was mediated through the Jak-signal transducer and activator of transcription 6 signaling pathway, increased paracellular flux of 4-kDa dextran. Benvitimod, a new drug for atopic dermatitis, upregulated CLDN4 and OCLN through the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator pathway. Benvitimod induced nuclear translocation of NRF2 and reduced production of ROS in keratinocytes, thus inhibiting IL-4-/IL-13-induced CLDN1 downregulation and signal transducer and activator of transcription 6 phosphorylation. These results indicate that T helper 2 cytokines are involved in tight junction impairment, and benvitimod can inhibit these effects.
Assuntos
Dermatite Atópica , Interleucina-13 , Resorcinóis , Estilbenos , Humanos , Interleucina-13/metabolismo , Dermatite Atópica/patologia , Junções Íntimas/metabolismo , Interleucina-4/farmacologia , Interleucina-4/metabolismo , Fosforilação , Fator de Transcrição STAT6/metabolismo , Queratinócitos/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/farmacologiaRESUMO
The mammalian family of basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factors possess the ability to sense and respond to diverse environmental and physiological cues. These proteins all share a common structural framework, comprising a bHLH domain, two PAS domains, and transcriptional activation or repression domain. To function effectively as transcription factors, members of the family must form dimers, bringing together bHLH segments to create a functional unit that allows for DNA response element binding. The significance of bHLH-PAS family is underscored by their involvement in many major human diseases, offering potential avenues for therapeutic intervention. Notably, the clear identification of ligand-binding cavities within their PAS domains enables the development of targeted small molecules. Two examples are Belzutifan, targeting hypoxia-inducible factor (HIF)-2α, and Tapinarof, targeting the aryl hydrocarbon receptor (AHR), both of which have gained regulatory approval recently. Here, we focus on the HIF subfamily. The crystal structures of all three HIF-α proteins have been elucidated, revealing their bHLH and tandem PAS domains are used to engage their dimerization partner aryl hydrocarbon receptor nuclear translocator (ARNT, also called HIF-1ß). A broad range of recent findings point to a shared allosteric modulation mechanism among these proteins, whereby small-molecules at the PAS-B domains exert direct influence over the HIF-α transcriptional functions. As our understanding of the architectural and allosteric mechanisms of bHLH-PAS proteins continues to advance, the possibility of discovering new therapeutic drugs becomes increasingly promising.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Sequências Hélice-Alça-Hélice , Animais , Humanos , Translocador Nuclear Receptor Aril Hidrocarboneto/química , Ativação Transcricional , Multimerização Proteica , Regulação Alostérica , Domínios ProteicosRESUMO
Nitric oxide (NO) regulates large swaths of animal physiology including wound healing, vasodilation, memory formation, odor detection, sexual function, and response to infectious disease. The primary NO receptor is soluble guanyly/guanylate cyclase (sGC), a dimeric protein of â¼150 kDa that detects NO through a ferrous heme, leading to a large change in conformation and enhanced production of cGMP from GTP. In humans, loss of sGC function contributes to multiple disease states, including cardiovascular disease and cancer, and is the target of a new class of drugs, sGC stimulators, now in clinical use. sGC evolved through the fusion of four ancient domains, a heme nitric oxide / oxygen (H-NOX) domain, a Per-ARNT-Sim (PAS) domain, a coiled coil, and a cyclase domain, with catalysis occurring at the interface of the two cyclase domains. In animals, the predominant dimer is the α1ß1 heterodimer, with the α1 subunit formed through gene duplication of the ß1 subunit. The PAS domain provides an extensive dimer interface that remains unchanged during sGC activation, acting as a core anchor. A large cleft formed at the PAS-PAS dimer interface tightly binds the N-terminal end of the coiled coil, keeping this region intact and unchanged while the rest of the coiled coil repacks, and the other domains reposition. This interface buries â¼3000 Å2 of monomer surface and includes highly conserved apolar and hydrogen bonding residues. Herein, we discuss the evolutionary history of sGC, describe the role of PAS domains in sGC function, and explore the regulatory factors affecting sGC function.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Óxido Nítrico , Guanilil Ciclase Solúvel , Animais , Humanos , Translocador Nuclear Receptor Aril Hidrocarboneto/química , Heme/metabolismo , Óxido Nítrico/metabolismo , Guanilil Ciclase Solúvel/química , Guanilil Ciclase Solúvel/genética , Transdução de Sinais , Domínios ProteicosRESUMO
The PAS (PER, ARNT, SIM) protein family plays a vital role in mammalian biology and human disease. This analysis arose from an interest in the signaling mechanics by the Ah receptor (AHR) and the Ah receptor nuclear translocator (ARNT). After more than fifty years by studying this and related mammalian sensor systems, describing the role of PAS domains in signal transduction is still challenging. In this perspective, we attempt to interpret recent studies of mammalian PAS protein structure and consider how this new insight might explain how these domains are employed in human signal transduction with an eye towards developing strategies to target and engineer these molecules for a new generation of therapeutics. Our approach is to integrate our understanding of PAS protein history, cell biology, and molecular biology with recent structural discoveries to help explain the mechanics of mammalian PAS protein signaling. As a learning set, we focus on sequences and crystal structures of mammalian PAS protein dimers that can be visualized using readily available software.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Receptores de Hidrocarboneto Arílico , Animais , Humanos , Translocador Nuclear Receptor Aril Hidrocarboneto/química , Receptores de Hidrocarboneto Arílico/química , Multimerização ProteicaRESUMO
The aryl hydrocarbon receptor (AHR) signaling pathway is a complex regulatory network that plays a critical role in various biological processes, including cellular metabolism, development, and immune responses. The complexity of AHR signaling arises from multiple factors, including the diverse ligands that activate the receptor, the expression level of AHR itself, and its interaction with the AHR nuclear translocator (ARNT). Additionally, the AHR crosstalks with the AHR repressor (AHRR) or other transcription factors and signaling pathways and it can also mediate non-genomic effects. Finally, posttranslational modifications of the AHR and its interaction partners, epigenetic regulation of AHR and its target genes, as well as AHR-mediated induction of enzymes that degrade AHR-activating ligands may contribute to the context-specificity of AHR activation. Understanding the complexity of AHR signaling is crucial for deciphering its physiological and pathological roles and developing therapeutic strategies targeting this pathway. Ongoing research continues to unravel the intricacies of AHR signaling, shedding light on the regulatory mechanisms controlling its diverse functions.
Assuntos
Neoplasias , Receptores de Hidrocarboneto Arílico , Humanos , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Epigênese Genética , Neoplasias/genética , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismoRESUMO
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.
Assuntos
Dibenzodioxinas Policloradas , Receptores de Hidrocarboneto Arílico , Receptores de Hidrocarboneto Arílico/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Ligantes , Dibenzodioxinas Policloradas/química , DNA/metabolismoRESUMO
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.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Receptores de Hidrocarboneto Arílico , Translocador Nuclear Receptor Aril Hidrocarboneto/química , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Cisteína/metabolismo , Ligação Proteica , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismoRESUMO
Circular RNAs, which have covalently closed ends, are in the class of non-coding RNAs. Recent studies reveal that they are associated with various biochemical pathways. One such involvement of circular RNAs is in the onset of different types of cancers. Though the circular RNAs are known as non-coding RNAs, some of them are found to possess the capacities to code for proteins. One such circular RNA is hsa-circ-0000437 which is known to code for a short peptide referred to as CORO1C-47aa. The peptide has anti-angiogenic activity and is associated with the prevention of endometrial cancer. The peptide binds to the PAS-B domain of the Aryl hydrocarbon Receptor Nuclear Translocator (ARNT). However, till date only the amino acid sequence of the peptide is known and no structural details of the peptide are available. Therefore, in this work, our aim was to predict how the peptide would fold and what could be its possible ligand binding sites. We used computational tools to determine the structure of the peptide refined further by molecular dynamics simulations. We then performed molecular docking simulations of the peptide with its known binding partner ARNT to gain an insight into the modes of binding as the process is associated with endometrial cancer. The possible ligand binding sites along-with the natures of the possible other different ligands of the peptide were analyzed further. From this structure function analysis study, we tried to elucidate the plausible mechanism of the involvements of the peptide in the onset of endometrial cancer. This is the first report on the structural characterization of the peptide and its modes of interactions with the partner protein ARNT. This study may therefore be useful in determining the structures of new drug candidates for the treatment of endometrial cancer.