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1.
Genes (Basel) ; 12(6)2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34073716

RESUMO

The current SARS-CoV-2 pandemic is still threatening humankind. Despite first successes in vaccine development and approval, no antiviral treatment is available for COVID-19 patients. The success is further tarnished by the emergence and spreading of mutation variants of SARS-CoV-2, for which some vaccines have lower efficacy. This highlights the urgent need for antiviral therapies even more. This article describes how the genome-scale metabolic model (GEM) of the host-virus interaction of human alveolar macrophages and SARS-CoV-2 was refined by incorporating the latest information about the virus's structural proteins and the mutant variants B.1.1.7, B.1.351, B.1.28, B.1.427/B.1.429, and B.1.617. We confirmed the initially identified guanylate kinase as a potential antiviral target with this refined model and identified further potential targets from the purine and pyrimidine metabolism. The model was further extended by incorporating the virus' lipid requirements. This opened new perspectives for potential antiviral targets in the altered lipid metabolism. Especially the phosphatidylcholine biosynthesis seems to play a pivotal role in viral replication. The guanylate kinase is even a robust target in all investigated mutation variants currently spreading worldwide. These new insights can guide laboratory experiments for the validation of identified potential antiviral targets. Only the combination of vaccines and antiviral therapies will effectively defeat this ongoing pandemic.


Assuntos
COVID-19/metabolismo , COVID-19/virologia , Metabolismo Energético , Genoma Viral , Guanilato Quinases/metabolismo , Interações Hospedeiro-Patógeno , Mutação , SARS-CoV-2/genética , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/genética , Técnicas de Silenciamento de Genes , Humanos , Metabolismo dos Lipídeos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , SARS-CoV-2/efeitos dos fármacos , Carga Viral , Replicação Viral
2.
Nat Commun ; 12(1): 3253, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059674

RESUMO

Muscle stem cell function has been suggested to be regulated by Acetyl-CoA and NAD+ availability, but the mechanisms remain unclear. Here we report the identification of two acetylation sites on PAX7 that positively regulate its transcriptional activity. Lack of PAX7 acetylation reduces DNA binding, specifically to the homeobox motif. The acetyltransferase MYST1 stimulated by Acetyl-CoA, and the deacetylase SIRT2 stimulated by NAD +, are identified as direct regulators of PAX7 acetylation and asymmetric division in muscle stem cells. Abolishing PAX7 acetylation in mice using CRISPR/Cas9 mutagenesis leads to an expansion of the satellite stem cell pool, reduced numbers of asymmetric stem cell divisions, and increased numbers of oxidative IIA myofibers. Gene expression analysis confirms that lack of PAX7 acetylation preferentially affects the expression of target genes regulated by homeodomain binding motifs. Therefore, PAX7 acetylation status regulates muscle stem cell function and differentiation potential to facilitate metabolic adaptation of muscle tissue.


Assuntos
Autorrenovação Celular/genética , Músculo Esquelético/lesões , Fator de Transcrição PAX7/metabolismo , Regeneração/genética , Células Satélites de Músculo Esquelético/fisiologia , Acetilação , Animais , Células COS , Sistemas CRISPR-Cas , Cardiotoxinas/administração & dosagem , Cardiotoxinas/toxicidade , Diferenciação Celular/genética , Chlorocebus aethiops , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Mutagênese , Cultura Primária de Células , Regiões Promotoras Genéticas , Células Sf9 , Sirtuína 2/genética , Sirtuína 2/metabolismo , Spodoptera , Ativação Transcricional
3.
Nat Commun ; 12(1): 3258, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059679

RESUMO

Autophagy can selectively target protein aggregates, pathogens, and dysfunctional organelles for the lysosomal degradation. Aberrant regulation of autophagy promotes tumorigenesis, while it is far less clear whether and how tumor-specific alterations result in autophagic aberrance. To form a link between aberrant autophagy selectivity and human cancer, we establish a computational pipeline and prioritize 222 potential LIR (LC3-interacting region) motif-associated mutations (LAMs) in 148 proteins. We validate LAMs in multiple proteins including ATG4B, STBD1, EHMT2 and BRAF that impair their interactions with LC3 and autophagy activities. Using a combination of transcriptomic, metabolomic and additional experimental assays, we show that STBD1, a poorly-characterized protein, inhibits tumor growth via modulating glycogen autophagy, while a patient-derived W203C mutation on LIR abolishes its cancer inhibitory function. This work suggests that altered autophagy selectivity is a frequently-used mechanism by cancer cells to survive during various stresses, and provides a framework to discover additional autophagy-related pathways that influence carcinogenesis.


Assuntos
Carcinogênese/genética , Macroautofagia/genética , Proteínas de Membrana/genética , Modelos Genéticos , Proteínas Musculares/genética , Neoplasias/genética , Algoritmos , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Simulação por Computador , Análise Mutacional de DNA , Conjuntos de Dados como Assunto , Técnicas de Silenciamento de Genes , Glicogênio/metabolismo , Humanos , Estimativa de Kaplan-Meier , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Musculares/metabolismo , Mutação , Neoplasias/mortalidade , Neoplasias/patologia , Via de Pentose Fosfato/genética , Domínios e Motivos de Interação entre Proteínas/genética , Proteoma/genética , RNA-Seq , Análise Serial de Tecidos , Efeito Warburg em Oncologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072570

RESUMO

Sorafenib, a multi-kinase inhibitor, is the first-line treatment for advanced hepatocellular carcinoma (HCC) patients. However, this drug only provides a short improvement of patients' overall survival, and drug resistance is commonly developed. Thus, the identification of resistant factor(s) or biomarker(s) is needed to develop more efficient therapeutic strategies. Long, non-coding RNAs (lncRNAs) have recently been viewed as attractive cancer biomarkers and drive many important cancer phenotypes. A lncRNA, ZFAS1 (ZNFX1 antisense RNA 1) has been found to promote HCC metastasis. This study found that sorafenib induced ZFAS1 expression specifically in sorafenib-resistant HCC cells. Although ZFAS1 knockdown did not restore the sensitivity of HCC cells to sorafenib, its expression may act as a resistant biomarker for sorafenib therapy. Bioinformatics analysis predicted that sorafenib tended to induce pathways related to endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in sorafenib-resistant HCC cells. In vitro experimental evidence suggested that sorafenib induced protein kinase RNA-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-dependent ZFAS1 expression, and sorafenib resistance could be overcome by PERK/ATF inhibitors. Therefore, PERK/ATF4/ZFAS1 signaling axis might be an attractive therapeutic and prognostic biomarker for sorafenib therapy in HCC.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Inibidores de Proteínas Quinases/farmacologia , RNA Longo não Codificante/genética , Sorafenibe/farmacologia , eIF-2 Quinase/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Prognóstico , Análise de Sequência de RNA
5.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070207

RESUMO

In domestic ruminants, endometrial receptivity is related to successful pregnancy and economic efficiency. Despite several molecules having been reported in the past regarding endometrial receptivity regulation, much regarding the mechanism of endometrial receptivity regulation remains unknown due to the complex nature of the trait. In this work, we demonstrated that the cysteine-rich transmembrane bone morphogenetic protein (BMP) regulator 1 (CRIM1) served as a novel regulator in the regulation of goat endometrial receptivity in vitro. Our results showed that hormones and IFN-τ increased the expression of CRIM1 in goat endometrial epithelial cells (EECs). Knockdown of CRIM1 via specific shRNA hindered cell proliferation, cell adhesion and prostaglandins (PGs) secretion and thus derailed normal endometrial receptivity. We further confirmed that receptivity defect phenotypes due to CRIM1 interference were restored by ATG7 overexpression in EECs while a loss of ATG7 further impaired receptivity phenotypes. Moreover, our results showed that changing the expression of ATG7 affected the reactive oxygen species (ROS) production. Moreover, mR-143-5p was shown to be a potential upstream factor of CRIM1-regulated endometrial receptivity in EECs. Overall, these results suggest that CRIM1, as the downstream target of miR-143-5p, has effects on ATG7-dependent autophagy, regulating cell proliferation, cell adhesion and PG secretion, and provides a new target for the diagnosis and treatment of early pregnancy failure and for improving the success rates of artificial reproduction.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas/fisiologia , Implantação do Embrião/genética , Endométrio/fisiologia , Cabras/fisiologia , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Autofagia/fisiologia , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/fisiologia , Receptores de Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Receptores de Proteínas Morfogenéticas Ósseas/genética , Adesão Celular , Proliferação de Células , Células Cultivadas , Implantação do Embrião/fisiologia , Endométrio/citologia , Endométrio/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Estradiol/farmacologia , Feminino , Técnicas de Silenciamento de Genes , Cabras/genética , Interferon Tipo I/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Gravidez , Proteínas da Gravidez/farmacologia , Progesterona/farmacologia , Prostaglandinas/metabolismo , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima
6.
Nat Commun ; 12(1): 3397, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099665

RESUMO

It is known that an RNA's structure determines its biological function, yet current RNA structure probing methods only capture partial structure information. The ability to measure intact (i.e., full length) RNA structures will facilitate investigations of the functions and regulation mechanisms of small RNAs and identify short fragments of functional sites. Here, we present icSHAPE-MaP, an approach combining in vivo selective 2'-hydroxyl acylation and mutational profiling to probe intact RNA structures. We further showcase the RNA structural landscape of substrates bound by human Dicer based on the combination of RNA immunoprecipitation pull-down and icSHAPE-MaP small RNA structural profiling. We discover distinct structural categories of Dicer substrates in correlation to both their binding affinity and cleavage efficiency. And by tertiary structural modeling constrained by icSHAPE-MaP RNA structural data, we find the spatial distance measuring as an influential parameter for Dicer cleavage-site selection.


Assuntos
RNA Helicases DEAD-box/metabolismo , Conformação de Ácido Nucleico , RNA/química , Ribonuclease III/metabolismo , Biologia Computacional , RNA Helicases DEAD-box/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , RNA/genética , RNA/metabolismo , Sondas RNA , RNA-Seq , Ribonuclease III/genética , Especificidade por Substrato/genética
7.
Int J Mol Sci ; 22(9)2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-34067147

RESUMO

Stress resistance mechanisms include upregulation of heat shock proteins (HSPs) and formation of granules. Stress-induced granules are classified into stress granules and nuclear stress bodies (nSBs). The present study examined the involvement of nSB formation in thermal resistance. We used chemical compounds that inhibit heat shock transcription factor 1 (HSF1) and scaffold attachment factor B (SAFB) granule formation and determined their effect on granule formation and HSP expression in HeLa cells. We found that formation of HSF1 and SAFB granules was inhibited by 2,5-hexanediol. We also found that suppression of HSF1 and SAFB granule formation enhanced heat stress-induced apoptosis. In addition, the upregulation of HSP27 and HSP70 during heat stress recovery was suppressed by 2,5-hexanediol. Our results suggested that the formation of HSF1 and SAFB granules was likely to be involved in the upregulation of HSP27 and HSP70 during heat stress recovery. Thus, the formation of HSF1 and SAFB granules was involved in thermal resistance.


Assuntos
Apoptose , Fatores de Transcrição de Choque Térmico/antagonistas & inibidores , Resposta ao Choque Térmico , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Receptores de Estrogênio/metabolismo , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Técnicas de Silenciamento de Genes , Glicóis/farmacologia , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Fatores de Transcrição de Choque Térmico/metabolismo , Resposta ao Choque Térmico/efeitos dos fármacos , Humanos , Modelos Biológicos , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismo , Temperatura , Regulação para Cima/efeitos dos fármacos
8.
Zool Res ; 42(3): 377-388, 2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-33998185

RESUMO

LIN28A, an RNA-binding protein, plays an important role in porcine induced pluripotent stem cells (piPSCs). However, the molecular mechanism underlying the function of LIN28A in the maintenance of pluripotency in piPSCs remains unclear. Here, we explored the function of LIN28A in piPSCs based on its overexpression and knockdown. We performed total RNA sequencing (RNA-seq) of piPSCs and detected the expression levels of relevant genes by quantitative real-time polymerase chain reaction (qRT-PCR), western blot analysis, and immunofluorescence staining. Results indicated that piPSC proliferation ability decreased following LIN28A knockdown. Furthermore, when LIN28A expression in the shLIN28A2 group was lower (by 20%) than that in the negative control knockdown group ( shNC), the pluripotency of piPSCs disappeared and they differentiated into neuroectoderm cells. Results also showed that LIN28A overexpression inhibited the expression of DUSP (dual-specificity phosphatases) family phosphatases and activated the mitogen-activated protein kinase (MAPK) signaling pathway. Thus, LIN28A appears to activate the MAPK signaling pathway to maintain the pluripotency and proliferation ability of piPSCs. Our study provides a new resource for exploring the functions of LIN28A in piPSCs.


Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proliferação de Células , Fosfatases de Especificidade Dupla/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas de Ligação a RNA/genética , Suínos
9.
Nat Commun ; 12(1): 2813, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001876

RESUMO

Apicomplexa are obligate intracellular parasites responsible for major human diseases. Their intracellular survival relies on intense lipid synthesis, which fuels membrane biogenesis. Parasite lipids are generated as an essential combination of fatty acids scavenged from the host and de novo synthesized within the parasite apicoplast. The molecular and metabolic mechanisms allowing regulation and channeling of these fatty acid fluxes for intracellular parasite survival are currently unknown. Here, we identify an essential phosphatidic acid phosphatase in Toxoplasma gondii, TgLIPIN, as the central metabolic nexus responsible for controlled lipid synthesis sustaining parasite development. Lipidomics reveal that TgLIPIN controls the synthesis of diacylglycerol and levels of phosphatidic acid that regulates the fine balance of lipids between storage and membrane biogenesis. Using fluxomic approaches, we uncover the first parasite host-scavenged lipidome and show that TgLIPIN prevents parasite death by 'lipotoxicity' through effective channeling of host-scavenged fatty acids to storage triacylglycerols and membrane phospholipids.


Assuntos
Membrana Celular/metabolismo , Lipidômica/métodos , Fosfatidato Fosfatase/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/parasitologia , Prepúcio do Pênis/citologia , Técnicas de Silenciamento de Genes , Homeostase/genética , Interações Hospedeiro-Parasita , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Fosfatidato Fosfatase/genética , Proteínas de Protozoários/genética , Toxoplasma/genética , Toxoplasma/ultraestrutura
10.
Nat Commun ; 12(1): 2628, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976159

RESUMO

Thoracic aortic aneurysm, as occurs in Marfan syndrome, is generally asymptomatic until dissection or rupture, requiring surgical intervention as the only available treatment. Here, we show that nitric oxide (NO) signaling dysregulates actin cytoskeleton dynamics in Marfan Syndrome smooth muscle cells and that NO-donors induce Marfan-like aortopathy in wild-type mice, indicating that a marked increase in NO suffices to induce aortopathy. Levels of nitrated proteins are higher in plasma from Marfan patients and mice and in aortic tissue from Marfan mice than in control samples, indicating elevated circulating and tissue NO. Soluble guanylate cyclase and cGMP-dependent protein kinase are both activated in Marfan patients and mice and in wild-type mice treated with NO-donors, as shown by increased plasma cGMP and pVASP-S239 staining in aortic tissue. Marfan aortopathy in mice is reverted by pharmacological inhibition of soluble guanylate cyclase and cGMP-dependent protein kinase and lentiviral-mediated Prkg1 silencing. These findings identify potential biomarkers for monitoring Marfan Syndrome in patients and urge evaluation of cGMP-dependent protein kinase and soluble guanylate cyclase as therapeutic targets.


Assuntos
Aneurisma da Aorta Torácica/patologia , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Síndrome de Marfan/complicações , Guanilil Ciclase Solúvel/metabolismo , Animais , Aorta/citologia , Aorta/diagnóstico por imagem , Aorta/efeitos dos fármacos , Aorta/patologia , Aneurisma da Aorta Torácica/diagnóstico , Aneurisma da Aorta Torácica/etiologia , Aneurisma da Aorta Torácica/prevenção & controle , Biomarcadores/sangue , Biomarcadores/metabolismo , Carbazóis/administração & dosagem , GMP Cíclico/sangue , GMP Cíclico/metabolismo , Modelos Animais de Doenças , Feminino , Fibrilina-1/genética , Técnicas de Silenciamento de Genes , Humanos , Masculino , Síndrome de Marfan/sangue , Síndrome de Marfan/genética , Síndrome de Marfan/patologia , Camundongos , Músculo Liso Vascular/citologia , Mutação , Miócitos de Músculo Liso , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/administração & dosagem , Cultura Primária de Células , Guanilil Ciclase Solúvel/antagonistas & inibidores , Ultrassonografia
11.
Nat Commun ; 12(1): 2617, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33976169

RESUMO

Disruption of the transcription factor FoxP2, which is enriched in the basal ganglia, impairs vocal development in humans and songbirds. The basal ganglia are important for the selection and sequencing of motor actions, but the circuit mechanisms governing accurate sequencing of learned vocalizations are unknown. Here, we show that expression of FoxP2 in the basal ganglia is vital for the fluent initiation and termination of birdsong, as well as the maintenance of song syllable sequencing in adulthood. Knockdown of FoxP2 imbalances dopamine receptor expression across striatal direct-like and indirect-like pathways, suggesting a role of dopaminergic signaling in regulating vocal motor sequencing. Confirming this prediction, we show that phasic dopamine activation, and not inhibition, during singing drives repetition of song syllables, thus also impairing fluent initiation and termination of birdsong. These findings demonstrate discrete circuit origins for the dysfluent repetition of vocal elements in songbirds, with implications for speech disorders.


Assuntos
Corpo Estriado/metabolismo , Tentilhões/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/fisiologia , Vocalização Animal/fisiologia , Adulto , Animais , Animais Geneticamente Modificados , Dopamina/metabolismo , Técnicas de Silenciamento de Genes , Centro Vocal Superior , Humanos , Masculino , Modelos Animais , Vias Neurais/fisiologia , Optogenética , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/metabolismo , Fala/fisiologia , Técnicas Estereotáxicas
12.
Nat Commun ; 12(1): 2538, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953160

RESUMO

Group 2 innate lymphoid cells (ILC2s) play a critical role in protection against helminths and in diverse inflammatory diseases by responding to soluble factors such as the alarmin IL-33, that is often overexpressed in cancer. Nonetheless, regulatory factors that dictate ILC2 functions remain poorly studied. Here, we show that peroxisome proliferator-activated receptor gamma (PPARγ) is selectively expressed in ILC2s in humans and in mice, acting as a central functional regulator. Pharmacologic inhibition or genetic deletion of PPARγ in ILC2s significantly impair IL-33-induced Type-2 cytokine production and mitochondrial fitness. Further, PPARγ blockade in ILC2s disrupts their pro-tumoral effect induced by IL-33-secreting cancer cells. Lastly, genetic ablation of PPARγ in ILC2s significantly suppresses tumor growth in vivo. Our findings highlight a crucial role for PPARγ in supporting the IL-33 dependent pro-tumorigenic role of ILC2s and suggest that PPARγ can be considered as a druggable pathway in ILC2s to inhibit their effector functions. Hence, PPARγ targeting might be exploited in cancer immunotherapy and in other ILC2-driven mediated disorders, such as asthma and allergy.


Assuntos
Imunidade Inata/imunologia , Interleucina-33/metabolismo , Linfócitos/metabolismo , Neoplasias/terapia , PPAR gama/metabolismo , Animais , Asma , Citocinas/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Hipersensibilidade , Imunoterapia , Linfócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias , Neoplasias/patologia , PPAR gama/genética
13.
Nat Commun ; 12(1): 2551, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953173

RESUMO

Endogenous cardiac pacemaker function regulates the rate and rhythm of cardiac contraction. The mutation p.Lys23Glu in the cohesin protein Shugoshin-1 causes severe heart arrhythmias due to sinoatrial node dysfunction and a debilitating gastrointestinal motility disorder, collectively termed the Chronic Atrial and Intestinal Dysrhythmia Syndrome, linking Shugoshin-1 and pacemaker activity. Hyperpolarization-activated, cyclic nucleotide-gated cation channel 4 (HCN4) is the predominant pacemaker ion-channel in the adult heart and carries the majority of the "funny" current, which strongly contributes to diastolic depolarization in pacemaker cells. Here, we study the mechanism by which Shugoshin-1 affects cardiac pacing activity with two cell models: neonatal rat ventricular myocytes and Chronic Atrial and Intestinal Dysrhythmia Syndrome patient-specific human induced pluripotent stem cell derived cardiomyocytes. We find that Shugoshin-1 interacts directly with HCN4 to promote and stabilize cardiac pacing. This interaction enhances funny-current by optimizing HCN4 cell-surface expression and function. The clinical p.Lys23Glu mutation leads to an impairment in the interaction between Shugoshin-1 and HCN4, along with depressed funny-current and dysrhythmic activity in induced pluripotent stem cell derived cardiomyocytes derived from Chronic Atrial and Intestinal Dysrhythmia Syndrome patients. Our work reveals a critical non-canonical, cohesin-independent role for Shugoshin-1 in maintaining cardiac automaticity and identifies potential therapeutic avenues for cardiac pacemaking disorders, in particular Chronic Atrial and Intestinal Dysrhythmia Syndrome.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Proteínas Musculares/metabolismo , Canais de Potássio/metabolismo , Animais , Arritmias Cardíacas , Proteínas de Ciclo Celular/genética , Linhagem Celular , Sobrevivência Celular , Proteínas Cromossômicas não Histona/genética , Técnicas de Silenciamento de Genes , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Transporte de Íons/fisiologia , Proteínas Musculares/genética , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Marca-Passo Artificial , Canais de Potássio/genética , Ratos
14.
Nat Commun ; 12(1): 2587, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972537

RESUMO

Host cells use several anti-bacterial pathways to defend against pathogens. Here, using a uropathogenic Escherichia coli (UPEC) infection model, we demonstrate that bacterial infection upregulates RhoB, which subsequently promotes intracellular bacteria clearance by inducing LC3 lipidation and autophagosome formation. RhoB binds with Beclin 1 through its residues at 118 to 140 and the Beclin 1 CCD domain, with RhoB Arg133 being the key binding residue. Binding of RhoB to Beclin 1 enhances the Hsp90-Beclin 1 interaction, preventing Beclin 1 degradation. RhoB also directly interacts with Hsp90, maintaining RhoB levels. UPEC infections increase RhoB, Beclin 1 and LC3 levels in bladder epithelium in vivo, whereas Beclin 1 and LC3 levels as well as UPEC clearance are substantially reduced in RhoB+/- and RhoB-/- mice upon infection. We conclude that when stimulated by UPEC infections, host cells promote UPEC clearance through the RhoB-Beclin 1-HSP90 complex, indicating RhoB may be a useful target when developing UPEC treatment strategies.


Assuntos
Autofagossomos/metabolismo , Proteína Beclina-1/metabolismo , Infecções por Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Infecções Urinárias/metabolismo , Escherichia coli Uropatogênica/crescimento & desenvolvimento , Proteína rhoB de Ligação ao GTP/metabolismo , Animais , Autofagossomos/genética , Autofagossomos/ultraestrutura , Proteína Beclina-1/genética , Linhagem Celular , Epitélio/metabolismo , Epitélio/microbiologia , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Feminino , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP90/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica , Estabilidade Proteica , RNA Interferente Pequeno , Proteínas Recombinantes , Bexiga Urinária/metabolismo , Bexiga Urinária/microbiologia , Infecções Urinárias/genética , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/patogenicidade , Proteína rhoB de Ligação ao GTP/genética
15.
Nat Commun ; 12(1): 2829, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990559

RESUMO

Polycomb group (PcG) proteins maintain cell identity by repressing gene expression during development. Surprisingly, emerging studies have recently reported that a number of PcG proteins directly activate gene expression during cell fate determination process. However, the mechanisms by which they direct gene activation in pluripotency remain poorly understood. Here, we show that Phc1, a subunit of canonical polycomb repressive complex 1 (cPRC1), can exert its function in pluripotency maintenance via a PRC1-independent activation of Nanog. Ablation of Phc1 reduces the expression of Nanog and overexpression of Nanog partially rescues impaired pluripotency caused by Phc1 depletion. We find that Phc1 interacts with Nanog and activates Nanog transcription by stabilizing the genome-wide chromatin interactions of the Nanog locus. This adds to the already known canonical function of PRC1 in pluripotency maintenance via a PRC1-dependent repression of differentiation genes. Overall, our study reveals a function of Phc1 to activate Nanog transcription through regulating chromatin architecture and proposes a paradigm for PcG proteins to maintain pluripotency.


Assuntos
Cromatina/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Proteína Homeobox Nanog/genética , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/fisiologia , Animais , Células Cultivadas , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Genoma Humano , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Genéticos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/fisiologia , Complexo Repressor Polycomb 1/antagonistas & inibidores , Complexo Repressor Polycomb 1/deficiência
16.
Nat Commun ; 12(1): 2830, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990564

RESUMO

Coffee and tea are extensively consumed beverages worldwide which have received considerable attention regarding health. Intake of these beverages is consistently linked to, among others, reduced risk of diabetes and liver diseases; however, the mechanisms of action remain elusive. Epigenetics is suggested as a mechanism mediating the effects of dietary and lifestyle factors on disease onset. Here we report the results from epigenome-wide association studies (EWAS) on coffee and tea consumption in 15,789 participants of European and African-American ancestries from 15 cohorts. EWAS meta-analysis of coffee consumption reveals 11 CpGs surpassing the epigenome-wide significance threshold (P-value <1.1×10-7), which annotated to the AHRR, F2RL3, FLJ43663, HDAC4, GFI1 and PHGDH genes. Among them, cg14476101 is significantly associated with expression of the PHGDH and risk of fatty liver disease. Knockdown of PHGDH expression in liver cells shows a correlation with expression levels of genes associated with circulating lipids, suggesting a role of PHGDH in hepatic-lipid metabolism. EWAS meta-analysis on tea consumption reveals no significant association, only two CpGs annotated to CACNA1A and PRDM16 genes show suggestive association (P-value <5.0×10-6). These findings indicate that coffee-associated changes in DNA methylation levels may explain the mechanism of action of coffee consumption in conferring risk of diseases.


Assuntos
Café/efeitos adversos , Metilação de DNA , Epigenoma , Chá/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Ilhas de CpG , Epigênese Genética , Feminino , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Humanos , Fígado/enzimologia , Masculino , Pessoa de Meia-Idade , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Fosfoglicerato Desidrogenase/genética , Fatores de Risco
17.
Nat Commun ; 12(1): 2812, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990570

RESUMO

Trastuzumab is the backbone of HER2-directed gastric cancer therapy, but poor patient response due to insufficient cell sensitivity and drug resistance remains a clinical challenge. Here, we report that HER2 is involved in cell mitotic promotion for tumorigenesis by hyperactivating a crucial HER2-SHCBP1-PLK1 axis that drives trastuzumab sensitivity and is targeted therapeutically. SHCBP1 is an Shc1-binding protein but is detached from scaffold protein Shc1 following HER2 activation. Released SHCBP1 responds to HER2 cascade by translocating into the nucleus following Ser273 phosphorylation, and then contributing to cell mitosis regulation through binding with PLK1 to promote the phosphorylation of the mitotic interactor MISP. Meanwhile, Shc1 is recruited to HER2 for MAPK or PI3K pathways activation. Also, clinical evidence shows that increased SHCBP1 prognosticates a poor response of patients to trastuzumab therapy. Theaflavine-3, 3'-digallate (TFBG) is identified as an inhibitor of the SHCBP1-PLK1 interaction, which is a potential trastuzumab sensitizing agent and, in combination with trastuzumab, is highly efficacious in suppressing HER2-positive gastric cancer growth. These findings suggest an aberrant mitotic HER2-SHCBP1-PLK1 axis underlies trastuzumab sensitivity and offer a new strategy to combat gastric cancer.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptor ErbB-2/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Trastuzumab/farmacologia , Animais , Antineoplásicos Imunológicos/farmacologia , Biflavonoides/farmacologia , Catequina/análogos & derivados , Catequina/farmacologia , Proteínas de Ciclo Celular/química , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Camundongos , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Mitose/efeitos dos fármacos , Modelos Biológicos , Modelos Moleculares , Fosfoproteínas/metabolismo , Prognóstico , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/química , Proteínas Proto-Oncogênicas/química , Receptor ErbB-2/antagonistas & inibidores , Proteínas Adaptadoras da Sinalização Shc/antagonistas & inibidores , Proteínas Adaptadoras da Sinalização Shc/química , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Nat Commun ; 12(1): 2999, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016966

RESUMO

The proper functional interaction between different tissues represents a key component in systemic metabolic control. Indeed, disruption of endocrine inter-tissue communication is a hallmark of severe metabolic dysfunction in obesity and diabetes. Here, we show that the FNDC4-GPR116, liver-white adipose tissue endocrine axis controls glucose homeostasis. We found that the liver primarily controlled the circulating levels of soluble FNDC4 (sFNDC4) and lowering of the hepatokine FNDC4 led to prediabetes in mice. Further, we identified the orphan adhesion GPCR GPR116 as a receptor of sFNDC4 in the white adipose tissue. Upon direct and high affinity binding of sFNDC4 to GPR116, sFNDC4 promoted insulin signaling and insulin-mediated glucose uptake in white adipocytes. Indeed, supplementation with FcsFNDC4 in prediabetic mice improved glucose tolerance and inflammatory markers in a white-adipocyte selective and GPR116-dependent manner. Of note, the sFNDC4-GPR116, liver-adipose tissue axis was dampened in (pre) diabetic human patients. Thus our findings will now allow for harnessing this endocrine circuit for alternative therapeutic strategies in obesity-related pre-diabetes.


Assuntos
Tecido Adiposo Branco/metabolismo , Proteínas de Membrana/metabolismo , Estado Pré-Diabético/metabolismo , Proteínas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Tecido Adiposo Branco/citologia , Adolescente , Adulto , Idoso , Animais , Células CHO , Estudos de Coortes , Cricetulus , Estudos Transversais , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/prevenção & controle , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Insulina/metabolismo , Resistência à Insulina , Ilhotas Pancreáticas/metabolismo , Fígado/metabolismo , Masculino , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/sangue , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Células NIH 3T3 , Estado Pré-Diabético/sangue , Estado Pré-Diabético/tratamento farmacológico , Estado Pré-Diabético/etiologia , Cultura Primária de Células , Proteínas/análise , Receptores Acoplados a Proteínas G/sangue , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Adulto Jovem
19.
Nat Commun ; 12(1): 2970, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016972

RESUMO

Activation of MAVS, an adaptor molecule in Rig-I-like receptor (RLR) signaling, is indispensable for antiviral immunity, yet the molecular mechanisms modulating MAVS activation are not completely understood. Ubiquitination has a central function in regulating the activity of MAVS. Here, we demonstrate that a mitochondria-localized deubiquitinase USP18 specifically interacts with MAVS, promotes K63-linked polyubiquitination and subsequent aggregation of MAVS. USP18 upregulates the expression and production of type I interferon following infection with Sendai virus (SeV) or Encephalomyocarditis virus (EMCV). Mice with a deficiency of USP18 are more susceptible to RNA virus infection. USP18 functions as a scaffold protein to facilitate the re-localization of TRIM31 and enhances the interaction between TRIM31 and MAVS in mitochondria. Our results indicate that USP18 functions as a post-translational modulator of MAVS-mediated antiviral signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Infecções por Cardiovirus/imunologia , Infecções por Respirovirus/imunologia , Ubiquitina Tiolesterase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/isolamento & purificação , Animais , Infecções por Cardiovirus/virologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Vírus da Encefalomiocardite/imunologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Imunidade Inata , Interferon Tipo I/metabolismo , Lisina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Processamento de Proteína Pós-Traducional/imunologia , Células RAW 264.7 , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Infecções por Respirovirus/virologia , Vírus Sendai/imunologia , Transdução de Sinais/imunologia , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/isolamento & purificação , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/imunologia
20.
Nat Commun ; 12(1): 2969, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016988

RESUMO

Chromatin accessibility profiling can identify putative regulatory regions genome wide; however, pooled single-cell methods for assessing the effects of regulatory perturbations on accessibility are limited. Here, we report a modified droplet-based single-cell ATAC-seq protocol for perturbing and evaluating dynamic single-cell epigenetic states. This method (Spear-ATAC) enables simultaneous read-out of chromatin accessibility profiles and integrated sgRNA spacer sequences from thousands of individual cells at once. Spear-ATAC profiling of 104,592 cells representing 414 sgRNA knock-down populations reveals the temporal dynamics of epigenetic responses to regulatory perturbations in cancer cells and the associations between transcription factor binding profiles.


Assuntos
Sistemas CRISPR-Cas/genética , Sequenciamento de Cromatina por Imunoprecipitação , Cromatina/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Neoplasias/genética , Sítios de Ligação/genética , Linhagem Celular Tumoral , Epigênese Genética , Epigenômica/métodos , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Humanos , RNA Guia/genética , Análise de Célula Única/métodos , Fatores de Transcrição/metabolismo
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