RESUMEN
Sterol regulatory element-binding proteins (SREBPs) activate genes involved in the synthesis and trafficking of cholesterol and other lipids and are critical for maintaining lipid homeostasis. Aberrant SREBP activity, however, can contribute to obesity, fatty liver disease, and insulin resistance, hallmarks of metabolic syndrome. Our studies identify a conserved regulatory circuit in which SREBP-1 controls genes in the one-carbon cycle, which produces the methyl donor S-adenosylmethionine (SAMe). Methylation is critical for the synthesis of phosphatidylcholine (PC), a major membrane component, and we find that blocking SAMe or PC synthesis in C. elegans, mouse liver, and human cells causes elevated SREBP-1-dependent transcription and lipid droplet accumulation. Distinct from negative regulation of SREBP-2 by cholesterol, our data suggest a feedback mechanism whereby maturation of nuclear, transcriptionally active SREBP-1 is controlled by levels of PC. Thus, nutritional or genetic conditions limiting SAMe or PC production may activate SREBP-1, contributing to human metabolic disorders.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Humanos , Lipogénesis , Ratones , Modelos Animales , Fosfatidilcolinas/biosíntesis , Interferencia de ARN , S-Adenosilmetionina/biosíntesisRESUMEN
Excessive long-term consumption of dietary carbohydrates, including glucose, sucrose, or fructose, has been shown to have significant impact on genome-wide gene expression, which likely results from changes in metabolic substrate flux. However, there has been no comprehensive study on the acute effects of individual sugars on the genome-wide gene expression that may reveal the genetic changes altering signaling pathways, subsequent metabolic processes, and ultimately physiological/pathological responses. Considering that gene expressions in response to acute carbohydrate ingestion might be different in nutrient sensitive and insensitive mammals, we conducted comparative studies of genome-wide gene expression by deep mRNA sequencing of the liver in nutrient sensitive C57BL/6J and nutrient insensitive BALB/cJ mice. Furthermore, to determine the temporal responses, we compared livers from mice in the fasted state and following ingestion of standard laboratory mouse chow supplemented with plain drinking water or water containing 20% glucose, sucrose, or fructose. Supplementation with these carbohydrates induced unique extents and temporal changes in gene expressions in a strain specific manner. Fructose and sucrose stimulated gene changes peaked at 3 h postprandial, whereas glucose effects peaked at 12 h and 6 h postprandial in C57BL/6J and BABL/cJ mice, respectively. Network analyses revealed that fructose changed genes were primarily involved in lipid metabolism and were more complex in C57BL/6J than in BALB/cJ mice. These data demonstrate that there are qualitative and antitative differences in the normal physiological responses of the liver between these two strains of mice and C57BL/6J is more sensitive to sugar intake than BALB/cJ.
Asunto(s)
Carbohidratos de la Dieta/administración & dosificación , Suplementos Dietéticos , Hígado/metabolismo , Transcriptoma/efectos de los fármacos , Transcriptoma/genética , Animales , Carbohidratos de la Dieta/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Ingestión de Alimentos , Ayuno , Fructosa/administración & dosificación , Fructosa/metabolismo , Glucosa/administración & dosificación , Glucosa/metabolismo , Metabolismo de los Lípidos/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Transducción de Señal/genética , Especificidad de la Especie , Sacarosa/administración & dosificación , Sacarosa/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
The liver maintains metabolic homeostasis by integrating the regulation of nutrient status with both hormonal and neural signals. Many studies on hepatic signaling in response to nutrients have been conducted in mice. However, no in-depth study is currently available that has investigated genome-wide changes in gene expression during the normal physiological fasting-feeding cycle in nutrient-sensitive and -insensitive mice. Using two strains of mice, C57BL/6J and BALB/cJ, and deploying deep RNA-Seq complemented with quantitative RT-PCR, we found that feeding causes substantial and transient changes in gene expression in the livers of both mouse strains. The majority of significantly changed transcripts fell within the areas of biological regulation and cellular and metabolic processes. Among the metabolisms of three major types of macronutrients (i.e. carbohydrates, proteins, and lipids), feeding affected lipid metabolism the most. We also noted that the C57BL/6J and BALB/cJ mice significantly differed in gene expression and in changes in gene expression in response to feeding. In both fasted and fed states, both mouse strains shared common expression patterns for about 10,200 genes, and an additional 400-600 genes were differentially regulated in one strain but not the other. Among the shared genes, more lipogenic genes were induced upon feeding in BABL/cJ than in C57BL/6J mice. In contrast, in the population of differentially enriched genes, C57BL/6J mice expressed more genes involved in lipid metabolism than BALB/cJ mice. In summary, these results reveal that the two mouse strains used here exhibit several differences in feeding-induced hepatic responses in gene expression, especially in lipogenic genes.
Asunto(s)
Biomarcadores/metabolismo , Ingestión de Alimentos , Ayuno , Regulación de la Expresión Génica , Metabolismo de los Lípidos , Hígado/metabolismo , Animales , Perfilación de la Expresión Génica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Especificidad de la EspecieRESUMEN
The sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors best known for stimulating the expression of genes encoding key lipogenic enzymes. However, SREBP functions beyond lipid metabolism are less understood. Here, we show that hepcidin antimicrobial peptide (Hamp), encoding the hormone hepcidin essential for iron homeostasis and regulated by dietary iron and inflammation, is a target gene of the two SREBP isoforms SREBP-1a/c. We found that in tissue culture, mature, active, and nuclear forms of the SREBP-1a/c proteins induce endogenous Hamp gene expression and increase the Hamp promoter activity primarily via three regulatory sequences, including an E-box. Moreover, ChIP experiments revealed that SREBP-1a binds to the Hamp gene promoter. Overexpression of nuclear SREBP-1a under the control of the phosphoenolpyruvate carboxylase-1 (Pck1) promoter in mice increased hepatic Hamp mRNA and blood hepcidin levels, and as expected, caused fatty liver. Consistent with the known effects of Hamp up-regulation, SREBP-1a-overexpressing mice displayed signs of dysregulation in iron metabolism, including reduced serum iron and increased hepatic and splenic iron storage. Conversely, liver-specific depletion of the nuclear forms of SREBPs, as in SREBP cleavage-activating protein knockout mice, impaired lipopolysaccharide-induced up-regulation of hepatic Hamp Together, these results indicate that the SREBP-1a/c transcription regulators activate hepcidin expression and thereby contribute to the control of mammalian iron metabolism.
Asunto(s)
Hepcidinas/metabolismo , Hierro/metabolismo , Lípidos/química , Lipogénesis , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Animales , Células Cultivadas , Células HEK293 , Células Hep G2 , Humanos , Lipogénesis/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genéticaRESUMEN
The Mediator complex plays a critical role in the regulation of transcription by linking transcription factors to RNA polymerase II. By examining mouse livers, we have found that in the fasted state, the Mediator complex exists primarily as an approximately 1.2-MDa complex, consistent with the size of the large Mediator complex, whereas following feeding, it converts to an approximately 600-kDa complex, consistent with the size of the core Mediator complex. This dynamic change is due to the dissociation and degradation of the kinase module that includes the MED13, MED12, cyclin-dependent kinase 8 (CDK8), and cyclin C (CCNC) subunits. The dissociation and degradation of the kinase module are dependent upon nutrient activation of mTORC1 that is necessary for the induction of lipogenic gene expression because pharmacological or genetic inhibition of mTORC1 in the fed state restores the kinase module. The degradation but not dissociation of the kinase module depends upon the E3 ligase, SCFFBW7 In addition, genetically insulin-resistant and obese db/db mice in the fasted state displayed elevated lipogenic gene expression and loss of the kinase module that was reversed following mTORC1 inhibition. These data demonstrate that the assembly state of the Mediator complex undergoes physiologic regulation during normal cycles of fasting and feeding in the mouse liver. Furthermore, the assembly state of the Mediator complex is dysregulated in states of obesity and insulin resistance.
Asunto(s)
Resistencia a la Insulina , Complejo Mediador/metabolismo , Obesidad/patología , Animales , Núcleo Celular/metabolismo , Ciclina C/metabolismo , Quinasa 8 Dependiente de Ciclina/metabolismo , Hígado/metabolismo , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Nutrientes/administración & dosificación , Obesidad/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Ligasas SKP Cullina F-box/deficiencia , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacologíaRESUMEN
Dysregulation of lipid metabolism is common in cancer cells, but the underlying mechanisms are poorly understood. Sterol regulatory element-binding proteins (SREBPs) stimulate lipid biosynthesis through transcriptional activation of lipogenic enzymes. However, SREBPs' roles and potential interacting partners in cancer cells are not fully defined. Using a biochemical approach, we found here that pyruvate kinase M2 (PKM2) physically interacts with the nuclear form of SREBP-1a (nBP1a), by binding to amino acids 43-56 in nBP1a. We also found that PKM2 activates SREBP target gene expression and lipid biosynthesis by stabilizing nBP1a proteins. Using a competitive peptide inhibitor to block the formation of the SREBP-1a/PKM2 complex, we observed that this blockade inhibited lipogenic gene expression. Of note, nBP1a phosphorylation at Thr-59 enhanced the binding to PKM2 and promoted cancer cell growth. Moreover, we show that PKM2 phosphorylates Thr-59 in vitro Lastly, in human patients with hepatocellular carcinoma, nBP1a phosphorylation at Thr-59 was negatively correlated with clinical outcomes. Together, our results reveal that nBP1a/PKM2 interaction activates lipid metabolism genes in cancer cells and that Thr-59 phosphorylation of SREBP-1a plays an important role in cancer cell proliferation.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Proteínas Portadoras/metabolismo , Proliferación Celular , Lipogénesis , Neoplasias Hepáticas/metabolismo , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Hormonas Tiroideas/metabolismo , Células A549 , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proteínas Portadoras/genética , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Células MCF-7 , Proteínas de la Membrana/genética , Complejos Multiproteicos/genética , Proteínas de Neoplasias/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Hormonas Tiroideas/genética , Proteínas de Unión a Hormona TiroideRESUMEN
Epigenetic regulation of gene expression by histone-modifying corepressor complexes is central to normal animal development. The NAD(+)-dependent deacetylase and gene repressor SIRT1 removes histone H4K16 acetylation marks and facilitates heterochromatin formation. However, the mechanistic contribution of SIRT1 to epigenetic regulation at euchromatic loci and whether it acts in concert with other chromatin-modifying activities to control developmental gene expression programs remain unclear. We describe here a SIRT1 corepressor complex containing the histone H3K4 demethylase LSD1/KDM1A and several other LSD1-associated proteins. SIRT1 and LSD1 interact directly and play conserved and concerted roles in H4K16 deacetylation and H3K4 demethylation to repress genes regulated by the Notch signaling pathway. Mutations in Drosophila SIRT1 and LSD1 orthologs result in similar developmental phenotypes and genetically interact with the Notch pathway in Drosophila. These findings offer new insights into conserved mechanisms of epigenetic gene repression and regulation of development by SIRT1 in metazoans.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila melanogaster/genética , Oxidorreductasas N-Desmetilantes/fisiología , Receptores Notch/genética , Sirtuina 1/fisiología , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Inmunoprecipitación , Mutación , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/metabolismo , Fenotipo , Receptores Notch/metabolismo , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
The worldwide increase in type 2 diabetes (T2D) is becoming a major health concern, thus searching for novel preventive and therapeutic strategies has become urgent. In last decade, the paralogous transcription factors MondoA and carbohydrate response element-binding protein (ChREBP) have been revealed to be central mediators of glucose sensing in multiple metabolic organs. Under normal nutrient conditions, MondoA/ChREBP plays vital roles in maintaining glucose homeostasis. However, under chronic nutrient overload, the dysregulation of MondoA/ChREBP contributes to metabolic disorders, such as insulin resistance (IR) and T2D. In this review, we aim to provide an overview of recent advances in the understanding of MondoA/ChREBP and its roles in T2D development. Specifically, we will briefly summarize the functional similarities and differences between MondoA and ChREBP. Then, we will update the roles of MondoA/ChREBP in four T2D-associated metabolic organs (i.e., the skeletal muscle, liver, adipose tissue, and pancreas) in physiological and pathological conditions. Finally, we will discuss the opportunities and challenges of MondoA/ChREBP as drug targets for anti-diabetes. By doing so, we highlight the potential use of therapies targeting MondoA/ChREBP to counteract T2D and its complications.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Transducción de Señal , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Biomarcadores , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/etiología , Regulación de la Expresión Génica , Humanos , Insulina/metabolismo , Resistencia a la Insulina , Hígado/metabolismo , Páncreas/metabolismoRESUMEN
Brown adipose tissue is important for maintaining energy homeostasis and adaptive thermogenesis in rodents and humans. As disorders arising from dysregulated energy metabolism, such as obesity and metabolic diseases, have increased, so has interest in the molecular mechanisms of adipocyte biology. Using a functional screen, we identified cyclin C (CycC), a conserved subunit of the Mediator complex, as a novel regulator for brown adipocyte formation. siRNA-mediated CycC knockdown (KD) in brown preadipocytes impaired the early transcriptional program of differentiation, and genetic KO of CycC completely blocked the differentiation process. RNA sequencing analyses of CycC-KD revealed a critical role of CycC in activating genes co-regulated by peroxisome proliferator activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα). Overexpression of PPARγ2 or addition of the PPARγ ligand rosiglitazone rescued the defects in CycC-KO brown preadipocytes and efficiently activated the PPARγ-responsive promoters in both WT and CycC-KO cells, suggesting that CycC is not essential for PPARγ transcriptional activity. In contrast, CycC-KO significantly reduced C/EBPα-dependent gene expression. Unlike for PPARγ, overexpression of C/EBPα could not induce C/EBPα target gene expression in CycC-KO cells or rescue the CycC-KO defects in brown adipogenesis, suggesting that CycC is essential for C/EBPα-mediated gene activation. CycC physically interacted with C/EBPα, and this interaction was required for C/EBPα transactivation domain activity. Consistent with the role of C/EBPα in white adipogenesis, CycC-KD also inhibited differentiation of 3T3-L1 cells into white adipocytes. Together, these data indicate that CycC activates adipogenesis in part by stimulating the transcriptional activity of C/EBPα.
Asunto(s)
Adipocitos Marrones/metabolismo , Adipogénesis , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Diferenciación Celular , Ciclina C/metabolismo , Activación Transcripcional , Células 3T3-L1 , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Ciclina C/genética , Humanos , Ratones , Ratones Noqueados , PPAR gamma/genética , PPAR gamma/metabolismoRESUMEN
The steroid hormone ecdysone and its receptor (EcR) play critical roles in orchestrating developmental transitions in arthropods. However, the mechanism by which EcR integrates nutritional and developmental cues to correctly activate transcription remains poorly understood. Here, we show that EcR-dependent transcription, and thus, developmental timing in Drosophila, is regulated by CDK8 and its regulatory partner Cyclin C (CycC), and the level of CDK8 is affected by nutrient availability. We observed that cdk8 and cycC mutants resemble EcR mutants and EcR-target genes are systematically down-regulated in both mutants. Indeed, the ability of the EcR-Ultraspiracle (USP) heterodimer to bind to polytene chromosomes and the promoters of EcR target genes is also diminished. Mass spectrometry analysis of proteins that co-immunoprecipitate with EcR and USP identified multiple Mediator subunits, including CDK8 and CycC. Consistently, CDK8-CycC interacts with EcR-USP in vivo; in particular, CDK8 and Med14 can directly interact with the AF1 domain of EcR. These results suggest that CDK8-CycC may serve as transcriptional cofactors for EcR-dependent transcription. During the larval-pupal transition, the levels of CDK8 protein positively correlate with EcR and USP levels, but inversely correlate with the activity of sterol regulatory element binding protein (SREBP), the master regulator of intracellular lipid homeostasis. Likewise, starvation of early third instar larvae precociously increases the levels of CDK8, EcR and USP, yet down-regulates SREBP activity. Conversely, refeeding the starved larvae strongly reduces CDK8 levels but increases SREBP activity. Importantly, these changes correlate with the timing for the larval-pupal transition. Taken together, these results suggest that CDK8-CycC links nutrient intake to developmental transitions (EcR activity) and fat metabolism (SREBP activity) during the larval-pupal transition.
Asunto(s)
Ciclina C/metabolismo , Quinasa 8 Dependiente de Ciclina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Receptores de Esteroides/metabolismo , Animales , Animales Modificados Genéticamente , Ciclina C/genética , Quinasa 8 Dependiente de Ciclina/genética , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Ecdisteroides/biosíntesis , Femenino , Privación de Alimentos , Regulación de la Expresión Génica , Larva/crecimiento & desarrollo , Larva/metabolismo , Mutación , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The sterol regulatory element-binding protein (SREBP) transcription factor family is a critical regulator of lipid and sterol homeostasis in eukaryotes. In mammals, SREBPs are highly active in the fed state to promote the expression of lipogenic and cholesterogenic genes and facilitate fat storage. During fasting, SREBP-dependent lipid/cholesterol synthesis is rapidly diminished in the mouse liver; however, the mechanism has remained incompletely understood. Moreover, the evolutionary conservation of fasting regulation of SREBP-dependent programs of gene expression and control of lipid homeostasis has been unclear. We demonstrate here a conserved role for orthologs of the NAD(+)-dependent deacetylase SIRT1 in metazoans in down-regulation of SREBP orthologs during fasting, resulting in inhibition of lipid synthesis and fat storage. Our data reveal that SIRT1 can directly deacetylate SREBP, and modulation of SIRT1 activity results in changes in SREBP ubiquitination, protein stability, and target gene expression. In addition, chemical activators of SIRT1 inhibit SREBP target gene expression in vitro and in vivo, correlating with decreased hepatic lipid and cholesterol levels and attenuated liver steatosis in diet-induced and genetically obese mice. We conclude that SIRT1 orthologs play a critical role in controlling SREBP-dependent gene regulation governing lipid/cholesterol homeostasis in metazoans in response to fasting cues. These findings may have important biomedical implications for the treatment of metabolic disorders associated with aberrant lipid/cholesterol homeostasis, including metabolic syndrome and atherosclerosis.
Asunto(s)
Regulación hacia Abajo , Ayuno/fisiología , Sirtuina 1/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Acetilación , Animales , Benzamidas/farmacología , Caenorhabditis elegans , Línea Celular , Colesterol/biosíntesis , Regulación hacia Abajo/efectos de los fármacos , Células HeLa , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Lípidos/biosíntesis , Ratones , Naftoles/farmacología , Niacinamida/farmacología , Estabilidad Proteica/efectos de los fármacos , Sirtuinas/antagonistas & inhibidoresRESUMEN
Dysregulation of lipid homeostasis is a common feature of several major human diseases, including type 2 diabetes and cardiovascular disease. However, because of the complex nature of lipid metabolism, the regulatory mechanisms remain poorly defined at the molecular level. As the key transcriptional activators of lipogenic genes, such as fatty acid synthase (FAS), sterol regulatory element-binding proteins (SREBPs) play a pivotal role in stimulating lipid biosynthesis. Several studies have shown that SREBPs are regulated by the NAD(+)-dependent histone deacetylase SIRT1, which forms a complex with the lysine-specific histone demethylase LSD1. Here, we show that LSD1 plays a role in regulating SREBP1-mediated gene expression. Multiple lines of evidence suggest that LSD1 is required for SREBP1-dependent activation of the FAS promoter in mammalian cells. LSD1 knockdown decreases SREBP-1a at the transcription level. Although LSD1 affects nuclear SREBP-1 abundance indirectly through SIRT1, it is also required for SREBP1 binding to the FAS promoter. As a result, LSD1 knockdown decreases triglyceride levels in hepatocytes. Taken together, these results show that LSD1 plays a role in regulating lipogenic gene expression, suggesting LSD1 as a potential target for treating dysregulation of lipid metabolism.
Asunto(s)
Regulación de la Expresión Génica , Histona Demetilasas/metabolismo , Lipogénesis/genética , Animales , Núcleo Celular/metabolismo , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Células HEK293 , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Masculino , Ratones , Regiones Promotoras Genéticas , Unión Proteica , Sirtuina 1/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Transcripción GenéticaRESUMEN
Macroautophagy is a physiological cellular response to nutrient stress, which leads to the engulfment of cytosolic contents by a double-walled membrane structure, the phagophore. Phagophores seal to become autophagosomes, which then fuse with lysosomes to deliver their contents for degradation. Macroautophagy is regulated by numerous cellular factors, including the Class III PI3K (phosphoinositide 3-kinase) Vps34 (vacuolar protein sorting 34). The autophagic functions of Vps34 require its recruitment to a complex that includes Vps15, Beclin-1 and Atg14L (autophagy-related 14-like protein) and is known as Vps34 Complex I. We have now identified NRBF2 (nuclear receptor-binding factor 2) as a new member of Vps34 Complex I. NRBF2 binds to complexes that include Vps34, Vps15, Beclin-1 and ATG-14L, but not the Vps34 Complex II component UVRAG (UV radiation resistance-associated gene). NRBF2 directly interacts with Vps15 via the Vps15 WD40 domain as well as other regions of Vps15. The formation of GFP-LC3 (light chain 3) punctae and PE (phosphatidylethanolamine)-conjugated LC3 (LC3-II) in serum-starved cells was inhibited by NRBF2 knockdown in the absence and presence of lysosomal inhibitors, and p62 levels were increased. Thus NRBF2 plays a critical role in the induction of starvation-induced autophagy as a specific member of Vps34 Complex I.
Asunto(s)
Autofagia/genética , Fosfatidilinositol 3-Quinasas Clase III/genética , Transactivadores/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Relacionadas con la Autofagia , Beclina-1 , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fagosomas/metabolismo , Transporte de Proteínas , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Transactivadores/antagonistas & inhibidores , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína de Clasificación Vacuolar VPS15/genética , Proteína de Clasificación Vacuolar VPS15/metabolismo , Vacuolas/metabolismoRESUMEN
Pyruvate kinase M2 (PKM2) is a key player in the Warburg effect of cancer cells. However, the mechanisms of regulating PKM2 are not fully elucidated. Here, we identified the protein-serine/threonine kinase PIM2, a known oncogene, as a novel binding partner of PKM2. The interaction between PIM2 and PKM2 was confirmed by multiple biochemical approaches in vitro and in cultured cells. Importantly, we found that PIM2 could directly phosphorylate PKM2 on the Thr-454 residue, resulting in an increase of PKM2 protein levels. Compared with wild type, PKM2 with the phosphorylation-defective mutation displayed a reduced effect on glycolysis, co-activating HIF-1α and ß-catenin, and cell proliferation, while enhancing mitochondrial respiration of cancer cells. These findings demonstrate that PIM2-dependent phosphorylation of PKM2 is critical for regulating the Warburg effect in cancer, highlighting PIM2 as a potential therapeutic target.
Asunto(s)
Neoplasias/genética , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Piruvato Quinasa/metabolismo , Integración Viral/genética , Línea Celular Tumoral , Glucólisis , Células HEK293 , Células Hep G2 , Humanos , Oncogenes , Fosforilación , Unión Proteica , Piruvato Quinasa/química , Piruvato Quinasa/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Treonina/química , Técnicas del Sistema de Dos HíbridosRESUMEN
The E2F1 transcription factor can promote proliferation or apoptosis when activated, and is a key downstream target of the retinoblastoma tumour suppressor protein (pRB). Here we show that E2F1 is a potent and specific inhibitor of beta-catenin/T-cell factor (TCF)-dependent transcription, and that this function contributes to E2F1-induced apoptosis. E2F1 deregulation suppresses beta-catenin activity in an adenomatous polyposis coli (APC)/glycogen synthase kinase-3 (GSK3)-independent manner, reducing the expression of key beta-catenin targets including c-MYC. This interaction explains why colorectal tumours, which depend on beta-catenin transcription for their abnormal proliferation, keep RB1 intact. Remarkably, E2F1 activity is also repressed by cyclin-dependent kinase-8 (CDK8), a colorectal oncoprotein. Elevated levels of CDK8 protect beta-catenin/TCF-dependent transcription from inhibition by E2F1. Thus, by retaining RB1 and amplifying CDK8, colorectal tumour cells select conditions that collectively suppress E2F1 and enhance the activity of beta-catenin.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Factor de Transcripción E2F1/antagonistas & inhibidores , Factor de Transcripción E2F1/metabolismo , Proteína de Retinoblastoma/metabolismo , Transcripción Genética , beta Catenina/antagonistas & inhibidores , beta Catenina/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Apoptosis , Línea Celular , Quinasa 8 Dependiente de Ciclina , Regulación de la Expresión Génica , Genes myc/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Proteína de Retinoblastoma/genética , Transducción de Señal , Factores de Transcripción TCF/metabolismo , Proteínas Wnt/metabolismoRESUMEN
Multidrug resistance (MDR) is a serious complication during treatment of opportunistic fungal infections that frequently afflict immunocompromised individuals, such as transplant recipients and cancer patients undergoing cytotoxic chemotherapy. Improved knowledge of the molecular pathways controlling MDR in pathogenic fungi should facilitate the development of novel therapies to combat these intransigent infections. MDR is often caused by upregulation of drug efflux pumps by members of the fungal zinc-cluster transcription-factor family (for example Pdr1p orthologues). However, the molecular mechanisms are poorly understood. Here we show that Pdr1p family members in Saccharomyces cerevisiae and the human pathogen Candida glabrata directly bind to structurally diverse drugs and xenobiotics, resulting in stimulated expression of drug efflux pumps and induction of MDR. Notably, this is mechanistically similar to regulation of MDR in vertebrates by the PXR nuclear receptor, revealing an unexpected functional analogy of fungal and metazoan regulators of MDR. We have also uncovered a critical and specific role of the Gal11p/MED15 subunit of the Mediator co-activator and its activator-targeted KIX domain in antifungal/xenobiotic-dependent regulation of MDR. This detailed mechanistic understanding of a fungal nuclear receptor-like gene regulatory pathway provides novel therapeutic targets for the treatment of multidrug-resistant fungal infections.
Asunto(s)
Candida glabrata/metabolismo , Farmacorresistencia Fúngica , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Receptores de Esteroides/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Antifúngicos/metabolismo , Antifúngicos/farmacología , Candida glabrata/efectos de los fármacos , Candida glabrata/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/genética , Genes Fúngicos/genética , Complejo Mediador , Familia de Multigenes , Receptor X de Pregnano , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transactivadores/química , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Xenobióticos/metabolismoRESUMEN
The liver acts as a master regulator of metabolic homeostasis in part by performing gluconeogenesis. This process is dysregulated in type 2 diabetes, leading to elevated hepatic glucose output. The parenchymal cells of the liver (hepatocytes) are heterogeneous, existing on an axis between the portal triad and the central vein, and perform distinct functions depending on location in the lobule. Here, using single cell analysis of hepatocytes across the liver lobule, we demonstrate that gluconeogenic gene expression ( Pck1 and G6pc ) is relatively low in the fed state and gradually increases first in the periportal hepatocytes during the initial fasting period. As the time of fasting progresses, pericentral hepatocyte gluconeogenic gene expression increases, and following entry into the starvation state, the pericentral hepatocytes show similar gluconeogenic gene expression to the periportal hepatocytes. Similarly, pyruvate-dependent gluconeogenic activity is approximately 10-fold higher in the periportal hepatocytes during the initial fasting state but only 1.5-fold higher in the starvation state. In parallel, starvation suppresses canonical beta-catenin signaling and modulates expression of pericentral and periportal glutamine synthetase and glutaminase, resulting in an enhanced pericentral glutamine-dependent gluconeogenesis. These findings demonstrate that hepatocyte gluconeogenic gene expression and gluconeogenic activity are highly spatially and temporally plastic across the liver lobule, underscoring the critical importance of using well-defined feeding and fasting conditions to define the basis of hepatic insulin resistance and glucose production.
RESUMEN
Aging, chronic high-fat diet feeding, or housing at thermoneutrality induces brown adipose tissue (BAT) involution, a process characterized by reduction of BAT mass and function with increased lipid droplet size. Single nuclei RNA sequencing of aged mice identifies a specific brown adipocyte population of Ucp1-low cells that are pyroptotic and display a reduction in the longevity gene syntaxin 4 (Stx4a). Similar to aged brown adipocytes, Ucp1-STX4KO mice display loss of brown adipose tissue mass and thermogenic dysfunction concomitant with increased pyroptosis. Restoration of STX4 expression or suppression of pyroptosis activation protects against the decline in both mass and thermogenic activity in the aged and Ucp1-STX4KO mice. Mechanistically, STX4 deficiency reduces oxidative phosphorylation, glucose uptake, and glycolysis leading to reduced ATP levels, a known triggering signal for pyroptosis. Together, these data demonstrate an understanding of rapid brown adipocyte involution and that physiologic aging and thermogenic dysfunction result from pyroptotic signaling activation.
Asunto(s)
Tejido Adiposo Pardo , Piroptosis , Animales , Ratones , Adipocitos Marrones/metabolismo , Tejido Adiposo Pardo/metabolismo , Transducción de Señal , Termogénesis/fisiología , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMEN
Myocardial ischemia/reperfusion (I/R) elicits an acute inflammatory response involving complement factors. Recently, we reported that myocardial necrosis was decreased in complement C3-/- mice after heart I/R. The current study used the same heart model to test the effect of C3 on myocardial apoptosis and investigated if C3 regulation of apoptosis occurred in human cardiomyocytes. Comparative proteomics analyses found that cytochrome c was present in the myocardial C3 complex of WT mice following I/R. Incubation of exogenous human C3 reduced apoptosis in a cell culture system of human cardiomyocytes that did not inherently express C3. In addition, human C3 inhibited the intrinsic apoptosis pathway in a cell-free apoptosis system. Finally, human pro-C3 was found to bind with an apoptotic factor, pro-caspase 3, in a cell-free system. Thus, we present firsthand evidence showing that C3 readily reduces myocardial apoptosis via interaction with the intrinsic apoptotic pathway.
Asunto(s)
Isquemia Miocárdica , Daño por Reperfusión Miocárdica , Ratones , Humanos , Animales , Complemento C3/metabolismo , Complemento C3/farmacología , Daño por Reperfusión Miocárdica/metabolismo , Apoptosis , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Isquemia Miocárdica/metabolismo , Isquemia/metabolismoRESUMEN
Objective: To investigate short-term effectiveness and clinical application advantages of orthopedic robot-assisted resection for osteoid osteoma compared with traditional open surgery. Methods: A retrospective analysis was conducted on clinical data of 48 osteoid osteoma patients who met the selection criteria between July 2022 and April 2023. Among them, 23 patients underwent orthopedic robot-assisted resection (robot-assisted surgery group), and 25 patients received traditional open surgery (traditional surgery group). There was no significant difference ( P>0.05) in gender, age, disease duration, lesion location and size, and preoperative visual analogue scale (VAS) score, and musculoskeletal tumor society (MSTS) score between the two groups. The surgical time, intraoperative blood loss, intraoperative lesion localization time, initial localization success rate, infection, and recurrence were recorded and compared. VAS scores before surgery and at 24 hours, 1, 3, 6, and 9 months after surgery and MSTS score before surgery and at 3 months after surgery were assessed. Results: All patients completed the surgery successfully, with no significant difference in surgical time between the two groups ( P>0.05). Compared to the traditional surgery group, the robot-assisted surgery group had less intraoperative blood loss, shorter lesion localization time, and shorter hospitalization time, with significant differences ( P<0.05). The initial localization success rate was higher in the robot-assisted surgery group than in the traditional surgery group, but the difference between the two groups was not significant ( P>0.05). All patients in both groups were followed up, with the follow-up time of 3-12 months in the robot-assisted surgery group (median, 6 months) and 3-14 months in the traditional surgery group (median, 6 months). The postoperative MSTS scores of both groups improved significantly when compared to those before surgery ( P<0.05), but there was no significant difference in the changes in MSTS scores between the two groups ( P>0.05). The postoperative VAS scores of both groups showed a gradually decreasing trend over time ( P<0.05), but there was no significant difference between the two groups after surgery ( P>0.05). During follow-up, except for 1 case of postoperative infection in the traditional surgery group, there was no infections or recurrences in other cases. There was no significant difference in the incidence of postoperative infection between the two groups ( P>0.05). Conclusion: Orthopedic robot-assisted osteoid osteoma resection achieves similar short-term effectiveness when compared to traditional open surgery, with shorter lesion localization time.