RESUMEN
Dysregulation of lipid metabolism could lead to the development of metabolic disorders. We report here that the F-box protein JFK promotes excessive lipid accumulation in adipose tissue and contributes to the development of metabolic syndrome. JFK transgenic mice develop spontaneous obesity, accompanied by dyslipidemia, hyperglycemia, and insulin resistance, phenotypes that are further exacerbated under high-fat diets. In contrast, Jfk knockout mice are lean and resistant to diet-induced metabolic malfunctions. Liver-specific reconstitution of JFK expression in Jfk knockout mice leads to hepatic lipid accumulation resembling human hepatic steatosis and nonalcoholic fatty liver disease. We show that JFK interacts with and destabilizes ING5 through assembly of the SCF complex. Integrative transcriptomic and genomic analysis reveals that the SCFJFK -ING5 axis interferes with AMPK activity and fatty acid ß-oxidation, leading to the suppression of hepatic lipid catabolism. Significantly, JFK is upregulated and AMPKα1 is down-regulated in liver tissues from NAFLD patients. These results reveal that SCFJFK is a bona fide E3 ligase for ING5 and link the SCFJFK -ING5 axis to the development of obesity and metabolic syndrome.
Asunto(s)
Resistencia a la Insulina , Síndrome Metabólico , Enfermedad del Hígado Graso no Alcohólico , Animales , Dieta Alta en Grasa/efectos adversos , Humanos , Metabolismo de los Lípidos/genética , Hígado/metabolismo , Síndrome Metabólico/genética , Síndrome Metabólico/metabolismo , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/genética , Obesidad/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Loss of function/dysregulation of inhibitor of growth 4 (ING4) and hyperactivation of NF-κB are frequent events in many types of human malignancies. However, the molecular mechanisms underlying these remarkable aberrations are not understood. Here, we report that ING4 is physically associated with JFK. We demonstrated that JFK targets ING4 for ubiquitination and degradation through assembly of an Skp1-Cul1-F-box (SCF) complex. We showed that JFK-mediated ING4 destabilization leads to the hyperactivation of the canonical NF-κB pathway and promotes angiogenesis and metastasis of breast cancer. Significantly, the expression of JFK is markedly up-regulated in breast cancer, and the level of JFK is negatively correlated with that of ING4 and positively correlated with an aggressive clinical behavior of breast carcinomas. Our study identified SCF(JFK) as a bona fide E3 ligase for ING4 and unraveled the JFK-ING4-NF-κB axis as an important player in the development and progression of breast cancer, supporting the pursuit of JFK as a potential target for breast cancer intervention.
Asunto(s)
Neoplasias de la Mama/enzimología , Neoplasias de la Mama/fisiopatología , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Proteínas de Homeodominio/metabolismo , Neovascularización Patológica/enzimología , Proteínas Supresoras de Tumor/metabolismo , Neoplasias de la Mama/irrigación sanguínea , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Complejos Multiproteicos , FN-kappa B/metabolismo , Metástasis de la Neoplasia , Neovascularización Patológica/genética , Proteolisis , Transducción de Señal , UbiquitinaciónRESUMEN
Histone H3K4 demethylase LSD1 plays an important role in stem cell biology, especially in the maintenance of the silencing of differentiation genes. However, how the function of LSD1 is regulated and the differentiation genes are derepressed are not understood. Here, we report that elimination of LSD1 promotes embryonic stem cell (ESC) differentiation toward neural lineage. We showed that the destabilization of LSD1 occurs posttranscriptionally via the ubiquitin-proteasome pathway by an E3 ubiquitin ligase, Jade-2. We demonstrated that Jade-2 is a major LSD1 negative regulator during neurogenesis in vitro and in vivo in both mouse developing cerebral cortices and zebra fish embryos. Apparently, Jade-2-mediated degradation of LSD1 acts as an antibraking system and serves as a quick adaptive mechanism for re-establishing epigenetic landscape without more laborious transcriptional regulations. As a potential anticancer strategy, Jade-2-mediated LSD1 degradation could potentially be used in neuroblastoma cells to induce differentiation toward postmitotic neurons.
Asunto(s)
Células Madre Embrionarias/metabolismo , Histona Demetilasas/metabolismo , Neuroblastoma/metabolismo , Neurogénesis , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Diferenciación Celular , Línea Celular Tumoral , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Células HeLa , Histona Demetilasas/genética , Humanos , Ratones , Neuroblastoma/fisiopatología , Oxidorreductasas N-Desmetilantes/genética , Oxidorreductasas N-Desmetilantes/metabolismo , Ubiquitina-Proteína Ligasas/genética , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Jumonji domain-containing 6 (JMJD6) is a member of the Jumonji C domain-containing family of proteins. Compared to other members of the family, the cellular activity of JMJD6 is still not clearly defined and its biological function is still largely unexplored. Here we report that JMJD6 is physically associated with the tumor suppressor p53. We demonstrated that JMJD6 acts as an α-ketoglutarate- and Fe(II)-dependent lysyl hydroxylase to catalyze p53 hydroxylation. We found that p53 indeed exists as a hydroxylated protein in vivo and that the hydroxylation occurs mainly on lysine 382 of p53. We showed that JMJD6 antagonizes p53 acetylation, promotes the association of p53 with its negative regulator MDMX, and represses transcriptional activity of p53. Depletion of JMJD6 enhances p53 transcriptional activity, arrests cells in the G1 phase, promotes cell apoptosis, and sensitizes cells to DNA damaging agent-induced cell death. Importantly, knockdown of JMJD6 represses p53-dependent colon cell proliferation and tumorigenesis in vivo, and significantly, the expression of JMJD6 is markedly up-regulated in various types of human cancer especially in colon cancer, and high nuclear JMJD6 protein is strongly correlated with aggressive clinical behaviors of colon adenocarcinomas. Our results reveal a novel posttranslational modification for p53 and support the pursuit of JMJD6 as a potential biomarker for colon cancer aggressiveness and a potential target for colon cancer intervention.
Asunto(s)
Neoplasias del Colon/genética , Histona Demetilasas con Dominio de Jumonji/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Carcinogénesis/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Hidroxilación , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Estudios Retrospectivos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
ATP-binding cassette (ABC) transporters are implicated in a diverse range of physiological and pathophysiological processes, such as cholesterol and lipid transportation and multidrug resistance. Despite the considerable efforts made in understanding of the cellular function of ABC proteins, the regulation mechanism of this type of protein is still poorly defined. Here we report the identification and functional characterization of a novel ATPase protein, protein associated with ABC transporters (PAAT), in humans. PAAT contains a nucleotide-binding domain (NBD)-like domain and a signal for intramitochondrial sorting. We showed that PAAT is localized in both the cytoplasm and the mitochondria and has an intrinsic ATPase activity. PAAT physically interacts with the 3 known mitochondrial inner membrane ABC proteins, ABCB7, ABCB8, and ABCB10, but not ABCB1, ABCB6, or ABCG2, and functionally regulates the transport of ferric nutrients and heme biosynthesis. Significantly, PAAT deficiency promotes cell death, reduces mitochondrial potential, and sensitizes mitochondria to oxidative stress-induced DNA damages. Our experiments revealed that PAAT is a novel ATPase and a trans-regulator of mitochondrial ABC transporters that plays an important role in the maintenance of mitochondrial homeostasis and cell survival.
Asunto(s)
Sistema de Transporte de Aminoácidos A/metabolismo , Homeostasis/fisiología , Mitocondrias/metabolismo , Células Cultivadas , Humanos , Unión Proteica , Transporte de Proteínas/fisiologíaRESUMEN
SET8 (SET domain containing 8) is a histone H4 lysine 20 (H4K20)-specific monomethyltransferase in higher eukaryotes that exerts diverse functions in transcription regulation, DNA repair, tumor metastasis, and genome integrity. The activity of SET8 is tightly controlled during cell cycle through post-translational modifications, including ubiquitination, phosphorylation, and sumoylation. However, how the expression of SET8 is regulated is not fully understood. Here, we report that microRNA-7 is a negative regulator of SET8. We demonstrated that microRNA-7 inhibits H4K20 monomethylation and suppresses epithelial-mesenchymal transition and the invasive potential of breast cancer cells. We showed that microRNA-7 promotes spontaneous DNA damages and sensitizes cells to induced DNA damages. Our experiments provide a molecular mechanism for the regulation of SET8 and extend the biological function of microRNA-7 to DNA damage response, supporting the pursuit of microRNA-7 as a potential target for breast cancer intervention.
Asunto(s)
Neoplasias de la Mama/metabolismo , Daño del ADN , N-Metiltransferasa de Histona-Lisina/metabolismo , MicroARNs/metabolismo , Proteínas de Neoplasias/metabolismo , ARN Neoplásico/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/prevención & control , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/genética , Femenino , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Histonas/metabolismo , Humanos , Metilación , MicroARNs/genética , Invasividad Neoplásica , Proteínas de Neoplasias/genética , ARN Neoplásico/genéticaRESUMEN
The efficacy and safety of gene-therapy strategies for indications like tissue damage hinge on precision; yet, current methods afford little spatial or temporal control of payload delivery. Here, we find that tissue-regeneration enhancer elements (TREEs) isolated from zebrafish can direct targeted, injury-associated gene expression from viral DNA vectors delivered systemically in small and large adult mammalian species. When employed in combination with CRISPR-based epigenome editing tools in mice, zebrafish TREEs stimulated or repressed the expression of endogenous genes after ischemic myocardial infarction. Intravenously delivered recombinant AAV vectors designed with a TREE to direct a constitutively active YAP factor boosted indicators of cardiac regeneration in mice and improved the function of the injured heart. Our findings establish the application of contextual enhancer elements as a potential therapeutic platform for spatiotemporally controlled tissue regeneration in mammals.
Asunto(s)
Elementos de Facilitación Genéticos , Terapia Genética , Corazón , Infarto del Miocardio , Miocitos Cardíacos , Regeneración , Animales , Ratones , Proliferación Celular , Corazón/fisiología , Infarto del Miocardio/genética , Infarto del Miocardio/terapia , Miocitos Cardíacos/metabolismo , Pez Cebra/genética , Terapia Genética/métodos , Regeneración/genéticaRESUMEN
BACKGROUND: The optimal delivery route to enhance effectiveness of regenerative therapeutics to the human heart is poorly understood. Direct intra-myocardial (IM) injection is the gold standard, however, it is relatively invasive. We thus compared targeted IM against less invasive, catheter-based intra-coronary (IC) delivery to porcine myocardium for the acute retention of nanoparticles using cardiac magnetic resonance (CMR) imaging and viral vector transduction using qPCR. METHODS: Ferumoxytol iron oxide (IO) nanoparticles (5 ml) were administered to Yorkshire swine (n = 13) by: (1) IM via thoracotomy, (2) catheter-based IC balloon-occlusion (BO) with infusion into the distal left anterior descending (LAD) coronary artery, (3) IC perforated side-wall (SW) infusion into the LAD, or (4) non-selective IC via left main (LM) coronary artery infusion. Hearts were harvested and imaged using at 3T whole-body MRI scanner. In separate Yorkshire swine (n = 13), an adeno-associated virus (AAV) vector was similarly delivered, tissue harvested 4-6 weeks later, and viral DNA quantified from predefined areas at risk (apical LV/RV) vs. not at risk in a potential mid-LAD infarct model. Results were analyzed using pairwise Student's t-test. RESULTS: IM delivery yielded the highest IO retention (16.0 ± 4.6% of left ventricular volume). Of the IC approaches, BO showed the highest IO retention (8.7 ± 2.2% vs. SW = 5.5 ± 4.9% and LM = 0%) and yielded consistent uptake in the porcine distal LAD territory, including the apical septum, LV, and RV. IM delivery was limited to the apex and anterior wall, without septal retention. For the AAV delivery, the BO was most efficient in the at risk territory (Risk: BO = 6.0 × 10-9, IM = 1.4 × 10-9, LM = 3.2 × 10-10 viral copies per µg genomic DNA) while all delivery routes were comparable in the non-risk territory (BO = 1.7 × 10-9, IM = 8.9 × 10-10, LM = 1.2 × 10-9). CONCLUSIONS: Direct IM injection has the highest local retention, while IC delivery with balloon occlusion and distal infusion is the most effective IC delivery technique to target therapeutics to a heart territory most in risk from an infarct.
RESUMEN
Although SIRT7 is a member of sirtuin family proteins that are described as NAD(+)-dependent class III histone deacetylases, the intrinsic enzymatic activity of this sirtuin protein remains to be investigated and the cellular function of SIRT7 remains to be explored. Here we report that SIRT7 is an NAD(+)-dependent histone desuccinylase. We show that SIRT7 is recruited to DNA double-strand breaks (DSBs) in a PARP1-dependent manner and catalyses desuccinylation of H3K122 therein, thereby promoting chromatin condensation and DSB repair. We demonstrate that depletion of SIRT7 impairs chromatin compaction during DNA-damage response and sensitizes cells to genotoxic stresses. Our study indicates SIRT7 is a histone desuccinylase, providing a molecular basis for the understanding of epigenetic regulation by this sirtuin protein. Our experiments reveal that SIRT7-catalysed H3K122 desuccinylation is critically implemented in DNA-damage response and cell survival, providing a mechanistic insight into the cellular function of SIRT7.
Asunto(s)
Cromatina/metabolismo , Inestabilidad Genómica , Histonas/metabolismo , Sirtuinas/metabolismo , Succinatos/metabolismo , Biocatálisis , Línea Celular , Supervivencia Celular , Daño del ADN , Reparación del ADN , Humanos , Lisina/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismoRESUMEN
FOXA1 functions in epigenetic reprogramming and is described as a 'pioneer factor'. However, exactly how FOXA1 achieves these remarkable biological functions is not fully understood. Here we report that FOXA1 associates with DNA repair complexes and is required for genomic targeting of DNA polymerase ß (POLB) in human cells. Genome-wide DNA methylomes demonstrate that the FOXA1 DNA repair complex is functionally linked to DNA demethylation in a lineage-specific fashion. Depletion of FOXA1 results in localized reestablishment of methylation in a large portion of FOXA1-bound regions, and the regions with the most consistent hypermethylation exhibit the greatest loss of POLB and are represented by active promoters and enhancers. Consistently, overexpression of FOXA1 commits its binding sites to active DNA demethylation in a POLB-dependent manner. Finally, FOXA1-associated DNA demethylation is tightly coupled with estrogen receptor genomic targeting and estrogen responsiveness. Together, these results link FOXA1-associated DNA demethylation to transcriptional pioneering by FOXA1.
Asunto(s)
Metilación de ADN , ADN Polimerasa beta/metabolismo , Enzimas Reparadoras del ADN/genética , Reparación del ADN/genética , Epigenómica , Factor Nuclear 3-alfa del Hepatocito/genética , Neoplasias/genética , Transcripción Genética , Sitios de Unión , Enzimas Reparadoras del ADN/metabolismo , Estrógenos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Humanos , Células MCF-7 , Neoplasias/metabolismo , Regiones Promotoras Genéticas/genéticaRESUMEN
How loss-of-function of GATA3 contributes to the development of breast cancer is poorly understood. Here, we report that GATA3 nucleates a transcription repression program composed of G9A and MTA3-, but not MTA1- or MTA2-, constituted NuRD complex. Genome-wide analysis of the GATA3/G9A/NuRD(MTA3) targets identified a cohort of genes including ZEB2 that are critically involved in epithelial-to-mesenchymal transition and cell invasion. We demonstrate that the GATA3/G9A/NuRD(MTA3) complex inhibits the invasive potential of breast cancer cells in vitro and suppresses breast cancer metastasis in vivo. Strikingly, the expression of GATA3, G9A, and MTA3 is concurrently downregulated during breast cancer progression, leading to an elevated expression of ZEB2, which, in turn, represses the expression of G9A and MTA3 through the recruitment of G9A/NuRD(MTA1).
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Factor de Transcripción GATA3/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Represoras/metabolismo , Animales , Neoplasias de la Mama/genética , Transición Epitelial-Mesenquimal , Femenino , Factor de Transcripción GATA3/genética , Xenoinjertos , Proteínas de Homeodominio/genética , Humanos , Células MCF-7 , Ratones , Ratones Desnudos , Ratones SCID , Metástasis de la Neoplasia , Proteínas Represoras/genética , Transfección , Caja Homeótica 2 de Unión a E-Box con Dedos de ZincRESUMEN
The effects of dimethyl sulfoxide (DMSO) on the activity of polyphenol oxidase (PPO, EC 1.14.18.1) from blowfly pupae for the oxidation of L-3,4-dihydroxyphenylalanine were studied. The results showed that low concentrations of DMSO could lead to reversible inactivation to the enzyme. The IC(50) value, the inactivator concentration leading to 50% activity lost, was estimated to be 2.35 M. Inactivation of the enzyme by DMSO was classified as mixed type. The kinetics of inactivation of PPO from blowfly pupae in the low concentrations of DMSO solution was studied using the kinetic method of the substrate reaction. The rate constants of inactivation were determined. The results show that k(+0) was much larger than k'(+0), indicating that the free enzyme molecule was more fragile than the enzyme-substrate complex in the DMSO solution. It was suggested that the presence of the substrate offers marked protection of this enzyme against inactivation by DMSO.