RESUMO
Retinoic acid receptor-related orphan receptor α (RORα) is a transcription factor involved in nuclear gene expression and a known tumor suppressor. RORα was the first identified substrate of lysine methylation-dependent degradation. However, the mechanisms of other post-translational modifications (PTMs) that occur in RORα remain largely unknown, especially in liver cancer. Arginine methylation is a common PTM in arginine residues of nonhistone and histone proteins and affects substrate protein function and fate. We found an analogous amino acid disposition containing R37 at the ROR N-terminus compared to histone H3 residue, which is arginine methylated. Here, we provide evidence that R37 methylation-dependent degradation is carried out by protein arginine methyltransferase 5 (PRMT5). Further, we discovered that PRMT5 regulated the interaction between the E3 ubiquitin ligase ITCH and RORα through RORα arginine methylation. Arginine methylation-dependent ubiquitination-mediated RORα degradation reduced downstream target gene activation. H2 O2 -induced reactive oxygen species (ROS) decreased PRMT5 protein levels, consequently increasing RORα protein levels in HepG2 liver cancer cells. In addition, ROS inhibited liver cancer progression by inducing apoptosis via PRMT5-mediated RORα methylation and the ITCH axis. Our results potentiate PRMT5 as an elimination target in cancer therapy, and this additional regulatory level within ROS signaling may help identify new targets for therapeutic intervention in liver cancer.
Assuntos
Arginina , Neoplasias Hepáticas , Humanos , Metilação , Espécies Reativas de Oxigênio/metabolismo , Arginina/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Neoplasias Hepáticas/genéticaRESUMO
The Gram-positive pathogen Staphylococcus aureus is the only bacterium known to synthesize arginine from proline via the arginine-proline interconversion pathway despite having genes for the well-conserved glutamate pathway. Since the proline-arginine interconversion pathway is repressed by CcpA-mediated carbon catabolite repression (CCR), CCR has been attributed to the arginine auxotrophy of S. aureus. Using ribose as a secondary carbon source, here, we demonstrate that S. aureus arginine auxotrophy is not due to CCR but due to the inadequate concentration of proline degradation product. Proline is degraded by proline dehydrogenase (PutA) into pyrroline-5-carboxylate (P5C). Although the PutA expression was fully induced by ribose, the P5C concentration remained insufficient to support arginine synthesis because P5C was constantly consumed by the P5C reductase ProC. When the P5C concentration was artificially increased by either PutA overexpression or proC deletion, S. aureus could synthesize arginine from proline regardless of carbon source. In contrast, when the P5C concentration was reduced by overexpression of proC, it inhibited the growth of the ccpA deletion mutant without arginine. Intriguingly, the ectopic expression of the glutamate pathway enzymes converted S. aureus into arginine prototroph. In an animal experiment, the arginine-proline interconversion pathway was not required for the survival of S. aureus. Based on these results, we concluded that S. aureus does not synthesize arginine from proline under physiological conditions. We also propose that arginine auxotrophy of S. aureus is not due to the CcpA-mediated CCR but due to the inactivity of the conserved glutamate pathway. IMPORTANCE Staphylococcus aureus is a versatile Gram-positive human pathogen infecting various human organs. The bacterium's versatility is partly due to efficient metabolic regulation via the carbon catabolite repression system (CCR). S. aureus is known to interconvert proline and arginine, and CCR represses the synthesis of both amino acids. However, when CCR is released by a nonpreferred carbon source, S. aureus can synthesize proline but not arginine. In this study, we show that, in S. aureus, the intracellular concentration of pyrroline-5-carboxylate (P5C), the degradation product of proline and the substrate of proline synthesis, is too low to synthesize arginine from proline. These results call into question the notion that S. aureus synthesizes arginine from proline.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Animais , Arginina/metabolismo , Carbono/metabolismo , Ácido Glutâmico/metabolismo , Mutação , Prolina/genética , Prolina/metabolismo , Ribose/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMO
Inexorable increases in insulin resistance, lipolysis, and hepatic glucose production (HGP) are hallmarks of type 2 diabetes. Previously, we showed that peripheral delivery of exogenous fibroblast growth factor 1 (FGF1) has robust anti-diabetic effects mediated by the adipose FGF receptor (FGFR) 1. However, its mechanism of action is not known. Here, we report that FGF1 acutely lowers HGP by suppressing adipose lipolysis. On a molecular level, FGF1 inhibits the cAMP-protein kinase A axis by activating phosphodiesterase 4D (PDE4D), which separates it mechanistically from the inhibitory actions of insulin via PDE3B. We identify Ser44 as an FGF1-induced regulatory phosphorylation site in PDE4D that is modulated by the feed-fast cycle. These findings establish the FGF1/PDE4 pathway as an alternate regulator of the adipose-HGP axis and identify FGF1 as an unrecognized regulator of fatty acid homeostasis.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Fator 1 de Crescimento de Fibroblastos/metabolismo , Humanos , Insulina/metabolismo , Lipólise/fisiologiaRESUMO
In bacteria, chromosomal DNA resides in the cytoplasm, and most transcription factors are also found in the cytoplasm. However, some transcription factors, called membrane-bound transcription factors (MTFs), reside in the cytoplasmic membrane. Here, we report the identification of a new MTF in the Gram-positive pathogen Staphylococcus aureus and its regulation by the protease FtsH. The MTF, named MbtS (membrane-bound transcription factor of Staphylococcus aureus), is encoded by SAUSA300_2640 and predicted to have an N-terminal DNA binding domain and three transmembrane helices. The MbtS protein was degraded by membrane vesicles containing FtsH or by the purified FtsH. MbtS bound to an inverted repeat sequence in its promoter region, and the DNA binding was essential for its transcription. Transcriptional comparison between the ftsH deletion mutant and the ftsH mbtS double mutant showed that MbtS could alter the transcription of over 200 genes. Although the MbtS protein was not detected in wild-type (WT) cells grown in a liquid medium, the protein was detected in some isolated colonies on an agar plate. In a murine model of a skin infection, the disruption of mbtS increased the lesion size. Based on these results, we concluded that MbtS is a new S. aureus MTF whose activity is proteolytically regulated by FtsH.IMPORTANCEStaphylococcus aureus is an important pathogenic bacterium causing various diseases in humans. In the bacterium, transcription is typically regulated by the transcription factors located in the cytoplasm. In this study, we report an atypical transcription factor identified in S. aureus Unlike most other transcription factors, the newly identified transcription factor is located in the cytoplasmic membrane, and its activity is proteolytically controlled by the membrane-bound AAA+ protease FtsH. The newly identified MTF, named MbtS, has the potential to regulate the transcription of over 200 genes. This study provides a molecular mechanism by which a protease affects bacterial transcription and illustrates the diversity of the bacterial transcriptional regulation.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/enzimologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia , Fatores de Transcrição/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Proteínas de Bactérias/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteólise , Staphylococcus aureus/genética , Fatores de Transcrição/genéticaRESUMO
Retinoic acid-related orphan receptor α (RORα) functions as a transcription factor for various biological processes, including circadian rhythm, cancer, and metabolism. Here, we generate intestinal epithelial cell (IEC)-specific RORα-deficient (RORαΔIEC) mice and find that RORα is crucial for maintaining intestinal homeostasis by attenuating nuclear factor κB (NF-κB) transcriptional activity. RORαΔIEC mice exhibit excessive intestinal inflammation and highly activated inflammatory responses in the dextran sulfate sodium (DSS) mouse colitis model. Transcriptome analysis reveals that deletion of RORα leads to up-regulation of NF-κB target genes in IECs. Chromatin immunoprecipitation analysis reveals corecruitment of RORα and histone deacetylase 3 (HDAC3) on NF-κB target promoters and subsequent dismissal of CREB binding protein (CBP) and bromodomain-containing protein 4 (BRD4) for transcriptional repression. Together, we demonstrate that RORα/HDAC3-mediated attenuation of NF-κB signaling controls the balance of inflammatory responses, and therapeutic strategies targeting this epigenetic regulation could be beneficial to the treatment of chronic inflammatory diseases, including inflammatory bowel disease (IBD).
Assuntos
Homeostase/fisiologia , Inflamação/metabolismo , Intestinos/fisiologia , Receptores Nucleares Órfãos/metabolismo , Animais , Epigênese Genética/fisiologia , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Transcriptoma/fisiologiaRESUMO
Branched-chain amino acid (BCAA; valine, leucine and isoleucine) supplementation is often beneficial to energy expenditure; however, increased circulating levels of BCAA are linked to obesity and diabetes. The mechanisms of this paradox remain unclear. Here we report that, on cold exposure, brown adipose tissue (BAT) actively utilizes BCAA in the mitochondria for thermogenesis and promotes systemic BCAA clearance in mice and humans. In turn, a BAT-specific defect in BCAA catabolism attenuates systemic BCAA clearance, BAT fuel oxidation and thermogenesis, leading to diet-induced obesity and glucose intolerance. Mechanistically, active BCAA catabolism in BAT is mediated by SLC25A44, which transports BCAAs into mitochondria. Our results suggest that BAT serves as a key metabolic filter that controls BCAA clearance via SLC25A44, thereby contributing to the improvement of metabolic health.
Assuntos
Tecido Adiposo Marrom/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Metabolismo Energético , Homeostase , Proteínas Mitocondriais/metabolismo , Proteínas Carreadoras de Solutos/metabolismo , Termogênese , Tecido Adiposo Marrom/citologia , Animais , Temperatura Baixa , Intolerância à Glucose/metabolismo , Humanos , Masculino , Camundongos , Mitocôndrias/metabolismo , Obesidade/metabolismoRESUMO
Environmental cues profoundly affect cellular plasticity in multicellular organisms. For instance, exercise promotes a glycolytic-to-oxidative fibre-type switch in skeletal muscle, and cold acclimation induces beige adipocyte biogenesis in adipose tissue. However, the molecular mechanisms by which physiological or pathological cues evoke developmental plasticity remain incompletely understood. Here we report a type of beige adipocyte that has a critical role in chronic cold adaptation in the absence of ß-adrenergic receptor signalling. This beige fat is distinct from conventional beige fat with respect to developmental origin and regulation, and displays enhanced glucose oxidation. We therefore refer to it as glycolytic beige fat. Mechanistically, we identify GA-binding protein α as a regulator of glycolytic beige adipocyte differentiation through a myogenic intermediate. Our study reveals a non-canonical adaptive mechanism by which thermal stress induces progenitor cell plasticity and recruits a distinct form of thermogenic cell that is required for energy homeostasis and survival.
Assuntos
Tecido Adiposo Bege/citologia , Tecido Adiposo Bege/metabolismo , Temperatura Baixa , Resposta ao Choque Frio , Glicólise , Desenvolvimento Muscular , Aclimatação , Tecido Adiposo Branco/citologia , Tecido Adiposo Branco/metabolismo , Animais , Diferenciação Celular , Sobrevivência Celular , Metabolismo Energético , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Homeostase , Masculino , Camundongos , Proteína MyoD/metabolismo , Mioblastos/citologia , Receptores Adrenérgicos beta/metabolismoRESUMO
Retinoic acid-related orphan receptor α (RORα) regulates diverse physiological processes, including inflammatory responses, lipid metabolism, circadian rhythm, and cancer biology. RORα has four different isoforms which have distinct N-terminal domains but share identical DNA binding domain and ligand binding domain in human. However, lack of specific antibody against each RORα isoform makes biochemical studies on each RORα isoform remain unclear. Here, we generate RORα2-specific antibody and characterize the role of RORα2 in promoting tumor progression in breast cancer. RORα2 requires lysine specific demethylase 1 (LSD1/KDM1A) as a coactivator for transcriptional activation of RORα2 target genes, exemplified by CTNND1. Intriguingly, RORα2 and LSD1 protein levels are dramatically elevated in human breast cancer specimens compared to normal counterparts. Taken together, our studies indicate that LSD1-mediated RORα2 transcriptional activity is important to promote tumor cell migration in human breast cancer as well as breast cancer cell lines. Therefore, our data establish that suppression of LSD1-mediated RORα2 transcriptional activity may be potent therapeutic strategy to attenuate tumor cell migration in human breast cancer.
Assuntos
Neoplasias da Mama/patologia , Regulação da Expressão Gênica , Histona Desmetilases/metabolismo , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Feminino , Humanos , Transcrição GênicaRESUMO
The retinoic acid receptor-related orphan receptor-α (RORα) is an important regulator of various biological processes, including cerebellum development, circadian rhythm and cancer. Here, we show that hepatic RORα controls lipid homeostasis by negatively regulating transcriptional activity of peroxisome proliferators-activated receptor-γ (PPARγ) that mediates hepatic lipid metabolism. Liver-specific Rorα-deficient mice develop hepatic steatosis, obesity and insulin resistance when challenged with a high-fat diet (HFD). Global transcriptome analysis reveals that liver-specific deletion of Rorα leads to the dysregulation of PPARγ signaling and increases hepatic glucose and lipid metabolism. RORα specifically binds and recruits histone deacetylase 3 (HDAC3) to PPARγ target promoters for the transcriptional repression of PPARγ. PPARγ antagonism restores metabolic homeostasis in HFD-fed liver-specific Rorα deficient mice. Our data indicate that RORα has a pivotal role in the regulation of hepatic lipid homeostasis. Therapeutic strategies designed to modulate RORα activity may be beneficial for the treatment of metabolic disorders.Hepatic steatosis development may result from dysregulation of lipid metabolism, which is finely tuned by several transcription factors including the PPAR family. Here Kim et al. show that the nuclear receptor RORα inhibits PPARγ-mediated transcriptional activity by interacting with HDAC3 and competing for the promoters of lipogenic genes.
Assuntos
Regulação da Expressão Gênica/genética , Histona Desacetilases/metabolismo , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , PPAR gama/genética , Animais , Dieta Hiperlipídica , Fígado Gorduroso/genética , Redes Reguladoras de Genes , Glucose/metabolismo , Homeostase , Resistência à Insulina/genética , Lipogênese/genética , Camundongos , Obesidade/genética , PPAR gama/antagonistas & inibidores , Regiões Promotoras Genéticas/genéticaRESUMO
The actions of transcription factors, chromatin modifiers and noncoding RNAs are crucial for the programming of cell states. Although the importance of various epigenetic machineries for controlling pluripotency of embryonic stem (ES) cells has been previously studied, how chromatin modifiers cooperate with specific transcription factors still remains largely elusive. Here, we find that Pontin chromatin remodelling factor plays an essential role as a coactivator for Oct4 for maintenance of pluripotency in mouse ES cells. Genome-wide analyses reveal that Pontin and Oct4 share a substantial set of target genes involved in ES cell maintenance. Intriguingly, we find that the Oct4-dependent coactivator function of Pontin extends to the transcription of large intergenic noncoding RNAs (lincRNAs) and in particular linc1253, a lineage programme repressing lincRNA, is a Pontin-dependent Oct4 target lincRNA. Together, our findings demonstrate that the Oct4-Pontin module plays critical roles in the regulation of genes involved in ES cell fate determination.
Assuntos
DNA Helicases/genética , Epigênese Genética , Células-Tronco Embrionárias Murinas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , RNA Longo não Codificante/genética , Animais , Diferenciação Celular , Cromatina/química , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA Helicases/deficiência , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Fator 3 de Transcrição de Octâmero/deficiência , Receptores Patched , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , RNA Longo não Codificante/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Tropomiosina/genética , Tropomiosina/metabolismoRESUMO
Ubiquitination plays a major role in protein degradation. Although phosphorylation-dependent ubiquitination is well known for the regulation of protein stability, methylation-dependent ubiquitination machinery has not been characterized. Here, we provide evidence that methylation-dependent ubiquitination is carried out by damage-specific DNA binding protein 1 (DDB1)/cullin4 (CUL4) E3 ubiquitin ligase complex and a DDB1-CUL4-associated factor 1 (DCAF1) adaptor, which recognizes monomethylated substrates. Molecular modeling and binding affinity studies reveal that the putative chromo domain of DCAF1 directly recognizes monomethylated substrates, whereas critical binding pocket mutations of the DCAF1 chromo domain ablated the binding from the monomethylated substrates. Further, we discovered that enhancer of zeste homolog 2 (EZH2) methyltransferase has distinct substrate specificities for histone H3K27 and nonhistones exemplified by an orphan nuclear receptor, RORα. We propose that EZH2-DCAF1/DDB1/CUL4 represents a previously unrecognized methylation-dependent ubiquitination machinery specifically recognizing "methyl degron"; through this, nonhistone protein stability can be dynamically regulated in a methylation-dependent manner.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Humanos , Células MCF-7 , Metilação , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Proteínas Serina-Treonina Quinases , Especificidade por SubstratoRESUMO
A critical component of the DNA damage response is the p53 tumor suppressor, and aberrant p53 function leads to uncontrolled cell proliferation and malignancy. Several molecules have been shown to regulate p53 stability; however, genome-wide systemic approaches for determining the affected, specific downstream target genes have not been extensively studied. Here, we first identified an orphan nuclear receptor, RORα, as a direct target gene of p53, which contains functional p53 response elements. The functional consequences of DNA damage-induced RORα are to stabilize p53 and activate p53 transcription in a HAUSP/Usp7-dependent manner. Interestingly, microarray analysis revealed that RORα-mediated p53 stabilization leads to the activation of a subset of p53 target genes that are specifically involved in apoptosis. We further confirmed that RORα enhances p53-dependent, in vivo apoptotic function in the Drosophila model system. Together, we determined that RORα is a p53 regulator that exerts its role in increased apoptosis via p53.
Assuntos
Apoptose , Dano ao DNA , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Estabilidade Proteica , Proteína Supressora de Tumor p53/metabolismo , Animais , Apoptose/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética , Ubiquitina Tiolesterase/metabolismo , UbiquitinaçãoRESUMO
Wnt family members play diverse roles in development and disease. Noncanonical Wnt ligands can inhibit canonical Wnt signaling depending on the cellular context; however, the underlying mechanism of this antagonism remains poorly understood. Here we identify a specific mechanism of orphan nuclear receptor RORalpha-mediated inhibition of canonical Wnt signaling in colon cancer. Wnt5a/PKCalpha-dependent phosphorylation on serine residue 35 of RORalpha is crucial to link RORalpha to Wnt/beta-catenin signaling, which exerts inhibitory function of the expression of Wnt/beta-catenin target genes. Intriguingly, there is a significant correlation of reduction of RORalpha phosphorylation in colorectal tumor cases compared to their normal counterpart, providing the clinical relevance of the findings. Our data provide evidence for a role of RORalpha, functioning at the crossroads between the canonical and the noncanonical Wnt signaling pathways, in mediating transrepression of the Wnt/beta-catenin target genes, thereby providing new approaches for the development of therapeutic agents for human cancers.