RESUMEN
Cancer cachexia is a complex metabolic disorder accounting for ~20% of cancer-related deaths, yet its metabolic landscape remains unexplored. Here, we report a decrease in B vitamin-related liver enzymes as a hallmark of systemic metabolic changes occurring in cancer cachexia. Metabolomics of multiple mouse models highlights cachexia-associated reductions of niacin, vitamin B6, and a glycine-related subset of one-carbon (C1) metabolites in the liver. Integration of proteomics and metabolomics reveals that liver enzymes related to niacin, vitamin B6, and glycine-related C1 enzymes dependent on B vitamins decrease linearly with their associated metabolites, likely reflecting stoichiometric cofactor-enzyme interactions. The decrease of B vitamin-related enzymes is also found to depend on protein abundance and cofactor subtype. These metabolic/proteomic changes and decreased protein malonylation, another cachexia feature identified by protein post-translational modification analysis, are reflected in blood samples from mouse models and gastric cancer patients with cachexia, underscoring the clinical relevance of our findings.
Asunto(s)
Niacina , Neoplasias Gástricas , Complejo Vitamínico B , Ratones , Animales , Humanos , Caquexia/etiología , Caquexia/metabolismo , Proteómica , Piridoxina , Vitamina B 6 , Hígado/metabolismo , Glicina/metabolismoRESUMEN
Signal-induced proliferation-associated 1 (SIPA1)-like 1 (SIPA1L1; also known as SPAR1) has been proposed to regulate synaptic functions that are important in maintaining normal neuronal activities, such as regulating spine growth and synaptic scaling, as a component of the PSD-95/NMDA-R-complex. However, its physiological role remains poorly understood. Here, we performed expression analyses using super-resolution microscopy (SRM) in mouse brain and demonstrated that SIPA1L1 is mainly localized to general submembranous regions in neurons, but surprisingly, not to PSD. Our screening for physiological interactors of SIPA1L1 in mouse brain identified spinophilin and neurabin-1, regulators of G-protein-coupled receptor (GPCR) signaling, but rejected PSD-95/NMDA-R-complex components. Furthermore, Sipa1l1-/- mice showed normal spine size distribution and NMDA-R-dependent synaptic plasticity. Nevertheless, Sipa1l1-/- mice showed aberrant responses to α2-adrenergic receptor (a spinophilin target) or adenosine A1 receptor (a neurabin-1 target) agonist stimulation, and striking behavioral anomalies, such as hyperactivity, enhanced anxiety, learning impairments, social interaction deficits, and enhanced epileptic seizure susceptibility. Male mice were used for all experiments. Our findings revealed unexpected properties of SIPA1L1, suggesting a possible association of SIPA1L1 deficiency with neuropsychiatric disorders related to dysregulated GPCR signaling, such as epilepsy, attention deficit hyperactivity disorder (ADHD), autism, or fragile X syndrome (FXS).SIGNIFICANCE STATEMENT Signal-induced proliferation-associated 1 (SIPA1)-like 1 (SIPA1L1) is thought to regulate essential synaptic functions as a component of the PSD-95/NMDA-R-complex. In our screening for physiological SIPA1L1-interactors, we identified G-protein-coupled receptor (GPCR)-signaling regulators. Moreover, SIPA1L1 knock-out (KO) mice showed striking behavioral anomalies, which may be relevant to GPCR signaling. Our findings revealed an unexpected role of SIPA1L1, which may open new avenues for research on neuropsychiatric disorders that involve dysregulated GPCR signaling. Another important aspect of this paper is that we showed effective methods for checking PSD association and identifying native protein interactors that are difficult to solubilize. These results may serve as a caution for future claims about interacting proteins and PSD proteins, which could eventually save time and resources for researchers and avoid confusion in the field.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , N-Metilaspartato , Proteínas del Tejido Nervioso , Animales , Homólogo 4 de la Proteína Discs Large , Masculino , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Receptor de Adenosina A1 , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Autophagy is a degradation process of cytoplasmic proteins and organelles trafficked to degradation vesicles known as autophagosomes. The conversion of LC3-I to LC3-II is an essential step of autophagosome formation, and FYCO1 is a LC3-binding protein that mediates autophagosome transport. The p62 protein also directly binds to LC3 and is degraded by autophagy. In the present study, we demonstrated that disrupting the FYCO1 gene in mice resulted in cataract formation. LC3 conversion decreased in eyes from FYCO1 knockout mice. Further, FYCO1 interacted with αA- and αB-crystallin, as demonstrated by yeast two-hybrid screening and immunoprecipitation analyses. In eyes from knockout mice, the soluble forms of αA- and αB-crystallin, the lens's major protein components, decreased. In addition, p62 accumulated in eyes from FYCO1 knockout mice. Collectively, these findings suggested that FYCO1 recruited damaged α-crystallin into autophagosomes to protect lens cells from cataract formation.
Asunto(s)
Autofagia/genética , Catarata/genética , Proteínas Asociadas a Microtúbulos/genética , Proteína Sequestosoma-1/genética , Animales , Autofagosomas/genética , Catarata/patología , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Noqueados , Cadena A de alfa-Cristalina/genética , Cadena B de alfa-Cristalina/genéticaRESUMEN
BACKGROUND: Cisplatin (CDDP) significantly prolongs survival in various cancers, but many patients also develop resistance that results in treatment failure. Thus, this study aimed to elucidate the underlying mechanisms by which ovarian cancer cells acquire CDDP resistance. METHODS: We evaluated the metabolic profiles in CDDP-sensitive ovarian cancer A2780 cells and CDDP-resistant A2780cis cells using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). We further examined the expression of glutamine metabolism enzymes using real-time PCR and Western blot analyses. Cell viability was accessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The results showed that levels of glutamine, glutamate, and glutathione (GSH), a key drug resistance mediator synthesized from glutamate, were significantly elevated in A2780cis cells than those in A2780 cells. Furthermore, glutamine starvation decreased the GSH levels and CDDP resistance in A2780cis cells. Interestingly, the expression of glutamine synthetase (GS/GLUL), which synthesizes glutamine from glutamate and thereby negatively regulates GSH production, was almost completely suppressed in resistant A2780cis cells. In addition, treatment of A2780cis cells with 5-aza-2'-deoxycytidine, a DNA-demethylating agent, restored GS expression and reduced CDDP resistance. In contrast, GS knockdown in CDDP-sensitive A2780 cells induced CDDP resistance. CONCLUSIONS: The results indicate that upregulation of GSH synthesis from glutamine via DNA methylation-mediated silencing of GS causes CDDP resistance in A2780cis cells. Therefore, glutamine metabolism could be a novel therapeutic target against CDDP resistance.
Asunto(s)
Cisplatino/farmacología , Resistencia a Antineoplásicos , Silenciador del Gen , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Glutamina/metabolismo , Metaboloma , Neoplasias Ováricas/tratamiento farmacológico , Antineoplásicos/farmacología , Apoptosis , Proliferación Celular , Reprogramación Celular , Femenino , Glutamato-Amoníaco Ligasa/genética , Humanos , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Células Tumorales CultivadasRESUMEN
Cancer cells alter their metabolism for the production of precursors of macromolecules. However, the control mechanisms underlying this reprogramming are poorly understood. Here we show that metabolic reprogramming of colorectal cancer is caused chiefly by aberrant MYC expression. Multiomics-based analyses of paired normal and tumor tissues from 275 patients with colorectal cancer revealed that metabolic alterations occur at the adenoma stage of carcinogenesis, in a manner not associated with specific gene mutations involved in colorectal carcinogenesis. MYC expression induced at least 215 metabolic reactions by changing the expression levels of 121 metabolic genes and 39 transporter genes. Further, MYC negatively regulated the expression of genes involved in mitochondrial biogenesis and maintenance but positively regulated genes involved in DNA and histone methylation. Knockdown of MYC in colorectal cancer cells reset the altered metabolism and suppressed cell growth. Moreover, inhibition of MYC target pyrimidine synthesis genes such as CAD, UMPS, and CTPS blocked cell growth, and thus are potential targets for colorectal cancer therapy.
Asunto(s)
Adenoma/metabolismo , Neoplasias Colorrectales/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Adenoma/genética , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Proliferación Celular/fisiología , Neoplasias Colorrectales/genética , Modelos Animales de Enfermedad , Femenino , Genes myc , Humanos , Masculino , Metabolómica/métodos , Ratones , Proteínas Proto-Oncogénicas c-myc/genética , Pirimidinas/biosíntesis , TranscriptomaRESUMEN
Dishevelled (DVL) is a central factor in the Wnt signaling pathway, which is highly conserved among various organisms. DVL plays important roles in transcriptional activation in the nucleus, but the molecular mechanisms underlying their nuclear localization remain unclear. In the present study, we identified IQGAP1 as a regulator of DVL function. In Xenopus embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of DVL, and expression of Wnt target genes during early embryogenesis. The domains in DVL and IQGAP1 that mediated their interaction are also required for their nuclear localization. Endogenous expression of Wnt target genes was reduced by depletion of IQGAP1 during early embryogenesis, but notably not by depletion of other IQGAP family genes. Moreover, expression of Wnt target genes caused by depletion of endogenous IQGAP1 could be rescued by expression of wild-type IQGAP1, but not IQGAP1 deleting DVL binding region. These results provide the first evidence that IQGAP1 functions as a modulator in the canonical Wnt signaling pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Núcleo Celular/metabolismo , Fosfoproteínas/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Transporte Activo de Núcleo Celular , Animales , Proteínas Dishevelled , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones , Células 3T3 NIH , Unión Proteica , Xenopus/embriología , Proteínas de XenopusRESUMEN
Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine of key importance for controlling embryogenesis and tissue homeostasis. How TGF-beta signals are attenuated and terminated is not well understood. Here, we show that TMEPAI, a direct target gene of TGF-beta signaling, antagonizes TGF-beta signaling by interfering with TGF-beta type I receptor (TbetaRI)-induced R-Smad phosphorylation. TMEPAI can directly interact with R-Smads via a Smad interaction motif. TMEPAI competes with Smad anchor for receptor activation for R-Smad binding, thereby sequestering R-Smads from TbetaRI kinase activation. In mammalian cells, ectopic expression of TMEPAI inhibited TGF-beta-dependent regulation of plasminogen activator inhibitor-1, JunB, cyclin-dependent kinase inhibitors, and c-myc expression, whereas specific knockdown of TMEPAI expression prolonged duration of TGF-beta-induced Smad2 and Smad3 phosphorylation and concomitantly potentiated cellular responsiveness to TGF-beta. Consistently, TMEPAI inhibits activin-mediated mesoderm formation in Xenopus embryos. Therefore, TMEPAI participates in a negative feedback loop to control the duration and intensity of TGF-beta/Smad signaling.
Asunto(s)
Proteínas de la Membrana/fisiología , Transducción de Señal , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Activinas/metabolismo , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Mesodermo/crecimiento & desarrollo , Ratones , Modelos Biológicos , Células 3T3 NIH , ARN Mensajero/metabolismo , XenopusRESUMEN
Malignant mesothelioma is an asbestos-related fatal disease with no effective cure. Recently, high dose of ascorbate in cancer treatment has been reexamined. We studied whether high dose of ascorbic acid induced cell death of four human mesothelioma cell lines. High dose of ascorbic acid induced cell death of all mesothelioma cell lines in a dose-dependent manner. We further clarified the cell killing mechanism that ascorbic acid induced reactive oxygen species and impaired mitochondrial membrane potential. In vivo experiment, intravenous administration of ascorbic acid significantly decreased the growth rate of mesothelioma tumor inoculated in mice. These data suggest that ascorbic acid may have benefits for patients with mesothelioma.
Asunto(s)
Apoptosis , Ácido Ascórbico/administración & dosificación , Mesotelioma/tratamiento farmacológico , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones SCID , Especies Reactivas de Oxígeno/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
ICAT, inhibitor of beta-catenin and T cell factor, or Ctnnbip1, is a negative regulator of the Wnt signaling pathway that interferes with the interaction between beta-catenin and T cell factor. Some ICAT-deficient (ICAT-/-) embryos exhibit unilateral or bilateral renal agenesis. In this study, we investigated developmental processes in the ICAT-/- kidney. ICAT was highly expressed in both the ureteric bud (UB) and the surrounding metanephric mesenchymal (MM) cells in the metanephros of embryonic day E11.5-E13.5 wild-type (ICAT+/+) mouse. In the E12.5-ICAT-/- metanephros, UB branching was delayed, and a T-shaped, bifurcated UB was frequently seen; this was never seen in the E12.5-ICAT+/+ metanephros. More apoptotic MM cells were detected in the ICAT-/- metanephros than in the ICAT+/+ metanephros. These results suggest that the loss of ICAT gene function causes the arrest of UB branching and the apoptotic death of MM cells, resulting in renal agenesis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Desarrollo Embrionario , Riñón/anomalías , Riñón/metabolismo , Morfogénesis , Factores de Transcripción/metabolismo , Uréter/anomalías , Uréter/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Riñón/embriología , Ratones , Ratones Noqueados , Proteínas Represoras , Uréter/embriologíaRESUMEN
The development of anterior neural structure in Xenopus laevis requires the inhibition of bone morphogenic protein 4 and Wnt signaling. We previously reported that Nemo-like kinase (NLK) negatively regulates Wnt signaling via the phosphorylation of T-cell factor/lymphoid enhancer factor. However, the molecular events occurring downstream of NLK pathways in early neural development remain unclear. In the present study, we identified the transcription factor myocyte enhancer factor 2A (MEF2A) as a novel substrate for NLK. NLK regulates the function of Xenopus MEF2A (xMEF2A) via phosphorylation, and this modification can be inhibited by the depletion of endogenous NLK. In Xenopus embryos, the depletion of either NLK or MEF2A results in a severe defect in anterior development. The endogenous expression of anterior markers was blocked by the depletion of endogenous Xenopus NLK (xNLK) or xMEF2A but, notably, not by the depletion of other xMEF2 family proteins, xMEF2C and xMEF2D. Defects in head formation or the expression of the anterior marker genes caused by the depletion of endogenous xMEF2A could be eliminated by the expression of wild-type xMEF2A, but not xMEF2A containing mutated xNLK phosphorylation sites. Furthermore, the expression of xNLK-induced anterior markers was efficiently blocked by the depletion of endogenous xMEF2A in animal pole explants. These results show that NLK specifically regulates the MEF2A activity required for anterior formation in Xenopus development.
Asunto(s)
Tipificación del Cuerpo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factores Reguladores Miogénicos/metabolismo , Transducción de Señal , Xenopus/embriología , Animales , Línea Celular , Embrión no Mamífero/enzimología , Regulación del Desarrollo de la Expresión Génica , Humanos , Factores de Transcripción MEF2 , Proteínas Quinasas Activadas por Mitógenos/genética , Factores Reguladores Miogénicos/genética , Sistema Nervioso/embriología , Sistema Nervioso/enzimología , Fosforilación , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Xenopus/genética , Proteínas de XenopusRESUMEN
beta-Catenin is a key player in the Wnt signaling pathway, and interacts with cofactor T cell factor/lymphoid enhancer factor (TCF/LEF) to generate a transcription activator complex that activates Wnt-induced genes. We previously reported that Nemo-like kinase (NLK) negatively regulates Wnt signaling via phosphorylation of TCF/LEF. To further evaluate the physiological roles of NLK, we performed yeast two-hybrid screening to identify NLK-interacting proteins. From this screen, we isolated a novel RING finger protein that we term NARF (NLK associated RING finger protein). Here, we show that NARF induces the ubiquitylation of TCF/LEF in vitro and in vivo, and functions as an E3 ubiquitin-ligase that specifically cooperates with the E2 conjugating enzyme E2-25K. We found that NLK augmented NARF binding and ubiquitylation of TCF/LEF, and this required NLK kinase activity. The ubiquitylated TCF/LEF was subsequently degraded by the proteasome. Furthermore, NARF inhibited formation of the secondary axis induced by the ectopic expression of beta-catenin in Xenopus embryos. Collectively, our findings raise the possibility that NARF functions as a novel ubiquitin-ligase to suppress the Wnt-beta-catenin signaling.
Asunto(s)
Proteínas Nucleares/biosíntesis , Factores de Transcripción TCF/metabolismo , Ubiquitina/química , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Oocitos/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Técnicas del Sistema de Dos Híbridos , Ubiquitina/metabolismo , Xenopus , beta Catenina/metabolismoRESUMEN
In the mammalian central nervous system, neurogenesis precedes gliogenesis; neurons are primarily generated at the neural stage, whereas most glial cells are generated perinatally and postnatally. However, the signals that regulate this sequence of events remain unclear. Here we show that Wnt signaling induces neuronal and astroglial differentiation but suppresses oligodendroglial differentiation. We observed that precursor cells infected with a retrovirus encoding beta-catenin differentiated into neurons, while astrocytes developed from uninfected precursor cells surrounding infected cells. As neurogenesis proceeded, expression of the bone morphogenetic proteins (BMPs), BMP2, 4 and 7, progressively increased in the cells infected with the retrovirus encoding beta-catenin. Furthermore, treatment of cells with Noggin, a BMP antagonist, completely inhibited astroglial differentiation but partially restored oligodendroglial differentiation. These results suggest that Wnt signaling indirectly regulates gliogenesis by inducing BMPs in neuronal cells. Thus, cooperation between Wnt and BMP signaling may play a key role in determining the sequence of neurogenesis and gliogenesis.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Animales , Astrocitos/metabolismo , Proteína Morfogenética Ósea 2 , Proteína Morfogenética Ósea 4 , Proteína Morfogenética Ósea 7 , Proteínas Morfogenéticas Óseas/antagonistas & inhibidores , Encéfalo/embriología , Proteínas Portadoras/farmacología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica , Ratones , Neuroglía/fisiología , Neuronas/fisiología , Oligodendroglía/metabolismo , Retroviridae/genética , Transducción de Señal , Células Madre/fisiología , Transfección , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Inhibitor of beta-catenin and T cell factor (ICAT) inhibits Wnt signaling by interfering with the interaction between beta-catenin and T cell factor. Here we show that ICAT(-/-) embryos exhibit malformation of the forebrain and craniofacial bones and lack the kidney. Analysis of the neuronal differentiation of embryonic stem cells revealed that Wnt3a redirects the fate of neural progenitors to a posterior character, whereas ICAT induces forebrain cells by inhibiting Wnt signaling. Furthermore, ICAT(-/-) embryonic stem cells were found to differentiate into neuronal cells possessing a posterior character. These results suggest that ICAT plays an important role in the anteriorization of neural cells by inhibiting the posteriorizing activity of Wnt signaling.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Neuronas/citología , Prosencéfalo/embriología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Transducción de Señal , Células Madre/citología , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Ciclo Celular/genética , Linaje de la Célula , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Prosencéfalo/citología , Prosencéfalo/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras , Células Madre/metabolismo , Factores de Transcripción/genética , Proteínas WntRESUMEN
DLG, the mammalian homolog of the Drosophila Discs Large suppressor protein, functions as a scaffolding protein that facilitates the transmission of diverse downstream signals. In the present study, we attempted to identify partner proteins for DLG, and found that DLG interacts through its PDZ domains with the ribosome receptor. The ribosome receptor is an integral endoplasmic reticulum protein that has been suggested to be involved in secretion. Our finding raises the possibility that DLG plays a role in the regulation of secretion by interacting with the ribosome receptor.
Asunto(s)
Proteínas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Homólogo 1 de la Proteína Discs Large , Células HeLa , Humanos , Proteínas de la Membrana , Datos de Secuencia Molecular , Receptores de Aminoácidos , Receptores Citoplasmáticos y Nucleares/química , PorcinosRESUMEN
It has been suggested that the LGN protein is associated with Galphai2 by the yeast two-hybrid system and in vitro pull-down assay. To determine the functions of LGN in the central nervous system, we examined the expression and localization of LGN in mouse brain by immunoblotting and immunofluorescence microscopy. By immunoblotting, almost similar amounts of LGN were detected in the olfactory bulb, cerebral cortex, hippocampus, and cerebellum of the adult mouse brain, and the levels of the postnatal LGN expression in the whole brain were fairly constant. Immunofluorescence microscopy showed that LGN is localized in nuclei of the neurons in the olfactory bulb, cerebral cortex, and hippocampus, but in both nuclei and cytoplasm of Purkinje cells in the cerebellum. On the other hand, Galphai2 was distributed throughout the neuronal elements except for the nuclei. Thus, LGN and Galphai2 were colocalized in the cytoplasm of Purkinje cells, but not in other neurons examined. These results suggest that LGN may be involved not only in the Galphai2-mediated signaling but also in other signaling pathways.