RESUMEN
Skp1, a subunit of E3 Skp1/Cullin-1/F-box protein ubiquitin ligases, is modified by a prolyl hydroxylase that mediates O2 regulation of the social amoeba Dictyostelium and the parasite Toxoplasma gondii The full effect of hydroxylation requires modification of the hydroxyproline by a pentasaccharide that, in Dictyostelium, influences Skp1 structure to favor assembly of Skp1/F-box protein subcomplexes. In Toxoplasma, the presence of a contrasting penultimate sugar assembled by a different glycosyltransferase enables testing of the conformational control model. To define the final sugar and its linkage, here we identified the glycosyltransferase that completes the glycan and found that it is closely related to glycogenin, an enzyme that may prime glycogen synthesis in yeast and animals. However, the Toxoplasma enzyme catalyzes formation of a Galα1,3Glcα linkage rather than the Glcα1,4Glcα linkage formed by glycogenin. Kinetic and crystallographic experiments showed that the glycosyltransferase Gat1 is specific for Skp1 in Toxoplasma and also in another protist, the crop pathogen Pythium ultimum The fifth sugar is important for glycan function as indicated by the slow-growth phenotype of gat1Δ parasites. Computational analyses indicated that, despite the sequence difference, the Toxoplasma glycan still assumes an ordered conformation that controls Skp1 structure and revealed the importance of nonpolar packing interactions of the fifth sugar. The substitution of glycosyltransferases in Toxoplasma and Pythium by an unrelated bifunctional enzyme that assembles a distinct but structurally compatible glycan in Dictyostelium is a remarkable case of convergent evolution, which emphasizes the importance of the terminal α-galactose and establishes the phylogenetic breadth of Skp1 glycoregulation.
Asunto(s)
Galactosa/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Dictyostelium/metabolismo , Proteínas F-Box/metabolismo , Glucosiltransferasas/metabolismo , Glicoproteínas/metabolismo , Glicosilación , Glicosiltransferasas/metabolismo , Hidroxilación , Hidroxiprolina/metabolismo , Filogenia , Procolágeno-Prolina Dioxigenasa/genética , Prolil Hidroxilasas/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Proteínas Ligasas SKP Cullina F-box/fisiología , Toxoplasma/metabolismoRESUMEN
BACKGROUND: F-box proteins are substrate-recognition components of the Skp1-Rbx1-Cul1-F-box protein (SCF) ubiquitin ligases. By selectively targeting the key regulatory proteins or enzymes for ubiquitination and 26S proteasome mediated degradation, F-box proteins play diverse roles in plant growth/development and in the responses of plants to both environmental and endogenous signals. Studies of F-box proteins from the model plant Arabidopsis and from many additional plant species have demonstrated that they belong to a super gene family, and function across almost all aspects of the plant life cycle. However, systematic exploration of F-box family genes in the important fiber crop cotton (Gossypium hirsutum) has not been previously performed. The genome-wide analysis of the cotton F-box gene family is now possible thanks to the completion of several cotton genome sequencing projects. RESULTS: In current study, we first conducted a genome-wide investigation of cotton F-box family genes by reference to the published F-box protein sequences from other plant species. 592 F-box protein encoding genes were identified in the Gossypium hirsutume acc.TM-1 genome and, subsequently, we were able to present their gene structures, chromosomal locations, syntenic relationships with their parent species. In addition, duplication modes analysis showed that cotton F-box genes were distributed to 26 chromosomes, with the maximum number of genes being detected on chromosome 5. Although the WGD (whole-genome duplication) mode seems play a dominant role during cotton F-box gene expansion process, other duplication modes including TD (tandem duplication), PD (proximal duplication), and TRD (transposed duplication) also contribute significantly to the evolutionary expansion of cotton F-box genes. Collectively, these bioinformatic analysis suggest possible evolutionary forces underlying F-box gene diversification. Additionally, we also conducted analyses of gene ontology, and expression profiles in silico, allowing identification of F-box gene members potentially involved in hormone signal transduction. CONCLUSION: The results of this study provide first insights into the Gossypium hirsutum F-box gene family, which lays the foundation for future studies of functionality, particularly those involving F-box protein family members that play a role in hormone signal transduction.
Asunto(s)
Proteínas F-Box/genética , Gossypium/genética , Proteínas de Plantas/genética , Proteínas F-Box/clasificación , Proteínas F-Box/metabolismo , Duplicación de Gen , Ontología de Genes , Genoma de Planta , Gossypium/metabolismo , Familia de Multigenes , Reguladores del Crecimiento de las Plantas/fisiología , Proteínas de Plantas/clasificación , Proteínas de Plantas/metabolismo , Proteínas Ligasas SKP Cullina F-box/fisiología , Transducción de SeñalRESUMEN
The mechanism of maternal protein degradation during preimplantation development has not been clarified yet. It is thought that a lot of maternal proteins are degraded by the ubiquitin-proteasome system. In this study, we focused on the role of the SCF (Skp1-Cullin-F-box) complexes during early bovine embryogenesis. We inhibited them using MLN4924, an inhibitor of SCF complex ligases controlled by neddylation. Oocytes maturated in MLN4924 could be fertilized, but we found no cumulus cell expansion and a high number of polyspermy after in vitro fertilization. We also found a statistically significant deterioration of development after MLN4924 treatment. After treatment with MLN4924 from the four-cell to late eight-cell stage, we found a statistically significant delay in their development; some of the treated embryos were, however, able to reach the blastocyst stage later. We found reduced levels of mRNA of EGA markers PAPOLA and U2AF1A, which can be related to this developmental delay. The cultivation with MLN4924 caused a significant increase in protein levels in MLN4924-treated oocytes and embryos; no such change was found in cumulus cells. To detect the proteins affected by MLN4924 treatment, we performed a Western blot analysis of selected proteins (SMAD4, ribosomal protein S6, centromeric protein E, P27, NFKB inhibitor alpha, RNA-binding motif protein 19). No statistically significant increase in protein levels was detected in either treated embryos or oocytes. In summary, our study shows that SCF ligases are necessary for the correct maturation of oocytes, cumulus cell expansion, fertilization, and early preimplantation development of cattle.
Asunto(s)
Blastocisto/efectos de los fármacos , Ciclopentanos/farmacología , Desarrollo Embrionario/efectos de los fármacos , Oocitos/efectos de los fármacos , Oogénesis/efectos de los fármacos , Pirimidinas/farmacología , Proteínas Ligasas SKP Cullina F-box/antagonistas & inhibidores , Animales , Blastocisto/citología , Blastocisto/fisiología , Bovinos , Células Cultivadas , Embrión de Mamíferos , Femenino , Técnicas de Maduración In Vitro de los Oocitos/métodos , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/metabolismo , Oocitos/citología , Oocitos/fisiología , Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteínas Ligasas SKP Cullina F-box/fisiología , Factores de TiempoRESUMEN
The tumor suppressor F-box protein 31 (FBXO31) is indispensable for maintaining genomic stability. Its levels drastically increase following DNA damage, leading to cyclin D1 and MDM2 degradation and G1 and G2/M arrest. Prolonged arrest in these phases leads to cellular senescence. Accordingly, FBXO31 needs to be kept at low basal levels in unstressed conditions for normal cell cycle progression during growth and development. However, the molecular mechanism maintaining these basal FBXO31 levels has remained unclear. Here, we identified the F-box family SCF-E3 ubiquitin ligase FBXO46 (SCFFBXO46) as an important proteasomal regulator of FBXO31 and found that FBXO46 helps maintain basal FBXO31 levels under unstressed conditions and thereby prevents premature senescence. Using molecular docking and mutational studies, we showed that FBXO46 recognizes an RXXR motif located at the FBXO31 C terminus to direct its polyubiquitination and thereby proteasomal degradation. Furthermore, FBXO46 depletion enhanced the basal levels of FBXO31, resulting in senescence induction. In response to genotoxic stress, ATM (ataxia telangiectasia-mutated) Ser/Thr kinase-mediated phosphorylation of FBXO31 at Ser-278 maintained FBXO31 levels. In contrast, activated ATM phosphorylated FBXO46 at Ser-21/Ser-67, leading to its degradation via FBXO31. Thus, ATM-catalyzed phosphorylation after DNA damage governs FBXO31 levels and FBXO46 degradation via a negative feedback loop. Collectively, our findings reveal that FBXO46 is a crucial proteasomal regulator of FBXO31 and thereby prevents senescence in normal growth conditions. They further indicate that FBXO46-mediated regulation of FBXO31 is abrogated following genotoxic stress to promote increased FBXO31 levels for maintenance of genomic stability.
Asunto(s)
Senescencia Celular , Proteínas F-Box/metabolismo , Proteínas Ligasas SKP Cullina F-box/fisiología , Proteínas Supresoras de Tumor/metabolismo , Inestabilidad Genómica , Humanos , Simulación del Acoplamiento Molecular , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , UbiquitinaciónRESUMEN
PHR (PAM/Highwire/RPM-1) proteins are conserved RING E3 ubiquitin ligases that function in developmental processes, such as axon termination and synapse formation, as well as axon degeneration. At present, our understanding of how PHR proteins form ubiquitin ligase complexes remains incomplete. Although genetic studies indicate NMNAT2 is an important mediator of PHR protein function in axon degeneration, it remains unknown how PHR proteins inhibit NMNAT2. Here, we decipher the biochemical basis for how the human PHR protein PAM, also called MYCBP2, forms a noncanonical Skp/Cullin/F-box (SCF) complex that contains the F-box protein FBXO45 and SKP1 but lacks CUL1. We show FBXO45 does not simply function in substrate recognition but is important for assembly of the PAM/FBXO45/SKP1 complex. Interestingly, we demonstrate a novel role for SKP1 as an auxiliary component of the target recognition module that enhances binding of FBXO45 to NMNAT2. Finally, we provide biochemical evidence that PAM polyubiquitinates NMNAT2 and regulates NMNAT2 protein stability and degradation by the proteasome.
Asunto(s)
Amidina-Liasas/química , Oxigenasas de Función Mixta/química , Nicotinamida-Nucleótido Adenililtransferasa/química , Proteínas Ligasas SKP Cullina F-box/química , Ubiquitinación , Proteínas Adaptadoras Transductoras de Señales , Animales , Caenorhabditis elegans , Proteínas F-Box/metabolismo , Humanos , Complejos Multiproteicos/química , Complejos Multiproteicos/fisiología , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Unión Proteica , Proteínas Quinasas Asociadas a Fase-S , Proteínas Ligasas SKP Cullina F-box/fisiología , Ubiquitina-Proteína LigasasRESUMEN
Molecular oscillators are important cellular regulators of, for example, circadian clocks, oscillations of immune regulators, and short-period (ultradian) rhythms during embryonic development. The Notch signaling factor HES1 (hairy and enhancer of split 1) is a well-known repressor of proneural genes, and HES1 ultradian oscillation is essential for keeping cells in an efficiently proliferating progenitor state. HES1 oscillation is driven by both transcriptional self-repression and ubiquitin-dependent proteolysis. However, the E3 ubiquitin ligase targeting HES1 for proteolysis remains unclear. Based on siRNA-mediated gene silencing screening, co-immunoprecipitation, and ubiquitination assays, we discovered that the E3 ubiquitin ligase SCFFBXL14 complex regulates HES1 ubiquitination and proteolysis. siRNA-mediated knockdown of the Cullin-RING E3 ubiquitin ligases RBX1 or CUL1 increased HES1 protein levels, prolonged its half-life, and dampened its oscillation. FBXL14, an F-box protein for SCF ubiquitin ligase, associates with HES1. FBXL14 silencing stabilized HES1, whereas FBXL14 overexpression decreased HES1 protein levels. Of note, the SCFFBXL14 complex promoted the ubiquitination of HES1 in vivo, and a conserved WRPW motif in HES1 was essential for HES1 binding to FBXL14 and for ubiquitin-dependent HES1 degradation. HES1 knockdown promoted neuronal differentiation, but FBXL14 silencing inhibited neuronal differentiation induced by HES1 ablation in mES and F9 cells. Our results suggest that SCFFBXL14 promotes neuronal differentiation by targeting HES1 for ubiquitin-dependent proteolysis and that the C-terminal WRPW motif in HES1 is required for this process.
Asunto(s)
Proteínas F-Box/fisiología , Neuronas/citología , Proteínas Ligasas SKP Cullina F-box/fisiología , Factor de Transcripción HES-1/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Secuencias de Aminoácidos/fisiología , Animales , Relojes Biológicos , Diferenciación Celular , Ratones , Unión Proteica , Proteolisis , Proteínas Ligasas SKP Cullina F-box/metabolismo , UbiquitinaciónRESUMEN
Physical training has been shown to be important to the control of muscle mass during aging, through the activation of several pathways including, IGF1-AKT and PGC-1α. Also, it was demonstrated that LRP130, a component of the PGC-1α complex, is important for the PGC-1α-dependent transcription of several mitochondrial genes in vivo. To explore the role of physical training during aging, we investigated the effects on muscle recovery after short-term immobilization followed by 3 or 7 days with aerobic or resistance training. Using morphological (myofibrillar adenosine triphosphatase activity, to assess the total muscle fiber cross-sectional area (CSA) and the frequency of specific fiber types), biochemical (myosin heavy chain), and molecular analyses (quantitative real-time PCR, functional pathways analyses, and Western blot), our results indicated that after an atrophic stimulus, only animals subjected to aerobic training showed entire recovery of cross-sectional area; aerobic training reduced the ubiquitin-proteasome system components involved in muscle atrophy after 3 days of recovery, and the upregulation in PGC-1α expression enhanced the process of muscle recovery by inhibiting the FoxO pathway, with the possible involvement of LRP130. These results suggest that aerobic training enhanced the muscle regeneration process after disuse-induced atrophy in aged rats possibly through of the LRP130/PGC-1α complex by inhibiting the ubiquitin-proteasome system.
Asunto(s)
Atrofia Muscular/terapia , Recuperación de la Función/fisiología , Entrenamiento de Fuerza , Factores de Transcripción/fisiología , Factores de Edad , Animales , Factores de Transcripción Forkhead/fisiología , Inmovilización , Masculino , Proteínas Musculares/fisiología , Músculo Esquelético/fisiopatología , Atrofia Muscular/etiología , Proteínas del Tejido Nervioso/fisiología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Ratas , Ratas Wistar , Proteínas Ligasas SKP Cullina F-box/fisiología , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/fisiologíaRESUMEN
Deficiency in repair of damaged DNA leads to genomic instability and is closely associated with tumorigenesis. Most DNA double-strand-breaks (DSBs) are repaired by two major mechanisms, homologous-recombination (HR) and non-homologous-end-joining (NHEJ). Although Akt has been reported to suppress HR, its role in NHEJ remains elusive. Here, we report that Akt phosphorylates XLF at Thr181 to trigger its dissociation from the DNA ligase IV/XRCC4 complex, and promotes its interaction with 14-3-3ß leading to XLF cytoplasmic retention, where cytosolic XLF is subsequently degraded by SCF(ß-TRCP) in a CKI-dependent manner. Physiologically, upon DNA damage, XLF-T181E expressing cells display impaired NHEJ and elevated cell death. Whereas a cancer-patient-derived XLF-R178Q mutant, deficient in XLF-T181 phosphorylation, exhibits an elevated tolerance of DNA damage. Together, our results reveal a pivotal role for Akt in suppressing NHEJ and highlight the tight connection between aberrant Akt hyper-activation and deficiency in timely DSB repair, leading to genomic instability and tumorigenesis.
Asunto(s)
Reparación del ADN por Unión de Extremidades/genética , Enzimas Reparadoras del ADN/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteínas 14-3-3/metabolismo , Secuencia de Aminoácidos , Carcinogénesis/genética , Citoplasma/metabolismo , Roturas del ADN de Doble Cadena , ADN Ligasa (ATP) , ADN Ligasas/metabolismo , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Inestabilidad Genómica , Humanos , Datos de Secuencia Molecular , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Ligasas SKP Cullina F-box/fisiología , Alineación de SecuenciaRESUMEN
Cadmium-induced cell death is associated with endoplasmic reticulum (ER) stress. We previously found that inhibition of FBXO6 expression, which is a ubiquitin ligase involved in ER-associated protein degradation (ERAD), induces high sensitivity to cadmium in HEK293 cells. However, the precise role of FBXO6 in ER stress remains unexplored. In this study, we investigated the role of FBXO6 in cadmium-induced ER stress in HEK293 cells. Our results showed that the cadmium-induced increase in expression of the ER stress marker proteins, BiP and CHOP, was further enhanced by inhibiting FBXO6 expression. Cadmium-induced c-Jun phosphorylation was also markedly increased by inhibition of FBXO6 expression. However, this c-Jun phosphorylation was almost entirely abolished by inhibition of c-Jun N-terminal kinase 1 (JNK1) expression. The level of high cadmium sensitivity induced by inhibition of FBXO6 expression was markedly lower in the JNK1-ablated cells than in the control cells. In addition, cadmium elevated the cellular level of ERAD substrate proteins, and this elevation was further enhanced by inhibiting FBXO6 expression. These results suggest that FBXO6 might inhibit cadmium-induced ER stress by functioning as a ubiquitin ligase in the ERAD system, thereby attenuating the cell death induced by subsequent JNK1 activation.
Asunto(s)
Cloruro de Cadmio/toxicidad , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Expresión Génica , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/fisiología , Células HEK293 , Humanos , Proteolisis , Proteínas Proto-Oncogénicas c-junRESUMEN
Cullin-RING ubiquitin ligases (CRLs) are critical in ubiquitinating Myc, while COP9 signalosome (CSN) controls neddylation of Cullin in CRL. The mechanistic link between Cullin neddylation and Myc ubiquitination/degradation is unclear. Here we show that Myc is a target of the CSN subunit 6 (CSN6)-Cullin signalling axis and that CSN6 is a positive regulator of Myc. CSN6 enhanced neddylation of Cullin-1 and facilitated autoubiquitination/degradation of Fbxw7, a component of CRL involved in Myc ubiquitination, thereby stabilizing Myc. Csn6 haplo-insufficiency decreased Cullin-1 neddylation but increased Fbxw7 stability to compromise Myc stability and activity in an Eµ-Myc mouse model, resulting in decelerated lymphomagenesis. We found that CSN6 overexpression, which leads to aberrant expression of Myc target genes, is frequent in human cancers. Together, these results define a mechanism for the regulation of Myc stability through the CSN-Cullin-Fbxw7 axis and provide insights into the correlation of CSN6 overexpression with Myc stabilization/activation during tumorigenesis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Carcinogénesis/genética , Péptido Hidrolasas/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Animales , Complejo del Señalosoma COP9 , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Linfoma/metabolismo , Linfoma/fisiopatología , Ratones , Ratones Transgénicos/genética , Neoplasias Experimentales/genética , Péptido Hidrolasas/biosíntesis , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Proteínas Ligasas SKP Cullina F-box/fisiología , Transcripción Genética/fisiología , UbiquitinaciónRESUMEN
Muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx)/atrogin-1 were identified more than 10 years ago as two muscle-specific E3 ubiquitin ligases that are increased transcriptionally in skeletal muscle under atrophy-inducing conditions, making them excellent markers of muscle atrophy. In the past 10 years much has been published about MuRF1 and MAFbx with respect to their mRNA expression patterns under atrophy-inducing conditions, their transcriptional regulation, and their putative substrates. However, much remains to be learned about the physiological role of both genes in the regulation of mass and other cellular functions in striated muscle. Although both MuRF1 and MAFbx are enriched in skeletal, cardiac, and smooth muscle, this review will focus on the current understanding of MuRF1 and MAFbx in skeletal muscle, highlighting the critical questions that remain to be answered.
Asunto(s)
Proteínas Musculares/fisiología , Atrofia Muscular/enzimología , Proteínas Ligasas SKP Cullina F-box/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Animales , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Atrofia Muscular/genética , Atrofia Muscular/patología , Tamaño de los Órganos/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The Wallenda (Wnd)/dual leucine zipper kinase (DLK)-Jnk pathway is an evolutionarily conserved MAPK signaling pathway that functions during neuronal development and following axonal injury. Improper pathway activation causes defects in axonal guidance and synaptic growth, whereas loss-of-function mutations in pathway components impairs axonal regeneration and degeneration after injury. Regulation of this pathway is in part through the E3 ubiquitin ligase Highwire (Hiw), which targets Wnd/DLK for degradation to limit MAPK signaling. To explore mechanisms controlling Wnd/DLK signaling, we performed a large-scale genetic screen in Drosophila to identify negative regulators of the pathway. Here we describe the identification and characterization of SkpA, a core component of SCF E3 ubiquitin ligases. Mutants in SkpA display synaptic overgrowth and an increase in Jnk signaling, similar to hiw mutants. The combination of hypomorphic alleles of SkpA and hiw leads to enhanced synaptic growth. Mutants in the Wnd-Jnk pathway suppress the overgrowth of SkpA mutants demonstrating that the synaptic overgrowth is due to increased Jnk signaling. These findings support the model that SkpA and the E3 ligase Hiw function as part of an SCF-like complex that attenuates Wnd/DLK signaling. In addition, SkpA, like Hiw, is required for synaptic and axonal responses to injury. Synapses in SkpA mutants are more stable following genetic or traumatic axonal injury, and axon loss is delayed in SkpA mutants after nerve crush. As in highwire mutants, this axonal protection requires Nmnat. Hence, SkpA is a novel negative regulator of the Wnd-Jnk pathway that functions with Hiw to regulate both synaptic development and axonal maintenance.
Asunto(s)
Axones/metabolismo , Proteínas de Drosophila/fisiología , Degeneración Nerviosa/metabolismo , Terminales Presinápticos/metabolismo , Proteínas Ligasas SKP Cullina F-box/fisiología , Sinapsis/metabolismo , Animales , Animales Modificados Genéticamente , Axones/patología , Drosophila melanogaster , Femenino , Masculino , Mutación/genética , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Terminales Presinápticos/patología , Sinapsis/genética , Sinapsis/patologíaRESUMEN
Cardiomyocyte proteostasis is mediated by the ubiquitin/proteasome system (UPS) and autophagy/lysosome system and is fundamental for cardiac adaptation to both physiologic (e.g., exercise) and pathologic (e.g., pressure overload) stresses. Both the UPS and autophagy/lysosome system exhibit reduced efficiency as a consequence of aging, and dysfunction in these systems is associated with cardiomyopathies. The muscle-specific ubiquitin ligase atrogin-1 targets signaling proteins involved in cardiac hypertrophy for degradation. Here, using atrogin-1 KO mice in combination with in vivo pulsed stable isotope labeling of amino acids in cell culture proteomics and biochemical and cellular analyses, we identified charged multivesicular body protein 2B (CHMP2B), which is part of an endosomal sorting complex (ESCRT) required for autophagy, as a target of atrogin-1-mediated degradation. Mice lacking atrogin-1 failed to degrade CHMP2B, resulting in autophagy impairment, intracellular protein aggregate accumulation, unfolded protein response activation, and subsequent cardiomyocyte apoptosis, all of which increased progressively with age. Cellular proteostasis alterations resulted in cardiomyopathy characterized by myocardial remodeling with interstitial fibrosis, with reduced diastolic function and arrhythmias. CHMP2B downregulation in atrogin-1 KO mice restored autophagy and decreased proteotoxicity, thereby preventing cell death. These data indicate that atrogin-1 promotes cardiomyocyte health through mediating the interplay between UPS and autophagy/lysosome system and its alteration promotes development of cardiomyopathies.
Asunto(s)
Autofagia/fisiología , Cardiomiopatías/etiología , Proteínas Musculares/deficiencia , Proteínas Ligasas SKP Cullina F-box/deficiencia , Animales , Apoptosis/fisiología , Cardiomiopatías/patología , Cardiomiopatías/fisiopatología , Modelos Animales de Enfermedad , Electrocardiografía , Estrés del Retículo Endoplásmico , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Lisosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Musculares/genética , Proteínas Musculares/fisiología , Miocitos Cardíacos/patología , Miocitos Cardíacos/fisiología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/fisiología , Taquicardia Ventricular/etiología , Ubiquitina/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
BACKGROUND: Congestive heart failure (CHF) is a leading cause of morbidity and mortality, and oxidative stress has been implicated in the pathogenesis of cachexia (muscle wasting) and the hallmark symptom, exercise intolerance. We have previously shown that a nitric oxide-dependent antioxidant defense renders oxidative skeletal muscle resistant to catabolic wasting. Here, we aimed to identify and determine the functional role of nitric oxide-inducible antioxidant enzyme(s) in protection against cardiac cachexia and exercise intolerance in CHF. METHODS AND RESULTS: We demonstrated that systemic administration of endogenous nitric oxide donor S-nitrosoglutathione in mice blocked the reduction of extracellular superoxide dismutase (EcSOD) protein expression, as well as the induction of MAFbx/Atrogin-1 mRNA expression and muscle atrophy induced by glucocorticoid. We further showed that endogenous EcSOD, expressed primarily by type IId/x and IIa myofibers and enriched at endothelial cells, is induced by exercise training. Muscle-specific overexpression of EcSOD by somatic gene transfer or transgenesis (muscle creatine kinase [MCK]-EcSOD) in mice significantly attenuated muscle atrophy. Importantly, when crossbred into a mouse genetic model of CHF (α-myosin heavy chain-calsequestrin), MCK-EcSOD transgenic mice had significant attenuation of cachexia with preserved whole body muscle strength and endurance capacity in the absence of reduced HF. Enhanced EcSOD expression significantly ameliorated CHF-induced oxidative stress, MAFbx/Atrogin-1 mRNA expression, loss of mitochondria, and vascular rarefaction in skeletal muscle. CONCLUSIONS: EcSOD plays an important antioxidant defense function in skeletal muscle against cardiac cachexia and exercise intolerance in CHF.
Asunto(s)
Caquexia/prevención & control , Caquexia/fisiopatología , Tolerancia al Ejercicio/fisiología , Insuficiencia Cardíaca/complicaciones , Insuficiencia Cardíaca/fisiopatología , Músculo Esquelético/fisiopatología , Superóxido Dismutasa/fisiología , Animales , Antioxidantes/fisiología , Forma MM de la Creatina-Quinasa/fisiología , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Musculares/fisiología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Condicionamiento Físico Animal/fisiología , ARN Mensajero/fisiología , S-Nitrosoglutatión/farmacología , Proteínas Ligasas SKP Cullina F-box/fisiología , Superóxido Dismutasa/deficiencia , Superóxido Dismutasa/genéticaRESUMEN
The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Proteínas Musculares/antagonistas & inhibidores , Factores de Transcripción Paired Box/metabolismo , Complejo Represivo Polycomb 2/fisiología , Rabdomiosarcoma Alveolar/metabolismo , Proteínas Ligasas SKP Cullina F-box/antagonistas & inhibidores , Adolescente , Apoptosis , Diferenciación Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , Supervivencia Celular , Niño , Proteína Potenciadora del Homólogo Zeste 2 , Femenino , Proteína Forkhead Box O1 , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , Homeostasis , Humanos , Masculino , Proteínas Musculares/fisiología , Factor de Transcripción PAX3 , Proteínas Ligasas SKP Cullina F-box/fisiologíaRESUMEN
During ageing skeletal muscles undergo a process of structural and functional remodelling that leads to sarcopenia, a syndrome characterized by loss of muscle mass and force and a major cause of physical frailty. To determine the causes of sarcopenia and identify potential targets for interventions aimed at mitigating ageing-dependent muscle wasting, we focussed on the main signalling pathway known to control protein turnover in skeletal muscle, consisting of the insulin-like growth factor 1 (IGF1), the kinase Akt and its downstream effectors, the mammalian target of rapamycin (mTOR) and the transcription factor FoxO. Expression analyses at the transcript and protein level, carried out on well-characterized cohorts of young, old sedentary and old active individuals and on mice aged 200, 500 and 800 days, revealed only modest age-related differences in this pathway. Our findings suggest that during ageing there is no downregulation of IGF1/Akt pathway and that sarcopenia is not due to FoxO activation and upregulation of the proteolytic systems. A potentially interesting result was the increased phosphorylation of the ribosomal protein S6, indicative of increased activation of mTOR complex1 (mTORC1), in aged mice. This result may provide the rationale why rapamycin treatment and caloric restriction promote longevity, since both interventions blunt activation of mTORC1; however, this change was not statistically significant in humans. Finally, genetic perturbation of these pathways in old mice aimed at promoting muscle hypertrophy via Akt overexpression or preventing muscle loss through inactivation of the ubiquitin ligase atrogin1 were found to paradoxically cause muscle pathology and reduce lifespan, suggesting that drastic activation of the IGF1-Akt pathway may be counterproductive, and that sarcopenia is accelerated, not delayed, when protein degradation pathways are impaired.
Asunto(s)
Envejecimiento/fisiología , Factores de Transcripción Forkhead/fisiología , Factor I del Crecimiento Similar a la Insulina/fisiología , Músculo Esquelético/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/fisiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Proteína 7 Relacionada con la Autofagia , Femenino , Proteína Forkhead Box O1 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/fisiología , Modelos Animales , Proteínas Musculares/genética , Proteínas Musculares/fisiología , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/fisiología , Sarcopenia/fisiopatología , Serpina E2/genética , Serpina E2/fisiología , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/fisiología , Adulto JovenRESUMEN
The distribution of the phytohormone auxin regulates many aspects of plant development including growth response to gravity. Gravitropic root curvature involves coordinated and asymmetric cell elongation between the lower and upper side of the root, mediated by differential cellular auxin levels. The asymmetry in the auxin distribution is established and maintained by a spatio-temporal regulation of the PIN-FORMED (PIN) auxin transporter activity. We provide novel insights into the complex regulation of PIN abundance and activity during root gravitropism. We show that PIN2 turnover is differentially regulated on the upper and lower side of gravistimulated roots by distinct but partially overlapping auxin feedback mechanisms. In addition to regulating transcription and clathrin-mediated internalization, auxin also controls PIN abundance at the plasma membrane by promoting their vacuolar targeting and degradation. This effect of elevated auxin levels requires the activity of SKP-Cullin-F-box(TIR1/AFB) (SCF(TIR1/AFB))-dependent pathway. Importantly, also suboptimal auxin levels mediate PIN degradation utilizing the same signalling pathway. These feedback mechanisms are functionally important during gravitropic response and ensure fine-tuning of auxin fluxes for maintaining as well as terminating asymmetric growth.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Gravitropismo/genética , Ácidos Indolacéticos/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Proteínas Ligasas SKP Cullina F-box/fisiología , Vacuolas/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Proteínas F-Box/fisiología , Regulación de la Expresión Génica de las Plantas , Raíces de Plantas/metabolismo , Raíces de Plantas/fisiología , Plantas Modificadas Genéticamente , Transporte de Proteínas , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/fisiología , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
AIMS: Increased cardiac sympathetic neuron (SN) activity has been associated with pathologies such as heart failure and hypertrophy, suggesting that cardiac innervation regulates cardiomyocyte trophism. Whether continuous input from the SNs is required for the maintenance of the cardiomyocyte size has not been determined thus far. METHODS AND RESULTS: To address the role of cardiac innervation in cardiomyocyte size regulation, we monitored the effect of pharmacological sympathetic denervation in mice on cardiac structure, function, and signalling from 24 h to 30 days in the absence of other pathological stimuli. SN ablation caused an immediate reduction in the cardiomyocyte size with minimal consequences on the resting contractile function. Atrophic remodelling was mediated by the ubiquitin-proteasome system through FOXO-dependent early induction of the muscle-specific E3 ubiquitin ligases Atrogin-1/MAFbx and MuRF1, which was followed by activation of the autophagy-lysosome system. MuRF1 was found to be determinant in denervation atrophy as remodelling did not develop in denervated MuRF1 knock-out (KO) hearts. These effects were caused by decreased basal stimulation of cardiomyocyte ß2-adrenoceptor (AR), as atrophy was prevented by treatment of denervated mice with the ß2-AR agonist clenbuterol. Consistent with these data, we also observed that ß2-AR KO mice showed cardiac atrophy at rest. CONCLUSION: Cardiac SNs are strong regulators of the cardiomyocyte size via ß2-AR-dependent repression of proteolysis, demonstrating that the neuro-cardiac axis operates constitutively for the determination of the physiological cardiomyocyte size. These results are of great clinical relevance given the role of ß-AR in cardiovascular diseases and their modulation in therapy.
Asunto(s)
Corazón/inervación , Miocitos Cardíacos/patología , Proteínas/metabolismo , Receptores Adrenérgicos beta 2/fisiología , Sistema Nervioso Simpático/fisiología , Animales , Atrofia , Autofagia , Células Cultivadas , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/fisiología , Ratones , Ratones Endogámicos C57BL , Proteínas Musculares/fisiología , Norepinefrina/farmacología , Proteínas Ligasas SKP Cullina F-box/fisiología , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/fisiologíaRESUMEN
Alterations in muscle play an important role in common diseases and conditions. Reactive oxygen species (ROS) are generated during hindlimb unloading due, at least in part, to the activation of xanthine oxidase (XO). The major aim of this study was to determine the mechanism by which XO activation causes unloading-induced muscle atrophy in rats, and its possible prevention by allopurinol, a well-known inhibitor of this enzyme. For this purpose we studied one of the main redox sensitive signalling cascades involved in skeletal muscle atrophy i.e. p38 MAPKinase, and the expression of two well known muscle specific E3 ubiquitin ligases involved in proteolysis, the Muscle atrophy F-Box (MAFbx; also known as atrogin-1) and Muscle RING (Really Interesting New Gene) Finger-1 (MuRF-1). We found that hindlimb unloading induced a significant increase in XO activity and in the protein expression of the antioxidant enzymes CuZnSOD and Catalase in skeletal muscle. The most relevant new fact reported in this paper is that inhibition of XO with allopurinol, a drug widely used in clinical practice, prevents soleus muscle atrophy by ~20% after hindlimb unloading. This was associated with the inhibition of the p38 MAPK-MAFbx pathway. Our data suggest that XO was involved in the loss of muscle mass via the activation of the p38MAPK-MAFbx pathway in unloaded muscle atrophy. Thus, allopurinol may have clinical benefits to combat skeletal muscle atrophy in bedridden, astronauts, sarcopenic, and cachexic patients.