RESUMEN
Dystonia type 1 (DYT1) is a dominantly inherited neurological disease caused by mutations in TOR1A, the gene encoding the endoplasmic reticulum (ER)-resident protein torsinA. Previous work mostly completed in cell-based systems suggests that mutant torsinA alters protein processing in the secretory pathway. We hypothesized that inducing ER stress in the mammalian brain in vivo would trigger or exacerbate mutant torsinA-induced dysfunction. To test this hypothesis, we crossed DYT1 knock-in with p58(IPK)-null mice. The ER co-chaperone p58(IPK) interacts with BiP and assists in protein maturation by helping to fold ER cargo. Its deletion increases the cellular sensitivity to ER stress. We found a lower generation of DYT1 knock-in/p58 knock-out mice than expected from this cross, suggesting a developmental interaction that influences viability. However, surviving animals did not exhibit abnormal motor function. Analysis of brain tissue uncovered dysregulation of eiF2α and Akt/mTOR translational control pathways in the DYT1 brain, a finding confirmed in a second rodent model and in human brain. Finally, an unbiased proteomic analysis identified relevant changes in the neuronal protein landscape suggesting abnormal ER protein metabolism and calcium dysregulation. Functional studies confirmed the interaction between the DYT1 genotype and neuronal calcium dynamics. Overall, these findings advance our knowledge on dystonia, linking translational control pathways and calcium physiology to dystonia pathogenesis and identifying potential new pharmacological targets. SIGNIFICANCE STATEMENT: Dystonia type 1 (DYT1) is one of the different forms of inherited dystonia, a neurological disorder characterized by involuntary, disabling movements. DYT1 is caused by mutations in the gene that encodes the endoplasmic reticulum (ER)-resident protein torsinA. How mutant torsinA causes neuronal dysfunction remains unknown. Here, we show the behavioral and molecular consequences of stressing the ER in DYT1 mice by increasing the amount of misfolded proteins. This resulted in the generation of a reduced number of animals, evidence of abnormal ER protein processing and dysregulation of translational control pathways. The work described here proposes a shared mechanism for different forms of dystonia, links for the first time known biological pathways to dystonia pathogenesis, and uncovers potential pharmacological targets for its treatment.
Asunto(s)
Distonía/genética , Distonía/fisiopatología , Retículo Endoplásmico/genética , Chaperonas Moleculares/genética , Animales , Conducta Animal , Señalización del Calcio/genética , Cerebelo/fisiopatología , Distonía/psicología , Estrés del Retículo Endoplásmico/genética , Regulación de la Expresión Génica/genética , Técnicas de Sustitución del Gen , Genotipo , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Humanos , Ratones , Ratones Noqueados , Neuronas/fisiología , Transducción de Señal/genéticaRESUMEN
Alpha-1 antitrypsin deficiency is the leading cause of childhood liver failure and one of the most common lethal genetic diseases. The disease-causing mutant A1AT-Z fails to fold correctly and accumulates in the endoplasmic reticulum (ER) of the liver, resulting in hepatic fibrosis and hepatocellular carcinoma in a subset of patients. Furthermore, A1AT-Z sequestration in hepatocytes leads to a reduction in A1AT secretion into the serum, causing panacinar emphysema in adults. The purpose of this work was to elucidate the details by which A1AT-Z is degraded in hepatic cell lines. We identified the ubiquitin ligase FBG1, which has been previously shown to degrade proteins by both the ubiquitin proteasome pathway and autophagy, as being key to A1AT-Z degradation. Using chemical and genetic approaches we show that FBG1 degrades A1AT-Z through both the ubiquitin proteasome system and autophagy. Overexpression of FBG1 decreases the half-life of A1AT-Z and knocking down FBG1 in a hepatic cell line, and in mice results in an increase in ATAT. Finally, we show that FBG1 degrades A1AT-Z through a Beclin1-dependent arm of autophagy. In our model, FBG1 acts as a safety ubiquitin ligase, whose function is to re-ubiquitinate ER proteins that have previously undergone de-ubiquitination to ensure they are degraded.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Autofagia/genética , Proteínas de la Membrana/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Ligasas SKP Cullina F-box/genética , Deficiencia de alfa 1-Antitripsina/genética , alfa 1-Antitripsina/genética , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Beclina-1 , Línea Celular Tumoral , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Femenino , Regulación de la Expresión Génica , Semivida , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Mutación , Unión Proteica , Proteolisis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Ligasas SKP Cullina F-box/antagonistas & inhibidores , Proteínas Ligasas SKP Cullina F-box/metabolismo , Transducción de Señal , Ubiquitinación , alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/metabolismo , Deficiencia de alfa 1-Antitripsina/patologíaRESUMEN
A significant fraction of all proteins are misfolded and must be degraded. The ubiquitin-proteasome pathway provides an essential protein quality control function necessary for normal cellular homeostasis. Substrate specificity is mediated by proteins called ubiquitin ligases. In the endoplasmic reticulum (ER) a specialized pathway, the endoplasmic reticulum associated degradation (ERAD) pathway provides means to eliminate misfolded proteins from the ER. One marker used by the ER to identify misfolded glycoproteins is the presence of a high-mannose (Man5-8GlcNAc2) glycan. Recently, FBXO2 was shown to bind high mannose glycans and participate in ERAD. Using glycan arrays, immobilized glycoprotein pulldowns, and glycan competition assays we demonstrate that FBXO2 preferentially binds unfolded glycoproteins. Using recombinant, bacterially expressed GST-FBXO2 as an unfolded protein sensor we demonstrate it can be used to monitor increases in misfolded glycoproteins after physiological or pharmaceutical stressors.
Asunto(s)
Técnicas Biosensibles , Proteínas de Ciclo Celular/química , Retículo Endoplásmico/metabolismo , Proteínas F-Box/química , Glicoproteínas/química , Proteínas del Tejido Nervioso/química , Desplegamiento Proteico , Ubiquitina-Proteína Ligasas/química , Animales , Células COS , Chlorocebus aethiops , Ratones , Oligosacáridos/química , Polisacáridos/química , Proteolisis , Proteínas Recombinantes de Fusión/química , Estrés FisiológicoRESUMEN
DYT1 dystonia is caused by a glutamic acid deletion (ΔE) in the endoplasmic reticulum (ER) protein torsinA. Previous studies suggest that torsinA modulates the aggregation of cytosolic misfolded proteins and ER stress responses, although the mechanisms underlying those effects remain unclear. In order to investigate the bases of these observations, we analyzed the interaction between torsinA expression, protein aggregation and ER stress in PC6.3 cells. Unexpectedly, we found that expression of torsinA(wt) or (ΔE) does not influence the inclusion formation by an expanded polyglutamine reporter protein in this cellular model. Furthermore, torsinA does not prevent the activation of ER stress induced by thapsigargin or the reducing agent DTT. Interestingly, DTT induces post-translational changes in torsinA, more prominently for torsinA(wt) than (ΔE). This work highlights the importance of model system selection for the study of torsinA function. Furthermore, it provides additional evidence suggesting that torsinA is sensitive to changes in the cellular redox potential.
Asunto(s)
Chaperonas Moleculares/metabolismo , Animales , Línea Celular , Distonía Muscular Deformante , Retículo Endoplásmico/metabolismo , Humanos , Chaperonas Moleculares/genética , Mutación , Péptidos/metabolismo , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Estrés FisiológicoRESUMEN
During cell proliferation, protein degradation is strictly regulated by the cell cycle and involves two complementary ubiquitin ligase complexes, the SCF (Skp, Cullin, F-box) and APC/C (Anaphase Promoting Complex/Cyclosome) ubiquitin ligases. SCF ligases are constitutively active and generally target only proteins after they have been selected for degradation, usually by phosphorylation. In contrast, APC/C complexes are themselves activated by phosphorylation and their substrates contain a targeting signal known as degron, a consensus amino acid sequence such as a D-Box. SCF complexes degrade proteins during the G1 phase. However, as DNA synthesis begins, the SCF complexes are degraded and APC/C complexes are activated. APC-2, a protein crucial to cell division, initiates anaphase by triggering the degradation of multiple proteins. This study explores an unexpected interaction between APC-2 and SCFFBG1. We found that FBG1 is a promiscuous ubiquitin ligase with many partners. Immunoprecipitation experiments demonstrate that FBG1 and APC2 interact directly. Mutagenesis-based experiments show that this interaction requires a D-Box found within the FBG1 F-box domain. Unexpectedly, we demonstrate that co-expression with FBG1 increases total APC2 levels. However, free APC2 is decreased, inhibiting cell proliferation. Finally, FACS analysis of cell populations expressing different forms of FBG1 demonstrate that this ubiquitin ligase induces S-phase arrest, illustrating the functional consequences of the interaction described. In summary, we have discovered a novel APC2 inhibitory activity of FBG1 independent from its function as ubiquitin ligase, providing the basis for future studies of FBG1 in aging and cancer.
Asunto(s)
Proteínas F-Box/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Animales , Células COS , Chlorocebus aethiops , Proteínas Cullin/química , Proteínas Cullin/metabolismo , Proteínas F-Box/química , Humanos , Datos de Secuencia Molecular , Mutagénesis , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Fase S , Proteínas Quinasas Asociadas a Fase-S/química , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Alineación de Secuencia , Complejos de Ubiquitina-Proteína Ligasa/antagonistas & inhibidoresRESUMEN
Post-translational modification of proteins regulates many cellular processes. Some modifications, including N-linked glycosylation, serve multiple functions. For example, the attachment of N-linked glycans to nascent proteins in the endoplasmic reticulum facilitates proper folding, whereas retention of high mannose glycans on misfolded glycoproteins serves as a signal for retrotranslocation and ubiquitin-mediated proteasomal degradation. Here we examine the substrate specificity of the only family of ubiquitin ligase subunits thought to target glycoproteins through their attached glycans. The five proteins comprising this FBA family (FBXO2, FBXO6, FBXO17, FBXO27, and FBXO44) contain a conserved G domain that mediates substrate binding. Using a variety of complementary approaches, including glycan arrays, we show that each family member has differing specificity for glycosylated substrates. Collectively, the F-box proteins in the FBA family bind high mannose and sulfated glycoproteins, with one FBA protein, FBX044, failing to bind any glycans on the tested arrays. Site-directed mutagenesis of two aromatic amino acids in the G domain demonstrated that the hydrophobic pocket created by these amino acids is necessary for high affinity glycan binding. All FBA proteins co-precipitated components of the canonical SCF complex (Skp1, Cullin1, and Rbx1), yet FBXO2 bound very little Cullin1, suggesting that FBXO2 may exist primarily as a heterodimer with Skp1. Using subunit-specific antibodies, we further demonstrate marked divergence in tissue distribution and developmental expression. These differences in substrate recognition, SCF complex formation, and tissue distribution suggest that FBA proteins play diverse roles in glycoprotein quality control.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Lectinas/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Animales , Línea Celular , Chlorocebus aethiops , Proteínas F-Box/clasificación , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Glicoproteínas/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Lectinas/clasificación , Manosa/metabolismo , Ratones , Modelos Biológicos , Familia de Multigenes , Polisacáridos/metabolismo , Unión Proteica , Especificidad por SustratoRESUMEN
Little is known about the role of protein quality control in the inner ear. We now report selective cochlear degeneration in mice deficient in Fbx2, a ubiquitin ligase F-box protein with specificity for high-mannose glycoproteins (Yoshida et al., 2002). Originally described as a brain-enriched protein (Erhardt et al., 1998), Fbx2 is also highly expressed in the organ of Corti, in which it has been called organ of Corti protein 1 (Thalmann et al., 1997). Mice with targeted deletion of Fbxo2 develop age-related hearing loss beginning at 2 months. Cellular degeneration begins in the epithelial support cells of the organ of Corti and is accompanied by changes in cellular membrane integrity and early increases in connexin 26, a cochlear gap junction protein previously shown to interact with Fbx2 (Henzl et al., 2004). Progressive degeneration includes hair cells and the spiral ganglion, but the brain itself is spared despite widespread CNS expression of Fbx2. Cochlear Fbx2 binds Skp1, the common binding partner for F-box proteins, and is an unusually abundant inner ear protein. Whereas cochlear Skp1 levels fall in parallel with the loss of Fbx2, other components of the canonical SCF (Skp1, Cullin1, F-box, Rbx1) ubiquitin ligase complex remain unchanged and show little if any complex formation with Fbx2/Skp1, suggesting that cochlear Fbx2 and Skp1 form a novel, heterodimeric complex. Our findings demonstrate that components of protein quality control are essential for inner ear homeostasis and implicate Fbx2 and Skp1 as potential genetic modifiers in age-related hearing loss.
Asunto(s)
Enfermedades Cocleares/metabolismo , Sordera/metabolismo , Proteínas F-Box/genética , Células Ciliadas Auditivas/metabolismo , Degeneración Nerviosa/metabolismo , Envejecimiento/genética , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/patología , Enfermedades Cocleares/genética , Enfermedades Cocleares/fisiopatología , Conexina 26 , Conexinas/genética , Conexinas/metabolismo , Sordera/genética , Sordera/fisiopatología , Glicoproteínas/metabolismo , Células Ciliadas Auditivas/patología , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/metabolismo , Pérdida Auditiva Sensorineural/fisiopatología , Células Laberínticas de Soporte/metabolismo , Células Laberínticas de Soporte/patología , Sustancias Macromoleculares/metabolismo , Ratones , Ratones Noqueados , Degeneración Nerviosa/genética , Degeneración Nerviosa/fisiopatología , Unión Proteica/fisiología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
In SCF (Skp1/Cullin/F-box protein) ubiquitin ligases, substrate specificity is conferred by a diverse array of F-box proteins. Only in fully assembled SCF complexes, it is believed, can substrates bound to F-box proteins become ubiquitinated. Here we show that Fbx2, a brain-enriched F-box protein implicated in the ubiquitination of glycoproteins discarded from the endoplasmic reticulum, binds the co-chaperone/ubiquitin ligase CHIP (C terminus of Hsc-70-interacting protein) through a unique N-terminal PEST domain in Fbx2. CHIP facilitates the ubiquitination and degradation of Fbx2-bound glycoproteins, including unassembled NMDA receptor subunits. These findings indicate that CHIP acts with Fbx2 in a novel ubiquitination pathway that links CHIP to glycoprotein quality control in neurons. In addition, they expand the repertoire of pathways by which F-box proteins can regulate ubiquitination and suggest a new role for PEST domains as a protein interaction motif.