RESUMEN
Asparagine (N)-linked glycosylation is a protein modification critical for glycoprotein folding, stability, and cellular localization. To identify small molecules that inhibit new targets in this biosynthetic pathway, we initiated a cell-based high-throughput screen and lead-compound-optimization campaign that delivered a cell-permeable inhibitor, NGI-1. NGI-1 targets oligosaccharyltransferase (OST), a hetero-oligomeric enzyme that exists in multiple isoforms and transfers oligosaccharides to recipient proteins. In non-small-cell lung cancer cells, NGI-1 blocks cell-surface localization and signaling of the epidermal growth factor receptor (EGFR) glycoprotein, but selectively arrests proliferation in only those cell lines that are dependent on EGFR (or fibroblast growth factor, FGFR) for survival. In these cell lines, OST inhibition causes cell-cycle arrest accompanied by induction of p21, autofluorescence, and cell morphology changes, all hallmarks of senescence. These results identify OST inhibition as a potential therapeutic approach for treating receptor-tyrosine-kinase-dependent tumors and provides a chemical probe for reversibly regulating N-linked glycosylation in mammalian cells.
Asunto(s)
Benzamidas/farmacología , Senescencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hexosiltransferasas/antagonistas & inhibidores , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Sulfonamidas/farmacología , Benzamidas/química , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Hexosiltransferasas/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Proteínas de la Membrana/metabolismo , Estructura Molecular , Proteínas Tirosina Quinasas Receptoras/metabolismo , Relación Estructura-Actividad , Sulfonamidas/químicaRESUMEN
Activation of the unfolded protein response (UPR) can be either adaptive or pathological. We term the pathological UPR that causes fatty liver disease a "stressed UPR." Here we investigate the mechanism of stressed UPR activation in zebrafish bearing a mutation in thetrappc11gene, which encodes a component of the transport protein particle (TRAPP) complex.trappc11mutants are characterized by secretory pathway defects, reflecting disruption of the TRAPP complex. In addition, we uncover a defect in protein glycosylation intrappc11mutants that is associated with reduced levels of lipid-linked oligosaccharides (LLOs) and compensatory up-regulation of genes in the terpenoid biosynthetic pathway that produces the LLO anchor dolichol. Treating wild-type larvae with terpenoid or LLO synthesis inhibitors phenocopies the stressed UPR seen intrappc11mutants and is synthetically lethal withtrappc11mutation. We propose that reduced LLO level causing hypoglycosylation is a mechanism of stressed UPR induction intrappc11mutants. Of importance, in human cells, depletion of TRAPPC11, but not other TRAPP components, causes protein hypoglycosylation, and lipid droplets accumulate in fibroblasts from patients with theTRAPPC11mutation. These data point to a previously unanticipated and conserved role for TRAPPC11 in LLO biosynthesis and protein glycosylation in addition to its established function in vesicle trafficking.
Asunto(s)
Oligosacáridos/metabolismo , Respuesta de Proteína Desplegada , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Atorvastatina/farmacología , Dolicoles/biosíntesis , Dolicoles/genética , Glicosilación , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Humanos , Larva/efectos de los fármacos , Larva/metabolismo , Lípidos/química , Hígado/metabolismo , Hígado/patología , Mutación , Oligosacáridos/química , Terpenos/metabolismo , Terpenos/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , Respuesta de Proteína Desplegada/genética , Proteínas de Transporte Vesicular/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
TREX1 is an endoplasmic reticulum (ER)-associated negative regulator of innate immunity. TREX1 mutations are associated with autoimmune and autoinflammatory diseases. Biallelic mutations abrogating DNase activity cause autoimmunity by allowing immunogenic self-DNA to accumulate, but it is unknown how dominant frameshift (fs) mutations that encode DNase-active but mislocalized proteins cause disease. We found that the TREX1 C terminus suppressed immune activation by interacting with the ER oligosaccharyltransferase (OST) complex and stabilizing its catalytic integrity. C-terminal truncation of TREX1 by fs mutations dysregulated the OST complex, leading to free glycan release from dolichol carriers, as well as immune activation and autoantibody production. A connection between OST dysregulation and immune disorders was demonstrated in Trex1(-/-) mice, TREX1-V235fs patient lymphoblasts, and TREX1-V235fs knock-in mice. Inhibiting OST with aclacinomycin corrects the glycan and immune defects associated with Trex1 deficiency or fs mutation. This function of the TREX1 C terminus suggests a potential therapeutic option for TREX1-fs mutant-associated diseases.
Asunto(s)
Citosol/enzimología , Exodesoxirribonucleasas/metabolismo , Hexosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Aclarubicina/análogos & derivados , Aclarubicina/farmacología , Animales , Células Cultivadas , Embrión de Mamíferos/citología , Exodesoxirribonucleasas/antagonistas & inhibidores , Exodesoxirribonucleasas/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Mutación del Sistema de Lectura , Células HEK293 , Células HeLa , Hexosiltransferasas/genética , Humanos , Inmunidad Innata/genética , Immunoblotting , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Fluorescente , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Polisacáridos/metabolismo , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BCR-ABL positive (+) acute lymphoblastic leukemia (ALL) accounts for â¼30% of cases of ALL. We recently demonstrated that 2-deoxy-d-glucose (2-DG), a dual energy (glycolysis inhibition) and ER-stress (N-linked-glycosylation inhibition) inducer, leads to cell death in ALL via ER-stress/UPR-mediated apoptosis. Among ALL subtypes, BCR-ABL+ ALL cells exhibited the highest sensitivity to 2-DG suggesting BCR-ABL expression may be linked to this increased vulnerability. To confirm the role of BCR-ABL, we constructed a NALM6/BCR-ABL stable cell line and found significant increase in 2-DG-induced apoptosis compared to control. We found that Mcl-1 was downregulated by agents inducing ER-stress and Mcl-1 levels correlated with ALL sensitivity. In addition, we showed that Mcl-1 expression is positively regulated by the MEK/ERK pathway, dependent on BCR-ABL, and further downregulated by combining ER-stressors with TKIs. We determined that energy/ER stressors led to translational repression of Mcl-1 via the AMPK/mTOR and UPR/PERK/eIF2α pathways. Taken together, our data indicate that BCR-ABL+ ALL exhibits heightened sensitivity to induction of energy and ER-stress through inhibition of the MEK/ERK pathway, and translational repression of Mcl-1 expression via AMPK/mTOR and UPR/PERK/eIF2α pathways. This study supports further consideration of strategies combining energy/ER-stress inducers with BCR-ABL TKIs for future clinical translation in BCR-ABL+ ALL patients.
RESUMEN
The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress.
Asunto(s)
Vías Biosintéticas , Proteínas de Unión al ADN/metabolismo , Hexosaminas/metabolismo , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada , Animales , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora) , Humanos , Masculino , Ratones , Ratones Transgénicos , Isquemia Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , Transferasas de Grupos Nitrogenados/genética , Factores de Transcripción del Factor Regulador X , Proteína 1 de Unión a la X-BoxRESUMEN
BACKGROUND: As tumors evolve, they upregulate glucose metabolism while also encountering intermittent periods of glucose deprivation. Here, we investigate mechanisms by which pancreatic cancer cells respond to therapeutic (2-deoxy-D-glucose, 2-DG) and physiologic (glucose starvation, GS) forms of glucose restriction. METHODS: From a tumor cell line (1420) that is unusually sensitive to 2-DG under normoxia, low (14DG2)- and high (14DG5)-dose resistant cell lines were selected and used to probe the metabolic pathways involved with their response to different forms of glucose deprivation. RESULTS: Muted induction of the unfolded protein response was found to correlate with resistance to 2-DG. Additionally, 14DG2 displayed reduced 2-DG uptake, while 14DG5 was cross-resistant to tunicamycin, suggesting it has enhanced ability to manage glycosylation defects. Conversely, 2-DG-resistant cell lines were more sensitive than their parental cell line to GS, which coincided with lowered levels of glycogen phosphorylase (PYGB) and reduced breakdown of glycogen to glucose in the 2-DG-resistant cell lines. Moreover, by inhibiting PYGB in the parental cell line, sensitivity to GS was increased. CONCLUSIONS: Overall, the data demonstrate that the manner in which glucose is restricted in tumor cells, i.e., therapeutic or physiologic, leads to differential biological responses involving distinct glucose metabolic pathways. Moreover, in evolving tumors where glucose restriction occurs, the identification of PYGB as a metabolic target may have clinical application.
Asunto(s)
Desoxiglucosa/farmacología , Glucosa/deficiencia , Glucosa/metabolismo , Glucógeno Fosforilasa/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Glucólisis , Humanos , Isoenzimas , Neoplasias Pancreáticas/enzimología , Respuesta de Proteína DesplegadaRESUMEN
Oligosaccharyltransferase (OT) catalyzes the signature reaction of the asparagine-linked glycosylation pathway, namely, the transfer of preformed glycans from the lipid-linked oligosaccharide Glc3Man9GlcNAc2-P-P-Dolichol (G3M9Gn2-LLO) to appropriate asparaginyl residues on acceptor polypeptides. We have identified a reaction, possibly catalyzed by OT, that results in the hydrolysis or "transfer to water" of host LLOs in response to viral infection with release of a free G3M9Gn2 glycan. The loss of LLO ostensibly hinders N-glycosylation of viral polypeptides. This response is achieved by a novel stress-activated signaling pathway in which free mannose-6-phosphate (M6P) acts as a second-messenger. Here, we describe methods with permeabilized mammalian cells for activation of the M6P-regulated LLO hydrolysis, or transfer of glycan to water, in vitro.
Asunto(s)
Lipopolisacáridos/metabolismo , Manosafosfatos/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Técnicas de Cultivo de Célula/métodos , Permeabilidad de la Membrana Celular , Glicosilación , Hidrólisis , Azúcares de Poliisoprenil Fosfato/metabolismo , Polisacáridos/metabolismo , Estreptolisinas/metabolismo , Agua/metabolismoRESUMEN
Mature dolichol-linked oligosaccharides (mDLOs) needed for eukaryotic protein N-glycosylation are synthesized by a multistep pathway in which the biosynthetic lipid intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) flips from the cytoplasmic to the luminal face of the endoplasmic reticulum. The endoplasmic reticulum membrane protein Rft1 is intimately involved in mDLO biosynthesis. Yeast genetic analyses implicated Rft1 as the M5-DLO flippase, but because biochemical tests challenged this assignment, the function of Rft1 remains obscure. To understand the role of Rft1, we sought to analyze mDLO biosynthesis in vivo in the complete absence of the protein. Rft1 is essential for yeast viability, and no Rft1-null organisms are currently available. Here, we exploited Trypanosoma brucei (Tb), an early diverging eukaryote whose Rft1 homologue functions in yeast. We report that TbRft1-null procyclic trypanosomes grow nearly normally. They have normal steady-state levels of mDLO and significant N-glycosylation, indicating robust M5-DLO flippase activity. Remarkably, the mutant cells have 30-100-fold greater steady-state levels of M5-DLO than wild-type cells. All N-glycans in the TbRft1-null cells originate from mDLO indicating that the M5-DLO excess is not available for glycosylation. These results suggest that rather than facilitating M5-DLO flipping, Rft1 facilitates conversion of M5-DLO to mDLO by another mechanism, possibly by acting as an M5-DLO chaperone.
Asunto(s)
Células Eucariotas/metabolismo , Glicoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismo , Electroforesis en Gel de Poliacrilamida , Retículo Endoplásmico/metabolismo , Citometría de Flujo , Glucosa/farmacología , Glicoproteínas/genética , Glicosilación , Proteínas de Membrana de los Lisosomas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Mutación , Oligosacáridos de Poliisoprenil Fosfato/metabolismo , Polisacáridos/metabolismo , Biosíntesis de Proteínas , Proteínas Protozoarias/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transformación Genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
Individuals with congenital disorders of glycosylation (CDG) have recessive mutations in genes required for protein N-glycosylation, resulting in multi-systemic disease. Despite the well-characterized biochemical consequences in these individuals, the underlying cellular defects that contribute to CDG are not well understood. Synthesis of the lipid-linked oligosaccharide (LLO), which serves as the sugar donor for the N-glycosylation of secretory proteins, requires conversion of fructose-6-phosphate to mannose-6-phosphate via the phosphomannose isomerase (MPI) enzyme. Individuals who are deficient in MPI present with bleeding, diarrhea, edema, gastrointestinal bleeding and liver fibrosis. MPI-CDG patients can be treated with oral mannose supplements, which is converted to mannose-6-phosphate through a minor complementary metabolic pathway, restoring protein glycosylation and ameliorating most symptoms, although liver disease continues to progress. Because Mpi deletion in mice causes early embryonic lethality and thus is difficult to study, we used zebrafish to establish a model of MPI-CDG. We used a morpholino to block mpi mRNA translation and established a concentration that consistently yielded 13% residual Mpi enzyme activity at 4 days post-fertilization (dpf), which is within the range of MPI activity detected in fibroblasts from MPI-CDG patients. Fluorophore-assisted carbohydrate electrophoresis detected decreased LLO and N-glycans in mpi morphants. These deficiencies resulted in 50% embryonic lethality by 4 dpf. Multi-systemic abnormalities, including small eyes, dysmorphic jaws, pericardial edema, a small liver and curled tails, occurred in 82% of the surviving larvae. Importantly, these phenotypes could be rescued with mannose supplementation. Thus, parallel processes in fish and humans contribute to the phenotypes caused by Mpi depletion. Interestingly, mannose was only effective if provided prior to 24 hpf. These data provide insight into treatment efficacy and the broader molecular and developmental abnormalities that contribute to disorders associated with defective protein glycosylation.
Asunto(s)
Trastornos Congénitos de Glicosilación/dietoterapia , Trastornos Congénitos de Glicosilación/enzimología , Manosa-6-Fosfato Isomerasa/deficiencia , Manosa-6-Fosfato Isomerasa/genética , Manosa/administración & dosificación , Animales , Secuencia de Bases , Trastornos Congénitos de Glicosilación/genética , Suplementos Dietéticos , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Manosa-6-Fosfato Isomerasa/antagonistas & inhibidores , Ratones , Morfolinos/administración & dosificación , Morfolinos/genética , Mutación , Fenotipo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Postprandially, the liver experiences an extensive metabolic reprogramming that is required for the switch from glucose production to glucose assimilation. Upon refeeding, the unfolded protein response (UPR) is rapidly, though only transiently, activated. Activation of the UPR results in a cessation of protein translation, increased chaperone expression, and increased ER-mediated protein degradation, but it is not clear how the UPR is involved in the postprandial switch to alternate fuel sources. Activation of the inositol-requiring enzyme 1 (IRE1) branch of the UPR signaling pathway triggers expression of the transcription factor Xbp1s. Using a mouse model with liver-specific inducible Xbp1s expression, we demonstrate that Xbp1s is sufficient to provoke a metabolic switch characteristic of the postprandial state, even in the absence of caloric influx. Mechanistically, we identified UDP-galactose-4-epimerase (GalE) as a direct transcriptional target of Xbp1s and as the key mediator of this effect. Our results provide evidence that the Xbp1s/GalE pathway functions as a novel regulatory nexus connecting the UPR to the characteristic postprandial metabolic changes in hepatocytes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Degradación Asociada con el Retículo Endoplásmico/fisiología , Hepatocitos/metabolismo , Hígado/metabolismo , Periodo Posprandial/fisiología , Factores de Transcripción/metabolismo , UDPglucosa 4-Epimerasa/metabolismo , Animales , Células Cultivadas , Proteínas de Unión al ADN/genética , Hepatocitos/citología , Hígado/citología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Conejos , Factores de Transcripción del Factor Regulador X , Factores de Transcripción/genética , UDPglucosa 4-Epimerasa/genética , Proteína 1 de Unión a la X-BoxRESUMEN
Congenital disorder of glycosylation (PMM2-CDG) results from mutations in pmm2, which encodes the phosphomannomutase (Pmm) that converts mannose-6-phosphate (M6P) to mannose-1-phosphate (M1P). Patients have wide-spectrum clinical abnormalities associated with impaired protein N-glycosylation. Although it has been widely proposed that Pmm2 deficiency depletes M1P, a precursor of GDP-mannose, and consequently suppresses lipid-linked oligosaccharide (LLO) levels needed for N-glycosylation, these deficiencies have not been demonstrated in patients or any animal model. Here we report a morpholino-based PMM2-CDG model in zebrafish. Morphant embryos had developmental abnormalities consistent with PMM2-CDG patients, including craniofacial defects and impaired motility associated with altered motor neurogenesis within the spinal cord. Significantly, global N-linked glycosylation and LLO levels were reduced in pmm2 morphants. Although M1P and GDP-mannose were below reliable detection/quantification limits, Pmm2 depletion unexpectedly caused accumulation of M6P, shown earlier to promote LLO cleavage in vitro. In pmm2 morphants, the free glycan by-products of LLO cleavage increased nearly twofold. Suppression of the M6P-synthesizing enzyme mannose phosphate isomerase within the pmm2 background normalized M6P levels and certain aspects of the craniofacial phenotype and abrogated pmm2-dependent LLO cleavage. In summary, we report the first zebrafish model of PMM2-CDG and uncover novel cellular insights not possible with other systems, including an M6P accumulation mechanism for underglycosylation.
Asunto(s)
Trastornos Congénitos de Glicosilación/enzimología , Trastornos Congénitos de Glicosilación/patología , Neurogénesis , Fosfotransferasas (Fosfomutasas)/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Cartílago/efectos de los fármacos , Cartílago/embriología , Cartílago/patología , Forma de la Célula/efectos de los fármacos , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Condrocitos/patología , Trastornos Congénitos de Glicosilación/genética , Anomalías Craneofaciales/embriología , Anomalías Craneofaciales/patología , Modelos Animales de Enfermedad , Embrión no Mamífero/anomalías , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/enzimología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Glicosilación/efectos de los fármacos , Lipopolisacáridos/metabolismo , Manosa-6-Fosfato Isomerasa/metabolismo , Manosafosfatos/metabolismo , Morfolinos/farmacología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Movimiento/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Fosfotransferasas (Fosfomutasas)/deficiencia , Fosfotransferasas (Fosfomutasas)/genética , Cráneo/anomalías , Cráneo/efectos de los fármacos , Cráneo/embriología , Médula Espinal/efectos de los fármacos , Médula Espinal/embriología , Médula Espinal/patología , Especificidad por Sustrato/efectos de los fármacos , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genéticaRESUMEN
In a cell-based assay for novel inhibitors, we have discovered that two glycosides of 5-thiomannose, each containing an interglycosidic nitrogen atom, prevented the correct zymogen processing of the prohormone proopiomelanocortinin (POMC) and the transcription factor sterol-regulatory element-binding protein-2 (SREBP-2) in mouse pituitary cells and Chinese hamster ovary (CHO) cells, respectively. In the case of SREBP-2, these effects were correlated with the altered N-linked glycosylation of subtilisin/kexin-like isozyme-1 (SKI-1), the protease responsible for SREBP-2 processing under sterol-limiting conditions. Further examination of the effects of these compounds in CHO cells showed that they cause extensive protein hypoglycosylation in a manner similar to type I congenital disorders of glycosylation (CDGs) since the remaining N-glycans in treated cells were complete (normal) structures. The under-glycosylation of glycoproteins in 5-thiomannoside-treated cells is now shown to be caused by the compromised biosynthesis of the dolichol-linked oligosaccharide (DLO) N-glycosylation donor, although the nucleotide sugars required for the synthesis of DLOs were neither reduced under these conditions, nor were their effects reversed upon the addition of exogenous mannose. Analysis of DLO intermediates by fluorophore-assisted carbohydrate electrophoresis demonstrated that 5-thiomannose-containing glycosides block DLO biosynthesis most likely at a stage prior to the GlcNAc(2) Man(3) intermediate, on the cytosolic face of the endoplasmic reticulum.
Asunto(s)
Trastornos Congénitos de Glicosilación/metabolismo , Dolicoles/antagonistas & inhibidores , Manosa/farmacología , Oligosacáridos/antagonistas & inhibidores , Animales , Células CHO , Células Cultivadas , Trastornos Congénitos de Glicosilación/prevención & control , Cricetinae , Modelos Animales de Enfermedad , Dolicoles/biosíntesis , Dolicoles/química , Manosa/análogos & derivados , Manosa/química , Ratones , Oligosacáridos/biosíntesis , Oligosacáridos/químicaRESUMEN
Mannose-6-phosphate (M6P) is an essential precursor for mannosyl glycoconjugates, including lipid-linked oligosaccharides (LLO; glucose(3)mannose(9)GlcNAc(2)-P-P-dolichol) used for protein N-glycosylation. In permeabilized mammalian cells, M6P also causes specific LLO cleavage. However, the context and purpose of this paradoxical reaction are unknown. In this study, we used intact mouse embryonic fibroblasts to show that endoplasmic reticulum (ER) stress elevates M6P concentrations, leading to cleavage of the LLO pyrophosphate linkage with recovery of its lipid and lumenal glycan components. We demonstrate that this M6P originates from glycogen, with glycogenolysis activated by the kinase domain of the stress sensor IRE1-α. The apparent futility of M6P causing destruction of its LLO product was resolved by experiments with another stress sensor, PKR-like ER kinase (PERK), which attenuates translation. PERK's reduction of N-glycoprotein synthesis (which consumes LLOs) stabilized steady-state LLO levels despite continuous LLO destruction. However, infection with herpes simplex virus 1, an N-glycoprotein-bearing pathogen that impairs PERK signaling, not only caused LLO destruction but depleted LLO levels as well. In conclusion, the common metabolite M6P is also part of a novel mammalian stress-signaling pathway, responding to viral stress by depleting host LLOs required for N-glycosylation of virus-associated polypeptides. Apparently conserved throughout evolution, LLO destruction may be a response to a variety of environmental stresses.
Asunto(s)
Herpes Simple/metabolismo , Lipopolisacáridos/metabolismo , Manosafosfatos/metabolismo , Amidas/farmacología , Aminoacridinas/química , Animales , Células CHO , Trastornos Congénitos de Glicosilación/metabolismo , Cricetinae , Fosfatos de Dolicol/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/virología , Colorantes Fluorescentes/química , Glucógeno/metabolismo , Glucógeno Fosforilasa/antagonistas & inhibidores , Herpesvirus Humano 1 , Interacciones Huésped-Patógeno , Inmunidad Celular , Indoles/farmacología , Manosafosfatos/farmacología , Ratones , Ratones Noqueados , Fosfotransferasas (Fosfomutasas)/deficiencia , Polisacáridos/metabolismo , Respuesta de Proteína Desplegada/efectos de los fármacos , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
Dolichol monophosphate (Dol-P) functions as an obligate glycosyl carrier lipid in protein glycosylation reactions. Dol-P is synthesized by the successive condensation of isopentenyl diphosphate (IPP), with farnesyl diphosphate catalysed by a cis-isoprenyltransferase (cis-IPTase) activity. Despite the recognition of cis-IPTase activity 40 years ago and the molecular cloning of the human cDNA encoding the mammalian enzyme, the molecular machinery responsible for regulating this activity remains incompletely understood. Here, we identify Nogo-B receptor (NgBR) as an essential component of the Dol-P biosynthetic machinery. Loss of NgBR results in a robust deficit in cis-IPTase activity and Dol-P production, leading to diminished levels of dolichol-linked oligosaccharides and a broad reduction in protein N-glycosylation. NgBR interacts with the previously identified cis-IPTase hCIT, enhances hCIT protein stability, and promotes Dol-P production. Identification of NgBR as a component of the cis-IPTase machinery yields insights into the regulation of dolichol biosynthesis.
Asunto(s)
Dolicoles/biosíntesis , Receptores de Superficie Celular/fisiología , Transferasas Alquil y Aril/antagonistas & inhibidores , Transferasas Alquil y Aril/deficiencia , Transferasas Alquil y Aril/metabolismo , Animales , Células COS , Proteínas Portadoras/metabolismo , Chlorocebus aethiops , Fosfatos de Dolicol/biosíntesis , Fosfatos de Dolicol/deficiencia , Dolicoles/deficiencia , Activación Enzimática/genética , Glicoproteínas/metabolismo , Humanos , Conformación Proteica , Receptores de Superficie Celular/química , Receptores de Superficie Celular/deficiencia , Proteínas de Transporte VesicularRESUMEN
BACKGROUND: During tumor angiogenesis, endothelial cells (ECs) are engaged in a number of energy consuming biological processes, such as proliferation, migration, and capillary formation. Since glucose uptake and metabolism are increased to meet this energy need, the effects of the glycolytic inhibitor 2-deoxy-D-glucose (2-DG) on in vitro and in vivo angiogenesis were investigated. METHODOLOGY/PRINCIPAL FINDINGS: In cell culture, 2-DG inhibited EC growth, induced cytotoxicity, blocked migration, and inhibited actively forming but not established endothelial capillaries. Surprisingly, 2-DG was a better inhibitor of these EC properties than two more efficacious glycolytic inhibitors, 2-fluorodeoxy-D-glucose and oxamate. As an alternative to a glycolytic inhibitory mechanism, we considered 2-DG's ability to interfere with endothelial N-linked glycosylation. 2-DG's effects were reversed by mannose, an N-linked glycosylation precursor, and at relevant concentrations 2-DG also inhibited synthesis of the lipid linked oligosaccharide (LLO) N-glycosylation donor in a mannose-reversible manner. Inhibition of LLO synthesis activated the unfolded protein response (UPR), which resulted in induction of GADD153/CHOP and EC apoptosis (TUNEL assay). Thus, 2-DG's effects on ECs appeared primarily due to inhibition of LLOs synthesis, not glycolysis. 2-DG was then evaluated in two mouse models, inhibiting angiogenesis in both the matrigel plug assay and the LH(BETA)T(AG) transgenic retinoblastoma model. CONCLUSIONS/SIGNIFICANCE: In conclusion, 2-DG inhibits endothelial cell angiogenesis in vitro and in vivo, at concentrations below those affecting tumor cells directly, most likely by interfering with N-linked glycosylation rather than glycolysis. Our data underscore the importance of glucose metabolism on neovascularization, and demonstrate a novel approach for anti-angiogenic strategies.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Desoxiglucosa/farmacología , Animales , Apoptosis , Western Blotting , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones , Ratones TransgénicosRESUMEN
To further evaluate the role of Rft1 in the transbilayer movement of Man(5)GlcNAc(2)-P-P-dolichol (M5-DLO), a series of experiments was conducted with intact cells and sealed microsomal vesicles. First, an unexpectedly large accumulation (37-fold) of M5-DLO was observed in Rft1-depleted cells (YG1137) relative to Glc(3)Man(9)GlcNAc(2)-P-P-Dol in wild type (SS328) cells when glycolipid levels were compared by fluorophore-assisted carbohydrate electrophoresis analysis. When sealed microsomes from wild type cells and cells depleted of Rft1 were incubated with GDP-[(3)H]mannose or UDP-[(3)H]GlcNAc in the presence of unlabeled GDP-Man, no difference was observed in the rate of synthesis of [(3)H]Man(9)GlcNAc(2)-P-P-dolichol or Man(9)[(3)H]GlcNAc(2)-P-P-dolichol, respectively. In addition, no difference was seen in the level of M5-DLO flippase activity in sealed wild type and Rft1-depleted microsomal vesicles when the activity was assessed by the transport of GlcNAc(2)-P-P-Dol(15), a water-soluble analogue. The entry of the analogue into the lumenal compartment was confirmed by demonstrating that [(3)H]chitobiosyl units were transferred to endogenous peptide acceptors via the yeast oligosaccharyltransferase when sealed vesicles were incubated with [(3)H]GlcNAc(2)-P-P-Dol(15) in the presence of an exogenously supplied acceptor peptide. In addition, several enzymes involved in Dol-P and lipid intermediate biosynthesis were found to be up-regulated in Rft1-depleted cells. All of these results indicate that although Rft1 may play a critical role in vivo, depletion of this protein does not impair the transbilayer movement of M5-DLO in sealed microsomal fractions prepared from disrupted cells.
Asunto(s)
Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Microsomas/metabolismo , Oligosacáridos de Poliisoprenil Fosfato/análisis , Oligosacáridos de Poliisoprenil Fosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transferasas Alquil y Aril/metabolismo , Transporte Biológico , Monofosfato de Dolicol Manosa/metabolismo , Regulación Bacteriana de la Expresión Génica , Glucosa/metabolismo , Hexosiltransferasas/metabolismo , Manosa/metabolismo , Proteínas de la Membrana/metabolismo , Microsomas/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Monosacáridos de Poliisoprenil Fosfato/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
GlcNAc-1-P transferase (GPT) transfers GlcNAc-1-P from UDP-GlcNAc to dolichol-P (Dol-P), forming GlcNAc-P-PDol to initiate synthesis of the lipid-linked oligosaccharide Glc3Man9GlcNAc2-P-P-dolichol (G3M9Gn2-P-P-Dol). Elevated expression of GPT in CHO-K1 cells is known to cause accumulation of the intermediate M5Gn2-P-P-Dol, presumably by excessively consuming Dol-P and thereby hindering Dol-P-dependent synthesis of Man-P-Dol (MPD) and Glc-P-Dol (GPD), which provide the residues for extending M5Gn2-P-P-Dol to G3M9Gn2-P-P-Dol. If so, elevated GPT expression should increase oligosaccharide-P-P-Dol quantities and reduce monosaccharide-P-Dol quantities, while requiring GPT enzymatic activity. Here we report that elevated GPT expression failed to appreciably alter the quantities of the two classes of dolichol-linked saccharide, and that neither a GPT inhibitor nor introduction of an inactivating mutation into GPT prevented M5Gn2-P-P-Dol accumulation,arguing against excessive Dol-P consumption. Unexpectedly,we noticed similarities between the phenotypes of GPT overexpressers and of CHO-K1 cells lacking Lec35p (encoded by MPDU1, the congenital disorder of glycosylation(CDG)-If locus), which is required for utilization of MPD and GPD. By compensatory overexpression of Lec35p, G3M9Gn2-P-P-Dol synthesis in GPT overexpressers could be restored. However, GPT overexpression did not affect the levels of Lec35 mRNA or protein. These results suggest that GPT may impair Lec35p function, and imply that upper as well as lower limits on GPT expression exist in normal cells. Since the mammalian GPT gene can undergo spontaneous amplification, the data also indicate a potential basis for forms of pseudo-CDG-If.
Asunto(s)
Azúcares de Poliisoprenil Fosfato/biosíntesis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Animales , Células CHO , Cricetinae , Cricetulus , Expresión Génica , Glicosilación , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoRESUMEN
During protein N-glycosylation, dolichyl pyrophosphate (Dol-P-P) is discharged in the lumenal monolayer of the endoplasmic reticulum (ER). Dol-P-P is then cleaved to Dol-P by Dol-P-P phosphatase (DPPase). Studies with the yeast mutant cwh8Delta, lacking DPPase activity, indicate that recycling of Dol-P produced by DPPase contributes significantly to the pool of Dol-P utilized for lipid intermediate biosynthesis on the cytoplasmic leaflet. Whether Dol-P formed in the lumen diffuses directly back to the cytoplasmic leaflet or is first dephosphorylated to dolichol has not been determined. Incubation of sealed ER vesicles from calf brain with acetyl-Asn-Tyr-Thr-NH(2), an N-glycosylatable peptide, to generate Dol-P-P in the lumenal monolayer produced corresponding increases in the rates of Man-P-Dol, Glc-P-Dol, and GlcNAc-P-P-Dol synthesis in the absence of CTP. No changes in dolichol kinase activity were observed. When streptolysin-O permeabilized CHO cells were incubated with an acceptor peptide, N-glycopeptide synthesis, requiring multiple cycles of the dolichol pathway, occurred in the absence of CTP. The results obtained with sealed microsomes and CHO cells indicate that Dol-P, formed from Dol-P-P, returns to the cytoplasmic leaflet where it can be reutilized for lipid intermediate biosynthesis, and dolichol kinase is not required for recycling. It is possible that the flip-flopping of the carrier lipid is mediated by a flippase, which would provide a mechanism for the recycling of Dol-P derived from Man-P-Dol-mediated reactions in N-, O-, and C-mannosylation of proteins, GPI anchor assembly, and the three Glc-P-Dol-mediated reactions in Glc(3)Man(9)GlcNAc(2)-P-P-Dol (DLO) biosynthesis.
Asunto(s)
Citoplasma/metabolismo , Fosfatos de Dolicol/metabolismo , Retículo Endoplásmico/metabolismo , Animales , Bacitracina/administración & dosificación , Encéfalo/metabolismo , Bovinos , Hidrólisis , Microsomas/metabolismoRESUMEN
In tumor cells growing under hypoxia, inhibiting glycolysis with 2-deoxy-d-glucose (2-DG) leads to cell death, whereas under normoxic conditions cells similarly treated survive. Surprisingly, here we find that 2-DG is toxic in select tumor cell lines growing under normal oxygen tension. In contrast, a more potent glycolytic inhibitor, 2-fluorodeoxy-d-glucose, shows little or no toxicity in these cell types, indicating that a mechanism other than inhibition of glycolysis is responsible for their sensitivity to 2-DG under normoxia. A clue to this other mechanism comes from previous studies in which it was shown that 2-DG interferes with viral N-linked glycosylation and is reversible by exogenous addition of mannose. Similarly, we find that 2-DG interferes with N-linked glycosylation more potently in the tumor cell types that are sensitive to 2-DG under normoxia, which can be reversed by exogenous mannose. Additionally, 2-DG induces an unfolded protein response, including up-regulation of GADD153 (C/EBP-homologous protein), an unfolded protein response-specific mediator of apoptosis, more effectively in 2-DG-sensitive cells. We conclude that 2-DG seems to be toxic in select tumor cell types growing under normoxia by inhibition of N-linked glycosylation and not by glycolysis. Because in a phase I study 2-DG is used in combination with an anticancer agent to target hypoxic cells, our results raise the possibility that in certain cases, 2-DG could be used as a single agent to selectively kill both the aerobic (via interference with glycosylation) and hypoxic (via inhibition of glycolysis) cells of a solid tumor.