RESUMO
Mutations affecting isocitrate dehydrogenase (IDH) enzymes are prevalent in glioma, leukemia, and other cancers. Although mutant IDH inhibitors are effective against leukemia, they seem to be less active in aggressive glioma, underscoring the need for alternative treatment strategies. Through a chemical synthetic lethality screen, we discovered that IDH1-mutant glioma cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). We developed a genetically engineered mouse model of mutant IDH1-driven astrocytoma and used it and multiple patient-derived models to show that the brain-penetrant DHODH inhibitor BAY 2402234 displays monotherapy efficacy against IDH-mutant gliomas. Mechanistically, this reflects an obligate dependence of glioma cells on the de novo pyrimidine synthesis pathway and mutant IDH's ability to sensitize to DNA damage upon nucleotide pool imbalance. Our work outlines a tumor-selective, biomarker-guided therapeutic strategy that is poised for clinical translation.
Assuntos
Neoplasias Encefálicas , Glioma , Leucemia , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Inibidores Enzimáticos/uso terapêutico , Glioma/tratamento farmacológico , Glioma/genética , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Camundongos , Mutação , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Salicilanilidas , TriazóisRESUMO
Herpes simplex virus 1 (HSV-1) is a contagious neurotropic herpesvirus responsible for oral lesions and herpesviral encephalitis. The HSV-1 envelope contains N-glycosylated proteins involved in infection and that are candidate drug targets. NGI-1 is a small-molecule inhibitor of oligosaccharyltransferase (OST) complexes STT3A-OST and STT3B-OST, which catalyze cotranslational and post-translational N-glycosylation, respectively. Because host OSTs attach HSV-1 glycans, NGI-1 might have anti-HSV-1 activity. We evaluated HSV-1 function using NGI-1 and human embryonic kidney 293 knockout lines for OST isoform-specific catalytic and accessory subunits. N-glycosylation of 2 representative envelope proteins (gC and gD) was primarily dependent upon STT3A-OST, but to a large extent replaceable by STT3B-OST. Knockouts impairing STT3A- or STT3B-OST activity, by themselves, did not appreciably affect HSV-1 function (plaque-forming units, normalized to viral particles measured by unglycosylated capsid protein VP5 content). However, with cells lacking STT3B-OST activity (missing the catalytic subunit STT3B or the oxidoreductase subunits magnesium transporter 1/tumor suppressor candidate 3) and thus solely dependent upon STT3A-OST for N-glycosylation, NGI-1 treatment resulted in HSV-1 having cell type-dependent dysfunction (affecting infectivity with Vero cells much more than with the 293 lines). Ablation of post-translational N-glycosylation can therefore make HSV-1 infectivity, and possibly masking of immunogenic peptide epitopes by glycans, highly sensitive to pharmacological inhibition of cotranslational N-glycosylation.-Lu, H., Cherepanova, N. A., Gilmore, R., Contessa, J. N., Lehrman, M. A. Targeting STT3A-oligosaccharyltransferase with NGI-1 causes herpes simplex virus 1 dysfunction.
Assuntos
Benzamidas/farmacologia , Herpes Simples/tratamento farmacológico , Herpesvirus Humano 1/efeitos dos fármacos , Hexosiltransferases/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Sulfonamidas/farmacologia , Animais , Chlorocebus aethiops , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Glicosilação , Células HEK293 , Herpes Simples/metabolismo , Herpes Simples/virologia , Humanos , Células VeroRESUMO
Discovered 40 years ago, the Lec5 glycosylation mutant cell line has a complex recessive genotype and is characterized by accumulation of lipid-linked oligosaccharide assembly intermediates, reduced conversion of polyprenols to dolichols, and an unusual phenotypic dependence upon cell culture conditions such as temperature, plating density and medium quality. The heritable defect in Lec5 is unknown. Here we demonstrate an unexpected epigenetic basis for Lec5, with a surprising linkage to increased expression of homeobox genes, which in turn is associated with increased transcription of cholesterol biosynthesis genes. These results suggest testable hypotheses for the biochemical abnormalities of the Lec5 mutant.
Assuntos
Colesterol/genética , Genes Homeobox/genética , Lectinas/genética , Lipopolissacarídeos/genética , Colesterol/metabolismo , Glicosilação , Humanos , Lectinas/metabolismo , Lipopolissacarídeos/metabolismo , MutaçãoRESUMO
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.
Assuntos
Benzamidas/farmacologia , Senescência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hexosiltransferases/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Sulfonamidas/farmacologia , Benzamidas/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Hexosiltransferases/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Proteínas de Membrana/metabolismo , Estrutura Molecular , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
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.
RESUMO
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.
Assuntos
Desoxiglucose/farmacologia , Glucose/deficiência , Glucose/metabolismo , Glicogênio Fosforilase/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Glicólise , Humanos , Isoenzimas , Neoplasias Pancreáticas/enzimologia , Resposta a Proteínas não DobradasRESUMO
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.
Assuntos
Lipopolissacarídeos/metabolismo , Manosefosfatos/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Técnicas de Cultura de Células/métodos , Permeabilidade da Membrana Celular , Glicosilação , Hidrólise , Açúcares de Poli-Isoprenil Fosfato/metabolismo , Polissacarídeos/metabolismo , Estreptolisinas/metabolismo , Água/metabolismoRESUMO
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.
Assuntos
Herpes Simples/metabolismo , Lipopolissacarídeos/metabolismo , Manosefosfatos/metabolismo , Amidas/farmacologia , Aminoacridinas/química , Animais , Células CHO , Defeitos Congênitos da Glicosilação/metabolismo , Cricetinae , Fosfatos de Dolicol/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/virologia , Corantes Fluorescentes/química , Glicogênio/metabolismo , Glicogênio Fosforilase/antagonistas & inibidores , Herpesvirus Humano 1 , Interações Hospedeiro-Patógeno , Imunidade Celular , Indóis/farmacologia , Manosefosfatos/farmacologia , Camundongos , Camundongos Knockout , Fosfotransferases (Fosfomutases)/deficiência , Polissacarídeos/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
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.
Assuntos
Inibidores da Angiogênese/farmacologia , Desoxiglucose/farmacologia , Animais , Apoptose , Western Blotting , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos TransgênicosRESUMO
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.
Assuntos
Açúcares de Poli-Isoprenil Fosfato/biossíntese , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Animais , Células CHO , Cricetinae , Cricetulus , Expressão Gênica , Glicosilação , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
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.
Assuntos
Desoxiglucose/farmacologia , Glicólise/efeitos dos fármacos , Neoplasias/patologia , Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Aerobiose/efeitos dos fármacos , Anaerobiose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fluordesoxiglucose F18/farmacologia , Glicosilação/efeitos dos fármacos , Humanos , Manose/farmacologia , Modelos Biológicos , Oligossacarídeos/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Dobramento de Proteína , Fator de Transcrição CHOP/genética , Regulação para Cima/efeitos dos fármacosRESUMO
The dolichol cycle involves synthesis of the lipid-linked oligosaccharide (LLO) Glc(3)Man(9)GlcNAc(2)-P-P-dolichol (G(3)M(9)Gn(2)-P-P-Dol), transfer of G(3)M(9)Gn(2) to asparaginyl residues of nascent endoplasmic reticulum (ER) polypeptides by oligosaccharyltransferase (OT), and recycling of the resultant Dol-P-P to Dol-P for new rounds of LLO synthesis. The importance of the dolichol cycle in secretory and membrane protein biosynthesis, ER function, and human genetic disease is now widely accepted. Elucidation of the fundamental properties of the dolichol cycle in intact cells was achieved through the use of radioactive sugar precursors, typically [(3)H]-labeled or [(14)C]-labeled d-mannose, d-galactose, or d-glucosamine. However, difficulties were encountered with cells or tissues not amenable to metabolic labeling, or in experiments influenced by isotope dilution, variable rates of LLO turnover, or special culture conditions required for the use of radioactive sugars. This article will review recently developed alternatives for LLO analysis that do not rely upon metabolic labeling with radioactive precursors, and thereby circumvent these problems. New information revealed by these methods with regard to regulation, genetic disorders, and evolution of the dolichol cycle, as well as caveats of radiolabeling techniques, will be discussed.
Assuntos
Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Açúcares de Poli-Isoprenil Fosfato/química , Açúcares de Poli-Isoprenil Fosfato/metabolismo , Polissacarídeos/análise , Radioisótopos de Carbono , Luminescência , Modelos Biológicos , Peptídeos/química , Peptídeos/metabolismo , Polissacarídeos/química , Sefarose/análogos & derivados , Sefarose/química , Sefarose/metabolismo , TrítioRESUMO
Endoplasmic reticulum (ER) homeostasis requires transfer and subsequent processing of the glycan Glc(3)Man(9)GlcNAc(2) (G(3)M(9)Gn(2)) from the lipid-linked oligosaccharide (LLO) glucose(3)mannose(9)N-acetylglucosamine(2)-P-P-dolichol (G(3)M(9)Gn(2)-P-P-Dol) to asparaginyl residues of nascent glycoprotein precursor polypeptides. However, it is unclear how the ER is protected against dysfunction from abnormal accumulation of LLO intermediates and aberrant N-glycosylation, as occurs in certain metabolic diseases. In metazoans phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) on Ser(51) by PERK (PKR-like ER kinase), which is activated by ER stress, attenuates translation initiation. We use brief glucose deprivation to simulate LLO biosynthesis disorders, and show that attenuation of polypeptide synthesis by PERK promotes extension of LLO intermediates to G(3)M(9)Gn(2)-P-P-Dol under these substrate-limiting conditions, as well as counteract abnormal N-glycosylation. This simple mechanism requires eIF2alpha Ser(51) phosphorylation by PERK, and is mimicked by agents that stimulate cytoplasmic stress-responsive Ser(51) kinase activity. Thus, by sensing ER stress from defective glycosylation, PERK can restore ER homeostasis by balancing polypeptide synthesis with flux through the LLO pathway.
Assuntos
Retículo Endoplasmático/metabolismo , Glicoproteínas/biossíntese , Lipopolissacarídeos/metabolismo , Biossíntese de Proteínas/fisiologia , eIF-2 Quinase/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Cicloeximida/farmacologia , Ativação Enzimática , Fator de Iniciação 2 em Eucariotos/metabolismo , Glicosilação , Homeostase , FosforilaçãoRESUMO
The experimental evaluation of the contribution of glycogen phosphorylase (GP) to biochemical pathways is limited to methods that raise cAMP, activating the cAMP-dependent protein kinase/phosphorylase kinase/GP cascade. Such methods convert the unphosphorylated form, "GPb," which catalyzes glycogenolysis only in the presence of appropriate allosteric activators such as AMP, to the phosphorylated, constitutively activated form, "GPa." However, activation of GP in this way is indirect, requires a functional cAMP kinase cascade, and is complicated by other actions of cAMP. Here, we demonstrate a strategy for the experimental manipulation of GP in intact dermal fibroblasts, involving activation by the membrane-permeable adenosine analog 5-aminoimidazole-4-carboxamide riboside (AICAR) and inhibition by caffeine and Pfizer compound CP-91149, which bind to GP at distinct sites. Potential complications because of activation of AMP-activated protein kinase by AICAR were assessed with metformin, which activates this kinase but does not activate GP. Using this strategy, we show that glycogen can be a significant and regulatable precursor of mannosyl units in lipid-linked oligosaccharides and glycoproteins.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Glicogênio Fosforilase/metabolismo , Glicogênio/metabolismo , Hipoglicemiantes/farmacologia , Manose/metabolismo , Ribonucleotídeos/farmacologia , Amidas/farmacologia , Sítios de Ligação , Cafeína/farmacologia , Catálise , Células Cultivadas , Cromatografia Líquida de Alta Pressão , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Ativação Enzimática , Fibroblastos/metabolismo , Humanos , Indóis/farmacologia , Metabolismo dos Lipídeos , Manose/química , Metformina/farmacologia , Oligossacarídeos/química , Fosforilação , Ligação Proteica , Pele/citologia , Fatores de TempoRESUMO
Endoplasmic reticulum (ER) stress is associated with misfolding of ER proteins and triggers the unfolded protein response (UPR). The UPR, in turn, helps restore normal ER function. Since fastidious N-linked glycosylation is critical for folding of most ER proteins, this study examined whether metabolic interconversions of precursors used for glycan assembly were controlled by the UPR. Thus, eight enzymes and factors with key roles in hexose phosphate metabolism were assayed in cytoplasmic extracts from primary dermal fibroblasts treated with UPR inducers. Stimulation of only one activity by the UPR was detected, AMP-independent glycogen phosphorylase (GP). GP activation required only 20 min of ER stress, with concurrent decreases in cellular glycogen and elevations of its metabolites Glc-1-P and Glc-6-P. Addition of phosphatase inhibitors to enzyme extracts from unstressed cells mimicked the effect of ER stress on GP activity, suggesting that phosphorylation of GP or a regulatory factor was involved. These data show that the UPR can modulate hexose metabolism in a manner beneficial for protein glycosylation. Since activation of GP appears to occur by a rapid post-translational process, it may be part of a general strategy of ER damage control, preceding the well-known transcription-dependent processes of the UPR that are manifested hours after the occurrence of ER stress.
Assuntos
Citoplasma/enzimologia , Retículo Endoplasmático/metabolismo , Glicogênio Fosforilase/metabolismo , Polissacarídeos/metabolismo , Dobramento de Proteína , Monofosfato de Adenosina/farmacologia , Cafeína/farmacologia , Células Cultivadas , Citoplasma/química , Derme/citologia , Ditiotreitol/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Ativação Enzimática , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Glucose-6-Fosfato/metabolismo , Glicogênio/metabolismo , Hexosefosfatos/metabolismo , Humanos , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Tunicamicina/farmacologiaRESUMO
In mammalian cells, inhibition of translation interferes with synthesis of the lipid-linked oligosaccharide (LLO) Glc3Man9GlcNAc2-P-P-dolichol as measured with radioactive sugar precursors. Conflicting hypotheses have been proposed, and the fundamental basis for this regulation has remained elusive. Here, fluorophore-assisted carbohydrate electrophoresis (FACE) was used to measure LLO concentrations directly in cells treated with translation blockers. Further, LLO biosynthetic enzymes were assayed in vitro with endogenous acceptor substrates using either cells gently permeabilized with streptolysin-O (SLO) or microsomes from homogenized cells. In Chinese hamster ovary (CHO)-K1 cells treated with translation blockers, FACE did not detect changes in concentrations of Glc3Man9GlcNAc2-P-P-dolichol or early LLO intermediates. These results do not support earlier proposals for feedback repression of LLO initiation by accumulated Glc3Man9GlcNAc2-P-P-dolichol, or inhibition of a GDP-mannose dependent transferase. With microsomes from cells treated with translation blockers, there was no interference with LLO initiation by GlcNAc-1-P transferase (GPT), mannose-P-dolichol synthase, glucose-P-dolichol synthase, or LLO synthesis in vitro, as reported previously. Surprisingly, inhibition of all of these was detected with the SLO in vitro system. Additional experiments with the SLO system showed that the three transferases shared a limited pool of dolichol-P that was trapped as Glc3Man9GlcNAc2-P-P-dolichol by translation arrest. Overexpression of GPT was unable to reverse the effects of translation arrest on LLO initiation, and experiments with FACE and the SLO system showed that overexpressed GPT was not functional in vivo, although it was highly active in microsomal assays. Thus, the combined use of the SLO in vitro system and FACE showed that LLO biosynthesis depends upon a limited primary pool of dolichol-P. Physical perturbation associated with microsome preparation appears to make available a secondary pool of dolichol-P, masking inhibition by translation arrest, as well as activating a nonfunctional fraction of GPT. The implications of these results for the organization of the LLO pathway are discussed.