RESUMO
Adaptation to the shortage in free amino acids (AA) is mediated by 2 pathways, the integrated stress response (ISR) and the mechanistic target of rapamycin (mTOR). In response to reduced levels, primarily of leucine or arginine, mTOR in its complex 1 configuration (mTORC1) is suppressed leading to a decrease in translation initiation and elongation. The eIF2α kinase general control nonderepressible 2 (GCN2) is activated by uncharged tRNAs, leading to induction of the ISR in response to a broader range of AA shortage. ISR confers a reduced translation initiation, while promoting the selective synthesis of stress proteins, such as ATF4. To efficiently adapt to AA starvation, the 2 pathways are cross-regulated at multiple levels. Here we identified a new mechanism of ISR/mTORC1 crosstalk that optimizes survival under AA starvation, when mTORC1 is forced to remain active. mTORC1 activation during acute AA shortage, augmented ATF4 expression in a GCN2-dependent manner. Under these conditions, enhanced GCN2 activity was not dependent on tRNA sensing, inferring a different activation mechanism. We identified a labile physical interaction between GCN2 and mTOR that results in a phosphorylation of GCN2 on serine 230 by mTOR, which promotes GCN2 activity. When examined under prolonged AA starvation, GCN2 phosphorylation by mTOR promoted survival. Our data unveils an adaptive mechanism to AA starvation, when mTORC1 evades inhibition.
RESUMO
AIMS/HYPOTHESIS: Second-generation antipsychotic (SGA) drugs have been associated with the development of type 2 diabetes and the metabolic syndrome in patients with schizophrenia. In this study, we aimed to investigate the effects of two different SGA drugs, olanzapine and aripiprazole, on metabolic state and islet function and plasticity. METHODS: We analysed the functional adaptation of beta cells in 12-week-old B6;129 female mice fed an olanzapine- or aripiprazole-supplemented diet (5.5-6.0 mg kg-1 day-1) for 6 months. Glucose and insulin tolerance tests, in vivo glucose-stimulated insulin secretion and indirect calorimetry were performed at the end of the study. The effects of SGAs on beta cell plasticity and islet serotonin levels were assessed by transcriptomic analysis and immunofluorescence. Insulin secretion was assessed by static incubations and Ca2+ fluxes by imaging techniques. RESULTS: Treatment of female mice with olanzapine or aripiprazole for 6 months induced weight gain (p<0.01 and p<0.05, respectively), glucose intolerance (p<0.01) and impaired insulin secretion (p<0.05) vs mice fed a control chow diet. Aripiprazole, but not olanzapine, induced serotonin production in beta cells vs controls, likely by increasing tryptophan hydroxylase 1 (TPH1) expression, and inhibited Ca2+ flux. Of note, aripiprazole increased beta cell size (p<0.05) and mass (p<0.01) vs mice fed a control chow diet, along with activation of mechanistic target of rapamycin complex 1 (mTORC1)/S6 signalling, without preventing beta cell dysfunction. CONCLUSIONS/INTERPRETATION: Both SGAs induced weight gain and beta cell dysfunction, leading to glucose intolerance; however, aripiprazole had a more potent effect in terms of metabolic alterations, which was likely a result of its ability to modulate the serotonergic system. The deleterious metabolic effects of SGAs on islet function should be considered while treating patients as these drugs may increase the risk for development of the metabolic syndrome and diabetes.
Assuntos
Antipsicóticos , Diabetes Mellitus Tipo 2 , Ilhotas Pancreáticas , Animais , Antipsicóticos/efeitos adversos , Aripiprazol/metabolismo , Aripiprazol/farmacologia , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Humanos , Ilhotas Pancreáticas/metabolismo , Camundongos , Olanzapina/efeitos adversos , Olanzapina/metabolismoRESUMO
Protective MHC class I-dependent immune responses require an overlap between repertoires of proteins directly presented on target cells and cross-presented by professional APC, specifically dendritic cells. How stable proteins that rely on defective ribosomal proteins for direct presentation are captured for cell-to-cell transfer remains enigmatic. In this study, we address this issue using a combination of in vitro (C57BL/6-derived mouse cell lines) and in vivo (C57BL/6 mouse strains) approaches involving stable and unstable versions of OVA model Ags displaying defective ribosomal protein-dependent and -independent Ag presentation, respectively. Apoptosis, but not necrosis, of donor cells was found associated with robust global protein aggregate formation and captured stable proteins permissive for cross-presentation. Potency of aggregates to serve as Ag source was directly demonstrated using polyglutamine-equipped model substrates. Collectively, our data implicate global protein aggregation in apoptotic cells as a mechanism that ensures the overlap between MHC class I epitopes presented directly or cross-presented by APC and demonstrate the unusual ability of dendritic cells to process stable protein aggregates.
Assuntos
Apresentação de Antígeno , Antígenos/imunologia , Células Dendríticas/imunologia , Peptídeos/imunologia , Agregados Proteicos/imunologia , Animais , Antígenos/genética , Linhagem Celular , Células Dendríticas/metabolismo , Epitopos/imunologia , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Camundongos , Camundongos Transgênicos , Ovalbumina/genética , Ovalbumina/imunologia , Peptídeos/metabolismoRESUMO
The endoplasmic reticulum (ER) is a dynamic organelle that responds to demand in secretory proteins by undergoing expansion. The mechanisms that control the homeostasis of ER size and function involve the activation of the unfolded protein response (UPR). The UPR plays a role in various effector functions of immune cells. Mast cells (MCs) are highly granular tissue-resident cells and key drivers of allergic inflammation. Their diverse secretory functions in response to activation through the high-affinity receptor for IgE (FcεRI) suggest a role for the UPR in their function. Using human cord blood-derived MCs, we found that FcεRI triggering elevated the expression level and induced activation of the UPR transducers IRE1α and PERK, accompanied by expansion of the ER. In mouse bone marrow-derived MCs and peritoneal MCs, the ER underwent a more moderate expansion, and the UPR was not induced following MC activation. The deletion of IRE1α in mouse MCs did not affect proliferation, survival, degranulation, or cytokine stimulation following FcεRI triggering, but it did diminish the surface expression of TLR4 and the consequent response to LPS. A similar phenotype was observed in human MCs using an IRE1α inhibitor. Our data indicate that the ER of MCs, primarily of humans, undergoes a rapid remodeling in response to activation that promotes responses to TLR4. We suggest that IRE1α inhibition can be a strategy for inhibiting the hyperactivation of MCs by LPS over the course of allergic responses.
Assuntos
Retículo Endoplasmático , Endorribonucleases , Proteínas Serina-Treonina Quinases , Receptor 4 Toll-Like , Animais , Citocinas/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Homeostase , Humanos , Imunoglobulina E/metabolismo , Lipopolissacarídeos , Mastócitos/metabolismo , Camundongos , Proteínas Serina-Treonina Quinases/genética , Receptores de IgE/metabolismo , Receptor 4 Toll-Like/metabolismo , Resposta a Proteínas não DobradasRESUMO
Rapidly proliferating cells reshape their metabolism to satisfy their ever-lasting need for cellular building blocks. This phenomenon is exemplified in certain malignant conditions such as cancer but also during embryonic development when cells rely heavily on glycolytic metabolism to exploit its metabolic intermediates for biosynthetic processes. How cells reshape their metabolism is not fully understood. Here we report that loss of cathepsin L (Cts L) is associated with a fast proliferation rate and enhanced glycolytic metabolism that depend on lactate dehydrogenase A (LDHA) activity. Using mass spectrometry analysis of cells treated with a pan cathepsin inhibitor, we observed an increased abundance of proteins involved in central carbon metabolism. Further inspection of putative Cts L targets revealed an enrichment for glycolytic metabolism that was independently confirmed by metabolomic and biochemical analyses. Moreover, proteomic analysis of Cts L-knockout cells identified LDHA overexpression that was demonstrated to be a key metabolic junction in these cells. Lastly, we show that Cts L inhibition led to increased LDHA protein expression, suggesting a causal relationship between LDHA expression and function. In conclusion, we propose that Cts L regulates this metabolic circuit to keep cell division under control, suggesting the therapeutic potential of targeting this protein and its networks in cancer.
Assuntos
Catepsina L/metabolismo , Redes e Vias Metabólicas , Animais , Proliferação de Células , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Deleção de Genes , Glicólise , Células HeLa , Humanos , Lactato Desidrogenase 5/genética , Lactato Desidrogenase 5/metabolismo , Lipogênese , Espectrometria de Massas , Metabolômica , Camundongos , Células NIH 3T3 , Fenótipo , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
AMPK-mTORC1 signaling senses nutrient availability, thereby regulating autophagy. Surprisingly, we found that, in ß-cells, the AMPK activator 5-amino-4-imidazolecarboxamide ribofuranoside (AICAR) inhibited, rather than stimulated, autophagy. AICAR is an intermediate in the generation of inosine monophosphate, with subsequent conversion to other purine nucleotides. Adenosine regulated autophagy in a concentration-dependent manner: at high concentrations, it mimicked the AICAR effect on autophagy, whereas at low concentrations it stimulated autophagy through its cognate A1 receptor. Adenosine regulation of autophagy was independent of AMPK or mTORC1 activity. Adenosine kinase (ADK) is the principal enzyme for metabolic adenosine clearance. ADK knockdown and pharmacological inhibition of the enzyme markedly stimulated autophagy in an adenosine A1 receptor-dependent manner. High-concentration adenosine increased insulin secretion in a manner sensitive to treatment with the autophagy inducer Tat-beclin1, and inhibition of autophagy augmented secretion. In conclusion, high concentrations of AICAR or adenosine inhibit autophagy, whereas physiological concentrations of adenosine or inhibition of adenosine clearance by ADK stimulate autophagy via the adenosine receptor. Adenosine might thus be an autocrine regulator of autophagy, independent of AMPK-mTORC1 signaling. Adenosine regulates insulin secretion, in part, through modulation of autophagy.
Assuntos
Nucleotídeos de Adenina/metabolismo , Autofagia/fisiologia , Células Secretoras de Insulina/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina , Animais , Western Blotting , Linhagem Celular , Imunofluorescência , Células Hep G2 , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Schizophrenia is a mental disease that results in decreased life expectancy and well-being by promoting obesity and sedentary lifestyles. Schizophrenia is treated by antipsychotic drugs. Although the second-generation antipsychotics (SGA), Olanzapine and Aripiprazole, are more effective in treating schizophrenia, they display a higher risk of metabolic side effects, mostly by development of diabetes and insulin resistance, weight gain, and dyslipidemia. Endoplasmic reticulum (ER) stress is induced when ER homeostasis of lipid biosynthesis and protein folding is impaired. This leads to the activation of the unfolded protein response (UPR), a signaling cascade that aims to restore ER homeostasis or initiate cell death. Chronic conditions of ER stress in the liver are associated with diabetes and perturbed lipid metabolism. These metabolic dysfunctions resemble the pharmacological side effects of SGAs. We therefore investigated whether SGAs promote the UPR in human and mouse hepatocytes. We observed full-fledged activation of ER stress by Aripiprazole not by Olanzapine. This occurred at low micromolar concentrations and to variable intensities in different cell types, such as hepatocellular carcinoma, melanoma, and glioblastoma. Mechanistically, Aripiprazole caused depletion of ER calcium, leading to activation of inositol-requiring enzyme 1 (IRE1)and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), two major transducers of the UPR. Cells underwent apoptosis with Aripiprazole treatment, which coincided with UPR induction, and this effect was reduced by adding glutathione without affecting UPR itself. Deletion of IRE1 from HepG2, a human liver cancer cell line, protected cells from Aripiprazole toxicity. Our study reveals for the first time a cytotoxic effect of Aripiprazole that involves the induction of ER stress. SIGNIFICANCE STATEMENT: The antischizophrenic drug Aripiprazole exerts cytotoxic properties at high concentrations. This study shows that this cytotoxicity is associated with the induction of endoplasmic reticulum (ER) stress and IRE1 activation, mechanisms involved in diet-induced obesity. Aripiprazole induced ER stress and calcium mobilization from the ER in human and mouse hepatocytes. Our study highlights a new mechanism of Aripiprazole that is not related to its effect on dopamine signaling.
RESUMO
The unfolded protein response (UPR) is an adaptive signaling pathway activated in response to endoplasmic reticulum (ER) stress. The effectors of the UPR are potent transcription activators; however, some genes are suppressed by ER stress at the mRNA level. The mechanisms underlying UPR-mediated gene suppression are less known. Exploration of the effect of UPR on NK cells ligand expression found that the transcription of NK group 2 member D (NKG2D) ligand major histocompatibility complex class I polypeptide-related sequence A/B (MICA/B) is suppressed by the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) pathway of the UPR. Deletion of IRE1 or XBP1 was sufficient to promote mRNA and surface levels of MICA. Accordingly, NKG2D played a greater role in the killing of IRE1/XBP1 knockout target cells. Analysis of effectors downstream to XBP1s identified E2F transcription factor 1 (E2F1) as linking UPR and MICA transcription. The inverse correlation between XBP1 and E2F1 or MICA expression was corroborated in RNA-Seq analysis of 470 primary melanoma tumors. While mechanisms that connect XBP1 to E2F1 are not fully understood, we implicate a few microRNA molecules that are modulated by ER stress and possess dual suppression of E2F1 and MICA. Because of the importance of E2F1 and MICA in cancer progression and recognition, these observations could be exploited for cancer therapy by manipulating the UPR in tumor cells.-Obiedat, A., Seidel, E., Mahameed, M., Berhani, O., Tsukerman, P., Voutetakis, K., Chatziioannou, A., McMahon, M., Avril, T., Chevet, E., Mandelboim, O., Tirosh, B. Transcription of the NKG2D ligand MICA is suppressed by the IRE1/XBP1 pathway of the unfolded protein response through the regulation of E2F1.
Assuntos
Fator de Transcrição E2F1/genética , Endorribonucleases/genética , Antígenos de Histocompatibilidade Classe I/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Proteínas Serina-Treonina Quinases/genética , Resposta a Proteínas não Dobradas/genética , Proteína 1 de Ligação a X-Box/genética , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/genética , Humanos , Ligantes , RNA Mensageiro/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
Poorly structured domains in proteins enhance their susceptibility to proteasomal degradation. To learn whether the presence of such a domain near either end of a protein determines its direction of entry into the proteasome, directional translocation was enforced on several proteasome substrates. Using archaeal PAN-20S complexes, mammalian 26S proteasomes, and cultured cells, we identified proteins that are degraded exclusively from either the C or N terminus and some showing no directional preference. This property results from interactions of the substrate's termini with the regulatory ATPase and could be predicted based on the calculated relative stabilities of the N and C termini. Surprisingly, the direction of entry into the proteasome affected markedly the spectrum of peptides released and consequently influenced the efficiency of MHC class I presentation. Thus, easily unfolded termini are translocated first, and the direction of translocation influences the peptides generated and presented to the immune system.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Desdobramento de Proteína , Proteínas/química , Proteínas/metabolismo , Animais , Calmodulina/química , Calmodulina/imunologia , Calmodulina/metabolismo , Caseínas/química , Caseínas/imunologia , Caseínas/metabolismo , Linhagem Celular Tumoral , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/imunologia , Proteínas Ligantes de Maltose/metabolismo , Camundongos , Ovalbumina/química , Ovalbumina/imunologia , Ovalbumina/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Transporte Proteico , Proteínas/imunologiaRESUMO
Recombinant glycoproteins produced in mammalian cells are clinically indispensable drugs used to treat a broad spectrum of diseases. Their bio-manufacturing process is laborious, time consuming, and expensive. Investment in expediting the process and reducing its cost is the subject of continued research. The PI3K/Akt/mTOR signaling pathway is a key regulator of diverse physiological functions such as proliferation, global protein, and lipid synthesis as well as many metabolic pathways interacting to increase secretory capabilities. In this review we detail various strategies previously employed to increase glycoprotein production yields via either genetic or pharmacological over-activation of the PI3K/Akt/mTOR pathway, and we discuss their potential and limitations.Key words: mTORC1, CRISPR, specific productivity, translation.
Assuntos
Glicoproteínas/biossíntese , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Humanos , Proteínas Recombinantes/biossínteseRESUMO
Protein translation has emerged as a critical bottleneck for overall productivity of biological molecules. An augmentation of protein translation can be achieved by cell line engineering or by sophisticated vector design. However, for industrial process development purposes, identification of media additives that promote translation will be of great value, obviating the generation of new host platforms. Here, we examined the effect of low cadmium chloride concentrations on protein synthesis and cell line productivity. At low micromolar concentrations, cadmium chloride induced the mTOR pathway and promoted total protein synthesis in HEK 293T and CHO-K1 cells with minimal toxicity. In a parallel screening of kinase inhibitors for promoting protein expression, we identified the RSK1 inhibitor, BI-D1870, as having a transcription promoting activity on cytomegalovirus promoter-driven transgenes. Fed-batch analyses of CHO-K1 cells producing the anticoagulant factor tissue plasminogen activator (tPA) demonstrated that inclusion of cadmium chloride alone and particularly in combination with BI-D1870 improved overall yields of tPA by more than two-fold with minimal effect on cell growth. We, therefore, underscore the use of cadmium alone and in combination with BI-D1870 for improving bioproduction yields.
Assuntos
Cloreto de Cádmio/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Recombinantes , Animais , Células CHO , Cloreto de Cádmio/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Células HEK293 , Humanos , Pteridinas/farmacologia , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Ativador de Plasminogênio Tecidual/análise , Ativador de Plasminogênio Tecidual/genética , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell metabolism and lymphocyte proliferation. It is inhibited by the tuberous sclerosis complex (TSC), a heterodimer of TSC1 and TSC2. Deletion of either gene results in robust activation of mTORC1. Mature B cells reside in the spleen at two major anatomical locations, the marginal zone (MZ) and follicles. The MZ constitutes the first line of humoral response against blood-borne pathogens and undergoes atrophy in chronic inflammation. In previous work, we showed that mice deleted for TSC1 in their B cells (TSC1BKO ) have almost no MZ B cells, whereas follicular B cells are minimally affected. To explore potential underlying mechanisms for MZ B-cell loss, we have analysed the spleen MZ architecture of TSC1BKO mice and found it to be severely impaired. Examination of lymphotoxins (LTα and LTß) and lymphotoxin receptor (LTßR) expression indicated that LTßR levels in spleen stroma were reduced by TSC1 deletion in the B cells. Furthermore, LTα transcripts in B cells were reduced. Because LTßR is sensitive to proteolysis, we analysed cathepsin activity in TSC1BKO . A higher cathepsin activity, particularly of cathepsin B, was observed, which was reduced by mTORC1 inhibition with rapamycin in vivo. Remarkably, in vivo administration of a pan-cathepsin inhibitor restored LTßR expression, LTα mRNA levels and the MZ architecture. Our data identify a novel connection, although not elucidated at the molecular level, between mTORC1 and cathepsin activity in a manner relevant to MZ dynamics.
Assuntos
Linfócitos B/imunologia , Catepsinas/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Baço/imunologia , Animais , Células CHO , Catepsinas/antagonistas & inibidores , Linhagem Celular , Cricetulus , Receptor beta de Linfotoxina/biossíntese , Linfotoxina-alfa/biossíntese , Linfotoxina-beta/biossíntese , Camundongos , Camundongos Transgênicos , Sirolimo/farmacologia , Baço/citologia , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/genéticaRESUMO
An optimized biomanufacturing process in mammalian cells is contingent on the ability of the producing cells to reach high viable cell densities. In addition, at the peak of growth, cells need to continue producing the biological entity at a consistent quality. Thus, engineering cells with robust growth performance and resilience to variable stress conditions is highly desirable. The tyrosine kinase receptor, KIT, plays a key role in cell differentiation and the survival of several immune cell types. Its oncogenic mutant, D816V, endows cells with high proliferation capacity, and resistance to kinase inhibitors. Importantly, this onco-KIT mutant when introduced into various cell types is arrested in the endoplasmic reticulum in a constitutively active form. Here, we investigated the effect of oncogenic D816V KIT on the performance of CHO-K1 cells under conventional tissue culture growth settings and when adapted, to shaking conditions. The onco-KIT promoted global protein synthesis, elevated the expression of a secretable transgene, enhanced proliferation, and improved the overall titers of a model glycoprotein. Moreover, the expression of the onco-KIT endowed the cells with a remarkable resistance to various stress conditions. Our data suggest that the introduction of onco-KIT can serve as a strategy for improving glycoprotein biomanufacturing. Biotechnol. Bioeng. 2017;114: 2560-2570. © 2017 Wiley Periodicals, Inc.
Assuntos
Células CHO/citologia , Células CHO/fisiologia , Proliferação de Células/fisiologia , Melhoramento Genético/métodos , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Recombinantes/biossíntese , Estresse Fisiológico/fisiologia , Animais , Técnicas de Cultura Celular por Lotes/métodos , CricetulusRESUMO
Antibody secretion is executed by plasma cells that are generated in the periphery and migrate to the bone marrow to establish a long lived pool. The terminal differentiation of B lymphocytes into plasma cells is executed by a network of transcription factors that cross-regulate each other in order to irreversibly promote this transition. While major progress has been made in the understanding the transcriptional activity of the underlying master regulators, much less is known on the metabolic regulation of plasma cell differentiation that is required to support antibody synthesis, folding and secretion at high levels and allow their long-lasting survival. In this review we will address the known cross talks between the transcription and metabolic control of plasma cells and elaborate on the gaps of knowledge in the field.
Assuntos
Diferenciação Celular , Metabolismo Energético , Plasmócitos/citologia , Plasmócitos/metabolismo , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Plasmócitos/imunologia , Transdução de Sinais , Estresse Fisiológico/genética , Estresse Fisiológico/imunologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
The kinase mTOR operates in two cellular complexes, mTORC1 and mTORC2. mTORC1 adjusts metabolic activity according to external growth conditions and nutrients availability. When conditions are prosperous, mTOR facilitates protein and lipid biosyntheses and inhibits autophagy, while under metabolic constraints, however, its attenuation induces a catabolic program, energy preservation and autophagy. CHO is a key cell line for manufacturing of biologics owing to its remarkable ability to grow to high densities and maintain protein production and secretion for extended times. While high mTOR activity has been associated with high productivity in CHO cells, its inhibition by rapamycin has also been documented to augment productivity via promotion of viability. Here using CRISPR/Cas9 editing we engineered CHO cells to enforce high mTORC1 activity by knocking-out TSC2, a major mTOR inhibitory protein, or PTEN, a phosphatase that attenuates the PI3K/AKT/mTOR pathway. Only TSC2-deleted cells exhibited a constitutive activation of mTORC1 under fed batch conditions. Cells grew larger in size, synthesized more proteins and displayed an over twofold elevation in their specific productivity. While peak viable cell density was compromised, overall titers increased to an extent dependent upon the parental clone. Our data underscore manipulation of TSC as a strategy to improve performance of CHO cell in bioreactors. Biotechnol. Bioeng. 2016;113: 1942-1952. © 2016 Wiley Periodicals, Inc.
Assuntos
Anticorpos/metabolismo , Reatores Biológicos , Técnicas de Inativação de Genes/métodos , Engenharia Metabólica/métodos , Proteínas Supressoras de Tumor/genética , Animais , Anticorpos/análise , Anticorpos/química , Anticorpos/genética , Células CHO , Sistemas CRISPR-Cas/genética , Cricetinae , Cricetulus , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , PTEN Fosfo-Hidrolase/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose TuberosaRESUMO
Inositol-requiring enzyme 1 (IRE1) is a kinase and ribonuclease that executes the splicing of X box binding protein 1 (XBP-1) mRNA in response to the accumulation of unfolded protein in the ER, a signal cascade termed the unfolded protein response. Recently, IRE1 has been implicated in mRNA and miRNA cleavage and degradation, a pathway termed regulated IRE1-dependent decay (RIDD). Deletion of XBP-1 in the liver and pancreas strongly enhances RIDD by upregulating IRE1 protein levels and enhancing its ribo-nuclease activity. Because XBP-1 is essential for generating plasma cells with developed secretory capacity, we sought to evaluate the contribution of RIDD to this regulation. Mice were conditionally deleted for XBP-1 and/or IRE1 in their B-cell lineage. Similarly to the liver, deletion of XBP-1 induces IRE1 expression in LPS-treated B cells. In vitro, IRE1 cleaves the mRNA of secretory µ chains, which explains the reduction in secretory µ mRNA and its synthesis in XBP-1 KO plasma cells. In accordance, the IgM response is partially restored in XBP-1/IRE1 double KO mice relative to XBP-1 KO mice. Interestingly, the IgG1 response is reduced to a similar level in XBP-1 KO, IRE1 KO, and their double knockout animals. Our data demonstrate a specific contribution by RIDD in curtailing immunoglobulin synthesis and secretion.
Assuntos
Formação de Anticorpos/fisiologia , Imunoglobulinas/biossíntese , Proteínas de Membrana/metabolismo , Plasmócitos/imunologia , Plasmócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Plasmócitos/ultraestrutura , Proteínas Serina-Treonina Quinases/genética , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição de Fator Regulador X , Linfócitos T/imunologia , Fatores de Transcrição/metabolismo , Proteína 1 de Ligação a X-BoxRESUMO
During viral infection, a massive demand for viral glycoproteins can overwhelm the capacity of the protein folding and quality control machinery, leading to an accumulation of unfolded proteins in the endoplasmic reticulum (ER). To restore ER homeostasis, cells initiate the unfolded protein response (UPR) by activating three ER-to-nucleus signaling pathways, of which the inositol-requiring enzyme 1 (IRE1)-dependent pathway is the most conserved. To reduce ER stress, the UPR decreases protein synthesis, increases degradation of unfolded proteins, and upregulates chaperone expression to enhance protein folding. Cytomegaloviruses, as other viral pathogens, modulate the UPR to their own advantage. However, the molecular mechanisms and the viral proteins responsible for UPR modulation remained to be identified. In this study, we investigated the modulation of IRE1 signaling by murine cytomegalovirus (MCMV) and found that IRE1-mediated mRNA splicing and expression of the X-box binding protein 1 (XBP1) is repressed in infected cells. By affinity purification, we identified the viral M50 protein as an IRE1-interacting protein. M50 expression in transfected or MCMV-infected cells induced a substantial downregulation of IRE1 protein levels. The N-terminal conserved region of M50 was found to be required for interaction with and downregulation of IRE1. Moreover, UL50, the human cytomegalovirus (HCMV) homolog of M50, affected IRE1 in the same way. Thus we concluded that IRE1 downregulation represents a previously undescribed viral strategy to curb the UPR.
Assuntos
Infecções por Citomegalovirus/metabolismo , Citomegalovirus/metabolismo , Endorribonucleases/biossíntese , Proteínas de Membrana/biossíntese , Muromegalovirus/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Resposta a Proteínas não Dobradas , Animais , Linhagem Celular Transformada , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/genética , Endorribonucleases/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Muromegalovirus/genética , Células NIH 3T3 , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Proteína 1 de Ligação a X-BoxRESUMO
Protein elimination by the ubiquitin-proteasome system requires the presence of a cis-acting degradation signal. Efforts to discern degradation signals of misfolded proteasome substrates thus far revealed a general mechanism whereby the exposure of cryptic hydrophobic motifs provides a degradation determinant. We have previously characterized such a determinant, employing the yeast kinetochore protein Ndc10 as a model substrate. Ndc10 is essentially a stable protein that is rapidly degraded upon exposure of a hydrophobic motif located at the C-terminal region. The degradation motif comprises two distinct and essential elements: DegA, encompassing two amphipathic helices, and DegB, a hydrophobic sequence within the loosely structured C-terminal tail of Ndc10. Here we show that the hydrophobic nature of DegB is irrelevant for the ubiquitylation of substrates containing the Ndc10 degradation motif, but is essential for proteasomal degradation. Mutant DegB, in which the hydrophobic sequence was disrupted, acted as a dominant degradation inhibitory element when expressed at the C-terminal regions of ubiquitin-dependent and -independent substrates of the 26S proteasome. This mutant stabilized substrates in both yeast and mammalian cells, indicative of a modular recognition moiety. The dominant function of the mutant DegB provides a powerful experimental tool for evaluating the physiological implications of stabilization of specific proteasome substrates in intact cells and for studying the associated pathological effects.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Cinetocoros/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitinação/fisiologia , Motivos de Aminoácidos , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Very long chain (C22-C24) ceramides are synthesized by ceramide synthase 2 (CerS2). A CerS2 null mouse displays hepatopathy because of depletion of C22-C24 ceramides, elevation of C16-ceramide, and/or elevation of sphinganine. Unexpectedly, CerS2 null mice were resistant to acetaminophen-induced hepatotoxicity. Although there were a number of biochemical changes in the liver, such as increased levels of glutathione and multiple drug-resistant protein 4, these effects are unlikely to account for the lack of acetaminophen toxicity. A number of other hepatotoxic agents, such as d-galactosamine, CCl4, and thioacetamide, were also ineffective in inducing liver damage. All of these drugs and chemicals require connexin (Cx) 32, a key gap junction protein, to induce hepatotoxicity. Cx32 was mislocalized to an intracellular location in hepatocytes from CerS2 null mice, which resulted in accelerated rates of its lysosomal degradation. This mislocalization resulted from the altered membrane properties of the CerS2 null mice, which was exemplified by the disruption of detergent-resistant membranes. The lack of acetaminophen toxicity and Cx32 mislocalization were reversed upon infection with recombinant adeno-associated virus expressing CerS2. We establish that Gap junction function is compromised upon altering the sphingolipid acyl chain length composition, which is of relevance for understanding the regulation of drug-induced liver injury.
Assuntos
Ceramidas/biossíntese , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Hepatócitos/metabolismo , Microdomínios da Membrana/metabolismo , Esfingosina N-Aciltransferase/metabolismo , Acetaminofen/efeitos adversos , Acetaminofen/farmacologia , Analgésicos não Narcóticos/efeitos adversos , Analgésicos não Narcóticos/farmacologia , Animais , Tetracloreto de Carbono/toxicidade , Intoxicação por Tetracloreto de Carbono/genética , Intoxicação por Tetracloreto de Carbono/metabolismo , Intoxicação por Tetracloreto de Carbono/patologia , Ceramidas/genética , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Conexinas/genética , Galactosamina/toxicidade , Junções Comunicantes/genética , Junções Comunicantes/patologia , Glutationa/genética , Glutationa/metabolismo , Hepatócitos/patologia , Microdomínios da Membrana/genética , Microdomínios da Membrana/patologia , Camundongos , Camundongos Mutantes , Esfingosina N-Aciltransferase/genética , Proteína beta-1 de Junções ComunicantesRESUMO
New agents are needed that selectively kill cancer cells without harming normal tissues. The TRAIL ligand and its receptors, DR5 and DR4, exhibit cancer-selective toxicity, but TRAIL analogs or agonistic antibodies targeting these receptors have not received FDA approval for cancer therapy. Small molecules for activating DR5 or DR4 independently of protein ligands may bypass some of the pharmacological limitations of these protein drugs. Previously described Disulfide bond Disrupting Agents (DDAs) activate DR5 by altering its disulfide bonding through inhibition of the Protein Disulfide Isomerases (PDIs) ERp44, AGR2, and PDIA1. Work presented here extends these findings by showing that disruption of single DR5 disulfide bonds causes high-level DR5 expression, disulfide-mediated clustering, and activation of Caspase 8-Caspase 3 mediated pro-apoptotic signaling. Recognition of the extracellular domain of DR5 by various antibodies is strongly influenced by the pattern of DR5 disulfide bonding, which has important implications for the use of agonistic DR5 antibodies for cancer therapy. Disulfide-defective DR5 mutants do not activate the ER stress response or stimulate autophagy, indicating that these DDA-mediated responses are separable from DR5 activation and pro-apoptotic signaling. Importantly, other ER stressors, including Thapsigargin and Tunicamycin also alter DR5 disulfide bonding in various cancer cell lines and in some instances, DR5 mis-disulfide bonding is potentiated by overriding the Integrated Stress Response (ISR) with inhibitors of the PERK kinase or the ISR inhibitor ISRIB. These observations indicate that the pattern of DR5 disulfide bonding functions as a sensor of ER stress and serves as an effector of proteotoxic stress by driving extrinsic apoptosis independently of extracellular ligands.