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BACKGROUND AND AIMS: Antimicrobial proteins of the regenerating family member 3 alpha (REG3A) family provide a first line of protection against infections and transformed cells. Their expression is inducible by inflammation, which makes their role in cancer biology less clear since an immune-inflammatory context may preexist or coexist with cancer, as occurs in HCC. The aim of this study is to clarify the role of REG3A in liver carcinogenesis and to determine whether its carbohydrate-binding functions are involved. APPROACH AND RESULTS: This study provides evidence for a suppressive role of REG3A in HCC by reducing O -GlcNAcylation in 2 mouse models of HCC, in vitro cell studies, and clinical samples. REG3A expression in hepatocytes significantly reduced global O -GlcNAcylation and O -GlcNAcylation of c-MYC in preneoplastic and tumor livers and markedly inhibited HCC development in REG3A-c-MYC double transgenic mice and mice exposed to diethylnitrosamine. REG3A modified O -GlcNAcylation without altering the expression or activity of O-linked N-acetylglucosaminyltransferase, O-linked N-acetylglucosaminyl hydrolase, or glutamine fructose-6-phosphate amidotransferase. Reduced O -GlcNAcylation was consistent with decreased levels of UDP-GlcNAc in precancerous and cancerous livers. This effect was linked to the ability of REG3A to bind glucose and glucose-6 phosphate, suggested by a REG3A mutant unable to bind glucose and glucose-6 phosphate and alter O -GlcNAcylation. Importantly, patients with cirrhosis with high hepatic REG3A expression had lower levels of O -GlcNAcylation and longer cancer-free survival than REG3A-negative cirrhotic livers. CONCLUSIONS: REG3A helps fight liver cancer by reducing O -GlcNAcylation. This study suggests a new paradigm for the regulation of O -GlcNAc signaling in cancer-related pathways through interactions with the carbohydrate-binding function of REG3A.
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O-GlcNAcylation corresponds to the addition of N-Acetylglucosamine (GlcNAc) on serine or threonine residues of cytosolic, nuclear and mitochondrial proteins. This reversible modification is catalysed by a unique couple of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). OGT uses UDP-GlcNAc produced in the hexosamine biosynthesis pathway, to modify proteins. UDP-GlcNAc is at the cross-roads of several cellular metabolisms, including glucose, amino acids and fatty acids. Therefore, OGT is considered as a metabolic sensor that post-translationally modifies proteins according to nutrient availability. O-GlcNAcylation can modulate protein-protein interactions and regulate protein enzymatic activities, stability or subcellular localization. In addition, it can compete with phosphorylation on the same serine or threonine residues, or regulate positively or negatively the phosphorylation of adjacent residues. As such, O-GlcNAcylation is a major actor in the regulation of cell signaling and has been implicated in numerous physiological and pathological processes. A large body of evidence have indicated that increased O-GlcNAcylation participates in the deleterious effects of glucose (glucotoxicity) in metabolic diseases. However, recent studies using mice models with OGT or OGA knock-out in different tissues have shown that O-GlcNAcylation protects against various cellular stresses, and indicate that both increase and decrease in O-GlcNAcylation have deleterious effects on the regulation of energy homeostasis.
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Acetilglucosamina , N-Acetilglucosaminiltransferasas , Acetilglucosamina/metabolismo , Animales , Glucosa , Homeostasis , Ratones , Ratones Noqueados , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , Proteínas , Serina , Treonina , Uridina DifosfatoRESUMEN
Glycosylation with O-linked ß-N-acetylglucosamine (O-GlcNAcylation) is a reversible posttranslational modification that regulates the activity of intracellular proteins according to glucose availability and its metabolism through the hexosamine biosynthesis pathway. This modification has been involved in the regulation of various immune cell types, including macrophages. However, little is known concerning the mechanisms that regulate the protein O-GlcNAcylation level in these cells. In the present work, we demonstrate that LPS treatment induces a marked increase in protein O-GlcNAcylation in RAW264.7 cells, bone marrow-derived and peritoneal mouse macrophages, as well as human monocyte-derived macrophages. Targeted deletion of OGT in macrophages resulted in an increased effect of LPS on NOS2 expression and cytokine production, suggesting that O-GlcNAcylation may restrain inflammatory processes induced by LPS. The effect of LPS on protein O-GlcNAcylation in macrophages was associated with an increased expression and activity of glutamine fructose 6-phosphate amidotransferase (GFAT), the enzyme that catalyzes the rate-limiting step of the hexosamine biosynthesis pathway. More specifically, we observed that LPS potently stimulated GFAT2 isoform mRNA and protein expression. Genetic or pharmacological inhibition of FoxO1 impaired the LPS effect on GFAT2 expression, suggesting a FoxO1-dependent mechanism. We conclude that GFAT2 should be considered a new LPS-inducible gene involved in regulation of protein O-GlcNAcylation, which permits limited exacerbation of inflammation upon macrophage activation.
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Acetilglucosamina/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Animales , Vías Biosintéticas/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Glicosilación/efectos de los fármacos , Humanos , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Células RAW 264.7RESUMEN
The viral Tax oncoprotein plays a key role in both Human T-cell lymphotropic virus type 1 (HTLV-1)-replication and HTLV-1-associated pathologies, notably adult T-cell leukemia. Tax governs the transcription from the viral 5'LTR, enhancing thereby its own expression, via the recruitment of dimers of phosphorylated CREB to cAMP-response elements located within the U3 region (vCRE). In addition to phosphorylation, CREB is also the target of O-GlcNAcylation, another reversible post-translational modification involved in a wide range of diseases, including cancers. O-GlcNAcylation consists in the addition of O-linked-N-acetylglucosamine (O-GlcNAc) on Serine or Threonine residues, a process controlled by two enzymes: O-GlcNAc transferase (OGT), which transfers O-GlcNAc on proteins, and O-GlcNAcase (OGA), which removes it. In this study, we investigated the status of O-GlcNAcylation enzymes in HTLV-1-transformed T cells. We found that OGA mRNA and protein expression levels are increased in HTLV-1-transformed T cells as compared to control T cell lines while OGT expression is unchanged. However, higher OGA production coincides with a reduction in OGA specific activity, showing that HTLV-1-transformed T cells produce high level of a less active form of OGA. Introducing Tax into HEK-293T cells or Tax-negative HTLV-1-transformed TL-om1 T cells is sufficient to inhibit OGA activity and increase total O-GlcNAcylation, without any change in OGT activity. Furthermore, Tax interacts with the OGT/OGA complex and inhibits the activity of OGT-bound OGA. Pharmacological inhibition of OGA increases CREB O-GlcNAcylation as well as HTLV-1-LTR transactivation by Tax and CREB recruitment to the LTR. Moreover, overexpression of wild-type CREB but not a CREB protein mutated on a previously described O-GlcNAcylation site enhances Tax-mediated LTR transactivation. Finally, both OGT and OGA are recruited to the LTR. These findings reveal the interplay between Tax and the O-GlcNAcylation pathway and identify new key molecular actors involved in the assembly of the Tax-dependent transactivation complex.
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Productos del Gen tax/metabolismo , Infecciones por HTLV-I/virología , Virus Linfotrópico T Tipo 1 Humano/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Linfocitos T/virología , beta-N-Acetilhexosaminidasas/metabolismo , Acetilglucosamina/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación Viral de la Expresión Génica , Productos del Gen tax/genética , Infecciones por HTLV-I/enzimología , Infecciones por HTLV-I/genética , Infecciones por HTLV-I/metabolismo , Interacciones Huésped-Patógeno , Virus Linfotrópico T Tipo 1 Humano/genética , Humanos , N-Acetilglucosaminiltransferasas/genética , Procesamiento Proteico-Postraduccional , Linfocitos T/enzimología , Linfocitos T/metabolismo , Transcripción Genética , beta-N-Acetilhexosaminidasas/genéticaRESUMEN
O-GlcNAcylation is a reversible post-translational modification that regulates cytosolic and nuclear proteins. We and others previously demonstrated that FoxO1 is O-GlcNAcylated in different cell types, resulting in an increase in its transcriptional activity. Four O-GlcNAcylation sites were identified in human FOXO1 but directed mutagenesis of each site individually had modest (T317) or no effect (S550, T648, S654) on its O-GlcNAcylation status and transcriptional activity. Moreover, the consequences of mutating all four sites had not been investigated. In the present work, we mutated these sites in the mouse Foxo1 and found that mutation of all four sites did not decrease Foxo1 O-GlcNAcylation status and transcriptional activity, and would even tend to increase them. In an attempt to identify other O-GlcNAcylation sites, we immunoprecipitated wild-type O-GlcNAcylated Foxo1 and analysed the tryptic digest peptides by mass spectrometry using High-energy Collisional Dissociation. We identified T646 as a new O-GlcNAcylation site on Foxo1. However, site directed mutagenesis of this site individually or together with all four previously identified residues did not impair Foxo1 O-GlcNAcylation and transcriptional activity. These results suggest that residues important for the control of Foxo1 activity by O-GlcNAcylation still remain to be identified.
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Factores de Transcripción Forkhead/química , Acetilglucosamina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Glicosilación , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Espectrometría de Masas en TándemRESUMEN
O-GlcNAcylation on serine/threonine is a post-translational modification that controls the activity of nucleocytoplasmic proteins according to glucose availability. We previously showed that O-GlcNAcylation of FoxO1 in liver cells increases its transcriptional activity. In the present study, we evaluated the potential involvement of FoxO1 O-GlcNAcylation in the context of pancreatic ß-cell glucotoxicity. FoxO1 was O-GlcNAcylated in INS-1 832/13 ß cells and isolated rat pancreatic islets. O-GlcNAcylation of FoxO1 resulted in a 2-fold increase in its transcriptional activity toward a FoxO1 reporter gene and a 3-fold increase in the expression of the insulin-like growth factor-binding protein 1 (Igfbp1) gene at the mRNA level, resulting in IGFBP1 protein oversecretion by the cells. Of note, increased IGFBP1 in the culture medium inhibited the activity of the insulin-like growth factor 1 receptor (IGF1R)/phosphatidyl inositol 3 kinase (PI3K)/Akt pathway. We reveal in this report a novel mechanism by which O-GlcNAcylation inhibits Akt activity through an autocrine mechanism. However, although inhibition of IGFBP1 expression using siRNA restored the PI3 kinase/Akt pathway, it did not rescue INS-1 832/13 cells from high-glucose- or O-glcNAcylation-induced cell death. In contrast, FoxO1 down-regulation by siRNA led to 30 to 60% protection of INS-1 832/13 cells from death mediated by glucotoxic conditions. Therefore, whereas FoxO1 O-GlcNAcylation inhibits Akt through an IGFBP1-mediated autocrine pathway, the deleterious effects of FoxO1 O-GlcNAcylation on cell survival appeared to be independent of this pathway.
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Factores de Transcripción Forkhead/metabolismo , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Apoptosis/genética , Glucosa/farmacología , Inmunoprecipitación , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , RatasRESUMEN
ß-Arrestins are multifunctional proteins that play central roles in G protein-coupled receptor (GPCR) trafficking and signaling. ß-Arrestin1 is also recruited to the insulin-like growth factor-1 receptor (IGF-1R), a receptor tyrosine kinase (RTK), mediating receptor degradation and signaling. Because GPCR phosphorylation by GPCR-kinases (GRKs) governs interactions of the receptors with ß-arrestins, we investigated the regulatory roles of the four widely expressed GRKs on IGF-1R signaling/degradation. By suppressing GRK expression with siRNA, we demonstrated that lowering GRK5/6 abolishes IGF1-mediated ERK and AKT activation, whereas GRK2 inhibition increases ERK activation and partially inhibits AKT signaling. Conversely, ß-arrestin-mediated ERK signaling is enhanced by overexpression of GRK6 and diminished by GRK2. Similarly, we demonstrated opposing effects of GRK2 and -6 on IGF-1R degradation: GRK2 decreases whereas GRK6 enhances ligand-induced degradation. GRK2 and GRK6 coimmunoprecipitate with IGF-1R and increase IGF-1R serine phosphorylation, promoting ß-arrestin1 association. Using immunoprecipitation, confocal microscopy, and FRET analysis, we demonstrated ß-arrestin/IGF-1R association to be transient for GRK2 and stable for GRK6. Using bioinformatic studies we identified serines 1248 and 1291 as the major serine phosphorylation sites of the IGF-1R, and subsequent mutation analysis demonstrated clear effects on IGF-1R signaling and degradation, mirroring alterations by GRKs. Targeted mutation of S1248 recapitulates GRK2 modulation, whereas S1291 mutation resembles GRK6 effects on IGF-1R signaling/degradation, consistent with GRK isoform-specific serine phosphorylation. This study demonstrates distinct roles for GRK isoforms in IGF-1R signaling through ß-arrestin binding with divergent functional outcomes.
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Quinasas de Receptores Acoplados a Proteína-G/metabolismo , Receptor IGF Tipo 1/metabolismo , Secuencia de Aminoácidos , Animales , Arrestinas/metabolismo , Secuencia de Bases , Línea Celular , Transferencia Resonante de Energía de Fluorescencia , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Quinasas de Receptores Acoplados a Proteína-G/antagonistas & inhibidores , Quinasas de Receptores Acoplados a Proteína-G/genética , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , ARN Interferente Pequeño/genética , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/genética , Serina/química , Transducción de Señal , Especificidad por Sustrato , beta-ArrestinasRESUMEN
The debate between reductionism and anti-reductionism, dealing with the ultimate constituents of the world, is one of the fundamental issues in the philosophy of science. However, in biology, reductionism is less of an ontological and more of an epistemological question: it argues that the explanation of biological processes resides in deciphering the genetic code of living entities. This position is still prevalent in cancer biology, which has long been defined as a cellular process where genetic alterations are responsible for aberrant proliferation. While the hypothesis of somatic mutations remains the central theoretical model, a bundle of experimental data reveals how important the disturbances of tissue organisation are in cancer development, leading to a renewal of holistic and organicist approaches. This latter perspective in particular attempts to contextualise and rethink the centrality of the genetic level by proposing a new conception of cancerogenesis as a tissue disease.
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Investigación Biomédica , Modelos Teóricos , Neoplasias , Animales , Investigación Biomédica/métodos , Investigación Biomédica/tendencias , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Microambiente Celular , Femenino , Humanos , Ratones , Neoplasias/etiología , Neoplasias/patología , Neoplasias/terapia , Oncogenes/fisiología , Filosofía , Probabilidad , RiesgoRESUMEN
Type 2 diabetes is characterised by the disruption of insulin and insulin-like growth factor (IGF) signalling. The key hubs of these signalling cascades - the Insulin receptor (IR) and Insulin-like growth factor 1 receptor (IGF1R) - are known to form functional IR-IGF1R hybrid receptors which are insulin resistant. However, the mechanisms underpinning IR-IGF1R hybrid formation are not fully understood, hindering the ability to modulate this for future therapies targeting this receptor. To pinpoint suitable sites for intervention, computational hotspot prediction was utilised to identify promising epitopes for targeting with point mutagenesis. Specific IGF1R point mutations F450A, R391A and D555A show reduced affinity of the hybrid receptor in a BRET based donor-saturation assay, confirming hybrid formation could be modulated at this interface. These data provide the basis for rational design of more effective hybrid receptor modulators, supporting the prospect of identifying a small molecule that specifically interacts with this target.
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Mutagénesis Sitio-Dirigida , Receptor IGF Tipo 1 , Receptor de Insulina , Receptor de Insulina/genética , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Humanos , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/metabolismo , Multimerización de Proteína , Péptidos Similares a la Insulina , Antígenos CDRESUMEN
Background & Aims: O-GlcNAcylation is a reversible post-translational modification controlled by the activity of two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). In the liver, O-GlcNAcylation has emerged as an important regulatory mechanism underlying normal liver physiology and metabolic disease. Methods: To address whether OGT acts as a critical hepatic nutritional node, mice with a constitutive hepatocyte-specific deletion of OGT (OGTLKO) were generated and challenged with different carbohydrate- and lipid-containing diets. Results: Analyses of 4-week-old OGTLKO mice revealed significant oxidative and endoplasmic reticulum stress, and DNA damage, together with inflammation and fibrosis, in the liver. Susceptibility to oxidative and endoplasmic reticulum stress-induced apoptosis was also elevated in OGTLKO hepatocytes. Although OGT expression was partially recovered in the liver of 8-week-old OGTLKO mice, hepatic injury and fibrosis were not rescued but rather worsened with time. Interestingly, weaning of OGTLKO mice on a ketogenic diet (low carbohydrate, high fat) fully prevented the hepatic alterations induced by OGT deletion, indicating that reduced carbohydrate intake protects an OGT-deficient liver. Conclusions: These findings pinpoint OGT as a key mediator of hepatocyte homeostasis and survival upon carbohydrate intake and validate OGTLKO mice as a valuable model for assessing therapeutical approaches of advanced liver fibrosis. Impact and Implications: Our study shows that hepatocyte-specific deletion of O-GlcNAc transferase (OGT) leads to severe liver injury, reinforcing the importance of O-GlcNAcylation and OGT for hepatocyte homeostasis and survival. Our study also validates the Ogt liver-deficient mouse as a valuable model for the study of advanced liver fibrosis. Importantly, as the severe hepatic fibrosis of Ogt liver-deficient mice could be fully prevented upon feeding on a ketogenic diet (i.e. very-low-carbohydrate, high-fat diet) this work underlines the potential interest of nutritional intervention as antifibrogenic strategies.
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The insulin receptor (IR) plays an important role in insulin signal transduction, the defect of which is believed to be the root cause of type 2 diabetes. In 3T3-L1 adipocytes as in other cell types, the mature IR is a heterotetrameric cell surface glycoprotein composed of two α subunits and two ß subunits. Our objective in our study, is to understand how the desialylation of N-glycan chains, induced by elastin-derived peptides, plays a major role in the function of the IR. Using the 3T3-L1 adipocyte line, we show that removal of the sialic acid from N-glycan chains (N893 and N908), induced by the elastin receptor complex (ERC) and elastin derived-peptides (EDPs), leads to a decrease in the autophosphorylation activity of the insulin receptor. We demonstrate by molecular dynamics approaches that the absence of sialic acids on one of these two sites is sufficient to generate local and general modifications of the structure of the IR. Biochemical approaches highlight a decrease in the interaction between insulin and its receptor when ERC sialidase activity is induced by EDPs. Therefore, desialylation by EDPs is synonymous with a decrease of IR sensitivity in adipocytes and could thus be a potential source of insulin resistance associated with diabetic conditions.
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Células 3T3-L1 , Adipocitos , Elastina , Insulina , Receptor de Insulina , Receptores de Superficie Celular , Ácidos Siálicos , Animales , Receptor de Insulina/metabolismo , Ratones , Adipocitos/metabolismo , Insulina/metabolismo , Elastina/metabolismo , Ácidos Siálicos/metabolismo , Fosforilación , Resistencia a la Insulina , Simulación de Dinámica Molecular , Péptidos/metabolismo , Péptidos/farmacología , Péptidos/química , Ácido N-Acetilneuramínico/metabolismo , Transducción de SeñalRESUMEN
[This corrects the article DOI: 10.1016/j.jhepr.2023.100878.].
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PTP1B is a protein tyrosine-phosphatase located on the cytosolic side of the endoplasmic reticulum that plays an important role in the regulation of the insulin receptor (IR). Replacement of the conserved Asp-181 by alanine is known to convert PTP1B into a substrate-trapping protein that binds to but cannot dephosphorylate its substrates. In this work, we have studied the effect of an additional mutation (Y46F) on the substrate-trapping efficiency of PTP1B-D181A. We observed that this mutation converts PTP1B-D181A into a highly efficient substrate-trapping mutant, resulting in much higher recovery of tyrosine-phosphorylated proteins coimmunoprecipitated with PTP1B. Bioluminescence resonance energy transfer (BRET) experiments were also performed to compare the dynamics of interaction of the IR with these mutants. Basal BRET, which mainly reflects the interaction of PTP1B with the IR precursor during its biosynthesis in the endoplasmic reticulum, was markedly increased with the PTP1B-D181A-Y46F mutant. In contrast, insulin-induced BRET was markedly reduced with PTP1B-D181A-Y46F. I(125) insulin binding experiments indicated that PTP1B-D181-Y46F reduced the expression of IR at the plasma membrane. Reduced expression at the cell surface was associated with higher amounts of the uncleaved IR precursor in the cell. Moreover, we observed that substantial amounts of the uncleaved IR precursor reached the Tris-phosphorylated, fully activated form in an insulin independent fashion. These results support the notion that PTP1B plays a crucial role in the control of the activity of the IR precursor during its biosynthesis. In addition, this new substrate-trapping mutant may be a valuable tool for the identification of new PTP1B substrates.
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Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica/fisiología , Insulina/metabolismo , Precursores de Proteínas/biosíntesis , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Receptor de Insulina/biosíntesis , Sustitución de Aminoácidos , Retículo Endoplásmico/genética , Activación Enzimática , Células HEK293 , Humanos , Insulina/genética , Mutación Missense , Precursores de Proteínas/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 1/genética , Receptor de Insulina/genéticaRESUMEN
O-GlcNAcylation is a reversible post-translational modification involved in the regulation of cytosolic, nuclear, and mitochondrial proteins. Only two enzymes, OGT (O-GlcNAc transferase) and OGA (O-GlcNAcase), control the attachment and removal of O-GlcNAc on proteins, respectively. Whereas a variant OGT (mOGT) has been proposed as the main isoform that O-GlcNAcylates proteins in mitochondria, identification of a mitochondrial OGA has not been performed yet. Two splice variants of OGA (short and long isoforms) have been described previously. In this work, using cell fractionation experiments, we show that short-OGA is preferentially recovered in mitochondria-enriched fractions from HEK-293T cells and RAW 264.7 cells, as well as mouse embryonic fibroblasts. Moreover, fluorescent microscopy imaging confirmed that GFP-tagged short-OGA is addressed to mitochondria. In addition, using a Bioluminescence Resonance Energy Transfer (BRET)-based mitochondrial O-GlcNAcylation biosensor, we show that co-transfection of short-OGA markedly reduced O-GlcNAcylation of the biosensor, whereas long-OGA had no significant effect. Finally, using genetically encoded or chemical fluorescent mitochondrial probes, we show that short-OGA overexpression increases mitochondrial ROS levels, whereas long-OGA has no significant effect. Together, our work reveals that the short-OGA isoform is targeted to the mitochondria where it regulates ROS homoeostasis.
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Fibroblastos , Mitocondrias , Animales , Fibroblastos/metabolismo , Células HEK293 , Humanos , Ratones , Mitocondrias/metabolismo , Isoformas de Proteínas/metabolismo , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , beta-N-AcetilhexosaminidasasRESUMEN
O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification on serine and threonine residues of cytosolic, nuclear and mitochondrial proteins. O-GlcNAcylation level is regulated by OGT (O-GlcNAc transferase), which adds GlcNAc on proteins, and OGA (O-GlcNAcase), which removes it. Abnormal level of protein O-GlcNAcylation has been observed in numerous cancer cell types, including cervical cancer cells. In the present study, we have evaluated the effect of increasing protein O-GlcNAcylation on cervical cancer-derived CaSki cells. We observed that pharmacological enhancement of protein O-GlcNAcylation by Thiamet G (an inhibitor of OGA) and glucosamine (which provides UDP-GlcNAc substrate to OGT) increases CaSki cells proliferation, migration and survival. Moreover, we showed that increased O-GlcNAcylation promotes IGF-1 receptor (IGF1R) autophosphorylation, possibly through inhibition of protein tyrosine-phosphatase 1B activity. This was associated with increased IGF-1-induced phosphatidyl-Inositol 3-phosphate production at the plasma membrane and increased Akt activation in CaSki cells. Finally, we showed that protein O-GlcNAcylation and Akt phosphorylation levels were higher in human cervical cancer samples compared to healthy cervix tissues, and a highly positive correlation was observed between O-GlcNAcylation level and Akt phosphorylation in theses tissues. Together, our results indicate that increased O-GlcNAcylation, by activating IGF1R/ Phosphatidyl inositol 3-Kinase (PI-3K)/Akt signaling, may participate in cervical cancer cell growth and proliferation.
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Acetilglucosamina , Neoplasias del Cuello Uterino , Acetilglucosamina/metabolismo , Cuello del Útero/metabolismo , Femenino , Humanos , Inositol/metabolismo , N-Acetilglucosaminiltransferasas/genética , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor IGF Tipo 1/metabolismo , Neoplasias del Cuello Uterino/metabolismoRESUMEN
TxNIP (Thioredoxin-interacting protein) is considered as a potential drug target for type 2 diabetes. Although TxNIP expression is correlated with hyperglycemia and glucotoxicity in pancreatic ß cells, its regulation in liver cells has been less investigated. In the current study, we aim at providing a better understanding of Txnip regulation in hepatocytes in response to physiological stimuli and in the context of hyperglycemia in db/db mice. We focused on regulatory pathways governed by ChREBP (Carbohydrate Responsive Element Binding Protein) and FoxO1 (Forkhead box protein O1), transcription factors that play central roles in mediating the effects of glucose and fasting on gene expression, respectively. Studies using genetically modified mice reveal that hepatic TxNIP is up-regulated by both ChREBP and FoxO1 in liver cells and that its expression strongly correlates with fasting, suggesting a major role for this protein in the physiological adaptation to nutrient restriction.
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AIM: Metabolic sources switch from carbohydrates in utero, to fatty acids after birth and then a mix once adults. O-GlcNAcylation (O-GlcNAc) is a post-translational modification considered as a nutrient sensor. The purpose of this work was to assess changes in protein O-GlcNAc levels, regulatory enzymes and metabolites during the first periods of life and decipher the impact of O-GlcNAcylation on cardiac proteins. METHODS: Heart, brain and liver were harvested from rats before and after birth (D-1 and D0), in suckling animals (D12), after weaning with a standard (D28) or a low-carbohydrate diet (D28F), and adults (D84). O-GlcNAc levels and regulatory enzymes were evaluated by western blots. Mass spectrometry (MS) approaches were performed to quantify levels of metabolites regulating O-GlcNAc and identify putative cardiac O-GlcNAcylated proteins. RESULTS: Protein O-GlcNAc levels decrease drastically and progressively from D-1 to D84 (13-fold, P < .05) in the heart, whereas the changes were opposite in liver and brain. O-GlcNAc levels were unaffected by weaning diet in any tissues. Changes in expression of enzymes and levels of metabolites regulating O-GlcNAc were tissue-dependent. MS analyses identified changes in putative cardiac O-GlcNAcylated proteins, namely those involved in the stress response and energy metabolism, such as ACAT1, which is only O-GlcNAcylated at D0. CONCLUSION: Our results demonstrate that protein O-GlcNAc levels are not linked to dietary intake and regulated in a time and tissue-specific manner during postnatal development. We have identified by untargeted MS putative proteins with a particular O-GlcNAc signature across the development process suggesting specific role of these proteins.
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Acetilglucosamina , Procesamiento Proteico-Postraduccional , Animales , Ingestión de Alimentos , Espectrometría de Masas , RatasAsunto(s)
Antígenos de Neoplasias/biosíntesis , Enfermedad de la Arteria Coronaria/enzimología , Vasos Coronarios/enzimología , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Tipo 1/enzimología , Angiopatías Diabéticas/enzimología , Células Endoteliales/enzimología , Endotelio Vascular/enzimología , Histona Acetiltransferasas/biosíntesis , Hialuronoglucosaminidasa/biosíntesis , beta-N-Acetilhexosaminidasas/biosíntesis , Animales , Humanos , MasculinoRESUMEN
The protein tyrosine phosphatase-1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) have been implicated in down-regulation of tyrosine kinase receptors, conferring anti-oncogenic functions to these PTPases. However, recent work has shown that PTP1B is positively implicated in oncogenic properties of breast cancer cells by regulating the ERK pathway. Here, we studied the function of PTP1B and TC-PTP in IGF-2-induced growth, survival and migration of MCF-7 breast cancer cells. Using siRNA, we showed that reduction in the expression of these PTPases decreased cell growth and ERK phosphorylation. Reduction in the expression of these PTPases did not impair IGF-2 effects on cell survival to acute treatment with 4-OH Tamoxifen. In contrast, IGF-2-induced MCF-7 cell migration was markedly impaired by reduction of PTP1B or TC-PTP expression, independently of the ERK pathway. This novel finding reinforces the potential role of these PTPases as therapeutic targets for treatment of breast cancer.
Asunto(s)
Neoplasias de la Mama/patología , Factor II del Crecimiento Similar a la Insulina/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 2/metabolismo , Neoplasias de la Mama/enzimología , Línea Celular Tumoral , Movimiento Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Factor II del Crecimiento Similar a la Insulina/farmacología , Metástasis de la Neoplasia , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 1/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 2/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
O-GlcNAcylation corresponds to the addition of N-acetylglucosamine on serine and threonine residues of cytosolic and nuclear proteins. O-GlcNAcylation is a dynamic post-translational modification, analogous to phosphorylation, that regulates the stability, the activity or the sub-cellular localisation of proteins. This reversible modification depends on the availability of glucose and therefore constitutes a powerful means by which cellular activities are regulated according to the nutritional environment of the cell. O-GlcNAcylation has been implicated in important human pathologies including Alzheimer disease and type-2 diabetes. Only two enzymes, OGT and O-GlcNAcase, control the O-GlcNAcylation level on proteins, and thereby regulate signaling pathways. Several lines of evidence indicate that OGT attenuates insulin signal by O-GlcNAcylation of proteins involved in proximal and distal steps in the signaling pathway. This negative feedback may be exacerbated when cells are exposed to elevated glucose concentrations as observed in diabetic patients, and could thereby contribute to insulin resistance and worsening of hyperglycaemia. double dagger.