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1.
Mayo Clin Proc ; 96(3): 577-591, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33673911

RESUMO

OBJECTIVE: To describe the clinical and pathological phenotype of membranous nephropathy (MN) associated with M-type-phospholipase-A2-receptor (PLA2R), thrombospondin-type-1-domain-containing-7A (THSD7A), semaphorin 3B (SEMA3B), neural-epidermal-growth-factor-like-1-protein (NELL-1), protocadherin 7 (PCDH7), exostosin 1/exostosin 2 (EXT1/EXT2) and neural cell adhesion molecule 1 (NCAM-1) as target antigens. METHODS: A retrospective cohort of 270 adult patients with biopsy-proven MN diagnosed between January 2015 and April 2020 was classified as PLA2R-, THSD7A-, SEMA3B-, NELL-1-, PCDH7-, EXT1/EXT2-, NCAM-1-associated or septuple-negative MN using serologic tests, immunostaining, and/or mass spectrometry. Clinical, biochemical, pathologic, and follow-up data were systematically abstracted from the medical records, including disease activity of conditions traditionally associated with MN and occurring within 5 years of MN diagnosis. RESULTS: Patients with PLA2R-associated MN were predominantly middle-aged white men without associated disease. The presence of associated disease did not affect the clinical and pathologic characteristics of PLA2R-associated MN, suggesting that they were coincidental rather than causally linked. THSD7A-, NELL-1-, PCDH7-, and NCAM-1-associated MN were rare and SEMA3B-associated MN was not discovered in our cohort. EXT1/EXT2-associated MN was primarily diagnosed in younger women with active systemic autoimmunity. A significant proportion of septuple-negative patients had associated malignancy or systemic autoimmunity. CONCLUSION: The widely used distinction between primary and secondary MN has limitations. We propose a refined terminology that combines the target antigen and associated disease to better classify MN and guide clinical decision making.


Assuntos
Antígenos/metabolismo , Autoanticorpos/metabolismo , Glomerulonefrite Membranosa/imunologia , Adulto , Idoso , Caderinas/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , N-Acetilglucosaminiltransferases/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Receptores da Fosfolipase A2/metabolismo , Índice de Gravidade de Doença , Trombospondinas/metabolismo
2.
Molecules ; 26(4)2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33669256

RESUMO

O-GlcNAcylation is a posttranslational modification that occurs at serine and threonine residues of protein substrates by the addition of O-linked ß-d-N-acetylglucosamine (GlcNAc) moiety. Two enzymes are involved in this modification: O-GlcNac transferase (OGT), which attaches the GlcNAc residue to the protein substrate, and O-GlcNAcase (OGA), which removes it. This biological balance is important for many biological processes, such as protein expression, cell apoptosis, and regulation of enzyme activity. The extent of this modification has sparked interest in the medical community to explore OGA and OGT as therapeutic targets, particularly in degenerative diseases. While some OGA inhibitors are already in phase 1 clinical trials for the treatment of Alzheimer's disease, OGT inhibitors still have a long way to go. Due to complex expression and instability, the discovery of potent OGT inhibitors is challenging. Over the years, the field has grappled with this problem, and scientists have developed a number of techniques and assays. In this review, we aim to highlight assays and techniques for OGT inhibitor discovery, evaluate their strength for the field, and give us direction for future bioassay methods.


Assuntos
Bioensaio/métodos , N-Acetilglucosaminiltransferases/metabolismo , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Fenômenos Biofísicos , Química Click , Ligação Proteica
3.
Medicine (Baltimore) ; 100(9): e24887, 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33655949

RESUMO

RATIONALE: Membranous glomerulonephritis (MN) is the leading cause of nephrotic syndrome in adults and is classified as primary or secondary. Secondary MN accounts for 20% to 30% of all MN cases and can arise from a number of conditions, including autoimmune diseases. Recently exostosin 1/exostosin 2 (EXT1/EXT2) have been identified as the common antigens in secondary autoimmune MN and are present in cases of pure membranous lupus nephritis (LN). The treatment of EXT1/EXT2-associated MN remains elusive. PATIENT CONCERNS: We present the case of a 15-year-old female who presented with nephrotic syndrome, positive ANA and dsDNA, and low serum complements. A renal biopsy revealed pure membranous nephritis with IgG and C3 deposition. EXT1 was found along the glomerular capillary walls and stained positive, while phospholipase A2 receptor (PLA2R) and thrombospondin type-1 domain-containing 7A (THSD7A) were negative. DIAGNOSIS: The patient was diagnosed with ETX1-associated membranous LN. INTERVENTIONS: She was treated with prednisone and multiple low-dose rituximab (4 200 mg doses, approximately every 2 months, based on CD19+ cells counts). OUTCOMES: The patient had complete remission within 8 months later, and she remained in remission for the 16-month period of follow-up. LESSONS: To our knowledge, this is the first case of EXT1-associated MN that has been successfully treated by multiple low-dose rituximab. Further studies can investigate the optimal dosage and treatment protocol.


Assuntos
Autoanticorpos/imunologia , Nefrite Lúpica/tratamento farmacológico , N-Acetilglucosaminiltransferases/imunologia , Rituximab/administração & dosagem , Adolescente , Autoanticorpos/metabolismo , Biomarcadores/metabolismo , Biópsia , Relação Dose-Resposta a Droga , Feminino , Humanos , Fatores Imunológicos/administração & dosagem , Glomérulos Renais/patologia , Nefrite Lúpica/imunologia , Nefrite Lúpica/metabolismo , N-Acetilglucosaminiltransferases/metabolismo
4.
Nat Commun ; 12(1): 945, 2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33574257

RESUMO

O-GlcNAc modification plays important roles in metabolic regulation of cellular status. Two homologs of O-GlcNAc transferase, SECRET AGENT (SEC) and SPINDLY (SPY), which have O-GlcNAc and O-fucosyl transferase activities, respectively, are essential in Arabidopsis but have largely unknown cellular targets. Here we show that AtACINUS is O-GlcNAcylated and O-fucosylated and mediates regulation of transcription, alternative splicing (AS), and developmental transitions. Knocking-out both AtACINUS and its distant paralog AtPININ causes severe growth defects including dwarfism, delayed seed germination and flowering, and abscisic acid (ABA) hypersensitivity. Transcriptomic and protein-DNA/RNA interaction analyses demonstrate that AtACINUS represses transcription of the flowering repressor FLC and mediates AS of ABH1 and HAB1, two negative regulators of ABA signaling. Proteomic analyses show AtACINUS's O-GlcNAcylation, O-fucosylation, and association with splicing factors, chromatin remodelers, and transcriptional regulators. Some AtACINUS/AtPININ-dependent AS events are altered in the sec and spy mutants, demonstrating a function of O-glycosylation in regulating alternative RNA splicing.


Assuntos
Processamento Alternativo/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Ácido Abscísico/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Glicosilação , N-Acetilglucosaminiltransferases/metabolismo , Proteômica
5.
PLoS Pathog ; 17(2): e1009282, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33556147

RESUMO

Receptor binding studies on sarbecoviruses would benefit from an available toolkit of recombinant spike proteins, or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric Receptor Binding Domain (RBD) proteins would be suitable candidates to study receptor binding properties of SARS-CoV-1 and -2. Here we created monomeric and trimeric fluorescent RBD proteins, derived from adherent HEK293T, as well as in GnTI-/- mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. The results demonstrate that trimeric, complex glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. Although differences in binding to commonly used cell lines were minimal between the different RBD preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. The RBD trimers demonstrated distinct ACE2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results show that complex glycosylated trimeric RBD proteins are attractive to analyze sarbecovirus receptor binding and explore ACE2 expression profiles in tissues.


Assuntos
/metabolismo , Multimerização Proteica , Glicoproteína da Espícula de Coronavírus/metabolismo , Células A549 , Animais , Chlorocebus aethiops , Cães , Glicosilação , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Mesocricetus , Camundongos , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Ligação Proteica , Glicoproteína da Espícula de Coronavírus/genética , Células Vero
6.
Anticancer Res ; 41(2): 845-858, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33517290

RESUMO

BACKGROUND/AIM: Triple-negative breast cancer (TNBC) remains difficult to treat and new molecular targets are needed. Here, we investigated the impact of glycosyltransferase genes on TNBC patient survival. PATIENTS AND METHODS: mRNA expression levels of 101 glycosyltransferase genes in TNBC patients were compared for correlation with patient survival using The Cancer Genome Atlas data. An antibody to ß-3-N-acetylgluco-saminyltransferase 8 (B3GNT8) was applied to investigate B3GNT8 protein distribution and expression levels in 23 TNBC surgical specimens. RESULTS: B3GNT8 mRNA levels inversely correlated with relapse-free survival (p<0.01) and overall survival (p<0.05) in TNBC patients. Anti-B3GNT8 antibody binding was observed as dots in the cytoplasm of cancer cells. These dots were supposed to correspond to B3GNT8 protein in tumour cells, but their number was smaller in relapsed patients than in non-relapsed patients. CONCLUSION: B3GNT8 mRNA expression levels in TNBC tumour tissues are potentially useful in distinguishing patients with favourable and poor clinical outcomes.


Assuntos
Citoplasma/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Neoplasias de Mama Triplo Negativas/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Recidiva , Análise de Sobrevida , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo
7.
J Biomed Sci ; 27(1): 57, 2020 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32349769

RESUMO

O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a type of glycosylation that occurs when a monosaccharide, O-GlcNAc, is added onto serine or threonine residues of nuclear or cytoplasmic proteins by O-GlcNAc transferase (OGT) and which can be reversibly removed by O-GlcNAcase (OGA). O-GlcNAcylation couples the processes of nutrient sensing, metabolism, signal transduction and transcription, and plays important roles in development, normal physiology and physiopathology. Cumulative studies have indicated that O-GlcNAcylation affects the functions of protein substrates in a number of ways, including protein cellular localization, protein stability and protein/protein interaction. Particularly, O-GlcNAcylation has been shown to have intricate crosstalk with phosphorylation as they both modify serine or threonine residues. Aberrant O-GlcNAcylation on various protein substrates has been implicated in many diseases, including neurodegenerative diseases, diabetes and cancers. However, the role of protein O-GlcNAcylation in immune cell lineages has been less explored. This review summarizes the current understanding of the fundamental biochemistry of O-GlcNAcylation, and discusses the molecular mechanisms by which O-GlcNAcylation regulates the development, maturation and functions of immune cells. In brief, O-GlcNAcylation promotes the development, proliferation, and activation of T and B cells. O-GlcNAcylation regulates inflammatory and antiviral responses of macrophages. O-GlcNAcylation promotes the function of activated neutrophils, but inhibits the activity of nature killer cells.


Assuntos
Sistema Imunitário/fisiologia , N-Acetilglucosaminiltransferases/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Acilação , Animais , Humanos , Sistema Imunitário/crescimento & desenvolvimento
8.
PLoS One ; 15(5): e0233492, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469948

RESUMO

Glycosylation can affect various protein properties such as stability, biological activity, and immunogenicity. To produce human therapeutic proteins, a host that can produce glycoproteins with correct glycan structures is required. Microbial expression systems offer economical, rapid and serum-free production and are more amenable to genetic manipulation. In this study, we developed a protocol for CRISPR/Cas9 multiple gene knockouts and knockins in Kluyveromyces marxianus, a probiotic yeast with a rapid growth rate. As hyper-mannosylation is a common problem in yeast, we first knocked out the α-1,3-mannosyltransferase (ALG3) and α-1,6-mannosyltransferase (OCH1) genes to reduce mannosylation. We also knocked out the subunit of the telomeric Ku domain (KU70) to increase the homologous recombination efficiency of K. marxianus. In addition, we knocked in the MdsI (α-1,2-mannosidase) gene to reduce mannosylation and the GnTI (ß-1,2-N-acetylglucosaminyltransferase I) and GnTII genes to produce human N-glycan structures. We finally obtained two strains that can produce low amounts of the core N-glycan Man3GlcNAc2 and the human complex N-glycan Man3GlcNAc4, where Man is mannose and GlcNAc is N-acetylglucosamine. This study lays a cornerstone of glycosylation engineering in K. marxianus toward producing human glycoproteins.


Assuntos
Kluyveromyces/genética , Kluyveromyces/metabolismo , Engenharia Metabólica/métodos , Polissacarídeos/biossíntese , Polissacarídeos/química , Biotecnologia , Sistemas CRISPR-Cas , Técnicas de Introdução de Genes , Técnicas de Inativação de Genes , Genes Fúngicos , Glicoproteínas/biossíntese , Glicoproteínas/química , Glicoproteínas/genética , Glicosilação , Humanos , Manosidases/genética , Manosidases/metabolismo , Manosiltransferases/antagonistas & inibidores , Manosiltransferases/genética , Manosiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
9.
Nucleic Acids Res ; 48(10): 5656-5669, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32329777

RESUMO

Intron detention in precursor RNAs serves to regulate expression of a substantial fraction of genes in eukaryotic genomes. How detained intron (DI) splicing is controlled is poorly understood. Here, we show that a ubiquitous post-translational modification called O-GlcNAc, which is thought to integrate signaling pathways as nutrient conditions fluctuate, controls detained intron splicing. Using specific inhibitors of the enzyme that installs O-GlcNAc (O-GlcNAc transferase, or OGT) and the enzyme that removes O-GlcNAc (O-GlcNAcase, or OGA), we first show that O-GlcNAc regulates splicing of the highly conserved detained introns in OGT and OGA to control mRNA abundance in order to buffer O-GlcNAc changes. We show that OGT and OGA represent two distinct paradigms for how DI splicing can control gene expression. We also show that when DI splicing of the O-GlcNAc-cycling genes fails to restore O-GlcNAc homeostasis, there is a global change in detained intron levels. Strikingly, almost all detained introns are spliced more efficiently when O-GlcNAc levels are low, yet other alternative splicing pathways change minimally. Our results demonstrate that O-GlcNAc controls detained intron splicing to tune system-wide gene expression, providing a means to couple nutrient conditions to the cell's transcriptional regime.


Assuntos
Acetilglucosamina/metabolismo , Glicosídeo Hidrolases/genética , Íntrons , N-Acetilglucosaminiltransferases/genética , Processamento de RNA , Linhagem Celular , Glicosídeo Hidrolases/metabolismo , Células HEK293 , Humanos , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/metabolismo , Fosforilação , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/metabolismo , RNA-Seq
10.
PLoS Genet ; 16(4): e1008730, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251422

RESUMO

O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) is the only enzyme catalyzing O-GlcNAcylation. Although it has been shown that OGT plays an essential role in maintaining postnatal heart function, its role in heart development remains unknown. Here we showed that loss of OGT in early fetal cardiomyocytes led to multiple heart developmental defects including hypertrabeculation, biventricular dilation, atrial septal defects, ventricular septal defects, and defects in coronary vessel development. In addition, RNA sequencing revealed that Angiopoietin-1, required within cardiomyocytes for both myocardial and coronary vessel development, was dramatically downregulated in cardiomyocyte-specific OGT knockout mouse hearts. In conclusion, our data demonstrated that OGT plays an essential role in regulating heart development through activating expression of cardiomyocyte Angiopoietin-1.


Assuntos
Coração/embriologia , Miócitos Cardíacos/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Angiopoietina-1/genética , Angiopoietina-1/metabolismo , Animais , Células Cultivadas , Coração/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , N-Acetilglucosaminiltransferases/genética
11.
Development ; 147(7)2020 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165492

RESUMO

Although the developing pancreas is exquisitely sensitive to nutrient supply in utero, it is not entirely clear how nutrient-driven post-translational modification of proteins impacts the pancreas during development. We hypothesized that the nutrient-sensing enzyme O-GlcNAc transferase (Ogt), which catalyzes an O-GlcNAc-modification onto key target proteins, integrates nutrient-signaling networks to regulate cell survival and development. In this study, we investigated the heretofore unknown role of Ogt in exocrine and endocrine islet development. By genetic manipulation in vivo and by using morphometric and molecular analyses, such as immunofluorescence imaging and single cell RNA sequencing, we show the first evidence that Ogt regulates pancreas development. Genetic deletion of Ogt in the pancreatic epithelium (OgtKOPanc) causes pancreatic hypoplasia, in part by increased apoptosis and reduced levels of of Pdx1 protein. Transcriptomic analysis of single cell and bulk RNA sequencing uncovered cell-type heterogeneity and predicted upstream regulator proteins that mediate cell survival, including Pdx1, Ptf1a and p53, which are putative Ogt targets. In conclusion, these findings underscore the requirement of O-GlcNAcylation during pancreas development and show that Ogt is essential for pancreatic progenitor survival, providing a novel mechanistic link between nutrients and pancreas development.


Assuntos
Acetilglucosamina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Nutrientes/farmacologia , Pâncreas Exócrino/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Animais , Embrião de Mamíferos , Feminino , Ilhotas Pancreáticas/embriologia , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , N-Acetilglucosaminiltransferases/efeitos dos fármacos , N-Acetilglucosaminiltransferases/metabolismo , Pâncreas Exócrino/embriologia , Pâncreas Exócrino/metabolismo , Transdução de Sinais/efeitos dos fármacos
12.
BMC Cancer ; 20(1): 192, 2020 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-32143591

RESUMO

BACKGROUND: Altered glycosylation associated with hepatocellular carcinoma (HCC) is well documented. However, few reports have investigated the association between dedifferentiation and glycosylation. Therefore, the aim of this study was to analyze glycosylation associated with dedifferentiation of HCC within the same nodule and to investigate glycosyltransferase related to the glycosylation. METHODS: We analyzed resected HCC specimens (n = 50) using lectin microarray to comprehensively and sensitively analyze glycan profiles, and identify changes to glycosylation between well- and moderately-differentiated components within the same nodule. Moreover, we performed immunohistochemical staining of mannosyl(α-1,3-)-glycoprotein ß-1,2-N-acetylglucosaminyltransferase (MGAT1), which is an essential glycosyltransferase that converts high-mannose glycans to complex- or hybrid-type N-glycans. RESULTS: Four lectins from Narcissus pseudonarcissus agglutinin (NPA), Concanavalin A, Galanthus nivalis agglutinin, and Calystegia sepium agglutinin were significantly elevated in moderately-differentiated components of HCC compared with well-differentiated components, and all lectins showed binding specificity to high-mannose glycans. Therefore, these structures were represented to a greater extent in moderately-differentiated components than in well-differentiated ones. Immunohistochemical staining revealed significantly increased NPA expression and decreased MGAT1 expression in moderately-differentiated components. Low MGAT1 expression in moderately-differentiated components of tumors was associated with intrahepatic metastasis and had tendency for poor prognosis. CONCLUSION: Dedifferentiation of well-differentiated HCC is associated with an increase in high-mannose glycans. MGAT1 may play a role in the dedifferentiation of HCC.


Assuntos
Carcinoma Hepatocelular/metabolismo , Concanavalina A/metabolismo , Neoplasias Hepáticas/metabolismo , Lectinas de Ligação a Manose/metabolismo , Lectinas de Plantas/metabolismo , Idoso , Calystegia/química , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Intervalo Livre de Doença , Feminino , Glicosilação , Humanos , Imuno-Histoquímica/métodos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Masculino , N-Acetilglucosaminiltransferases/metabolismo , Narcissus/química , Imagem Óptica/métodos , Polissacarídeos/química , Coloração e Rotulagem/métodos
13.
Zhonghua Gan Zang Bing Za Zhi ; 28(2): 147-151, 2020 Feb 20.
Artigo em Chinês | MEDLINE | ID: mdl-32164066

RESUMO

Objective: To investigate the effect of knockdown of O-GlcNAc transferase (OGT) on hepatocyte fat synthesis. Methods: Liver cell line L02 were used to established the model of hepatic steatosis. The levels of OGT and O-GlcNAc protein were detected by Western blot. The OGT knockdown cell line of L02 cells was established, and its lipid formation ability was detected after induction of oleic acid (OA). Real-time quantitative PCR (qRT-PCR) and Western blot were used to detect mRNA and protein expression of enzymes related to fat synthesis. An independent sample t test was used. Results: Western blot showed that the expression of OGT and O-GlcNAc was increased in L02 cells after adipogenesis (P < 0.05). After shOGT lentivirus infects L02 cells, OGT mRNA levels were down-regulated (P < 0.01). Oil red O staining showed that the lipid in L02 shOGT cells decreased, qRT-PCR showed that the mRNA expressions of fat synthase (ACC1), (FASN) and (SCD1) were decreased, the difference was statistically significant (P < 0.05), protein Expression is consistent with mRNA expression. Conclusion: Knockdown of OGT can inhibit hepatocyte fat synthesis by reducing O-GlcNAc levels.


Assuntos
Antígenos de Neoplasias/metabolismo , Fígado Gorduroso , Hepatócitos/metabolismo , Histona Acetiltransferases/metabolismo , Hialuronoglucosaminidase/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Linhagem Celular , Humanos
14.
Biochem Biophys Res Commun ; 526(1): 184-190, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32201074

RESUMO

The Notch signaling pathway is highly conserved and essential in animal development and tissue homeostasis. Regulation of Notch signaling is a crucial process for human health. Ligands initiate a signal cascade by binding to Notch receptors expressed on the neighboring cell. Notch receptors interact with ligands through their epidermal growth factor-like repeats (EGF repeats). Most EGF repeats are modified by O-glycosylation with residues, such as O-linked N-acetylglucosamine (O-GlcNAc), O-fucose, and O-glucose. A recent study revealed the distinct roles of these O-glycans in ligand binding, processing, and trafficking of Notch receptors. In particular, O-GlcNAc glycans are essential for Delta-like (DLL) ligand-mediated Notch signaling. In this study, we showed that O-GlcNAc promotes Notch1 trafficking to the cell surfaces under the condition that O-fucose and O-glucose are removed from consecutive EGF repeats of Notch1. Through in vitro experiments, we showed that O-GlcNAc mediates the stability of EGF domains in the same manner as O-fucose and O-glucose. Thus, O-GlcNAc on EGF domains possesses a shared function in the stability of EGF domains and Notch1 trafficking.


Assuntos
Fator de Crescimento Epidérmico/química , Espaço Extracelular/metabolismo , Glucosamina/metabolismo , Dobramento de Proteína , Receptores Notch/química , Receptores Notch/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Drosophila/metabolismo , Fucose/metabolismo , Glucose/metabolismo , Células HEK293 , Humanos , Camundongos , Proteínas Mutantes/metabolismo , Mutação/genética , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/metabolismo , Domínios Proteicos , Estabilidade Proteica , Transporte Proteico
15.
Invest Ophthalmol Vis Sci ; 61(2): 24, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32068794

RESUMO

Purpose: O-GlcNAcylation of cellular proteins contributes to the pathophysiology of diabetes and evidence supports a role for augmented O-GlcNAcylation in diabetic retinopathy. The aim of this study was to investigate the impact of the renin-angiotensin system on retinal protein O-GlcNAcylation. Methods: Mice fed a high-fat diet were treated chronically with the angiotensin-converting enzyme inhibitor captopril or captopril plus the angiotensin-(1-7) Mas receptor antagonist A779. Western blotting and quantitative polymerase chain reaction were used to analyze retinal homogenates. Similar analyses were performed on lysates from human MIO-M1 retinal Müller cell cultures exposed to media supplemented with angiotensin-(1-7). Culture conditions were manipulated to influence the hexosamine biosynthetic pathway and/or signaling downstream of the Mas receptor. Results: In the retina of mice fed a high-fat diet, captopril attenuated protein O-GlcNAcylation in a manner dependent on Mas receptor activation. In MIO-M1 cells, angiotensin-(1-7) or adenylate cyclase activation were sufficient to enhance cyclic AMP (cAMP) levels and inhibit O-GlcNAcylation. The repressive effect of cAMP on O-GlcNAcylation was dependent on exchange protein activated by cAMP (EPAC), but not protein kinase A, and was recapitulated by a constitutively active variant of the small GTPase Rap1. We provide evidence that cAMP and angiotensin-(1-7) act to suppress O-GlcNAcylation by inhibition of O-GlcNAc transferase (OGT) activity. In cells exposed to an O-GlcNAcase inhibitor or hyperglycemic culture conditions, mitochondrial superoxide levels were elevated; however, angiotensin-(1-7) signaling prevented the effect. Conclusions: Angiotensin-(1-7) inhibits retinal protein O-GlcNAcylation via an EPAC/Rap1/OGT signaling axis.


Assuntos
Angiotensina I/farmacologia , N-Acetilglucosaminiltransferases/metabolismo , Fragmentos de Peptídeos/farmacologia , Retina/metabolismo , Animais , Captopril/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Retinopatia Diabética/metabolismo , Camundongos , Sistema Renina-Angiotensina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
16.
J Immunol ; 204(6): 1674-1688, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-32060138

RESUMO

Notch signaling is emerging as a critical regulator of T cell activation and function. However, there is no reliable cell surface indicator of Notch signaling across activated T cell subsets. In this study, we show that Notch signals induce upregulated expression of the Gcnt1 glycosyltransferase gene in T cells mediating graft-versus-host disease after allogeneic bone marrow transplantation in mice. To determine if Gcnt1-mediated O-glycosylation could be used as a Notch signaling reporter, we quantified the core-2 O-glycoform of CD43 in multiple T cell subsets during graft-versus-host disease. Pharmacological blockade of Delta-like Notch ligands abrogated core-2 O-glycosylation in a dose-dependent manner after allogeneic bone marrow transplantation, both in donor-derived CD4+ and CD8+ effector T cells and in Foxp3+ regulatory T cells. CD43 core-2 O-glycosylation depended on cell-intrinsic canonical Notch signals and identified CD4+ and CD8+ T cells with high cytokine-producing ability. Gcnt1-deficient T cells still drove lethal alloreactivity, showing that core-2 O-glycosylation predicted, but did not cause, Notch-dependent T cell pathogenicity. Using core-2 O-glycosylation as a marker of Notch signaling, we identified Ccl19-Cre+ fibroblastic stromal cells as critical sources of Delta-like ligands in graft-versus-host responses irrespective of conditioning intensity. Core-2 O-glycosylation also reported Notch signaling in CD8+ T cell responses to dendritic cell immunization, Listeria infection, and viral infection. Thus, we uncovered a role for Notch in controlling core-2 O-glycosylation and identified a cell surface marker to quantify Notch signals in multiple immunological contexts. Our findings will help refine our understanding of the regulation, cellular source, and timing of Notch signals in T cell immunity.


Assuntos
Transplante de Medula Óssea/efeitos adversos , Linfócitos T CD8-Positivos/metabolismo , Doença Enxerto-Hospedeiro/imunologia , N-Acetilglucosaminiltransferases/metabolismo , Receptores Notch/metabolismo , Animais , Biomarcadores/metabolismo , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Estudos de Viabilidade , Feminino , Citometria de Fluxo/métodos , Glicosilação/efeitos dos fármacos , Humanos , Leucossialina/metabolismo , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Sensibilidade e Especificidade , Sialomucinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Células Estromais/imunologia , Células Estromais/metabolismo , Transplante Homólogo/efeitos adversos , Regulação para Cima
17.
Biochim Biophys Acta Mol Basis Dis ; 1866(5): 165712, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32014551

RESUMO

The post-translational modification of serine and threonine residues of nuclear, cytosolic, and mitochondrial proteins by O-linked ß-N-acetyl glucosamine (O-GlcNAc) has long been seen as an important regulatory mechanism in the cardiovascular system. O-GlcNAcylation of cardiac proteins has been shown to contribute to the regulation of transcription, metabolism, mitochondrial function, protein quality control and turnover, autophagy, and calcium handling. In the heart, acute increases in O-GlcNAc have been associated with cardioprotection, such as those observed during ischemia/reperfusion. Conversely, chronic increases in O-GlcNAc, often associated with diabetes and nutrient excess, have been shown to contribute to cardiac dysfunction. Traditionally, many studies have linked changes in O-GlcNAc with nutrient availability and as such O-GlcNAcylation is often seen as a nutrient driven process. However, emerging evidence suggests that O-GlcNAcylation may also be regulated by non-nutrient dependent mechanisms, such as transcriptional and post-translational regulation. Therefore, the goals of this review are to provide an overview of the impact of O-GlcNAcylation in the cardiovascular system, how this is regulated and to discuss the emergence of regulatory mechanisms other than nutrient availability.


Assuntos
Acetilglucosamina/metabolismo , Doenças Cardiovasculares/patologia , Coração/fisiologia , Miocárdio/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/fisiopatologia , Modelos Animais de Doenças , Comportamento Alimentar/fisiologia , Humanos , Miocárdio/patologia , N-Acetilglucosaminiltransferases/metabolismo , Nutrientes/metabolismo , Estresse Fisiológico
18.
Lab Invest ; 100(5): 777-785, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31896813

RESUMO

TRAIL-activating therapy is promising in treating various cancers, including pancreatic cancer, a highly malignant neoplasm with poor prognosis. However, many pancreatic cancer cells are resistant to TRAIL-induced apoptosis despite their expression of intact death receptors (DRs). Protein O-GlcNAcylation is a versatile posttranslational modification that regulates various biological processes. Elevated protein O-GlcNAcylation has been recently linked to cancer cell growth and survival. In this study, we evaluated the role of protein O-GlcNAcylation in pancreatic cancer TRAIL resistance, and identified higher levels of O-GlcNAcylation in TRAIL-resistant pancreatic cancer cells. With gain- and loss-of-function of the O-GlcNAc-adding enzyme, O-GlcNActransferase (OGT), we determined that increasing O-GlcNAcylation rendered TRAIL-sensitive cells more resistant to TRA-8-induced apoptosis, while inhibiting O-GlcNAcylation promoted TRA-8-induced apoptosis in TRAIL-resistance cells. Furthermore, we demonstrated that OGT knockdown sensitized TRAIL-resistant cells to TRA-8 therapy in a mouse model in vivo. Mechanistic studies revealed direct O-GlcNAc modifications of DR5, which regulated TRA-8-induced DR5 oligomerization. We further defined that DR5 O-GlcNAcylation was independent of FADD, the adapter protein for the downstream death-inducing signaling. These studies have demonstrated an important role of protein O-GlcNAcylation in regulating TRAIL resistance of pancreatic cancer cells; and uncovered the contribution of O-GlcNAcylation to DR5 oligomerization and thus mediating DR-inducing signaling.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , N-Acetilglucosaminiltransferases , Neoplasias Pancreáticas , Ligante Indutor de Apoptose Relacionado a TNF , Acetilglucosamina/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Nus , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais/genética , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
19.
DNA Cell Biol ; 39(3): 417-427, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31968179

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadly tumors in digestive tract tumors. Although there has been advancement in PDAC treatment, its prognosis still remains unsatisfactory, mainly because of dismal diagnosis. This article aims to develop new prognostic factors related to energy metabolism in PDAC and to use these genes for novel risk stratification. Hundred fifty messenger RNA (mRNA) expression profiles and clinicopathological data of PDAC were downloaded from The Cancer Genome Atlas dataset. The glycolysis pathway was the significant pathway based on the gene set enrichment analysis. We chose the glycolysis pathway-related 176 genes for further analysis. Multivariate Cox regression analysis and forward stepwise Cox regression model established a novel three-gene glycolytic signature (including MET, B3GNT3, and SPAG4) for PDAC patients' prognosis prediction. All 150 patients were classified into two groups by the median risk score. High-risk group had a worse outcome compared to the low-risk group. The risk score was also significantly correlated with age and radiotherapy. A nomogram, including the glycolytic gene signature, has shown some clinical net benefit for overall survival prediction. We also validated the validity and reliability in the Puleo dataset. This novel gene expression signature may be involved in the pathophysiology and used for risk stratification and prognosis prediction in PDAC.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , Glicólise/genética , Neoplasias Pancreáticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Transcriptoma
20.
Proc Natl Acad Sci U S A ; 117(4): 2004-2013, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31932432

RESUMO

Environmental cues such as nutrients alter cellular behaviors by acting on a wide array of molecular sensors inside cells. Of emerging interest is the link observed between effects of dietary sugars on cancer proliferation. Here, we identify the requirements of hexosamine biosynthetic pathway (HBP) and O-GlcNAc transferase (OGT) for Drosophila homeodomain-interacting protein kinase (Hipk)-induced growth abnormalities in response to a high sugar diet. On a normal diet, OGT is both necessary and sufficient for inducing Hipk-mediated tumor-like growth. We further show that OGT maintains Hipk protein stability by blocking its proteasomal degradation and that Hipk is O-GlcNAcylated by OGT. In mammalian cells, human HIPK2 proteins accumulate posttranscriptionally upon OGT overexpression. Mass spectrometry analyses reveal that HIPK2 is at least O-GlcNAc modified at S852, T1009, and S1147 residues. Mutations of these residues reduce HIPK2 O-GlcNAcylation and stability. Together, our data demonstrate a conserved role of OGT in positively regulating the protein stability of HIPKs (fly Hipk and human HIPK2), which likely permits the nutritional responsiveness of HIPKs.


Assuntos
Carcinogênese/patologia , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Glucose/farmacologia , N-Acetilglucosaminiltransferases/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Acetilglucosamina/metabolismo , Animais , Carcinogênese/induzido quimicamente , Carcinogênese/metabolismo , Proteínas de Transporte/genética , Proliferação de Células , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Células HEK293 , Humanos , Células MCF-7 , Camundongos , N-Acetilglucosaminiltransferases/genética , Fosforilação , Proteínas Quinases/genética , Estabilidade Proteica , Proteínas Serina-Treonina Quinases/genética , Edulcorantes/farmacologia
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