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1.
Sci Rep ; 9(1): 12569, 2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31467334

RESUMO

Small numbers of hematopoietic stem cells (HSCs) balance self-renewal and differentiation to produce the diversity and abundance of cell types that make up the blood system. How nutrients are recruited to support this massive differentiation and proliferation process remains largely unknown. The unique metabolism of adult HSCs, which rely on glycolysis and glutaminolysis, suggests a potential role for the post-translational modification O-GlcNAc as a critical nutrient signal in these cells. Glutamine, glucose, and other metabolites drive the hexosamine biosynthetic pathway (HBP) ultimately leading to the O-GlcNAc modification of critical intracellular targets. Here, we used a conditional targeted genetic deletion of the enzyme that removes O-GlcNAc, O-GlcNAcase (OGA), to determine the consequences of blocked O-GlcNAc cycling on HSCs. Oga deletion in mouse HSCs resulted in greatly diminished progenitor pools, impaired stem cell self-renewal and nearly complete loss of competitive repopulation capacity. Further, early T cell specification was particularly sensitive to Oga deletion. Loss of Oga resulted in a doubling of apoptotic cells within the bone marrow and transcriptional deregulation of key genes involved in adult stem cell maintenance and lineage specification. These findings suggest that O-GlcNAc cycling plays a critical role in supporting HSC homeostasis and early thymocyte development.

2.
Front Microbiol ; 10: 1360, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31281295

RESUMO

Non-nutritive sweeteners (NNS) are marketed as sugar alternatives providing sweet taste with few or no calories. Yet their consumption has been linked to metabolic dysfunction and changes in the gut microbiome. NNS exposure mostly originates from diet beverages and sweetener packages in adults or breastmilk in infants. Consequences of early life exposure remain largely unknown. We exposed pregnant and lactating mice to NNS (sucralose, acesulfame-K) at doses relevant for human consumption. While the pups' exposure was low, metabolic changes were drastic, indicating extensive downregulation of hepatic detoxification mechanisms and changes in bacterial metabolites. Microbiome profiling confirmed a significant increase in firmicutes and a striking decrease of Akkermansia muciniphila. Similar microbiome alterations in humans have been linked to metabolic disease and obesity. While our findings need to be reproduced in humans, they suggest that NNS consumption during pregnancy and lactation may have adverse effects on infant metabolism.

3.
J Nucl Med ; 60(1): 129-134, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30213846

RESUMO

Accumulation of hyperphosphorylated tau, a microtubule-associated protein, plays an important role in the progression of Alzheimer disease. Animal studies suggest that one strategy for treating Alzheimer disease and related tauopathies may be inhibition of O-GlcNAcase (OGA), which may subsequently decrease pathologic tau phosphorylation. Here, we report the pharmacokinetics of a novel PET radioligand, 18F-LSN3316612, which binds with high affinity and selectivity to OGA. Methods: PET imaging was performed on rhesus monkeys at baseline and after administration of either thiamet-G, a potent OGA inhibitor, or nonradioactive LSN3316612. The density of the enzyme was calculated as distribution volume using a 2-tissue-compartment model and serial concentrations of parent radioligand in arterial plasma. The radiation burden for future studies was based on whole-body imaging of monkeys. Oga ∆Br, a mouse brain-specific knockout of Oga, was also scanned to assess the specificity of the radioligand for its target enzyme. Results: Uptake of radioactivity in monkey brain was high (∼5 SUV) and followed by slow washout. The highest uptake was in the amygdala, followed by striatum and hippocampus. Pretreatment with thiamet-G or nonradioactive LSN3316612 reduced brain uptake to a low and uniform concentration in all regions, corresponding to an approximately 90% decrease in distribution volume. Whole-body imaging of rhesus monkeys showed high uptake in kidney, spleen, liver, and testes. In Oga ∆Br mice, brain uptake of 18F-LSN3316612 was reduced by 82% compared with control mice. Peripheral organs were unaffected in Oga ∆Br mice, consistent with loss of OGA expression exclusively in the brain. The effective dose of 18F-LSN3316612 in humans was calculated to be 22 µSv/MBq, which is typical for 18F-labeled radioligands. Conclusion: These results show that 18F-LSN3316612 is an excellent radioligand for imaging and quantifying OGA in rhesus monkeys and mice. On the basis of these data, 18F-LSN3316612 merits evaluation in humans.


Assuntos
Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Piperidinas/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Tiazóis/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Animais , Transporte Biológico , Processamento de Imagem Assistida por Computador , Cinética , Ligantes , Macaca mulatta , Camundongos , Camundongos Knockout , Piperidinas/farmacocinética , Radiometria , Tiazóis/farmacocinética , Distribuição Tecidual
4.
Artigo em Inglês | MEDLINE | ID: mdl-30250452

RESUMO

Nutrient-driven O-GlcNAcylation has been linked to epigenetic regulation of gene expression in metazoans. In C. elegans, O-GlcNAc marks the promoters of over 800 developmental, metabolic, and stress-related genes; these O-GlcNAc marked genes show a strong 5', promoter-proximal bias in the distribution of RNA Polymerase II (Pol II). In response to starvation or feeding, the steady state distribution of O-GlcNAc at promoters remain nearly constant presumably due to dynamic cycling mediated by the transferase OGT-1 and the O-GlcNAcase OGA-1. However, in viable mutants lacking either of these enzymes of O-GlcNAc metabolism, the nutrient-responsive GlcNAcylation of promoters is dramatically altered. Blocked O-GlcNAc cycling leads to a striking nutrient-dependent accumulation of O-GlcNAc on RNA Pol II. O-GlcNAc cycling mutants also show an exaggerated, nutrient-responsive redistribution of promoter-proximal RNA Pol II isoforms and extensive transcriptional deregulation. Our findings suggest a complex interplay between the O-GlcNAc modification at promoters, the kinase-dependent "CTD-code," and co-factors regulating RNA Pol II dynamics. Nutrient-responsive O-GlcNAc cycling may buffer the transcriptional apparatus from dramatic swings in nutrient availability by modulating promoter activity to meet metabolic and developmental needs.

5.
J Biol Chem ; 293(35): 13673-13681, 2018 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-29954943

RESUMO

O-GlcNAcylation is an abundant posttranslational protein modification in which the monosaccharide O-GlcNAc is added to Ser/Thr residues by O-GlcNAc transferase and removed by O-GlcNAcase. Analyses of O-GlcNAc-mediated signaling and metabolic phenomena are complicated by factors including unsatisfactory inhibitors and loss-of-function cell lines lacking identical genetic backgrounds. In this work, we generated immortalized WT, Oga knockout, and Ogt floxed allele (Ogt floxed) mouse embryonic fibroblast (MEF) cell lines with similar genetic backgrounds. These lines will facilitate experiments and serve as a platform to study O-GlcNAc cycling in mammals. As a test paradigm, we used the immortalized MEF lines to investigate how changes in O-GlcNAcylation affected pathological phosphorylation of the tau protein. The activity of glycogen synthase kinase 3ß (GSK3ß), a kinase that phosphorylates tau, decreases when expressed in Oga knockout MEFs compared with WT cells. Phosphorylation at Thr231 in recombinant, tauopathy-associated tau with a proline-to-leucine mutation at position 301 (P301L) was altered when expressed in MEFs with altered O-GlcNAc cycling. In aggregate, our data support that O-GlcNAc cycling indirectly affects tau phosphorylation at Thr231, but tau phosphorylation was highly variable, even in genetically stable, immortalized MEF cells. The variable nature of tau phosphorylation observed here supports the need to use cells akin to those generated here with genetically defined lesions and similar backgrounds to study complex biological processes.


Assuntos
Acetilglucosamina/metabolismo , Fibroblastos/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Transdução de Sinais , beta-N-Acetil-Hexosaminidases/metabolismo , Acetilglucosamina/genética , Alelos , Animais , Células Cultivadas , Feminino , Técnicas de Inativação de Genes , Glicogênio Sintase Quinase 3 beta/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , N-Acetilglucosaminiltransferases/genética , beta-N-Acetil-Hexosaminidases/genética , Proteínas tau/metabolismo
6.
FEBS Lett ; 592(23): 3943-3949, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29904918

RESUMO

O-GlcNAcylation is an essential post-translational modification important for integrating metabolism with cell physiology. Using diverse model systems, studies of this evolutionarily conserved intracellular glycosylation have highlighted its role in stem cell maintenance, lineage specification, and disease. Although discovered over 30 years ago, the study of O-GlcNAc continues to evolve and uncover surprising roles for O-GlcNAc and the enzymes of O-GlcNAc cycling: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). In this review, using the immune system as a model of stem cell biology and cell fate determination, we discuss how O-GlcNAc is at the nexus of metabolism, proliferation, and disease.


Assuntos
Acetilglucosamina/metabolismo , Sistema Imunitário/metabolismo , Processamento de Proteína Pós-Traducional , Linfócitos T/metabolismo , Diferenciação Celular , Glicosilação , Humanos , Modelos Biológicos , N-Acetilglucosaminiltransferases/metabolismo , Células-Tronco/metabolismo
7.
J Bioenerg Biomembr ; 50(3): 155-173, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29594839

RESUMO

Cancer cells exhibit unregulated growth, altered metabolism, enhanced metastatic potential and altered cell surface glycans. Fueled by oncometabolism and elevated uptake of glucose and glutamine, the hexosamine biosynthetic pathway (HBP) sustains glycosylation in the endomembrane system. In addition, the elevated pools of UDP-GlcNAc drives the O-GlcNAc modification of key targets in the cytoplasm, nucleus and mitochondrion. These targets include transcription factors, kinases, key cytoplasmic enzymes of intermediary metabolism, and electron transport chain complexes. O-GlcNAcylation can thereby alter epigenetics, transcription, signaling, proteostasis, and bioenergetics, key 'hallmarks of cancer'. In this review, we summarize accumulating evidence that many cancer hallmarks are linked to dysregulation of O-GlcNAc cycling on cancer-relevant targets. We argue that onconutrient and oncometabolite-fueled elevation increases HBP flux and triggers O-GlcNAcylation of key regulatory enzymes in glycolysis, Kreb's cycle, pentose-phosphate pathway, and the HBP itself. The resulting rerouting of glucose metabolites leads to elevated O-GlcNAcylation of oncogenes and tumor suppressors further escalating elevation in HBP flux creating a 'vicious cycle'. Downstream, elevated O-GlcNAcylation alters DNA repair and cellular stress pathways which influence oncogenesis. The elevated steady-state levels of O-GlcNAcylated targets found in many cancers may also provide these cells with a selective advantage for sustained growth, enhanced metastatic potential, and immune evasion in the tumor microenvironment.


Assuntos
Acetilglucosamina/metabolismo , Neoplasias/metabolismo , Animais , Vias Biossintéticas , Glicosilação , Humanos
8.
Genet Med ; 20(6): 664-668, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29176683

RESUMO

PurposeAdults with Turner syndrome (TS) have an increased predisposition to ischemic heart disease. The quantitative relationship between coronary atherosclerosis and TS has yet to be established.MethodsA total of 128 females (62 with TS) participated in this prospective study. Coronary computed tomography angiography was performed to measure coronary calcified plaque burden, and prevalent noncalcified plaque burden. Regression analysis was used to study the effects of TS and traditional cardiovascular disease risk factors on coronary plaque burden.ResultsAdults with TS were 63% more likely to have coronary calcifications than controls (odds ratio 1.63, 95% confidence interval: 1.02, 2.61, P = 0.04), with an age cutoff of 51.7 years for a probability of >50% for the presence of coronary calcifications, when compared to 55.7 years in female controls. The average age of TS patients with calcified plaques was significantly lower than that of controls with calcified plaques (51.5 ± 8.9 years vs. 60.5 ± 7.0 years, P < 0.001). Age increased the likelihood of coronary calcifications by 13% per year (odds ratio 1.13, confidence interval 95%: 1.07-1.19, P < 0.001).ConclusionThis study demonstrates a higher prevalence and earlier onset of calcified coronary plaques in TS. These findings have important implications for cardiovascular risk assessment and the management of patients with TS.


Assuntos
Calcinose/fisiopatologia , Cardiomiopatias/fisiopatologia , Doença da Artéria Coronariana/fisiopatologia , Adulto , Calcificação Fisiológica/fisiologia , Calcinose/metabolismo , Angiografia por Tomografia Computadorizada/métodos , Feminino , Humanos , Pessoa de Meia-Idade , Placa Aterosclerótica/fisiopatologia , Prevalência , Estudos Prospectivos , Medição de Risco , Fatores de Risco , Síndrome de Turner/fisiopatologia
9.
J Neurochem ; 144(1): 7-34, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29049853

RESUMO

Proteostasis is essential in the mammalian brain where post-mitotic cells must function for decades to maintain synaptic contacts and memory. The brain is dependent on glucose and other metabolites for proper function and is spared from metabolic deficits even during starvation. In this review, we outline how the nutrient-sensitive nucleocytoplasmic post-translational modification O-linked N-acetylglucosamine (O-GlcNAc) regulates protein homeostasis. The O-GlcNAc modification is highly abundant in the mammalian brain and has been linked to proteopathies, including neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's. C. elegans, Drosophila, and mouse models harboring O-GlcNAc transferase- and O-GlcNAcase-knockout alleles have helped define the role O-GlcNAc plays in development as well as age-associated neurodegenerative disease. These enzymes add and remove the single monosaccharide from protein serine and threonine residues, respectively. Blocking O-GlcNAc cycling is detrimental to mammalian brain development and interferes with neurogenesis, neural migration, and proteostasis. Findings in C. elegans and Drosophila model systems indicate that the dynamic turnover of O-GlcNAc is critical for maintaining levels of key transcriptional regulators responsible for neurodevelopment cell fate decisions. In addition, pathways of autophagy and proteasomal degradation depend on a transcriptional network that is also reliant on O-GlcNAc cycling. Like the quality control system in the endoplasmic reticulum which uses a 'mannose timer' to monitor protein folding, we propose that cytoplasmic proteostasis relies on an 'O-GlcNAc timer' to help regulate the lifetime and fate of nuclear and cytoplasmic proteins. O-GlcNAc-dependent developmental alterations impact metabolism and growth of the developing mouse embryo and persist into adulthood. Brain-selective knockout mouse models will be an important tool for understanding the role of O-GlcNAc in the physiology of the brain and its susceptibility to neurodegenerative injury.


Assuntos
Acetilglucosamina/metabolismo , N-Acetilglucosaminiltransferases/fisiologia , Degeneração Neural/metabolismo , Proteostase/fisiologia , beta-N-Acetil-Hexosaminidases/fisiologia , Animais , Autofagia/fisiologia , Química Encefálica , Proteínas de Caenorhabditis elegans/fisiologia , Ciclo Celular/fisiologia , Movimento Celular/fisiologia , Proteínas de Drosophila/fisiologia , Epigênese Genética , Glicoproteínas/metabolismo , Hexosaminas/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Mamíferos/metabolismo , Camundongos Knockout , Mitocôndrias/metabolismo , Modelos Moleculares , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/deficiência , N-Acetilglucosaminiltransferases/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Agregação Patológica de Proteínas/metabolismo , Conformação Proteica , Domínios Proteicos , Isoformas de Proteínas , beta-N-Acetil-Hexosaminidases/química , beta-N-Acetil-Hexosaminidases/deficiência , beta-N-Acetil-Hexosaminidases/genética
10.
Biochem Soc Trans ; 45(2): 427-436, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28408483

RESUMO

The dynamic carbohydrate post-translational modification (PTM) O-linked ß-N-acetyl glucosamine (O-GlcNAc) is found on thousands of proteins throughout the nucleus and cytoplasm, and rivals phosphorylation in terms of the number of substrates and pathways influenced. O-GlcNAc is highly conserved and essential in most organisms, with disruption of O-GlcNAc cycling linked to diseases ranging from cancer to neurodegeneration. Nuclear pore proteins were the first identified O-GlcNAc-modified substrates, generating intense and ongoing interest in understanding the role of O-GlcNAc cycling in nuclear pore complex structure and function. Recent advances in detecting and altering O-GlcNAcylation levels have provided insights into many mechanisms by which O-GlcNAcylation influences the nucleocytoplasmic localization and stability of protein targets. The emerging view is that the multifunctional enzymes of O-GlcNAc cycling are critical nutrient-sensing components of a complex network of signaling cascades involving multiple PTMs. Furthermore, O-GlcNAc plays a role in maintaining the structural integrity of the nuclear pore and regulating its function as the gatekeeper of nucleocytoplasmic trafficking.


Assuntos
Acetilglucosamina/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Nucleares/química , Acilação , Animais , Humanos , Poro Nuclear/fisiologia , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Transdução de Sinais
11.
Genetics ; 206(2): 939-952, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28428286

RESUMO

Oxidative damage contributes to human diseases of aging including diabetes, cancer, and cardiovascular disorders. Reactive oxygen species resulting from xenobiotic and endogenous metabolites are sensed by a poorly understood process, triggering a cascade of regulatory factors and leading to the activation of the transcription factor Nrf2 (Nuclear factor-erythroid-related factor 2, SKN-1 in Caenorhabditis elegans). Nrf2/SKN-1 activation promotes the induction of the phase II detoxification system that serves to limit oxidative stress. We have extended a previous C. elegans genetic approach to explore the mechanisms by which a phase II enzyme is induced by endogenous and exogenous oxidants. The xrep (xenobiotics response pathway) mutants were isolated as defective in their ability to properly regulate the induction of a glutathione S-transferase (GST) reporter. The xrep-1 gene was previously identified as wdr-23, which encodes a C. elegans homolog of the mammalian ß-propeller repeat-containing protein WDR-23 Here, we identify and confirm the mutations in xrep-2, xrep-3, and xrep-4 The xrep-2 gene is alh-6, an ortholog of a human gene mutated in familial hyperprolinemia. The xrep-3 mutation is a gain-of-function allele of skn-1 The xrep-4 gene is F46F11.6, which encodes a F-box-containing protein. We demonstrate that xrep-4 alters the stability of WDR-23 (xrep-1), a key regulator of SKN-1 (xrep-3). Epistatic relationships among the xrep mutants and their interacting partners allow us to propose an ordered genetic pathway by which endogenous and exogenous stressors induce the phase II detoxification response.


Assuntos
Aldeído Desidrogenase/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ligação a DNA/genética , Glutationa Transferase/genética , Inativação Metabólica/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/genética , Humanos , Redes e Vias Metabólicas/genética , Mutação , Estresse Oxidativo/efeitos dos fármacos , Proteínas Repressoras , Xenobióticos/metabolismo
12.
Cilia ; 6: 2, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28344780

RESUMO

BACKGROUND: The discovery of disease pathogenesis requires systematic agnostic screening of multiple homeostatic processes that may become deregulated. We illustrate this principle in the evaluation and diagnosis of a 5-year-old boy with Joubert syndrome type 10 (JBTS10). He carried the OFD1 mutation p.Gln886Lysfs*2 (NM_003611.2: c.2656del) and manifested features of Joubert syndrome. METHODS: We integrated exome sequencing, MALDI-TOF mass spectrometry analyses of plasma and cultured dermal fibroblasts glycomes, and full clinical evaluation of the proband. Analyses of cilia formation and lectin staining were performed by immunofluorescence. Measurement of cellular nucleotide sugar levels was performed with high-performance anion-exchange chromatography with pulsed amperometric detection. Statistical analyses utilized the Student's and Fisher's exact t tests. RESULTS: Glycome analyses of plasma and cultured dermal fibroblasts identified abnormal N- and O-linked glycosylation profiles. These findings replicated in two unrelated males with OFD1 mutations. Cultured fibroblasts from affected individuals had a defect in ciliogenesis. The proband's fibroblasts also had an abnormally elevated nuclear sialylation signature and increased total cellular levels of CMP-sialic acid. Ciliogenesis and each glycosylation anomaly were rescued by expression of wild-type OFD1. CONCLUSIONS: The rescue of ciliogenesis and glycosylation upon reintroduction of WT OFD1 suggests that both contribute to the pathogenesis of JBTS10.

13.
J Biol Chem ; 292(15): 6076-6085, 2017 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-28246173

RESUMO

Nutrient-driven O-GlcNAcylation is strikingly abundant in the brain and has been linked to development and neurodegenerative disease. We selectively targeted the O-GlcNAcase (Oga) gene in the mouse brain to define the role of O-GlcNAc cycling in the central nervous system. Brain knockout animals exhibited dramatically increased brain O-GlcNAc levels and pleiotropic phenotypes, including early-onset obesity, growth defects, and metabolic dysregulation. Anatomical defects in the Oga knockout included delayed brain differentiation and neurogenesis as well as abnormal proliferation accompanying a developmental delay. The molecular basis for these defects included transcriptional changes accompanying differentiating embryonic stem cells. In Oga KO mouse ES cells, we observed pronounced changes in expression of pluripotency markers, including Sox2, Nanog, and Otx2. These findings link the O-GlcNAc modification to mammalian neurogenesis and highlight the role of this nutrient-sensing pathway in developmental plasticity and metabolic homeostasis.


Assuntos
Acetilglucosamina/metabolismo , Encéfalo/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Neurogênese/fisiologia , Acetilglucosamina/genética , Animais , Encéfalo/citologia , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , N-Acetilglucosaminiltransferases/genética , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Especificidade de Órgãos/fisiologia , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
15.
G3 (Bethesda) ; 7(1): 257-268, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-27856697

RESUMO

Human exome sequencing has dramatically increased the rate of identification of disease-associated polymorphisms. However, examining the functional consequences of those variants has created an analytic bottleneck. Insulin-like signaling in Caenorhabditis elegans has long provided a model to assess consequences of human insulin signaling mutations, but this has not been evaluated in the context of current genetic tools. We have exploited strains derived from the Million Mutation Project (MMP) and gene editing to explore further the evolutionary relationships and conservation between the human and C. elegans insulin receptors. Of 40 MMP alleles analyzed in the C. elegans insulin-like receptor gene DAF-2, 35 exhibited insulin-like signaling indistinguishable from wild-type animals, indicating tolerated mutations. Five MMP alleles proved to be novel dauer-enhancing mutations, including one new allele in the previously uncharacterized C-terminus of DAF-2 CRISPR-Cas9 genome editing was used to confirm the phenotypic consequence of six of these DAF-2 mutations and to replicate an allelic series of known human disease mutations in a highly conserved tyrosine kinase active site residue, demonstrating the utility of C. elegans for directly modeling human disease. Our results illustrate the challenges associated with prediction of the phenotypic consequences of amino acid substitutions, the value of assaying mutant isoform function in vivo, and how recently developed tools and resources afford the opportunity to expand our understanding even of highly conserved regulatory modules such as insulin signaling. This approach may prove generally useful for modeling phenotypic consequences of candidate human pathogenic mutations in conserved signaling and developmental pathways.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Insulina/metabolismo , Longevidade/genética , Receptor de Insulina/genética , Substituição de Aminoácidos/genética , Animais , Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Insulina/genética , Mutação
16.
Mol Genet Metab ; 119(1-2): 144-50, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27471012

RESUMO

Fabry disease is a glycosphingolipid storage disorder that is caused by a genetic deficiency of the enzyme alpha-galactosidase A (AGA, EC 3.2.1.22). It is a multisystem disease that affects the vascular, cardiac, renal, and nervous systems. One of the hallmarks of this disorder is neuropathic pain and sympathetic and parasympathetic nervous dysfunction. The exact mechanism by which changes in AGA activity result in change in neuronal function is not clear, partly due to of a lack of relevant model systems. In this study, we report the development of an in vitro model system to study neuronal dysfunction in Fabry disease by using short-hairpin RNA to create a stable knock-down of AGA in the human cholinergic neuronal cell line, LA-N-2. We show that gene-silenced cells show specifically reduced AGA activity and store globotriaosylceramide. In gene-silenced cells, release of the neurotransmitter acetylcholine is significantly reduced, demonstrating that this model may be used to study specific neuronal functions such as neurotransmitter release in Fabry disease.


Assuntos
Neurônios Colinérgicos/patologia , Doença de Fabry/genética , Neuralgia/metabolismo , alfa-Galactosidase/genética , Neurônios Colinérgicos/metabolismo , Doença de Fabry/metabolismo , Doença de Fabry/patologia , Técnicas de Silenciamento de Genes , Terapia Genética , Humanos , Rim/metabolismo , Rim/patologia , Neuralgia/genética , Neuralgia/patologia , Sistema Nervoso Parassimpático/metabolismo , Sistema Nervoso Parassimpático/patologia , RNA Interferente Pequeno/genética , Sistema Nervoso Simpático/metabolismo , Sistema Nervoso Simpático/patologia , Triexosilceramidas/metabolismo , alfa-Galactosidase/biossíntese
17.
J Biol Chem ; 291(19): 9906-19, 2016 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-26957542

RESUMO

Gene expression during Drosophila development is subject to regulation by the Polycomb (Pc), Trithorax (Trx), and Compass chromatin modifier complexes. O-GlcNAc transferase (OGT/SXC) is essential for Pc repression suggesting that the O-GlcNAcylation of proteins plays a key role in regulating development. OGT transfers O-GlcNAc onto serine and threonine residues in intrinsically disordered domains of key transcriptional regulators; O-GlcNAcase (OGA) removes the modification. To pinpoint genomic regions that are regulated by O-GlcNAc levels, we performed ChIP-chip and microarray analysis after OGT or OGA RNAi knockdown in S2 cells. After OGA RNAi, we observed a genome-wide increase in the intensity of most O-GlcNAc-occupied regions including genes linked to cell cycle, ubiquitin, and steroid response. In contrast, O-GlcNAc levels were strikingly insensitive to OGA RNAi at sites of polycomb repression such as the Hox and NK homeobox gene clusters. Microarray analysis suggested that altered O-GlcNAc cycling perturbed the expression of genes associated with morphogenesis and cell cycle regulation. We then produced a viable null allele of oga (oga(del.1)) in Drosophila allowing visualization of altered O-GlcNAc cycling on polytene chromosomes. We found that trithorax (TRX), absent small or homeotic discs 1 (ASH1), and Compass member SET1 histone methyltransferases were O-GlcNAc-modified in oga(del.1) mutants. The oga(del.1) mutants displayed altered expression of a distinct set of cell cycle-related genes. Our results show that the loss of OGA in Drosophila globally impacts the epigenetic machinery allowing O-GlcNAc accumulation on RNA polymerase II and numerous chromatin factors including TRX, ASH1, and SET1.


Assuntos
Acetilglucosamina/metabolismo , Cromatina/metabolismo , Drosophila/enzimologia , Epigênese Genética/genética , N-Acetilglucosaminiltransferases/genética , Processamento de Proteína Pós-Traducional , Deleção de Sequência , Animais , Western Blotting , Células Cultivadas , Cromatina/genética , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Imunoprecipitação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma
18.
J Biol Methods ; 3(2): e41, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-31453208

RESUMO

The genetically amenable mouse model has led to a large collection of genetically defined lines from which mouse embryonic fibroblasts (MEFs) have been derived. Despite their widespread use, MEFs are time consuming to generate and have a limited lifespan. Immortalizing primary MEFs with the desired genetic manipulations greatly reduces culture maintenance time, enables the generation of near limitless amounts of protein lysate, and facilitates biological replicates during experimentation. In this work, we have evaluated several approaches for MEF immortalization. When cultivated at 3% O2, some primary MEF lines could be proliferated for > 40 passages with a median doubling rate of 45 ± 55 h (n = 8). However, serial passaging at 3% O2 achieved spontaneous immortalization with varying success. If cultures seemed to be reaching their Hayflick limit when cultivated at 3% O2, supplementing the culture media with 5 µM ROCK inhibitor Y-27632 helped to extend proliferation and achieve spontaneous immortalization. MEFs immortalized via SV40 Ta infection reliably produced cell lines with a median doubling rate of 25 ± 9 h (n = 9) and viability greater than 90%. In addition to a discussion of the characteristics of cell lines generated with various immortalization strategies, pros and cons of each strategy are included as are recommendations for generating immortalized MEFs.

19.
Curr Opin Clin Nutr Metab Care ; 18(4): 339-45, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26049631

RESUMO

PURPOSE OF REVIEW: The O-linked N-acetylglucosamine (O-GlcNAc) modification is both responsive to nutrient availability and capable of altering intracellular cellular signalling. We summarize data defining a role for O-GlcNAcylation in metabolic homeostasis and epigenetic regulation of development in the intrauterine environment. RECENT FINDINGS: O-GlcNAc transferase (OGT) catalyzes nutrient-driven O-GlcNAc addition and is subject to random X-inactivation. OGT plays key roles in growth factor signalling, stem cell biology, epigenetics and possibly imprinting. The O-GlcNAcase, which removes O-GlcNAc, is subject to tight regulation by higher order chromatin structure. O-GlcNAc cycling plays an important role in the intrauterine environment wherein OGT expression is an important biomarker of placental stress. SUMMARY: Regulation of O-GlcNAc cycling by X-inactivation, epigenetic regulation and nutrient-driven processes makes it an ideal candidate for a nutrient-dependent epigenetic regulator of human disease. In addition, O-GlcNAc cycling influences chromatin modifiers critical to the regulation and timing of normal development including the polycomb repression complex and the ten-eleven translocation proteins mediating DNA methyl cytosine demethylation. The pathway also impacts the hypothalamic-pituitary-adrenal axis critical to intrauterine programming influencing disease susceptibility in later life.


Assuntos
Acetilglucosamina/administração & dosagem , Acetilglucosamina/efeitos adversos , Epigênese Genética , Comportamento Alimentar , Doença de Alzheimer/etiologia , Doença de Alzheimer/genética , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/genética , Cromatina/genética , Cromatina/metabolismo , Doença Crônica , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/genética , Dieta , Feminino , Regulação da Expressão Gênica , Loci Gênicos , Impressão Genômica , Homeostase/efeitos dos fármacos , Humanos , Sistema Hipotálamo-Hipofisário/metabolismo , Lúpus Eritematoso Sistêmico/etiologia , Lúpus Eritematoso Sistêmico/genética , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Neoplasias/etiologia , Neoplasias/genética , Neurogênese/efeitos dos fármacos , Obesidade/etiologia , Obesidade/genética , Processamento de Proteína Pós-Traducional , Inativação do Cromossomo X/fisiologia
20.
J Cell Biol ; 208(7): 869-80, 2015 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-25825515

RESUMO

Unlike the complex glycans decorating the cell surface, the O-linked ß-N-acetyl glucosamine (O-GlcNAc) modification is a simple intracellular Ser/Thr-linked monosaccharide that is important for disease-relevant signaling and enzyme regulation. O-GlcNAcylation requires uridine diphosphate-GlcNAc, a precursor responsive to nutrient status and other environmental cues. Alternative splicing of the genes encoding the O-GlcNAc cycling enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) yields isoforms targeted to discrete sites in the nucleus, cytoplasm, and mitochondria. OGT and OGA also partner with cellular effectors and act in tandem with other posttranslational modifications. The enzymes of O-GlcNAc cycling act preferentially on intrinsically disordered domains of target proteins impacting transcription, metabolism, apoptosis, organelle biogenesis, and transport.


Assuntos
Acetilglucosamina/química , Mitocôndrias/metabolismo , N-Acetilglucosaminiltransferases/genética , Acetilglucosamina/análogos & derivados , Acetilglucosamina/biossíntese , Processamento Alternativo , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Células-Tronco/metabolismo , Difosfato de Uridina/análogos & derivados , Difosfato de Uridina/química
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