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1.
J Biol Chem ; 300(5): 107271, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38588813

RESUMO

Lafora disease (LD) is an autosomal recessive myoclonus epilepsy with onset in the teenage years leading to death within a decade of onset. LD is characterized by the overaccumulation of hyperphosphorylated, poorly branched, insoluble, glycogen-like polymers called Lafora bodies. The disease is caused by mutations in either EPM2A, encoding laforin, a dual specificity phosphatase that dephosphorylates glycogen, or EMP2B, encoding malin, an E3-ubiquitin ligase. While glycogen is a widely accepted laforin substrate, substrates for malin have been difficult to identify partly due to the lack of malin antibodies able to detect malin in vivo. Here we describe a mouse model in which the malin gene is modified at the C-terminus to contain the c-myc tag sequence, making an expression of malin-myc readily detectable. Mass spectrometry analyses of immunoprecipitates using c-myc tag antibodies demonstrate that malin interacts with laforin and several glycogen-metabolizing enzymes. To investigate the role of laforin in these interactions we analyzed two additional mouse models: malin-myc/laforin knockout and malin-myc/LaforinCS, where laforin was either absent or the catalytic Cys was genomically mutated to Ser, respectively. The interaction of malin with partner proteins requires laforin but is not dependent on its catalytic activity or the presence of glycogen. Overall, the results demonstrate that laforin and malin form a complex in vivo, which stabilizes malin and enhances interaction with partner proteins to facilitate normal glycogen metabolism. They also provide insights into the development of LD and the rescue of the disease by the catalytically inactive phosphatase.


Assuntos
Doença de Lafora , Proteínas Tirosina Fosfatases não Receptoras , Ubiquitina-Proteína Ligases , Doença de Lafora/metabolismo , Doença de Lafora/genética , Doença de Lafora/patologia , Animais , Camundongos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/genética , Humanos , Fosfatases de Especificidade Dupla/metabolismo , Fosfatases de Especificidade Dupla/genética , Modelos Animais de Doenças , Glicogênio/metabolismo , Glicogênio/genética
2.
J Biol Chem ; 292(25): 10455-10464, 2017 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-28483921

RESUMO

Disruption of the Gys2 gene encoding the liver isoform of glycogen synthase generates a mouse strain (LGSKO) that almost completely lacks hepatic glycogen, has impaired glucose disposal, and is pre-disposed to entering the fasted state. This study investigated how the lack of liver glycogen increases fat accumulation and the development of liver insulin resistance. Insulin signaling in LGSKO mice was reduced in liver, but not muscle, suggesting an organ-specific defect. Phosphorylation of components of the hepatic insulin-signaling pathway, namely IRS1, Akt, and GSK3, was decreased in LGSKO mice. Moreover, insulin stimulation of their phosphorylation was significantly suppressed, both temporally and in an insulin dose response. Phosphorylation of the insulin-regulated transcription factor FoxO1 was somewhat reduced and insulin treatment did not elicit normal translocation of FoxO1 out of the nucleus. Fat overaccumulated in LGSKO livers, showing an aberrant distribution in the acinus, an increase not explained by a reduction in hepatic triglyceride export. Rather, when administered orally to fasted mice, glucose was directed toward hepatic lipogenesis as judged by the activity, protein levels, and expression of several fatty acid synthesis genes, namely, acetyl-CoA carboxylase, fatty acid synthase, SREBP1c, chREBP, glucokinase, and pyruvate kinase. Furthermore, using cultured primary hepatocytes, we found that lipogenesis was increased by 40% in LGSKO cells compared with controls. Of note, the hepatic insulin resistance was not associated with increased levels of pro-inflammatory markers. Our results suggest that loss of liver glycogen synthesis diverts glucose toward fat synthesis, correlating with impaired hepatic insulin signaling and glucose disposal.


Assuntos
Núcleo Celular/metabolismo , Fígado Gorduroso/metabolismo , Glicogênio/deficiência , Hepatócitos/metabolismo , Resistência à Insulina , Transdução de Sinais , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Transporte Ativo do Núcleo Celular/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Núcleo Celular/genética , Núcleo Celular/patologia , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Glicogênio/genética , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Hepatócitos/patologia , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
Biochemistry ; 56(1): 179-188, 2017 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-27935293

RESUMO

Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS is negatively regulated by covalent phosphorylation and allosterically activated by glucose-6-phosphate (G-6-P). To gain structural insights into the inhibited state of the enzyme, we solved the crystal structure of yGsy2-R589A/R592A to a resolution of 3.3 Å. The double mutant has an activity ratio similar to the phosphorylated enzyme and also retains the ability to be activated by G-6-P. When compared to the 2.88 Å structure of the wild-type G-6-P activated enzyme, the crystal structure of the low-activity mutant showed that the N-terminal domain of the inhibited state is tightly held against the dimer-related interface thereby hindering acceptor access to the catalytic cleft. On the basis of these two structural observations, we developed a reversible redox regulatory feature in yeast GS by substituting cysteine residues for two highly conserved arginine residues. When oxidized, the cysteine mutant enzyme exhibits activity levels similar to the phosphorylated enzyme but cannot be activated by G-6-P. Upon reduction, the cysteine mutant enzyme regains normal activity levels and regulatory response to G-6-P activation.


Assuntos
Glicogênio Sintase/genética , Mutação , Saccharomyces cerevisiae/genética , Cristalização , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Glucose-6-Fosfato/metabolismo , Glucose-6-Fosfato/farmacologia , Glicogênio/metabolismo , Glicogênio Sintase/química , Glicogênio Sintase/metabolismo , Cinética , Modelos Moleculares , Oxirredução , Fosforilação , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Uridina Difosfato Glucose/metabolismo
4.
Glycobiology ; 27(5): 416-424, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28077463

RESUMO

y: Glycogen, a branched polymer of glucose, functions as an energy reserve in many living organisms. Abnormalities in glycogen metabolism, usually excessive accumulation, can be caused genetically, most often through mutation of the enzymes directly involved in synthesis and degradation of the polymer leading to a variety of glycogen storage diseases (GSDs). Microscopic visualization of glycogen deposits in cells and tissues is important for the study of normal glycogen metabolism as well as diagnosis of GSDs. Here, we describe a method for the detection of glycogen using a renewable, recombinant protein which contains the carbohydrate-binding module (CBM) from starch-binding domain containing protein 1 (Stbd1). We generated a fusion protein containing g lutathione S-transferase, a cM c eptitope and the tbd1 BM (GYSC) for use as a glycogen-binding probe, which can be detected with secondary antibodies against glutathione S-transferase or cMyc. By enzyme-linked immunosorbent assay, we demonstrate that GYSC binds glycogen and two other polymers of glucose, amylopectin and amylose. Immunofluorescence staining of cultured cells indicate a GYSC-specific signal that is co-localized with signals obtained with anti-glycogen or anti-glycogen synthase antibodies. GYSC-positive staining inside of lysosomes is observed in individual muscle fibers isolated from mice deficient in lysosomal enzyme acid alpha-glucosidase, a well-characterized model of GSD II (Pompe disease). Co-localized GYSC and glycogen signals are also found in muscle fibers isolated from mice deficient in malin, a model for Lafora disease. These data indicate that GYSC is a novel probe that can be used to study glycogen metabolism under normal and pathological conditions.


Assuntos
Glucose/metabolismo , Doença de Depósito de Glicogênio/diagnóstico , Glicogênio/isolamento & purificação , Doença de Lafora/diagnóstico , Animais , Ensaio de Imunoadsorção Enzimática , Glutationa Transferase/química , Glicogênio/química , Glicogênio/metabolismo , Doença de Depósito de Glicogênio/metabolismo , Humanos , Doença de Lafora/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/química , Camundongos , Proteínas Musculares/química , Proteínas Recombinantes/química
5.
J Biol Chem ; 290(2): 841-50, 2015 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-25416783

RESUMO

Glycogen is a branched polymer of glucose that acts as an energy reserve in many cell types. Glycogen contains trace amounts of covalent phosphate, in the range of 1 phosphate per 500-2000 glucose residues depending on the source. The function, if any, is unknown, but in at least one genetic disease, the progressive myoclonic epilepsy Lafora disease, excessive phosphorylation of glycogen has been implicated in the pathology by disturbing glycogen structure. Some 90% of Lafora cases are attributed to mutations of the EPM2A or EPM2B genes, and mice with either gene disrupted accumulate hyperphosphorylated glycogen. It is, therefore, of importance to understand the chemistry of glycogen phosphorylation. Rabbit skeletal muscle glycogen contained covalent phosphate as monoesters of C2, C3, and C6 carbons of glucose residues based on analyses of phospho-oligosaccharides by NMR. Furthermore, using a sensitive assay for glucose 6-P in hydrolysates of glycogen coupled with measurement of total phosphate, we determined the proportion of C6 phosphorylation in rabbit muscle glycogen to be ∼20%. C6 phosphorylation also accounted for ∼20% of the covalent phosphate in wild type mouse muscle glycogen. Glycogen phosphorylation in Epm2a(-/-) and Epm2b(-/-) mice was increased 8- and 4-fold compared with wild type mice, but the proportion of C6 phosphorylation remained unchanged at ∼20%. Therefore, our results suggest that C2, C3, and/or C6 phosphate could all contribute to abnormal glycogen structure or to Lafora disease.


Assuntos
Glicogênio/genética , Glicogênio/metabolismo , Doença de Lafora/genética , Doença de Lafora/metabolismo , Animais , Modelos Animais de Doenças , Fosfatases de Especificidade Dupla/genética , Glucose-6-Fosfato/metabolismo , Glicogênio/química , Humanos , Doença de Lafora/patologia , Camundongos , Camundongos Transgênicos , Mutação , Fosforilação , Proteínas Tirosina Fosfatases não Receptoras , Coelhos , Ubiquitina-Proteína Ligases/genética
6.
J Biol Chem ; 290(37): 22686-98, 2015 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-26216881

RESUMO

Glycogen, the repository of glucose in many cell types, contains small amounts of covalent phosphate, of uncertain function and poorly understood metabolism. Loss-of-function mutations in the laforin gene cause the fatal neurodegenerative disorder, Lafora disease, characterized by increased glycogen phosphorylation and the formation of abnormal deposits of glycogen-like material called Lafora bodies. It is generally accepted that the phosphate is removed by the laforin phosphatase. To study the dynamics of skeletal muscle glycogen phosphorylation in vivo under physiological conditions, mice were subjected to glycogen-depleting exercise and then monitored while they resynthesized glycogen. Depletion of glycogen by exercise was associated with a substantial reduction in total glycogen phosphate and the newly resynthesized glycogen was less branched and less phosphorylated. Branching returned to normal on a time frame of days, whereas phosphorylation remained suppressed over a longer period of time. We observed no change in markers of autophagy. Exercise of 3-month-old laforin knock-out mice caused a similar depletion of glycogen but no loss of glycogen phosphate. Furthermore, remodeling of glycogen to restore the basal branching pattern was delayed in the knock-out animals. From these results, we infer that 1) laforin is responsible for glycogen dephosphorylation during exercise and acts during the cytosolic degradation of glycogen, 2) excess glycogen phosphorylation in the absence of laforin delays the normal remodeling of the branching structure, and 3) the accumulation of glycogen phosphate is a relatively slow process involving multiple cycles of glycogen synthesis-degradation, consistent with the slow onset of the symptoms of Lafora disease.


Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Glicogênio/metabolismo , Músculo Esquelético/metabolismo , Condicionamento Físico Animal , Animais , Fosfatases de Especificidade Dupla/genética , Glicogênio/genética , Doença de Lafora/genética , Doença de Lafora/metabolismo , Doença de Lafora/patologia , Camundongos , Camundongos Knockout , Músculo Esquelético/patologia , Fosforilação/genética , Proteínas Tirosina Fosfatases não Receptoras
7.
Arch Biochem Biophys ; 597: 21-9, 2016 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-27036853

RESUMO

The storage polymer glycogen normally contains small amounts of covalently attached phosphate as phosphomonoesters at C2, C3 and C6 atoms of glucose residues. In the absence of the laforin phosphatase, as in the rare childhood epilepsy Lafora disease, the phosphorylation level is elevated and is associated with abnormal glycogen structure that contributes to the pathology. Laforin therefore likely functions in vivo as a glycogen phosphatase. The mechanism of glycogen phosphorylation is less well-understood. We have reported that glycogen synthase incorporates phosphate into glycogen via a rare side reaction in which glucose-phosphate rather than glucose is transferred to a growing polyglucose chain (Tagliabracci et al. (2011) Cell Metab13, 274-282). We proposed a mechanism to account for phosphorylation at C2 and possibly at C3. Our results have since been challenged (Nitschke et al. (2013) Cell Metab17, 756-767). Here we extend the evidence supporting our conclusion, validating the assay used for the detection of glycogen phosphorylation, measurement of the transfer of (32)P from [ß-(32)P]UDP-glucose to glycogen by glycogen synthase. The (32)P associated with the glycogen fraction was stable to ethanol precipitation, SDS-PAGE and gel filtration on Sephadex G50. The (32)P-signal was not affected by inclusion of excess unlabeled UDP before analysis or by treatment with a UDPase, arguing against the signal being due to contaminating [ß-(32)P]UDP generated in the reaction. Furthermore, [(32)P]UDP did not bind non-covalently to glycogen. The (32)P associated with glycogen was released by laforin treatment, suggesting that it was present as a phosphomonoester. The conclusion is that glycogen synthase can mediate the introduction of phosphate into glycogen, thereby providing a possible mechanism for C2, and perhaps C3, phosphorylation.


Assuntos
Glicogênio Sintase/química , Glicogênio/química , Fosfatos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Glicogênio/biossíntese , Glicogênio Sintase/metabolismo , Humanos , Fosfatos/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/química , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Açúcares de Uridina Difosfato/química , Açúcares de Uridina Difosfato/metabolismo
8.
Proc Natl Acad Sci U S A ; 110(52): 20976-81, 2013 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-24324135

RESUMO

Glycogen is a glucose polymer that contains minor amounts of covalently attached phosphate. Hyperphosphorylation is deleterious to glycogen structure and can lead to Lafora disease. Recently, it was demonstrated that glycogen synthase catalyzes glucose-phosphate transfer in addition to its characteristic glucose transfer reaction. Glucose-1,2-cyclic-phosphate (GCP) was proposed to be formed from UDP-Glc breakdown and subsequently transferred, thus providing a source of phosphate found in glycogen. To gain further insight into the molecular basis for glucose-phosphate transfer, two structures of yeast glycogen synthase were determined; a 3.0-Å resolution structure of the complex with UMP/GCP and a 2.8-Å resolution structure of the complex with UDP/glucose. Structural superposition of the complexes revealed that the bound ligands and most active site residues are positioned similarly, consistent with the use of a common transfer mechanism for both reactions. The N-terminal domain of the UDP-glucose complex was found to be 13.3° more closed compared with a UDP complex. However, the UMP · GCP complex was 4.8° less closed than the glucose complex, which may explain the low efficiency of GCP transfer. Modeling of either α- or ß-glucose or a mixture of both anomers can account for the observed electron density of the UDP-glucose complex. NMR studies of UDP-Glc hydrolysis by yeast glycogen synthase were used to verify the stereochemistry of the product, and they also showed synchronous GCP accumulation. The similarities in the active sites of glycogen synthase and glycogen phosphorylase support the idea of a common catalytic mechanism in GT-B enzymes independent of the specific reaction catalyzed.


Assuntos
Glicogênio Sintase/metabolismo , Glicogênio/química , Modelos Moleculares , Fosfatos/química , Cristalografia , Glicogênio/metabolismo , Glicogênio Sintase/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Mutagênese , Fosfatos/metabolismo
9.
J Biol Chem ; 289(30): 20606-14, 2014 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-24914213

RESUMO

Lafora disease is a progressive myoclonus epilepsy caused by mutations in the EPM2A or EPM2B genes that encode a glycogen phosphatase, laforin, and an E3 ubiquitin ligase, malin, respectively. Lafora disease is characterized by accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle, heart, and liver. The laforinmalin complex has been proposed to play a role in the regulation of glycogen metabolism and protein quality control. We evaluated three arms of the protein degradation/ quality control process (the autophago-lysosomal pathway, the ubiquitin-proteasomal pathway, and the endoplasmic reticulum (ER) stress response) in mouse embryonic fibroblasts from Epm2a(-/-), Epm2b(-/-), and Epm2a(-/-) Epm2b(-/-) mice. The levels of LC3-II, a marker of autophagy, were decreased in all knock-out cells as compared with wild type even though they still showed a slight response to starvation and rapamycin. Furthermore, ribosomal protein S6 kinase and S6 phosphorylation were increased. Under basal conditions there was no effect on the levels of ubiquitinated proteins in the knock-out cells, but ubiquitinated protein degradation was decreased during starvation or stress. Lack of malin (Epm2b(-/-) and Epm2a(-/-) Epm2b(-/-) cells) but not laforin (Epm2a(-/-) cells) decreased LAMP1, a lysosomal marker. CHOP expression was similar in wild type and knock-out cells under basal conditions or with ER stress-inducing agents. In conclusion, both laforin and malin knock-out cells display mTOR-dependent autophagy defects and reduced proteasomal activity but no defects in the ER stress response. We speculate that these defects may be secondary to glycogen overaccumulation. This study also suggests a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal.


Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Lisossomos/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Animais , Fosfatases de Especificidade Dupla/genética , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/genética , Camundongos , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Tirosina Fosfatases não Receptoras , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Ubiquitina-Proteína Ligases/genética
10.
J Biol Chem ; 289(9): 5510-7, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24398675

RESUMO

Sterol regulatory element-binding protein-1 (SREBP-1) is a key transcription factor that regulates genes in the de novo lipogenesis and glycolysis pathways. The levels of SREBP-1 are significantly elevated in obese patients and in animal models of obesity and type 2 diabetes, and a vast number of studies have implicated this transcription factor as a contributor to hepatic lipid accumulation and insulin resistance. However, its role in regulating carbohydrate metabolism is poorly understood. Here we have addressed whether SREBP-1 is needed for regulating glucose homeostasis. Using RNAi and a new generation of adenoviral vector, we have silenced hepatic SREBP-1 in normal and obese mice. In normal animals, SREBP-1 deficiency increased Pck1 and reduced glycogen deposition during fed conditions, providing evidence that SREBP-1 is necessary to regulate carbohydrate metabolism during the fed state. Knocking SREBP-1 down in db/db mice resulted in a significant reduction in triglyceride accumulation, as anticipated. However, mice remained hyperglycemic, which was associated with up-regulation of gluconeogenesis gene expression as well as decreased glycolysis and glycogen synthesis gene expression. Furthermore, glycogen synthase activity and glycogen accumulation were significantly reduced. In conclusion, silencing both isoforms of SREBP-1 leads to significant changes in carbohydrate metabolism and does not improve insulin resistance despite reducing steatosis in an animal model of obesity and type 2 diabetes.


Assuntos
Regulação da Expressão Gênica/fisiologia , Gluconeogênese/fisiologia , Glicogênio/biossíntese , Fígado/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Animais , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Técnicas de Silenciamento de Genes , Glicogênio/genética , Masculino , Camundongos , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , Proteína de Ligação a Elemento Regulador de Esterol 1/genética
11.
Hum Mol Genet ; 21(7): 1604-10, 2012 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-22186021

RESUMO

Lafora disease is a fatal, progressive myoclonus epilepsy caused in ~90% of cases by mutations in the EPM2A or EPM2B genes. Characteristic of the disease is the formation of Lafora bodies, insoluble deposits containing abnormal glycogen-like material in many tissues, including neurons, muscle, heart and liver. Because glycogen is important for glucose homeostasis, the aberrant glycogen metabolism in Lafora disease might disturb whole-body glucose handling. Indeed, Vernia et al. [Vernia, S., Heredia, M., Criado, O., Rodriguez de Cordoba, S., Garcia-Roves, P.M., Cansell, C., Denis, R., Luquet, S., Foufelle, F., Ferre, P. et al. (2011) Laforin, a dual-specificity phosphatase involved in Lafora disease, regulates insulin response and whole-body energy balance in mice. Hum. Mol. Genet., 20, 2571-2584] reported that Epm2a-/- mice had enhanced glucose disposal and insulin sensitivity, leading them to suggest that laforin, the Epm2a gene product, is involved in insulin signaling. We analyzed 3-month- and 6-7-month-old Epm2a-/- mice and observed no differences in glucose tolerance tests (GTTs) or insulin tolerance tests (ITTs) compared with wild-type mice of matched genetic background. At 3 months, Epm2b-/- mice also showed no differences in GTTs and ITTs. In the 6-7-month-old Epm2a-/- mice, there was no evidence for increased insulin stimulation of the phosphorylation of Akt, GSK-3 or S6 in skeletal muscle, liver and heart. From metabolic analyses, these animals were normal with regard to food intake, oxygen consumption, energy expenditure and respiratory exchange ratio. By dual-energy X-ray absorptiometry scan, body composition was unaltered at 3 or 6-7 months of age. Echocardiography showed no defects of cardiac function in Epm2a-/- or Epm2b-/- mice. We conclude that laforin and malin have no effect on whole-body glucose metabolism and insulin sensitivity, and that laforin is not involved in insulin signaling.


Assuntos
Glicemia/análise , Fosfatases de Especificidade Dupla/genética , Resistência à Insulina , Ubiquitina-Proteína Ligases/genética , Animais , Coração/fisiologia , Insulina/farmacologia , Camundongos , Camundongos Knockout , Proteínas Tirosina Fosfatases não Receptoras , Transdução de Sinais
12.
PLoS Genet ; 7(4): e1002037, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21552327

RESUMO

Lafora disease is the most common teenage-onset neurodegenerative disease, the main teenage-onset form of progressive myoclonus epilepsy (PME), and one of the severest epilepsies. Pathologically, a starch-like compound, polyglucosan, accumulates in neuronal cell bodies and overtakes neuronal small processes, mainly dendrites. Polyglucosan formation is catalyzed by glycogen synthase, which is activated through dephosphorylation by glycogen-associated protein phosphatase-1 (PP1). Here we remove PTG, one of the proteins that target PP1 to glycogen, from mice with Lafora disease. This results in near-complete disappearance of polyglucosans and in resolution of neurodegeneration and myoclonic epilepsy. This work discloses an entryway to treating this fatal epilepsy and potentially other glycogen storage diseases.


Assuntos
Glucanos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Doença de Lafora/fisiopatologia , Animais , Encéfalo/fisiopatologia , Modelos Animais de Doenças , Glucanos/análise , Glicogênio Sintase/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Doença de Lafora/genética , Camundongos , Camundongos Knockout
13.
Nat Metab ; 6(3): 494-513, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38443593

RESUMO

Long-lasting pain stimuli can trigger maladaptive changes in the spinal cord, reminiscent of plasticity associated with memory formation. Metabolic coupling between astrocytes and neurons has been implicated in neuronal plasticity and memory formation in the central nervous system, but neither its involvement in pathological pain nor in spinal plasticity has been tested. Here we report a form of neuroglia signalling involving spinal astrocytic glycogen dynamics triggered by persistent noxious stimulation via upregulation of the Protein Targeting to Glycogen (PTG) in spinal astrocytes. PTG drove glycogen build-up in astrocytes, and blunting glycogen accumulation and turnover by Ptg gene deletion reduced pain-related behaviours and promoted faster recovery by shortening pain maintenance in mice. Furthermore, mechanistic analyses revealed that glycogen dynamics is a critically required process for maintenance of pain by facilitating neuronal plasticity in spinal lamina 1 neurons. In summary, our study describes a previously unappreciated mechanism of astrocyte-neuron metabolic communication through glycogen breakdown in the spinal cord that fuels spinal neuron hyperexcitability.


Assuntos
Astrócitos , Dor , Camundongos , Animais , Astrócitos/metabolismo , Dor/metabolismo , Dor/patologia , Neurônios/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia , Glicogênio/metabolismo
14.
Sci Transl Med ; 16(730): eadf1691, 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38232139

RESUMO

Glycogen synthase 1 (GYS1), the rate-limiting enzyme in muscle glycogen synthesis, plays a central role in energy homeostasis and has been proposed as a therapeutic target in multiple glycogen storage diseases. Despite decades of investigation, there are no known potent, selective small-molecule inhibitors of this enzyme. Here, we report the preclinical characterization of MZ-101, a small molecule that potently inhibits GYS1 in vitro and in vivo without inhibiting GYS2, a related isoform essential for synthesizing liver glycogen. Chronic treatment with MZ-101 depleted muscle glycogen and was well tolerated in mice. Pompe disease, a glycogen storage disease caused by mutations in acid α glucosidase (GAA), results in pathological accumulation of glycogen and consequent autophagolysosomal abnormalities, metabolic dysregulation, and muscle atrophy. Enzyme replacement therapy (ERT) with recombinant GAA is the only approved treatment for Pompe disease, but it requires frequent infusions, and efficacy is limited by suboptimal skeletal muscle distribution. In a mouse model of Pompe disease, chronic oral administration of MZ-101 alone reduced glycogen buildup in skeletal muscle with comparable efficacy to ERT. In addition, treatment with MZ-101 in combination with ERT had an additive effect and could normalize muscle glycogen concentrations. Biochemical, metabolomic, and transcriptomic analyses of muscle tissue demonstrated that lowering of glycogen concentrations with MZ-101, alone or in combination with ERT, corrected the cellular pathology in this mouse model. These data suggest that substrate reduction therapy with GYS1 inhibition may be a promising therapeutic approach for Pompe disease and other glycogen storage diseases.


Assuntos
Doença de Depósito de Glicogênio Tipo II , Camundongos , Animais , Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Glicogênio Sintase/metabolismo , Glicogênio Sintase/farmacologia , Camundongos Knockout , Glicogênio/metabolismo , Músculo Esquelético/metabolismo , Terapia de Reposição de Enzimas/métodos
15.
Biochem J ; 441(3): 763-87, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22248338

RESUMO

Glycogen is a branched polymer of glucose that acts as a store of energy in times of nutritional sufficiency for utilization in times of need. Its metabolism has been the subject of extensive investigation and much is known about its regulation by hormones such as insulin, glucagon and adrenaline (epinephrine). There has been debate over the relative importance of allosteric compared with covalent control of the key biosynthetic enzyme, glycogen synthase, as well as the relative importance of glucose entry into cells compared with glycogen synthase regulation in determining glycogen accumulation. Significant new developments in eukaryotic glycogen metabolism over the last decade or so include: (i) three-dimensional structures of the biosynthetic enzymes glycogenin and glycogen synthase, with associated implications for mechanism and control; (ii) analyses of several genetically engineered mice with altered glycogen metabolism that shed light on the mechanism of control; (iii) greater appreciation of the spatial aspects of glycogen metabolism, including more focus on the lysosomal degradation of glycogen; and (iv) glycogen phosphorylation and advances in the study of Lafora disease, which is emerging as a glycogen storage disease.


Assuntos
Biologia/tendências , Glicogênio/metabolismo , Glicogênio/fisiologia , Sequência de Aminoácidos , Animais , Metabolismo dos Carboidratos/genética , Metabolismo dos Carboidratos/fisiologia , Formação de Conceito , Gluconeogênese/fisiologia , Glicogênio/química , Glicogenólise/fisiologia , Humanos , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/fisiologia , Camundongos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
16.
Proc Natl Acad Sci U S A ; 107(41): 17563-8, 2010 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-20876143

RESUMO

Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to the basal activity state and glucose-6-phosphate activated state of yeast glycogen synthase-2. The enzyme is assembled into an unusual tetramer by an insertion unique to the eukaryotic enzymes, and this subunit interface is rearranged by the binding of glucose-6-phosphate, which frees the active site cleft and facilitates catalysis. Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we demonstrate that the enzyme's response to glucose-6-phosphate is controlled by Arg583 and Arg587, while four additional arginine residues present within the same regulatory helix regulate the response to phosphorylation.


Assuntos
Ativação Enzimática/fisiologia , Glucose-6-Fosfato/fisiologia , Glicogênio Sintase/química , Glicogênio Sintase/metabolismo , Modelos Moleculares , Conformação Proteica , Arginina/metabolismo , Cristalização , Glucose-6-Fosfato/metabolismo , Mutagênese , Fosforilação , Ligação Proteica , Saccharomyces cerevisiae
17.
J Biol Chem ; 286(39): 33999-4006, 2011 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-21835915

RESUMO

Glycogen synthase is a rate-limiting enzyme in the biosynthesis of glycogen and has an essential role in glucose homeostasis. The three-dimensional structures of yeast glycogen synthase (Gsy2p) complexed with maltooctaose identified four conserved maltodextrin-binding sites distributed across the surface of the enzyme. Site-1 is positioned on the N-terminal domain, site-2 and site-3 are present on the C-terminal domain, and site-4 is located in an interdomain cleft adjacent to the active site. Mutation of these surface sites decreased glycogen binding and catalytic efficiency toward glycogen. Mutations within site-1 and site-2 reduced the V(max)/S(0.5) for glycogen by 40- and 70-fold, respectively. Combined mutation of site-1 and site-2 decreased the V(max)/S(0.5) for glycogen by >3000-fold. Consistent with the in vitro data, glycogen accumulation in glycogen synthase-deficient yeast cells (Δgsy1-gsy2) transformed with the site-1, site-2, combined site-1/site-2, or site-4 mutant form of Gsy2p was decreased by up to 40-fold. In contrast to the glycogen results, the ability to utilize maltooctaose as an in vitro substrate was unaffected in the site-2 mutant, moderately affected in the site-1 mutant, and almost completely abolished in the site-4 mutant. These data show that the ability to utilize maltooctaose as a substrate can be independent of the ability to utilize glycogen. Our data support the hypothesis that site-1 and site-2 provide a "toehold mechanism," keeping glycogen synthase tightly associated with the glycogen particle, whereas site-4 is more closely associated with positioning of the nonreducing end during catalysis.


Assuntos
Glicogênio Sintase/química , Glicogênio/química , Oligossacarídeos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Glicogênio/genética , Glicogênio/metabolismo , Glicogênio Sintase/genética , Glicogênio Sintase/metabolismo , Mutação , Oligossacarídeos/genética , Oligossacarídeos/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
Cell Rep ; 40(1): 111041, 2022 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-35793618

RESUMO

Glycogen is the primary energy reserve in mammals, and dysregulation of glycogen metabolism can result in glycogen storage diseases (GSDs). In muscle, glycogen synthesis is initiated by the enzymes glycogenin-1 (GYG1), which seeds the molecule by autoglucosylation, and glycogen synthase-1 (GYS1), which extends the glycogen chain. Although both enzymes are required for proper glycogen production, the nature of their interaction has been enigmatic. Here, we present the human GYS1:GYG1 complex in multiple conformations representing different functional states. We observe an asymmetric conformation of GYS1 that exposes an interface for close GYG1 association, and propose this state facilitates handoff of the GYG1-associated glycogen chain to a GYS1 subunit for elongation. Full activation of GYS1 widens the GYG1-binding groove, enabling GYG1 release concomitant with glycogen chain growth. This structural mechanism connecting chain nucleation and extension explains the apparent stepwise nature of glycogen synthesis and suggests distinct states to target for GSD-modifying therapeutics.


Assuntos
Glicogênio Sintase , Glicogenólise , Glicoproteínas , Glucosiltransferases/metabolismo , Glicogênio/metabolismo , Glicogênio Sintase/metabolismo , Glicoproteínas/metabolismo , Humanos
19.
J Biol Chem ; 285(33): 25372-81, 2010 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-20538597

RESUMO

Approximately 90% of cases of Lafora disease, a fatal teenage-onset progressive myoclonus epilepsy, are caused by mutations in either the EPM2A or the EPM2B genes that encode, respectively, a glycogen phosphatase called laforin and an E3 ubiquitin ligase called malin. Lafora disease is characterized by the formation of Lafora bodies, insoluble deposits containing poorly branched glycogen or polyglucosan, in many tissues including skeletal muscle, liver, and brain. Disruption of the Epm2b gene in mice resulted in viable animals that, by 3 months of age, accumulated Lafora bodies in the brain and to a lesser extent in heart and skeletal muscle. Analysis of muscle and brain of the Epm2b(-/-) mice by Western blotting indicated no effect on the levels of glycogen synthase, PTG (type 1 phosphatase-targeting subunit), or debranching enzyme, making it unlikely that these proteins are targeted for destruction by malin, as has been proposed. Total laforin protein was increased in the brain of Epm2b(-/-) mice and, most notably, was redistributed from the soluble, low speed supernatant to the insoluble low speed pellet, which now contained 90% of the total laforin. This result correlated with elevated insolubility of glycogen and glycogen synthase. Because up-regulation of laforin cannot explain Lafora body formation, we conclude that malin functions to maintain laforin associated with soluble glycogen and that its absence causes sequestration of laforin to an insoluble polysaccharide fraction where it is functionally inert.


Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Doença de Lafora/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Western Blotting , Encéfalo/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Fosfatases de Especificidade Dupla/genética , Humanos , Doença de Lafora/genética , Doença de Lafora/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Músculos/metabolismo , Reação em Cadeia da Polimerase , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Ubiquitina-Proteína Ligases/genética
20.
J Biol Chem ; 285(17): 12851-61, 2010 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-20178984

RESUMO

Conversion to glycogen is a major fate of ingested glucose in the body. A rate-limiting enzyme in the synthesis of glycogen is glycogen synthase encoded by two genes, GYS1, expressed in muscle and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase). Defects in GYS2 cause the inherited monogenic disease glycogen storage disease 0. We have generated mice with a liver-specific disruption of the Gys2 gene (liver glycogen synthase knock-out (LGSKO) mice), using Lox-P/Cre technology. Conditional mice carrying floxed Gys2 were crossed with mice expressing Cre recombinase under the albumin promoter. The resulting LGSKO mice are viable, develop liver glycogen synthase deficiency, and have a 95% reduction in fed liver glycogen content. They have mild hypoglycemia but dispose glucose less well in a glucose tolerance test. Fed, LGSKO mice also have a reduced capacity for exhaustive exercise compared with mice carrying floxed alleles, but the difference disappears after an overnight fast. Upon fasting, LGSKO mice reach within 4 h decreased blood glucose levels attained by control floxed mice only after 24 h of food deprivation. The LGSKO mice maintain this low blood glucose for at least 24 h. Basal gluconeogenesis is increased in LGSKO mice, and insulin suppression of endogenous glucose production is impaired as assessed by euglycemic-hyperinsulinemic clamp. This observation correlates with an increase in the liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase expression and activity. This mouse model mimics the pathophysiology of glycogen storage disease 0 patients and highlights the importance of liver glycogen stores in whole body glucose homeostasis.


Assuntos
Jejum , Doença de Depósito de Glicogênio/enzimologia , Glicogênio Sintase/metabolismo , Fígado/enzimologia , Animais , Glicemia/genética , Glicemia/metabolismo , Cruzamentos Genéticos , Gluconeogênese/genética , Técnica Clamp de Glucose/métodos , Teste de Tolerância a Glucose , Glicogênio/genética , Glicogênio/metabolismo , Doença de Depósito de Glicogênio/genética , Glicogênio Sintase/genética , Hipoglicemia/genética , Hipoglicemia/metabolismo , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Fatores de Tempo
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