[Show abstract][Hide abstract] ABSTRACT: 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 laforin-malin 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 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 knockout cells as compared to wild type, even though they still showed a slight response to starvation and rapamycin. Furthermore, S6K and S6 phosphorylation were increased. Under basal conditions, there was no effect on the levels of ubiquitinated proteins in the knockout 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 knockout cells under basal conditions or with ER stress-inducing agents. In conclusion, both laforin and malin knockout cells display mTOR-dependent autophagy defects, reduced proteasomal activity but no defects in the ER stress response. We speculate that these defects may be secondary to glycogen overaccumulation. The study also suggests a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal.
Preview · Article · Jun 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Jan 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: 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 β-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.
No preview · Article · Dec 2013 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Feb 2012 · Biochemical Journal
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Dec 2011 · Human Molecular Genetics
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Aug 2011 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Author Summary
Lafora disease (LD) is a fatal epilepsy that afflicts previously normal teenagers. It is caused by mutations in the EPM2A or EPM2B genes encoding the laforin carbohydrate-binding phosphatase and the malin E3 ubiquitin ligase. LD is the most common neurodegenerative epilepsy of adolescents. Affected children suffer an ordeal lasting 10 years, consisting of escalating seizures, constant body jerking, particularly frightening epileptic visual hallucinations, and later on dementia. They die of massive convulsion. Brain biopsies reveal accumulation of a starch-like compound, polyglucosan, overtaking dendrites and likely causing the disease, and neurodegeneration. Glycogen synthase (GS), the enzyme that forms normal glycogen, is also responsible for synthesizing these polyglucosans. We reasoned that reducing GS activity might prevent polyglucosan formation. Mice deficient of Epm2a replicate LD and are a standard model. Members of our group generated mice deficient of PTG, a protein involved in activating GS. By breeding LD mice with PTG-lacking mice, we generated LD mice lacking the GS-activating effect of PTG. This resulted in a cure. The double knockout mice have almost no polyglucosan, no neurodegeneration, and no seizures. Our work opens an avenue of treatment for this fatal epilepsy, which may also be applicable to other glycogen storage diseases.
[Show abstract][Hide abstract] ABSTRACT: Glycogen is a branched polymer of glucose that serves as an energy store. Phosphate, a trace constituent of glycogen, has profound effects on glycogen structure, and phosphate hyperaccumulation is linked to Lafora disease, a fatal progressive myoclonus epilepsy that can be caused by mutations of laforin, a glycogen phosphatase. However, little is known about the metabolism of glycogen phosphate. We demonstrate here that the biosynthetic enzyme glycogen synthase, which normally adds glucose residues to glycogen, is capable of incorporating the β-phosphate of its substrate UDP-glucose at a rate of one phosphate per approximately 10,000 glucoses, in what may be considered a catalytic error. We show that the phosphate in glycogen is present as C2 and C3 phosphomonoesters. Since hyperphosphorylation of glycogen causes Lafora disease, phosphate removal by laforin may thus be considered a repair or damage control mechanism.
[Show abstract][Hide abstract] ABSTRACT: Stbd1 is a protein of previously unknown function that is most prevalent in liver and muscle, the major sites for storage of the energy reserve glycogen. The protein is predicted to contain a hydrophobic N terminus and a C-terminal CBM20 glycan binding domain. Here, we show that Stbd1 binds to glycogen in vitro and that endogenous Stbd1 locates to perinuclear compartments in cultured mouse FL83B or Rat1 cells. When overexpressed in COSM9 cells, Stbd1 concentrated at enlarged perinuclear structures, co-localized with glycogen, the late endosomal/lysosomal marker LAMP1 and the autophagy protein GABARAPL1. Mutant Stbd1 lacking the N-terminal hydrophobic segment had a diffuse distribution throughout the cell. Point mutations in the CBM20 domain did not change the perinuclear localization of Stbd1, but glycogen was no longer concentrated in this compartment. Stable overexpression of glycogen synthase in Rat1WT4 cells resulted in accumulation of glycogen as massive perinuclear deposits, where a large fraction of the detectable Stbd1 co-localized. Starvation of Rat1WT4 cells for glucose resulted in dissipation of the massive glycogen stores into numerous and much smaller glycogen deposits that retained Stbd1. In vitro, in cells, and in animal models, Stbd1 consistently tracked with glycogen. We conclude that Stbd1 is involved in glycogen metabolism by binding to glycogen and anchoring it to membranes, thereby affecting its cellular localization and its intracellular trafficking to lysosomes.
Full-text · Article · Nov 2010 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Increased activity of Ser/Thr protein phosphatases types 1 (PP1) and 2A (PP2A) during maladaptive cardiac hypertrophy contributes to cardiac dysfunction and eventual failure, partly through effects on calcium metabolism. A second maladaptive feature of pressure overload cardiac hypertrophy that instead leads to heart failure by interfering with cardiac contraction and intracellular transport is a dense microtubule network stabilized by decoration with microtubule-associated protein 4 (MAP4). In an earlier study we showed that the major determinant of MAP4-microtubule affinity, and thus microtubule network density and stability, is site-specific MAP4 dephosphorylation at Ser-924 and to a lesser extent at Ser-1056; this was found to be prominent in hypertrophied myocardium. Therefore, in seeking the etiology of this MAP4 dephosphorylation, we looked here at PP2A and PP1, as well as the upstream p21-activated kinase 1, in maladaptive pressure overload cardiac hypertrophy. The activity of each was increased persistently during maladaptive hypertrophy, and overexpression of PP2A or PP1 in normal hearts reproduced both the microtubule network phenotype and the dephosphorylation of MAP4 Ser-924 and Ser-1056 seen in hypertrophy. Given the major microtubule-based abnormalities of contractile and transport function in maladaptive hypertrophy, these findings constitute a second important mechanism for phosphatase-dependent pathology in the hypertrophied and failing heart.
Preview · Article · Oct 2010 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Sep 2010 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Aug 2010 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Feb 2010 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Entry into mitosis requires the activation of mitotic kinases, including Aurora A and Polo-like kinase 1 (Plk1). Increased levels of these kinases are frequently found associated with human cancers, and therefore it is imperative to understand the processes leading to their activation. We demonstrate that TPX2, but neither Ajuba nor Inhibitor-2, can activate Aurora A directly. Moreover, Plx1 can induce Aurora A T-loop phosphorylation indirectly in vivo during oocyte maturation. We identify Ser204 in TPX2 as a Plx1 phosphorylation site. Mutating Ser204 to alanine decreases activation of Aurora A, whereas a phosphomimetic Asp mutant exhibits enhanced activating ability. Finally, we show that phosphorylation of TPX2 with Plx1 increases its ability to activate Aurora A. Taken together, our data indicate that Plx1 promotes activation of Aurora A, most likely through TPX2. In light of the current literature, we propose a model in which Plx1 and Aurora A activate each other in a positive feedback loop.
[Show abstract][Hide abstract] ABSTRACT: Lafora disease is a progressive myoclonus epilepsy with onset in the teenage years followed by neurodegeneration and death within 10 years. A characteristic is the widespread formation of poorly branched, insoluble glycogen-like polymers (polyglucosan) known as Lafora bodies, which accumulate in neurons, muscle, liver, and other tissues. Approximately half of the cases of Lafora disease result from mutations in the EPM2A gene, which encodes laforin, a member of the dual specificity protein phosphatase family that is able to release the small amount of covalent phosphate normally present in glycogen. In studies of Epm2a(-/-) mice that lack laforin, we observed a progressive change in the properties and structure of glycogen that paralleled the formation of Lafora bodies. At three months, glycogen metabolism remained essentially normal, even though the phosphorylation of glycogen has increased 4-fold and causes altered physical properties of the polysaccharide. By 9 months, the glycogen has overaccumulated by 3-fold, has become somewhat more phosphorylated, but, more notably, is now poorly branched, is insoluble in water, and has acquired an abnormal morphology visible by electron microscopy. These glycogen molecules have a tendency to aggregate and can be recovered in the pellet after low speed centrifugation of tissue extracts. The aggregation requires the phosphorylation of glycogen. The aggregrated glycogen sequesters glycogen synthase but not other glycogen metabolizing enzymes. We propose that laforin functions to suppress excessive glycogen phosphorylation and is an essential component of the metabolism of normally structured glycogen.
Preview · Article · Nov 2008 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Cardiac-specific overexpression of the catalytic subunit of protein phosphatase type 1 (PP1) in mice results in hypertrophy, depressed contractility, propensity to heart failure, and premature death. To further address the role of PP1 in heart function, PP1 mice were crossed with mice that overexpress a functional COOH-terminally truncated form of PP1 inhibitor-2 (I-2(140)). Protein phosphatase activity was increased in PP1 mice but was normalized in double transgenic (DT) mice. The maximal rates of contraction (+dP/dt) and of relaxation (-dP/dt) were reduced in catheterized PP1 mice but normalized in DT mice. Similar contractile abnormalities were observed in isolated, perfused work-performing hearts and in whole animals by means of echocardiography. The increased absolute and relative heart weights observed in PP1 mice were normalized in DT mice. Histological analyses indicated that PP1 mice had significant cardiac fibrosis, which was absent in DT mice. Furthermore, PP1 mice exhibited an age-dependent increase in mortality, which was abrogated in DT mice. These results indicate that I-2 overexpression prevents the detrimental effects of PP1 overexpression in the heart and further underscore the fundamental role of PP1 in cardiac function. Therefore, PP1 inhibitors such as I-2 could offer new therapeutic options to ameliorate the deleterious effects of heart failure.