Browsing by Subject "Laforin"

Now showing 1 - 8 of 8
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    Accurate and sensitive quantitation of glucose and glucose phosphates derived from storage carbohydrates by mass spectrometry
    (Elsevier, 2020-02-15) Young, Lyndsay E.A.; Brizzee, Corey O.; Macedo, Jessica K. A.; Murphy, Robert D.; Contreras, Christopher J.; DePaoli-Roach, Anna A.; Roach, Peter J.; Gentry, Matthew S.; Sun, Ramon C.; Biochemistry and Molecular Biology, School of Medicine
    The addition of phosphate groups into glycogen modulates its branching pattern and solubility which all impact its accessibility to glycogen interacting enzymes. As glycogen architecture modulates its metabolism, it is essential to accurately evaluate and quantify its phosphate content. Simultaneous direct quantitation of glucose and its phosphate esters requires an assay with high sensitivity and a robust dynamic range. Herein, we describe a highly-sensitive method for the accurate detection of both glycogen-derived glucose and glucose-phosphate esters utilizing gas-chromatography coupled mass spectrometry. Using this method, we observed higher glycogen levels in the liver compared to skeletal muscle, but skeletal muscle contained many more phosphate esters. Importantly, this method can detect femtomole levels of glucose and glucose phosphate esters within an extremely robust dynamic range with excellent accuracy and reproducibility. The method can also be easily adapted for the quantification of plant starch, amylopectin or other biopolymers.
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    The effects of laforin, malin, Stbd1, and Ptg deficiencies on heart glycogen levels in Pompe disease mouse models
    (2015-08) Conway, Betsy Ann; Roach, Peter J.; DePaoli-Roach, Anna; Hurley, Thomas
    Pompe disease (PD) is a rare metabolic myopathy characterized by loss of acid alpha-glucosidase (GAA), the enzyme responsible for breaking down glycogen to glucose within the lysosomes. PD cells accumulate massive quantities of glycogen within their lysosomes, and as such, PD is classified as a “lysosomal storage disease” (LSD). GAA-deficient cells also exhibit accumulation of autophagic debris. Symptoms of severe infantile PD include extreme muscle weakness, hypotonia, and hypertrophic cardiomyopathy, resulting in death before one year of age. Certain LSDs are currently being successfully treated with enzyme replacement therapy (ERT), which involves intravenous infusion of a recombinant enzyme to counteract the endogenous deficiency. ERT has been less successful in PD, however, due to ineffective delivery of the recombinant enzyme. Alternatively, specific genes deletion may reduce lysosomal glycogen load, and could thus be targeted in PD therapy development. Absence of malin (EPM2B) or laforin (EPM2A) has been proposed to impair autophagy, which could reduce lysosomal glycogen levels. Additionally, deficiency of Stbd1 has been postulated to disable lysosomal glycogen import. Furthermore, Ptg deficiency was previously reported to abrogate Lafora body formation and correct neurological abnormalities in Lafora disease mouse models and could have similar effects on PD pathologies. The goal of this study was to characterize the effects of homozygous disruption of Epm2a, Epm2b, Stbd1, and Ptg loci on total glycogen levels in PD mouse model heart tissue, as in severe infantile PD, it is accumulation of glycogen in the heart that results in fatal hypertrophic cardiomyopathy. Gaa-/- mice were intercrossed with Epm2a-/-, Epm2b-/-, Stbd1-/-, and Ptg-/- mice to generate wildtype (WT), single knockout, and double knockout mice. The results indicated that Gaa-/- hearts accumulated up to 100-fold more glycogen than the WT. These mice also displayed cardiac hypertrophy. However, deficiency of Epm2a, Epm2b, Stbd1, or PTG in the Gaa-/- background did not reveal changes of statistical significance in either heart glycogen or cardiac hypertrophy. Nevertheless, since total glycogen was measured, these deficiencies should not be discarded in future discussions of PD therapy, as increasing sample sizes and/or distinguishing cytosolic from lysosomal glycogen content may yet reveal differences of greater significance.
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    Glycogen metabolism in Lafora disease
    (2018-02) Contreras, Christopher J.; Roach, Peter J.; DePaoli-Roach, Anna A.; Hurley, Thomas D.; Herring, B. Paul
    Glycogen, a branched polymer of glucose, serves as an osmotically neutral means of storing glucose. Covalent phosphate is a trace component of mammalian glycogen and has been a point of interest with respect to Lafora disease, a fatal form of juvenile myoclonus epilepsy. Mutations in either the EPM2A or EPM2B genes, which encode laforin and malin respectively, account for ~90% of disease cases. A characteristic of Lafora disease is the formation of Lafora bodies, which are mainly composed of an excess amount of abnormal glycogen that is poorly branched and insoluble. Laforin-/- and malin-/- knockout mice share several characteristics of the human disease, formation of Lafora bodies in various tissues, increased glycogen phosphorylation and development of neurological symptoms. The source of phosphate in glycogen has been an area of interest and here we provide evidence that glycogen synthase is capable of incorporating phosphate into glycogen. Mice lacking the glycogen targeting subunit PTG of the PP1 protein phosphatase have decreased glycogen stores in a number of tissues. When crossed with mice lacking either laforin or malin, the double knockout mice no longer over-accumulate glycogen, Lafora body formation is almost absent and the neurological disorders are normalized. Another question has been whether the abnormal glycogen in the Lafora disease mouse models can be metabolized. Using exercise to provoke glycogen degradation, we show that in laforin-/- and malin-/- mice the insoluble, abnormal glycogen appears to be metabolically inactive. These studies suggest that a therapeutic approach to Lafora disease may be to reduce the overall glycogen levels in cells so that insoluble, metabolically inert pools of the polysaccharide do not accumulate.
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    Glycogen phosphorylation and Lafora disease
    (Elsevier, 2015-12) Roach, Peter J.; Department of Biochemistry & Molecular Biology, IU School of Medicine
    Covalent phosphorylation of glycogen, first described 35 years ago, was put on firm ground through the work of the Whelan laboratory in the 1990s. But glycogen phosphorylation lay fallow until interest was rekindled in the mid 2000s by the finding that it could be removed by a glycogen-binding phosphatase, laforin, and that mutations in laforin cause a fatal teenage-onset epilepsy, called Lafora disease. Glycogen phosphorylation is due to phosphomonoesters at C2, C3 and C6 of glucose residues. Phosphate is rare, ranging from 1:500 to 1:5000 phosphates/glucose depending on the glycogen source. The mechanisms of glycogen phosphorylation remain under investigation but one hypothesis to explain C2 and perhaps C3 phosphate is that it results from a rare side reaction of the normal synthetic enzyme glycogen synthase. Lafora disease is likely caused by over-accumulation of abnormal glycogen in insoluble deposits termed Lafora bodies in neurons. The abnormality in the glycogen correlates with elevated phosphorylation (at C2, C3 and C6), reduced branching, insolubility and an enhanced tendency to aggregate and become insoluble. Hyperphosphorylation of glycogen is emerging as an important feature of this deadly childhood disease.
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    Impaired malin expression and interaction with partner proteins in Lafora disease
    (Elsevier, 2024) Skurat, Alexander V.; Segvich, Dyann M.; Contreras, Christopher J.; Hu, Yueh-Chiang; Hurley, Thomas D.; DePaoli-Roach, Anna A.; Roach, Peter J.; Biochemistry and Molecular Biology, School of Medicine
    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.
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    Incorporation of phosphate into glycogen by glycogen synthase
    (Elsevier, 2016-05-01) Contreras, Christopher J.; Segvich, Dyann M.; Mahalingan, Krishna; Chikwana, Vimbai M.; Kirley, Terence L.; Hurley, Thomas D.; DePaoli-Roach, Anna A.; Roach, Peter J.; Department of Biochemistry & Molecular Biology, IU School of Medicine
    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 [β-(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 [β-(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.
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    Muscle glycogen remodeling and glycogen phosphate metabolism following exhaustive exercise of wild type and laforin knockout mice
    (American Society for Biochemistry & Molecular Biology, 2015-09-11) Irimia, Jose M.; Tagliabracci, Vincent S.; Meyer, Catalina M.; Segvich, Dyann M.; DePaoli-Roach, Anna A.; Roach, Peter J.; Department of Biochemistry & Molecular Biology, IU School of Medicine
    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.
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    Protein degradation and quality control in cells from laforin and malin knockout mice
    (ASBMB, 2014-07-25) Garyali, Punitee; Segvich, Dyann M.; DePaoli-Roach, Anna A.; Roach, Peter J.; Department of Biochemistry & Molecular Biology, IU School of Medicine
    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.