Explore the vast range of titles available.

Glycogen, an α-glucan polymer serving as an energy storage compound in microorganisms, is synthesized through distinct pathways (GlgC-GlgA or GlgE pathway). Both pathways involve multiple enzymes, with a shared glycogen branching enzyme (GBE). GBEs play a pivotal role in establishing α-1,6-linkages within the glycogen structure. GBEs are also used for starch modification. Understanding how these enzymes work is interesting for both glycogen synthesis in microorganisms, as well as novel applications for starch modification. This study focuses on a putative enzyme GH13_9 GBE (PoGBE13), present in a polysaccharide utilization locus (PUL) of Pontibacter sp. SGAir0037, and related to the GlgE glycogen synthesis pathway. While the PUL of Pontibacter sp. SGAir0037 contains glycogen-degrading enzymes, the branching enzyme (PoGBE13) was also found due to genetic closeness. Characterization revealed that PoGBE13 functions as a typical branching enzyme, exhibiting a relatively high branching over non-branching (hydrolysis and α-1,4-transferase activity) ratio on linear maltooctadecaose (3.0 ± 0.4). Besides the GH13_9 GBE, a GH57 (PoGH57) enzyme was selected for characterization from the same PUL due to its undefined function. The combined action of both GH13 and GH57 enzymes suggested 4-α-glucanotransferase activity for PoGH57. The characterization of these unique enzymes related to a GlgE glycogen synthesis pathway provides a more profound understanding of their interactions and synergistic roles in glycogen synthesis and are potential enzymes for use in starch modification processes. Due to the structural similarity between glycogen and starch, PoGBE13 can potentially be used for starch modification with different applications, for example, in functional food ingredients.

Glycogen is a highly branched α-glucan polymer widely used as energy and carbon reserve by many microorganisms. The branches are introduced by glycogen branching enzymes (EC 2.4.1.18), that are classified into glycoside hydrolase families 13 (GH13) and 57 (GH57). Most microorganisms have typically only a single glycogen branching enzyme (gbe) gene. Only a few microorganisms carry both GH13 and GH57 gbe genes, such as Petrotoga mobilis and Mycobacterium tuberculosis. Here we report the basic characteristics of the GH13 and GH57 GBE of P. mobilis, both heterologously expressed in E. coli. The GH13 GBE has a considerably higher branching activity towards the linear α-glucan amylose, and produces a highly branched α-glucan with a high molecular weight which is very similar to glycogen. The GH57 GBE, on the contrary, makes a much smaller branched α-glucan. While the GH13 GBE acts as a classical glycogen branching enzyme involved in glycogen synthesis, the role of GH57 GBE remains unclear.

At least five enzymes including three E3 ubiquitin ligases are dedicated to glycogen’s spherical structure. Absence of any reverts glycogen to a structure resembling amylopectin of the plant kingdom. This amylopectinosis (polyglucosan body formation) causes fatal neurological diseases including adult polyglucosan body disease (APBD) due to glycogen branching enzyme deficiency, Lafora disease (LD) due to deficiencies of the laforin glycogen phosphatase or the malin E3 ubiquitin ligase and type 1 polyglucosan body myopathy (PGBM1) due to RBCK1 E3 ubiquitin ligase deficiency. Little is known about these enzymes’ functions in glycogen structuring. Toward understanding these functions, we undertake a comparative murine study of the amylopectinoses of APBD, LD and PGBM1. We discover that in skeletal muscle, polyglucosan bodies form as two main types, small and multitudinous (‘pebbles’) or giant and single (‘boulders’), and that this is primarily determined by the myofiber types in which they form, ‘pebbles’ in glycolytic and ‘boulders’ in oxidative fibers. This pattern recapitulates what is known in the brain in LD, innumerable dust-like in astrocytes and single giant sized in neurons. We also show that oxidative myofibers are relatively protected against amylopectinosis, in part through highly increased glycogen branching enzyme expression. We present evidence of polyglucosan body size-dependent cell necrosis. We show that sex influences amylopectinosis in genotype, brain region and myofiber-type-specific fashion. RBCK1 is a component of the linear ubiquitin chain assembly complex (LUBAC), the only known cellular machinery for head-to-tail linear ubiquitination critical to numerous cellular pathways. We show that the amylopectinosis of RBCK1 deficiency is not due to loss of linear ubiquitination, and that another function of RBCK1 or LUBAC must exist and operate in the shaping of glycogen. This work opens multiple new avenues toward understanding the structural determinants of the mammalian carbohydrate reservoir critical to neurologic and neuromuscular function and disease.

Iron Regulatory Protein 1 (IRP1) is a bifunctional cytosolic iron sensor. When iron levels are normal, IRP1 harbours an iron-sulphur cluster (holo-IRP1), an enzyme with aconitase activity. When iron levels fall, IRP1 loses the cluster (apo-IRP1) and binds to iron-responsive elements (IREs) in messenger RNAs (mRNAs) encoding proteins involved in cellular iron uptake, distribution, and storage. Here we show that mutations in the Drosophila 1,4-Alpha-Glucan Branching Enzyme ( AGBE ) gene cause porphyria. AGBE was hitherto only linked to glycogen metabolism and a fatal human disorder known as glycogen storage disease type IV. AGBE binds specifically to holo-IRP1 and to mitoNEET, a protein capable of repairing IRP1 iron-sulphur clusters. This interaction ensures nuclear translocation of holo-IRP1 and downregulation of iron-dependent processes, demonstrating that holo-IRP1 functions not just as an aconitase, but throttles target gene expression in anticipation of declining iron requirements. Higher organisms regulate cellular iron concentrations through Iron Regulatory Proteins (IRPs), which regulate specific messenger RNAs. Here Huynh et al. show that IRP1 requires a Glycogen Branching Enzyme for proper function, and that IRP1 has additional regulatory roles in cell nuclei.

Adult polyglucosan body disease (APBD) and Lafora disease (LD) are autosomal recessive glycogen storage neurological disorders. APBD is caused by mutations in the glycogen branching enzyme (GBE1) gene and is characterized by progressive upper and lower motor neuron dysfunction and premature death. LD is a fatal progressive myoclonus epilepsy caused by loss of function mutations in the EPM2A or EPM2B gene. These clinically distinct neurogenetic diseases share a common pathology. This consists of time-dependent formation, precipitation, and accumulation of an abnormal form of glycogen (polyglucosan) into gradually enlarging inclusions, polyglucosan bodies (PBs) in ever-increasing numbers of neurons and astrocytes. The growth and spread of PBs are followed by astrogliosis, microgliosis, and neurodegeneration. The key defect in polyglucosans is that their glucan branches are longer than those of normal glycogen, which prevents them from remaining in solution. Since the lengths of glycogen branches are determined by the enzyme glycogen synthase, we hypothesized that downregulating this enzyme could prevent or hinder the generation of the pathogenic PBs. Here, we pursued an adeno-associated virus vector (AAV) mediated RNA-interference (RNAi) strategy. This approach resulted in approximately 15% reduction of glycogen synthase mRNA and an approximately 40% reduction of PBs across the brain in the APBD and both LD mouse models. This was accompanied by improvements in early neuroinflammatory markers of disease. This work represents proof of principle toward developing a single lifetime dose therapy for two fatal neurological diseases: APBD and LD. The approach is likely applicable to other severe and common diseases of glycogen storage.

This work employs adult polyglucosan body disease (APBD) models to explore the efficacy and mechanism of action of the polyglucosan‐reducing compound 144DG11. APBD is a glycogen storage disorder (GSD) caused by glycogen branching enzyme (GBE) deficiency causing accumulation of poorly branched glycogen inclusions called polyglucosans. 144DG11 improved survival and motor parameters in a GBE knockin (Gbe ys/ys ) APBD mouse model. 144DG11 reduced polyglucosan and glycogen in brain, liver, heart, and peripheral nerve. Indirect calorimetry experiments revealed that 144DG11 increases carbohydrate burn at the expense of fat burn, suggesting metabolic mobilization of pathogenic polyglucosan. At the cellular level, 144DG11 increased glycolytic, mitochondrial, and total ATP production. The molecular target of 144DG11 is the lysosomal membrane protein LAMP1, whose interaction with the compound, similar to LAMP1 knockdown, enhanced autolysosomal degradation of glycogen and lysosomal acidification. 144DG11 also enhanced mitochondrial activity and modulated lysosomal features as revealed by bioenergetic, image‐based phenotyping and proteomics analyses. As an effective lysosomal targeting therapy in a GSD model, 144DG11 could be developed into a safe and efficacious glycogen and lysosomal storage disease therapy. SYNOPSIS Adult‐onset glycogen storage disorder type 4, or Adult Polyglucosan Body Disease (APBD), is an incurable progressive axonopathy caused by accumulation of poorly branched glycogen inclusions called polyglucosans. Here we test in APBD models the in vivo , ex vivo and in vitro therapeutic efficacy of the new polyglucosan reducing small molecule 144DG11. 144DG11 was demonstrated to improve survival and motor capacities compromised in glycogen branching enzyme (GBE) knockin, APBD modeling mice. 144DG11 reduced polyglucosans and glycogen in brain, liver, heart, and peripheral nerve of Gbe knockin mice. 144DG11 increased carbohydrate catabolism in vivo and correspondingly the relative contribution of glycolysis to ATP production as well as total ATP production. The molecular target of 144DG11 is the lysosomal membrane protein LAMP1. Autophagic flux and autolysosomal glycogen degradation were enhanced by the 144DG11‐LAMP1 interaction. At cellular and proteomic levels, 144DG11 modulated bioenergetic, mitochondrial and lysosomal features. Graphical Abstract Adult‐onset glycogen storage disorder type 4, or Adult Polyglucosan Body Disease (APBD), is an incurable progressive axonopathy caused by accumulation of poorly branched glycogen inclusions called polyglucosans. Here we test in APBD models the in vivo , ex vivo and in vitro therapeutic efficacy of the new polyglucosan reducing small molecule 144DG11.

Major stores of glucose are found as glycogen in skeletal muscle and liver. Skeletal muscle is a heterogenous tissue, with cellular metabolic and contractile distinctions dependent on whether the cell (fibre) is slow-twitch (Type I) or fast-twitch (Type II). We hypothesised that proteins important for glycogen metabolism would be differentially abundant between these diverse fibres. We further hypothesised that the cellular location of these proteins would be different in muscle samples between control (CON) and individuals with type 2 diabetes (T2D). We dissected individual muscle fibre segments from vastus lateralis skeletal muscle biopsy samples from CON and T2D and used cell-type-specific approaches to address muscle heterogeneity. We measured glycogen and glycogen-related proteins by immunoblotting techniques. A lower proportion of Type I fibres was found in muscle in T2D compared with CON. AMPK-β2, glycogen branching enzyme (GBE), glycogen debranching enzyme (GDE), and glycogen phosphorylase (GP) were differentially localized between fibre types and in fibres from CON and T2D individuals. A key novel finding was that the majority of glycogen is loosely bound or cytosolic in location in human skeletal muscle. The proportion of this diffusible pool of glycogen was significantly lower in Type I fibres in T2D compared to CON. A hyperinsulinaemic, euglycaemic clamp in people with type 2 diabetes had no effect on the proportion of diffusible glycogen. We identify cell-type as an important consideration when assessing glycogen metabolism in muscle. Our findings demonstrate varying glucose handling abilities in specific muscle fibre types in type 2 diabetes. A model is presented to provide an overview of the cell-specific differences in glycogen metabolism in type 2 diabetes.

Some acute exercise effects are influenced by postexercise (PEX) diet, and these diet-effects are attributed to differential glycogen resynthesis. However, this idea is challenging to test rigorously. Therefore, we devised a novel genetic model to modify muscle glycogen synthase 1 (GS1) expression in rat skeletal muscle with an adeno-associated virus (AAV) short hairpin RNA knockdown vector targeting GS1 (shRNA-GS1). Contralateral muscles were injected with scrambled shRNA (shRNA-Scr). Muscles from exercised (2-hour-swim) and time-matched sedentary (Sed) rats were collected immediately postexercise (IPEX), 5-hours-PEX (5hPEX), or 9-hours-PEX (9hPEX). Rats in 5hPEX and 9hPEX experiments were refed (RF) or not-refed (NRF) chow. Muscles were analyzed for glycogen, abundance of metabolic proteins (pyruvate dehydrogenase kinase 4, PDK4; peroxisome proliferator-activated receptor γ coactivator-1α, PGC1α; hexokinase II, HKII; glucose transporter 4, GLUT4), AMP-activated protein kinase phosphorylation (pAMPK), and glycogen metabolism-related enzymes (glycogen phosphorylase, PYGM; glycogen debranching enzyme, AGL; glycogen branching enzyme, GBE1). shRNA-GS1 versus paired shRNA-Scr muscles had markedly lower GS1 abundance. IPEX versus Sed rats had lower glycogen and greater pAMPK, and neither of these IPEX-values differed for shRNA-GS1 versus paired shRNA-Scr muscles. IPEX versus Sed groups did not differ for abundance of metabolic proteins, regardless of GS1 knockdown. Glycogen in RF-rats was lower for shRNA-GS1 versus paired shRNA-Scr muscles at both 5hPEX and 9hPEX. HKII protein abundance was greater for 5hPEX versus Sed groups, regardless of GS1 knockdown or diet, and despite differing glycogen levels. At 9hPEX, shRNA-GS1 versus paired shRNA-Scr muscles had greater PDK4 and PGC1α abundance within each diet group. However, the magnitude of PDK4 or PGC1α changes was similar in each diet group regardless of GS1 knockdown although glycogen differed between paired muscles only in RF-rats. In summary, we established a novel genetic approach to investigate the relationship between muscle glycogen and other exercise effects. Our results suggest that exercise-effects on abundance of several metabolic proteins did not uniformly correspond to differences in postexercise glycogen.

Background Changes in glycogen metabolism is an essential feature among the various metabolic adaptations used by cancer cells to adjust to the conditions imposed by the tumor microenvironment. Our previous study showed that glycogen branching enzyme (GBE1) is downstream of the HIF1 pathway in hypoxia-conditioned lung cancer cells. In the present study, we investigated whether GBE1 is involved in the immune regulation of the tumor microenvironment in lung adenocarcinoma (LUAD). Methods We used RNA-sequencing analysis and the multiplex assay to determine changes in GBE1 knockdown cells. The role of GBE1 in LUAD was evaluated both in vitro and in vivo. Results GBE1 knockdown increased the expression of chemokines CCL5 and CXCL10 in A549 cells. CD8 expression correlated positively with CCL5 and CXCL10 expression in LUAD. The supernatants from the GBE1 knockdown cells increased recruitment of CD8 + T lymphocytes. However, the neutralizing antibodies of CCL5 or CXCL10 significantly inhibited cell migration induced by shGBE1 cell supernatants. STING/IFN-I pathway mediated the effect of GBE1 knockdown for CCL5 and CXCL10 upregulation. Moreover, PD-L1 increased significantly in shGBE1 A549 cells compared to those in control cells. Additionally, in LUAD tumor tissues, a negative link between PD-L1 and GBE1 was observed. Lastly, blockade of GBE1 signaling combined with anti-PD-L1 antibody significantly inhibited tumor growth in vivo. Conclusions GBE1 blockade promotes the secretion of CCL5 and CXCL10 to recruit CD8 + T lymphocytes to the tumor microenvironment via the IFN-I/STING signaling pathway, accompanied by upregulation of PD-L1 in LUAD cells, suggesting that GBE1 could be a promising target for achieving tumor regression through cancer immunotherapy in LUAD.

AmyC, a glycoside hydrolase family 57 (GH57) enzyme of Thermotoga maritima MSB8, has previously been identified as an intracellular α-amylase playing a role in either maltodextrin utilization or storage polysaccharide metabolism. However, the α-amylase specificity of AmyC is questionable as extensive phylogenetic analysis of GH57 and tertiary structural comparison suggest that AmyC could actually be a glycogen-branching enzyme (GBE), a key enzyme in the biosynthesis of glycogen. This communication presents phylogenetic and biochemical evidence that AmyC is a GBE with a relatively high hydrolytic (α-amylase) activity (up to 30% of the total activity), creating a branched α-glucan with 8.5% α-1,6-glycosidic bonds. The high hydrolytic activity is explained by the fact that AmyC has a considerably shorter catalytic loop (residues 213–220) not reaching the acceptor side. Secondly, in AmyC, the tryptophan residue (W 246) near the active site has its side chain buried in the protein interior, while the side chain is at the surface in Tk1436 and Tt1467 GBEs. The putative GBEs from three other Thermotogaceae , with very high sequence similarities to AmyC, were found to have the same structural elements as AmyC, suggesting that GH57 GBEs with relatively high hydrolytic activity may be widespread in nature.