ABSTRACT
Glucose-6-phosphatase-α (G6Pase-α) catalyzes the hydrolysis of glucose-6-phosphate to glucose and functions as a key regulator in maintaining blood glucose homeostasis. Deficiency in G6Pase-α causes glycogen storage disease 1a (GSD1a), an inherited disorder characterized by life-threatening hypoglycemia and other long-term complications. We have developed a potential mRNA-based therapy for GSD1a and demonstrated that a human G6Pase-α (hG6Pase-α) variant harboring a single serine (S) to cysteine (C) substitution at the amino acid site 298 (S298C) had > twofold increase in protein expression, resulting in improved in vivo efficacy. Here, we sought to investigate the mechanisms contributing to the increased expression of the S298C variant. Mutagenesis of hG6Pase-α identified distinct protein variants at the 298 amino acid position with substantial reduction in protein expression in cultured cells. Kinetic analysis of expression and subcellular localization in mammalian cells, combined with cell-free in vitro translation assays, revealed that altered protein expression stemmed from differences in cellular protein stability rather than biosynthetic rates. Site-specific mutagenesis studies targeting other cysteines of the hG6Pase-α S298C variant suggest the observed improvements in stability are not due to additional disulfide bond formation. The glycosylation at Asparagine (N)-96 is critical in maintaining enzymatic activity and mutations at position 298 mainly affected glycosylated forms of hG6Pase-α. Finally, proteasome inhibition by lactacystin improved expression levels of unstable hG6Pase-α variants. Taken together, these data uncover a critical role for a single amino acid substitution impacting the stability of G6Pase-α and provide insights into the molecular genetics of GSD1a and protein engineering for therapeutic development.
Subject(s)
Glucose-6-Phosphatase , Glycogen Storage Disease Type I , Animals , Humans , Glucose-6-Phosphatase/genetics , Glucose-6-Phosphatase/chemistry , Glucose-6-Phosphatase/metabolism , Glycogen Storage Disease Type I/genetics , Glycogen Storage Disease Type I/metabolism , Kinetics , Glucose/metabolism , Amino Acids , Mammals/metabolismABSTRACT
Glucose-6-phosphatase catalytic subunit 1 (G6PC1) plays a critical role in hepatic glucose production during fasting by mediating the terminal step of the gluconeogenesis and glycogenolysis pathways. In concert with accessory transport proteins, this membrane-integrated enzyme catalyzes glucose production from glucose-6-phosphate (G6P) to support blood glucose homeostasis. Consistent with its metabolic function, dysregulation of G6PC1 gene expression contributes to diabetes, and mutations that impair phosphohydrolase activity form the clinical basis of glycogen storage disease type 1a. Despite its relevance to health and disease, a comprehensive view of G6PC1 structure and mechanism has been limited by the absence of expression and purification strategies that isolate the enzyme in a functional form. In this report, we apply a suite of biophysical and biochemical tools to fingerprint the in vitro attributes of catalytically active G6PC1 solubilized in lauryl maltose neopentyl glycol (LMNG) detergent micelles. When purified from Sf9 insect cell membranes, the glycosylated mouse ortholog (mG6PC1) recapitulated functional properties observed previously in intact hepatic microsomes and displayed the highest specific activity reported to date. Additionally, our results establish a direct correlation between the catalytic and structural stability of mG6PC1, which is underscored by the enhanced thermostability conferred by phosphatidylcholine and the cholesterol analog cholesteryl hemisuccinate. In contrast, the N96A variant, which blocks N-linked glycosylation, reduced thermostability. The methodologies described here overcome long-standing obstacles in the field and lay the necessary groundwork for a detailed analysis of the mechanistic structural biology of G6PC1 and its role in complex metabolic disorders.
Subject(s)
Glucose-6-Phosphatase , Glycogen Storage Disease Type I , Animals , Catalytic Domain , Glucose/metabolism , Glucose-6-Phosphatase/chemistry , Glucose-6-Phosphatase/metabolism , Glycogen Storage Disease Type I/enzymology , Glycogen Storage Disease Type I/metabolism , Mice , Microsomes, Liver/enzymology , Microsomes, Liver/metabolismABSTRACT
Protein structures can provide invaluable information, both for reasoning about biological processes and for enabling interventions such as structure-based drug development or targeted mutagenesis. After decades of effort, 17% of the total residues in human protein sequences are covered by an experimentally determined structure1. Here we markedly expand the structural coverage of the proteome by applying the state-of-the-art machine learning method, AlphaFold2, at a scale that covers almost the entire human proteome (98.5% of human proteins). The resulting dataset covers 58% of residues with a confident prediction, of which a subset (36% of all residues) have very high confidence. We introduce several metrics developed by building on the AlphaFold model and use them to interpret the dataset, identifying strong multi-domain predictions as well as regions that are likely to be disordered. Finally, we provide some case studies to illustrate how high-quality predictions could be used to generate biological hypotheses. We are making our predictions freely available to the community and anticipate that routine large-scale and high-accuracy structure prediction will become an important tool that will allow new questions to be addressed from a structural perspective.
Subject(s)
Computational Biology/standards , Deep Learning/standards , Models, Molecular , Protein Conformation , Proteome/chemistry , Datasets as Topic/standards , Diacylglycerol O-Acyltransferase/chemistry , Glucose-6-Phosphatase/chemistry , Humans , Membrane Proteins/chemistry , Protein Folding , Reproducibility of ResultsABSTRACT
tRNA-derived fragments (tRFs) are a novel class of small non-coding RNAs whose biological roles are not well defined. Here, using multiple approaches, we investigated its role in human triple-negative breast cancer (TNBC). Our genome-wide transcriptome analysis of small non-coding RNAs revealed that tRFLys-CTT-010 was significantly increased in human TNBC. It promoted TNBC proliferation and migration. It also closely associated with starch and sucrose metabolism pathways (Kyoto Encyclopedia of Genes and Genomes analysis) and positively regulated the expression of glucose-6-phosphatase catalytic subunit (G6PC), one of the related genes in the pathway. G6PC, a complex of glucose-6-phosphatase in gluconeogenesis and glycogenolysis, is upregulated in human TNBC samples. Further studies demonstrated that overexpression of G6PC in tRFLys-CTT-010 inhibitor-transfected TNBC cell lines can reverse malignant biological behavior and knockdown of G6PC in TNBC cell lines inhibited tumor progression and reversed the oncogenic function of tRFLys-CTT-010. In addition, tRFLys-CTT-010 interacted with G6PC to regulate cellular lactate production and glycogen consumption, resulting in cell survival and proliferation. Thus, fine-tuning glucose metabolism and the tRFLys-CTT-010/G6PC axis may provide a therapeutic target for TNBC treatment.
Subject(s)
Glucose-6-Phosphatase/metabolism , Glucose/metabolism , RNA, Transfer/metabolism , Triple Negative Breast Neoplasms/pathology , Catalytic Domain , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Disease Progression , Female , Gene Knockdown Techniques , Glucose-6-Phosphatase/chemistry , Humans , Neoplasm Invasiveness/genetics , RNA, Transfer/chemistry , RNA, Transfer/genetics , Sequence Analysis, RNA/methods , Triple Negative Breast Neoplasms/enzymology , Triple Negative Breast Neoplasms/metabolismABSTRACT
OBJECTIVE: Liver glycogen levels are dynamic and highly regulated by nutrient availability as the levels decrease during fasting and are restored during the feeding cycle. However, feeding in the presence of fructose in water suppresses glycogen accumulation in the liver by upregulating the expression of the glucose-6-phosphatase catalytic subunit (G6pc) gene, although the exact mechanism is unknown. We generated liver-specific knockout MED13 mice that lacked the transcriptional Mediator complex kinase module to examine its effect on the transcriptional activation of inducible target gene expression, such as the ChREBP- and FOXO1-dependent control of the G6pc gene promoter. METHODS: The relative changes in liver expression of lipogenic and gluconeogenic genes as well as glycogen levels were examined in response to feeding standard low-fat laboratory chow supplemented with water or water containing sucrose or fructose in control (Med13fl/fl) and liver-specific MED13 knockout (MED13-LKO) mice. RESULTS: Although MED13 deficiency had no significant effect on constitutive gene expression, all the dietary inducible gene transcripts were significantly reduced despite the unchanged insulin sensitivity in the MED13-LKO mice compared to that in the control mice. G6pc gene transcription displayed the most significant difference between the Med13 fl/fl and MED13-LKO mice, particularly when fed fructose. Following fasting that depleted liver glycogen, feeding induced the restoration of glycogen levels except in the presence of fructose. MED13 deficiency rescued the glycogen accumulation defect in the presence of fructose. This resulted from the suppression of G6pc expression and thus G6PC enzymatic activity. Among two transcriptional factors that regulate G6pc gene expression, FOXO1 binding to the G6pc promoter was not affected, whereas ChREBP binding was dramatically reduced in MED13-LKO hepatocytes. In addition, there was a marked suppression of FOXO1 and ChREBP-ß transcriptional activities in MED13-LKO hepatocytes. CONCLUSIONS: Taken together, our data suggest that the kinase module of the Mediator complex is necessary for the transcriptional activation of metabolic genes such as G6pc and has an important role in regulating glycogen levels in the liver through altering transcription factor binding and activity at the G6pc promoter.
Subject(s)
Catalytic Domain/genetics , Fructose/metabolism , Glucose-6-Phosphatase/chemistry , Glucose-6-Phosphatase/metabolism , Liver Glycogen/biosynthesis , Liver/metabolism , Mediator Complex/metabolism , Signal Transduction/genetics , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Cells, Cultured , Enzyme Activation/genetics , Fasting , Fructose/pharmacology , Gene Expression , Gluconeogenesis/drug effects , Gluconeogenesis/genetics , Glucose-6-Phosphatase/genetics , Hepatocytes/metabolism , Insulin Resistance/genetics , Lipogenesis/drug effects , Lipogenesis/genetics , Male , Mediator Complex/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Up-Regulation/drug effects , Up-Regulation/geneticsABSTRACT
BACKGROUND: Glycogen storage disease type-1a is an inherited, autosomal recessive disorder caused by mutations in G6PC1 gene leading to deficiency of glucose-6-phosphatase-α specifically in the liver/kidney/intestine. PATIENTS AND METHODS: DNA of six unrelated Indian GSD-1a patients were screened for mutations in the entire coding region of G6PC1 gene followed by direct DNA sequencing and functional was tested using glucose-6-phosphatase assay. RESULTS: Mutational screening of GSD-1a patients identified five novel mutations, viz., 1) p.V99Cfs*3, 2) p.G125R, 3) IVS1-2Aâ¯>â¯T, 4) IVS3â¯+â¯39Gâ¯>â¯A and 5) IVS3â¯+â¯42Gâ¯>â¯A along with three previously reported mutations p.G118D, p.R149Q and p.A331V. Interestingly, each of the p.V99Cfs*3, IVS1-2Aâ¯>â¯T and p.G118D mutations are identified in two unrelated GSD-1a cases. Further allelic distribution of p.V99Cfs*3 and p.A331V mutations were confirmed by RFLP analysis, consistent with autosomal recessive inheritance. Functional characterization revealed that glucose-6-phosphatase activity was completely abrogated with the mutant proteins p.G125R, p.R149Q, p.G118D, p.A331V and p.V99Cfs*3 than wild-type. However, no significant changes were observed in the expression of mutant constructs at transcription and translation level. CONCLUSION: Five novel mutations, p.V99Cfs*3, p.G125R, IVS1-2Aâ¯>â¯T, IVS3â¯+â¯39Gâ¯>â¯A and IVS3â¯+â¯42Gâ¯>â¯A are reported first time to cause GSD-1a among Indian ethnicity and are not yet reported elsewhere, suggesting separate ethnic founder effects for some mutations among Indian ethnicity.
Subject(s)
Down-Regulation , Glucose-6-Phosphatase/genetics , Glucose-6-Phosphatase/metabolism , Glycogen Storage Disease Type I/genetics , Mutation , Sequence Analysis, DNA/methods , Amplified Fragment Length Polymorphism Analysis , Child , Child, Preschool , DNA Mutational Analysis , Female , Gene Frequency , Glucose-6-Phosphatase/chemistry , Glycogen Storage Disease Type I/metabolism , HEK293 Cells , Humans , India , Infant , Male , Models, Molecular , Protein ConformationABSTRACT
Type 1 diabetes (T1D) is mediated by destruction of pancreatic ß cells by autoantigen-specific CD4+ and CD8+ T cells, thus the ideal solution for T1D is the restoration of immune tolerance to ß cell antigens. We demonstrate the ability of carboxylated 500 nm biodegradable poly(lactide-co-glycolide) (PLG) nanoparticles PLG nanoparticles (either surface coupled with or encapsulating the cognate diabetogenic peptides) to rapidly and efficiently restore tolerance in NOD.SCID recipients of both activated diabetogenic CD4+ BDC2.5 chromagranin A-specific and CD8+ NY8.3 islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific TCR transgenic T cells in an antigen-specific manner. Further, initiation and maintenance of Ag-PLG tolerance operates via several overlapping, but independent, pathways including regulation via negative-co-stimulatory molecules (CTLA-4 and PD-1) and the systemic induction of peptide-specific Tregs which were critical for long-term maintenance of tolerance by controlling both trafficking of effector T cells to, and their release of pro-inflammatory cytokines within the pancreas, concomitant with selective retention of effector cells in the spleens of recipient mice.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Diabetes Mellitus, Type 1/immunology , Insulin-Secreting Cells/pathology , Nanoparticles/therapeutic use , Animals , Autoantigens/chemistry , Autoantigens/immunology , Cells, Cultured , Diabetes Mellitus, Type 1/therapy , Disease Models, Animal , Female , Glucose-6-Phosphatase/chemistry , Glucose-6-Phosphatase/genetics , Glucose-6-Phosphatase/immunology , Immune Tolerance , Mice , Mice, Inbred NOD , Mice, Transgenic , Nanoparticles/chemistry , Peptides/chemistry , Peptides/immunology , Polylactic Acid-Polyglycolic Acid Copolymer , Receptors, Antigen, T-Cell, alpha-beta/geneticsABSTRACT
CD8+ T cells directed against beta cell autoantigens are considered relevant for the pathogenesis of type 1 diabetes. Using single cell T cell receptor sequencing of CD8+ T cells specific for the IGRP265-273 epitope, we examined whether there was expansion of clonotypes and sharing of T cell receptor chains in autoreactive CD8+ T cell repertoires. HLA-A*0201 positive type 1 diabetes patients (n = 19) and controls (n = 18) were analysed. TCR α- and ß-chain sequences of 418 patient-derived IGRP265-273-multimer+ CD8+ T cells representing 48 clonotypes were obtained. Expanded populations of IGRP265-273-specific CD8+ T cells with dominant clonotypes that had TCR α-chains shared across patients were observed. The SGGSNYKLTF motif corresponding to TRAJ53 was contained in 384 (91.9%) cells, and in 20 (41.7%) patient-derived clonotypes. TRAJ53 together with TRAV29/DV5 was found in 15 (31.3%) clonotypes. Using next generation TCR α-chain sequencing, we found enrichment of one of these TCR α-chains in the memory CD8+ T cells of patients as compared to healthy controls. CD8+ T cell clones bearing the enriched motifs mediated antigen-specific target cell lysis. We provide the first evidence for restriction of T cell receptor motifs in the alpha chain of human CD8+ T cells with specificity to a beta cell antigen.
Subject(s)
Autoantigens/metabolism , CD8-Positive T-Lymphocytes/immunology , Glucose-6-Phosphatase/metabolism , Islets of Langerhans/metabolism , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Amino Acid Motifs , Amino Acid Sequence , Clone Cells , Female , Glucose-6-Phosphatase/chemistry , Humans , Male , Sequence Analysis, Protein , Young AdultABSTRACT
AIM: Small molecule activators of glucokinase (GKAs) have been explored extensively as potential anti-hyperglycaemic drugs for type 2 diabetes (T2D). Several GKAs were remarkably effective in lowering blood glucose during early therapy but then lost their glycaemic efficacy chronically during clinical trials. MATERIALS AND METHODS: We used rat hepatocytes to test the hypothesis that GKAs raise hepatocyte glucose 6-phosphate (G6P, the glucokinase product) and down-stream metabolites with consequent repression of the liver glucokinase gene ( Gck). We compared a GKA with metformin, the most widely prescribed drug for T2D. RESULTS: Treatment of hepatocytes with 25 mM glucose raised cell G6P, concomitantly with Gck repression and induction of G6pc (glucose 6-phosphatase) and Pklr (pyruvate kinase). A GKA mimicked high glucose by raising G6P and fructose-2,6-bisphosphate, a regulatory metabolite, causing a left-shift in glucose responsiveness on gene regulation. Fructose, like the GKA, repressed Gck but modestly induced G6pc. 2-Deoxyglucose, which is phosphorylated by glucokinase but not further metabolized caused Gck repression but not G6pc induction, implicating the glucokinase product in Gck repression. Metformin counteracted the effect of high glucose on the elevated G6P and fructose 2,6-bisphosphate and on Gck repression, recruitment of Mlx-ChREBP to the G6pc and Pklr promoters and induction of these genes. CONCLUSIONS: Elevation in hepatocyte G6P and downstream metabolites, with consequent liver Gck repression, is a potential contributing mechanism to the loss of GKA efficacy during chronic therapy. Cell metformin loads within the therapeutic range attenuate the effect of high glucose on G6P and on glucose-regulated gene expression.
Subject(s)
Enzyme Activators/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Glucokinase/metabolism , Hepatocytes/drug effects , Hypoglycemic Agents/pharmacology , Metformin/pharmacology , Thiazoles/pharmacology , Active Transport, Cell Nucleus/drug effects , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Cells, Cultured , Diet, Western/adverse effects , Fructose/administration & dosage , Fructose/adverse effects , Fructosediphosphates/metabolism , Glucokinase/antagonists & inhibitors , Glucokinase/chemistry , Glucokinase/genetics , Glucose-6-Phosphatase/antagonists & inhibitors , Glucose-6-Phosphatase/chemistry , Glucose-6-Phosphatase/genetics , Glucose-6-Phosphatase/metabolism , Glucose-6-Phosphate/metabolism , Hepatocytes/cytology , Hepatocytes/metabolism , Hepatocytes/pathology , Male , Mice, Inbred C3H , Overweight/enzymology , Overweight/metabolism , Overweight/pathology , Promoter Regions, Genetic/drug effects , Pyruvate Kinase/antagonists & inhibitors , Pyruvate Kinase/chemistry , Pyruvate Kinase/genetics , Pyruvate Kinase/metabolism , Rats, WistarABSTRACT
Elevated fasting blood glucose (FBG) has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs) in G6PC2 are the most important common determinants of variations in FBG in humans. Studies using G6pc2 knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. G6PC2 and the related G6PC1 and G6PC3 genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous G6PC2 SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel in situ functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human G6PC2 rs144254880 (Arg79Gln), rs149663725 (Gly114Arg) and rs2232326 (Ser324Pro) SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu), rs138726309 (His177Tyr), rs2232323 (Tyr207Ser) rs374055555 (Arg293Trp), rs2232326 (Ser324Pro), rs137857125 (Pro313Leu) and rs2232327 (Pro340Leu) SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple G6PC2 variants that have the potential to be associated with altered FBG in humans.
Subject(s)
Amino Acid Substitution , Gene Expression , Glucose-6-Phosphatase/genetics , Glucose-6-Phosphatase/metabolism , Mutation , Amino Acid Sequence , Animals , Blood Glucose , Cell Line , Codon , Conserved Sequence , Enzyme Activation , Fasting/blood , Glucose-6-Phosphatase/chemistry , Humans , Mice , Polymorphism, Single Nucleotide , RatsABSTRACT
Cross-presentation by CD8(+) conventional dendritic cells (cDCs) is involved in the maintenance of peripheral tolerance and this process is termed cross-tolerance. Previous reports showed that non-obese diabetic (NOD) mice have reduced number of splenic CD8(+) cDCs compared with non-diabetic strains, and that the administration of Flt3L to enhance DC development resulted in reduced diabetes incidence. As CD8(+) cDCs are the most efficient antigen cross-presenting cells, it was assumed that reduced cross-presentation by non-activated, tolerogenic CD8(+) cDC predisposes to autoimmune diabetogenesis. Here we show for the first time that indeed NOD mice have a defect in autoantigen cross-presentation capacity. First, we showed that NOD CD8(+) cDCs were less sensitive to iatrogenic cytochrome c, which had previously been shown to selectively deplete CD8(+) cDCs that functionally cross-present. Second, we found that proliferation of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific CD8(+) T cells was impaired in NOD compared with non-obese diabetes resistant mice after immunization with cell associated recombinant fusion protein containing the cognate IGRP peptide. This study, therefore, suggests that the reduced number of CD8(+) cDCs in NOD mice, coupled with the reduced capacity to cross-present self-antigens, reduces the overall capacity to maintain peripheral tolerance in the spontaneous autoimmune type 1 diabetes mice.
Subject(s)
Antigen Presentation/immunology , Cross-Priming/immunology , Animals , Autoantigens/immunology , CD8 Antigens/metabolism , Cell Count , Cell Differentiation , Dendritic Cells/cytology , Dendritic Cells/immunology , Dendritic Cells/metabolism , Epitopes, T-Lymphocyte/immunology , Glucose-6-Phosphatase/chemistry , Glucose-6-Phosphatase/immunology , Histocompatibility Antigens Class I/immunology , Immunophenotyping , Mice , Mice, Inbred NOD , Peptides/immunology , Spleen/cytology , Spleen/immunologyABSTRACT
Membrane-integrated type II phosphatidic acid phosphatases (PAP2s) are important for numerous bacterial to human biological processes, including glucose transport, lipid metabolism, and signaling. Escherichia coli phosphatidylglycerol-phosphate phosphatase B (ecPgpB) catalyzes removing the terminal phosphate group from a lipid carrier, undecaprenyl pyrophosphate, and is essential for transport of many hydrophilic small molecules across the membrane. We determined the crystal structure of ecPgpB at a resolution of 3.2 Å. This structure shares a similar folding topology and a nearly identical active site with soluble PAP2 enzymes. However, the substrate binding mechanism appears to be fundamentally different from that in soluble PAP2 enzymes. In ecPgpB, the potential substrate entrance to the active site is located in a cleft formed by a V-shaped transmembrane helix pair, allowing lateral movement of the lipid substrate entering the active site from the membrane lipid bilayer. Activity assays of point mutations confirmed the importance of the catalytic residues and potential residues involved in phosphate binding. The structure also suggests an induced-fit mechanism for the substrate binding. The 3D structure of ecPgpB serves as a prototype to study eukaryotic PAP2 enzymes, including human glucose-6-phosphatase, a key enzyme in the homeostatic regulation of blood glucose concentrations.
Subject(s)
Escherichia coli/enzymology , Models, Molecular , Phosphoric Monoester Hydrolases/chemistry , Cloning, Molecular , Crystallography, X-Ray , Glucose-6-Phosphatase/chemistry , Humans , Protein ConformationABSTRACT
CD8(+) T cells are prominent in autoimmune diabetes of both humans and non-obese diabetic (NOD) mice. For example, CD8(+) T cells against islet-specific glucose 6-phosphatase catalytic subunit-related protein (IGRP) can be detected readily in older NOD mice. It has been suggested that the enumeration of islet-specific CD8(+) T cells in the peripheral blood may be a predictive biomarker for autoimmune type 1 diabetes (T1D). Here, we determined the natural history of the functional endogenous IGRP(206-214)-specific cytotoxic T lymphocytes (CTLs) in NOD mice with regard to age (3- to 15-week-old pre-diabetic mice and diabetic mice) and sex. We demonstrated that in vivo IGRP(206-214)-specific CTLs significantly increased after 12 weeks of age and in vivo cytotoxicity in female NOD mice was significantly higher than in male NOD mice. To determine the in vivo IGRP(206-214)-specific CTL frequency without killing the mice, we performed splenectomies on a cohort of mice after injecting IGRP(206-214)-coated targets and then followed their diabetes progression. We found that CTL frequency correlated with future of disease onset. Thus, our data support that IGRP(206-214)-specific CTLs may be a potent biomarker for T1D.
Subject(s)
Diabetes Mellitus, Experimental/immunology , Epitopes, T-Lymphocyte/immunology , Glucose-6-Phosphatase/immunology , Peptides/immunology , T-Lymphocytes, Cytotoxic/immunology , Age Factors , Animals , Cytotoxicity, Immunologic , Diabetes Mellitus, Experimental/diagnosis , Diabetes Mellitus, Type 1/diagnosis , Diabetes Mellitus, Type 1/immunology , Epitopes, T-Lymphocyte/chemistry , Female , Glucose-6-Phosphatase/chemistry , Male , Mice , Mice, Inbred NOD , Peptides/chemistry , PrognosisABSTRACT
The G6PC3 gene encodes the ubiquitously expressed glucose-6-phosphatase enzyme (G-6-Pase ß or G-6-Pase 3 or G6PC3). Bi-allelic G6PC3 mutations cause a multi-system autosomal recessive disorder of G6PC3 deficiency (also called severe congenital neutropenia type 4, MIM 612541). To date, at least 57 patients with G6PC3 deficiency have been described in the literature.G6PC3 deficiency is characterized by severe congenital neutropenia, recurrent bacterial infections, intermittent thrombocytopenia in many patients, a prominent superficial venous pattern and a high incidence of congenital cardiac defects and uro-genital anomalies. The phenotypic spectrum of the condition is wide and includes rare manifestations such as maturation arrest of the myeloid lineage, a normocellular bone marrow, myelokathexis, lymphopaenia, thymic hypoplasia, inflammatory bowel disease, primary pulmonary hypertension, endocrine abnormalities, growth retardation, minor facial dysmorphism, skeletal and integument anomalies amongst others. Dursun syndrome is part of this extended spectrum. G6PC3 deficiency can also result in isolated non-syndromic severe neutropenia. G6PC3 mutations in result in reduced enzyme activity, endoplasmic reticulum stress response, increased rates of apoptosis of affected cells and dysfunction of neutrophil activity.In this review we demonstrate that loss of function in missense G6PC3 mutations likely results from decreased enzyme stability. The condition can be diagnosed by sequencing the G6PC3 gene. A number of G6PC3 founder mutations are known in various populations and a possible genotype-phenotype relationship also exists. G6PC3 deficiency should be considered as part of the differential diagnoses in any patient with unexplained congenital neutropenia.Treatment with G-CSF leads to improvement in neutrophil numbers, prevents infections and improves quality of life. Mildly affected patients can be managed with prophylactic antibiotics. Untreated G6PC3 deficiency can be fatal. Echocardiogram, renal and pelvic ultrasound scans should be performed in all cases of suspected or confirmed G6PC3 deficiency. Routine assessment should include biochemical profile, growth profile and monitoring for development of varicose veins or venous ulcers.
Subject(s)
Glucose-6-Phosphatase/genetics , Glycogen Storage Disease Type I/genetics , Glycogen Storage Disease Type I/physiopathology , Mutation , Neutropenia , Amino Acid Sequence , Female , Glucose-6-Phosphatase/chemistry , Glucose-6-Phosphatase/metabolism , Glycogen Storage Disease Type I/diagnosis , Glycogen Storage Disease Type I/epidemiology , Heart Defects, Congenital/etiology , Heart Defects, Congenital/genetics , Heart Septal Defects, Atrial/genetics , Humans , Hypertension, Pulmonary/genetics , Leukopenia/genetics , Male , Molecular Sequence Data , Neutropenia/congenital , Neutropenia/genetics , Sequence Analysis, DNAABSTRACT
Ultrasound-mediated delivery (USMD) of novel therapeutic agents in the presence of microbubbles is a potentially safe and effective method for gene therapy offering many desired characteristics, such as low toxicity, potential for repeated treatment, and organ specificity. In this study, we tested the capability of USMD to improve gene expression in mice livers using glycogen storage disease Type Ia as a model disease under systemic administration of naked plasmid DNA. Image-guided therapeutic ultrasound was used in two studies to provide therapeutic ultrasound to mice livers. In the first study, involving wild-type mice, control animals received naked plasmid DNA (pG6Pase 150 µg) via the tail vein, followed by an infusion of microbubbles; the treated animals additionally received therapeutic ultrasound (1 MHz). Following the procedure, the animals were left to recover and were subsequently euthanized after 2 d and liver samples were extracted. Reverse transcription polymerase chain reaction (RT-PCR) assays were performed on the samples to quantify mRNA expression. In addition, Western blot assays of FLAG-tagged glucose-6-phosphatase (G6Pase) were performed to evaluate protein expression. Ultrasound-exposed animals showed a 4-fold increase in G6Pase RNA in the liver, in comparison with control animals. Furthermore, results from Western blot analysis demonstrated a 2-fold increased protein expression in ultrasound-exposed animals after two days ( p < 0.05). A second pilot study was performed with G6Pase knockout mice, and the animals were monitored for correction of hypoglycemia over a period of 3 weeks before tissue analysis. The RT-PCR assays of samples from these animals demonstrated increased G6Pase RNA in the liver following ultrasound treatment. These results demonstrate that USMD can increase gene expression of systemically injected naked pDNA in the liver and also provide insight into the development of realistic approaches that can be translated into clinical practice.
Subject(s)
DNA/administration & dosage , DNA/genetics , Liver/metabolism , Microbubbles , Plasmids/administration & dosage , Plasmids/genetics , Transfection/methods , Ultrasonics/methods , Animals , Blotting, Western , DNA/pharmacokinetics , Genetic Therapy/methods , Glucose-6-Phosphatase/chemistry , Glucose-6-Phosphatase/genetics , Glucose-6-Phosphatase/metabolism , Liver/chemistry , Mice , Mice, Knockout , Pilot Projects , Plasmids/pharmacokineticsABSTRACT
Mutations in the glucose-6-phosphatase (G6Pase) gene are responsible for glycogen storage disease type Ia (GSD Ia). By genotype analysis of the affected pedigree, we identified a novel type mutation in a Chinese patient with GSD Ia. Mutation analysis was performed for the coding region of G6Pase gene using DNA sequencing and TaqMan gene expression assay was used to further confirm the novel mutation. The proband was compound heterozygous for c.311A>T/c.648G>T. Our report expands the spectrum of G6Pase gene mutation in China.
Subject(s)
Glucose-6-Phosphatase/genetics , Glycogen Storage Disease Type I/enzymology , Glycogen Storage Disease Type I/genetics , Amino Acid Substitution , Asian People/genetics , Base Sequence , DNA Mutational Analysis , DNA Primers/genetics , Glucose-6-Phosphatase/chemistry , Glycogen Storage Disease Type I/pathology , Heterozygote , Humans , Hydroxybutyrates , Indoles , Male , Mutation, Missense , RNA Splice Sites , Young AdultABSTRACT
The manifestations of glycogen storage disease type 1a (GSD 1a) are usually so prominent in childhood that it is readily diagnosed by pediatricians. However, a mild form of the disease may only become apparent during adolescence or adulthood. We observed a brother and sister with subtle manifestations of the disease, which was discovered after the brother's son was diagnosed with typical GSD 1a. The adult siblings never suffered from hypoglycemia, had normal fasting blood glucose and liver transaminases at the time of diagnosis, and were taller than average for Chinese. Their only notable disease manifestations were recurrent gouty arthritis associated with hyperuricemia and hyperlipidemia during adolescence. When diagnosed, the brother had multiple benign and malignant hepatic tumors, and died of fulminant metastatic hepatocellular carcinoma 6 months after liver transplantation. p.M121V/p.R83H and p.M121V/p.M121V genotypic constellations of the G6PC gene were identified in this family. Both siblings were homozygous for the newly identified p.M121V mutation. The infant had compound heterozygous mutations, p.R83H and p.M121V. We recommend that mild GSD should be considered in the adolescents with unexplained hyperuricemia and hyperlipidemia, despite the presence of normal blood glucose levels. This report also reminds us that hepatocellular carcinoma could develop even in very mild GSD 1a patients.
Subject(s)
Diagnostic Errors , Fatty Liver/diagnosis , Glycogen Storage Disease Type I/diagnosis , Glycogen Storage Disease Type I/genetics , Adolescent , Adult , Arthritis, Gouty/enzymology , Arthritis, Gouty/genetics , Base Sequence , Carcinoma, Hepatocellular/enzymology , Carcinoma, Hepatocellular/genetics , Child, Preschool , Consanguinity , DNA, Complementary/genetics , Female , Glucose-6-Phosphatase/chemistry , Glucose-6-Phosphatase/genetics , Glucose-6-Phosphatase/metabolism , Glycogen Storage Disease Type I/enzymology , Heterozygote , Homozygote , Humans , Hyperlipidemias/enzymology , Hyperlipidemias/genetics , Hyperuricemia/enzymology , Hyperuricemia/genetics , Liver Function Tests , Liver Neoplasms/enzymology , Liver Neoplasms/genetics , Male , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Mutation, Missense , Pedigree , Phenotype , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Type 1 diabetes is characterized by the autoimmune destruction of pancreatic ß-cells. Recognition of major histocompatibility complex (MHC)-bound peptides is critical for both the initiation and progression of disease. In this study, MHC peptide complexes were purified from NIT-1 ß-cells, interferon-γ (IFN-γ)-treated NIT-1 cells, splenic and thymic tissue of 12-week-old NOD mice, and peptides identified by mass spectrometry. In addition to global liquid chromatography-tandem mass spectrometry analysis, the targeted approach of multiple-reaction monitoring was used to quantitate the immunodominant K(d)-restricted T-cell epitope islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206â214. We identified >2,000 MHC-bound peptides; 1,100 of these presented by ß-cells grown under normal conditions or after exposure to IFN-γ. These include sequences from a number of known autoantigens. Quantitation of IGRP206â214 revealed low-level presentation by K(d) (~25 complexes/cell) on NIT-1 cells after IFN-γ treatment compared with the simultaneous presentation of the endogenously processed K(d)-restricted peptide Janus kinase-1355â363 (~15,000 copies/cell). We have successfully sequenced peptides from NIT-1 ß-cells under basal and inflammatory conditions. We have shown the feasibility of quantitating disease-associated peptides and provide the first direct demonstration of the disparity between presentation of a known autoantigenic epitope and a common endogenously presented peptide.
Subject(s)
Autoantigens/metabolism , Diabetes Mellitus, Type 1/metabolism , Immunodominant Epitopes/metabolism , Insulin-Secreting Cells/metabolism , Peptide Fragments/metabolism , Animals , Autoantigens/chemistry , Autoantigens/isolation & purification , Cell Line , Chromatography, High Pressure Liquid , Diabetes Mellitus, Type 1/immunology , Female , Glucose-6-Phosphatase/chemistry , Glucose-6-Phosphatase/isolation & purification , Glucose-6-Phosphatase/metabolism , Histocompatibility Antigens/chemistry , Histocompatibility Antigens/isolation & purification , Histocompatibility Antigens/metabolism , Immunodominant Epitopes/chemistry , Immunodominant Epitopes/isolation & purification , Inflammation Mediators/chemistry , Inflammation Mediators/isolation & purification , Inflammation Mediators/metabolism , Insulin-Secreting Cells/immunology , Interferon-gamma/metabolism , Janus Kinase 1/chemistry , Janus Kinase 1/isolation & purification , Janus Kinase 1/metabolism , Mice , Mice, Inbred NOD , Organ Specificity , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Proteins/chemistry , Proteins/isolation & purification , Proteins/metabolism , Spleen/immunology , Spleen/metabolism , Tandem Mass Spectrometry , Thymus Gland/immunology , Thymus Gland/metabolismABSTRACT
Glucose-6 phosphatase (G6Pase), a key enzyme of glucose homeostasis, catalyses the hydrolysis of glucose-6 phosphate (G6P) to glucose and inorganic phosphate. A deficiency in G6Pase activity causes type 1 glycogen storage disease (GSD-1), mainly characterised by hypoglycaemia. Genetic analyses of the two forms of this rare disease have shown that the G6Pase system consists of two proteins, a catalytic subunit (G6PC) responsible for GSD-1a, and a G6P translocase (G6PT), responsible for GSD-1b. However, since their identification, few investigations concerning their structural relationship have been made. In this study, we investigated the localisation and membrane organisation of the G6Pase complex. To this aim, we developed chimera proteins by adding a fluorescent protein to the C-terminal ends of both subunits. The G6PC and G6PT fluorescent chimeras were both addressed to perinuclear membranes as previously suggested, but also to vesicles throughout the cytoplasm. We demonstrated that both proteins strongly colocalised in perinuclear membranes. Then, we studied G6PT organisation in the membrane. We highlighted FRET between the labelled C and N termini of G6PT. The intramolecular FRET of this G6PT chimera was 27%. The coexpression of unlabelled G6PC did not modify this FRET intensity. Finally, the chimera constructs generated in this work enabled us for the first time to analyze the relationship between GSD-1 mutations and the intracellular localisation of both G6Pase subunits. We showed that GSD1 mutations did neither alter the G6PC or G6PT chimera localisation, nor the interaction between G6PT termini. In conclusion, our results provide novel information on the intracellular distribution and organisation of the G6Pase complex.
Subject(s)
Glucose-6-Phosphatase/metabolism , Protein Subunits/metabolism , Antiporters/chemistry , Antiporters/genetics , Antiporters/metabolism , Fluorescence Resonance Energy Transfer , Glucose-6-Phosphatase/chemistry , Glucose-6-Phosphatase/genetics , Glycogen Storage Disease Type I/enzymology , Glycogen Storage Disease Type I/genetics , HeLa Cells , Hep G2 Cells , Humans , Immunoblotting , Immunohistochemistry , Intracellular Membranes , Monosaccharide Transport Proteins/chemistry , Monosaccharide Transport Proteins/genetics , Monosaccharide Transport Proteins/metabolism , Protein Structure, Secondary , Protein Subunits/chemistry , Protein Subunits/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Gluconeogenesis contributes to insulin resistance in type 1 and type 2 diabetes, but its regulation and the underlying molecular mechanisms remain unclear. Recently, calcium-regulated heat-stable protein 1 (CARHSP1) was identified as a biomarker for diabetic complications. In this study, we investigated the role of CARHSP1 in hepatic gluconeogenesis. We assessed the regulation of hepatic CARHSP1 expression under conditions of fasting and refeeding. Adenovirus-mediated CARHSP1 overexpression and siRNA-mediated knockdown experiments were performed to characterize the role of CARHSP1 in the regulation of gluconeogenic gene expression. Here, we document for the first time that CARHSP1 is regulated by nutrient status in the liver and functions at the transcriptional level to negatively regulate gluconeogenic genes, including the glucose-6-phosphatase catalytic subunit (G6Pc) and phosphoenolpyruvate carboxykinase 1 (PEPCK1). In addition, we found that CARHSP1 can physically interact with peroxisome proliferator-activated receptor-α (PPARα) and inhibit its transcriptional activity. Both pharmacological and genetic ablations of PPARα attenuate the inhibitory effect of CARHSP1 on gluconeogenic gene expression in hepatocytes. Our data suggest that CARHSP1 inhibits hepatic gluconeogenic gene expression via repression of PPARα and that CARHSP1 may be a molecular target for the treatment of diabetes.