Highly accurate protein structure prediction for the human proteome.

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.

Biophysical and functional properties of purified glucose-6-phosphatase catalytic subunit 1.

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.

Amnio acid substitution at position 298 of human glucose-6 phosphatase-α significantly impacts its stability in mammalian cells.

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.

tRNA-derived fragment tRFLys-CTT-010 promotes triple-negative breast cancer progression by regulating glucose metabolism via G6PC.

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.

Reversal of autoimmunity by boosting memory-like autoregulatory T cells.

Blunting autoreactivity without compromising immunity remains an elusive goal in the treatment of autoimmunity. We show that progression to autoimmune diabetes results in the conversion of naive low-avidity autoreactive CD8(+) T cells into memory-like autoregulatory cells that can be expanded in vivo with nanoparticles coated with disease-relevant peptide-major histocompatibility complexes (pMHC-NP). Treatment of NOD mice with monospecific pMHC-NPs expanded cognate autoregulatory T cells, suppressed the recruitment of noncognate specificities, prevented disease in prediabetic mice, and restored normoglycemia in diabetic animals. pMHC-NP therapy was inconsequential in mice engineered to bear an immune system unresponsive to the corresponding epitope, owing to absence of epitope-experienced autoregulatory T cells. pMHC-NP-expanded autoregulatory T cells suppressed local presentation of autoantigens in an interferon-gamma-, indoleamine 2,3-dioxygenase-, and perforin-dependent manner. Nanoparticles coated with human diabetes-relevant pHLA complexes restored normoglycemia in a humanized model of diabetes. These observations expose a paradigm in the pathogenesis of autoimmunity amenable for therapeutic intervention.

Tolerogenic Ag-PLG nanoparticles induce tregs to suppress activated diabetogenic CD4 and CD8 T cells.

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.

Opposite effects of a glucokinase activator and metformin on glucose-regulated gene expression in hepatocytes.

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.

Crystal structure of lipid phosphatase Escherichia coli phosphatidylglycerophosphate phosphatase B.

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.

NOD mice are functionally deficient in the capacity of cross-presentation.

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.

Functional cytotoxic T lymphocytes against IGRP206-214 predict diabetes in the non-obese diabetic mouse.

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.

Inhibition of gluconeogenic genes by calcium-regulated heat-stable protein 1 via repression of peroxisome proliferator-activated receptor α.

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.

The Mediator complex kinase module is necessary for fructose regulation of liver glycogen levels through induction of glucose-6-phosphatase catalytic subunit (G6pc).

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.

Mutations in the G6PC3 gene cause Dursun syndrome.

Dursun syndrome is a triad of familial primary pulmonary hypertension, leucopenia, and atrial septal defect. Here we demonstrate that mutations in G6PC3 cause Dursun syndrome. Mutations in G6PC3 are known to also cause severe congenital neutropenia type 4. Identification of the genetic basis of Dursun syndrome expands the pre-existing knowledge about the phenotypic effects of mutations in G6PC3. We propose that Dursun syndrome should now be considered as a subset of severe congenital neutropenia type 4 with pulmonary hypertension as an important clinical feature.

[Activities and properties of glucose-6-phosphatase of turbellaria Phagocata sibirica and cestodes Bothriocephalus scorpii].

There were studied activities and properties of mitochondrial and microsomal glucose-6-phosphatases (G6Pases) in free living turbellaria Phagocata sibirica and cestodes Bothriocephalus scorpii. Action of various effectors (sodium fluoride, glucose, HCO3-, citrate, Cu2+, DTT, EDTA, ATP, AFP) on the enzyme activity was studied. The obtained results and literature data demonstrate that G6Pase is present in various muscles of representatives of the animal kingdom. The conclusion can be made that invertebrate G6Pase releases glucose from glycogen and gluconeogenic precursors.

Mutational spectrum and identification of five novel mutations in G6PC1 gene from a cohort of Glycogen Storage Disease Type 1a.

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) IVS1-2A > T, 4) IVS3 + 39G > A and 5) IVS3 + 42G > A along with three previously reported mutations p.G118D, p.R149Q and p.A331V. Interestingly, each of the p.V99Cfs*3, IVS1-2A > 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, IVS1-2A > T, IVS3 + 39G > A and IVS3 + 42G > 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.

Cloning and comparative bioinformatic analysis of feline glucose-6-phosphatase catalytic subunit cDNA.

Glucose-6-phosphatase is a multicomponent enzyme composed of a transporter subunit and a catalytic subunit that is involved in hepatic glucose production. The objective of the present study was to determine the complete nucleotide sequence of feline hepatic glucose-6-phosphatase catalytic subunit (G6Pc) cDNA and to perform comparative analysis of the molecular features of the feline G6Pc cDNA and protein. Feline G6Pc cDNA contains 2261 bases and encodes a 357 aa protein. The feline cDNA and protein are highly conserved with overall identity ranging from 73-86% to 86-95%, respectively, among mammalian species. Membrane topology, phosphatase consensus sequence, ER retention sequence, N-glycosylation sites and active site residues are conserved in the feline protein. Analysis of the putative feline G6Pc protein did not reveal any species-specific features to explain the unusual in vivo regulation of G6Pase activity reported in feline liver.

CD8+ T cells specific for the islet autoantigen IGRP are restricted in their T cell receptor chain usage.

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.

Functional Analysis of Mouse G6pc1 Mutations Using a Novel In Situ Assay for Glucose-6-Phosphatase Activity and the Effect of Mutations in Conserved Human G6PC1/G6PC2 Amino Acids on G6PC2 Protein Expression.

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.

Effect of reductive alkylation on catalytic properties of glycogen phosphorylase B. enzyme derivatives with changed nucleotide affinities.

Glycogen phosphorylase b modified by NaBH4 and aliphatic aldehydes of varying chain length: ranging from acetaldehyde to n-heptanaldehyde were purified by heat-treatment. Kinetic studies showed that the various purified enzyme derivatives were similar to native phosphorylase b in kinetic properties with respect to glucose-I-P. They, however, exhibited different affinities toward AMP. For acetaldehyde, propionaldehyde and butyraldehyde modified phosphorylases b, increase in chain length of the modifying aldehyde resulted in a decrease in AMP affinity of the modified enzyme. For the other aldehydes increase in chain length resulted in an increased AMP affinity of the modified enzyme. Consequently, the apparent values of butyraldehyde and heptanaldehyde modified phosphorylase b exhibited greatest deviation from that of native phosphorylase b. These two enzyme derivatives were studied in more detail. The activation of both enzyme derivatives by AMP could be greatly stimulated by spermine at suboptimal levels of AMP. Both derivatives could be activated by IMP, UMP or CMP in addition to AMP. The extents of activation of heptanaldehyde modified phosphorylase b by these nucleotides were higher than those found for native phosphorylase b. Thus, under certain conditions which included using suboptimal AMP or using IMP, UMP or GMP instead of AMP, heptanaldehyde modified phosphorylase b had much higher catalytic activity than native phosphorylase b. The interactions between burtyraldehyde or heptanaldehyde modified phosphorylases b with IMP were respectively weaker or stronger than that between native phosphorylase b and IMP. The interactions between butyraldehyde or heptanaldehyde modified phosphorylase b with glucose-6-P, an inhibitor of phosphorylase b partially competitive with respective to AMP, were also respectively weaker or stronger than that between the inhibitor and the native enzyme.

Basal level glucose-6-phosphatase gene transcription requires binding sites for Sp family proteins within the gene promoter.

The significance of two regions (SpA: -19 to -11 and SpB: -63 to -55) within the human glucose-6-phosphatase (G6Pase) gene promoter for gene expression was examined. The mutation of SpA and SpB together, but not alone, decreased G6Pase promoter activity. Electromobility shift assays showed that SpA and SpB were able to bind the transcription factors Sp1 and Sp3.