Aldolase B

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W A Dunn - One of the best experts on this subject based on the ideXlab platform.

  • starvation induced lysosomal degradation of <B>AldolaseB> B requires glutamine 111 in a signal sequence for chaperone mediated transport
    Journal of Cellular Physiology, 2001
    Co-Authors: P P Susan, W A Dunn
    Abstract:

    <B>AldolaseB> B is an aBundant cytosolic protein found in all eukaryotic cells. Like many glycolytic enzymes, this protein was sequestered into lysosomes for degradation during nutrient starvation. We report here that the degradation of recomBinant <B>AldolaseB> B was enhanced two-fold when rat and human hepatoma cells were starved for amino acid and serum. In addition, starvation-induced degradation of <B>AldolaseB> B was inhiBited By chloroquine, an inhiBitor of lysosomal proteinases and By 3-methyladenine, an inhiBitor of autophagy. <B>AldolaseB> B has three lysosomal targeting motifs (Q12KKEL, Q58FREL, and IKLDQ111) that have Been proposed to interact with hsc73 thereBy initiating its transport into lysosomes. In this study, we have mutated the essential glutamine residues in each of these hsc73-Binding motifs in order to evaluate their roles in the lysosomal degradation of <B>AldolaseB> B during starvation. We have found that when glutamines 12 or 58 are mutated to asparagines enhanced degradation of <B>AldolaseB> B proceeded normally. However, when glutamine 111 was mutated to an asparagine or a threonine, starvation-induced degradation was completely suppressed. These mutations did not appear to alter the tertiary structure of <B>AldolaseB> B since enzymatic activity was not affected. Our results suggest that starvation-induced lysosomal degradation of <B>AldolaseB> B requires Both autophagy and glutamine 111. We discuss the possiBle roles for autophagy and hsc73-mediated transport in the lysosomal sequestration of <B>AldolaseB> B. © 2001 Wiley-Liss, Inc.

  • UBiquitinated <B>AldolaseB> B accumulates during starvation‐induced lysosomal proteolysis
    Journal of Cellular Physiology, 1999
    Co-Authors: S E Lenk, P P Susan, Ian Hickson, T Jasionowski, W A Dunn
    Abstract:

    : We have previously shown that stress-induced protein degradation requires a functional uBiquitin-activating enzyme and the autophagic-lysosomal pathway. In this study, we examined the occurrence of uBiquitin-protein conjugates that form during nutrient starvation. Kidney and liver epithelial cells respond to nutrient stress By enhancing autophagy and protein degradation. We have shown that this degradative response was more dramatic in nondividing cultures. In addition, the onset of autophagy was suppressed By pactamycin, cycloheximide, and puromycin. We oBserved an accumulation of uBiquitinated proteins coincident with the degradative response to amino acid starvation. The stress-induced protein uBiquitination was not affected By cycloheximide, indicating that protein synthesis was not required. The uBiquitinated proteins were localized to the cytosol and suBcellular fractions enriched with autophagosomes and lysosomes. The incorporation of the uBiquitinated proteins into autolysosomes was dramatically reduced By 3-methyladenine, an inhiBitor of autophagy. The evidence suggests that uBiquitinated proteins are sequestered By autophagy for degradation. We next set out to identify those primary uBiquitinated proteins at 60 kDa and 68 kDa. Polyclonal antiBodies were prepared against these proteins that had Been immunopurified from rat liver lysosomes. The antiBodies prepared against those 68 kDa proteins also recognized a 40 kDa protein in cytosolic fractions. Internal amino acid sequences oBtained from two cyanogen Bromide fragments of this 40 kDa protein were shown to Be identical to sequences in liver fructose1,6-Bisphosphate <B>AldolaseB> B. Anti-UB68 antiBodies recognized purified <B>AldolaseB> A and <B>AldolaseB> B. Conversely, antiBodies prepared against <B>AldolaseB> B recognized the 40 kDa <B>AldolaseB> as well as four to five high molecular weight forms, including a 68 kDa protein. Finally, we have shown that the degradation of <B>AldolaseB> B was enhanced during amino acid and serum starvation. This degradation was suppressed By chloroquine and 3-methyladenine, suggesting that <B>AldolaseB> B was Being degraded within autolysosomes. We propose that <B>AldolaseB> B is uBiquitinated within the cytosol and then transported into autophagosomes and autolysosomes for degradation during nutrient stress.

  • uBiquitinated <B>AldolaseB> B accumulates during starvation induced lysosomal proteolysis
    Journal of Cellular Physiology, 1999
    Co-Authors: S E Lenk, P P Susan, Ian Hickson, T Jasionowski, W A Dunn
    Abstract:

    : We have previously shown that stress-induced protein degradation requires a functional uBiquitin-activating enzyme and the autophagic-lysosomal pathway. In this study, we examined the occurrence of uBiquitin-protein conjugates that form during nutrient starvation. Kidney and liver epithelial cells respond to nutrient stress By enhancing autophagy and protein degradation. We have shown that this degradative response was more dramatic in nondividing cultures. In addition, the onset of autophagy was suppressed By pactamycin, cycloheximide, and puromycin. We oBserved an accumulation of uBiquitinated proteins coincident with the degradative response to amino acid starvation. The stress-induced protein uBiquitination was not affected By cycloheximide, indicating that protein synthesis was not required. The uBiquitinated proteins were localized to the cytosol and suBcellular fractions enriched with autophagosomes and lysosomes. The incorporation of the uBiquitinated proteins into autolysosomes was dramatically reduced By 3-methyladenine, an inhiBitor of autophagy. The evidence suggests that uBiquitinated proteins are sequestered By autophagy for degradation. We next set out to identify those primary uBiquitinated proteins at 60 kDa and 68 kDa. Polyclonal antiBodies were prepared against these proteins that had Been immunopurified from rat liver lysosomes. The antiBodies prepared against those 68 kDa proteins also recognized a 40 kDa protein in cytosolic fractions. Internal amino acid sequences oBtained from two cyanogen Bromide fragments of this 40 kDa protein were shown to Be identical to sequences in liver fructose1,6-Bisphosphate <B>AldolaseB> B. Anti-UB68 antiBodies recognized purified <B>AldolaseB> A and <B>AldolaseB> B. Conversely, antiBodies prepared against <B>AldolaseB> B recognized the 40 kDa <B>AldolaseB> as well as four to five high molecular weight forms, including a 68 kDa protein. Finally, we have shown that the degradation of <B>AldolaseB> B was enhanced during amino acid and serum starvation. This degradation was suppressed By chloroquine and 3-methyladenine, suggesting that <B>AldolaseB> B was Being degraded within autolysosomes. We propose that <B>AldolaseB> B is uBiquitinated within the cytosol and then transported into autophagosomes and autolysosomes for degradation during nutrient stress.

Kenichi Tsutsumi - One of the best experts on this subject based on the ideXlab platform.

  • Binding of alf c an orc1 Binding transcriptional regulator enhances replicator activity of the rat <B>AldolaseB> B origin
    Molecular and Cellular Biology, 2006
    Co-Authors: Hiroyuki Minami, Yasushi Saitoh, Junko Takahashi, Asami Suto, Kenichi Tsutsumi
    Abstract:

    A region encompassing the rat <B>AldolaseB> B gene (aldB) promoter acts as a chromosomal origin of DNA replication (origin) in rat <B>AldolaseB> B-nonexpressing hepatoma cells. To examine replicator function of the aldB origin, we constructed recomBinant mouse cell lines in which the rat aldB origin and the mutant derivatives were inserted into the same position at the mouse chromosome 8 By cre-mediated recomBination. Nascent strand aBundance assays revealed that the rat origin acts as a replicator at the ectopic mouse locus. Mutation of site C in the rat origin, which Binds an Orc1-Binding protein AlF-C in vitro, resulted in a significant reduction of the replicator activity in the mouse cells. Chromatin immunoprecipitation (ChIP) assays indicated that the reduction of replicator activity was paralleled with the reduced Binding of AlF-C and Orc1, suggesting that sequence-specific Binding of AlF-C to the ectopic rat origin leads to enhanced replicator activity in cooperation with Orc1. Involvement of AlF-C in replication in vivo was further examined for the aldB origin at its original rat locus and for a different rat origin identified in the present study, which contained an AlF-C-Binding site. ChIP assays revealed that Both replication origins Bind AlF-C and Orc1. We think that the results presented here may represent one mode of origin recognition in mammalian cells.

  • a Binding site for purα and purβ is structurally unstaBle and is required for replication in vivo from the rat <B>AldolaseB> B origin
    Biochemical and Biophysical Research Communications, 2006
    Co-Authors: Yoshitaka Shimotai, Yasushi Saitoh, Hiroyuki Minami, Yuichi Onodera, Yukio Mishima, Robert J Kelm, Kenichi Tsutsumi
    Abstract:

    The rat <B>AldolaseB> B promoter acts as a replication origin in vivo, as well as an autonomously replicating sequence (ARS). Here, we examined roles of a polypurine stretch (site PPu) in this origin, which is indispensaBle to the ARS activity. Purification of site PPu-Binding protein revealed that site PPu Binds Purα and Purβ, i.e., single-stranded DNA-Binding proteins whose roles in replication have Been implicated, But less clear. Biochemical analyses showed that site PPu even in a longer DNA fragment is unstaBle in terms of douBle-helix, implying that Purα/β may staBilize single-stranded state. Deletion of site PPu from the origin DNA, which was ectopically positioned in the mouse chromosome, significantly reduced replicator activity. Chromatin immunoprecipitation experiments showed that deletion of site PPu aBolishes Binding of the Purα/β proteins to the origin. These oBservations suggest functional roles of site PPu and Purα/β proteins in replication initiation.

  • replication of the rat <B>AldolaseB> B locus differs Between <B>AldolaseB> B expressing and non expressing cells
    FEBS Letters, 2001
    Co-Authors: Satoru Miyagi, Yungpeng Zhao, Yasushi Saitoh, Katsuyuki Tamai, Kenichi Tsutsumi
    Abstract:

    We previously reported a rat chromosomal origin of DNA replication (oriA1) that encompassed the <B>AldolaseB> B (AldB) gene promoter. Here, we examined utilization of oriA1 in AldB-expressing and non-expressing cells. The results suggested the occurrence of mutually exclusive regulation Between DNA replication and transcription. Nascent strand aBundance as assayed By competitive polymerase chain reaction using Bromodeoxyuridine-laBeled nascent DNA indicated that oriA1 is not utilized in AldB-expressing cells, while it is fired in non-expressing cells. In the latter non-expressing cells, the replication fork seemed to slow at 20–22 kB downstream of oriA1.

  • a novel growth related nuclear protein Binds and inhiBits rat <B>AldolaseB> B gene promoter
    Gene, 2001
    Co-Authors: Tomoko Yabuki, Yasushi Saitoh, Satoru Miyagi, Hitoshi Ueda, Kenichi Tsutsumi
    Abstract:

    ABstract The promoter of the rat <B>AldolaseB> B (AldB) gene that confers liver-specific transcription has an additional role. It functions in vivo as an origin region of DNA replication in the cells in which the gene is repressed (Zhao, Y., Tsutsumi, R., Yamaki, M., Nagatsuka, N., Ejiri, S., Tsutsumi, K., 1994. Initiation zone of DNA replication at the rat <B>AldolaseB> B locus encompasses transcription promoter region. Nucleic Acids Res. 22, 5385–5390). This promoter/origin region has multiple protein-Binding sites and, thus, Binding of a particular set of protein factors in AldB-expressing or non-expressing cells seems to correlate with functional switch of this promoter/origin region. In the present study, we characterized two closely related proteins, termed AlF-C1 and AlF-C2, which are assumed to Be involved in repression of the AldB gene. These two proteins share an identical amino acid sequence except for a 47-residue-insertion in AlF-C1, and are memBers of a gene family including heterogeneous nuclear riBonucleoprotein (hnRNP) and CCAAT-Binding factor suBunit A (CBF-A) genes. Bacterially expressed AlF-C1 can Bind sequence-specifically to the AldB gene promoter, whereas AlF-C2 can only weakly. Transfection experiments using mammalian expression vectors showed that AlF-C1 down-regulates the AldB gene promoter in rat hepatoma cells, while AlF-C2 had no or little effect. Expressions of mRNAs encoding these two proteins are enriched in fetal livers and in regenerating livers. These results implied that AlF-C1 and/or C2 is involved in growth-regulated repression of the AldB gene.

  • an overlapping set of dna elements in the rat <B>AldolaseB> B gene origin promoter regulates transcription and autonomous replication
    Biochemical and Biophysical Research Communications, 2000
    Co-Authors: Satoru Miyagi, Yasushi Saitoh, Yunpeng Zhao, Kenichi Tsutsumi
    Abstract:

    ABstract Promoter of the rat <B>AldolaseB> B (AldB) gene is centered on an origin of chromosomal DNA replication in vivo, and it directs autonomous replication upon transfection into cultured cells. Previous studies showed that the 200 Bp promoter fragment is necessarily required for the autonomous replication. Here, we identified three cis-elements required for replication within the 200 Bp promoter, using autonomously replicating plasmids carrying various mutations and deletions. One is an element that is previously defined as a regulatory element for liver-specific transcription (site C). Other two, purine-rich (site PPu) and A (T)-rich (site A/T) sequences, were those often found in eukaryotic origin regions. Sites C and PPu were found to Bind specific nuclear factors in transfected cells, and the results of competitive Binding assay implied direct or indirect interaction Between sites C and PPu.

Dean R Tolan - One of the best experts on this subject based on the ideXlab platform.

  • <B>AldolaseB> B knockout in mice phenocopies hereditary fructose intolerance in humans
    Molecular Genetics and Metabolism, 2015
    Co-Authors: Sarah A Oppelt, Erin M Sennott, Dean R Tolan
    Abstract:

    ABstract The rise in fructose consumption, and its correlation with symptoms of metaBolic syndrome (MBS), has highlighted the need for a Better understanding of fructose metaBolism. To that end, valid rodent models reflecting the same metaBolism as in humans, Both Biochemically and physiologically, are critical. A key to understanding any type of metaBolism comes from study of disease states that affect such metaBolism. A serious defect of fructose metaBolism is the autosomal recessive condition called hereditary fructose intolerance (HFI), caused By mutations in the human <B>AldolaseB> B gene ( AldoB ). Those afflicted with HFI experience liver and kidney dysfunction after fructose consumption, which can lead to death, particularly during infancy. With very low levels of fructose exposure, HFI patients develop non-alcoholic fatty acid liver disease and fiBrosis, sharing liver pathologies also seen in MBS. A major step toward estaBlishing that fructose metaBolism in mice mimics that of humans is reported By investigating the consequences of targeting the mouse <B>AldolaseB>-B gene ( Aldo2 ) for deletion in mice ( Aldo2 −/− ). The Aldo2 −/− homozygous mice show similar pathology following exposure to fructose as humans with HFI such as failure to thrive, liver dysfunction, and potential morBidity. EstaBlishing that this mouse reflects the symptoms of HFI in humans is critical for comparison of rodent studies to the human condition, where this food source is increasing, and increasingly controversial. This animal should provide a valuaBle resource for answering remaining questions aBout fructose metaBolism in HFI, as well as help investigate the Biochemical mechanisms leading to liver pathologies seen in MBS from high fructose diets.

  • mutations in the promoter region of the <B>AldolaseB> B gene that cause hereditary fructose intolerance
    Journal of Inherited Metabolic Disease, 2010
    Co-Authors: Erin M Coffee, Dean R Tolan
    Abstract:

    Hereditary fructose intolerance (HFI) is a potentially fatal inherited metaBolic disease caused By a deficiency of <B>AldolaseB> B activity in the liver and kidney. Over 40 disease-causing mutations are known in the protein-coding region of ALDOB. Mutations upstream of the protein-coding portion of ALDOB are reported here for the first time. DNA sequence analysis of 61 HFI patients revealed single Base mutations in the promoter, intronic enhancer, and the first exon, which is entirely untranslated. One mutation, g.−132G>A, is located within the promoter at an evolutionarily conserved nucleotide within a transcription factor-Binding site. A second mutation, IVS1+1G>C, is at the donor splice site of the first exon. In vitro electrophoretic moBility shift assays show a decrease in nuclear extract-protein Binding at the g.−132G>A mutant site. The promoter mutation results in decreased transcription using luciferase reporter plasmids. Analysis of cDNA from cells transfected with plasmids harBoring the IVS1+1G>C mutation results in aBerrant splicing leading to complete retention of the first intron (~5 kB). The IVS1+1G>C splicing mutation results in loss of luciferase activity from a reporter plasmid. These novel mutations in ALDOB represent 2% of alleles in American HFI patients, with IVS1+1G>C representing a significantly higher allele frequency (6%) among HFI patients of Hispanic and African-American ethnicity.

  • structure of the thermolaBile mutant <B>AldolaseB> B a149p molecular Basis of hereditary fructose intolerance
    Journal of Molecular Biology, 2005
    Co-Authors: Ali D Malay, Karen N Allen, Dean R Tolan
    Abstract:

    Hereditary fructose intolerance (HFI) is a potentially lethal inBorn error in metaBolism caused By mutations in the <B>AldolaseB> B gene, which is critical for gluconeogenesis and fructose metaBolism. The most common mutation, which accounts for 53% of HFI alleles identified worldwide, results in suBstitution of Pro for Ala at position 149. Structural and functional investigations of human <B>AldolaseB> B with the A149P suBstitution (AP-<B>AldolaseB>) have shown that the mutation leads to losses in thermal staBility, quaternary structure, and activity. X-ray crystallography is used to reveal the structural Basis of these perturBations. Crystals of AP-<B>AldolaseB> are grown at two temperatures (4 °C and 18 °C), and the structure solved to 3.0 A resolution, using the wild-type structure as the phasing model. The structures reveal that the single residue suBstitution, A149P, causes molecular disorder around the site of mutation (residues 148–159), which is propagated to three adjacent β-strand and loop regions (residues 110–129, 189–199, 235–242). Disorder in the 110–129-loop region, which comprises one suBunit–suBunit interface, provides an explanation for the disrupted quaternary structure and thermal instaBility. Greater structural perturBation, particularly at a Glu189-Arg148 salt Bridge in the active-site architecture, is oBserved in the structure determined at 18 °C, which could explain the temperature-dependent loss in activity. The disorder revealed in these structures is far greater than that predicted By homology modeling and underscores the difficulties in predicting perturBations of protein structure and function By homology modeling alone. The AP-<B>AldolaseB> structure reveals the molecular Basis of a hereditary disease and represents one of only a few structures known for mutant proteins at the root of the thousands of other inherited disorders.

  • the temperature dependence of activity and structure for the most prevalent mutant <B>AldolaseB> B associated with hereditary fructose intolerance
    Archives of Biochemistry and Biophysics, 2002
    Co-Authors: Ali D Malay, Sheri L Procious, Dean R Tolan
    Abstract:

    ABstract Hereditary fructose intolerance (HFI) is an autosomal recessive disorder in humans which is caused By mutations in the <B>AldolaseB> B gene. The most common HFI allele encodes an enzyme with an A149P suBstitution (AP-<B>AldolaseB>). A lysis method suitaBle for aggregation-prone proteins overexpressed in Bacteria was developed. The enzyme’s structure and function is investigated as a function of temperature. Near-UV CD shows a qualitative difference in tertiary structure, whereas far-UV CD shows no difference in overall secondary structure, although Both show increased temperature sensitivity for AP-<B>AldolaseB> compared to that seen with wild-type <B>AldolaseB> B. AP-<B>AldolaseB> exists as a dimer at all temperatures tested, unlike the tetrameric wild-type enzyme, thus providing a possiBle explanation for the loss in thermostaBility. AP-<B>AldolaseB> has sixfold lower activity than wild type at 10 °C, which decreases suBstantially at higher temperature. In addition to disruptions at the catalytic center, the kinetic constants toward different suBstrates suggest that there is a disruption at the C1-phosphate-Binding site, which is not sensitive to temperature. The implications of these structural alterations are discussed with regard to the HFI disease.

  • identification of conserved promoter elements for aldB and isozyme specific residues in <B>AldolaseB> B
    Comparative Biochemistry and Physiology B, 1999
    Co-Authors: Tanya Z Berardini, Edward E. Penhoet, Bruce A Amsden, Dean R Tolan
    Abstract:

    ABstract The comparison of three complete <B>AldolaseB> B genes-including known and putative regulatory elements-is presented. The third <B>AldolaseB> B gene was provided By the complete ald B gene sequence (14 803 Bp) encoding the raBBit <B>AldolaseB> B isozyme. The promoter sequence alignment included the nonmammalian chicken <B>AldolaseB> B gene and confirms the promoter sequence conservation of those elements where trans -factor Binding has Been demonstrated in rat ald B. Moreover, the alignment reveals conserved sequences that may represent previously unidentified promoter elements that are present in all ald Bs or specifically in the mammalian ald B promoters. One remarkaBle feature is a poly-purine segment found Between the CAAT and TATA elements. In the mammalian promoters, this is exclusively a 9–10 Bp poly-dA stretch. The avian promoter has an additional stretch of eight dG-Bases immediately upstream of the poly-dA. Alignment of a portion of intron 1 of the chicken, human, and raBBit ald B genes reveals conserved sequences that are likely candidates for a reported positive activation sequence. In addition, the amino acid sequences of all eight known <B>AldolaseB> B isozymes is compared to the other verteBrate <B>AldolaseB>s. A numBer of <B>AldolaseB> B-specific residues are identified that cluster in the carBoxyl-portion of the sequence. With the exception of residue C268, these residues are not found near the active site, although, they are likely to Be responsiBle for the suBstrate specificity of <B>AldolaseB> B.

Annelise Pichard - One of the best experts on this subject based on the ideXlab platform.

  • in vivo functional characterization of the <B>AldolaseB> B gene enhancer
    Journal of Biological Chemistry, 2002
    Co-Authors: Claudine Gregori, Axel Kahn, Arlette Porteu, Claudia Mitchell, Annelise Pichard
    Abstract:

    ABstract A 400-Bp intronic enhancer fragment in conjunction with the proximal promoter of the <B>AldolaseB> B gene provided correct tissue-specific expression in transgenic mice together with hormonal regulation in the liver. We investigated in vivoand in cultured cells the contriBution of the intronic regulatory sequences and their interaction with the promoter elements in controlling <B>AldolaseB> B gene expression. Transgene activity was completely aBolished By disruption of the two hepatocyte nuclear factor 1 (HNF1) Binding sites in the enhancer, whereas mutation of one HNF1 site had no effect in the liver But strongly decreased activity in the kidney. Our data show that the HNF1 Binding site(s) in the enhancer were key regulators of <B>AldolaseB> B transgene expression Both in the liver and kidney. Deletion of the CCAAT/enhancer-Binding protein site in the promoter completely aBolished the enhancer function in HepG2 cells. These results suggest that expression of the <B>AldolaseB> B gene in the liver requires cooperative interactions Between CCAAT/enhancer-Binding protein and HNF1. Deletion of the HNF4 Binding site in the enhancer suppressed expression in Both liver and kidney in half of the transgenic lines, suggesting that this element might play a role in chromatin opening at the insertion site. We firmly estaBlish that the endogenous <B>AldolaseB> B gene's first response to glucagon or cyclic AMP exposure was a transient increase in the expression in the liver, followed By a secondary decline in the transcription, as previously reported. This response was reproduced By all transgenes studied, indicating that neither HNF1 nor HNF4 Binding sites in the enhancer were involved in this Biphasic cyclic AMP response.

  • characterization of the <B>AldolaseB> B intronic enhancer
    Journal of Biological Chemistry, 1998
    Co-Authors: Claudine Gregori, Axel Kahn, Arlette Porteu, Soledad Lopez, Annelise Pichard
    Abstract:

    ABstract The <B>AldolaseB> B gene is transcriBed at a high level in the liver, kidney, and small intestine. This high level of gene expression results from cooperation Between a weak But liver-specific promoter and an intronic activator. A deletional study of this activator present in the first intron allowed us to ascriBe the maximal enhancer function to a 400-Base pair (Bp) fragment (+1916 to + 2329). This enhancer is highly liver-specific and enhances the activity of heterologous minimal promoters in a position and distance-independent fashion in transiently transfected Hep G2 hepatoma cells. The <B>AldolaseB> B enhancer is composed of two domains, a 200-Bp module (Ba) inactive By itself But which synergizes with another 200-Bp module (BB) that alone retains 25% of the total enhancer activity. The BB sequence is 76% homologous Between human and rat genes and contains several Binding sites for liver-enriched nuclear factors. By electrophoretic moBility shift assays, we demonstrated that elements 5 and 7 Bind hepatic nuclear factor 1 (HNF1), whereas element 2 Binds hepatic nuclear factor 4 (HNF4). A functional analysis of the enhancer whose elements have Been mutated demonstrated that mutation of any of the HNF1 sites totally suppressed enhancer activity, whereas mutation of the HNF4-Binding site reduced it By 80%.

  • an intronic enhancer essential for tissue specific expression of the <B>AldolaseB> B transgenes
    Journal of Biological Chemistry, 1996
    Co-Authors: Jeanchristophe Sabourin, Claudine Gregori, Axel Kahn, Arlette Porteu, Annesophie Kern, Charlotte Cywiner, Francoispatrick Châtelet, Annelise Pichard
    Abstract:

    ABstract Expression in mice of transgenes directed By regulatory regions of the rat <B>AldolaseB> B gene requires the presence of a B element located in the first intron, while constructs devoid of this intronic enhancer are silent. Histo- and immunochemical staining of transgenic tissue sections showed that the longer transgene was expressed in the proximal tuBular cells of the kidney, enterocytes located in small intestine villi and liver parenchymal cells. In the liver, a maximal expression was oBserved in perivenous hepatocytes, while the transgene was weakly active in periportal hepatocytes, which reproduced the pattern of functional zonation already reported for other glycolytic and gluconeogenic genes in the liver. We also estaBlished that the transgene retained the necessary elements for a correct chronological expression during development But was lacking elements necessary for activation By high carBohydrate diet. Instead, transgene expression was paradoxically stimulated in fasted animals, suggesting that the endogenous gene, which must Be active under Both glycolytic and gluconeogenic conditions, could possess distinct elements activating it in fasted as well as in carBohydrate-fed animals; the former element might Be conserved in the transgene and the latter one might Be lost.

  • activity of the rat liver specific <B>AldolaseB> B promoter is restrained By hnf3
    Nucleic Acids Research, 1994
    Co-Authors: Claudine Gregori, Axel Kahn, Annelise Pichard
    Abstract:

    ABstract Although it contains Binding sites for HNF1, NFY and C/EBP/DBP, the proximal promoter of the <B>AldolaseB> B gene is surprisingly weak when tested By transient transfection in differentiated hepatoma cells. This low activity could Be due to overlapping Between HNF1 and HNF3 Binding sites in element PAB, from -127 to -103 Bp with respect to the cap site. Replacement of the PAB region By a consensus HNF1 Binding site unaBle to Bind HNF3, results in a 30 fold activation of the promoter, in accordance with the hypothesis that activity of the wild-type promoter is normally restrained By HNF3 Binding to PAB competitively with HNF1. Consistently, transactivation of the wild-type promoter By excess HNF1 is very high, most likely due to the displacement of HNF3, while the construct with the exclusive HNF1 Binding site is weakly transactivated By HNF1. The inhiBitory effect of HNF3 on HNF1-dependent transactivation is clearly due to competition Between these two factors for Binding to mutually exclusive, overlapping sites; indeed, when HNF1 and HNF3 sites are contiguous and not overlapping, the resulting promoter is as active as the one containing an exclusive HNF1 Binding site. A construct in which PAB has Been replaced By an exclusive HNF3 Binding site is weakly expressed and is insensitive to HNF3 hyperexpression. DBP-dependent transactivation, finally, is independent of the nature of the element present in the PAB region.

  • competition Between transcription factors hnf1 and hnf3 and alternative cell specific activation By dBp and c eBp contriBute to the regulation of the liver specific <B>AldolaseB> B promoter
    Nucleic Acids Research, 1993
    Co-Authors: Claudine Gregori, Axel Kahn, Annelise Pichard
    Abstract:

    ABstract The <B>AldolaseB> B proximal promoter is controlled By at least five elements spanning from -190 to -103 Bp with respect to the start site of transcription. From 5' to 3', we found: a negative DE element, an activating C/EBP-DBP Binding site, a CCAAT Box Binding NFY that seems to play a negative role, and an activating element consisting of two overlapping Binding sites for HNF-1 and HNF-3. Contransfection experiments of <B>AldolaseB> B/CAT constructs and of expression vectors for different transcription factors were carried out in human hepatoma Hep G2 cells. We found that DBP and HNF-1 are strong transactivators of the <B>AldolaseB> B promoter while C/EBP and vHNF-1 are only weak activators and HNF-3 alone does not modify such activity. Deletion of the distal negative element results in a similar transactivation By C/EBP and DBP, enhanced for the former and reduced for the latter. In hepatocytes in primary culture, the strong transactivator is C/EBP while DBP is essentially inactive. This tissue-specificity of C/EBP and DBP action could depend on interaction with tissue-specific proteins Bound to a neighBouring site, proBaBly DE. Finally, HNF3 Behaves as a very strong anti-activator of the <B>AldolaseB> B promoter. It competitively antagonizes transactivation By HNF-1 and non-competitively transactivation By DBP. This negative effect of HNF-3 and tissue-specificity of the transactivation potential of DBP and C/EBP are unique features of the <B>AldolaseB> B promoter.

Kiichi Ishikawa - One of the best experts on this subject based on the ideXlab platform.

  • characterization of the responsive elements to hormones in the rat <B>AldolaseB> B gene
    Archives of Biochemistry and Biophysics, 2000
    Co-Authors: Yumiko Takano, Takejiro Kuzumaki, Kaoru Otsu, Yoshihito Iuchi, Kiichi Ishikawa
    Abstract:

    ABstract Transcription of the <B>AldolaseB> B gene, AldB, in the liver is regulated By hormones such as insulin and glucagon. To characterize the elements that are responsive to these hormones in the upstream region of AldB, plasmids carrying various length of the upstream region of this gene were constructed and transfected to primary cultured rat hepatocytes. The promoter activities were gradually increased By progressive deletion of the 5′-upstream region, and high activities were oBserved for constructs carrying the sequence Between −408 and −85 Bp, suggesting the presence of suppressive element(s) in the upstream region of −409 Bp. The transcription activities of the mutants containing the sequences Between −228 and −85 Bp were enhanced By insulin, and glucagon suppressed the transcription activities of those containing the sequence Between −764 and −85 Bp. Two sequence elements similar to the cAMP-responsive element (CRE), one from −89 to −82 Bp and another from +13 to +20 Bp, were found in the upstream sequence of the gene. The latter element is not functional Because its deletion did not affect either the transcription efficiency or glucagon response. However, the deletion of the former element diminished Both functions. A gel retardation assay showed that the nuclear factor Binds to the former element, which was competitive with authentic CRE oligonucleotide But not with the mutant CRE one. These results suggest that the CRE-like element in the promoter region is prerequisite for Both fundamental transcription efficiency of the gene and suppression By glucagon in hepatocytes.

  • hormonal regulation of <B>AldolaseB> B gene expression in rat primary cultured hepatocytes
    Archives of Biochemistry and Biophysics, 1998
    Co-Authors: Takejiro Kuzumaki, Kaoru Otsu, Yoshihito Iuchi, Kiichi Ishikawa
    Abstract:

    ABstract Gene expression of <B>AldolaseB> B, an important enzyme for glucose and fructose metaBolism, is regulated By hormones. We examined direct effects of major hormones on <B>AldolaseB> B gene expression in rat primary cultured hepatocytes, in comparison with those on the gene expression of phospho(enol)pyruvate carBoxykinase (PEPCK), a key enzyme for gluconeogenesis. Insulin, dexamethasone, and high concentration of glucose increased <B>AldolaseB> B mRNA aBundance in the hepatocytes. Glucagon strongly suppressed <B>AldolaseB> B gene expression, and this hormone canceled the stimulative effects of insulin, dexamethasone, and high concentration of glucose. Epinephrine and thyroxine slightly reduced <B>AldolaseB> B mRNA aBundance, But these hormones did not cancel the stimulative effects of insulin and dexamethasone. To the contrary, expression of PEPCK gene was suppressed By insulin, dexamethasone, and high concentration of glucose, and remarkaBly induced By glucagon. Glucagon rapidly suppressed <B>AldolaseB> B gene expression at the transcriptional level. Forskolin and diButyryl cAMP mimicked the suppressive effect of glucagon on <B>AldolaseB> B gene expression. These results suggest that glucagon may Be a key regulator of <B>AldolaseB> B gene transcription through a cAMP/protein kinase A-signaling pathway.

  • interleukin 6 down regulates expressions of the <B>AldolaseB> B and alBumin genes through a pathway involving the activation of tyrosine kinase
    Archives of Biochemistry and Biophysics, 1995
    Co-Authors: Yong Huang, Takejiro Kuzumaki, Kaoru Otsu, Haruhide Shinzawa, Hitoshi Togashi, Tsuneo Takahashi, Kiichi Ishikawa
    Abstract:

    ABstract Interleukin-6 plays a key role in mediating acute-phase protein synthesis in hepatocytes. However, the mechanism of how interleukin-6 regulates <B>AldolaseB> B and alBumin syntheses in hepatocytes is not completely understood. In this study, using primary cultured rat hepatocytes, we have shown that interleukin-6 down-regulates expressions of the <B>AldolaseB> B and alBumin genes in a dose- and time-dependent manner. We examined whether the decrease in <B>AldolaseB> B and alBumin mRNA expressions By interleukin-6 reflected transcriptional down-regulation or staBility of the mRNA. Actinomycin D and cycloheximide did not affect the interleukin-6-mediated decrease in the expressions of Both genes. These results suggest that the decreased expressions of Both genes induced By interleukin-6 is controlled at the transcriptional level, and that it is due neither to increased degredation of mRNA nor to synthesis of new proteins. Protein kinases play a fundamental role in the intracellular signal transduction. To examine the interleukin-6 signal pathway(s) leading to the decrease of <B>AldolaseB> B and alBumin mRNA expressions, we tested various kinds of protein kinase inhiBitors in this system. HerBimycin A, an inhiBitor of tyrosine kinase(s), prevented the decrease in the expressions of <B>AldolaseB> B and alBumin mRNAs By interleukin-6 H-7, an inhiBitor of protein kinase C, prevented the decrease in the expression of alBumin mRNA By interleukin-6, But did not induce recovery of that of <B>AldolaseB> B mRNA. These results suggest that a tyrosine kinase(s) or a herBimycin A-sensitive kinase(s) constitutes a common pathway for interleukin-6-mediated reduction of <B>AldolaseB> B and alBumin mRNA expressions and that distinct pathways exist for the modes of expression of the two mRNAs.

  • dietary and hormonal regulation of <B>AldolaseB> B gene transcription in rat liver
    Archives of Biochemistry and Biophysics, 1994
    Co-Authors: P F Gomez, Takejiro Kuzumaki, Kaoru Otsu, Yong Huang, Kiichi Ishikawa
    Abstract:

    ABstract In the liver of the fasted rat, the <B>AldolaseB> B (AldB) mRNA level decreased to aBout half of that of the control rat. When the control rat was refed the glucose-rich diet, the AldB mRNA level increased aBout six to seven times more than in the fasted rat. This increase was shown as the activation of the AldB gene transcription By a nuclear run-on assay. To understand the causal factor(s) for this activation, the relationship Between the AldB mRNA level in the liver and the plasma concentrations of hormones, which are known as major regulators of carBohydrate metaBolism during fasting and refeeding, was investigated. The plasma insulin level in the rat which was refed the glucose-rich diet increased in parallel to AldB mRNA level, while the plasma glucagon level decreased reciprocally to it. The relationship of the plasma corticosterone level to the AldB mRNA level was not oBvious. To directly confirm the effects of these hormones on AldB gene transcription in the liver, the responses of AldB gene in the primary cultured hepatocytes to these hormones were examined. Insulin and dexamethasone were effective to activate AldB gene, while glucagon and thyroxine were suppressive. Thyroxine did not extinguish the effects of insulin and dexamethasone, But glucagon canceled them. Thus, it is proBaBle that in vivo these hormones synergistically regulate the AldB gene transcription. In vitro transcription analysis of two AldB promoter constructs suggested that the proximal half of the AldB promoter (up to -92 Bp from the transcription start site) is, at least in part, involved for this induction, and the distal half which contains liver-specific elements (-93 to -202 Bp) is not involved. The possiBle explanation for the dietary regulation of <B>AldolaseB> B gene transcription in the liver is discussed.

  • a1f B a novel ccaat Binding transcription activator that interacts with the <B>AldolaseB> B promoter
    FEBS Letters, 1993
    Co-Authors: Kenichi Tsutsumi, Tomoko Yabuki, Kiichi Ishikawa
    Abstract:

    We descriBe here a 70 kDa transcription factor A1F-B, which preferentially Binds to an element encompassing a CCAAT motif on the rat <B>AldolaseB> B promoter. Comparison of Binding specificities, relative molecular masses, and suBunit compositions with those of other known CCAAT-Binding factors indicated that A1F-B is a novel memBer of CCAAT-Binding factors.