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Aldolase B

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W A Dunn – 1st expert 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, W A Dunn, P P Susan, Ian Hickson, T Jasionowski

    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 – 2nd expert 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.

Dean R Tolan – 3rd expert 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.