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Roberta F. Colman - One of the best experts on this subject based on the ideXlab platform.

  • In vitro hybridization and separation of hybrids of human Adenylosuccinate Lyase from wild-type and disease-associated mutant enzymes.
    Biokhimiia (Moscow Russia), 2011
    Co-Authors: Lushanti De Zoysa Ariyananda, Christina Antonopoulos, Jenna M. Currier, Roberta F. Colman
    Abstract:

    Human Adenylosuccinate Lyase (ASL) deficiency is an inherited metabolic disease in which the majority of the patients are compound heterozygotes for the mutations that occur in the ASL gene. Starting with purified wild-type (WT) and single-mutant human ASL, we generated in vitro hybrids that mimic compound heterozygote ASL. For this study, we used His-tagged WT/non-His-tagged WT, His-tagged WT/non-His-tagged R396C, His-tagged WT/non-His-tagged R396H, His-tagged R194C/non-His-tagged R396C, and His-tagged L311V/non-His-tagged R396H enzyme pairs. We generated various hybrids by denaturing pairs of enzymes in 1 M guanidinium chloride and renaturing them by removing the denaturant. The hybrids were separated on a nickel−nitrilotriacetic acid−agarose column based on the number of His tags present in the enzyme tetramer. Analytical ultracentrifuge data indicate that the hybrids have predominant amounts of heterotetramers. Analysis of the Vmax values of the hybrids indicates that most of the subunits behave indep...

  • Biochemical and Biophysical Analysis of Five Disease-Associated Human Adenylosuccinate Lyase Mutants †
    Biochemistry, 2009
    Co-Authors: Lushanti De Zoysa Ariyananda, Peychii Lee, Christina Antonopoulos, Roberta F. Colman
    Abstract:

    Adenylosuccinate Lyase (ASL), a catalyst of key reactions in purine biosynthesis, is normally a homotetramer in which three subunits contribute to each of four active sites. Human ASL deficiency is an inherited metabolic disease associated with autism and mental retardation. We have characterized five disease-associated ASL mutants: R194C and K246E are located at subunit interfaces, L311V is in the central helical region away from the active site, and R396C and R396H are at the entrance to the active site. The Vmax (at 25 °C) for R194C is comparable to that of WT; while those of L311V, R396C, R396H and K246E are considerably reduced and affinity for Adenylosuccinate is retained. The mutant enzymes have decreased positive cooperativity as compared to WT. K246E exists mainly as dimer or monomer, accounting for its negligible activity; whereas the other mutant enzymes are similar to WT in the predominance of tetramer. At 37 °C, the specific activity of WT and these mutant enzymes slowly decreases 30-40% with time and reaches a limiting specific activity without changing significantly the amount of tetramer. Mutant R194C is unique in being rapidly inactivated at the harsher temperature of 60°C, indicating that it is the least stable enzyme in vitro. Conformational changes in the mutant enzymes are evident from protein fluorescence intensity at 25 °C and after incubation at 37 °C, which correlates with the loss of enzymatic activity. Thus, these disease-associated single mutations can yield enzyme with reduced activity either by affecting the active site or by perturbing the enzyme’s structure and/or native conformation which are required for catalytic function.

  • biochemical and biophysical analysis of five disease associated human Adenylosuccinate Lyase mutants
    Biochemistry, 2009
    Co-Authors: Lushanti De Zoysa Ariyananda, Peychii Lee, Christina Antonopoulos, Roberta F. Colman
    Abstract:

    Adenylosuccinate Lyase (ASL), a catalyst of key reactions in purine biosynthesis, is normally a homotetramer in which three subunits contribute to each of four active sites. Human ASL deficiency is an inherited metabolic disease associated with autism and mental retardation. We have characterized five disease-associated ASL mutants: R194C and K246E are located at subunit interfaces, L311V is in the central helical region away from the active site, and R396C and R396H are at the entrance to the active site. The Vmax (at 25 °C) for R194C is comparable to that of WT; while those of L311V, R396C, R396H and K246E are considerably reduced and affinity for Adenylosuccinate is retained. The mutant enzymes have decreased positive cooperativity as compared to WT. K246E exists mainly as dimer or monomer, accounting for its negligible activity; whereas the other mutant enzymes are similar to WT in the predominance of tetramer. At 37 °C, the specific activity of WT and these mutant enzymes slowly decreases 30-40% with time and reaches a limiting specific activity without changing significantly the amount of tetramer. Mutant R194C is unique in being rapidly inactivated at the harsher temperature of 60°C, indicating that it is the least stable enzyme in vitro. Conformational changes in the mutant enzymes are evident from protein fluorescence intensity at 25 °C and after incubation at 37 °C, which correlates with the loss of enzymatic activity. Thus, these disease-associated single mutations can yield enzyme with reduced activity either by affecting the active site or by perturbing the enzyme’s structure and/or native conformation which are required for catalytic function.

  • Effect of a new non-cleavable substrate analog on wild-type and serine mutants in the signature sequence of Adenylosuccinate Lyase of Bacillus subtilis and Homo sapiens.
    Protein science : a publication of the Protein Society, 2008
    Co-Authors: Sharmila Sivendran, Roberta F. Colman
    Abstract:

    Adenylosuccinate Lyase (ASL) catalyzes two β-elimination reactions in purine biosynthesis, leading to the question of whether the two substrates occupy the same or different active sites. Kinetic studies of Bacillus subtilis and human ASL with a new substrate analog, adenosine phosphonobutyric acid, 2′(3′), 5′-diphosphate (APBADP), show that it acts as a competitive inhibitor with respect to either substrate (KI ∼ 0.1 μM), indicating that the two substrates occupy the same active site. Binding studies show that both the B. subtilis and human ASLs bind up to 4 mol of APBADP per mole of enzyme tetramer and that both enzymes exhibit cooperativity: negative for B. subtilis ASL and positive for human ASL. Mutant B. subtilis ASLs, with replacements for residues previously identified as critical for catalysis, bind the substrate analog similarly to wild-type ASL. Two serines in a flexible loop of ASL have been proposed to play roles in catalysis because they are close to the substrate in the crystal structure of Escherichia coli ASL. We have now mutated the corresponding serines to alanines in B. subtilis and human ASL to evaluate their involvement in enzyme function. Kinetic data reveal that human Ser289 and B. subtilis Ser262 and Ser263 are essential for catalysis, while the ability of these Ser mutants to bind APBADP suggests that they do not contribute to substrate affinity. Although these serines are not visible in the crystal structure of human Adenylosuccinate Lyase complexed with substrate or products (PDB #2VD6), they may be interacting with the active sites.

  • Evaluation of types of interactions in subunit association in Bacillus subtilis Adenylosuccinate Lyase.
    Biochemistry, 2008
    Co-Authors: Lushanti De Zoysa Ariyananda, Roberta F. Colman
    Abstract:

    Adenylosuccinate Lyase (ASL) of Bacillus subtilis is a homotetramer in which three subunits contribute to each of four active sites. We sought to evaluate the types of interactions responsible for subunit association by studying the enzyme’s oligomeric structure at low temperatures as compared to 25 °C, in the presence of KBr and after mutagenesis. Analytical ultracentrifugation data reveal that at 25 °C ASL is active and exists as 100% tetramer, while at 8 and 4 °C, as hydrophobic interactions are weakened, the catalytic activity decreases strikingly and the enzyme dissociates to a mixture of monomer–dimer–trimer, with small amounts of tetramer. In the presence of increasing concentrations of KBr (0.1–2.5 M), which disrupts electrostatic interactions, ASL is dissociated initially to monomer–dimer, with small amounts of trimer−tetramer, and then the monomer species predominates along with small amounts of trimer−tetramer. Very low enzymatic activity was found under these conditions. Accordingly, we postul...

Ron A. Wevers - One of the best experts on this subject based on the ideXlab platform.

  • In vivo proton MR spectroscopy findings specific for Adenylosuccinate Lyase deficiency
    NMR in biomedicine, 2010
    Co-Authors: Marco Henneke, Marinette Van Der Graaf, Michèl A.a.p. Willemsen, Steffi Dreha-kulaczewski, Knut Brockmann, Udo F. H. Engelke, Peter Dechent, Arend Heerschap, Gunther Helms, Ron A. Wevers
    Abstract:

    Adenylosuccinate Lyase (ADSL) deficiency is an inherited metabolic disorder affecting predominantly the central nervous system. The disease is characterized by the accumulation of succinylaminoimidazolecarboxamide riboside and succinyladenosine (S-Ado) in tissue and body fluids. Three children presented with muscular hypotonia, psychomotor delay, behavioral abnormalities, and white matter changes on brain MRI. Two of them were affected by seizures. Screening for inborn errors of metabolism including in vitro high resolution proton MRS revealed an ADSL deficiency that was confirmed genetically in all cases. All patients were studied by in vivo proton MRS. In vitro high resolution proton MRS of patient cerebrospinal fluid showed singlet resonances at 8.27 and 8.29 ppm that correspond to accumulated S-Ado. In vivo proton MRS measurements also revealed a prominent signal at 8.3 ppm in gray and white matter brain regions of all patients. The resonance was undetectable in healthy human brain. In vivo proton MRS provides a conclusive finding in ADSL deficiency and represents a reliable noninvasive diagnostic tool for this neurometabolic disorder.

Costantino Salerno - One of the best experts on this subject based on the ideXlab platform.

  • Inhibition of defective Adenylosuccinate Lyase by HNE : A neurological disease that may be affected by oxidative stress
    BioFactors (Oxford England), 2005
    Co-Authors: Carlo Crifò, Werner Siems, S. Soro, Costantino Salerno
    Abstract:

    Adenylosuccinate Lyase is an enzyme of fumarase superfamily that participates in the purine biosynthetic pathway, catalysing the nonhydrolytic cleavage of succinyl groups from SAICA ribotide and Adenylosuccinate. Enzyme defects are associated with a human inherited disease, which arises from single point mutations to the gene and results in mild to severe psychomotor retardation, epilepsy, muscle wasting, and autistic features. Adenylosuccinate Lyase activity is lost to a different extent in the patients. Diminished levels of enzyme have been attributed to loss of catalytic activity, protein instability, or environmental factors. P100A/D422Y mutation represents a feasible model for studying the effect of cell milieu on the activity of the impaired enzyme. The defective enzyme is inhibited by micromolar concentrations of trans-4-hydroxy-2-nonenal (HNE), a major product of membrane peroxidation that has been found to accumulate in brain tissues of patients with neurodegenerative disorders. It is suggested that inactivation of defective Adenylosuccinate Lyase by HNE and other membrane peroxidation products may account, at least in part, for the impairment of neurological functions and recurrent worsening of the symptoms.

  • Neurologic aspects of Adenylosuccinate Lyase deficiency.
    Journal of child neurology, 2001
    Co-Authors: Francesca Ciardo, Costantino Salerno, Paolo Curatolo
    Abstract:

    Adenylosuccinate Lyase deficiency is an autosomal-recessive disorder of the purine de novo synthesis pathway, diagnosed up to now in approximately 40 patients. The clinical presentation is characterized by severe neurologic involvement including seizures, developmental delay, hypotonia, and autistic features. Neonatal seizures and a severe infantile epileptic encephalopathy are often the first manifestations of this disorder. The existence of genetic heterogeneity for the Adenylosuccinate Lyase defect could account for variability of the clinical presentation. Deficiency of purine nucleotides, impairment of energy metabolism, and toxic effects are potential mechanisms of cerebral damage. Laboratory investigations show the presence in urine and cerebrospinal fluid of succinylpurines, which are normally undetectable. Currently, no effective treatment is available for Adenylosuccinate Lyase deficiency. A search for this disorder should be included in the screening program of children with unexplained neonatal seizures or severe infantile epileptic encephalopathy.

  • Succinylpurinemic autism: increased sensitivity of defective Adenylosuccinate Lyase towards 4-hydroxy-2-nonenal
    Biochimica et Biophysica Acta, 2000
    Co-Authors: Costantino Salerno, Werner Siems, Carlo Crifò
    Abstract:

    Abstract We studied the effect of trans -4-hydroxy-2-nonenal on the wild-type human Adenylosuccinate Lyase and on the enzyme from a patient compound-heterozygous for two missense mutations (P75A/D397Y; McKusick 103050.0003/103050.0004). Both the enzymes were inhibited by 10–50 μM trans -4-hydroxy-2-nonenal in a concentration-dependent manner by means of a mixed-type co-operative mechanism. A significantly stronger inhibition was noticed in the presence of the defective enzyme. Nonanal and trans -2,3-nonenal inhibited the enzymes to a less extent and at about 10-times higher concentrations. Hydroxylamine reversed the inhibition by trans -4-hydroxy-2-nonenal, trans -2,3-nonenal or nonanal in the case of the wild-type enzyme, but it was ineffective to reverse the inhibition by trans -4-hydroxy-2-nonenal on the defective enzyme. Dithiothreitol slightly decreased the inhibition exerted by trans -4-hydroxy-2-nonenal on both the wild-type and the defective Adenylosuccinate Lyase, while it did not produce practically any change in the presence of trans -2,3-nonenal or nonanal.

  • Identification of new mutations in the Adenylosuccinate Lyase gene associated with impaired enzyme activity in lymphocytes and red blood cells.
    Biochimica et biophysica acta, 1998
    Co-Authors: Daniela Verginelli, Carlo Crifò, Costantino Salerno, Bruno Luckow, Manfred Gross
    Abstract:

    Abstract We determined the DNA sequence of the Adenylosuccinate Lyase (ASL) gene from a 13 year-old female, who showed a reduced ASL enzymatic activity in lymphocytes and red blood cells and suffered from severe psychomotor retardation. The patient was the offspring of a non-consanguineous marriage. She was found to be compound heterozygous for two missense-mutations located on different alleles (C 300 –G and G 1266 –T): the first mutation replaces Pro75 by Ala, the second mutation replaces Asp397 by Tyr.

  • Inhibition of Adenylosuccinate Lyase By 2′, 3′-Acyclic Substrate Analogs
    Advances in experimental medicine and biology, 1998
    Co-Authors: Carlo Crifò, A. Lomonte, Costantino Salerno
    Abstract:

    Adenylosuccinate Lyase (EC 4.3.2.2; ASase) catalyzes the trans-elimination of fumarate from Adenylosuccinate (S-AMP) to yield AMP. The other substrate in purine biosynthesis cleaved by ASase is 5-amino-4-imidazole-N-succinocarboxamide ribotide (SAICAR), which also has a succinyl group attached and closely resembles S-AMP.1 Moreover, the enzyme plays a pivotal role in the conversion of dideoxypurine nucleosides into dideoxynucleotide triphosphates with known anti-HIV (human immunodeficiency virus) activity.2–3 Increased ASase activities have been found in kidney tumors and in the liver following the administration of carcinogens.4–5

Marco Henneke - One of the best experts on this subject based on the ideXlab platform.

  • In vivo proton MR spectroscopy findings specific for Adenylosuccinate Lyase deficiency
    NMR in biomedicine, 2010
    Co-Authors: Marco Henneke, Marinette Van Der Graaf, Michèl A.a.p. Willemsen, Steffi Dreha-kulaczewski, Knut Brockmann, Udo F. H. Engelke, Peter Dechent, Arend Heerschap, Gunther Helms, Ron A. Wevers
    Abstract:

    Adenylosuccinate Lyase (ADSL) deficiency is an inherited metabolic disorder affecting predominantly the central nervous system. The disease is characterized by the accumulation of succinylaminoimidazolecarboxamide riboside and succinyladenosine (S-Ado) in tissue and body fluids. Three children presented with muscular hypotonia, psychomotor delay, behavioral abnormalities, and white matter changes on brain MRI. Two of them were affected by seizures. Screening for inborn errors of metabolism including in vitro high resolution proton MRS revealed an ADSL deficiency that was confirmed genetically in all cases. All patients were studied by in vivo proton MRS. In vitro high resolution proton MRS of patient cerebrospinal fluid showed singlet resonances at 8.27 and 8.29 ppm that correspond to accumulated S-Ado. In vivo proton MRS measurements also revealed a prominent signal at 8.3 ppm in gray and white matter brain regions of all patients. The resonance was undetectable in healthy human brain. In vivo proton MRS provides a conclusive finding in ADSL deficiency and represents a reliable noninvasive diagnostic tool for this neurometabolic disorder.

Jason W. Locasale - One of the best experts on this subject based on the ideXlab platform.

  • Prolyl hydroxylase substrate Adenylosuccinate Lyase is an oncogenic driver in triple negative breast cancer
    Nature communications, 2019
    Co-Authors: Giada Zurlo, Xijuan Liu, Mamoru Takada, Cheng Fan, Jeremy M. Simon, Travis S. Ptacek, Javier Rodriguez, Alex Von Kriegsheim, Juan Liu, Jason W. Locasale
    Abstract:

    Protein hydroxylation affects protein stability, activity, and interactome, therefore contributing to various diseases including cancers. However, the transiency of the hydroxylation reaction hinders the identification of hydroxylase substrates. By developing an enzyme-substrate trapping strategy coupled with TAP-TAG or orthogonal GST- purification followed by mass spectrometry, we identify Adenylosuccinate Lyase (ADSL) as an EglN2 hydroxylase substrate in triple negative breast cancer (TNBC). ADSL expression is higher in TNBC than other breast cancer subtypes or normal breast tissues. ADSL knockout impairs TNBC cell proliferation and invasiveness in vitro and in vivo. An integrated transcriptomics and metabolomics analysis reveals that ADSL activates the oncogenic cMYC pathway by regulating cMYC protein level via a mechanism requiring ADSL proline 24 hydroxylation. Hydroxylation-proficient ADSL, by affecting adenosine levels, represses the expression of the long non-coding RNA MIR22HG, thus upregulating cMYC protein level. Our findings highlight the role of ADSL hydroxylation in controlling cMYC and TNBC tumorigenesis.