Acta biochimica et biophysica; Academiae Scientiarum Hungaricae, 1983
The NAD-induced local conformational changes in the fluorescent dye-binding region of muscle glyc... more The NAD-induced local conformational changes in the fluorescent dye-binding region of muscle glyceraldehyde-3-phosphate dehydrogenase were studied (Ovádi et al., 1982). We have isolated a dominant peptide containing the fluorescent dye from the tryptic digest of labelled dehydrogenase, and identified this labelled amino acid residue. The data indicate that fluorescein isothiocyanate can react rather specifically with tyrosyl residue 91, which is not associated with the catalytic function of the enzyme. Tyrosyl-91 modified by the fluorescent dye does not interact directly with any part of the NAD bound at the active site of glyceraldehyde-3-phosphate dehydrogenase.
Publisher Summary This chapter discusses the control of metabolism by dynamic macromolecular inte... more Publisher Summary This chapter discusses the control of metabolism by dynamic macromolecular interactions. Metabolic pathways are controlled and directed by pacemaker, bottleneck, and key enzymes. In general, no single enzyme is responsible for the control of a whole metabolic pathway. In pursuing the pacemaker theory, attempts are made to quantify metabolic regulation, and one studies each enzyme in a sequence separately in situ , determines its kinetic properties in the greatest possible detail and accuracy, and then seeks to determine how it works when it is in the intact cell. In prokaryotes and in eukaryotes the largest macrocompartment is the cytoplasm, containing quantities of soluble enzymes and is full of membranes associated with a great variety of organelles. A theoretical analysis of glycolysis in human erythrocytes has been provided, implicitly assuming that it proceeds in a homogeneous bulk solution without any interaction among the participating enzymes.
Intrinsically disordered proteins (IDPs), which can interact with many partner proteins, are cent... more Intrinsically disordered proteins (IDPs), which can interact with many partner proteins, are central to many physiological functions and to various pathologies that include neurodegeneration. Here, we introduce the Sherpa hypothesis, according to which a subset of stable IDPs that we term Phenotype-Preserving Disordered Proteins (PPDP) play a central role in protecting cell phenotypes from perturbations. To illustrate and test this hypothesis, we computer-simulate some salient features of how cells evolve and differentiate in the presence of either a single PPDP or two incompatible PPDPs. We relate this virtual experiment to the pathological interactions between two PPDPs, α-synuclein and Tubulin Polymerization Promoting Protein/p25, in neurodegenerative disorders. Finally, we discuss the implications of the Sherpa hypothesis for aptamer-based therapies of such disorders.
In a Hungarian family with triosephosphate isomerase (TPI; D-gll aldehyde-3-phosphate keto-isomer... more In a Hungarian family with triosephosphate isomerase (TPI; D-gll aldehyde-3-phosphate keto-isomerase, EC 5.3.1.1) deficiency, germ-line identical, but phenotypically differing compound hel zygote brothers (one of them with neurological disorder) have I identified with the same very low ( brother without neurological d der > normal control. This distinct microcompartmentation of mi proteins may be relevant in the development of the neurodeger tive process in TPI deficiency and in other, more common neurolo diseases.
Sirtuins are NAD<sup>+</sup>-dependent protein deacylases capable of cleaving off ace... more Sirtuins are NAD<sup>+</sup>-dependent protein deacylases capable of cleaving off acetyl as well as other acyl groups from the ɛ-amino group of lysines in histones and other substrate proteins. They have been reported as promising drug targets, and thus modulators of their activity are needed as molecular tools to uncover their biological function and as potential therapeutics. Here, we present new assay formats that complement existing assays for sirtuin biochemistry and cellular target engagement. Firstly, we report the development of a homogeneous fluorescence-based activity assay using unlabelled acylated peptides. Upon deacylation, the free lysine residue reacts with fluorescamine to form a fluorophore. Secondly, using click chemistry with a TAMRA-azide on a propargylated sirtuin inhibitor, we prepared the first fluorescently labelled small-molecule inhibitor of Sirt2. This is used in a binding assay, which is based on fluorescence polarization. We used it successfully to map potential inhibitor-binding sites and also to show cellular Sirt2 engagement. By means of these new assays, we were able to identify and characterize novel Sirt2 inhibitors out of a focused library screen. The binding of the identified Sirt2 inhibitors was rationalized by molecular docking studies. These new chemical tools thus can enhance further sirtuin research.This article is part of the discussion meeting issue 'Frontiers in epigenetic chemical biology'.
Acta biochimica et biophysica; Academiae Scientiarum Hungaricae, 1983
The NAD-induced local conformational changes in the fluorescent dye-binding region of muscle glyc... more The NAD-induced local conformational changes in the fluorescent dye-binding region of muscle glyceraldehyde-3-phosphate dehydrogenase were studied (Ovádi et al., 1982). We have isolated a dominant peptide containing the fluorescent dye from the tryptic digest of labelled dehydrogenase, and identified this labelled amino acid residue. The data indicate that fluorescein isothiocyanate can react rather specifically with tyrosyl residue 91, which is not associated with the catalytic function of the enzyme. Tyrosyl-91 modified by the fluorescent dye does not interact directly with any part of the NAD bound at the active site of glyceraldehyde-3-phosphate dehydrogenase.
Publisher Summary This chapter discusses the control of metabolism by dynamic macromolecular inte... more Publisher Summary This chapter discusses the control of metabolism by dynamic macromolecular interactions. Metabolic pathways are controlled and directed by pacemaker, bottleneck, and key enzymes. In general, no single enzyme is responsible for the control of a whole metabolic pathway. In pursuing the pacemaker theory, attempts are made to quantify metabolic regulation, and one studies each enzyme in a sequence separately in situ , determines its kinetic properties in the greatest possible detail and accuracy, and then seeks to determine how it works when it is in the intact cell. In prokaryotes and in eukaryotes the largest macrocompartment is the cytoplasm, containing quantities of soluble enzymes and is full of membranes associated with a great variety of organelles. A theoretical analysis of glycolysis in human erythrocytes has been provided, implicitly assuming that it proceeds in a homogeneous bulk solution without any interaction among the participating enzymes.
Intrinsically disordered proteins (IDPs), which can interact with many partner proteins, are cent... more Intrinsically disordered proteins (IDPs), which can interact with many partner proteins, are central to many physiological functions and to various pathologies that include neurodegeneration. Here, we introduce the Sherpa hypothesis, according to which a subset of stable IDPs that we term Phenotype-Preserving Disordered Proteins (PPDP) play a central role in protecting cell phenotypes from perturbations. To illustrate and test this hypothesis, we computer-simulate some salient features of how cells evolve and differentiate in the presence of either a single PPDP or two incompatible PPDPs. We relate this virtual experiment to the pathological interactions between two PPDPs, α-synuclein and Tubulin Polymerization Promoting Protein/p25, in neurodegenerative disorders. Finally, we discuss the implications of the Sherpa hypothesis for aptamer-based therapies of such disorders.
In a Hungarian family with triosephosphate isomerase (TPI; D-gll aldehyde-3-phosphate keto-isomer... more In a Hungarian family with triosephosphate isomerase (TPI; D-gll aldehyde-3-phosphate keto-isomerase, EC 5.3.1.1) deficiency, germ-line identical, but phenotypically differing compound hel zygote brothers (one of them with neurological disorder) have I identified with the same very low ( brother without neurological d der > normal control. This distinct microcompartmentation of mi proteins may be relevant in the development of the neurodeger tive process in TPI deficiency and in other, more common neurolo diseases.
Sirtuins are NAD<sup>+</sup>-dependent protein deacylases capable of cleaving off ace... more Sirtuins are NAD<sup>+</sup>-dependent protein deacylases capable of cleaving off acetyl as well as other acyl groups from the ɛ-amino group of lysines in histones and other substrate proteins. They have been reported as promising drug targets, and thus modulators of their activity are needed as molecular tools to uncover their biological function and as potential therapeutics. Here, we present new assay formats that complement existing assays for sirtuin biochemistry and cellular target engagement. Firstly, we report the development of a homogeneous fluorescence-based activity assay using unlabelled acylated peptides. Upon deacylation, the free lysine residue reacts with fluorescamine to form a fluorophore. Secondly, using click chemistry with a TAMRA-azide on a propargylated sirtuin inhibitor, we prepared the first fluorescently labelled small-molecule inhibitor of Sirt2. This is used in a binding assay, which is based on fluorescence polarization. We used it successfully to map potential inhibitor-binding sites and also to show cellular Sirt2 engagement. By means of these new assays, we were able to identify and characterize novel Sirt2 inhibitors out of a focused library screen. The binding of the identified Sirt2 inhibitors was rationalized by molecular docking studies. These new chemical tools thus can enhance further sirtuin research.This article is part of the discussion meeting issue 'Frontiers in epigenetic chemical biology'.
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Papers by Judit Ovádi