Molecular basis of human glutamate dehydrogenase regulation under changing energy demands

Mastorodemos, Vasileios; Zaganas, Ioannis; Spanaki, Cleanthe; Bessa, Maria João; Plaitakis, Andreas

doi:10.1002/jnr.20353

Cited by 74 publications

(61 citation statements)

References 49 publications

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“…Within the cytoplasm of these cells, the anti-hGDH2 antibody labeled coarse structures resembling mitochondria. We have observed a similar pattern in cultured cells expressing hGDH1/EGFP and hGDH2/EGFP (21,22). These data accord with our Western blot results using subcellular fractions, which showed that hGDH2 localizes to mitochondria.…”

Section: Discussionsupporting

confidence: 90%

Human GLUD2 Glutamate Dehydrogenase Is Expressed in Neural and Testicular Supporting Cells

Spanaki

Zaganas

Kleopa

et al. 2010

Journal of Biological Chemistry

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Mammalian glutamate dehydrogenase (GDH) is an allosterically regulated enzyme that is expressed widely. Its activity is potently inhibited by GTP and thought to be controlled by the need of the cell for ATP. In addition to this housekeeping human (h) GDH1, humans have acquired (via a duplication event) a highly homologous isoenzyme (hGDH2) that is resistant to GTP. Although transcripts of GLUD2, the gene encoding hGDH2, have been detected in human neural and testicular tissues, data on the endogenous protein are lacking. Here, we developed an antibody specific for hGDH2 and used it to study human tissues. Western blot analyses revealed, to our surprise, that endogenous hGDH2 is more densely expressed in testis than in brain. At the subcellular level, hGDH2 localized to mitochondria. Study of testicular tissue using immunocytochemical and immunofluorescence methods revealed that the Sertoli cells were strongly labeled by our anti-hGDH2 antibody. In human cerebral cortex, a robust labeling of astrocytes was detected, with neurons showing faint hGDH2 immunoreactivity. Astrocytes and Sertoli cells are known to support neurons and germ cells, respectively, providing them with lactate that largely derives from the tricarboxylic acid cycle via conversion of glutamate to ␣-ketoglutarate (GDH reaction). As hGDH2 is not subject to GTP control, the enzyme is able to metabolize glutamate even when the tricarboxylic acid cycle generates GTP amounts sufficient to inactivate the housekeeping hGDH1 protein. Hence, the selective expression of hGDH2 by astrocytes and Sertoli cells may provide a significant biological advantage by facilitating metabolic recycling processes essential to the supportive role of these cells.Glutamate dehydrogenase (GDH 3 ; EC 1.4.1.3) catalyzes the reversible interconversion of glutamate to ␣-ketoglutarate and ammonia using NADP(H) and NAD(H) as cofactors, thus interconnecting amino acid and carbohydrate metabolism. Mammalian GDH is allosterically regulated, with GTP and ADP being the main negative and positive modulators, respectively (1, 2).Although GDH in mammals has long been thought to be encoded by a single gene, studies in human tissues revealed the presence of two GDH activities, differing in their regulatory properties and relative resistance to thermal inactivation (3). These observations led to the cloning of two genes encoding human GDH: an intron-containing GLUD1 gene that is located on the 10th chromosome and is expressed widely (housekeeping) (4) and an X-linked intronless GLUD2 gene that is expressed mainly in retina, brain, and testis (5). There is evidence that the GLUD2 gene originated via a duplication event Ͻ23,000,000 years ago, being highly homologous to the conserved GLUD1 gene (6). As a result, the proteins encoded by the GLUD1 and GLUD2 genes (hGDH1 and hGDH2, respectively) share in their mature form all but 15 of their 505 amino acid residues. Recombinant hGDH1 and hGDH2 isoproteins, obtained by expression of the corresponding GLUD1 and GLUD2 cDNAs in Sf21 cells, were f...

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Section: Discussionsupporting

confidence: 90%

Human GLUD2 Glutamate Dehydrogenase Is Expressed in Neural and Testicular Supporting Cells

Spanaki

Zaganas

Kleopa

et al. 2010

Journal of Biological Chemistry

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“…2B). Previously, we reported that single replacement of Ser by Arg at hGDH2 position 443 abolished the heat lability of hGDH2 and changed the half-life of hGDH2 to almost the same as that of hGDH1, whereas single mutagenesis at several other sites (L415M, A456G, and H470R) that are different between hGDH1 and hGDH2 did not show any change in thermal stability (5). Therefore, the influence of heat stability on hGDH isozymes caused by swapping amino acid segment 390 -448 is mainly due to the difference of the amino acid at the 443 site, which is Arg in hGDH1 and Ser in hGDH2.…”

Section: Construction Expression and Purification Of Hgdh1-(hgdh2mentioning

confidence: 86%

“…Although this enzyme does not exhibit allosteric regulation in plants, bacteria, or fungi, its activity is tightly controlled by a number of compounds in mammals (1)(2)(3)(4)(5)(6). All mammalian GDHs are homohexameric, exhibiting 32 symmetry, and the first 200 residues form the core "glutamate binding" domain.…”

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confidence: 99%

“…Reciprocally interchanging amino acids that are different between hGDH isozymes within the regulatory domain may reveal the residues important for preference in hGDH isozymes. Recent studies of structure-function relationships using site-directed mutagenesis of hGDH1 at single sites differing from hGDH2 showed that the R443S and the G456A change reproduced some but not all of the properties of hGDH2 (3)(4)(5)17). In addition, several other residues differing between hGDH isozymes also have been examined by many investigators (4,17).…”

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Amino Acid Changes within Antenna Helix Are Responsible for Different Regulatory Preferences of Human Glutamate Dehydrogenase Isozymes

Choi

Kim

Yang

et al. 2007

Journal of Biological Chemistry

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Human glutamate dehydrogenase (hGDH) exists in hGDH1 (housekeeping isozyme) and in hGDH2 (nerve-specific isozyme), which differ markedly in their allosteric regulation. Because they differ in only 16 of their 505 amino acids, the regulatory preferences must arise from amino acid residues that are not common between hGDH1 and hGDH2. To our knowledge none of the mutagenesis studies on the hGDH isozymes to date have identified the amino acid residues fully responsible for the different regulatory preferences between hGDH1 and hGDH2. In this study we constructed hGDH1(hGDH2 390 -448 )hGDH1 (amino acid segment 390 -448 of hGDH1 replaced by the corresponding hGDH2 segment) and hGDH2(hGDH1 390 -448 )hGDH2 (amino acid segment 390 -448 of hGDH2 replaced by the corresponding hGDH1 segment) by swapping the corresponding amino acid segments in hGDH1 and hGDH2. The chimeric enzymes by reciprocal swapping resulted in double mutations in amino acid sequences at 415 and 443 residues that are not common between hGDH1 and hGDH2 and are located in the C-terminal 48-residue "antenna" helix, which is thought to be part of the regulatory domain of mammalian GDHs. Functional analyses revealed that the doubly mutated chimeric enzymes almost completely acquired most of the different regulatory preferences between hGDH1 and hGDH2 for electrophoretic mobility, heat-stability, ADP activation, palmitoyl-CoA inhibition, and L-leucine activation, except for GTP inhibition. Our results indicate that substitutions of the residues in the antenna region may be important evolutionary changes that led to the adaptation of hGDH2 to the unique metabolic needs of the nerve tissue.

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“…Glutamate dehydrogenase is present in neurons and glia (McKenna et al, 2000), and catalyzes the deamination of glutamate to alpha-ketoglutarate and ammonia using either NAD or NADP as cofactors (Mastorodemos et al, 2005). It is possible that neuronal mitochondria preferentially use the aspartate aminotransferase reaction instead of the glutamate dehydrogenase reaction to generate the key metabolite alpha ketoglutarate in order to avoid the problem of ammonia toxicity (Madhavarao et al, 2003;Madhavarao et al, 2005;Madhavarao and Namboodiri, 2006).…”

Section: A Model Of Naa Synthesis Linked To Mitochondrial Energeticsmentioning

confidence: 99%

N-Acetylaspartate in the CNS: From neurodiagnostics to neurobiology

Moffett

Ross

Arun

et al. 2007

Progress in Neurobiology

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The brain is unique among organs in many respects, including its mechanisms of lipid synthesis and energy production. The nervous system-specific metabolite N-acetylaspartate (NAA), which is synthesized from aspartate and acetyl-coenzyme A in neurons, appears to be a key link in these distinct biochemical features of CNS metabolism. During early postnatal CNS development, the expression of lipogenic enzymes in oligodendrocytes, including the NAA-degrading enzyme aspartoacylase (ASPA), is increased along with increased NAA production in neurons. NAA is transported from neurons to the cytoplasm of oligodendrocytes, where ASPA cleaves the acetate moiety for use in fatty acid and steroid synthesis. The fatty acids and steroids produced then go on to be used as building blocks for myelin lipid synthesis. Mutations in the gene for ASPA result in the fatal leukodystrophy Canavan disease, for which there is currently no effective treatment. Once postnatal myelination is completed, NAA may continue to be involved in myelin lipid turnover in adults, but it also appears to adopt other roles, including a bioenergetic role in neuronal mitochondria. NAA and ATP metabolism appear to be linked indirectly, whereby acetylation of aspartate may facilitate its removal from neuronal mitochondria, thus favoring conversion of glutamate to alpha ketoglutarate which can enter the tricarboxylic acid cycle for energy production. In its role as a mechanism for enhancing mitochondrial energy production from glutamate, NAA is in a key position to act as a magnetic resonance spectroscopy marker for neuronal health, viability and number. Evidence suggests that NAA is a direct precursor for the enzymatic synthesis of the neuron specific dipeptide N-acetylaspartylglutamate, the most concentrated neuropeptide in the human brain. Other proposed roles for NAA include neuronal osmoregulation and axon-glial signaling. We propose that NAA may also be involved in brain nitrogen balance. Further research will be required to more fully understand the biochemical functions served by NAA in CNS development and activity, and additional functions are likely to be discovered.

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Molecular basis of human glutamate dehydrogenase regulation under changing energy demands

Cited by 74 publications

References 49 publications

Human GLUD2 Glutamate Dehydrogenase Is Expressed in Neural and Testicular Supporting Cells

Human GLUD2 Glutamate Dehydrogenase Is Expressed in Neural and Testicular Supporting Cells

Amino Acid Changes within Antenna Helix Are Responsible for Different Regulatory Preferences of Human Glutamate Dehydrogenase Isozymes

N-Acetylaspartate in the CNS: From neurodiagnostics to neurobiology

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