Human Glutamate Dehydrogenase Research Articles

Evolution of Glutamate Metabolism via GLUD2 Enhances Lactate-Dependent Synaptic Plasticity and Complex Cognition.

Human evolution is characterized by rapid brain enlargement and the emergence of unique cognitive abilities. Besides its distinctive cytoarchitectural organization and extensive inter-neuronal connectivity, the human brain is also defined by high rates of synaptic, mainly glutamatergic, transmission, and energy utilization. While these adaptations' origins remain elusive, evolutionary changes occurred in synaptic glutamate metabolism in the common ancestor of humans and apes via the emergence of GLUD2, a gene encoding the human glutamate dehydrogenase 2 (hGDH2) isoenzyme. Driven by positive selection, hGDH2 became adapted to function upon intense excitatory firing, a process central to the long-term strengthening of synaptic connections. It also gained expression in brain astrocytes and cortical pyramidal neurons, including the CA1-CA3 hippocampal cells, neurons crucial to cognition. In mice transgenic for GLUD2, theta-burst-evoked long-term potentiation (LTP) is markedly enhanced in hippocampal CA3-CA1 synapses, with patch-clamp recordings from CA1 pyramidal neurons revealing increased sNMDA receptor currents. D-lactate blocked LTP enhancement, implying that glutamate metabolism via hGDH2 potentiates L-lactate-dependent glia-neuron interaction, a process essential to memory consolidation. The transgenic (Tg) mice exhibited increased dendritic spine density/synaptogenesis in the hippocampus and improved complex cognitive functions. Hence, enhancement of neuron-glia communication, via GLUD2 evolution, likely contributed to human cognitive advancement by potentiating synaptic plasticity and inter-neuronal connectivity.

Evolutionary Changes in Primate Glutamate Dehydrogenases 1 and 2 Influence the Protein Regulation by Ligands, Targeting and Posttranslational Modifications.

There are two paralogs of glutamate dehydrogenase (GDH) in humans encoded by the GLUD1 and GLUD2 genes as a result of a recent retroposition during the evolution of primates. The two human GDHs possess significantly different regulation by allosteric ligands, which is not fully characterized at the structural level. Recent advances in identification of the GDH ligand binding sites provide a deeper perspective on the significance of the accumulated substitutions within the two GDH paralogs. In this review, we describe the evolution of GLUD1 and GLUD2 after the duplication event in primates using the accumulated sequencing and structural data. A new gibbon GLUD2 sequence questions the indispensability of ancestral R496S and G509A mutations for GLUD2 irresponsiveness to GTP, providing an alternative with potentially similar regulatory features. The data of both GLUD1 and GLUD2 evolution not only confirm substitutions enhancing GLUD2 mitochondrial targeting, but also reveal a conserved mutation in ape GLUD1 mitochondrial targeting sequence that likely reduces its transport to mitochondria. Moreover, the information of GDH interactors, posttranslational modification and subcellular localization are provided for better understanding of the GDH mutations. Medically significant point mutations causing deregulation of GDH are considered from the structural and regulatory point of view.

A comparative analysis of fruit fly and human glutamate dehydrogenases in Drosophila melanogaster sperm development.

Glutamate dehydrogenases are enzymes that take part in both amino acid and energy metabolism. Their role is clear in many biological processes, from neuronal function to cancer development. The putative testis-specific Drosophila glutamate dehydrogenase, Bb8, is required for male fertility and the development of mitochondrial derivatives in spermatids. Testis-specific genes are less conserved and could gain new functions, thus raising a question whether Bb8 has retained its original enzymatic activity. We show that while Bb8 displays glutamate dehydrogenase activity, there are significant functional differences between the housekeeping Gdh and the testis-specific Bb8. Both human GLUD1 and GLUD2 can rescue the bb8 ms mutant phenotype, with superior performance by GLUD2. We also tested the role of three conserved amino acids observed in both Bb8 and GLUD2 in Gdh mutants, which showed their importance in the glutamate dehydrogenase function. The findings of our study indicate that Drosophila Bb8 and human GLUD2 could be novel examples of convergent molecular evolution. Furthermore, we investigated the importance of glutamate levels in mitochondrial homeostasis during spermatogenesis by ectopic expression of the mitochondrial glutamate transporter Aralar1, which caused mitochondrial abnormalities in fly spermatids. The data presented in our study offer evidence supporting the significant involvement of glutamate metabolism in sperm development.

Evaluation of decursin and its isomer decursinol angelate as potential inhibitors of human glutamate dehydrogenase activity through in silico and enzymatic assay screening

Glutaminolysis is a typical hallmark of malignant tumors across different cancers. Glutamate dehydrogenase (GDH, GLUD1) is one such enzyme involved in the conversion of glutamate to α-ketoglutarate. High levels of GDH are associated with numerous diseases and is also a prognostic marker for predicting metastasis in colorectal cancer. Therefore, inhibiting GDH can be a crucial therapeutic target. Here in this study, we performed molecular docking analysis of 8 different plants derived single compounds collected from pubChem database for screening and selected decursin (DN) and decursinol angelate (DA). We performed molecular dynamics simulation (MD), monitored the stability, interaction for protein and docked ligand at 50 ns, and evaluated the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) free energy calculation on the twoselected compounds along with a standard inhibitor epigallocatechin gallate (EGCG) as reference. The final results showed the formation of stable hydrogen bond interactions by DN and DA in the residues of R400 and Y386 at the ADP activation site of GDH, which was important for the selective inhibition of GDH activity. Additionally, the total binding energy of DN and DA were −115.5 kJ/mol and −106.2 kJ/mol, which was higher than the standard reference GDH inhibitor EGCG (−92.8 kJ/mol). Furthermore, biochemical analysis for GDH inhibition substantiated our computational results and established DN and DA as novel GDH inhibitor. The percentage of IC50 inhibition for DN and DA were 1.035 μM and 1.432 μM. Conclusively, DN and DA can be a novel therapeutic drug for inhibition of glutamate dehydrogenase.

Mitochondria-Mediated Apoptosis of HCC Cells Triggered by Knockdown of Glutamate Dehydrogenase 1: Perspective for Its Inhibition through Quercetin and Permethylated Anigopreissin A.

Metabolic reprogramming is a hallmark of cancer cells required to ensure high energy needs and the maintenance of redox balance. A relevant metabolic change of cancer cell bioenergetics is the increase in glutamine metabolism. Hepatocellular carcinoma (HCC), one of the most lethal cancer and which requires the continuous development of new therapeutic strategies, shows an up-regulation of human glutamate dehydrogenase 1 (hGDH1). GDH1 function may be relevant in cancer cells (or HCC) to drive the glutamine catabolism from L-glutamate towards the synthesis of α-ketoglutarate (α-KG), thus supplying key tricarboxylic acid cycle (TCA cycle) metabolites. Here, the effects of hGLUD1 gene silencing (siGLUD1) and GDH1 inhibition were evaluated. Our results demonstrate that siGLUD1 in HepG2 cells induces a significant reduction in cell proliferation (58.8% ± 10.63%), a decrease in BCL2 expression levels, mitochondrial mass (75% ± 5.89%), mitochondrial membrane potential (30% ± 7.06%), and a significant increase in mitochondrial superoxide anion (25% ± 6.55%) compared to control/untreated cells. The inhibition strategy leads us to identify two possible inhibitors of hGDH1: quercetin and Permethylated Anigopreissin A (PAA). These findings suggest that hGDH1 could be a potential candidate target to impair the metabolic reprogramming of HCC cells.

Dissecting the Antenna in Human Glutamate Dehydrogenase: Understanding Its Role in Subunit Communication and Allosteric Regulation.

Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate. While GDH is found in all living organisms, only that from animals is highly allosterically regulated by a wide array of metabolites. Because only animal GDH has a 50-residue antenna domain, we hypothesized that it was critical for allostery. To this end, we previously replaced the antenna with the loop found in bacteria, and the resulting chimera was no longer regulated by purine nucleotides. Hence, it seemed logical that the purpose of the antenna is to exert the subunit communication necessary for heterotrophic allosteric regulation. Here, we revisit the antenna deletion studies by retaining 10 more of the human GDH (hGDH) residues without adding the bacterial loop. Unexpectedly, the results were profoundly different than before. The basal activity of the mutant is only ∼13% of that of the wild type but ∼100 times more sensitive to all allosteric activators. In contrast, the mutant is still affected by all of the tested inhibitors to approximately the same degree. The resulting antenna-less mutant retained its negative cooperativity with respect to the coenzyme, again suggesting that intersubunit communication is intact. Finally, the mutant still exhibits substrate inhibition, albeit there are differences in the details. We present a model in which the majority of the antenna is not directly involved in allosteric regulation per se but rather may be responsible for improving enzymatic efficiency by acting as a conduit for substrate binding energy between subunits.

Propylselen inhibits cancer cell growth by targeting glutamate dehydrogenase at the NADP+ binding site

High levels of glutamate dehydrogenase (GDH) activity are associated with hypoglycemia, cancer, and Parkinson's disease. Propylselen was synthesized to investigate its mechanism of GDH inhibition in comparison with Ebselen and Epigallocatechin gallate (EGCG). Because Ebselen was found to crosslink with the peptide (AA299-341) at the active site of E.coli GDH, the Cys, Pro, and Lys residues of the corresponding peptide were mutagenized to Ala residues. Using enzyme kinetics and biomolecular interaction assays, we found that the conserved GDH P320 residue is important for propylselen binding, C321 for Ebselen binding, and K341 for EGCG binding. In addition, these 3 mutations abolished NADP+ binding to E. coli GDH in the absence of glutamate substrate, but in the presence of glutamate, the catalytic activity of the mutants was reduced only by 2–4 fold, indicating that a substrate-induced fit mechanism exists in E. coli GDH. Furthermore, biochemical analysis showed that NADP+ had high affinity (Kd of 77 nM) for GDH; by targeting the NADP binding site, propylselen effectively inhibited both E. coli and human GDH activity and improved anticancer activity.

Localization of Human Glutamate Dehydrogenases Provides Insights into Their Metabolic Role and Their Involvement in Disease Processes.

Glutamate dehydrogenase (GDH) catalyzes the reversible deamination of L-glutamate to α-ketoglutarate and ammonia. In mammals, GDH contributes to important processes such as amino acid and carbohydrate metabolism, energy production, ammonia management, neurotransmitter recycling and insulin secretion. In humans, two isoforms of GDH are found, namely hGDH1 and hGDH2, with the former being ubiquitously expressed and the latter found mainly in brain, testis and kidney. These two iso-enzymes display highly divergent allosteric properties, especially concerning their basal activity, ADP activation and GTP inhibition. On the other hand, both enzymes are thought to predominantly localize in the mitochondrial matrix, even though alternative localizations have been proposed. To further study the subcellular localization of the two human iso-enzymes, we created HEK293 cell lines stably over-expressing hGDH1 and hGDH2. In these cell lines, immunofluorescence and enzymatic analyses verified the overexpression of both hGDH1 and hGDH2 iso-enzymes, whereas subcellular fractionation followed by immunoblotting showed their predominantly mitochondrial localization. Given that previous studies have only indirectly compared the subcellular localization of the two iso-enzymes, we co-expressed them tagged with different fluorescent dyes (green and red fluorescent protein for hGDH1 and hGDH2, respectively) and found them to co-localize. Despite the wealth of information related to the functional properties of hGDH1 and hGDH2 and the availability of the hGDH1 structure, there is still an ongoing debate concerning their metabolic role and their involvement in disease processes. Data on the localization of hGDHs, as the ones presented here, could contribute to better understanding of the function of these important human enzymes.

Ebselen Reversibly Inhibits Human Glutamate Dehydrogenase at the Catalytic Site.

Human glutamate dehydrogenase (GDH) plays an important role in neurological diseases, tumor metabolism, and hyperinsulinism-hyperammonemia syndrome (HHS). However, there are very few inhibitors known for human GDH. Recently, Ebselen was reported to crosslink with Escherichia coli GDH at the active site cysteine residue (Cys321), but the sequence alignment showed that the corresponding residue is Ala329 in human GDH. To investigate whether Ebselen could be an inhibitor for human GDH, we cloned and expressed an N-terminal His-tagged human GDH in E. coli. The recombinant human GDH enzyme showed expected properties such as adenosine diphosphate activation and nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate dual recognition. Further, we developed a 2-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl) benzenesulfonate sodium salt (EZMTT)-based assay for human GDH, which was highly sensitive and is suitable for high-throughput screening for potent GDH inhibitors. In addition, ForteBio binding assays demonstrated that Ebselen is a reversible active site inhibitor for human GDH. Since Ebselen is a multifunctional organoselenium compound in Phase III clinical trials for inflammation, an Ebselen-based GDH inhibitor might be valuable for future drug discovery for HHS patients.

Getting into mitochondria.

The human mitochondrial glutamate dehydrogenase isoenzymes (hGDH1 and hGDH2) are abundant matrix-localized proteins encoded by nuclear genes. The proteins are synthesized in the cytoplasm, with an atypically long N-terminal mitochondrial targeting sequence (MTS). The results of secondary structure predictions suggest the presence of two α-helices within the N-terminal region of the MTS. Results from deletion analyses indicate that individual helices have limited ability to direct protein import and matrix localization, but that there is a synergistic interaction when both helices are present [Biochem. J. (2016) 473: , 2813-2829]. Mutagenesis of the MTS cleavage sites blocked post-import removal of the presequences, but did not impede import. The authors propose that the high matrix levels of hGDH can be attributed to the unusual length and secondary structure of the MTS.

Widening Spectrum of Cellular and Subcellular Expression of Human GLUD1 and GLUD2 Glutamate Dehydrogenases Suggests Novel Functions.

Mammalian glutamate dehydrogenase1 (GDH1) (E.C. 1.4.1.3) is a mitochondrial enzyme that catalyzes the reversible oxidative deamination of glutamate to α-ketoglutarate and ammonia while reducing NAD+ and/or NADP+ to NADH and/or NADPH. It links amino acid with carbohydrate metabolism, contributing to Krebs cycle anaplerosis, energy production, ammonia handling and redox homeostasis. Although GDH1 was one of the first major metabolic enzymes to be studied decades ago, its role in cell biology is still incompletely understood. There is however growing interest in a novel GDH2 isoenzyme that emerged via duplication in primates and underwent rapid evolutionary selection concomitant with prefrontal human cortex expansion. Also, the anaplerotic function of GDH1 and GDH2 is currently under sharp focus as this relates to the biology of glial tumors and other neoplasias. Here we used antibodies specific for human GDH1 (hGDH1) and human GDH2 (hGDH2) to study the expression of these isoenzymes in human tissues. Results revealed that both hGDH1 and hGDH2 are expressed in human brain, kidney, testis and steroidogenic organs. However, distinct hGDH1 and hGDH2 expression patterns emerged. Thus, while the Sertoli cells of human testis were strongly positive for hGDH2, they were negative for hGDH1. Conversely, hGDH1 showed very high levels of expression in human liver, but hepatocytes were virtually devoid of hGDH2. In human adrenals, both hGDHs were densely expressed in steroid-producing cells, with hGDH2 expression pattern matching that of the cholesterol side chain cleavage system involved in steroid synthesis. Similarly in human ovaries and placenta, both hGDH1 and hGDH2 were densely expressed in estrogen producing cells. In addition, hGDH1, being a housekeeping enzyme, was also expressed in cells that lack endocrine function. Regarding human brain, study of cortical sections using immunofluorescence (IF) with confocal microscopy revealed that hGDH1 and hGDH2 were both expressed in the cytoplasm of gray and white matter astrocytes within coarse structures resembling mitochondria. Additionally, hGDH1 localized to the nuclear membrane of a subpopulation of astrocytes and of the vast majority of oligodendrocytes and their precursors. Remarkably, hGDH2-specific staining was detected in human cortical neurons, with different expression patterns having emerged. One pattern, observed in large cortical neurons (some with pyramidal morphology), was a hGDH2-specific labeling of cytoplasmic structures resembling mitochondria. These were distributed either in the cell body-axon or on the cell surface in close proximity to astrocytic end-feet that encircle glutamatergic synapses. Another pattern was observed in small cortical neurons with round dense nuclei in which the hGDH2-specific staining was found in the nuclear membrane. A detailed description of these observations and their functional implications, suggesting that the GDH flux is used by different cells to serve some of their unique functions, is presented below.

Expression of human GLUD1 and GLUD2 glutamate dehydrogenases in steroid producing tissues

Besides the housekeeping glutamate dehydrogenase1 (hGDH1), humans have acquired, via a recent duplication event, a hGDH2 isoenzyme with distinct functional properties and tissue expression profile. GDH catalyzes the reversible deamination of glutamate to α-ketoglutarate while reducing NAD(P) to NAD(P)H. As the generated NADPH can be used in bio-synthetic pathways, we studied here the expression of hGDH1 and hGDH2 in human steroidogenic tissues using specific antibodies. Results revealed high levels of hGDH1 and hGDH2 expression in steroid-producing cells in all tissues studied. While the cellular expression pattern of the two proteins was similar for the adrenal cortex, it was distinct for testis, ovaries and placenta. Functional analyses revealed that steroid hormones interacted differentially with the two isoenzymes. As synthesis of steroid hormones requires NADPH, expression of hGDH1 and hGDH2 in steroidogenic cells may serve their particular metabolic needs.

Differential interaction of hGDH1 and hGDH2 with manganese: Implications for metabolism and toxicity

Manganese (Mn) is an essential trace element that serves as co-factor for many important mammalian enzymes. In humans, the importance of this cation is highlighted by the fact that low levels of Mn cause developmental and metabolic abnormalities and, on the other hand, chronic exposure to excessive amounts of Mn is characterized by neurotoxicity, possibly mediated by perturbation of astrocytic mitochondrial energy metabolism. Here we sought to study the effect of Mn on the two human glutamate dehydrogenases (hGDH1 and hGDH2, respectively), key mitochondrial enzymes involved in numerous cellular processes, including mitochondrial metabolism, glutamate homeostasis and neurotransmission, and cell signaling. Our studies showed that, compared to magnesium (Mg) and calcium (Ca), Mn exerted a significant inhibitory effect on both human isoenzymes with hGDH2 being more sensitive than hGDH1, especially under conditions of low ADP levels. Specifically, in the presence of 0.25 mM ADP, the Mn IC50 was 1.14 ± 0.02 mM and 1.54 ± 0.08 mM for hGDH2 and for hGDH1, respectively (p = 0.0001). Increasing Mn levels potentiated this differential effect, with 3 mM Mn inhibiting hGDH2 by 96.5% and hGDH1 by 70.2%. At 1 mM ADP, the Mn IC50 was 1.84 ± 0.02 mM and 2.04 ± 0.07 mM (p = 0.01) for hGDH2 and hGDH1, respectively, with 3 mM Mn inhibiting hGDH2 by 93.6% and hGDH1 by 70.9%. These results were due to the sigmoidal inhibitory curve of Mn that was more pronounced for hGDH2 than for hGDH1. Indeed, at 0.25 mM, the Hill coefficient value was higher for hGDH2 (3.42 ± 0.20) than for hGDH1 (1.94 ± 0.25; p = 0.0002) indicating that interaction of Mn with hGDH2 was substantially more co-operative than for hGDH1. These findings, showing an enhanced sensitivity of the hGDH2 isoenzyme to Mn, especially at low ADP levels, might be of pathophysiological relevance under conditions of Mn neurotoxicity.

Side-chain interactions in the regulatory domain of human glutamate dehydrogenase determine basal activity and regulation.

Glutamate Dehydrogenase (GDH) is central to the metabolism of glutamate, a major excitatory transmitter in mammalian central nervous system (CNS). hGDH1 is activated by ADP and L-leucine and powerfully inhibited by GTP. Besides this housekeeping hGDH1, duplication led to an hGDH2 isoform that is expressed in the human brain dissociating its function from GTP control. The novel enzyme has reduced basal activity (4-6% of capacity) while remaining remarkably responsive to ADP/L-leucine activation. While the molecular basis of this evolutionary adaptation remains unclear, substitution of Ser for Arg443 in hGDH1 is shown to diminish basal activity (< 2% of capacity) and abrogate L-leucine activation. To explore whether the Arg443Ser mutation disrupts hydrogen bonding between Arg443 and Ser409 of adjacent monomers in the regulatory domain ('antenna'), we replaced Ser409 by Arg or Asp in hGDH1. The Ser409Arg-1 change essentially replicated the Arg443Ser-1 mutation effects. Molecular dynamics simulation predicted that Ser409 and Arg443 of neighboring monomers come in close proximity in the open conformation and that introduction of Ser443-1 or Arg409-1 causes them to separate with the swap mutation (Arg409/Ser443) reinstating their proximity. A swapped Ser409Arg/Arg443Ser-1 mutant protein, obtained in recombinant form, regained most of the wild-type hGDH1 properties. Also, when Ser443 was replaced by Arg443 in hGDH2 (as occurs in hGDH1), the Ser443Arg-2 mutant acquired most of the hGDH1 properties. Hence, side-chain interactions between 409 and 443 positions in the 'antenna' region of hGDHs are crucial for basal catalytic activity, allosteric regulation, and relative resistance to thermal inactivation.

The Odyssey of a Young Gene: Structure–Function Studies in Human Glutamate Dehydrogenases Reveal Evolutionary-Acquired Complex Allosteric Regulation Mechanisms

Mammalian glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia, interconnecting carbon skeleton and nitrogen metabolism. In addition, it functions as an energy switch by its ability to fuel the Krebs cycle depending on the energy status of the cell. As GDH lies at the intersection of several metabolic pathways, its activity is tightly regulated by several allosteric compounds that are metabolic intermediates. In contrast to other mammals that have a single GDH-encoding gene, humans and great apes possess two isoforms of GDH (hGDH1 and hGDH2, encoded by the GLUD1 and GLUD2 genes, respectively) with distinct regulation pattern, but remarkable sequence similarity (they differ, in their mature form, in only 15 of their 505 amino-acids). The GLUD2 gene is considered a very young gene, emerging from the GLUD1 gene through retro-position only recently (<23 million years ago). The new hGDH2 iso-enzyme, through random mutations and natural selection, is thought to have conferred an evolutionary advantage that helped its persistence through primate evolution. The properties of the two highly homologous human GDHs have been studied using purified recombinant hGDH1 and hGDH2 proteins obtained by expression of the corresponding cDNAs in Sf21 cells. According to these studies, in contrast to hGDH1 that maintains basal activity at 35-40% of its maximal, hGDH2 displays low basal activity that is highly responsive to activation by rising levels of ADP and/or L-leucine which can also act synergistically. While hGDH1 is inhibited potently by GTP, hGDH2 shows remarkable GTP resistance. Furthermore, the two iso-enzymes are differentially inhibited by estrogens, polyamines and neuroleptics, and also differ in heat-lability. To elucidate the molecular mechanisms that underlie these different regulation patterns of the two iso-enzymes (and consequently the evolutionary adaptation of hGDH2 to a new functional role), we have performed mutagenesis at sites of difference in their amino acid sequence. Results showed that the low basal activity, heat-lability and estrogen sensitivity of hGDH2 could be, at least partially, ascribed to the Arg443Ser evolutionary change, whereas resistance to GTP inhibition has been attributed to the Gly456Ala change. Other amino acid substitutions studied thus far cannot explain all the remaining functional differences between the two iso-enzymes. Also, the Arg443Ser/Gly456Ala double mutation in hGDH1 approached the properties of wild-type hGDH2, without being identical to it. The insights into the structural mechanism of enzymatic regulation and the implications in cell biology provided by these findings are discussed.

Chicken liver glutamate dehydrogenase (GDH) demonstrates a histone H3 specific protease (H3ase) activity in vitro

Site-specific proteolysis of the N or C-terminus of histone tails has emerged as a novel form of irreversible post-translational modifications assigned to histones. Though there are many reports describing histone specific proteolysis, there are very few studies on purification of a histone specific protease. Here, we demonstrate a histone H3 specific protease (H3ase) activity in chicken liver nuclear extract. H3ase was purified to homogeneity and identified as glutamate dehydrogenase (GDH) by sequencing. A series of biochemical experiments further confirmed that the H3ase activity was due to GDH. The H3ase clipped histone H3 products were sequenced by N-terminal sequencing and the precise clipping sites of H3ase were mapped. H3ase activity was only specific to chicken liver as it was not demonstrated in other tissues like heart, muscle and brain of chicken. We assign a novel serine like protease activity to GDH which is specific to histone H3.

Glutamate Dehydrogenase Isoforms with N-Terminal (His)6- or FLAG-Tag Retain Their Kinetic Properties and Cellular Localization

Glutamate dehydrogenase (GDH) is a crucial enzyme on the crossroads of amino acid and energy metabolism and it is operating in all domains of life. According to current knowledge GDH is present only in one functional isoform in most animals, including mice. In addition to this housekeeping enzyme (hGDH1 in humans), humans and apes have acquired a second isoform (hGDH2) with a distinct tissue expression profile. In the current study we have cloned both mouse and human GDH constructs containing FLAG and (His)6 small genetically-encoded tags, respectively. The hGDH1 and hGDH2 constructs containing N-terminal (His)6 tags were successfully expressed in Sf9 cells and the recombinant proteins were isolated to ≥95% purity in a two-step procedure involving ammonium sulfate precipitation and Ni(2+)-based immobilized metal ion affinity chromatography. To explore whether the presence of the FLAG and (His)6 tags affects the cellular localization and functionality of the GDH isoforms, we studied the subcellular distribution of the expressed enzymes as well as their regulation by adenosine diphosphate monopotassium salt (ADP) and guanosine-5'-triphosphate sodium salt (GTP). Through immunoblot analysis of the mitochondrial and cytosolic fraction of the HEK cells expressing the recombinant proteins we found that neither FLAG nor (His)6 tag disturbs the mitochondrial localization of GDH. The addition of the small tags to the N-terminus of the mature mitochondrial mouse GDH1 or human hGDH1 and hGDH2 did not change the ADP activation or GTP inhibition pattern of the proteins as compared to their untagged counterparts. However, the addition of FLAG tag to the C-terminus of the mouse GDH left the recombinant protein fivefold less sensitive to ADP activation. This finding highlights the necessity of the functional characterization of recombinant proteins containing even the smallest available tags.

The effect of pH and ADP on ammonia affinity for human glutamate dehydrogenases

Glutamate dehydrogenase (GDH) uses ammonia to reversibly convert α-ketoglutarate to glutamate using NADP(H) and NAD(H) as cofactors. While GDH in most mammals is encoded by a single GLUD1 gene, humans and other primates have acquired a GLUD2 gene with distinct tissue expression profile. The two human isoenzymes (hGDH1 and hGDH2), though highly homologous, differ markedly in their regulatory properties. Here we obtained hGDH1 and hGDH2 in recombinant form and studied their Km for ammonia in the presence of 1.0 mM ADP. The analyses showed that lowering the pH of the buffer (from 8.0 to 7.0) increased the Km for ammonia substantially (hGDH1: from 12.8 ± 1.4 mM to 57.5 ± 1.6 mM; hGDH2: from 14.7 ± 1.6 mM to 62.2 ± 1.7 mM), thus essentially precluding reductive amination. Moreover, lowering the ADP concentration to 0.1 mM not only increased the K0.5 [NH4 (+)] of hGDH2, but also introduced a positive cooperative binding phenomenon in this isoenzyme. Hence, intra-mitochondrial acidification, as occurring in astrocytes during glutamatergic transmission should favor the oxidative deamination of glutamate. Similar considerations apply to the handling of glutamate by the proximal convoluted tubules of the kidney during systemic acidosis. The reverse could apply for conditions of local or systemic hyperammonemia or alkalosis.

Roles of cysteine residues in the inhibition of human glutamate dehydrogenase by palmitoyl-CoA

Human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) have been known to be inhibited by palmitoyl-CoA with a high affinity. In this study, we have performed the cassette mutagenesis at six different Cys residues (Cys59, Cys93, Cys119, Cys201, Cys274, and Cys323) to identify palmitoyl-CoA binding sites within hGDH2. Four cysteine residues at positions of C59, C93, C201, or C274 may be involved, at least in part, in the inhibition of hGDH2 by palmitoyl-CoA. There was a biphasic relationship, depending on the levels of palmitoyl-CoA, between the binding of palmitoyl-CoA and the loss of enzyme activity during the inactivation process. The inhibition of hGDH2 by palmitoyl-CoA was not affected by the allosteric inhibitor GTP. Multiple mutagenesis studies on the hGDH2 are in progress to identify the amino acid residues fully responsible for the inhibition by palmitoyl-CoA. [BMB Reports 2012; 45(12): 707-712]

Alpha helical structures in the leader sequence of human GLUD2 glutamate dehydrogenase responsible for mitochondrial import

Human glutamate dehydrogenase (hGDH) exists in two highly homologous isoforms with a distinct regulatory and tissue expression profile: a housekeeping hGDH1 isoprotein encoded by the GLUD1 gene and an hGDH2 isoenzyme encoded by the GLUD2 gene. There is evidence that both isoenzymes are synthesized as pro-enzymes containing a 53 amino acid long N-terminal leader peptide that is cleaved upon translocation into the mitochondria. However, this GDH signal peptide is substantially larger than that of most nuclear DNA-encoded mitochondrial proteins, the leader sequence of which typically contains 17–35 amino acids and they often form a single amphipathic α-helix. To decode the structural elements that are essential for the mitochondrial targeting of human GDHs, we performed secondary structure analyses of their leader sequence. These analyses predicted, with 82% accuracy, that both leader peptides are positively charged and that they form two to three α-helices, separated by intermediate loops. The first α-helix of hGDH2 is strongly amphipathic, displaying both a positively charged surface and a hydrophobic plane. We then constructed GLUD2-EGFP deletion mutants and used them to transfect three mammalian cell lines (HEK293, COS 7 and SHSY-5Y). Confocal laser scanning microscopy, following co-transfection with pDsRed2-Mito mitochondrial targeting vector, revealed that deletion of the entire leader sequence prevented the enzyme from entering the mitochondria, resulting in its retention in the cytoplasm. Deletion of the first strongly amphipathic α-helix only was also sufficient to prevent the mitochondrial localization of the truncated protein. Moreover, truncated leader sequences, retaining the second and/or the third putative α-helix, failed to restore the mitochondrial import of hGDH2. As such, the first N-terminal alpha helical structure is crucial for the mitochondrial import of hGDH2 and these findings may have implications in understanding the evolutionary mechanisms that led to the large mitochondrial targeting signals of human GDHs.