Myung-Min Choi

Identification of ADP-ribosylation site in human glutamate dehydrogenase isozymes

When the influence of ADP‐ribosylation on the activities of the purified human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was measured in the presence of 100 μM NAD+ for 60 min, hGDH isozymes were inhibited by up to 75%. If incubations were performed for longer time periods up to 3 h, the inhibition of hGDH isozymes did not increased further. This phenomenon may be related to the reversibility of ADP‐ribosylation in mitochondria. ADP‐ribosylated hDGH isozymes were reactivated by Mg2+‐dependent mitochondrial ADP‐ribosylcysteine hydrolase. The stoichiometry between incorporated ADP‐ribose and GDH subunits shows a modification of one subunit per catalytically active homohexamer. Since ADP and GTP had no effects on the extent of modification, it would appear that the ADP‐ribosylation is unlikely to occur in allosteric sites. It has been proposed that Cys residue may be involved in the ADP‐ribosylation of GDH, although identification of the reactive Cys residue has not been reported. To identify the reactive Cys residue involved in the ADP‐ribosylation, we performed cassette mutagenesis at three different positions (Cys59, Cys119, and Cys274) using synthetic genes of hGDH isozymes. Among the Cys residues tested, only Cys119 mutants showed a significant reduction in the ADP‐ribosylation. These results suggest a possibility that the Cys119 residue has an important role in the regulation of hGDH isozymes by ADP‐ribosylation.

Inhibitory effects of gallic acid and quercetin on UDP-glucose dehydrogenase activity.

We have examined polyphenols as potential inhibitors of UDP‐glucose dehydrogenase (UGDH) activity. Gallic acid and quercetin decreased specific activities of UGDH and inhibited the proliferation of MCF‐7 human breast cancer cells. Western blot analysis showed that gallic acid and quercetin did not affect UGDH protein expression, suggesting that UGDH activity is inhibited by polyphenols at the post‐translational level. Kinetics studies using human UGDH revealed that gallic acid was a non‐competitive inhibitor with respect to UDP‐glucose and NAD+. In contrast, quercetin showed a competitive inhibition and a mixed‐type inhibition with respect to UDP‐glucose and NAD+, respectively. These results indicate that gallic acid and quercetin are effective inhibitors of UGDH that exert strong antiproliferative activity in breast cancer cells.

Regulation of glutamate level in rat brain through activation of glutamate dehydrogenase by Corydalis ternata.

When treated with protopine and alkalized extracts of the tuber of Corydalis ternata for one year, significant decrease in glutamate level and increase in glutamate dehydrogenase (GDH) activity was observed in rat brains. The expression of GDH between the two groups remained unchanged as determined by Western and Northern blot analysis, suggesting a post-translational regulation of GDH activity in alkalized extracts treated rat brains. The stimulatory effects of alkalized extracts and protopine on the GDH activity was further examined in vitro with two types of human GDH isozymes, hGDH1 (house-keeping GDH) and hGDH2 (nerve-specific GDH). Alkalized extracts and protopine activated the human GDH isozymes up to 4.8-fold. hGDH2 (nervespecific GDH) was more sensitively affected by 1 mM ADP than hGDH1 (house-keeping GDH) on the activation by alkalized extracts. Studies with cassette mutagenesis at ADP-binding site showed that hGDH2 was more sensitively regulated by ADP than hGDH1 on the activation by Corydalis ternata. Our results suggest that prolonged exposure to Corydalis ternata may be one of the ways to regulate glutamate concentration in brain through the activation of GDH.

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

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(hGDH2390–448)hGDH1 (amino acid segment 390–448 of hGDH1 replaced by the corresponding hGDH2 segment) and hGDH2(hGDH1390–448)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.

Inhibition of Human UDP-Glucose Dehydrogenase Expression Using siRNA Expression Vector in Breast Cancer Cells

UDP-glucose dehydrogenase (UGDH) catalyzes two oxidations of UDP-glucose to yield UDP-glucuronic acid. Pathological over-production of extracellular matrix components may be linked to the availability of UDP-glucuronic acid, therefore UGDH is a potential therapeutic target. RNA interference (RNAi) has been adapted to knock down the expression of human UGDH. A UGDH siRNA plasmid was constructed using a pRNA-U6.1/Neo vector and transfected into breast cancer cells, ZR-75-1, with an efficiency of up to 50%. Western blot analysis showed that the UGDH expression was efficiently knocked down at protein levels by RNAi in ZR-75-1 cells.

Small‐interfering‐RNA‐mediated silencing of human glutamate dehydrogenase induces apoptosis in neuroblastoma cells

In the nervous system, GDH (glutamate dehydrogenase) is enriched in astrocytes and is important for recycling glutamate, a major excitatory neurotransmitter. The function of hGDH (human GDH) may be important in neurodegenerative diseases such as Parkinsons disease. To test the effect of decreased hGDH expression, several vector‐based plasmidlinked hGDH siRNAs (small interfering RNAs) were expressed intracellularly in BE(2)C human neuroblastoma cells. Immunoblotting and reverse‐transcription–PCR confirmed that expression of hGDH protein and mRNA was knocked down by co‐transfection with phGDH‐siRNA vectors in BE(2)C human neuroblastoma cells. TUNEL (terminal uridine deoxynucleotidyl transferase dUTP nick‐end labelling) and DNA fragmentation assays 48 h after transfection of phGDH‐siRNAs revealed that inhibition of hGDH expression induced cellular apoptosis and activated phospho‐ERK1/2 (phospho‐extracellular‐signal‐regulated kinase 1/2). These findings show that inhibition of hGDH expression by siRNA is related to apoptosis in neuronal cells.

Identification of amino acid residues responsible for different GTP preferences of human glutamate dehydrogenase isozymes

Human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) differ markedly in their inhibition by GTP. These regulatory preferences must arise from amino acid residues that are not common between hGDH isozymes. We have constructed chimeric enzymes by reciprocally switching the corresponding amino acid segments 390-465 in hGDH isozymes that are located within or near the C-terminal 48-residue antenna helix, which is thought to be part of the regulatory domain of mammalian GDHs. These resulted in triple mutations in amino acid sequences at 415, 443, and 456 sites that are not common between hGDH1 and hGDH2. The chimeric enzymes did not change their enzyme efficiency (k(cat)/K(m)) and expression level. Functional analyses, however, revealed that the chimeric mutants almost completely acquired the different GTP regulatory preference between hGDH isozymes. These results suggest that the 415, 443, and 456 residues acting in concert are responsible for the GTP inhibitory properties of hGDH isozymes.