Important role of Ser443 in different thermal stability of human glutamate dehydrogenase isozymes

Molecular biological studies confirmed that two glutamate dehydrogenase isozymes (hGDH1 and hGDH2) of distinct genetic origin are expressed in human tissues. hGDH1 is heat-stable and expressed widely, whereas hGDH2 is heat-labile and specific for neural and testicular tissues. A selective deficiency of hGDH2 has been reported in patients with spinocerebellar ataxia. We have identified an amino acid residue involved in the different thermal stability of human GDH isozymes. At 45 degrees C (pH 7.0), heat inactivation proceeded faster for hGDH2 (half life=45 min) than for hGDH1 (half-life=310 min) in the absence of allosteric regulators. Both hGDH1 and hGDH2, however, showed much slower heat inactivation processes in the presence of 1 mM ADP or 3 mM L-Leu. Virtually most of the enzyme activity remained up to 100 min at 45 degrees C after treatment with ADP and L-Leu in combination. In contrast to ADP and L-Leu, the thermal stabilities of the hGDH isozymes were not affected by addition of substrates or coenzymes. In human GDH isozymes, the 443 site is Arg in hGDH1 and Ser in hGDH2. Replacement of Ser by Arg at the 443 site by cassette mutagenesis abolished the heat lability of hGDH2 with a similar half-life of hGDH1. The mutagenesis at several other sites (L415M, A456G, and H470R) having differences in amino acid sequence between the two GDH isozymes did not show any change in the thermal stability. These results suggest that the Ser443 residue plays an important role in the different thermal stability of human GDH isozymes.     

Reactive amino acid residues involved in glutamate-binding of human glutamate dehydrogenase isozymes

In the present study, the cassette mutagenesis at several putative positions (K94, G96, K118, K130, or D172) was performed to examine the residues involved in the glutamate-binding of the human glutamate dehydrogenase isozymes (hGDH1 and hGDH2). None of the mutations tested affected the expression or stability of the proteins. There was dramatic reduction in the catalytic efficiency in mutant proteins at K94, G96, K118, or K130 site, but not at D172 site. The K(M) values for glutamate were 4-10-fold greater for the mutants at K94, G96, or K118 site than for the wild-type hGDH1 and hGDH2, whereas no differences in the K(M) values for NAD(+) were detected between the mutant and wild-type enzymes. For K130Y mutant, the K(M) value for glutamate increased 1.6-fold, whereas the catalytic efficiency (k(cat)/K(M)) showed only 2-3% of the wild-type. Therefore, the decreased catalytic efficiency of the K130 mutant mainly results from the reduced k(cat) value, suggesting a possibility that the K130Y residue may be involved in the catalysis rather than in the glutamate-binding. The D172Y mutant did not show any changes in k(cat) value and K(M) values for glutamate and NAD(+), indicating that D172Y is not directly involved in catalysis and substrates binding of the hGDH isozymes. For sensitivity to ADP activation, only the D172Y mutant showed a reduced sensitivity to ADP activation. The reduction of ADP activation in D172Y mutant was more profoundly observed in hGDH2 than in hGDH1. There were no differences in their sensitivities to GTP inhibition between the wild-type and mutant GDHs at all positions tested. Our results suggest that K94, G96, and K118 residues play an important role, although at different degrees, in the binding of glutamate to hGDH isozymes.     

TGF-beta-induced protein beta ig-h3 is upregulated by high glucose in vascular smooth muscle cells

TGF-beta-induced gene-h3 (beta ig-h3) is an adhesive molecule that interacts with integrins. Because TGF-beta plays an important role in diabetic complications and beta ig-h3 serves as a cell substrate, we hypothesized that diabetic conditions might increase beta ig-h3 synthesis in vascular smooth muscle cells (VSMCs), which may subsequently contribute to the pathogenesis of diabetic angiopathy. The concentrations of beta ig-h3 and TGF-beta were measured in conditioned media using an enzyme-linked immunosorbent assay. An immunohistochemical study showed that beta ig-h3 was expressed in the VSMCs and the matrix of rat aortas. TGF-beta stimulated beta ig-h3 production, and high glucose induced beta ig-h3 as well as TGF-beta production in the VSMCs. The high glucose-induced beta ig-h3 expression was almost entirely blocked by an anti-TGF-beta antibody. beta ig-h3 protein mediated the adhesion, spreading, migration, and proliferation of rat VSMCs. These results suggest that the high glucose-induced beta ig-h3 in VSMCs regulates VSMC functions and may play an important role in diabetic angiopathy.     

Critical role of the cysteine 323 residue in the catalytic activity of human glutamate dehydrogenase isozymes

The role of residue C323 in catalysis by human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was examined by substituting Arg, Gly, Leu, Met, or Tyr at C323 by cassette mutagenesis using synthetic human GDH isozyme genes. As a result, the Km of the enzyme for NADH and alpha-ketoglutarate increased up to 1.6-fold and 1.1-fold, respectively. It seems likely that C323 is not responsible for substrate-binding or coenzyme-binding. The efficiency (kcat/Km) of the mutant enzymes was only 11-14% of that of the wild-type isozymes, mainly due to a decrease in kcat values. There was a linear relationship between incorporation of [14C]p-chloromercuribenzoic acid and loss of enzyme activity that extrapolated to a stoichiometry of one mol of [14C] incorporated per mol of monomer for wild type hGDHs. No incorporation of [14C]p-chloromer-curibenzoic acid was observed with the C323 mutants. ADP and GTP had no effect on the binding of p-chloromercuribenzoic acid, suggesting that C323 is not directly involved in allosteric regulation. There were no differences between the two hGDH isozymes in sensitivities to mutagenesis at C323. Our results suggest that C323 plays an important role in catalysis by human GDH isozymes.     

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 microM 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.     

Neuroprotective Effects of 2-Cyclopropylimino-3-Methyl-1,3-Thiazoline Hydrochloride Against Oxidative Stress

Oxidative stress, glutamate excitotoxicity, and inflammation are the important pathological mechanisms in neurodegenerative diseases. Recently, we reported that 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride protects rat glial cells against glutamate-induced excitotoxicity. In this study, we report the effects of 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride on primary cultured cortical astrocytes after exposure to hydrogen peroxide (H₂O₂). Pretreatment of cells with 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride prior to H₂O₂ exposure attenuated the H₂O₂-induced reductions in cell survival and superoxide dismutase, catalase, glutathione, and glutathione peroxidase activities. It also reduced H₂O₂-induced increases in reactive oxygen species levels, malondialdehyde content, and production of nitric oxide. These effects were all concentration-dependent. Our results suggest that 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride protects against oxidative stress.     

Over-production of β-carotene from metabolically engineered Escherichia coli

To produce recombinant β-carotene in vitro, synthetic operons encoding genes governing its biosynthesis were engineered into Escherichia coli. Constructs harboring these operons were introduced into either a high-copy or low-copy cloning vector. β-Carotene production from these recombinant E. coli cells was either constitutive or inducible depending upon plasmid copy number. The most efficient β-carotene production was with the low-copy based vector. The process was increased incrementally from a 5 l to a 50 l fermentor and finally into a 300 l fermentor. The maximal β-carotene yields achieved using the 50 l and 300 l fermentor were 390 mg l⁻¹ and 240 mg l⁻¹, respectively, with overall productivities of 7.8 mg l⁻¹ h⁻¹ and 4.8 mg l⁻¹ h⁻¹.     

Fatty acid binding protein 1 is related with development of aspirin-exacerbated respiratory disease

Background

Aspirin-exacerbated respiratory disease (AERD) refers to the development of bronchoconstriction in asthmatics following the ingestion of aspirin. Although alterations in eicosanoid metabolites play a role in AERD, other immune or inflammatory mechanisms may be involved. We aimed to identify proteins that were differentially expressed in nasal polyps between patients with AERD and aspirin-tolerant asthma (ATA).

Methodology/Principal Findings

Two-dimensional electrophoresis was adopted for differential display proteomics. Proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS). Western blotting and immunohistochemical staining were performed to compare the amount of fatty acid-binding protein 1 (FABP1) in the nasal polyps of patients with AERD and ATA. Fifteen proteins were significantly up- (seven spots) or down-regulated in the nasal polyps of patients with AERD (n = 5) compared to those with ATA (n = 8). LC-MS revealed an increase in seven proteins expression and a decrease in eight proteins expression in patients with AERD compared to those with ATA (P = 0.003–0.045). FABP1-expression based on immunoblotting and immunohistochemical analysis was significantly higher in the nasal polyps of patients with AERD compared to that in patients with ATA. FABP1 was observed in epithelial, eosinophils, macrophages, and the smooth-muscle cells of blood vessels in the polyps.

Conclusions/Significance

Our results indicate that alterations in 15 proteins, including FABP1, may be related to the development of AERD.     

2-Cyclopropylimino-3-methyl-1,3-thiazoline Hydrochloride Protects Against Beta-amyloid-induced Activation of the Apoptotic Cascade in Cultured Cortical Neurons

Aggregated β-amyloid, implicated in the pathogenesis of Alzheimer’s disease (AD), induces neurotoxicity by evoking a cascade of oxidative damage-dependent apoptosis in neurons. We investigated the molecular mechanisms underlying the protective effect of 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride (KHG26377) against the beta-amyloid (Aβ_25–35)-induced primary cortical neuronal cell neurotoxicity. Treatment with KHG26377 attenuated the Aβ_25–35-induced apoptosis by decreasing the Bax/Bcl-2 ratio and suppressing the activation of caspase-3. A marked increase in calcium influx and in the level of reactive oxygen species together with a decrease in glutathione levels was found after Aβ_25–35 exposure; however, KHG26377 treatment reversed these changes in a concentration-dependent manner. In addition, KHG26377 significantly suppressed Aβ_25–35-induced toxicity concomitant with a reduction in the activation of extracellular signal-regulated kinases 1 and 2 and nuclear factor kappa B. The KHG26377-induced protection of neuronal cells against Aβ toxicity was also mediated by suppressing the expression of glycogen synthase kinase-3β, increasing the levels of β-catenin, and reducing the levels of phosphorylated tau. Our findings suggest that KHG26377 may modulate the neurotoxic effects of β-amyloid and provide a rationale for treatment of AD.