Activity of Human Dihydrolipoamide Dehydrogenase Is Largely Reduced by Mutation at Isoleucine-51 to Alanine

Kim, Hakjung

doi:10.5483/bmbrep.2006.39.2.223

Cited by 6 publications

(4 citation statements)

References 7 publications

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“…In NMBR, the isoleucine 199 is located adjacent to Cys 198 in EC3, which is presumed to form a disulfide bridge with Cys 116 in EC2, which is important in determining EC3 conformation. The possibility that the substitution of isoleucine 199 in NMBR, with the equivalent amino acid alanine in GRPR, could be effecting the global conformation of NMBR or the receptor structure is supported by studies that show that the presence of isoleucine promotes formation of Cys-Cys bridges (Kim, 2006). Our data suggest that it is unlikely that the presence of an alanine instead of isoleucine 199 is altering the global structure of the mutated receptor because the mutated receptor retained affinity for other peptide ligands.…”

Section: Tablesupporting

confidence: 57%

Molecular Basis for the Selectivity of the Mammalian Bombesin Peptide, Neuromedin B, for Its Receptor

González

Nakagawa²,

Mantey³

et al. 2009

J Pharmacol Exp Ther

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The mammalian bombesin (Bn) peptides, neuromedin B (NMB) and gastrin-releasing peptide (GRP), have widespread actions in many tissues, and their effects are mediated by two closely related G-protein-coupled receptors, the NMBR and GRPR. Little is known about the structural determinants of NMBR selectivity for NMB, in contrast to GRP selectivity for the GRPR, which has been extensively studied. To provide insight, chimeric NMBR-GRPR loss-of-affinity and gain-of-affinity mutants were made, as well as NH 2 -terminally truncated NMBR and point mutants using site-directed mutagenesis. Receptors were expressed in Balb-3T3-cells or CHOP cells, and affinities were determined. NMB had 115-fold greater affinity for NMBR than GRPR. Receptor-chimeric studies showed that NMBR selectivity for NMB was primarily determined by differences in the third extracellular (EC3) regions of GRPR-NMBR and adjacent upper-transmembrane-5 (TM5) region. In this region, 24 NMB gain-of-affinity GRPR mutants or NMBR loss-of-affinity point/ combination mutants were made. Three gain-of-affinity mutant GRPRs [[A198I] (EC3), [H202Q] (EC3), [S215I] (upper TM5)]had increased NMB affinity (2.4 -21-fold), and these results were confirmed with NMBR loss-of-affinity mutants [I199A, Q203H,I215S-NMBR]. The combination mutant [A198I, S215]GRPR had the greatest effect causing a complete NMB gain-of-affinity. The importance of differences at position 199NMBR or 203NMBR was studied by substituting amino acids with various properties. Our results show that NMBR selectivity for NMB is due to differences in the EC3 of NMBR-GRPR and the adjacent upper-TM5 region. Within these regions, isoleucines in NMBR [position 199 (EC3)] (instead of A198GRPR) and in 215NMBR (TM5) (instead of S214GRPR), as well as Q203NMBR (instead of H202GRPR) are responsible for high NMB-affinity/selectivity of NMBR. The effect at position 199 is primarily due to differences in hydrophobicity of the substitution, whereas steric factors and charge of the substitution at position 203 were important determinants of NMB selectivity.

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Section: Tablesupporting

confidence: 57%

Molecular Basis for the Selectivity of the Mammalian Bombesin Peptide, Neuromedin B, for Its Receptor

González

Nakagawa²,

Mantey³

et al. 2009

J Pharmacol Exp Ther

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“…Kim et al . [ 29 ] observed that DLD expression increases with tumor progression, thus providing more energy for tumor cell growth. Therefore, high expression of LDH and DLD is a crucial factor for the development and progression of gastric cancer, and H .…”

Section: Discussionmentioning

confidence: 99%

Proteomics-Based Identification and Analysis of Proteins Associated with Helicobacter pylori in Gastric Cancer

et al. 2016

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Helicobacter pylori (H. pylori) is a spiral-shaped Gram-negative bacterium that causes the most common chronic infection in the human stomach. Approximately 1%-3% of infected individuals develop gastric cancer. However, the mechanisms by which H. pylori induces gastric cancer are not completely understood. The available evidence indicates a strong link between the virulence factor of H. pylori, cytotoxin-associated gene A (CagA), and gastric cancer. To further characterize H. pylori virulence, we established three cell lines by infecting the gastric cancer cell lines SGC-7901 and AGS with cagA+ H. pylori and transfecting SGC-7901 with a vector carrying the full-length cagA gene. We detected 135 differently expressed proteins from the three cell lines using proteome technology, and 10 differential proteins common to the three cell lines were selected and identified by LC-MS/MS as well as verified by western blot: β-actin, L-lactate dehydrogenase (LDH), dihydrolipoamide dehydrogenase (DLD), pre-mRNA-processing factor 19 homolog (PRPF19), ATP synthase, calmodulin (CaM), p64 CLCP, Ran-specific GTPase-activating protein (RanGAP), P43 and calreticulin. Detection of the expression of these proteins and genes encoding these proteins in human gastric cancer tissues by real-time PCR (RT-qPCR) and western blot revealed that the expression of β-ACTIN, LDH, DLD, PRPF19 and CaM genes were up-regulated and RanGAP was down-regulated in gastric cancer tissues and/or metastatic lymph nodes compared to peri-cancerous tissues. High gene expression was observed for H. pylori infection in gastric cancer tissues. Furthermore, the LDH, DLD and CaM genes were demethylated at the promoter -2325, -1885 and -276 sites, respectively, and the RanGAP gene was highly methylated at the promoter -570 and -170 sites in H. pylori-infected and cagA-overexpressing cells. These results provide new insights into the molecular pathogenesis and treatment targets for gastric cancer with H. pylori infection.

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“…In addition, nine artificial substitution mutations were created and characterized. Among them, three were located at FAD-binding domain [23][24][25], one was located at NAD + -binding domain [23], three were located at interface domain [26,27], and two were located at center domain [28]. The characterization of these mutants leads the way to understand the structure-function relationship of E3.…”

Section: Introductionmentioning

confidence: 98%

The role of amino acids T148 and R281 in human dihydrolipoamide dehydrogenase

Wang

et al. 2007

J Biomed Sci

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Human dihydrolipoamide dehydrogenase (hE3) is a common component of alpha-ketoacid dehydrogenase complexes. Mutations of this homodimeric protein cause E3 deficiency and are always fatal. To investigate its reaction mechanism, we first performed multiple sequence alignment with other 17 eukaryotic E3s. According to hE3 structure and the result of multiple sequence alignment, two amino acids, T148 and R281, were subjected to mutagenesis and four hE3 mutants, T148G, T148S, R281N, and R281K, were expressed and assayed. The specific activities of T148G, T148S, R281N, and R281K are 76.34%, 88.62%, 12.50%, and 11.93% to that of wild-type E3, respectively. The FAD content analysis indicated that the FAD content of these mutant E3s were about 71.0%, 92%, 96%, and 93% that of wild-type E3, respectively. The molecular weight analysis showed that these three mutant proteins form the dimer. Kinetic data demonstrated that the K(cat) of forward reaction of all mutants, except T148 mutants, were decreased dramatically. The results of kinetic study suggest that T148 is not important to E3 catalytic function and R281 play a role in the catalytic function of the E3.

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Activity of Human Dihydrolipoamide Dehydrogenase Is Largely Reduced by Mutation at Isoleucine-51 to Alanine

Cited by 6 publications

References 7 publications

Molecular Basis for the Selectivity of the Mammalian Bombesin Peptide, Neuromedin B, for Its Receptor

Molecular Basis for the Selectivity of the Mammalian Bombesin Peptide, Neuromedin B, for Its Receptor

Proteomics-Based Identification and Analysis of Proteins Associated with Helicobacter pylori in Gastric Cancer

The role of amino acids T148 and R281 in human dihydrolipoamide dehydrogenase

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