Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many ...Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. HNE-modified proteins are degraded by the ubiquitin-proteasome pathway or the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by cathepsin G. In the present study, we examined whether GAPDH in U937 cells treated with HNE in culture is degraded similarly to that incubated with HNE and U937 cell extract. Treatment with HNE for 10 min in culture decreased GAPDH activity in a concentration dependent manner, but did not affect GAPDH degradation. The proteasome activities were not affected by HNE, but culturing with HNE decreased cathepsin G activity and protein level in a concentration dependent manner. These results suggest that HNE-induced oxidative stress leads to decreased cathepsin G activity and results in the loss of GAPDH degradation. Taken together, our findings indicate that cathepsin G has an important role in the degradation of oxidatively modified GAPDH in U937 cells.展开更多
Glyceraldehyde-3-phosphate dehydro- genase (GAPDH) is a multifunctional protein well defined in eukaryotes, especially in mammalian and Saccharomyces cerevisiae. Using the method of suppression subtractive hybridizati...Glyceraldehyde-3-phosphate dehydro- genase (GAPDH) is a multifunctional protein well defined in eukaryotes, especially in mammalian and Saccharomyces cerevisiae. Using the method of suppression subtractive hybridization (SSH), we identified a Phytophthora sojae cDNA coding GAPDH, which was up-regulated during the early stage of soybean infection. The termed PsGapdh gene pos- sessed three copies in the P. sojae genome. Its amino acid sequence harbored overall conserved domain of GADPH, homologous closest to GapC1 of Achlya bisexualis (oomycete) and adjoined to GapC2s of Odontella sinensis and Phaeodactylum tricornutum (diatom), on the C-Ⅱ branch of subfamily GapC in phylogeny tree of GAPDH. The transcrip- tional level of PsGapdh was up-regulated throughout early infection. Heterogenous expression of PsGapdh in the yeast tdh1-deleted mutant could rescue growth arrest under continuous exposure to H2O2. These results indicated active roles of PsGapdh in patho- gen-host interaction and anti-oxidation.展开更多
It has been suggested that the energy required for sperm motility is produced by oxidative phosphorylation while glycolysis seems to be an important source for ATP transmission along the flagellum. Some studies have i...It has been suggested that the energy required for sperm motility is produced by oxidative phosphorylation while glycolysis seems to be an important source for ATP transmission along the flagellum. Some studies have investigated the chemical and kinetic properties of the enzyme glyceraldehyde 3-phosphate dehydrogenase to identify any changes in the regulation of glycolysis and sperm motility. In contrast, there are few studies analyzing the genetic basis of hypokinesis. For this reason, we investigated the glyceraldehyde 3-phosphate dehydrogenase gene in human sperm to evaluate whether asthenozoospermia was correlated with any changes in its expression. Semen examination and glyceraldehyde 3-phosphate dehydrogenase gene expression studies were carried out on 116 semen samples divided into two groups - Group A consisted of 58 normokinetic samples and Group B of 58 hypokinetic samples. Total RNA was extracted from spermatozoa, and real-time PCR quantification of mRNA was carried out using specific primers and probes. The expression profiles for the Groups A and B were very similar. The mean delta Ct was as follows - Group A, 5.79 ＋ 1.04; Group B, 5.47 ＋ 1.27. Our study shows that in human sperm, there is no difference in glyceraldehyde 3-phosphate dehydrogenase gene expression between samples with impaired motility and samples with normal kinetics. We believe that this study could help in the understanding of the molecular mechanisms of sperm kinetics, suggesting that hypomotility may be due to a possible posttranscriptional impairment of the control mechanism, such as mRNA splicing, or to posttranslational changes.展开更多
Phosphorous is one of the essential mineral elements for plant growth and development.Typically, the shoots of plant seedlings usually turn a dark-brown or purple colour under low-Pi stress. Using protein 2-D gel and ...Phosphorous is one of the essential mineral elements for plant growth and development.Typically, the shoots of plant seedlings usually turn a dark-brown or purple colour under low-Pi stress. Using protein 2-D gel and peptide mass fingerprinting mapping (PMF) methods, a cytoplasmic glyceralde-hyde-3-phosphate dehydrogenase GapC-2 was identified as a low-Pi responsive protein in Arabidopsisplants. Expression of AtGapC-2 protein was significantly decreased after 4 d of low-Pi stress. Two in-dependent T-DNA insertion lines of GapC-2 gene (At1g13440) showed a hypersensitive phenotype inresponse to low-Pi stress compared with wild type plants, while the transgenic complementation linesof the mutants showed a similar phenotype to the wild type. These results indicate that AtGapC-2 mayplay an important role in Arabidopsis responses to low-Pi stress, possibly by regulation of glycoly-sis-associated "Pi-pool" and accumulation of anthocyanin pigments in plants.展开更多
D-glyceraldehyde-3-phosphate dehydrogenase (holo-GAPDH) from Palinurus versicolor was crystallized in a novel crystal form by the method of sitting-drop vapor diffusion. The crystals have space group P4212, cell param...D-glyceraldehyde-3-phosphate dehydrogenase (holo-GAPDH) from Palinurus versicolor was crystallized in a novel crystal form by the method of sitting-drop vapor diffusion. The crystals have space group P4212, cell parameters a=15.49 nm, c=8.03 nm and two subunits per asymmetric unit. The crystal structure at 0.34 nm was determined by the molecular replacement method. The final model has crystallographic Rfree and R factors of 0.274 and 0.262, and r.m.s. deviations of 0.002 nm for bond lengths and 2.33?for bond angles. The two subunits in asymmetric unit are similar to each other not only in the three-dimensional structure, but also in average temperature factors. This result demonstrates that the obvious difference in average temperature factors for the different subunits in C2 crystal form reported previously may be attributed to the different crystallographic environments of the subunits. This further supports that holo-GAPDH has a good 222 molecular symmetry.展开更多
In contrast with the coezyme, two coenzyme analogs, ADP-ribose and SNAD, bind non-cooperatively to D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Palinurus versicolor (PV) GAPDH complexed with ADP-ribose and SNAD...In contrast with the coezyme, two coenzyme analogs, ADP-ribose and SNAD, bind non-cooperatively to D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Palinurus versicolor (PV) GAPDH complexed with ADP-ribose and SNAD has been crystallized by the method of sitting-drop vapor diffusion. X-ray diffraction data analysis reveals that both crystals belong to the same space group (C2), and have similar cell dimensions: a =152.80 A, b =100.35 A, c =128.31 A, β=110.28° and a =153.41 A, b =100.51 A, c =128.44 A, β =110.48°, respectively. It is estimated that the asymmetric unit in each crystal contains 4 subunits. This is a novel crystal form which is quite different from that previously reported for holo- and apo-GAPDH from the same spurce. The result suggests that the binding of the two coenzyme analogs to GAPDH may lead to some significant conformational changes, which are different from those induced by the coenzyme binding. The self-rotation function indicates that the tetramer of these two GAPDH展开更多
文摘Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. HNE-modified proteins are degraded by the ubiquitin-proteasome pathway or the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by cathepsin G. In the present study, we examined whether GAPDH in U937 cells treated with HNE in culture is degraded similarly to that incubated with HNE and U937 cell extract. Treatment with HNE for 10 min in culture decreased GAPDH activity in a concentration dependent manner, but did not affect GAPDH degradation. The proteasome activities were not affected by HNE, but culturing with HNE decreased cathepsin G activity and protein level in a concentration dependent manner. These results suggest that HNE-induced oxidative stress leads to decreased cathepsin G activity and results in the loss of GAPDH degradation. Taken together, our findings indicate that cathepsin G has an important role in the degradation of oxidatively modified GAPDH in U937 cells.
文摘Glyceraldehyde-3-phosphate dehydro- genase (GAPDH) is a multifunctional protein well defined in eukaryotes, especially in mammalian and Saccharomyces cerevisiae. Using the method of suppression subtractive hybridization (SSH), we identified a Phytophthora sojae cDNA coding GAPDH, which was up-regulated during the early stage of soybean infection. The termed PsGapdh gene pos- sessed three copies in the P. sojae genome. Its amino acid sequence harbored overall conserved domain of GADPH, homologous closest to GapC1 of Achlya bisexualis (oomycete) and adjoined to GapC2s of Odontella sinensis and Phaeodactylum tricornutum (diatom), on the C-Ⅱ branch of subfamily GapC in phylogeny tree of GAPDH. The transcrip- tional level of PsGapdh was up-regulated throughout early infection. Heterogenous expression of PsGapdh in the yeast tdh1-deleted mutant could rescue growth arrest under continuous exposure to H2O2. These results indicated active roles of PsGapdh in patho- gen-host interaction and anti-oxidation.
文摘It has been suggested that the energy required for sperm motility is produced by oxidative phosphorylation while glycolysis seems to be an important source for ATP transmission along the flagellum. Some studies have investigated the chemical and kinetic properties of the enzyme glyceraldehyde 3-phosphate dehydrogenase to identify any changes in the regulation of glycolysis and sperm motility. In contrast, there are few studies analyzing the genetic basis of hypokinesis. For this reason, we investigated the glyceraldehyde 3-phosphate dehydrogenase gene in human sperm to evaluate whether asthenozoospermia was correlated with any changes in its expression. Semen examination and glyceraldehyde 3-phosphate dehydrogenase gene expression studies were carried out on 116 semen samples divided into two groups - Group A consisted of 58 normokinetic samples and Group B of 58 hypokinetic samples. Total RNA was extracted from spermatozoa, and real-time PCR quantification of mRNA was carried out using specific primers and probes. The expression profiles for the Groups A and B were very similar. The mean delta Ct was as follows - Group A, 5.79 ＋ 1.04; Group B, 5.47 ＋ 1.27. Our study shows that in human sperm, there is no difference in glyceraldehyde 3-phosphate dehydrogenase gene expression between samples with impaired motility and samples with normal kinetics. We believe that this study could help in the understanding of the molecular mechanisms of sperm kinetics, suggesting that hypomotility may be due to a possible posttranscriptional impairment of the control mechanism, such as mRNA splicing, or to posttranslational changes.
基金the Chinese National Key Basic Research Project (Grant No.2006CB100100)
文摘Phosphorous is one of the essential mineral elements for plant growth and development.Typically, the shoots of plant seedlings usually turn a dark-brown or purple colour under low-Pi stress. Using protein 2-D gel and peptide mass fingerprinting mapping (PMF) methods, a cytoplasmic glyceralde-hyde-3-phosphate dehydrogenase GapC-2 was identified as a low-Pi responsive protein in Arabidopsisplants. Expression of AtGapC-2 protein was significantly decreased after 4 d of low-Pi stress. Two in-dependent T-DNA insertion lines of GapC-2 gene (At1g13440) showed a hypersensitive phenotype inresponse to low-Pi stress compared with wild type plants, while the transgenic complementation linesof the mutants showed a similar phenotype to the wild type. These results indicate that AtGapC-2 mayplay an important role in Arabidopsis responses to low-Pi stress, possibly by regulation of glycoly-sis-associated "Pi-pool" and accumulation of anthocyanin pigments in plants.
文摘D-glyceraldehyde-3-phosphate dehydrogenase (holo-GAPDH) from Palinurus versicolor was crystallized in a novel crystal form by the method of sitting-drop vapor diffusion. The crystals have space group P4212, cell parameters a=15.49 nm, c=8.03 nm and two subunits per asymmetric unit. The crystal structure at 0.34 nm was determined by the molecular replacement method. The final model has crystallographic Rfree and R factors of 0.274 and 0.262, and r.m.s. deviations of 0.002 nm for bond lengths and 2.33?for bond angles. The two subunits in asymmetric unit are similar to each other not only in the three-dimensional structure, but also in average temperature factors. This result demonstrates that the obvious difference in average temperature factors for the different subunits in C2 crystal form reported previously may be attributed to the different crystallographic environments of the subunits. This further supports that holo-GAPDH has a good 222 molecular symmetry.
文摘In contrast with the coezyme, two coenzyme analogs, ADP-ribose and SNAD, bind non-cooperatively to D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Palinurus versicolor (PV) GAPDH complexed with ADP-ribose and SNAD has been crystallized by the method of sitting-drop vapor diffusion. X-ray diffraction data analysis reveals that both crystals belong to the same space group (C2), and have similar cell dimensions: a =152.80 A, b =100.35 A, c =128.31 A, β=110.28° and a =153.41 A, b =100.51 A, c =128.44 A, β =110.48°, respectively. It is estimated that the asymmetric unit in each crystal contains 4 subunits. This is a novel crystal form which is quite different from that previously reported for holo- and apo-GAPDH from the same spurce. The result suggests that the binding of the two coenzyme analogs to GAPDH may lead to some significant conformational changes, which are different from those induced by the coenzyme binding. The self-rotation function indicates that the tetramer of these two GAPDH
