Cloning and Characterization of Glyceraldehyde-3-phosphate Dehydrogenase Encoding Gene in Gracilaria/Gracilariopsis lemaneiformis

Glyceraldehyde-3-phosphate dehydrogenase （GAPDH） plays important roles in various cellular processes. A cytosolic GAPDH encoding gene （gpd） of Gracilaria/Gracilariopsis lemaneiformis was cloned and characterized. Deduced amino acid sequence of the enzyme of G. lemaneiformis had high homology with those of seven red algae. The 5＇-untranslated regions of the GAPDHs encoding genes of these red algae varied greatly. GAPDHs of these red algae shared the highly conserved glyceraldehyde 3-phosphate dehydrogenase active site ASCTTNCL. However, such active site of Cyanidium caldarium was different from those of the other six algae at the last two residues （CL to LF）, thus the spatial structure of its GAPDH active center may be different from those of the other six. Phylogenetic analysis indicated that GAPDH of G. lemaneiformis might have undergone an evolution similar to those of Porphyra yezoensis, Chondrus crispus, and Gracilaria verrucosa. C. caldarium had a closer evolutionary relationship with Cyanidioschyzon merolae than with Cyanidium sp. Virtual Northern blot analysis revealed that gpd of G. lemaneiformis expressed constitutively, which suggested that it might be house-keeping and could be adapted as an inner control in gene expression analysis of G. lemaneiformis.

Purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from saline strain Idiomarina loihiensis

Idiomarina loihiensis was isolated from the salt works in Sfax (Tunisia), until now, the characterization of the GAPDH phosphorylante was never studied. Here, we report the isolation and the biochemical characterization of glyceralehyde-3-phosphate dehydrogenase (GAPDH) fromI. loihiensis saline’s bacteria on the basis of the apparent native and subunit molecular weights, physico-chemical and kinetic characterizations. The purification method consisted of two steps, ammonium sulfate fractionation followed by one chromatographic step, namely dye-affinity on Blue Sepharose CL-6B. Polyclonal antibodies against the purified enzyme were used to recognize theI. loihiensis GAPDH by Western blotting. The optimum pH of the purified enzyme was 8.5. Studies on the effect of temperatures revealed an enzyme increasing activity of about 45?C. The molecular weight of the purified enzyme was 36 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Non-denaturing polyacrylamide gels yield a molecular weight of 147 kDa. The Michaelis constants for NAD+ and D-glyceraldehyde-3-phosphate estimated was 19 μM and 3.1 μM, respectively. The maximal velocity of the purified enzyme was estimated to be 2.06 U/mg, approximately 6-fold increase in specific activity and a final yield of approximately 32.5%. The physicochemical properties of this GAPDH, being characterized, could be used in further studies.

Role of Cathepsin G in the Degradation of Glyceraldehyde-3-Phosphate Dehydrogenase Triggered by 4-Hydroxy-2-Nonenal in U937 Cells

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.

A homolog of glyceraldehyde-3-phosphate dehydrogenase from Riemerella anatipestifer is an extracellular protein and exhibits biological activity

Riemerella anatipestifer is the causative agent of septicemia anserum exsudativa in ducks. Its pathogenesis and virulence factors are still unclear. The glycelytic enzyme, glyceraldehyde-3-phosphate dehydrogenase （GAPDH）, an anchorless and multifunctional protein on the surface of several pathogenic microorganisms, is involved in virulence and adhesion. Whether homologs of GAPDH exist, and display similar characteristics in R. anatipestifer （RaGAPDH） has not been determined. In our research, the RaGAPDH activity from various R. anatipestifer isolates was confirmed. Twenty-two gapdh genes from genornic DNA of R. anatipestifer isolates were cloned and sequenced for phylogenetic analysis. The distribution of RaGAPDH in R. anatipestifer CZ2 strain was confirmed by antisera to recombinant RaGAPDH. The ability of purified RaGAPDH to bind host proteins was analyzed by solid-phase ligandbinding assay. Results revealed that all R. anatipestifer isolates showed different levels of GAPDH activity except four strains, which contained a gapdh-like gene. The gapdh of R. anatipestifer, which is located phylogenetically in the same branch as enterohemorrhagic Escherichia coil （EHEC）, belonged to class I GAPDH, and encoded a 36.7-kDa protein. All RaGAPDH-encoding gene sequences from field isolates of R. anatipestiferdisplayed 100% homology. The RaGAPDH localized on the extracellular membrane of several R. anatipestifer strains. Further, it was released into the culture medium, and exhibited GAPDH enzyme activity. We also confirmed the binding of RaGAPDH to plasminogen and fibrinogen. These results demonstrated that GAPDH was present in R. anatipestifer, although not in all strains, and that RaGAPDH might contribute to the microorganism＇s virulence.

Critical protein GAPDH and its regulatory mechanisms in cancer cells

Glyceraldehyde-3-phosphate dehydrogenase （GAPDH）, initially identified as a glycolytic enzyme and considered as a housekeeping gene, is widely used as an internal control in experiments on proteins, mRNA, and DNA. However, emerging evidence indicates that GAPDH is implicated in diverse functions independent of its role in energy metabolism; the expression status of GAPDH is also deregulated in various cancer cells. One of the most common effects of GAPDH is its inconsistent role in the determination of cancer cell fate. Furthermore, studies have described GAPDH as a regulator of cell death; other studies have suggested that GAPDH participates in tumor progression and serves as a new therapeutic target. However, related regulatory mechanisms of its numerous cellular functions and deregulated expression levels remain unclear. GAPDH is tightly regulated at transcriptional and pnsttranscriptional levels, which are involved in the regulation of diverse GAPDH functions. Several cancer-related factors, such as insulin, hypoxia inducible factor-1 （HIF-1）, p53, nitric oxide （NO）, and acetylated histone, not only modulate GAPDH gene expression but also affect protein functions via common pathways. Moreover, posttranslational modifications （PTMs） occurring in GAPDH in cancer cells result in new activities unrelated to the original glycnlytic function of GAPDH. In this review, recent findings related to GAPDH transcriptional regulation and PTMs are summarized. Mechanisms and pathways involved in GAPDH regulation and its different roles in cancer cells are also described.

Construction of an alternative NADPH regeneration pathway improves ethanol production in Saccharomyces cerevisiae with xylose metabolic pathway

Full conversion of glucose and xylose from lignocellulosic hydrolysates is required for obtaining a high ethanol yield.However,glucose and xylose share flux in the pentose phosphate pathway(PPP)and glycolysis pathway(EMP),with glucose having a competitive advantage in the shared metabolic pathways.In this work,we knocked down ZWF1 to preclude glucose from entering the PPP.This reduced the[NADPH]level and disturbed growth on both glucose or xylose,confirming that the oxidative PPP,which begins with Zwf1p and ultimately leads to CO_(2) production,is the primary source of NADPH in both glucose and xylose.Upon glucose depletion,gluconeogenesis is necessary to generate glucose-6-phosphate,the substrate of Zwf1p.We re-established the NADPH regeneration pathway by replacing the endogenous NAD^(+)-dependent glyceraldehyde-3-phosphate dehydrogenase(GAPDH)gene TDH3 with heterogenous NADP^(+)-GAPDH genes GDH,gapB,and GDP1.Among the resulting strains,the strain BZP1(zwf1Δ,tdh3::GDP1)exhibited a similar xylose consumption rate before glucose depletion,but a 1.6-fold increased xylose consumption rate following glucose depletion compared to the original strain BSGX001,and the ethanol yield for total consumed sugars of BZP1 was 13.5%higher than BSGX001.This suggested that using the EMP instead of PPP to generate NADPH reduces the wasteful metabolic cycle and excess CO_(2)^(++) release from oxidative PPP.Furthermore,we used a copper-repressing promoter to modulate the expression of ZWF1 and optimize the timing of turning off the ZWF1,therefore,to determine the competitive equilibrium between glucose-xylose co-metabolism.This strategy allowed fast growth in the early stage of fermentation and low waste in the following stages of fermentation.