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Glyceraldehyde-3-phosphate dehydrogenase [GAPDH, NAD + oxidoreductase (phosphorylating) 1.2.1.12] catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate coupled with the reduction of NAD(+) to NADH. In addition to its role in glycolysis, this enzyme has numerous alternate functions, in both prokaryotes and eukaryotes. In plants, additional functions have been reported from multiple species including Pisum sativum. A recent study has identified that GAPDH may play an important role in seed ageing and programmed cell death. Despite this the existing purification protocols are almost 40 years old, and only partial characterization of the enzyme has been reported. In the current study, we report a modified method for purification of enzymatically active pea seed GAPDH along with the characterization of the enzyme. Using 2D gel electrophoresis our study also demonstrates that pea seeds contain four isoforms of NAD(+) dependent GAPDH.
Abscisic acid (ABA) controls plant development and regulates plant responses to environmental stresses. A role for ABA in sugar regulation of plant development has also been well documented although the molecular mechanisms connecting the hormone with sugar signal transduction pathways are not well understood. In this work it is shown that Arabidopsis thaliana mutants deficient in plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase (gapcp1gapcp2) are ABA insensitive in growth, stomatal closure, and germination assays. The ABA levels of gapcp1gapcp2 were normal, suggesting that the ABA signal transduction pathway is impaired in the mutants. ABA modified gapcp1gapcp2 gene expression, but the mutant response to the hormone differed from that observed in wild-type plants. The gene expression of the transcription factor ABI4, involved in both sugar and ABA signalling, was altered in gapcp1gapcp2, suggesting that their ABA insensitivity is mediated, at least partially, through this transcriptional regulator. Serine supplementation was able partly to restore the ABA sensitivity of gapcp1gapcp2, indicating that amino acid homeostasis and/or serine metabolism may also be important determinants in the connections of ABA with primary metabolism. Overall, these studies provide new insights into the links between plant primary metabolism and ABA signalling, and demonstrate the importance of plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase in these interactions.
The E3 ubiquitin-protein ligases are associated to various processes such as cell cycle control and diverse developmental pathways. Arabidopsis thaliana SEVEN IN ABSENTIA like 7, which has ubiquitin ligase activity, is located in the nucleus and cytosol and is expressed at several stages in almost all plant tissues suggesting an important role in plant functions. However, the mechanism underlying the regulation of this protein is unknown. Since we found that the SEVEN IN ABSENTIA like 7 gene expression is altered in plants with impaired mitochondria, and in plants deficient in the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase 1, we decided to study the possible interactions between both proteins as potential partners in plant signaling functions. We found that SEVEN IN ABSENTIA like 7 is able to interact in vitro with glyceraldehyde-3-phosphate dehydrogenase and that the Lys231 residue of the last is essential for this function. Following the interaction, a concomitant increase in the glyceraldehyde-3-phosphate dehydrogenase catalytic activity was observed. However, when SEVEN IN ABSENTIA like 7 was supplemented with E1 and E2 proteins to form a complete E1-E2-E3 modifier complex, we observed the mono-ubiquitination of glyceraldehyde-3-phosphate dehydrogenase 1 at the Lys76 residue and a dramatic decrease of its catalytic activity. Moreover, we found that localization of glyceraldehyde-3-phosphate dehydrogenase 1 in the nucleus is dependent on the expression SEVEN IN ABSENTIA like 7. These observations suggest that the association of both proteins might result in different biological consequences in plants either through affecting the glycolytic flux or via cytoplasm-nucleus relocation.
Aerobic glycolysis is one of the important metabolic characteristics of many malignant tumors. Pyruvate dehydrogenase kinase (PDHK) plays a key role in aerobic glycolysis by phosphorylating the E1α subunit of pyruvate dehydrogenase (PDH). Hence, PDHK has been recognized as a molecular target for cancer treatment. Here, we report that huzhangoside A (Hu.A), a triterpenoid glycoside compound isolated from several plants of the Anemone genus, acts as a novel PDHK inhibitor. Hu.A was found to decrease the cell viability of human breast cancer MDA-MB-231, hepatocellular carcinoma Hep3B, colon cancer HT-29, DLD-1, and murine lewis lung carcinoma LLC cell lines. The activity of PDHK1 was decreased by Hu.A in both in vitro assays and in vivo assays in DLD-1 cells. Hu.A significantly increased the oxygen consumption and decreased the secretory lactate levels in DLD-1 cells. In addition, Hu.A interacted with the ATP-binding pocket of PDHK1 without affecting the interaction of PDHK1 and pyruvate dehydrogenase complex (PDC) subunits. Furthermore, Hu.A significantly induced mitochondrial reactive oxygen species (ROS) and depolarized the mitochondrial membrane potential in DLD-1 cells. Consistently, when Hu.A was intraperitoneally injected into LLC allograft mice, the tumor growth was significantly decreased. In conclusion, Hu.A suppressed the growth of tumors in both in vitro and in vivo models via inhibition of PDHK activity.
Mathematical models are key to systems biology where they typically describe the topology and dynamics of biological networks, listing biochemical entities and their relationships with one another. Some (hyper)thermophilic Archaea contain an enzyme, called non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN), which catalyzes the direct oxidation of glyceraldehyde-3-phosphate to 3-phosphoglycerate omitting adenosine 5'-triphosphate (ATP) formation by substrate-level-phosphorylation via phosphoglycerate kinase. In this study we formulate three hypotheses that could explain functionally why GAPN exists in these Archaea, and then construct and use mathematical models to test these three hypotheses. We used kinetic parameters of enzymes of Sulfolobus solfataricus (S. solfataricus) which is a thermo-acidophilic archaeon that grows optimally between 60 and 90 °C and between pH 2 and 4. For comparison, we used a model of Saccharomyces cerevisiae (S. cerevisiae), an organism that can live at moderate temperatures. We find that both the first hypothesis, i.e., that the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plus phosphoglycerate kinase (PGK) route (the alternative to GAPN) is thermodynamically too much uphill and the third hypothesis, i.e., that GAPDH plus PGK are required to carry the flux in the gluconeogenic direction, are correct. The second hypothesis, i.e., that the GAPDH plus PGK route delivers less than the 1 ATP per pyruvate that is delivered by the GAPN route, is only correct when GAPDH reaction has a high rate and 1,3-bis-phosphoglycerate (BPG) spontaneously degrades to 3PG at a high rate.
Effects of fructose 1,6-bisphosphate (F-1,6-P2) towards N-methyl-d-aspartate NMDA excitotoxicity were evaluated in rat organotypic hippocampal brain slice cultures (OHSC) challenged for 3 h with 30 μM NMDA, followed by incubations (24, 48, and 72 h) without (controls) and with F-1,6-P2 (0.5, 1 or 1.5 mM). At each time, cell necrosis was determined by measuring LDH in the medium. Energy metabolism was evaluated by measuring ATP, GTP, ADP, AMP, and ATP catabolites (nucleosides and oxypurines) in deproteinized OHSC extracts. Gene expressions of phosphofructokinase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase were also measured. F-1,6-P2 dose-dependently decreased NMDA excitotoxicity, abolishing cell necrosis at the highest concentration tested (1.5 mM). Additionally, F-1,6-P2 attenuated cell energy imbalance caused by NMDA, ameliorating the mitochondrial phosphorylating capacity (increase in ATP/ADP ratio) Metabolism normalization occurred when using 1.5 mM F-1,6-P2. Remarkable increase in expressions of phosphofructokinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase (up to 25 times over the values of controls) was also observed. Since this phenomenon was recorded even in OHSC treated with F-1,6-P2 with no prior challenge with NMDA, it is highly conceivable that F-1,6-P2 can enter into intact cerebral cells producing significant benefits on energy metabolism. These effects are possibly mediated by changes occurring at the gene level, thus opening new perspectives for F-1,6-P2 application as a useful adjuvant to rescue mitochondrial metabolism of cerebral cells under stressing conditions.
The Australian plague locust, Chortoicetes terminifera, is among the most promising species to unravel the suites of genes underling the density-dependent shift from shy and cryptic solitarious behaviour to the highly active and aggregating gregarious behaviour that is characteristic of locusts. This is because it lacks many of the major phenotypic changes in colour and morphology that accompany phase change in other locust species. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most sensitive method available for determining changes in gene expression. However, to accurately monitor the expression of target genes, it is essential to select an appropriate normalization strategy to control for non-specific variation between samples. Here we identify eight potential reference genes and examine their expression stability at different rearing density treatments in neural tissue of the Australian plague locust.
Stomatal movements require massive changes in guard cell osmotic content, and both stomatal opening and stomatal closure have been shown to be energy-requiring processes. A possible role for glycolysis in contributing to the energetic, reducing requirements, or signalling processes regulating stomatal movements has not been investigated previously. Glycolysis, oxidization of glucose to pyruvate, is a central metabolic pathway and yields a net gain of 2 ATP and 2 NADH. 2,3-biphosphoglycerate-independent phosphoglycerate mutase (iPGAM) is a key enzymatic activity in glycolysis and catalyses the reversible interconversion of 3-phosphoglycerate to 2-phosphoglycerate. To investigate functions of iPGAMs and glycolysis in stomatal function and plant growth, Arabidopsis insertional mutants in At1g09780 and At3g08590, both of which have been annotated as iPGAMs on the basis of sequence homology, were identified and characterized. While single mutants were indistinguishable from the wild type in all plant phenotypes assayed, double mutants had no detectable iPGAM activity and showed defects in blue light-, abscisic acid-, and low CO(2)-regulated stomatal movements. Vegetative plant growth was severely impaired in the double mutants and pollen was not produced. The data demonstrate that iPGAMs and glycolytic activity are critical for guard cell function and fertility in Arabidopsis.
Radiation (IR)-induced DNA damage triggers cell cycle arrest and has a suppressive effect on the tumor microenvironment (TME). Wee1, a cell cycle regulator, can eliminate G2/M arrest by phosphorylating cyclin-dependent kinase 1 (CDK1). Meanwhile, programed death-1/programed death ligand-1 (PD-1/PDL-1) blockade is closely related to TME. This study aims to investigate the effects and mechanisms of Wee1 inhibitor AZD1775 and anti-PD-1 antibody (anti-PD-1 Ab) on radiosensitization of hepatoma.
Perlecan Domain V (DV) promotes brain angiogenesis by inducing VEGF release from brain endothelial cells (BECs) following stroke. In this study, we define the specific mechanism of DV interaction with the α(5)β(1) integrin, identify the downstream signal transduction pathway, and further investigate the functional significance of resultant VEGF release. Interestingly, we found that the LG3 portion of DV, which has been suggested to possess most of DV's angio-modulatory activity outside of the brain, binds poorly to α(5)β(1) and induces less BEC proliferation compared to full length DV. Additionally, we implicate DV's DGR sequence as an important element for the interaction of DV with α(5)β(1). Furthermore, we investigated the importance of AKT and ERK signaling in DV-induced VEGF expression and secretion. We show that DV increases the phosphorylation of ERK, which leads to subsequent activation and stabilization of eIF4E and HIF-1α. Inhibition of ERK activity by U0126 suppressed DV-induced expression and secretion of VEGR in BECs. While DV was capable of phosphorylating AKT we show that AKT phosphorylation does not play a role in DV's induction of VEGF expression or secretion using two separate inhibitors, LY294002 and Akt IV. Lastly, we demonstrate that VEGF activity is critical for DV increases in BEC proliferation, as well as angiogenesis in a BEC-neuronal co-culture system. Collectively, our findings expand our understanding of DV's mechanism of action on BECs, and further support its potential as a novel stroke therapy.
Salinity is a serious threat to most land plants. Although seaweeds adapt to salty environments, intertidal species experience wide fluctuations in external salinities, including hyper- and hypo-saline stress. Bangia fuscopurpurea is an economic intertidal seaweed with a strong tolerance to hypo-salinity. Until now, the salt stress tolerance mechanism has remained elusive. Our previous study showed that the expression of B. fuscopurpurea plasma membrane H+-ATPase (BfPMHA) genes were the most upregulated under hypo-salinity. In this study, we obtained the complete sequence of BfPMHA, traced the relative expression of this BfPMHA gene in B. fuscopurpurea under hypo-salinity, and analyzed the protein structure and properties based on the gene's sequence. The result showed that the expression of BfPMHA in B. fuscopurpurea increased significantly with varying hypo-salinity treatments, and the higher the degree of low salinity stress, the higher the expression level. This BfPMHA had typical PMHA structures with a Cation-N domain, an E1-E2 ATPase domain, a Hydrolase domain, and seven transmembrane domains. In addition, through the membrane system yeast two-hybrid library, three candidate proteins interacting with BfPMHA during hypo-saline stress were screened, fructose-bisphosphate aldolase (BfFBA), glyceraldehyde 3-phosphate dehydrogenase (NADP+) (phosphorylating) (BfGAPDH), and manganese superoxide dismutase (BfMnSOD). The three candidates and BfPMHA genes were successfully transferred and overexpressed in a BY4741 yeast strain. All of them significantly enhanced the yeast tolerance to NaCl stress, verifying the function of BfPMHA in salt stress response. This is the first study to report the structure and topological features of PMHA in B. fuscopurpurea and its candidate interaction proteins in response to salt stress.
Heat shock factor 1 (HSF1) monitors the structural integrity of the proteome. Phosphorylation at S326 is a hallmark for HSF1 activation, but the identity of the kinase(s) phosphorylating this site has remained elusive. We show here that the dietary agent phenethyl isothiocyanate (PEITC) inhibits heat shock protein 90 (Hsp90), the main negative regulator of HSF1; activates p38 mitogen-activated protein kinase (MAPK); and increases S326 phosphorylation, trimerization, and nuclear translocation of HSF1, and the transcription of a luciferase reporter, as well as the endogenous prototypic HSF1 target Hsp70. In vitro, all members of the p38 MAPK family rapidly and stoichiometrically catalyze the S326 phosphorylation. The use of stable knockdown cell lines and inhibitors indicated that among the p38 MAPKs, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at S326 in cells. A protease-mass spectrometry approach confirmed S326 phosphorylation and unexpectedly revealed that p38 MAPK also catalyzes the phosphorylation of HSF1 at S303/307, previously known repressive posttranslational modifications. Thus, we have identified p38 MAPKs as highly efficient catalysts for the phosphorylation of HSF1. Furthermore, our findings suggest that the magnitude and persistence of activation of p38 MAPK are important determinants of the extent and duration of the heat shock response.
Accurate chromosome segregation requires kinetochores on duplicated chromatids to biorient by attaching to dynamic microtubules from opposite spindle poles, which exerts forces to bring kinetochores under tension. However, kinetochores initially bind to microtubules indiscriminately, resulting in errors that must be corrected. While the Aurora B protein kinase destabilizes low-tension attachments by phosphorylating kinetochores, low-tension attachments are intrinsically less stable than those under higher tension in vitro independent of Aurora activity. Intrinsic tension-sensitive behavior requires the microtubule regulator Stu2 (budding yeast Dis1/XMAP215 ortholog), which we demonstrate here is likely a conserved function for the TOG protein family. The human TOG protein, chTOG, localizes to kinetochores independent of microtubules by interacting with Hec1. We identify a chTOG mutant that regulates microtubule dynamics but accumulates erroneous kinetochore-microtubule attachments that are not destabilized by Aurora B. Thus, TOG proteins confer a unique, intrinsic error correction activity to kinetochores that ensures accurate chromosome segregation.
Missense mutations in the LRRK2 (Leucine-rich repeat protein kinase-2) and VPS35 genes result in autosomal dominant Parkinson's disease. The VPS35 gene encodes for the cargo-binding component of the retromer complex, while LRRK2 modulates vesicular trafficking by phosphorylating a subgroup of Rab proteins. Pathogenic mutations in LRRK2 increase its kinase activity. It is not known how the only thus far described pathogenic VPS35 mutation, [p.D620N] exerts its effects. We reveal that the VPS35[D620N] knock-in mutation strikingly elevates LRRK2-mediated phosphorylation of Rab8A, Rab10, and Rab12 in mouse embryonic fibroblasts. The VPS35[D620N] mutation also increases Rab10 phosphorylation in mouse tissues (the lung, kidney, spleen, and brain). Furthermore, LRRK2-mediated Rab10 phosphorylation is increased in neutrophils as well as monocytes isolated from three Parkinson's patients with a heterozygous VPS35[D620N] mutation compared with healthy donors and idiopathic Parkinson's patients. LRRK2-mediated Rab10 phosphorylation is significantly suppressed by knock-out or knock-down of VPS35 in wild-type, LRRK2[R1441C], or VPS35[D620N] cells. Finally, VPS35[D620N] mutation promotes Rab10 phosphorylation more potently than LRRK2 pathogenic mutations. Available data suggest that Parkinson's patients with VPS35[D620N] develop the disease at a younger age than those with LRRK2 mutations. Our observations indicate that VPS35 controls LRRK2 activity and that the VPS35[D620N] mutation results in a gain of function, potentially causing PD through hyperactivation of the LRRK2 kinase. Our findings suggest that it may be possible to elaborate compounds that target the retromer complex to suppress LRRK2 activity. Moreover, patients with VPS35[D620N] associated Parkinson's might benefit from LRRK2 inhibitor treatment that have entered clinical trials in humans.
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