The discovery of an anti-Candida xanthone with selective inhibition of Candida albicans GAPDH.

OBJECTIVES: This study aimed to discover novel antifungals targeting Candida albicans glyceraldehyde-3-phosphate dehydrogenase (CaGAPDH), have an insight into inhibitory mode, and provide evidence supporting CaGAPDH as a target for new antifungals. METHODS: Virtual screening was utilized to discover inhibitors of CaGAPDH. The inhibitory effect on cellular GAPDH was evaluated by determining the levels of ATP, NAD, NADH, etc., as well as examining GAPDH mRNA and protein expression. The role of GAPDH inhibition in C. albicans was supported by drug affinity responsive target stability and overexpression experiments. The mechanism of CaGAPDH inhibition was elucidated by Michaelis-Menten enzyme kinetics and site-specific mutagenesis based on docking. Chemical synthesis was used to produce an improved candidate. Different sources of GAPDH were used to evaluate inhibitory selectivity across species. In vitro and in vivo antifungal tests, along with anti-biofilm activity, were carried out to evaluate antifungal potential of GAPDH inhibitors. RESULTS: A natural xanthone was identified as the first competitive inhibitor of CaGAPDH. It demonstrated in vitro anti-C. albicans potential but also caused hemolysis. XP-W, a synthetic side-chain-optimized xanthone, demonstrated a better safety profile, exhibiting a 50-fold selectivity for CaGAPDH over human GAPDH. XP-W also exhibited potent anti-biofilm activity and displayed broad-spectrum anti-Candida activities in vitro and in vivo, including multi-azole-resistant C. albicans. CONCLUSIONS: These results demonstrate for the first time that CaGAPDH is a valuable target for antifungal drug discovery, and XP-W provides a promising lead.

Natural product 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose is a reversible inhibitor of glyceraldehyde 3-phosphate dehydrogenase.

Aerobic glycolysis, also known as the Warburg effect, is a hallmark of cancer cell glucose metabolism and plays a crucial role in the activation of various types of immune cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of D-glyceraldehyde 3-phosphate to D-glycerate 1,3-bisphosphate in the 6th critical step in glycolysis. GAPDH exerts metabolic flux control during aerobic glycolysis and therefore is an attractive therapeutic target for cancer and autoimmune diseases. Recently, GAPDH inhibitors were reported to function through common suicide inactivation by covalent binding to the cysteine catalytic residue of GAPDH. Herein, by developing a high-throughput enzymatic screening assay, we discovered that the natural product 1,2,3,4,6-penta-O-galloyl-ß-D-glucopyranose (PGG) is an inhibitor of GAPDH with Ki = 0.5 µM. PGG blocks GAPDH activity by a reversible and NAD+ and Pi competitive mechanism, suggesting that it represents a novel class of GAPDH inhibitors. In-depth hydrogen deuterium exchange mass spectrometry (HDX-MS) analysis revealed that PGG binds to a region that disrupts NAD+ and inorganic phosphate binding, resulting in a distal conformational change at the GAPDH tetramer interface. In addition, structural modeling analysis indicated that PGG probably reversibly binds to the center pocket of GAPDH. Moreover, PGG inhibits LPS-stimulated macrophage activation by specific downregulation of GAPDH-dependent glucose consumption and lactate production. In summary, PGG represents a novel class of GAPDH inhibitors that probably reversibly binds to the center pocket of GAPDH. Our study sheds new light on factors for designing a more potent and specific inhibitor of GAPDH for future therapeutic applications.

Glyceraldehyde-3-phosphate dehydrogenase and glutamine synthetase inhibition in the presence of pro-inflammatory cytokines contribute to the metabolic imbalance of diabetic retinopathy.

Diabetic retinopathy (DR) is the leading cause of vision impairment in working age adults. In addition to hyperglycemia, retinal inflammation is an important driving factor for DR development. Although DR is clinically described as diabetes-induced damage to the retinal blood vessels, several studies have reported that metabolic dysregulation occurs in the retina prior to the development of microvascular damage. The two most commonly affected metabolic pathways in diabetic conditions are glycolysis and the glutamate pathway. We investigated the role of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutamine synthetase (GS) in an in-vitro model of DR incorporating high glucose and pro-inflammatory cytokines. We found that GAPDH and GS enzyme activity were not significantly affected in hyperglycemic conditions or after exposure to cytokines alone, but were significantly decreased in the DR model. This confirmed that pro-inflammatory cytokines IL-1ß and TNFα enhance the hyperglycemic metabolic deficit. We further investigated metabolite and amino acid levels after specific pharmacological inhibition of GAPDH or GS in the absence/presence of pro-inflammatory cytokines. The results indicate that GAPDH inhibition increased glucose and addition of cytokines increased lactate and ATP levels and reduced glutamate levels. GS inhibition did not alter retinal metabolite levels but the addition of cytokines increased ATP levels and caused glutamate accumulation in Müller cells. We conclude that it is the action of pro-inflammatory cytokines concomitantly with the inhibition of the glycolytic or GS mediated glutamate recycling that contribute to metabolic dysregulation in DR. Therefore, in the absence of good glycemic control, therapeutic interventions aimed at regulating inflammation may prevent the onset of early metabolic imbalance in DR.

Glyceraldehyde-3-Phosphate Dehydrogenase Facilitates Macroautophagic Degradation of Mutant Huntingtin Protein Aggregates.

Protein aggregate accumulation is a pathological hallmark of several neurodegenerative disorders. Autophagy is critical for clearance of aggregate-prone proteins. In this study, we identify a novel role of the multifunctional glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in clearance of intracellular protein aggregates. Previously, it has been reported that though clearance of wild-type huntingtin protein is mediated by chaperone-mediated autophagy (CMA), however, degradation of mutant huntingtin (mHtt with numerous poly Q repeats) remains impaired by this route as mutant Htt binds with high affinity to Hsc70 and LAMP-2A. This delays delivery of misfolded protein to lysosomes and results in accumulation of intracellular aggregates which are degraded only by macroautophagy. Earlier investigations also suggest that mHtt causes inactivation of mTOR signaling, causing upregulation of autophagy. GAPDH had earlier been reported to interact with mHtt resulting in cellular toxicity. Utilizing a cell culture model of mHtt aggregates coupled with modulation of GAPDH expression, we analyzed the formation of intracellular aggregates and correlated this with autophagy induction. We observed that GAPDH knockdown cells transfected with N-terminal mutant huntingtin (103 poly Q residues) aggregate-prone protein exhibit diminished autophagy. GAPDH was found to regulate autophagy via the mTOR pathway. Significantly more and larger-sized huntingtin protein aggregates were observed in GAPDH knockdown cells compared to empty vector-transfected control cells. This correlated with the observed decrease in autophagy. Overexpression of GAPDH had a protective effect on cells resulting in a decreased load of aggregates. Our results demonstrate that GAPDH assists in the clearance of protein aggregates by autophagy induction. These findings provide a new insight in understanding the mechanism of mutant huntingtin aggregate clearance. By studying the molecular mechanism of protein aggregate clearance via GAPDH, we hope to provide a new approach in targeting and understanding several neurodegenerative disorders.

Tyr-Asp inhibition of glyceraldehyde 3-phosphate dehydrogenase affects plant redox metabolism.

How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr-Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr-Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr-Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched-chain amino acid-containing dipeptides, but not by Tyr-Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small-molecule regulators at the nexus of stress, protein degradation, and metabolism.

Antitumor Activity of Asperphenin B by Induction of Apoptosis and Regulation of Glyceraldehyde-3-phosphate Dehydrogenase in Human Colorectal Cancer Cells.

Colorectal cancer (CRC) is a common and intractable malignancy with a high mortality risk. Conventional chemotherapeutics are effective for patients with early stage CRC, but the majority of deaths of CRC patients are linked to acquired drug resistance or metastasis occurrence. Asperphenin B (1), a lipopeptidyl benzophenone isolated from a marine-derived Aspergillus sp. fungus, reportedly possesses antiproliferative activity against cancer cells. However, its antitumor activity and the underlying molecular mechanisms remain unexplored. In this study, 1 induced G2/M phase cell cycle arrest and subsequent apoptotic cell death and inhibited tumor growth in a xenograft model. The 1-induced G2/M phase arrest was associated with the regulation of checkpoint proteins, including Chk1/2 and Cdc25c. The 1-induced apoptosis was correlated with an upregulation of p53 and cleaved caspases and a downregulation of survivin. Further experiments revealed that 1-mediated suppression of migration and invasion of metastatic HCT116 cells was partially associated with the downregulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. The antimetastatic potential of 1 was also confirmed by E-cadherin upregulation and N-cadherin and Snail downregulation, which were in turn associated with the GAPDH regulation. These findings highlight the potential use of 1 as a novel candidate for treating metastatic CRC with the modulation of GAPDH function.

Molecular Docking, QSAR and Microscopic Studies of Anti-trypanosomal Compounds from the Pathogen Box.

BACKGROUND: Trypanosoma brucei (T. brucei) is the cause of the deadly human African trypanosomiasis (HAT) with a case fatality ratio of 10%. OBJECTIVE: Targeting the essential Trypanosomal glucose metabolism pathway through the inhibition of phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a valid strategy for anti-T. brucei drug development. METHODS: Here, quantitative structure activity relationship, molecular docking and microscopic studies were used to describe the mode of inhibition of selected compounds from the pathogen box PGK and GAPDH. RESULTS: We identified 4 hit compounds from the pathogen box with optimal binding and chemical interactions. Notably, it was identified that interacting charge surface and atomic mass were key aspects of both PGK and GAPDH inhibition. Also, novel anti-trypanosomal compounds were identified from the pathogen box and their half maximal inhibitory concentrations were described. CONCLUSION: Our study presents new anti-trypanosomal compounds with optimal pharmacological profiles and an optimization strategy for improving target specificity in the rational design of novel anti-trypanosomal compounds.

Covalent inhibitors of GAPDH: From unspecific warheads to selective compounds.

Targeting glycolysis is an attractive approach for the treatment of a wide range of pathologies, such as various tumors and parasitic infections. Due to its pivotal role in the glycolysis, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) represents a rate-limiting enzyme in those cells that mostly, or exclusively rely on this pathway for energy production. In this context, GAPDH inhibition can be a valuable approach for the development of anticancer and antiparasitic drugs. In addition to its glycolytic role, GAPDH possesses several moonlight functions, whose deregulation is involved in some pathological conditions. Covalent modification on different amino acids of GAPDH, in particular on cysteine residues, can lead to a modulation of the enzyme activity. The selectivity towards specific cysteine residues is essential to achieve a specific phenotypic effect. In this work we report an extensive overview of the latest advances on the numerous compounds able to inhibit GAPDH through the covalent binding to cysteine residues, ranging from endogenous metabolites and xenobiotics, which may serve as pharmacological tools to actual drug-like compounds with promising therapeutic perspectives. Furthermore, we focused on the potentialities of the different warheads, shedding light on the possibility to exploit a combination of a finely tuned electrophilic group with a well-designed recognition moiety. These findings can provide useful information for the rational design of novel covalent inhibitors of GAPDH, with the final goal to expand the current treatment options.

Discovery of novel glyceraldehyde-3-phosphate dehydrogenase inhibitor via docking-based virtual screening.

Glycolysis is enhanced in cancer cells. Cancer cells utilize glycolysis as their primary energy source, even under aerobic conditions. This is known as the Warburg effect. Thus, effective inhibition of the glycolytic pathway is a crucial component of cancer therapy. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an important enzyme in glycolysis and overexpresses in cancers. Therefore, targeting GAPDH to inhibit its role in glycolysis is important for GAPDH functional studies and the treatment of cancers. However, only a few GAPDH inhibitors have been reported. In our current study, we identified a GAPDH inhibitor, DC-5163, using docking-based virtual screening and biochemical and biophysical analysis. DC-5163 is a small molecule compound that inhibits GAPDH enzyme activity and cancer cell proliferation (normal cells were tolerant to it). It can inhibit glycolysis pathway partially, which was manifested by decreased glucose uptake and lactic acid production. And it also leaded to cell death through apoptotic pathways. This study reflects the pivotal role of GAPDH in cancer cells and demonstrates that DC-5163 is a useful inhibitor and can be of value in studying the role of GAPDH and the development of new clinical cancer treatments.

Structures of glyceraldehyde 3-phosphate dehydrogenase in Neisseria gonorrhoeae and Chlamydia trachomatis.

Neisseria gonorrhoeae (Ng) and Chlamydia trachomatis (Ct) are the most commonly reported sexually transmitted bacteria worldwide and usually present as co-infections. Increasing resistance of Ng to currently recommended dual therapy of azithromycin and ceftriaxone presents therapeutic challenges for syndromic management of Ng-Ct co-infections. Development of a safe, effective, and inexpensive dual therapy for Ng-Ct co-infections is an effective strategy for the global control and prevention of these two most prevalent bacterial sexually transmitted infections. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a validated drug target with two approved drugs for indications other than antibacterials. Nonetheless, any new drugs targeting GAPDH in Ng and Ct must be specific inhibitors of bacterial GAPDH that do not inhibit human GAPDH, and structural information of Ng and Ct GAPDH will aid in finding such selective inhibitors. Here, we report the X-ray crystal structures of Ng and Ct GAPDH. Analysis of the structures demonstrates significant differences in amino acid residues in the active sites of human GAPDH from those of the two bacterial enzymes suggesting design of compounds to selectively inhibit Ng and Ct is possible. We also describe an efficient in vitro assay of recombinant GAPDH enzyme activity amenable to high-throughput drug screening to aid in identifying inhibitory compounds and begin to address selectivity.

Design of Inhibitors for Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Enzyme of Leishmania mexicana.

BACKGROUND: Leishmaniosis is a neglected tropical disease and glyceraldehyde 3- phosphate dehydrogenase (GAPDH) is a key enzyme in the design of new drugs to fight this disease. OBJECTIVE: The present study aimed to evaluate potential inhibitors of GAPDH enzyme found in Leishmania mexicana (L. mexicana). METHODS: A search for novel antileishmanial molecules was carried out based on similarities from the pharmacophoric point of view related to the binding site of the crystallographic enzyme using the ZINCPharmer server. The molecules selected in this screening were subjected to molecular docking and molecular dynamics simulations. RESULTS: Consensual analysis of the docking energy values was performed, resulting in the selection of ten compounds. These ligand-receptor complexes were visually inspected in order to analyze the main interactions and subjected to toxicophoric evaluation, culminating in the selection of three compounds, which were subsequently submitted to molecular dynamics simulations. The docking results showed that the selected compounds interacted with GAPDH from L. mexicana, especially by hydrogen bonds with Cys166, Arg249, His194, Thr167, and Thr226. From the results obtained from molecular dynamics, it was observed that one of the loop regions, corresponding to the residues 195-222, can be related to the fitting of the substrate at the binding site, assisting in the positioning and the molecular recognition via residues responsible for the catalytic activity. CONCLUSION: The use of molecular modeling techniques enabled the identification of promising compounds as inhibitors of the GAPDH enzyme from L. mexicana, and the results obtained here can serve as a starting point to design new and more effective compounds than those currently available.

Inhibitors of Glyceraldehyde 3-Phosphate Dehydrogenase and Unexpected Effects of Its Reduced Activity.

The review describes the use of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) inhibitors to study the enzyme and to suppress its activity in various cell types. The main problem of selective GAPDH inhibition is a highly conserved nature of the enzyme active site and, especially, Cys150 environment important for the catalytic action of cysteine sulfhydryl group. Numerous attempts to find specific inhibitors of sperm GAPDH and enzymes from Trypanosoma sp. and Mycobacterium tuberculosis that would not inhibit GAPDH of somatic mammalian cells have failed, which has pushed researchers to search for new ways to solve this problem. The sections of the review are devoted to the studies of GAPDH inactivation by reactive oxygen species, glutathione, and glycating agents. The final section discusses possible effects of GAPDH inhibition and inactivation on glycolysis and related metabolic pathways (pentose phosphate pathway, uncoupling of the glycolytic oxidation and phosphorylation, etc.).

Steady-state kinetics of the tungsten containing aldehyde: ferredoxin oxidoreductases from the hyperthermophilic archaeon Pyrococcus furiosus.

The tungsten containing Aldehyde:ferredoxin oxidoreductases (AOR) offer interesting opportunities for biocatalytic approaches towards aldehyde oxidation and carboxylic acid reduction. The hyperthermophilic archaeon Pyrococcus furiosus encodes five different AOR family members: glyceraldehyde-3-phosphate oxidoreductase (GAPOR), aldehyde oxidoreductase (AOR), and formaldehyde oxidoreductase (FOR), WOR4 and WOR5. GAPOR functions as a glycolytic enzyme and is highly specific for the substrate glyceraldehyde-3-phosphate (GAP). AOR, FOR and WOR5 have a broad substrate spectrum, and for WOR4 no substrate has been identified to date. As ambiguous kinetic parameters have been reported for different AOR family enzymes the steady state kinetics under different physiologically relevant conditions was explored. The GAPOR substrate GAP was found to degrade at 60 °C by non-enzymatic elimination of the phosphate group to methylglyoxal with a half-life t1/2 = 6.5 min. Methylglyoxal is not a substrate or inhibitor of GAPOR. D-GAP was identified as the only substrate oxidized by GAPOR, and the kinetics of the enzyme was unaffected by the presence of L-GAP, which makes GAPOR the first enantioselective enzyme of the AOR family. The steady-state kinetics of GAPOR showed partial substrate inhibition, which assumes the GAP inhibited form of the enzyme retains some activity. This inhibition was found to be alleviated completely by a 1 M NaCl resulting in increased enzyme activity at high substrate concentrations. GAPOR activity was strongly pH dependent, with the optimum at pH 9. At pH 9, the substrate is a divalent anion and, therefore, positively charged amino acid residues are likely to be involved in the binding of the substrate. FOR exhibited a significant primary kinetic isotope effect of the apparent Vmax for the deuterated substrate, formaldehyde-d2, which shows that the rate-determining step involves a CH bond break from the aldehyde. The implications of these results for the reaction mechanism of tungsten-containing AORs, are discussed.

Interaction between Silver Nanoparticles and Two Dehydrogenases: Role of Thiol Groups.

Widely used silver nanoparticles (AgNPs) are readily accessible to biological fluids and then surrounded by proteins. However, interactions between AgNPs and proteins are poorly understood. Two dehydrogenases, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and malate dehydrogenase (MDH), are chosen to investigate these interactions. Ag bound to thiol groups of these enzymes significantly decreases the number of free thiols available. Dose-dependent inhibition of enzyme activities is observed in both AgNPs and Ag+ treatments. Based on the concentration required to inhibit 50% activity, GAPDH and MDH are 24-30 fold more sensitive to Ag+ than to AgNPs suggesting that the measured 4.2% Ag+ containing AgNPs can be responsible for the enzymes inhibition. GAPDH, with a thiol group in its active site, is more sensitive to Ag than MDH, displaying many thiol groups but none in its active site, suggesting that thiol groups at the active site strongly determines the sensitivity of enzymes toward AgNPs. In contrast, the dramatic changes of circular dichroism spectra show that the global secondary structure of MDH under AgNPs treatment is more altered than that of GAPDH. In summary, this study shows that the thiol groups and their location on these dehydrogenases are crucial for the AgNPs effects.

Moonlighting Protein Glyceraldehyde-3-Phosphate Dehydrogenase: A Cellular Rapid-Response Molecule for Maintenance of Iron Homeostasis in Hypoxia.

BACKGROUND/AIMS: Hypoxia triggers a rapid increase in iron demand to meet the requirements of enhanced erythropoiesis. The mobilization of iron stores from macrophage to plasma as holo-transferrin (Tf) from where it is accessible to erythroid precursor cells impacts iron homeostasis. Despite the immediate need for enhanced iron uptake by bone marrow cells, numerous studies have shown that transferrin receptor levels do not rise until more than 24 hours after the onset of hypoxia, suggesting the existence of heretofore unknown rapid response cellular machinery for iron acquisition in the early stages of cellular hypoxia. METHODS: We performed flow cytometry to measure cell surface levels of TfR1, GAPDH, and Tf binding after hypoxia treatment. We utilized FRET analysis and co-immunoprecipitation methods to establish the interaction between Tf and GAPDH. RESULTS: In the current study, we demonstrated that hypoxia induces K562 cells to translocate the cytosolic moonlighting protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) onto cell surfaces and into the extracellular milieu to acquire transferrin-bound iron, even while levels of the classical transferrin receptor TfR1 (CD71) remain suppressed. GAPDH knockdown confirmed this protein's role in transferrin acquisition. Interestingly, macrophages did not show enhanced levels of extracellular GAPDH under hypoxia. CONCLUSION: Our results suggest the role of GAPDH-mediated Tf uptake as a rapid response mechanism by which cells acquire iron during the early stages of hypoxia. This is a tissue-specific phenomenon for the distinct requirements of cells that are consumers of iron versus cells that play a role in iron storage and recycling. This rapid deployment of an abundantly available multipurpose molecule allows hypoxic cells to internalize more Tf and maintain enhanced iron supplies in the early stages of hypoxia before specialized receptors can be synthesized and deployed to the cell membrane.

Identification of a gene cluster for biosynthesis of the sesquiterpene antibiotic, heptelidic acid, in Aspergillus oryzae.

Heptelidic acid (HA), a sesquiterpene lactone, is a known inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Recently, we found that HA was produced by Aspergillus oryzae RIB40 and acted as the growth inhibitor of the salt-tolerant lactic acid bacteria in soy sauce brewing. Although several decades have passed since the discovery of HA, the genes involved in its biosynthesis and biosynthetic pathway have not yet been fully identified. In this study, we identified the HA biosynthetic gene cluster (HA cluster) using gene disruption and expression analysis. We also revealed that two transcription regulatory genes adjacent to the HA cluster were responsible for the expression of HA biosynthetic genes in A. oryzae. Interestingly, the HA cluster contained a gene encoding GAPDH (gpdB), which showed much higher resistance to HA than the GAPDH gene (gpdA) located at the other locus, but which did not seem to act as a self-resistant gene.

A metabolic control analysis approach to introduce the study of systems in biochemistry: the glycolytic pathway in the red blood cell.

Metabolic control analysis (MCA) is a promising approach in biochemistry aimed at understanding processes in a quantitative fashion. Here the contribution of enzymes and transporters to the control of a given pathway flux and metabolite concentrations is determined and expressed quantitatively by means of numerical coefficients. Metabolic flux can be influenced by a wide variety of modulators acting on one or more metabolic steps along the pathway. We describe a laboratory exercise to study metabolic regulation of human erythrocytes (RBCs). Within the framework of MCA, students use these cells to determine the sensitivity of the glycolytic flux to two inhibitors (iodoacetic acid: IA, and iodoacetamide: IAA) known to act on the enzyme glyceraldehyde-3-phosphate-dehydrogenase. Glycolytic flux was estimated by determining the concentration of extracellular lactate, the end product of RBC glycolysis. A low-cost colorimetric assay was implemented, that takes advantage of the straightforward quantification of the absorbance signal from the photographic image of the multi-well plate taken with a standard digital camera. Students estimate flux response coefficients for each inhibitor by fitting an empirical function to the experimental data, followed by analytical derivation of this function. IA and IAA exhibit qualitatively different patterns, which are thoroughly analyzed in terms of the physicochemical properties influencing their action on the target enzyme. IA causes highest glycolytic flux inhibition at lower concentration than IAA. This work illustrates the feasibility of using the MCA approach to study key variables of a simple metabolic system, in the context of an upper level biochemistry course. © 2018 International Union of Biochemistry and Molecular Biology, 46(5):502-515, 2018.

Oxidatively modified glyceraldehyde-3-phosphate dehydrogenase in neurodegenerative processes and the role of low molecular weight compounds in counteracting its aggregation and nuclear translocation.

A number of independent studies have shown the contribution of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the pathogenesis of several neurodegenerative disorders. Indeed, GAPDH aggregates have been found in many post-mortem samples of brains of patients diagnosed with Alzheimer's and Parkinson disease. Currently, it is accepted that GAPDH-mediated cell death pathways in the neurodegenerative processes are associated with apoptosis caused by GAPDH nuclear translocation and excessive aggregation under oxidative stress conditions. Also the role of GAPDH in neurodegenerative diseases is linked to it directly binding to specific amyloidogenic proteins and petides such as ß-amyloid precursor protein, ß-amyloid peptide and tau protein in Alzheimer's disease, huntingtin in Huntington's disease and α-synuclein in Parkinson disease. One of the latest studies indicated that GAPDH aggregates significantly accelerate amyloidogenesis of the ß-amyloid peptide, which implies that aggregates of GAPDH may act as a specific aggregation "seed" in vitro. Previous detailed studies revealed that the active-site cysteine (Cys152) of GAPDH plays an essential role in the oxidative stress-induced aggregation of GAPDH associated with cell death. Furthermore, oxidative modification of this cysteine residue initiates the translocation of the enzyme to the nucleus, subsequently leading to apoptosis. The crystallographic structure of GAPDH shows that the Cys152 residue is located close to the surface of the molecule in a hydrophilic environment, which means that it can react with low molecular weight compounds such as hydroxynonenal or piceatannol. Therefore, it is highly possible that GAPDH may serve as a target for small molecule compounds with the potential to slow down or prevent the progression of neurodegenerative disorders. Recently appearing new evidence has highlighted the significance of low molecular weight compounds in counteracting the oxidation of GAPDH and consequently its aggregation and other unfavourable pathological processes. Hence, this review aims to present all recent findings concerning molecules that are able to interact with GAPDH and counteract its aggregation and translocation to the nucleus.

Survival strategy of the salt-tolerant lactic acid bacterium, Tetragenococcus halophilus, to counteract koji mold, Aspergillus oryzae, in soy sauce brewing.

In soy sauce brewing, the results of the fermentation of lactic acid greatly affect the quality of soy sauce. The soy sauce moromi produced with Aspergillus oryzae RIB40 allows the growth of Tetragenococcus halophilus NBRC 12172 but not T. halophilus D10. We isolated and identified heptelidic acid (HA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), produced by A. oryzae RIB40 as the growth inhibitor of the salt-tolerant lactic acid bacteria. The growth inhibition of T. halophilus D10 by HA was suggested to be associated with the direct inhibition of GAPDH activity under high salt environment. The difference in the susceptibility to HA among various strains of T. halophilus was caused by the mutations in the gene encoding GAPDH.