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1.
Biochemistry (Mosc) ; 84(11): 1268-1279, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31760917

RESUMO

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.).


Assuntos
Inibidores Enzimáticos/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Antioxidantes/química , Antioxidantes/metabolismo , Inibidores Enzimáticos/metabolismo , Glutationa/química , Glutationa/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Glicosilação , Mycobacterium tuberculosis/enzimologia , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo , Trypanosoma/enzimologia
2.
Clin Nephrol ; 92(5): 263-272, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31496514

RESUMO

Infection-related glomerulonephritis (IRGN) was previously thought to be due mostly to Streptococcus species, but is now known to be caused by a variety of other pathogens. Nephritis-associated plasmin receptor (NAPlr) was originally isolated from group A streptococci as the protein responsible for acute poststreptococcal glomerulonephritis, and was shown to be identical to streptococcal glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Here, we describe a 7-year-old boy diagnosed with Mycoplasma pneumoniae IRGN presenting with acute nephritic syndrome. Laboratory data revealed a significant increase in serum anti-M. pneumoniae antibody titer. Renal biopsy revealed diffuse global endocapillary proliferation and cellular crescents in 5/43 glomeruli examined. Although antistreptolysin O antibody titer and serum complement C3 level were within the respective normal ranges, glomeruli showed positive staining for NAPlr and upregulation of plasmin activity. In addition, positive staining for NAPlr in the glomeruli was abolished by preabsorption of anti-NAPlr antibody with recombinant M. pneumoniae GAPDH. Western blotting analysis revealed anti-NAPlr antibody reactivity with a band at around the predicted size of GAPDH in the protein isolate of M. pneumoniae (37 kDa). Furthermore, immobilized M. pneumoniae GAPDH bound to anti-NAPlr antibody as well as plasmin in vitro. These data suggest that M. pneumoniae GAPDH has a function similar to streptococcal GAPDH (NAPlr) and may induce plasmin-related glomerular damage in M. pneumoniae IRGN. NAPlr could be a marker of glomerulonephritis related to infection not only by streptococci but also by &M. pneumoniae.


Assuntos
Antígenos de Bactérias , Proteínas de Bactérias , Glomerulonefrite/microbiologia , Gliceraldeído-3-Fosfato Desidrogenases , Infecções por Mycoplasma/microbiologia , Mycoplasma pneumoniae , Doença Aguda , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Criança , Gliceraldeído-3-Fosfato Desidrogenases/imunologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Masculino , Mycoplasma pneumoniae/enzimologia , Mycoplasma pneumoniae/imunologia
3.
Mol Cells ; 42(8): 597-603, 2019 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-31387164

RESUMO

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a core enzyme of the aerobic glycolytic pathway with versatile functions and is associated with cancer development. Recently, Kornberg et al . published the detailed correlation between GAPDH and di- or monomethyl fumarate (DMF or MMF), which are well-known GAPDH antagonists in the immune system. As an extension, herein, we report the crystal structure of MMF-bound human GAPDH at 2.29 Å. The MMF molecule is covalently linked to the catalytic Cys152 of human GAPDH, and inhibits the catalytic activity of the residue and dramatically reduces the enzymatic activity of GAPDH. Structural comparisons between NAD+bound GAPDH and MMF-bound GAPDH revealed that the covalently linked MMF can block the binding of the NAD+ cosubstrate due to steric hindrance of the nicotinamide portion of the NAD+ molecule, illuminating the specific mechanism by which MMF inhibits GAPDH. Our data provide insights into GAPDH antagonist development for GAPDH-mediated disease treatment.


Assuntos
Fumaratos/química , Gliceraldeído-3-Fosfato Desidrogenases/química , Maleatos/química , Domínio Catalítico , Proliferação de Células/efeitos dos fármacos , Fumaratos/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Humanos , Maleatos/farmacologia , Ligação Proteica , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos
4.
BMC Med Genet ; 20(1): 138, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409279

RESUMO

BACKGROUND: Reference genes are often interchangeably called housekeeping genes due to 1) the essential cellular functions their proteins provide and 2) their constitutive expression across a range of normal and pathophysiological conditions. However, given the proliferative drive of malignant cells, many reference genes such as beta-actin (ACTB) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) which play critical roles in cell membrane organization and glycolysis, may be dysregulated in tumors versus their corresponding normal controls METHODS: Because Next Generation Sequencing (NGS) technology has several advantages over hybridization-based technologies, such as independent detection and quantitation of transcription levels, greater sensitivity, and increased dynamic range, we evaluated colorectal cancers (CRC) and their histologically normal tissue counterparts by NGS to evaluate the expression of 21 "classical" reference genes used as normalization standards for PCR based methods. Seventy-nine paired tissue samples of CRC and their patient matched healthy colonic tissues were subjected to NGS analysis of their mRNAs. RESULTS: We affirmed that 17 out of 21 classical reference genes had upregulated expression in tumors compared to normal colonic epithelial tissue and dramatically so in some cases. Indeed, tumors were distinguished from normal controls in both unsupervised hierarchical clustering analyses (HCA) and principal component analyses (PCA). We then identified 42 novel potential reference genes with minimal coefficients of variation (CV) across 79 CRC tumor pairs. Though largely consistently expressed across tumors and normal control tissues, a subset of high stage tumors (HSTs) as well as some normal tissue samples (HSNs) located adjacent to these HSTs demonstrated dysregulated expression, thus identifying a subset of tumors with a potentially distinct and aggressive biological profile. CONCLUSION: While classical CRC reference genes were found to be differentially expressed between tumors and normal controls, novel reference genes, identified via NGS, were more consistently expressed across malignant and normal colonic tissues. Nonetheless, a subset of HST had profound dysregulation of such genes as did many of the histologically normal tissues adjacent to such HSTs, indicating that the HSTs so distinguished may have unique biological properties and that their histologically normal tissues likely harbor a small population of microscopically undetected but metabolically active tumors.


Assuntos
Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica/genética , Estudos de Associação Genética , Predisposição Genética para Doença/genética , Actinas/genética , Actinas/metabolismo , Biomarcadores Tumorais/genética , Colo/patologia , Neoplasias Colorretais/patologia , Feminino , Perfilação da Expressão Gênica , Genes Essenciais/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , RNA Mensageiro , Análise de Sequência de RNA
5.
Int J Radiat Biol ; 95(11): 1472-1483, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31290706

RESUMO

Purpose: This work investigates the effect of resveratrol and melatonin on structural and functional changes of two enzymes, lactate dehydrogenase (LDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), exposed to radiation-induced reactive oxygen species.Materials and methods: Solutions of dehydrogenases with or without antioxidants (resveratrol or melatonin) were irradiated with X-rays under the atmosphere of air and at room temperature (21 ± 2 °C). In order to determine the protective effect of melatonin and resveratrol in radiation-induced damage to GAPDH and LDH spectroscopy and HPLC methods were used. Furthermore, plausible binding sites of melatonin or resveratrol to the GAPDH or LDH molecule were analysed.Results and conclusions: Resveratrol shows better protective properties in the inactivation of GAPDH when compared to melatonin. LDH does not contain ‒SH groups in its active site, and is not inactivated by water radiolysis products other than hydroxyl radicals or the secondary radicals of the studied low-molecular-weight compounds. Resveratrol and melatonin protected the structure of LDH to a greater extent than GAPDH. This difference can be attributed to the fact that LDH potentially binds more resveratrol or melatonin molecules (27 binding sites for resveratrol and 40 for melatonin) than GAPDH (10 binding sites for resveratrol and 18 for melatonin).


Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , L-Lactato Desidrogenase/metabolismo , Melatonina/administração & dosagem , Proteção Radiológica/métodos , Resveratrol/administração & dosagem , Ar , Animais , Antioxidantes/química , Domínio Catalítico , Cisteína/química , Relação Dose-Resposta à Radiação , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Coelhos , Espécies Reativas de Oxigênio , Temperatura Ambiente , Raios X
6.
Mol Med Rep ; 20(3): 2227-2235, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31322210

RESUMO

Elevated plasma homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), is an independent risk factor for neurodegenerative diseases. Hcy, even at a low concentration, can promote free radical formation and increase oxidative stress, leading to neuronal death, which may be an important mechanism underlying the pathogenesis of neurodegenerative diseases. Although several reports have indicated that the nuclear translocation of glyceraldehyde 3­phosphate dehydrogenase (GAPDH) may be involved in Hcy­induced apoptosis, the exact mechanism remains to be fully elucidated. The siah E3 ubiquitin protein ligase 1 (siah­1) gene was found to be critical for the translocation of GAPDH from the cytoplasm to the nucleus. In the present study, the role of siah­1 was investigated in the nuclear translocation of GAPDH in rat C6 astroglioma cells treated with Hcy. C6 cells were treated with various concentrations of Hcy for 48 h and the expression level of siah­1 was examined using reverse transcription­quantitative polymerase chain reaction and western blotting analysis. In addition, the subcellular localization of siah­1 and GAPDH and the interaction between these two factors were investigated by immunofluorescence staining and co­immunoprecipitation assay, respectively. The results showed that Hcy at a high concentration increased the expression of siah­1 and induced nuclear translocation of siah­1 and GAPDH. In addition, siah­1 knockdown by siah­1 small interfering RNA significantly decreased the Hcy­induced nuclear accumulation of GAPDH and inhibited the impairment of C6 cells. These findings suggest that siah­1 is involved in Hcy­induced cell damage by promoting the nuclear translocation of GAPDH.


Assuntos
Astrocitoma/metabolismo , Núcleo Celular/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Homocisteína/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Astrocitoma/patologia , Linhagem Celular Tumoral , Ratos
7.
N Biotechnol ; 53: 24-34, 2019 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-31195158

RESUMO

The increasing demand for recombinant proteins for a wide range of applications, from biopharmaceutical protein complexes to industrial enzymes, is leading to important growth in this market. Among the different efficient host organism alternatives commonly used for protein production, the yeast Pichia pastoris (Komagataella phaffii) is currently considered to be one of the most effective and versatile expression platforms. The promising features of this cell factory are giving rise to interesting studies covering the different aspects that contribute to improving the bioprocess efficiency, from strain engineering to bioprocess engineering. The numerous drawbacks of using methanol in industrial processes are driving interest towards methanol-free alternatives, among which the GAP promoter-based systems stand out. The aim of this work is to present the most promising innovative developments in operational strategies based on rational approaches through bioprocess engineering tools. This rational design should be based on physiological characterization of the producing strains under bioprocess conditions and its interrelation with specific rates. This review focuses on understanding the key factors that can enhance recombinant protein production in Pichia pastoris; they are the basis for a further discussion on future industrial applications with the aim of developing scalable alternative strategies that maximize yields and productivity.


Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Engenharia Metabólica , Pichia/metabolismo , Pichia/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química
8.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067671

RESUMO

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.


Assuntos
Frutose-Bifosfatase/farmacologia , Hipocampo/efeitos dos fármacos , N-Metilaspartato/toxicidade , Fármacos Neuroprotetores/farmacologia , Animais , Metabolismo Energético , Frutose-Bifosfato Aldolase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Necrose , Fosfofrutoquinases/metabolismo , Nucleosídeos de Purina/metabolismo , Ratos , Ratos Wistar
9.
J Biotechnol ; 300: 20-31, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31095980

RESUMO

Increasing levels of antibiotic resistance in pathogens, including Staphylococcus aureus, remains a serious problem for public health, leading to the need for better alternative antimicrobial strategies. The antimicrobial proteins produced by Lactobacillus plantarum USM8613 attributed to its anti-staphylococcal activity were identified as extracellular transglycosylase and glyceraldehyde-3-phosphate dehydrogenase (GADPH), both with different mechanisms of action. Extracellular transglycosylase, which contains a LysM domain, exerts a cell wall-mediated killing mechanism, while GADPH penetrates into S. aureus cells and subsequently induces the overexpression of autolysis regulators, resulting in S. aureus autolysis. Both extracellular transglycosylase and GADPH exert anti-inflammatory effects in S. aureus-infected HaCaT cells by reducing the expression and production of TLR-2, hBDs and various pro-inflammatory cytokines (IL-1α, IL-1ß, IL-6, TNF-α, and IL-8). Taken together, extracellular transglycosylase and GADPH produced by L. plantarum USM8613 could potentially be applied as an alternative therapeutic agent to treat S. aureus skin infections and promote skin health.


Assuntos
Antibacterianos/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/farmacologia , Glicosiltransferases/farmacologia , Lactobacillus plantarum/enzimologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Linhagem Celular , Citocinas/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/isolamento & purificação , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicosiltransferases/química , Glicosiltransferases/isolamento & purificação , Glicosiltransferases/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia
10.
Int J Mol Sci ; 20(9)2019 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-31027346

RESUMO

Several studies indicate that the cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has pleiotropic functions independent of its canonical role in glycolysis. The GAPDH functional diversity is mainly due to post-translational modifications in different amino acid residues or due to protein-protein interactions altering its localization from cytosol to nucleus, mitochondria or extracellular microenvironment. Non-glycolytic functions of GAPDH include the regulation of cell death, autophagy, DNA repair and RNA export, and they are observed in physiological and pathological conditions as cancer and neurodegenerative disorders. In disease, the knowledge of the mechanisms regarding GAPDH-mediated cell death is becoming fundamental for the identification of novel therapies. Here, we elucidate the correlation between autophagy and GAPDH in cancer, describing the molecular mechanisms involved and its impact in cancer development. Since autophagy is a degradative pathway associated with the regulation of cell death, we discuss recent evidence supporting GAPDH as a therapeutic target for autophagy regulation in cancer therapy. Furthermore, we summarize the molecular mechanisms and the cellular effects of GAPDH aggregates, which are correlated with mitochondrial malfunctions and can be considered a potential therapeutic target for various diseases, including cancer and neurodegenerative disorders.


Assuntos
Autofagia/fisiologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Humanos , Modelos Biológicos
11.
Microb Cell Fact ; 18(1): 65, 2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30943966

RESUMO

BACKGROUND: NAD(H/+) and NADP(H/+) are the most important redox cofactors in bacteria. However, the intracellular redox balance is in advantage of the cell growth and production of NAD(P)H-dependent products. RESULTS: In this paper, we rationally engineered glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and isocitrate dehydrogenase (IDH) to switch the nucleotide-cofactor specificity resulting in an increase in final titer [from 85.6 to 121.4 g L-1] and carbon yield [from 0.33 to 0.46 g (g glucose)-1] of L-lysine in strain RGI in fed-batch fermentation. To do this, we firstly analyzed the production performance of original strain JL-6, indicating that the imbalance of intracellular redox was the limiting factor for L-lysine production. Subsequently, we modified the native GAPDH and indicated that recombinant strain RG with nonnative NADP-GAPDH dramatically changed the intracellular levels of NADH and NADPH. However, L-lysine production did not significantly increase because cell growth was harmed at low NADH level. Lastly, the nonnative NAD-IDH was introduced in strain RG to increase the NADH availability and to equilibrate the intracellular redox. The resulted strain RGI showed the stable ratio of NADPH/NADH at about 1.00, which in turn improved cell growth (µmax. = 0.31 h-1) and L-lysine productivity (qLys, max. = 0.53 g g-1 h-1) as compared with strain RG (µmax. = 0.14 h-1 and qLys, max. = 0.42 g g-1 h-1). CONCLUSIONS: This is the first report of balancing the intracellular redox state by switching the nucleotide-cofactor specificity of GAPDH and IDH, thereby improving cell growth and L-lysine production.


Assuntos
Coenzimas/metabolismo , Corynebacterium glutamicum/crescimento & desenvolvimento , Corynebacterium glutamicum/metabolismo , Lisina/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/química , Corynebacterium glutamicum/genética , Fermentação , Glucose/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Cinética , Engenharia Metabólica , NAD/metabolismo , NADP/metabolismo , Oxirredução
12.
Cell Physiol Biochem ; 52(3): 517-531, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30897319

RESUMO

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.


Assuntos
Hipóxia Celular , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Ferro/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Células K562 , Macrófagos/citologia , Macrófagos/metabolismo , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Transferrina/metabolismo
13.
Parasitol Res ; 118(3): 861-872, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30706165

RESUMO

Fasciola gigantica is an important food-borne trematode responsible for the hepatobiliary disease, commonly known as fascioliasis. In F. gigantica, the glyceraldehyde 3-phosphate dehydrogenase (FgGAPDH) is a key enzyme of the glycolytic pathway and catalyzes the reversible oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G-3-P) to 1,3-bisphosphoglycerate (1,3-BPG), with the simultaneous reduction of NAD+ to NADH. In the present study, we analyzed the sequence of FgGAPDH and investigated its structural, binding, and catalytic properties. Sequence alignment of FgGAPDH showed 100% identity with the sister fluke Fasciola hepatica GAPDH. The gapdh gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified. The purified FgGAPDH exists as a homo-tetramer, composed of a ~ 37-kDa subunit under non-dissociating conditions at 300 mM salt concentration indicating that higher salt stabilizes the tetrameric state. The binding of the cofactor NAD+ caused a conformational rearrangement in the enzyme structure, leading to the stabilization of the enzyme. A homology model of FgGAPDH was constructed, the cofactor (NAD+) and substrate (G-3-P) were docked, and the binding sites were identified in a single chain. The inter-subunit cleft of GAPDH that has been exploited for structure-based drug design in certain protozoan parasites is closed in the case of FgGAPDH, similar to the human GAPDH. Thus, the conformation of FgGAPDH in this region is similar to the human enzyme. Therefore, GAPDH may not be a suitable target for drug discovery against fascioliasis. Still, the analysis of the structural and functional attributes of GAPDH will be significant in understanding the various roles of this enzyme in the parasite as well as provide new insights into the biochemistry of flukes.


Assuntos
Fasciola/enzimologia , Fasciola/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fasciolíase/parasitologia , Humanos , Proteínas Recombinantes/genética , Alinhamento de Sequência
14.
Appl Microbiol Biotechnol ; 103(7): 3073-3083, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30734124

RESUMO

Microbial contamination of alcoholic fermentation processes (e.g. winemaking and fuel-ethanol production) is a serious problem for the industry since it may render the product unacceptable and/or reduce its productivity, leading to large economic losses. Brettanomyces/Dekkera bruxellensis is one of the most dangerous microbial contaminant of ethanol industrial fermentations. In the case of wine, this yeast species can produce phenolic compounds that confer off-flavours to the final product. In fuel-ethanol fermentations, D. bruxellensis is a persistent contaminant that affects ethanol yields and productivities. We recently found that Saccharomyces cerevisiae secretes a biocide, which we named saccharomycin, composed of antimicrobial peptides (AMPs) derived from the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Saccharomycin is active against several wine-related yeast species, namely D. bruxellensis. However, the levels of saccharomycin naturally secreted by S. cerevisiae during alcoholic fermentation are not sufficient to ensure the complete death of D. bruxellensis. Therefore, the aim of the present work was to construct genetically modified S. cerevisiae strains to overproduce these GAPDH-derived AMPs. The expression levels of the nucleotides sequences encoding the AMPs were evaluated in the modified S. cerevisiae strains by RT-qPCR, confirming the success of the recombinant approach. Furthermore, we confirmed by immunological tests that the modified S. cerevisiae strains secreted higher amounts of the AMPs by comparison with the non-modified strain, inducing total death of D. bruxellensis during alcoholic fermentations.


Assuntos
Agentes de Controle Biológico , Brettanomyces , Dekkera , Fermentação , Microbiologia de Alimentos , Saccharomyces cerevisiae/enzimologia , Etanol/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Microbiologia Industrial , Microrganismos Geneticamente Modificados , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vinho/microbiologia
15.
Int J Biol Macromol ; 129: 471-476, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30763643

RESUMO

In this study, chondroitinase (ChSase) AC II from Arthrobacter sp. CS01 was cloned, expressed in Escherichia coli BL21 (DE3), purified and characterised. To assist in protein folding and improve on high protein aggregation rates, two strategies involving chaperones and fusion tags were chosen to increase enzyme activity and improve enzymatic properties. ChSase AC II enzyme activity increased from 3.12 to 9.15 U/ml with chaperone GroEs-GroEL, and the specific activity increased from 19.8 to 25.74 U/mg with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) tag. ChSase AC II and GAPDH-ChSase AC II displayed maximum activities at 37 °C and 40 °C, at pH 6.5 and 7.0, respectively. GAPDH-ChSase AC II activity remained above 69.8% after incubation at 40 °C for 120 min, and ChSase AC II activity remained approximately 32.1% under the same conditions, indicating that ChSase AC II thermostability was enhanced by the GAPDH tag. These properties suggested that the enzymes are promising prospects in medical and industrial applications.


Assuntos
Arthrobacter/enzimologia , Chaperonina 60/metabolismo , Condroitina Liases/genética , Condroitina Liases/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Arthrobacter/genética , Clonagem Molecular , Estabilidade Enzimática , Expressão Gênica , Concentração de Íons de Hidrogênio , Metais/farmacologia , Tensoativos/farmacologia , Temperatura Ambiente
16.
Int J Biol Macromol ; 127: 278-285, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30658140

RESUMO

α-Synuclein was recently found to interact with moonlighting glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) involved in neurodegenerative diseases development. In the present work, we have analyzed influence of α-synuclein glycation on this interaction, because the literature data suggest relation between diabetes and Parkinson's disease. According to zeta potential measurement, glycation can shift the charge of α-synuclein to more negative values that was pronounced in case of modification by glyceraldehyde-3-phosphate. We selected carboxymethyl lysine as a typical advanced glycation end product and performed molecular dynamics simulations. The binding was found to be electrostatically driven and was significantly amplified after α-synuclein glycation because of increase the number of acidic residues. Since the main binding site was located in the anion-binding groove, which comprises the active site of GAPDH, enhanced binding of α-synuclein can result in GAPDH inactivation. This hypothesis was proven experimentally. Glycation of α-synuclein resulted in increase of GAPDH inactivation, and this effect was more pronounced in case of modification by glyceraldehyde-3-phosphate. The obtained results can reflect the probable relations between protein glycation and neurodegenerative diseases.


Assuntos
Produtos Finais de Glicação Avançada , Gliceraldeído 3-Fosfato , Gliceraldeído-3-Fosfato Desidrogenases , Simulação de Dinâmica Molecular , Doenças Neurodegenerativas/metabolismo , alfa-Sinucleína , Domínio Catalítico , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/metabolismo , Gliceraldeído 3-Fosfato/química , Gliceraldeído 3-Fosfato/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Glicosilação , Humanos , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo
17.
FEBS Open Bio ; 9(1): 53-73, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30652074

RESUMO

Protein CoAlation (S-thiolation by coenzyme A) has recently emerged as an alternative redox-regulated post-translational modification by which protein thiols are covalently modified with coenzyme A (CoA). However, little is known about the role and mechanism of this post-translational modification. In the present study, we investigated CoAlation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from a facultative anaerobic Gram-negative bacterium Citrobacter sp. S-77 (Cb GAPDH). GAPDH is a key glycolytic enzyme whose activity relies on the thiol-based redox-regulated post-translational modifications of active-site cysteine. LC-MS/MS analysis revealed that CoAlation of Cb GAPDH occurred in vivo under sodium hypochlorite (NaOCl) stress. The purified Cb GAPDH was highly sensitive to overoxidation by H2O2 and NaOCl, which resulted in irreversible enzyme inactivation. By contrast, treatment with coenzyme A disulphide (CoASSCoA) or H2O2/NaOCl in the presence of CoA led to CoAlation and inactivation of the enzyme; activity could be recovered after incubation with dithiothreitol, glutathione and CoA. CoAlation of the enzyme in vitro was confirmed by mass spectrometry. The presence of a substrate, glyceraldehyde-3-phosphate, fully protected Cb GAPDH from inactivation by CoAlation, suggesting that the inactivation is due to the formation of mixed disulphides between CoA and the active-site cysteine Cys149. A molecular docking study also supported the formation of mixed disulphide without steric constraints. These observations suggest that CoAlation is an alternative mechanism to protect the redox-sensitive thiol (Cys149) of Cb GAPDH against irreversible oxidation, thereby regulating enzyme activity under oxidative stress.


Assuntos
Citrobacter/enzimologia , Coenzima A/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Estresse Oxidativo , Processamento de Proteína Pós-Traducional
18.
Nat Commun ; 10(1): 338, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30659183

RESUMO

Macrophages undergo metabolic changes during activation that are coupled to functional responses. The gram negative bacterial product lipopolysaccharide (LPS) is especially potent at driving metabolic reprogramming, enhancing glycolysis and altering the Krebs cycle. Here we describe a role for the citrate-derived metabolite malonyl-CoA in the effect of LPS in macrophages. Malonylation of a wide variety of proteins occurs in response to LPS. We focused on one of these, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In resting macrophages, GAPDH binds to and suppresses translation of several inflammatory mRNAs, including that encoding TNFα. Upon LPS stimulation, GAPDH undergoes malonylation on lysine 213, leading to its dissociation from TNFα mRNA, promoting translation. We therefore identify for the first time malonylation as a signal, regulating GAPDH mRNA binding to promote inflammation.


Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Mediadores da Inflamação/farmacologia , Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Animais , Citocinas/metabolismo , Células HEK293 , Humanos , Lipopolissacarídeos/farmacologia , Lisina/metabolismo , Malonil Coenzima A/metabolismo , Camundongos Endogâmicos C57BL , Mutagênese , Polirribossomos , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
19.
Semin Cell Dev Biol ; 86: 162-173, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29574117

RESUMO

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays a key role in glycolysis but is also known for its involvement in a myriad of extra-glycolytic functions. While GAPDH is not the only enzyme with established moonlighting roles, it shows great diversity in terms of its functions, cellular localizations, protein partners, and post-translational modifications. This review focuses on GAPDH's role as a non-canonical RNA binding protein to regulate the stability and translation of cellular mRNAs. Despite the clear involvement of GAPDH in gene expression regulation, how and where GAPDH binds to its RNA targets is still unknown. In addition, the mechanism by which GAPDH switches among its various cellular functions is also unknown. This review will summarize our current understanding of GAPDH-mediated regulation of RNA function.


Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , RNA/genética , RNA/metabolismo , Sequência Rica em At , Gliceraldeído-3-Fosfato Desidrogenases/química , Humanos , Modelos Moleculares
20.
Curr Genet ; 65(1): 243-252, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30046843

RESUMO

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyses the sixth step of glycolysis, and is also known to perform other (moonlighting) activities in animal cells. We have earlier identified an additional GAPDH gene in Trichoderma virens genome. This gene is consistently associated with the vir cluster responsible for biosynthesis of a range of volatile sesquiterpenes in Trichoderma virens. This gene is also associated with an orthologous gene cluster in Aspergillus spp. Both glycolytic GAPDH and the vir cluster-associated GAPDH show more than 80% similarity with essentially conserved NAD+ cofactor- and substrate-binding sites. However, a conserved indel is consistently present only in GAPDH associated with the vir cluster, both in T. virens and Aspergillus spp. Using gene knockout, we demonstrate here that the vir cluster-associated GAPDH is involved in biosynthesis of volatile sesquiterpenes in T. virens. We thus, for the first time, elucidate the non-glycolytic role of a GAPDH in a fungal system, and also prove for the first time that a GAPDH, a primary metabolism protein, is involved in secondary metabolism.


Assuntos
Proteínas Fúngicas/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Mutação , Metabolismo Secundário/genética , Sesquiterpenos/metabolismo , Trichoderma/genética , Proteínas Fúngicas/classificação , Proteínas Fúngicas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Regulação Fúngica da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/classificação , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Família Multigênica/genética , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , Trichoderma/metabolismo , Compostos Orgânicos Voláteis/metabolismo
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