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1.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070501

RESUMO

Our goal was to analyze postmortem tissues of an adult patient with late-onset thymidine kinase 2 (TK2) deficiency who died of respiratory failure. Compared with control tissues, we found a low mtDNA content in the patient's skeletal muscle, liver, kidney, small intestine, and particularly in the diaphragm, whereas heart and brain tissue showed normal mtDNA levels. mtDNA deletions were present in skeletal muscle and diaphragm. All tissues showed a low content of OXPHOS subunits, and this was especially evident in diaphragm, which also exhibited an abnormal protein profile, expression of non-muscular ß-actin and loss of GAPDH and α-actin. MALDI-TOF/TOF mass spectrometry analysis demonstrated the loss of the enzyme fructose-bisphosphate aldolase, and enrichment for serum albumin in the patient's diaphragm tissue. The TK2-deficient patient's diaphragm showed a more profound loss of OXPHOS proteins, with lower levels of catalase, peroxiredoxin 6, cytosolic superoxide dismutase, p62 and the catalytic subunits of proteasome than diaphragms of ventilated controls. Strong overexpression of TK1 was observed in all tissues of the patient with diaphragm showing the highest levels. TK2 deficiency induces a more profound dysfunction of the diaphragm than of other tissues, which manifests as loss of OXPHOS and glycolytic proteins, sarcomeric components, antioxidants and overactivation of the TK1 salvage pathway that is not attributed to mechanical ventilation.


Assuntos
DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Diafragma/metabolismo , Mitocôndrias/metabolismo , Insuficiência Respiratória/metabolismo , Timidina Quinase/deficiência , Timidina Quinase/genética , Actinas/metabolismo , Adulto , Autopsia , Encéfalo/metabolismo , Catalase/metabolismo , Diafragma/enzimologia , Feminino , Frutose-Bifosfato Aldolase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Humanos , Intestino Delgado/metabolismo , Rim/metabolismo , Fígado/metabolismo , Espectrometria de Massas , Mitocôndrias/enzimologia , Mitocôndrias/genética , Músculo Esquelético/metabolismo , Fosforilação Oxidativa , Peroxirredoxina VI/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteoma/genética , Proteoma/metabolismo , Insuficiência Respiratória/genética , Insuficiência Respiratória/mortalidade , Superóxido Dismutase/metabolismo , Timidina Quinase/metabolismo , Regulação para Cima
2.
Enzyme Microb Technol ; 147: 109784, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33992412

RESUMO

Asymmetric CC bond formation catalyzed by aldolases requires the supplementation of nucleophiles and receptors in the reaction medium. However, aldol condensation using a single ketone as substrate has never been reported yet. In this work, we discovered that d-fructose-6-phosphate aldolase (FSA) could convert two 1-hydroxyalkanones, such as hydroxyacetone (HA) and 1-hydroxy-2-butanone, into two type of diketones. The initial product synthesis rate increased 3-fold and the yield reached to 56 %, when pure oxygen was directly inputted into the reaction medium. The results confirmed that oxygen participated in this reaction and hydrogen peroxide was generated. Metal ions Co2+ and Cu2+ remarkably increased the conversion yield compared with the control. For this reaction mechanism, we conjectured that HA may be oxidized to methylglyoxal by enzyme FSA in the presence of oxygen in the medium, and then FSA catalyzes the aldol addition between HA and its oxidative product MG to form diketone products. The obtained diketones could serve as important precursors for preparing furans and pyrroles.


Assuntos
Escherichia coli , Frutose-Bifosfato Aldolase , Aldeído Liases/metabolismo , Catálise , Escherichia coli/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Frutosefosfatos , Cetonas , Especificidade por Substrato
3.
Chem Commun (Camb) ; 57(43): 5306-5309, 2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-33912877

RESUMO

The retro-aldolase mechanism of methodol catalysed by the catalytic antibody 33F12 is described based on the exploration of the free energy landscape obtained with QM/MM methods. The amino acids involved in the reaction have been identified, as well as their specific role played in the active site and in the flexibility of the loops. Finally, the comparison with a de novo enzyme RA95.5-8F provides a deeper understanding of catalytic differences between such different protein scaffolds.


Assuntos
Anticorpos Catalíticos/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Teoria Quântica , Anticorpos Catalíticos/química , Biocatálise , Cristalografia por Raios X , Frutose-Bifosfato Aldolase/química , Metanol/química , Metanol/metabolismo , Simulação de Dinâmica Molecular
4.
J Biol Chem ; 296: 100699, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33895133

RESUMO

N-acetylneuraminate (Neu5Ac), an abundant sugar present in glycans in vertebrates and some bacteria, can be used as an energy source by several prokaryotes, including Escherichia coli. In solution, more than 99% of Neu5Ac is in cyclic form (≈92% beta-anomer and ≈7% alpha-anomer), whereas <0.5% is in the open form. The aldolase that initiates Neu5Ac metabolism in E. coli, NanA, has been reported to act on the alpha-anomer. Surprisingly, when we performed this reaction at pH 6 to minimize spontaneous anomerization, we found NanA and its human homolog NPL preferentially metabolize the open form of this substrate. We tested whether the E. coli Neu5Ac anomerase NanM could promote turnover, finding it stimulated the utilization of both beta and alpha-anomers by NanA in vitro. However, NanM is localized in the periplasmic space and cannot facilitate Neu5Ac metabolism by NanA in the cytoplasm in vivo. We discovered that YhcH, a cytoplasmic protein encoded by many Neu5Ac catabolic operons and belonging to a protein family of unknown function (DUF386), also facilitated Neu5Ac utilization by NanA and NPL and displayed Neu5Ac anomerase activity in vitro. YhcH contains Zn, and its accelerating effect on the aldolase reaction was inhibited by metal chelators. Remarkably, several transition metals accelerated Neu5Ac anomerization in the absence of enzyme. Experiments with E. coli mutants indicated that YhcH expression provides a selective advantage for growth on Neu5Ac. In conclusion, YhcH plays the unprecedented role of providing an aldolase with the preferred unstable open form of its substrate.


Assuntos
Frutose-Bifosfato Aldolase/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Escherichia coli/enzimologia , Frutose-Bifosfato Aldolase/química , Modelos Moleculares , Ácido N-Acetilneuramínico/química , Periplasma/metabolismo , Conformação Proteica , Transporte Proteico , Estereoisomerismo
5.
Sci Rep ; 11(1): 6589, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758324

RESUMO

The aim of this systematic review was to perform qualitative and quantitative analysis on the toxic effects of chloroquine (CQ) and hydroxychloroquine (HCQ) on skeletal muscles. We designed the study according to PRISMA guidelines. Studies for qualitative and quantitative analyses were selected according to the following inclusion criteria: English language; size of sample (> 5 patients), adult (> age of 18) patients, treated with CQ/HCQ for inflammatory diseases, and presenting and not presenting with toxic effects on skeletal muscles. We collected data published from 1990 to April 2020 using PubMed, Cochrane Library, EMBASE, and SciELO. Risk of bias for observational studies was assessed regarding the ROBIN-I scale. Studies with less than five patients (case reports) were selected for an additional qualitative analysis. We used the software Comprehensive Meta-Analysis at the confidence level of 0.05. We identified 23 studies for qualitative analysis (17 case-reports), and five studies were eligible for quantitative analysis. From case reports, 21 patients presented muscle weakness and confirmatory biopsy for CQ/HCQ induced myopathy. From observational studies, 37 patients out of 1,367 patients from five studies presented muscle weakness related to the use of CQ/HCQ, and 252 patients presented elevated levels of muscle enzymes (aldolase, creatine phosphokinase, and lactate dehydrogenase). Four studies presented data on 34 patients with confirmatory biopsy for drug-induced myopathy. No study presented randomized samples. The chronic use of CQ/HCQ may be a risk for drug-induced myopathy. There is substantiated need for proper randomized trials and controlled prospective studies needed to assess the clinical and subclinical stages of CQ/HCQ -induced muscle myopathy.


Assuntos
Hidroxicloroquina/toxicidade , Debilidade Muscular/etiologia , Músculo Esquelético/efeitos dos fármacos , Adulto , Idoso , Creatina Quinase/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Humanos , Hidroxicloroquina/administração & dosagem , Hidroxicloroquina/efeitos adversos , L-Lactato Desidrogenase/metabolismo , Pessoa de Meia-Idade , Debilidade Muscular/epidemiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Estudos Observacionais como Assunto
6.
Theranostics ; 11(10): 4825-4838, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33754030

RESUMO

Rationale: Sirtuins are NAD+-dependent protein deacylases known to have protective effects against age-related diseases such as diabetes, cancer, and neurodegenerative disease. SIRT2 is the only primarily cytoplasmic isoform and its overall role in glucose homeostasis remains uncertain. Methods: SIRT2-knockout (KO) rats were constructed to evaluate the role of SIRT2 in glucose homeostasis. The effect of SIRT2 on ß-cell function was detected by investigating the morphology, insulin secretion, and metabolomic state of islets. The deacetylation and stabilization of GKRP in ß-cells by SIRT2 were determined by western blot, adenoviral infection, and immunoprecipitation. Results: SIRT2-KO rats exhibited impaired glucose tolerance and glucose-stimulated insulin secretion (GSIS), without change in insulin sensitivity. SIRT2 deficiency or inhibition by AGK2 decreased GSIS in isolated rat islets, with lowered oxygen consumption rate. Adenovirus-mediated overexpression of SIRT2 enhanced insulin secretion from rat islets. Metabolomics analysis revealed a decrease in metabolites of glycolysis and tricarboxylic acid cycle in SIRT2-KO islets compared with control islets. Our study further demonstrated that glucokinase regulatory protein (GKRP), an endogenous inhibitor of glucokinase (GCK), was expressed in rat islets. SIRT2 overexpression deacetylated GKRP in INS-1 ß-cells. SIRT2 knockout or inhibition elevated GKRP protein stability in islet ß-cells, leading to an increase in the interaction of GKRP and GCK. On the contrary, SIRT2 inhibition promoted the protein degradation of ALDOA, a glycolytic enzyme. Conclusions: SIRT2 ablation inhibits GSIS through blocking GKRP protein degradation and promoting ALDOA protein degradation, resulting in a decrease in glycolytic flux.


Assuntos
Intolerância à Glucose/genética , Glicólise/genética , Resistência à Insulina/genética , Secreção de Insulina/genética , Sirtuína 2/genética , Animais , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ciclo do Ácido Cítrico/genética , Frutose-Bifosfato Aldolase/metabolismo , Furanos/farmacologia , Técnicas de Inativação de Genes , Intolerância à Glucose/metabolismo , Glicólise/efeitos dos fármacos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ilhotas Pancreáticas/metabolismo , Metabolômica , Quinolinas/farmacologia , Ratos , Ratos Transgênicos
7.
J Exp Bot ; 72(10): 3739-3755, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33684221

RESUMO

Plastid metabolism is critical in both photoautotrophic and heterotrophic plant cells. In chloroplasts, fructose-1,6-bisphosphate aldolase (FBA) catalyses the formation of both fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate within the Calvin-Benson cycle. Three Arabidopsis genes, AtFBA1-AtFBA3, encode plastidial isoforms of FBA, but the contribution of each isoform is unknown. Phylogenetic analysis indicates that FBA1 and FBA2 derive from a recently duplicated gene, while FBA3 is a more ancient paralog. fba1 mutants are phenotypically indistinguishable from the wild type, while both fba2 and fba3 have reduced growth. We show that FBA2 is the major isoform in leaves, contributing most of the measurable activity. Partial redundancy with FBA1 allows both single mutants to survive, but combining both mutations is lethal, indicating a block of photoautotrophy. In contrast, FBA3 is expressed predominantly in heterotrophic tissues, especially the leaf and root vasculature, but not in the leaf mesophyll. We show that the loss of FBA3 affects plastidial glycolytic metabolism of the root, potentially limiting the biosynthesis of essential compounds such as amino acids. However, grafting experiments suggest that fba3 is dysfunctional in leaf phloem transport, and we suggest that a block in photoassimilate export from leaves causes the buildup of high carbohydrate concentrations and retarded growth.


Assuntos
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Fotossíntese , Filogenia , Plastídeos/metabolismo
8.
Carbohydr Polym ; 255: 117532, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33436261

RESUMO

Aldolase A (ALDOA) facilitated aerobic glycolysis in cancer cells is a potential target in the treatment of hepatocellular carcinoma (HCC). However, only few effective inhibitors of ALDOA have been reported until now. In this research, we found a polysaccharide called HDPS-4II from Holotrichia diomphalia Bates, which can specifically bind to ALDOA with a dissociation constant of 2.86 µM. HDPS-4II with a molecular weight of 19 kDa was a linear triple-helix glucan composed of ɑ-d-1,4-Glcp and ɑ-d-1,6-Glcp in a ratio of 1.0:10.0. HDPS-4II significantly inhibited aldolase enzyme activity, glycolysis, and further inhibited the expression of phosphorylated AMPKα in HCC cells. Through analyzing ALDOA-overexpressing and -knockdown cells, it was confirmed that ALDOA mediated the viability and glycolysis inhibition of HDPS-4II. Moreover, HDPS-4II administration markedly inhibited tumor growth in mice xenografted with HCCs. These findings suggest that HDPS-4II, as an ALDOA antagonist, is a promising remedy in the treatment and prevention of HCC.


Assuntos
Antineoplásicos/farmacologia , Besouros/química , Frutose-Bifosfato Aldolase/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucanos/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Frutose-Bifosfato Aldolase/antagonistas & inibidores , Frutose-Bifosfato Aldolase/metabolismo , Glucanos/química , Glucanos/isolamento & purificação , Glicólise/efeitos dos fármacos , Glicólise/genética , Células Hep G2 , Hexoquinase/genética , Hexoquinase/metabolismo , Humanos , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Larva/química , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Piruvato Quinase/genética , Piruvato Quinase/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
9.
J Biosci Bioeng ; 131(3): 271-276, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33168471

RESUMO

Identification of the rate-limiting step in a metabolic pathway is an important challenge in metabolic engineering for enhancing pathway flow. Although specific enzyme activities (Vmax) provide valuable clues for the identification, it is time-consuming and difficult to measure multiple enzymes in the pathway because different assay protocols are required for each enzyme. In the present study, we propose a method to simultaneously determine the Vmax values of multiple enzymes using a kinetic model with a time course of the intermediate concentrations through an in vitro experiment. To demonstrate this method, nine glycolysis reactions for converting glucose-6-phosphate (G6P) to pyruvate in Escherichia coli were considered. In a reaction mixture containing G6P and cofactors, glycolysis was initiated by adding a crude cell extract obtained from stationary phase cells. The Vmax values were optimized to minimize the difference between the measured and simulated time-courses using a kinetic model. Metabolic control analysis using the kinetic model with the estimated Vmax values revealed that fructose bisphosphate aldolase (FBA) was the rate-limiting step in the upper part of glycolysis. The addition of FBA in the reaction mixture successfully increased the glycolytic flux in vitro. Furthermore, in vivo, the specific glucose consumption rate of an FBA overexpression strain was 1.4 times higher than that of the control strain during the stationary phase. These results confirmed that FBA was the rate-limiting step in glycolysis under the stationary phase. This approach provides Vmax values of multiple enzymes in a pathway for metabolic control analysis with a kinetic model.


Assuntos
Redes e Vias Metabólicas , Modelos Biológicos , Escherichia coli/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Glucose/metabolismo , Glicólise , Cinética
10.
Biochim Biophys Acta Gen Subj ; 1865(1): 129762, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33053413

RESUMO

BACKGROUND: Previous studies have demonstrated the formation of stable complexes between inorganic pyrophosphatase (PPase) and three other Escherichia coli enzymes - cupin-type phosphoglucose isomerase (cPGI), class I fructose-1,6-bisphosphate aldolase (FbaB) and l-glutamate decarboxylase (GadA). METHODS: Here, we determined by activity measurements how complex formation between these enzymes affects their activities and oligomeric structure. RESULTS: cPGI activity was modulated by all partner proteins, but none was reciprocally affected by cPGI. PPase activity was down-regulated upon complex formation, whereas all other enzymes were up-regulated. For cPGI, the activation was partially counteracted by a shift in dimer ⇆ hexamer equilibrium to inactive hexamer. Complex stoichiometry appeared to be 1:1 in most cases, but FbaB formed both 1:1 and 1:2 complexes with both GadA and PPase, FbaB activation was only observed in the 1:2 complexes. FbaB and GadA induced functional asymmetry (negative kinetic cooperativity) in hexameric PPase, presumably by favoring partial dissociation to trimers. CONCLUSIONS: These four enzymes form all six possible binary complexes in vitro, resulting in modulated activity of at least one of the constituent enzymes. In five complexes, the effects on activity were unidirectional, and in one complex (FbaB⋅PPase), the effects were reciprocal. The effects of potential physiological significance include inhibition of PPase by FbaB and GadA and activation of FbaB and cPGI by PPase. Together, they provide a mechanism for feedback regulation of FbaB and GadA biosynthesis. GENERAL SIGNIFICANCE: These findings indicate the complexity of functionally significant interactions between cellular enzymes, which classical enzymology treats as individual entities, and demonstrate their moonlighting activities as regulators.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Glucose-6-Fosfato Isomerase/metabolismo , Glutamato Descarboxilase/metabolismo , Pirofosfatase Inorgânica/metabolismo , Proteínas de Membrana/metabolismo , Escherichia coli/química , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/química , Frutose-Bifosfato Aldolase/química , Glucose-6-Fosfato Isomerase/química , Glutamato Descarboxilase/química , Humanos , Pirofosfatase Inorgânica/química , Cinética , Proteínas de Membrana/química , Multimerização Proteica
11.
PLoS Biol ; 18(12): e3000803, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33275593

RESUMO

Loss of hepatic fructose-1, 6-bisphosphate aldolase B (Aldob) leads to a paradoxical up-regulation of glucose metabolism to favor hepatocellular carcinogenesis (HCC), but the upstream signaling events remain poorly defined. Akt is highly activated in HCC, and targeting Akt is being explored as a potential therapy for HCC. Herein, we demonstrate that Aldob suppresses Akt activity and tumor growth through a protein complex containing Aldob, Akt, and protein phosphatase 2A (PP2A), leading to inhibition of cell viability, cell cycle progression, glucose uptake, and metabolism. Interestingly, Aldob directly interacts with phosphorylated Akt (p-Akt) and promotes the recruitment of PP2A to dephosphorylate p-Akt, and this scaffolding effect of Aldob is independent of its enzymatic activity. Loss of Aldob or disruption of Aldob/Akt interaction in Aldob R304A mutant restores Akt activity and tumor-promoting effects. Consistently, Aldob and p-Akt expression are inversely correlated in human HCC tissues, and Aldob down-regulation coupled with p-Akt up-regulation predicts a poor prognosis for HCC. We have further discovered that Akt inhibition or a specific small-molecule activator of PP2A (SMAP) efficiently attenuates HCC tumorigenesis in xenograft mouse models. Our work reveals a novel nonenzymatic role of Aldob in negative regulation of Akt activation, suggesting that directly inhibiting Akt activity or through reactivating PP2A may be a potential therapeutic approach for HCC treatment.


Assuntos
Carcinoma Hepatocelular/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/fisiopatologia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , China , Frutose-Bifosfato Aldolase/biossíntese , Frutose-Bifosfato Aldolase/genética , Glucose/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Nus , Fosforilação , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
J Vis Exp ; (166)2020 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-33346205

RESUMO

Cerebellum plays an important role in several key functions including control of movement, balance, cognition, reward, and affect. Imaging studies indicate that distinct cerebellar regions contribute to these different functions. Molecular studies examining regional cerebellar differences are lagging as they are mostly done on whole cerebellar extracts thereby masking any distinctions across specific cerebellar regions. Here we describe a technique to reproducibly and quickly dissect four different cerebellar regions: the deep cerebellar nuclei (DCN), anterior and posterior vermal cerebellar cortex, and the cerebellar cortex of the hemispheres. Dissecting out these distinct regions allows for the exploration of molecular mechanisms that may underlie their unique contributions to balance, movement, affect and cognition. This technique may also be used to explore differences in pathological susceptibility of these specific regions across various mouse disease models.


Assuntos
Cerebelo/metabolismo , Dissecação , Animais , Frutose-Bifosfato Aldolase/metabolismo , Regulação da Expressão Gênica , Humanos , Processamento de Imagem Assistida por Computador , Camundongos Endogâmicos C57BL , RNA/isolamento & purificação
13.
Biochemistry ; 59(48): 4573-4580, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33231431

RESUMO

Non-typhoidal Salmonella are capable of colonizing livestock and humans, where they can progressively cause disease. Previously, a library of targeted single-gene deletion mutants of Salmonella enterica serotype Typhimurium was inoculated to ligated ileal loops in calves to identify genes under selection. Of those genes identified, a cluster of genes is related to carbohydrate metabolism and transportation. It is proposed that an incoming carbohydrate is first phosphorylated by a phosphoenolpyruvate-dependent phosphotransferase system. The metabolite is further phosphorylated by the kinase STM3781 and then cleaved by the aldolase STM3780. STM3780 is functionally annotated as a class II fructose-bisphosphate aldolase. The aldolase was purified to homogeneity, and its aldol condensation activity with a range of aldehydes was determined. In the condensation reaction, STM3780 was shown to catalyze the abstraction of the pro-S hydrogen from C3 of dihydroxyacetone and subsequent formation of a carbon-carbon bond with S stereochemistry at C3 and R stereochemistry at C4. The best aldehyde substrate was identified as l-threouronate. Surprisingly, STM3780 was also shown to catalyze the condensation of two molecules of dihydroxyacetone phosphate to form the branched carbohydrate dendroketose bisphosphate.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Genes Bacterianos , Salmonella typhimurium/enzimologia , Salmonella typhimurium/genética , Animais , Biocatálise , Metabolismo dos Carboidratos , Carboidratos/química , Bovinos , Doenças dos Bovinos/microbiologia , Medição da Troca de Deutério , Fosfato de Di-Hidroxiacetona/metabolismo , Humanos , Família Multigênica , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salmonelose Animal/microbiologia , Sorogrupo , Estereoisomerismo , Especificidade por Substrato
14.
Infect Immun ; 88(12)2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-32900818

RESUMO

The obligate intracellular pathogen Chlamydia trachomatis is the leading cause of noncongenital blindness and causative agent of the most common sexually transmitted infection of bacterial origin. With a reduced genome, C. trachomatis is dependent on its host for survival, in part due to a need for the host cell to compensate for incomplete bacterial metabolic pathways. However, relatively little is known regarding how C. trachomatis is able to hijack host cell metabolism. In this study, we show that two host glycolytic enzymes, aldolase A and pyruvate kinase, as well as lactate dehydrogenase, are enriched at the C. trachomatis inclusion membrane during infection. Inclusion localization was not species specific, since a similar phenotype was observed with C. muridarum Time course experiments showed that the number of positive inclusions increased throughout the developmental cycle. In addition, these host enzymes colocalized to the same inclusion, and their localization did not appear to be dependent on sustained bacterial protein synthesis or on intact host actin, vesicular trafficking, or microtubules. Depletion of the host glycolytic enzyme aldolase A resulted in decreased inclusion size and infectious progeny production, indicating a role for host glycolysis in bacterial growth. Finally, quantitative PCR analysis showed that expression of C. trachomatis glycolytic enzymes inversely correlated with host enzyme localization at the inclusion. We discuss potential mechanisms leading to inclusion localization of host glycolytic enzymes and how it could benefit the bacteria. Altogether, our findings provide further insight into the intricate relationship between host and bacterial metabolism during Chlamydia infection.


Assuntos
Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Glicólise , Interações entre Hospedeiro e Microrganismos , Corpos de Inclusão/metabolismo , L-Lactato Desidrogenase/metabolismo , Piruvato Quinase/metabolismo , Actinas/metabolismo , Membrana Externa Bacteriana/enzimologia , Membrana Externa Bacteriana/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Chlamydia/enzimologia , Infecções por Chlamydia/genética , Chlamydia muridarum/metabolismo , Chlamydia trachomatis/enzimologia , Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydia trachomatis/patogenicidade , Frutose-Bifosfato Aldolase/genética , Células HeLa , Humanos , Corpos de Inclusão/enzimologia , Corpos de Inclusão/microbiologia , L-Lactato Desidrogenase/genética , Microtúbulos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Piruvato Quinase/genética
15.
Nat Metab ; 2(9): 893-901, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32719541

RESUMO

The mechanistic target of rapamycin complex 1 (mTORC1) kinase regulates cell growth by setting the balance between anabolic and catabolic processes. To be active, mTORC1 requires the environmental presence of amino acids and glucose. While a mechanistic understanding of amino acid sensing by mTORC1 is emerging, how glucose activates mTORC1 remains mysterious. Here, we used metabolically engineered human cells lacking the canonical energy sensor AMP-activated protein kinase to identify glucose-derived metabolites required to activate mTORC1 independent of energetic stress. We show that mTORC1 senses a metabolite downstream of the aldolase and upstream of the GAPDH-catalysed steps of glycolysis and pinpoint dihydroxyacetone phosphate (DHAP) as the key molecule. In cells expressing a triose kinase, the synthesis of DHAP from DHA is sufficient to activate mTORC1 even in the absence of glucose. DHAP is a precursor for lipid synthesis, a process under the control of mTORC1, which provides a potential rationale for the sensing of DHAP by mTORC1.


Assuntos
Fosfato de Di-Hidroxiacetona/fisiologia , Glucose/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Di-Hidroxiacetona/metabolismo , Fosfato de Di-Hidroxiacetona/biossíntese , Metabolismo Energético , Frutose-Bifosfato Aldolase/metabolismo , Glucose/deficiência , Glicólise , Células HEK293 , Humanos , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Serina-Treonina Quinases TOR/genética
16.
Biochemistry ; 59(32): 2962-2973, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32697085

RESUMO

l-2-Keto-3-deoxyarabinonate (l-KDA) dehydratase (AraD) catalyzes the hydration of l-KDA to α-ketoglutaric semialdehyde in the nonphosphorylative l-arabinose pathway from bacteria and belongs to the dihydrodipicolinate synthase (DHDPS)/N-acetylneuraminate lyase (NAL) protein superfamily. All members of this superfamily, including several aldolases for l-KDA, share a common catalytic mechanism of retro-aldol fission, in which a lysine residue forms a Schiff base with the carbonyl C2 atom of the substrate, followed by proton abstraction of the substrate by a tyrosine residue as the base catalyst. Only AraD possesses a glutamine residue instead of this active site tyrosine, suggesting its involvement in catalysis. We herein determined the crystal structures of AraD from the nitrogen-fixing bacterium Azospirillum brasilense and AraD in complex with ß-hydroxypyruvate and 2-oxobutyrate, two substrate analogues, at resolutions of 1.9, 1.6, and 2.2 Å, respectively. In both of the complexed structures, the ε-nitrogen of the conserved Lys171 was covalently linked to the carbonyl C2 atom of the ligand, which was consistent with the Schiff base intermediate form, similar to other DHDPS/NAL members. A site-directed mutagenic study revealed that Glu173 and Glu200 played important roles as base catalysts, whereas Gln143 was not absolutely essential. The abstraction of one of the C3 protons of the substrate (but not the O4 hydroxyl) by Glu173 was similar to that by the (conserved) tyrosine residues in the two DHDPS/NAL members that produce α-ketoglutaric semialdehyde (d-5-keto-4-deoxygalactarate dehydratase and Δ1-pyrroline-4-hydroxy-2-carboxylate deaminase), indicating that these enzymes evolved convergently despite similarities in the overall reaction.


Assuntos
Biocatálise , Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/metabolismo , Azospirillum brasilense/enzimologia , Frutose-Bifosfato Aldolase/genética , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica
17.
Exp Cell Res ; 394(1): 112118, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32502493

RESUMO

The MUC16 C-terminal (MUC16c) level is associated with tumor serum CA-125 levels, however, the roles remain unclear in gallbladder carcinoma (GBC). In this study, we found that MUC16c promoted glucose uptake and glycolysis for GBC cell proliferation. Mass spectrometry analysis suggested that MUC16c could combine with aldolase. The ALDOC mRNA and protein are overexpressed in GBC tumors. The IHC results also showed the consistent up-regulation of. ALDOC and MUC16c level in GBC tumor tissues than in peritumor tissues. We determined that MUC16c combining with ALDOC promoted ALDOC protein stability and disrupted the ability of ALDOC sensing glucose deficiency, which activated AMPK pathway and increased GBC cell proliferation. ALDOC knockdown significantly inhibited the glucose uptake and glycolysis induced by MUC16c. Our study established important roles of MUC16c promoting GBC cell glycolysis and proliferation and revealed the underlying mechanism of CA-125-related heavy tumor metabolic burden in GBC.


Assuntos
Antígeno Ca-125/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Frutose-Bifosfato Aldolase/metabolismo , Neoplasias da Vesícula Biliar/metabolismo , Proteínas de Membrana/metabolismo , Antígeno Ca-125/genética , Frutose-Bifosfato Aldolase/genética , Neoplasias da Vesícula Biliar/genética , Regulação Neoplásica da Expressão Gênica/genética , Glicólise/genética , Humanos , Proteínas de Membrana/genética
18.
Cancer Sci ; 111(8): 3071-3081, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32530543

RESUMO

Recent studies have revealed that metabolic reprogramming is closely associated with epithelial-mesenchymal transition (EMT) during cancer progression. Aldolase A (ALDOA) is a key glycolytic enzyme that is highly expressed in several types of cancer. In this study, we found that ALDOA is highly expressed in uterine cervical adenocarcinoma and that high ALDOA expression promotes EMT to increase malignant potentials, such as metastasis and invasiveness, in cervical adenocarcinoma cells. In human surgical specimens, ALDOA was highly expressed in cervical adenocarcinoma and high ALDOA expression was correlated with lymph node metastasis, lymphovascular infiltration, and short overall survival. Suppression of ALDOA expression significantly reduced cell growth, migration, and invasiveness of cervical cancer cells. Aldolase A expression was partially regulated by hypoxia-inducible factor-1α (HIF-1α). Shotgun proteome analysis revealed that cell-cell adhesion-related proteins were significantly increased in ALDOA-overexpressing cells. Interestingly, overexpression of ALDOA caused severe morphological changes, including a cuboidal-to-spindle shape shift and reduced microvilli formation, coincident with modulation of the expression of typical EMT-related proteins. Overexpression of ALDOA increased migration and invasion in vitro. Furthermore, overexpression of ALDOA induced HIF-1α, suggesting a positive feedback loop between ALDOA and HIF-1α. In conclusion, ALDOA is overexpressed in cervical adenocarcinoma and contributes to malignant potentials of tumor cells through modulation of HIF-1α signaling. The feedback loop between ALDOA and HIF-1α could become a therapeutic target to improve the prognosis of this malignancy.


Assuntos
Adenocarcinoma/patologia , Frutose-Bifosfato Aldolase/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias do Colo do Útero/patologia , Adenocarcinoma/mortalidade , Adenocarcinoma/cirurgia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Colo do Útero/patologia , Colo do Útero/cirurgia , Intervalo Livre de Doença , Transição Epitelial-Mesenquimal , Retroalimentação Fisiológica , Feminino , Frutose-Bifosfato Aldolase/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Estimativa de Kaplan-Meier , Prognóstico , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/genética , Neoplasias do Colo do Útero/mortalidade , Neoplasias do Colo do Útero/cirurgia
19.
Sheng Wu Gong Cheng Xue Bao ; 36(5): 942-948, 2020 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-32567277

RESUMO

It is of great significance to use biosynthesis to transform the inorganic substance formaldehyde into organic sugars. Most important in this process was to find a suitable catalyst combination to achieve the dimerization of formaldehyde. In a recent report, an engineered glycolaldehyde synthase was reported to catalyze this reaction. It could be combined with engineered D-fructose-6-phosphate aldolase, a "one-pot enzyme" method, to synthesize L-xylose using formaldehyde and the conversion rate could reach up to 64%. This process also provides a reference for the synthesis of other sugars. With the increasing consumption of non-renewable resources, it was of great significance to convert formaldehyde into sugar by biosynthesis.


Assuntos
Formaldeído , Xilose , Biocatálise , Formaldeído/química , Frutose-Bifosfato Aldolase/metabolismo , Xilose/síntese química
20.
PLoS One ; 15(5): e0233449, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32442224

RESUMO

Follicular lymphoma (FL) is an indolent neoplasia comprising approximately 20% of lymphomas. FL is generally considered incurable, with a median survival exceeding 10 years. A subset of FL patients experiences histological transformation (HT) to a more aggressive lymphoma, resulting in markedly poorer clinical outcome, with a reduced median survival after transformation of 1-2 years. Early, reliable prediction of HT would be valuable in the clinical setting, allowing pre-emptive therapeutic intervention. We previously used proteomics to identify the glycolytic enzymes fructose-bisphosphate aldolase A (aldolase A) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as candidate predictors of FL transformation. Now, we use immunohistochemistry to evaluate expression of these enzymes in paired primary FLs from patients with (n = 41) or without subsequent HT (n = 49), to test their value as predictive biomarkers. At initial FL diagnosis, patients with subsequent HT had significantly higher expression of aldolase A and GAPDH (p<0.001 and p<0.01) compared with patients without HT. Furthermore, high expression of aldolase A and GAPDH was associated with significantly shorter transformation free survival (p = 0.018, p = 0.001). These data suggest that high expression of aldolase A and GAPDH, may indicate increased metabolic turnover, and that these enzymes may be useful biomarkers in primary FL for predicting the risk of subsequent lymphoma transformation.


Assuntos
Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Frutose-Bifosfato Aldolase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Glicólise , Linfoma Folicular/metabolismo , Linfoma Folicular/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Estudos de Coortes , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Prognóstico
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