RESUMEN
Nucleotide sugars (NS) fulfil important roles in all living organisms and in humans, related defects result in severe clinical syndromes. NS can be seen as the "activated" sugars used for biosynthesis of a wide range of glycoconjugates and serve as substrates themselves for the synthesis of other nucleotide sugars. NS analysis is complicated by the presence of multiple stereoisomers without diagnostic transition ions, therefore requiring separation by liquid chromatography. In this paper, we explored weak anion-exchange/reversed-phase chromatography on a hybrid column for the separation of 17 nucleotide sugars that can occur in humans. A robust and reproducible method was established with intra- and inter-day coefficients of variation below 10% and a linear range spanning three orders of magnitude. Application to patient fibroblasts with genetic defects in mannose-1-phosphate guanylyltransferase beta, CDP-L-ribitol pyrophosphorylase A, and UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase showed abnormal levels of guanosine-5'-diphosphate-α-D-mannose (GDP-Man), cytidine-5'-diphosphate-L-ribitol (CDP-ribitol), and cytidine-5'-monophosphate-N-acetyl-ß-D-neuraminic acid (CMP-Neu5Ac), respectively, in consonance with expectations based on the diagnosis. In conclusion, a novel, semi-quantitative method was established for the analysis of nucleotide sugars that can be applied to diagnose several genetic glycosylation disorders in fibroblasts and beyond.
Asunto(s)
Cromatografía de Fase Inversa , Fibroblastos , Espectrometría de Masas en Tándem , Humanos , Fibroblastos/metabolismo , Espectrometría de Masas en Tándem/métodos , Cromatografía por Intercambio Iónico/métodos , Cromatografía de Fase Inversa/métodos , Nucleótidos/análisis , Nucleótidos/metabolismo , Aniones/análisis , Cromatografía Líquida con Espectrometría de MasasRESUMEN
Phosphoglucomutase 1 (PGM1) is a key enzyme for the regulation of energy metabolism from glycogen and glycolysis, as it catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. PGM1 deficiency is an autosomal recessive disorder characterized by a highly heterogenous clinical spectrum, including hypoglycemia, cleft palate, liver dysfunction, growth delay, exercise intolerance, and dilated cardiomyopathy. Abnormal protein glycosylation has been observed in this disease. Oral supplementation with D-galactose efficiently restores protein glycosylation by replenishing the lacking pool of UDP-galactose, and rescues some symptoms, such as hypoglycemia, hepatopathy, and growth delay. However, D-galactose effects on skeletal muscle and heart symptoms remain unclear. In this study, we established an in vitro muscle model for PGM1 deficiency to investigate the role of PGM1 and the effect of D-galactose on nucleotide sugars and energy metabolism. Genome-editing of C2C12 myoblasts via CRISPR/Cas9 resulted in Pgm1 (mouse homologue of human PGM1, according to updated nomenclature) knockout clones, which showed impaired maturation to myotubes. No difference was found for steady-state levels of nucleotide sugars, while dynamic flux analysis based on 13C6-galactose suggested a block in the use of galactose for energy production in knockout myoblasts. Subsequent analyses revealed a lower basal respiration and mitochondrial ATP production capacity in the knockout myoblasts and myotubes, which were not restored by D-galactose. In conclusion, an in vitro mouse muscle cell model has been established to study the muscle-specific metabolic mechanisms in PGM1 deficiency, which suggested that galactose was unable to restore the reduced energy production capacity.
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Hipoglucemia , Fosfoglucomutasa , Animales , Ratones , Galactosa/farmacología , Glucosa , Homeostasis , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Nucleótidos , Fosfatos , Fosfoglucomutasa/genética , Fosfoglucomutasa/metabolismoRESUMEN
Carbapenems, a family of ß-lactam antibiotics, are among the most powerful bactericidal compounds in clinical use. However, as rational engineering of native carbapenem-producing microbes is not currently possible, the present carbapenem supply relies upon total chemical synthesis of artificial carbapenem derivatives. To enable access to the full diversity of natural carbapenems, we have engineered production of a simple carbapenem antibiotic within Escherichia coli. By increasing concentrations of precursor metabolites and identifying a reducing cofactor of a bottleneck enzyme, we improved productivity by 60-fold over the minimal pathway and surpassed reported titers obtained from carbapenem-producing Streptomyces species. We stabilized E. coli metabolism against antibacterial effects of the carbapenem product by artificially inhibiting membrane synthesis, which further increased antibiotic productivity. As all known naturally occurring carbapenems are derived from a common intermediate, our engineered strain provides a platform for biosynthesis of tailored carbapenem derivatives in a genetically tractable and fast-growing species.
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Carbapenémicos/biosíntesis , Escherichia coli/metabolismo , Ingeniería Metabólica , Carbapenémicos/químicaRESUMEN
The production of fatty acids from simple nutrients occurs via a complex biosynthetic pathway with dozens of intermediate compounds and multiple branch points. Despite its importance for microbial physiology and biotechnology, critical aspects of fatty acid biosynthesis, especially dynamics of in vivo regulation, remain poorly characterized. We have developed a liquid chromatography/mass spectroscopy (LC-MS) method for relative quantification of fatty acid synthesis intermediates in Escherichia coli, a model organism for studies of fatty acid metabolism. The acyl carrier protein, a vehicle for the substrates and intermediates of fatty acid synthesis, is extracted from E. coli, proteolytically digested, resolved using reverse-phase LC, and detected using electrospray ionization coupled with a tandem MS. Our method reliably resolves 21 intermediates of fatty acid synthesis, with an average relative standard deviation in ratios of individual acyl-ACP species to total ACP concentrations of 20%. We demonstrate that fast sampling and quenching of cells is essential to accurately characterize intracellular concentrations of ACP species. We apply our method to examine the rapid response of fatty acid metabolism to the antibiotic cerulenin. We anticipate that our method will enable the characterization of in vivo regulation and kinetics of microbial fatty acid synthesis at unprecedented detail and will improve integration of fatty acid synthesis into models of microbial metabolism.
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Proteínas Bacterianas/metabolismo , Escherichia coli/química , Ácidos Grasos/metabolismo , Proteína Transportadora de Acilo/metabolismo , Vías Biosintéticas/efectos de los fármacos , Proteínas Portadoras/metabolismo , Cerulenina/farmacología , Ácidos Grasos/biosíntesis , Espectrometría de Masas , Unión ProteicaRESUMEN
Metabolism not only produces energy necessary for the cell but is also a key regulator of several cellular functions, including pluripotency and self-renewal. Nucleotide sugars (NSs) are activated sugars that link glucose metabolism with cellular functions via protein N-glycosylation and O-GlcNAcylation. Thus, understanding how different metabolic pathways converge in the synthesis of NSs is critical to explore new opportunities for metabolic interference and modulation of stem cell functions. Tracer-based metabolomics is suited for this challenge, however chemically-defined, customizable media for stem cell culture in which nutrients can be replaced with isotopically labeled analogs are scarcely available. Here, we established a customizable flux-conditioned E8 (FC-E8) medium that enables stem cell culture with stable isotopes for metabolic tracing, and a dedicated liquid chromatography mass-spectrometry (LC-MS/MS) method targeting metabolic pathways converging in NS biosynthesis. By 13C6-glucose feeding, we successfully traced the time-course of carbon incorporation into NSs directly via glucose, and indirectly via other pathways, such as glycolysis and pentose phosphate pathways, in induced pluripotent stem cells (hiPSCs) and embryonic stem cells. Then, we applied these tools to investigate the NS biosynthesis in hiPSC lines from a patient affected by deficiency of phosphoglucomutase 1 (PGM1), an enzyme regulating the synthesis of the two most abundant NSs, UDP-glucose and UDP-galactose.
Asunto(s)
Células Madre Pluripotentes , Espectrometría de Masas en Tándem , Humanos , Cromatografía Liquida , Glucosa/metabolismo , Células Madre Pluripotentes/metabolismo , Azúcares , Nucleótidos , Uridina DifosfatoRESUMEN
The sugar fucose is expressed on mammalian cell membranes as part of glycoconjugates and mediates essential physiological processes. The aberrant expression of fucosylated glycans has been linked to pathologies such as cancer, inflammation, infection, and genetic disorders. Tools to modulate fucose expression on living cells are needed to elucidate the biological role of fucose sugars and the development of potential therapeutics. Herein, we report a class of fucosylation inhibitors directly targeting de novo GDP-fucose biosynthesis via competitive GMDS inhibition. We demonstrate that cell permeable fluorinated rhamnose 1-phosphate derivatives (Fucotrim I & II) are metabolic prodrugs that are metabolized to their respective GDP-mannose derivatives and efficiently inhibit cellular fucosylation.
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Inhibidores Enzimáticos/farmacología , Fucosa/química , Guanosina Difosfato Fucosa/antagonistas & inhibidores , Hidroliasas/antagonistas & inhibidores , Profármacos/farmacología , Animales , Secuencia de Carbohidratos , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Expresión Génica , Glicosilación/efectos de los fármacos , Guanosina Difosfato Fucosa/biosíntesis , Halogenación , Humanos , Hidroliasas/genética , Hidroliasas/metabolismo , Células Jurkat , Linfocitos/citología , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Ratones , Profármacos/síntesis química , Relación Estructura-Actividad , Células THP-1RESUMEN
The molecule guanosine tetraphophosphate (ppGpp) is most commonly considered an alarmone produced during acute stress. However, ppGpp is also present at low concentrations during steady-state growth. Whether ppGpp controls the same cellular targets at both low and high concentrations remains an open question and is vital for understanding growth rate regulation. It is widely assumed that basal ppGpp concentrations vary inversely with growth rate, and that the main function of basal ppGpp is to regulate transcription of ribosomal RNA in response to environmental conditions. Unfortunately, studies to confirm this relationship and to define regulatory targets of basal ppGpp are limited by difficulties in quantifying basal ppGpp. In this Perspective we compare reported concentrations of basal ppGpp in E. coli and quantify ppGpp within several strains using a recently developed analytical method. We find that although the inverse correlation between ppGpp and growth rate is robust across strains and analytical methods, absolute ppGpp concentrations do not absolutely determine RNA synthesis rates. In addition, we investigated the consequences of two separate RNA polymerase mutations that each individually reduce (but do not abolish) sensitivity to ppGpp and find that the relationship between ppGpp, growth rate, and RNA content of single-site mutants remains unaffected. Both literature and our new data suggest that environmental conditions may be communicated to RNA polymerase via an additional regulator. We conclude that basal ppGpp is one of potentially several agents controlling ribosome abundance and DNA replication initiation, but that evidence for additional roles in controlling macromolecular synthesis requires further study.
RESUMEN
Every cell must produce enough membrane to contain itself. However, the mechanisms by which the rate of membrane synthesis is coupled with the rate of cell growth remain unresolved. By comparing substrate and enzyme concentrations of the fatty acid and phospholipid synthesis pathways of Escherichia coli across a 3-fold range of carbon-limited growth rates, we show that the rate of membrane phospholipid synthesis during steady-state growth is determined principally through allosteric control of a single enzyme, PlsB. Due to feedback regulation of the fatty acid pathway, PlsB activity also indirectly controls synthesis of lipopolysaccharide, a major component of the outer membrane synthesized from a fatty acid synthesis intermediate. Surprisingly, concentrations of the enzyme that catalyzes the committed step of lipopolysaccharide synthesis (LpxC) do not differ across steady-state growth conditions, suggesting that steady-state lipopolysaccharide synthesis is modulated primarily via indirect control by PlsB. In contrast to steady-state regulation, we found that responses to environmental perturbations are triggered directly via changes in acetyl coenzyme A (acetyl-CoA) concentrations, which enable rapid adaptation. Adaptations are further modulated by ppGpp, which regulates PlsB activity during slow growth and growth arrest. The strong reliance of the membrane synthesis pathway upon posttranslational regulation ensures both the reliability and the responsiveness of membrane synthesis.IMPORTANCE How do bacterial cells grow without breaking their membranes? Although the biochemistry of fatty acid and membrane synthesis is well known, how membrane synthesis is balanced with growth and metabolism has remained unclear. This is partly due to the many control points that have been discovered within the membrane synthesis pathways. By precisely establishing the contributions of individual pathway enzymes, our results simplify the model of membrane biogenesis in the model bacterial species Escherichia coli Specifically, we found that allosteric control of a single enzyme, PlsB, is sufficient to balance growth with membrane synthesis and to ensure that growing E. coli cells produce sufficient membrane. Identifying the signals that activate and deactivate PlsB will resolve the issue of how membrane synthesis is synchronized with growth.
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Acetiltransferasas/metabolismo , Membrana Celular/metabolismo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/genética , Fosfolípidos/biosíntesis , Acetiltransferasas/genética , Vías Biosintéticas , Lipopolisacáridos/biosíntesis , Espectrometría de Masas , Procesamiento Proteico-PostraduccionalRESUMEN
INTRODUCTION: Metabolic profiling of cerebrospinal fluid (CSF) is a promising technique for studying brain diseases. Measurements should reflect the in vivo situation, so ex vivo metabolism should be avoided. OBJECTIVE: To investigate the effects of temperature (room temperature vs. 4 °C), centrifugation and ethanol, as anti-enzymatic additive during CSF sampling on concentrations of glutamic acid, glutamine and other endogenous amines. METHODS: CSF samples from 21 individuals were processed using five different protocols. Isotopically-labeled alanine, isoleucine, glutamine, glutamic acid and dopamine were added prior to sampling to trace any degradation. Metabolomics analysis of endogenous amines, isotopically-labeled compounds and degradation products was performed with a validated LC-MS method. RESULTS: Thirty-six endogenous amines were quantified. There were no statistically significant differences between sampling protocols for 31 out of 36 amines. For GABA there was primarily an effect of temperature (higher concentrations at room temperature than at 4 °C) and a small effect of ethanol (lower concentrations if added) due to possible degradation. O-phosphoethanolamine concentrations were also lower when ethanol was added. Degradation of isotopically-labeled compounds (e.g. glutamine to glutamic acid) was minor with no differences between protocols. CONCLUSION: Most amines can be considered stable during sampling, provided that samples are cooled immediately to 4 °C, centrifuged, and stored at - 80 °C within 2 h. The effect of ethanol addition for more unstable metabolites needs further investigation. This was the first time that labeled compounds were used to monitor ex vivo metabolism during sampling. This is a useful strategy to study the stability of other metabolites of interest.
RESUMEN
The goal of bottom-up synthetic biology culminates in the assembly of an entire cell from separate biological building blocks. One major challenge resides in the in vitro production and implementation of complex genetic and metabolic pathways that can support essential cellular functions. Here, we show that phospholipid biosynthesis, a multiple-step process involved in cell membrane homeostasis, can be reconstituted starting from the genes encoding for all necessary proteins. A total of eight E. coli enzymes for acyl transfer and headgroup modifications were produced in a cell-free gene expression system and were co-translationally reconstituted in liposomes. Acyl-coenzyme A and glycerol-3-phosphate were used as canonical precursors to generate a variety of important bacterial lipids. Moreover, this study demonstrates that two-step acyl transfer can occur from enzymes synthesized inside vesicles. Besides clear implications for growth and potentially division of a synthetic cell, we postulate that gene-based lipid biosynthesis can become instrumental for ex vivo and protein purification-free production of natural and non-natural lipids.
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Aciltransferasas/genética , Aciltransferasas/metabolismo , Ingeniería Genética/métodos , Glicerol-3-Fosfato O-Aciltransferasa/genética , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Liposomas/metabolismo , Fosfolípidos/biosíntesis , Aciltransferasas/biosíntesis , Biocatálisis , Membrana Celular/metabolismo , Dihidroxifenilalanina/biosíntesis , Escherichia coli/enzimología , Escherichia coli/genética , Glicerol-3-Fosfato O-Aciltransferasa/biosíntesis , Homeostasis , Biología SintéticaRESUMEN
BACKGROUND: Phosphorylation is a key process regulating a large number of fundamental biochemical reactions in living organisms. It is known that many mechanisms of response to chronic drugs administration are regulated by phosphorylation. It can be assumed that some of the phosphorylation sites are known, but they represent only a small fraction of the regulatory phosphorylation events in this system. Therefore, it is important to investigate protein phosphorylation with high-throughput methods such as mass spectrometry, that allow for efficient global analysis. The aim of this work was to develop a robust workflow for quantitative phosphoproteomic analysis, which operates in a semi-automatic manner. METHODS: The proposed approach consists of two methods of phosphopeptides enrichment (TiO2, IMAC), stable isotope methyl labeling, data-dependent mass spectrometry acquisition with simultaneous CID/ETD fragmentation, and data analysis platform based on Trans-Proteomic Pipeline. We have applied our method to analyze selected brain structures from rat involved in morphine dependence. RESULTS: We have identified and quantified number of phosphoproteins that were up- or down-regulated as a result of morphine treatment. Finally, we have applied a three-step filtration process to emerge the most regulated candidates. In parallel, all of the regulated proteins were annotated with GO terms to follow global trends of protein regulation. CONCLUSIONS: The proposed MS-based workflow with following data analysis is efficient method for quantitative phosphoproteomic analysis:
Asunto(s)
Encéfalo/metabolismo , Dependencia de Morfina/fisiopatología , Fosfopéptidos/análisis , Proteómica/métodos , Animales , Ensayos Analíticos de Alto Rendimiento/métodos , Marcaje Isotópico/métodos , Espectrometría de Masas/métodos , Fosforilación/fisiología , Ratas , Flujo de TrabajoRESUMEN
Experimental Autoimmune Encephalomyelitis (EAE) is the most commonly used animal model for Multiple Sclerosis (MScl). CSF metabolomics in an acute EAE rat model was investigated using targetted LC-MS and GC-MS. Acute EAE in Lewis rats was induced by co-injection of Myelin Basic Protein with Complete Freund's Adjuvant. CSF samples were collected at two time points: 10 days after inoculation, which was during the onset of the disease, and 14 days after inoculation, which was during the peak of the disease. The obtained metabolite profiles from the two time points of EAE development show profound differences between onset and the peak of the disease, suggesting significant changes in CNS metabolism over the course of MBP-induced neuroinflammation. Around the onset of EAE the metabolome profile shows significant decreases in arginine, alanine and branched amino acid levels, relative to controls. At the peak of the disease, significant increases in concentrations of multiple metabolites are observed, including glutamine, O-phosphoethanolamine, branched-chain amino acids and putrescine. Observed changes in metabolite levels suggest profound changes in CNS metabolism over the course of EAE. Affected pathways include nitric oxide synthesis, altered energy metabolism, polyamine synthesis and levels of endogenous antioxidants. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0306-3) contains supplementary material, which is available to authorized users.
RESUMEN
A novel method for peptide sequencing by matrix-assisted laser desorption/ionization mass spectrometry with a time-of-flight/time-of-flight analyzer (MALDI-TOF/TOF) is presented. A stable isotope label introduced in the peptide N-terminus by derivatization, using a 1:1 mixture of acetic anhydride and deuterated acetic anhydride, allows for easy and unambiguous identification of ions belonging either to the N- or the C-terminal ion series in the product ion spectrum, making sequence assignment significantly simplified. The good performance of this technique was shown by successful sequencing of the contents of several peptide maps. A similar approach was recently applied to nanoelectrospray ionization (nanoESI) and nano-liquid chromatography/tandem mass spectrometry (LC/MS/MS). The MALDI-TOF/TOF technique allows for fast, direct sequencing of modified peptides in proteomics samples, and is complementary to the nanoESI and nanoLC/MS/MS approaches.
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Deuterio/química , Mapeo Peptídico/métodos , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem/métodos , Tritio/química , Acetilación , Microquímica/métodos , Proteómica/instrumentación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentaciónRESUMEN
An improved method for peptide sequencing based on acetylation/deuteroacetylation in conjunction with ESI MS is introduced. Derivatization with a 1:1 mixture of acetic anhydride and deuterated acetic anhydride incorporates a stable isotope label into the analyzed molecule. This approach has been initially applied to FAB. Using MS/MS, the technique provides a fast, highly sensitive and reliable determination of the primary structure of unknown peptides. This procedure labels N-terminal fragments formed during MS/MS analysis, resulting in a simplification and faster interpretation of the spectra. The performance of the method has been tested with several synthetic peptides and applied to an efficient sequencing of the peptide map, using a nano-scale LC coupled on-line to a tandem mass spectrometer.