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1.
Cell ; 184(18): 4651-4668.e25, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34450028

RESUMO

GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.


Assuntos
Produtos Biológicos/uso terapêutico , Encéfalo/metabolismo , Doenças por Armazenamento dos Lisossomos/terapia , Progranulinas/uso terapêutico , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Endossomos/metabolismo , Feminino , Demência Frontotemporal/sangue , Demência Frontotemporal/líquido cefalorraquidiano , Gliose/complicações , Gliose/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Inflamação/patologia , Metabolismo dos Lipídeos , Lipofuscina/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Degeneração Neural/patologia , Fenótipo , Progranulinas/deficiência , Progranulinas/metabolismo , Receptores Imunológicos/metabolismo , Receptores da Transferrina/metabolismo , Distribuição Tecidual
3.
Mass Spectrom Rev ; 41(5): 722-765, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-33522625

RESUMO

Researchers worldwide are taking advantage of novel, commercially available, technologies, such as ion mobility mass spectrometry (IM-MS), for metabolomics and lipidomics applications in a variety of fields including life, biomedical, and food sciences. IM-MS provides three main technical advantages over traditional LC-MS workflows. Firstly, in addition to mass, IM-MS allows collision cross-section values to be measured for metabolites and lipids, a physicochemical identifier related to the chemical shape of an analyte that increases the confidence of identification. Second, IM-MS increases peak capacity and the signal-to-noise, improving fingerprinting as well as quantification, and better defining the spatial localization of metabolites and lipids in biological and food samples. Third, IM-MS can be coupled with various fragmentation modes, adding new tools to improve structural characterization and molecular annotation. Here, we review the state-of-the-art in IM-MS technologies and approaches utilized to support metabolomics and lipidomics applications and we assess the challenges and opportunities in this growing field.


Assuntos
Espectrometria de Mobilidade Iônica , Lipidômica , Espectrometria de Mobilidade Iônica/métodos , Lipídeos/análise , Espectrometria de Massas/métodos , Metabolômica/métodos
4.
Int J Mol Sci ; 21(24)2020 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-33348903

RESUMO

Gangliosides constitute a subgroup of glycosphingolipids characterized by the presence of sialic acid residues in their structure. As constituents of cellular membranes, in particular of raft microdomains, they exert multiple functions, some of them capital in cell homeostasis. Their presence in cells is tightly regulated by a balanced expression and function of the enzymes responsible for their biosynthesis, ganglioside synthases, and their degradation, glycosidases. The dysregulation of their abundance results in rare and common diseases. In this review, we make a point on the relevance of gangliosides and some of their metabolic precursors, such as ceramides, in the function of podocytes, the main cellular component of the glomerular filtration barrier, as well as their implications in podocytopathies. The results presented in this review suggest the pertinence of clinical lipidomic studies targeting these metabolites.


Assuntos
Membrana Celular/metabolismo , Gangliosídeos/metabolismo , Barreira de Filtração Glomerular/metabolismo , Podócitos/patologia , Animais , Humanos , Podócitos/metabolismo
5.
Int J Mol Sci ; 21(15)2020 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-32707880

RESUMO

Mucopolysaccharidosis type II is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS) and characterized by the accumulation of the primary storage substrate, glycosaminoglycans (GAGs). Understanding central nervous system (CNS) pathophysiology in neuronopathic MPS II (nMPS II) has been hindered by the lack of CNS biomarkers. Characterization of fluid biomarkers has been largely focused on evaluating GAGs in cerebrospinal fluid (CSF) and the periphery; however, GAG levels alone do not accurately reflect the broad cellular dysfunction in the brains of MPS II patients. We utilized a preclinical mouse model of MPS II, treated with a brain penetrant form of IDS (ETV:IDS) to establish the relationship between markers of primary storage and downstream pathway biomarkers in the brain and CSF. We extended the characterization of pathway and neurodegeneration biomarkers to nMPS II patient samples. In addition to the accumulation of CSF GAGs, nMPS II patients show elevated levels of lysosomal lipids, neurofilament light chain, and other biomarkers of neuronal damage and degeneration. Furthermore, we find that these biomarkers of downstream pathology are tightly correlated with heparan sulfate. Exploration of the responsiveness of not only CSF GAGs but also pathway and disease-relevant biomarkers during drug development will be crucial for monitoring disease progression, and the development of effective therapies for nMPS II.


Assuntos
Encéfalo/metabolismo , Glicosaminoglicanos/metabolismo , Iduronato Sulfatase/metabolismo , Metabolismo dos Lipídeos , Lisossomos/metabolismo , Mucopolissacaridose II/sangue , Mucopolissacaridose II/líquido cefalorraquidiano , Adolescente , Animais , Biomarcadores/metabolismo , Encéfalo/patologia , Criança , Pré-Escolar , Dermatan Sulfato/sangue , Dermatan Sulfato/líquido cefalorraquidiano , Dermatan Sulfato/metabolismo , Terapia de Reposição de Enzimas , Feminino , Gangliosídeos/metabolismo , Glicosaminoglicanos/líquido cefalorraquidiano , Transplante de Células-Tronco Hematopoéticas , Heparitina Sulfato/sangue , Heparitina Sulfato/líquido cefalorraquidiano , Heparitina Sulfato/metabolismo , Humanos , Iduronato Sulfatase/genética , Iduronato Sulfatase/farmacologia , Lactente , Inflamação/metabolismo , Lisossomos/patologia , Masculino , Espectrometria de Massas , Camundongos , Camundongos Knockout , Mucopolissacaridose II/metabolismo , Mucopolissacaridose II/terapia , Proteínas de Neurofilamentos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
6.
Int J Mol Sci ; 21(15)2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32751752

RESUMO

We recently developed a blood-brain barrier (BBB)-penetrating enzyme transport vehicle (ETV) fused to the lysosomal enzyme iduronate 2-sulfatase (ETV:IDS) and demonstrated its ability to reduce glycosaminoglycan (GAG) accumulation in the brains of a mouse model of mucopolysaccharidosis (MPS) II. To accurately quantify GAGs, we developed a plate-based high-throughput enzymatic digestion assay coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously measure heparan sulfate and dermatan sulfate derived disaccharides in tissue, cerebrospinal fluid (CSF) and individual cell populations isolated from mouse brain. The method offers ultra-high sensitivity enabling quantitation of specific GAG species in as low as 100,000 isolated neurons and a low volume of CSF. With an LOD at 3 ng/mL and LLOQs at 5-10 ng/mL, this method is at least five times more sensitive than previously reported approaches. Our analysis demonstrated that the accumulation of CSF and brain GAGs are in good correlation, supporting the potential use of CSF GAGs as a surrogate biomarker for brain GAGs. The bioanalytical method was qualified through the generation of standard curves in matrix for preclinical studies of CSF, demonstrating the feasibility of this assay for evaluating therapeutic effects of ETV:IDS in future studies and applications in a wide variety of MPS disorders.


Assuntos
Biomarcadores/metabolismo , Glicosaminoglicanos/isolamento & purificação , Iduronato Sulfatase/genética , Mucopolissacaridose II/diagnóstico , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Cromatografia Líquida , Dermatan Sulfato/farmacologia , Dissacarídeos/química , Modelos Animais de Doenças , Glicosaminoglicanos/genética , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/farmacologia , Humanos , Iduronato Sulfatase/metabolismo , Camundongos , Mucopolissacaridose II/genética , Mucopolissacaridose II/patologia , Espectrometria de Massas em Tandem
7.
Anal Chem ; 91(14): 9266-9276, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31149811

RESUMO

Algae biomass is formed by an extremely complex set of metabolites, and its molecular characterization has been very challenging. We report the characterization of microalgae extracts via traveling wave ion mobility-mass spectrometry (TWIM-MS) by two different analysis strategies. First, the extracts were analyzed by direct infusion electrospray ionization (ESI) with no previous chromatographic separation (DI-ESI-TWIM-MS). Second, the samples were screened for metabolites and lipids using an untargeted high-throughput method that employs ultrahigh-performance liquid chromatography (UHPLC) using data-independent analysis (DIA) - MSE (UHPLC-HDMSE). Sixteen different microalgae biomasses were evaluated by both strategies. DI-ESI-TWIM-MS was able, via distinct drift times, to set apart different classes of metabolites, with the differences in the profiles of each microalga readily evident. With the UHPLC-HDMSE approach, 1251 different compounds were putatively annotated across 16 samples with 210 classified as lipids. From the normalized abundance for each annotated compound category, a detailed profiling in terms of metabolites, lipids, and lipid classes of each sample was performed. The reported workflow represents a powerful tool to determine the most suitable biotechnological applications for a given alga type and may allow for real-time monitoring of the algae composition distribution as a function of growth conditions, feedstocks, and the like. The determination of collision cross section results in improved confidence in the identification of triacylglycerols in samples, highly applicable to biofuels production. The two analysis strategies explored in this work offer powerful tools for the biomass industry by aiding in the identification of ideal strains and culture conditions for a specific application, saving analysis time and facilitating identification of a large number of constituents at once.


Assuntos
Biomassa , Espectrometria de Mobilidade Iônica/métodos , Lipídeos/análise , Metaboloma , Microalgas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Cromatografia Líquida de Alta Pressão , Lipidômica/métodos , Metabolômica/métodos
8.
Proc Natl Acad Sci U S A ; 112(8): 2431-6, 2015 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-25675482

RESUMO

Tetrahydrobiopterin is a cofactor synthesized from GTP with well-known roles in enzymatic nitric oxide synthesis and aromatic amino acid hydroxylation. It is used to treat mild forms of phenylketonuria. Less is known about the role of tetrahydrobiopterin in lipid metabolism, although it is essential for irreversible ether lipid cleavage by alkylglycerol monooxygenase. Here we found intracellular alkylglycerol monooxygenase activity to be an important regulator of alkylglycerol metabolism in intact murine RAW264.7 macrophage-like cells. Alkylglycerol monooxygenase was expressed and active also in primary mouse bone marrow-derived monocytes and "alternatively activated" M2 macrophages obtained by interleukin 4 treatment, but almost missing in M1 macrophages obtained by IFN-γ and lipopolysaccharide treatment. The cellular lipidome of RAW264.7 was markedly changed in a parallel way by modulation of alkylglycerol monooxygenase expression and of tetrahydrobiopterin biosynthesis affecting not only various ether lipid species upstream of alkylglycerol monooxygenase but also other more complex lipids including glycosylated ceramides and cardiolipins, which have no direct connection to ether lipid pathways. Alkylglycerol monooxygenase activity manipulation modulated the IFN-γ/lipopolysaccharide-induced expression of inducible nitric oxide synthase, interleukin-1ß, and interleukin 1 receptor antagonist but not transforming growth factor ß1, suggesting that alkylglycerol monooxygenase activity affects IFN-γ/lipopolysaccharide signaling. Our results demonstrate a central role of tetrahydrobiopterin and alkylglycerol monooxygenase in ether lipid metabolism of murine macrophages and reveal that alteration of alkylglycerol monooxygenase activity has a profound impact on the lipidome also beyond the class of ether lipids.


Assuntos
Biopterinas/análogos & derivados , Metabolismo dos Lipídeos/efeitos dos fármacos , Macrófagos/metabolismo , Oxigenases de Função Mista/metabolismo , Animais , Biopterinas/farmacologia , Células da Medula Óssea/citologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Análise por Conglomerados , GTP Cicloidrolase/metabolismo , Técnicas de Silenciamento de Genes , Interferon gama/farmacologia , Lentivirus/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Óxido Nítrico Sintase Tipo II/metabolismo
9.
J Proteome Res ; 16(10): 3805-3815, 2017 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-28825479

RESUMO

The potential for radiological accidents and nuclear terrorism has increased the need for the development of new rapid biodosimetry methods. In addition, in a clinical setting the issue of an individual's radiosensitivity should be taken into consideration during radiotherapy. We utilized metabolomics and lipidomics to investigate changes of metabolites in serum samples following exposure to total body ionizing radiation in humans. Serum was collected prior to irradiation, at 3-8 h after a single dose of 1.25-2 Gy, and at 24 h with a total delivered dose of 2-3.75 Gy. Metabolomics revealed perturbations in glycerophosphocholine, phenylalanine, ubiquinone Q2, and oxalic acid. Alterations were observed in circulating levels of lipids from monoacylglycerol, triacylglycerol, phosphatidylcholine, and phosphatidylglycerol lipid classes. Polyunsaturated fatty acids were some of the most dysregulated lipids, with increased levels linked to proinflammatory processes. A targeted metabolomics approach for eicosanoids was also employed. The results showed a rapid response for proinflammatory eicosanoids, with a dampening of the signal at the later time point. Sex differences were observed in the markers from the untargeted approach but not the targeted method. The ability to identify and quantify small molecules in blood can therefore be utilized to monitor radiation exposure in human populations.


Assuntos
Inflamação/sangue , Lipídeos/sangue , Metaboloma/genética , Irradiação Corporal Total/efeitos adversos , Biomarcadores/sangue , Relação Dose-Resposta à Radiação , Eicosanoides/sangue , Eicosanoides/genética , Feminino , Humanos , Inflamação/etiologia , Inflamação/genética , Inflamação/patologia , Lipídeos/efeitos da radiação , Masculino , Metaboloma/efeitos da radiação , Metabolômica/métodos , Exposição à Radiação/efeitos adversos
10.
J Biol Chem ; 291(44): 23318-23329, 2016 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-27634039

RESUMO

Liver regeneration offers a distinctive opportunity to study cell proliferation in vivo Mammalian silent information regulator 1 (SIRT1), a NAD+-dependent histone deacetylase, is an important regulator of various cellular processes, including proliferation, metabolism, and circadian rhythms. In the liver, SIRT1 coordinates the circadian oscillation of clock-controlled genes, including genes that encode enzymes involved in metabolic pathways. We performed partial hepatectomy in WT and liver-specific Sirt1-deficient mice and analyzed the expression of cell cycle regulators in liver samples taken at different times during the regenerative process, by real time PCR, Western blotting analysis, and immunohistochemistry. Lipidomic analysis was performed in the same samples by MS/HPLC. We showed that G1/S progression was significantly affected by absence of SIRT1 in the liver, as well as circadian gene expression. This was associated to lipid accumulation due to defective fatty acid beta-oxidation. Our study revealed for the first time the importance of SIRT1 in the regulation of hepatocellular proliferation, circadian rhythms, and lipid metabolism during liver regeneration in mice. These results represent an additional step toward the characterization of SIRT1 function in the liver.


Assuntos
Ciclo Celular , Proliferação de Células , Metabolismo dos Lipídeos , Hepatopatias/enzimologia , Regeneração Hepática , Fígado/enzimologia , Sirtuína 1/metabolismo , Animais , Humanos , Fígado/metabolismo , Fígado/fisiopatologia , Hepatopatias/genética , Hepatopatias/metabolismo , Hepatopatias/fisiopatologia , Camundongos , Camundongos Knockout , Sirtuína 1/genética
11.
Mar Drugs ; 15(2)2017 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-28241423

RESUMO

Twenty-eight sponge specimens were collected at a shallow water hydrothermal vent site north of Iceland. Extracts were prepared and tested in vitro for cytotoxic activity, and eight of them were shown to be cytotoxic. A mass spectrometry (MS)-based metabolomics approach was used to determine the chemical composition of the extracts. This analysis highlighted clear differences in the metabolomes of three sponge specimens, and all of them were identified as Haliclona (Rhizoniera) rosea (Bowerbank, 1866). Therefore, these specimens were selected for further investigation. Haliclona rosea metabolomes contained a class of potential key compounds, the 3-alkyl pyridine alkaloids (3-APA) responsible for the cytotoxic activity of the fractions. Several 3-APA compounds were tentatively identified including haliclamines, cyclostellettamines, viscosalines and viscosamines. Among these compounds, cyclostellettamine P was tentatively identified for the first time by using ion mobility MS in time-aligned parallel (TAP) fragmentation mode. In this work, we show the potential of applying metabolomics strategies and in particular the utility of coupling ion mobility with MS for the molecular characterization of sponge specimens.


Assuntos
Alcaloides/toxicidade , Fontes Hidrotermais/química , Metaboloma/efeitos dos fármacos , Poríferos/efeitos dos fármacos , Poríferos/metabolismo , Piridinas/toxicidade , Alcaloides/química , Animais , Haliclona/química , Haliclona/metabolismo , Islândia , Metabolômica/métodos , Piridinas/química , Piridinas/metabolismo , Compostos de Piridínio/química , Compostos de Piridínio/metabolismo , Água/química
12.
J Proteome Res ; 15(2): 608-18, 2016 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-26717242

RESUMO

Alzheimer's disease (AD) is the most common cause of adult dementia. Yet the complete set of molecular changes accompanying this inexorable, neurodegenerative disease remains elusive. Here we adopted an unbiased lipidomics and metabolomics approach to surveying frozen frontal cortex samples from clinically characterized AD patients (n = 21) and age-matched controls (n = 19), revealing marked molecular differences between them. Then, by means of metabolomic pathway analysis, we incorporated the novel molecular information into the known biochemical pathways and compared it with the results of a metabolomics meta-analysis of previously published AD research. We found six metabolic pathways of the central metabolism as well as glycerophospholipid metabolism predominantly altered in AD brains. Using targeted metabolomics approaches and MS imaging, we confirmed a marked dysregulation of mitochondrial aspartate metabolism. The altered metabolic pathways were further integrated with clinical data, showing various degrees of correlation with parameters of dementia and AD pathology. Our study highlights specific, altered biochemical pathways in the brains of individuals with AD compared with those of control subjects, emphasizing dysregulation of mitochondrial aspartate metabolism and supporting future venues of investigation.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Metaboloma , Metabolômica/métodos , Mitocôndrias/metabolismo , Idoso , Idoso de 80 Anos ou mais , Autopsia , Feminino , Humanos , Masculino , Redes e Vias Metabólicas , Mudanças Depois da Morte , Espectrometria de Massas por Ionização por Electrospray
13.
Biochim Biophys Acta ; 1851(4): 456-68, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25486530

RESUMO

Oxidation of polyunsaturated fatty acids (PUFA) through enzymatic or non-enzymatic free radical-mediated reactions can yield an array of lipid metabolites including eicosanoids, octadecanoids, docosanoids and related species. In mammals, these oxygenated PUFA mediators play prominent roles in the physiological and pathological regulation of many key biological processes in the cardiovascular, renal, reproductive and other systems including their pivotal contribution to inflammation. Mass spectrometry-based technology platforms have revolutionized our ability to analyze the complex mixture of lipid mediators found in biological samples, with increased numbers of metabolites that can be simultaneously quantified from a single sample in few analytical steps. The recent development of high-sensitivity and high-throughput analytical tools for lipid mediators affords a broader view of these oxygenated PUFA species, and facilitates research into their role in health and disease. In this review, we illustrate current analytical approaches for a high-throughput lipidomic analysis of eicosanoids and related mediators in biological samples. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance."


Assuntos
Ácidos Graxos Insaturados/metabolismo , Ensaios de Triagem em Larga Escala , Animais , Biomarcadores/metabolismo , Cromatografia Líquida , Humanos , Espectrometria de Massas , Oxirredução , Transdução de Sinais , Biologia de Sistemas
14.
Proc Natl Acad Sci U S A ; 110(9): 3333-8, 2013 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-23341587

RESUMO

Circadian rhythms govern a wide variety of physiological and metabolic functions in many organisms, from prokaryotes to humans. We previously reported that silent information regulator 1 (SIRT1), a NAD(+)-dependent deacetylase, contributes to circadian control. In addition, SIRT1 activity is regulated in a cyclic manner in virtue of the circadian oscillation of the coenzyme NAD(+). Here we used specific SIRT1 activator compounds both in vitro and in vivo. We tested a variety of compounds to show that the activation of SIRT1 alters CLOCK:BMAL1-driven transcription in different systems. Activation of SIRT1 induces repression of circadian gene expression and decreases H3 K9/K14 acetylation at corresponding promoters in a time-specific manner. Specific activation of SIRT1 was demonstrated in vivo using liver-specific SIRT1-deficient mice, where the effect of SIRT1 activator compounds was shown to be dependent on SIRT1. Our findings demonstrate that SIRT1 can fine-tune circadian rhythm and pave the way to the development of pharmacological strategies to address a broad range of therapeutic indications.


Assuntos
Ritmo Circadiano/genética , Ativadores de Enzimas/farmacologia , Sirtuína 1/metabolismo , Fatores de Transcrição ARNTL/metabolismo , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Linhagem Celular , Cromatina/metabolismo , Ritmo Circadiano/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Knockout , NAD/metabolismo , Transcrição Gênica/efeitos dos fármacos
15.
J Biol Chem ; 289(9): 6091-7, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24425865

RESUMO

The circadian clock regulates a wide range of physiological and metabolic processes, and its disruption leads to metabolic disorders such as diabetes and obesity. Accumulating evidence reveals that the circadian clock regulates levels of metabolites that, in turn, may regulate the clock. Here we demonstrate that the circadian clock regulates the intracellular levels of acetyl-CoA by modulating the enzymatic activity of acetyl-CoA Synthetase 1 (AceCS1). Acetylation of AceCS1 controls the activity of the enzyme. We show that acetylation of AceCS1 is cyclic and that its rhythmicity requires a functional circadian clock and the NAD(+)-dependent deacetylase SIRT1. Cyclic acetylation of AceCS1 contributes to the rhythmicity of acetyl-CoA levels both in vivo and in cultured cells. Down-regulation of AceCS1 causes a significant decrease in the cellular acetyl-CoA pool, leading to reduction in circadian changes in fatty acid elongation. Thus, a nontranscriptional, enzymatic loop is governed by the circadian clock to control acetyl-CoA levels and fatty acid synthesis.


Assuntos
Acetato-CoA Ligase/metabolismo , Relógios Circadianos/fisiologia , Ácidos Graxos/biossíntese , Sirtuína 1/metabolismo , Acetato-CoA Ligase/genética , Acetilação , Animais , Células Cultivadas , Ácidos Graxos/genética , Camundongos , Camundongos Knockout , NAD/genética , NAD/metabolismo , Sirtuína 1/genética
16.
Anal Chem ; 87(5): 2593-9, 2015 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-25647265

RESUMO

Here, we propose a novel strategy that combines a typical ultra high performance liquid chromatography (UHPLC), data-independent mass spectrometry (MS(E)) workflow with traveling wave ion mobility (TWIM) and UV detection, to improve the characterization of carotenoids and chlorophylls in complex biological matrices. UV detection selectively highlighted pigments absorbing at specific wavelengths, while TWIM coupled to MS was used to maximize the peak capacity. We applied this approach for the analysis of pigments in different microalgae samples, including Chlorella vulgaris, Dunaliella salina, and Phaeodactylum tricornutum. Using UHPLC-UV-MS(E) information (retention time, absorbance at 450 nm, and accurate masses of precursors and product ions), we tentatively identified 26 different pigments (carotenes, chlorophylls, and xanthophylls). By adding TWIM information (collision cross sections), we further resolved 5 isobaric pigments, not resolved by UHPLC-UV-MS(E) alone. The characterization of the molecular phenotypes allowed us to differentiate the microalgae species. Our results demonstrate that a combination of TWIM and UV detection with traditional analytical approaches increases the selectivity and specificity of analysis, providing a new tool to characterize pigments in biological samples. We anticipate that such an analytical approach will be extended to other lipidomics and metabolomics applications.


Assuntos
Produtos Biológicos/análise , Cromatografia Líquida de Alta Pressão/métodos , Microalgas/classificação , Microalgas/metabolismo , Pigmentos Biológicos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos
17.
Anal Chem ; 87(2): 1137-44, 2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25495617

RESUMO

Despite recent advances in analytical and computational chemistry, lipid identification remains a significant challenge in lipidomics. Ion-mobility spectrometry provides an accurate measure of the molecules' rotationally averaged collision cross-section (CCS) in the gas phase and is thus related to ionic shape. Here, we investigate the use of CCS as a highly specific molecular descriptor for identifying lipids in biological samples. Using traveling wave ion mobility mass spectrometry (MS), we measured the CCS values of over 200 lipids within multiple chemical classes. CCS values derived from ion mobility were not affected by instrument settings or chromatographic conditions, and they were highly reproducible on instruments located in independent laboratories (interlaboratory RSD < 3% for 98% of molecules). CCS values were used as additional molecular descriptors to identify brain lipids using a variety of traditional lipidomic approaches. The addition of CCS improved the reproducibility of analysis in a liquid chromatography-MS workflow and maximized the separation of isobaric species and the signal-to-noise ratio in direct-MS analyses (e.g., "shotgun" lipidomics and MS imaging). These results indicate that adding CCS to databases and lipidomics workflows increases the specificity and selectivity of analysis, thus improving the confidence in lipid identification compared to traditional analytical approaches. The CCS/accurate-mass database described here is made publicly available.


Assuntos
Encéfalo/metabolismo , Lipídeos/análise , Espectrometria de Massa de Íon Secundário/métodos , Idoso , Cromatografia Líquida , Humanos , Razão Sinal-Ruído
18.
Anal Bioanal Chem ; 407(17): 4995-5007, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25893801

RESUMO

The high chemical complexity of the lipidome is one of the major challenges in lipidomics research. Ion-mobility spectrometry (IMS), a gas-phase electrophoretic technique, makes possible the separation of ions in the gas phase according to their charge, shape, and size. IMS can be combined with mass spectrometry (MS), adding three major benefits to traditional lipidomic approaches. First, IMS-MS allows the determination of the collision cross section (CCS), a physicochemical measure related to the conformational structure of lipid ions. The CCS is used to improve the confidence of lipid identification. Second, IMS-MS provides a new set of hybrid fragmentation experiments. These experiments, which combine collision-induced dissociation with ion-mobility separation, improve the specificity of MS/MS-based approaches. Third, IMS-MS improves the peak capacity and signal-to-noise ratio of traditional analytical approaches. In doing so, it allows the separation of complex lipid extracts from interfering isobaric species. Developing in parallel with advances in instrumentation, informatics solutions enable analysts to process and exploit IMS-MS data for qualitative and quantitative applications. Here we review the current approaches for lipidomics research based on IMS-MS, including liquid chromatography-MS and direct-MS analyses of "shotgun" lipidomics and MS imaging.


Assuntos
Cromatografia Líquida/métodos , Lipídeos/análise , Espectrometria de Massas/métodos , Animais , Humanos , Íons/análise , Metabolômica/métodos , Modelos Moleculares
19.
Int J Mol Sci ; 16(6): 13678-91, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-26084047

RESUMO

The consumption of vegetables belonging to the family Brassicaceae (e.g., broccoli and cauliflower) is linked to a reduced incidence of cancer and cardiovascular diseases. The molecular composition of such plants is strongly affected by growing conditions. Here we developed an unbiased metabolomics approach to investigate the effect of light and dark exposure on the metabolome of broccoli sprouts and we applied such an approach to provide a bird's-eye view of the overall metabolic response after light exposure. Broccoli seeds were germinated and grown hydroponically for five days in total darkness or with a light/dark photoperiod (16 h light/8 h dark cycle). We used an ultra-performance liquid-chromatography system coupled to an ion-mobility, time-of-flight mass spectrometer to profile the large array of metabolites present in the sprouts. Differences at the metabolite level between groups were analyzed using multivariate statistical analyses, including principal component analysis and correlation analysis. Altered metabolites were identified by searching publicly available and in-house databases. Metabolite pathway analyses were used to support the identification of subtle but significant changes among groups of related metabolites that may have gone unnoticed with conventional approaches. Besides the chlorophyll pathway, light exposure activated the biosynthesis and metabolism of sterol lipids, prenol lipids, and polyunsaturated lipids, which are essential for the photosynthetic machinery. Our results also revealed that light exposure increased the levels of polyketides, including flavonoids, and oxylipins, which play essential roles in the plant's developmental processes and defense mechanism against herbivores. This study highlights the significant contribution of light exposure to the ultimate metabolic phenotype, which might affect the cellular physiology and nutritional value of broccoli sprouts. Furthermore, this study highlights the potential of an unbiased omics approach for the comprehensive study of the metabolism.


Assuntos
Brassica/efeitos da radiação , Metabolismo dos Lipídeos , Metaboloma , Luz Solar , Brassica/metabolismo , Plântula/metabolismo , Plântula/efeitos da radiação
20.
J Proteome Res ; 13(9): 4143-54, 2014 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-25126707

RESUMO

Exposure to ionizing radiation has dramatically increased in modern society, raising serious health concerns. The molecular response to ionizing radiation, however, is still not completely understood. Here, we screened mouse serum for metabolic alterations following an acute exposure to γ radiation using a multiplatform mass-spectrometry-based strategy. A global, molecular profiling revealed that mouse serum undergoes a series of significant molecular alterations following radiation exposure. We identified and quantified bioactive metabolites belonging to key biochemical pathways and low-abundance, oxygenated, polyunsaturated fatty acids (PUFAs) in the two groups of animals. Exposure to γ radiation induced a significant increase in the serum levels of ether phosphatidylcholines (PCs) while decreasing the levels of diacyl PCs carrying PUFAs. In exposed mice, levels of pro-inflammatory, oxygenated metabolites of arachidonic acid increased, whereas levels of anti-inflammatory metabolites of omega-3 PUFAs decreased. Our results indicate a specific serum lipidomic biosignature that could be utilized as an indicator of radiation exposure and as novel target for therapeutic intervention. Monitoring such a molecular response to radiation exposure might have implications not only for radiation pathology but also for countermeasures and personalized medicine.


Assuntos
Ácidos Graxos Ômega-3/sangue , Ácidos Graxos Ômega-3/metabolismo , Metaboloma/efeitos da radiação , Metabolômica/métodos , Radiação Ionizante , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo
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