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Diabetes, obesity, and cancer affect upward of 15% of the world's population. Interestingly, all three diseases juxtapose dysregulated intracellular signaling with altered metabolic state. Exactly which genetic factors define stable metabolic set points in vivo remains poorly understood. Here, we show that hedgehog signaling rewires cellular metabolism. We identify a cilium-dependent Smo-Ca(2+)-Ampk axis that triggers rapid Warburg-like metabolic reprogramming within minutes of activation and is required for proper metabolic selectivity and flexibility. We show that Smo modulators can uncouple the Smo-Ampk axis from canonical signaling and identify cyclopamine as one of a new class of "selective partial agonists," capable of concomitant inhibition of canonical and activation of noncanonical hedgehog signaling. Intriguingly, activation of the Smo-Ampk axis in vivo drives robust insulin-independent glucose uptake in muscle and brown adipose tissue. These data identify multiple noncanonical endpoints that are pivotal for rational design of hedgehog modulators and provide a new therapeutic avenue for obesity and diabetes.
Assuntos
Tecido Adiposo Marrom/metabolismo , Glicólise , Proteínas Hedgehog/metabolismo , Células Musculares/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Quinases Proteína-Quinases Ativadas por AMP , Adipócitos/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Cílios/metabolismo , Diabetes Mellitus/metabolismo , Humanos , Camundongos , Neoplasias/metabolismo , Obesidade/metabolismo , Proteínas Quinases/metabolismo , Receptor SmoothenedRESUMO
Even though they share many thematical overlaps, plant metabolomics and stable isotope ecology have been rather separate fields mainly due to different mass spectrometry demands. New high-resolution bioanalytical mass spectrometers are now not only offering high-throughput metabolite identification but are also suitable for compound- and intramolecular position-specific isotope analysis in the natural isotope abundance range. In plant metabolomics, label-free metabolic pathway and metabolic flux analysis might become possible when applying this new technology. This is because changes in the commitment of substrates to particular metabolic pathways and the activation or deactivation of others alter enzyme-specific isotope effects. This leads to differences in intramolecular and compound-specific isotope compositions. In plant isotope ecology, position-specific isotope analysis in plant archives informed by metabolic pathway analysis could be used to reconstruct and separate environmental impacts on complex metabolic processes. A technology-driven linkage between the two disciplines could allow to extract information on environment-metabolism interaction from plant archives such as tree rings but also within ecosystems. This would contribute to a holistic understanding of how plants react to environmental drivers, thus also providing helpful information on the trajectories of the vegetation under the conditions to come.
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Extreme droughts can have long-lasting effects on forest community dynamics and species interactions. Yet, our understanding of how drought legacy modulates ecological relationships is just unfolding. We tested the hypothesis that leaf chemistry and herbivory show long-term responses to premature defoliation caused by an extreme drought event in European beech (Fagus sylvatica L.). For two consecutive years after the extreme European summer drought in 2018, we collected leaves from the upper and lower canopy of adjacently growing drought-stressed and unstressed trees. Leaf chemistry was analyzed and leaf damage by different herbivore-feeding guilds was quantified. We found that drought had lasting impacts on leaf nutrients and on specialized metabolomic profiles. However, drought did not affect the primary metabolome. Drought-related phytochemical changes affected damage of leaf-chewing herbivores whereas damage caused by other herbivore-feeding guilds was largely unaffected. Drought legacy effects on phytochemistry and herbivory were often weaker than between-year or between-canopy strata variability. Our findings suggest that a single extreme drought event bears the potential to long-lastingly affect tree-herbivore interactions. Drought legacy effects likely become more important in modulating tree-herbivore interactions since drought frequency and severity are projected to globally increase in the coming decades.
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Secas , Fagus , Herbivoria , Compostos Fitoquímicos , Folhas de Planta , Fagus/fisiologia , Herbivoria/fisiologia , Folhas de Planta/fisiologia , Animais , MetabolomaRESUMO
The link between cancer and aberrant glycosylation has recently become evident. Glycans and their altered forms, known as tumour-associated carbohydrate antigens (TACAs), are diverse, complex and difficult to target therapeutically. Lectins are naturally occurring glycan-binding proteins that offer a unique opportunity to recognise TACAs. T cells expressing chimeric antigen receptors (CARs) have proven to be a successful immunotherapy against leukaemias, but so far have shown limited success in solid tumours. We developed a panel of lectin-CARs that recognise the glycosphingolipid globotriaosylceramide (Gb3), which is overexpressed in various cancers, such as Burkitt's lymphoma, colorectal, breast and pancreatic. We have selected the following lectins: Shiga toxin's B-subunit from Shigella dysenteriae, LecA from Pseudomonas aeruginosa, and the engineered lectin Mitsuba from Mytilus galloprovincialis as antigen-binding domains and fused them to a well-known second-generation CAR. The Gb3-binding lectin-CARs have demonstrated target-specific cytotoxicity against Burkitt's lymphoma-derived cell lines as well as solid tumour cells from colorectal and triple-negative breast cancer. Our findings reveal the big potential of lectin-based CARs as therapeutical applications to target Gb3 and other TACAs expressed in haematological malignancies and solid tumours.
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Linfoma de Burkitt , Neoplasias Colorretais , Receptores de Antígenos Quiméricos , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/terapia , Humanos , Lectinas/metabolismo , Polissacarídeos/metabolismo , Linfócitos TRESUMO
The opportunistic pathogen Pseudomonas aeruginosa has gained precedence over the years due to its ability to develop resistance to existing antibiotics, thereby necessitating alternative strategies to understand and combat the bacterium. Our previous work identified the interaction between the bacterial lectin LecA and its host cell glycosphingolipid receptor globotriaosylceramide (Gb3) as a crucial step for the engulfment of P. aeruginosa via the lipid zipper mechanism. In this study, we define the LecA-associated host cell membrane domain by pull-down and mass spectrometry analysis. We unraveled a predilection of LecA for binding to saturated, long fatty acyl chain-containing Gb3 species in the extracellular membrane leaflet and an induction of dynamic phosphatidylinositol (3,4,5)-trisphosphate (PIP3) clusters at the intracellular leaflet co-localizing with sites of LecA binding. We found flotillins and the GPI-anchored protein CD59 not only to be an integral part of the LecA-interacting membrane domain, but also majorly influencing bacterial invasion as depletion of either of these host cell proteins resulted in about 50% reduced invasiveness of the P. aeruginosa strain PAO1. In summary, we report that the LecA-Gb3 interaction at the extracellular leaflet induces the formation of a plasma membrane domain enriched in saturated Gb3 species, CD59, PIP3 and flotillin thereby facilitating efficient uptake of PAO1.
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Antígenos CD59/metabolismo , Membrana Celular/metabolismo , Interações Hospedeiro-Patógeno , Pulmão/microbiologia , Proteínas de Membrana/metabolismo , Pseudomonas aeruginosa/isolamento & purificação , Triexosilceramidas/metabolismo , Transporte Biológico , Antígenos CD59/genética , Endocitose , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Humanos , Pulmão/metabolismo , Pulmão/patologia , Proteínas de Membrana/genética , Pseudomonas aeruginosa/fisiologia , Transdução de SinaisRESUMO
Vascular endothelial growth factor A (VEGFA) secretion from podocytes is crucial for maintaining endothelial integrity within the glomerular filtration barrier. However, until now, the molecular mechanisms underlying podocyte secretory function remained unclear. Through podocyte-specific deletion of BECLIN1 (ATG6 or Becn1), a key protein in autophagy initiation, we identified a major role for this molecule in anterograde Golgi trafficking. The Becn1-deficient podocytes displayed aberrant vesicle formation in the trans-Golgi network (TGN), leading to dramatic vesicle accumulation and complex disrupted patterns of intracellular vesicle trafficking and membrane dynamics. Phenotypically, podocyte-specific deletion of Becn1 resulted in early-onset glomerulosclerosis, which rapidly progressed and dramatically reduced mouse life span. Further, in vivo and in vitro studies clearly showed that VEGFA secretion, and thereby endothelial integrity, greatly depended on BECLIN1 availability and function. Being the first to demonstrate the importance of a secretory pathway for podocyte integrity and function, we identified BECLIN1 as a key component in this complex cellular process. Functionally, by promoting VEGFA secretion, a specific secretory pathway emerged as an essential component for the podocyte-endothelial crosstalk that maintains the glomerular filtration barrier.
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Podócitos , Animais , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Barreira de Filtração Glomerular/metabolismo , Camundongos , Podócitos/metabolismo , Via Secretória , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Magic Spot Nucleotides (MSN) regulate the stringent response, a highly conserved bacterial stress adaptation mechanism, enabling survival under adverse external challenges. In times of antibiotic crisis, a detailed understanding of stringent response is essential, as potentially new targets for pharmacological intervention could be identified. In this study, we delineate the MSN interactome in Escherichia coli and Salmonella typhimurium applying a family of trifunctional photoaffinity capture compounds. We introduce MSN probes covering a diverse phosphorylation pattern, such as pppGpp, ppGpp, and pGpp. Our chemical proteomics approach provides datasets of putative MSN receptors both from cytosolic and membrane fractions that unveil new MSN targets. We find that the activity of the non-Nudix hydrolase ApaH is potently inhibited by pppGpp, which itself is converted to pGpp by ApaH. The capture compounds described herein will be useful to identify MSN interactomes across bacterial species.
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Regulação Bacteriana da Expressão Gênica , Guanosina Pentafosfato , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Guanosina Tetrafosfato , NucleotídeosRESUMO
PURPOSE: Dioxygenases are oxidoreductase enzymes with roles in metabolic pathways necessary for aerobic life. 4-hydroxyphenylpyruvate dioxygenase-like protein (HPDL), encoded by HPDL, is an orphan paralogue of 4-hydroxyphenylpyruvate dioxygenase (HPD), an iron-dependent dioxygenase involved in tyrosine catabolism. The function and association of HPDL with human diseases remain unknown. METHODS: We applied exome sequencing in a cohort of over 10,000 individuals with neurodevelopmental diseases. Effects of HPDL loss were investigated in vitro and in vivo, and through mass spectrometry analysis. Evolutionary analysis was performed to investigate the potential functional separation of HPDL from HPD. RESULTS: We identified biallelic variants in HPDL in eight families displaying recessive inheritance. Knockout mice closely phenocopied humans and showed evidence of apoptosis in multiple cellular lineages within the cerebral cortex. HPDL is a single-exonic gene that likely arose from a retrotransposition event at the base of the tetrapod lineage, and unlike HPD, HPDL is mitochondria-localized. Metabolic profiling of HPDL mutant cells and mice showed no evidence of altered tyrosine metabolites, but rather notable accumulations in other metabolic pathways. CONCLUSION: The mitochondrial localization, along with its disrupted metabolic profile, suggests HPDL loss in humans links to a unique neurometabolic mitochondrial infantile neurodegenerative condition.
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4-Hidroxifenilpiruvato Dioxigenase , Dioxigenases , 4-Hidroxifenilpiruvato Dioxigenase/genética , Animais , Éxons , Humanos , Camundongos , Camundongos Knockout , FenótipoRESUMO
INTRODUCTION: One approach to dampen the inflammatory reactions resulting from implantation surgery of cochlear implant hearing aids is to embed dexamethasone into the matrix of the electrode carrier. Possible side effects for sensory cells in the inner ear on the metabolomics have not yet been evaluated. OBJECTIVE: We examined changes in the metabolome of the HEI-OC1 cell line after dexamethasone incubation as a cell model of sensory cells of the inner ear. RESULTS AND CONCLUSION: Untargeted GC-MS-profiling of metabolic alterations after dexamethasone treatment showed that dexamethasone had antithetical effects on the metabolic signature of the cells depending on growth conditions. The differentiated state of HEI-OC1 cells is better suited for elucidating metabolic changes induced by external factors. Dexamethasone treatment of differentiated cells led to an increase in intracellular amino acids and enhanced glucose uptake and ß-oxidation in the cells. Increased availability of precursors for glycolysis and ATP production by ß-oxidation stabilizes the energy supply in the cells, which could be assumed to be beneficial in coping with cellular stress. We found no negative effects of dexamethasone on the metabolic level, and changes may even prepare sensory cells to better overcome cellular stress following implantation surgery.
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Orelha Interna , Linhagem Celular , Dexametasona/farmacologiaRESUMO
Vancomycin is a glycopeptide antibiotic used against multi-drug resistant gram-positive bacteria such as Staphylococcus aureus (MRSA). Although invaluable against resistant bacteria, vancomycin harbors adverse drug reactions including cytopenia, ototoxicity, as well as nephrotoxicity. Since nephrotoxicity is a rarely occurring side effect, its mechanism is incompletely understood. Only recently, the actual clinically relevant concentration the in kidneys of patients receiving vancomycin was investigated and were found to exceed plasma concentrations by far. We applied these clinically relevant vancomycin concentrations to murine and canine renal epithelial cell lines and assessed metabolic and lipidomic alterations by untargeted and targeted gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analyses. Despite marked differences in the lipidome, both cell lines increased anabolic glucose reactions, resulting in higher sorbitol and lactate levels. To the best of our knowledge, this is the first endometabolic profiling of kidney cells exposed to clinically relevant vancomycin concentrations. The presented study will provide a valuable dataset to nephrotoxicity researchers and might add to unveiling the nephrotoxic mechanism of vancomycin.
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Rim/efeitos dos fármacos , Lipidômica , Vancomicina/farmacologia , Animais , Antibacterianos/farmacologia , Cromatografia Líquida , Cães , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Cromatografia Gasosa-Espectrometria de Massas , Glutationa/metabolismo , Túbulos Renais Coletores/metabolismo , Lipídeos/química , Células Madin Darby de Rim Canino , Espectrometria de Massas , Metabolômica , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Infecções Estafilocócicas/tratamento farmacológicoRESUMO
Podocyte maintenance and stress resistance are exquisitely based on high basal rates of autophagy making these cells a unique model to unravel mechanisms of autophagy regulation. Polyamines have key cellular functions such as proliferation, nucleic acid biosynthesis and autophagy. Here we test whether endogenous spermidine signaling is a driver of basal and dynamic autophagy in podocytes by using genetic and pharmacologic approaches to interfere with different steps of polyamine metabolism. Translational studies revealed altered spermidine signaling in focal segmental glomerulosclerosis in vivo and in vitro. Exogenous spermidine supplementation emerged as new treatment strategy by successfully activating autophagy in vivo via inhibition of EP300, a protein with an essential role in controlling cell growth, cell division and prompting cells to differentiate to take on specialized functions. Surprisingly, gas chromatography-mass spectroscopy based untargeted metabolomics of wild type and autophagy deficient primary podocytes revealed a positive feedback mechanism whereby autophagy itself maintains polyamine metabolism and spermidine synthesis. The transcription factor MAFB acted as an upstream regulator of polyamine metabolism. Thus, our data highlight a novel positive feedback loop of autophagy and spermidine signaling allowing maintenance of high basal levels of autophagy as a key mechanism to sustain the filtration barrier. Hence, spermidine supplementation may emerge as a new therapeutic to restore autophagy in glomerular disease.
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Glomerulosclerose Segmentar e Focal , Podócitos , Espermidina , Autofagia , Proliferação de Células , Humanos , Espermidina/metabolismoRESUMO
The changes in the myocardial proteome and metabolome associated with left ventricular assist device (LVAD) therapy in patients with ischemic cardiomyopathy (ICM) are poorly characterized. We investigated the impact of mechanical unloading following LVAD therapy on the myocardial proteome and metabolome. Matched samples of 5 patients' myocardial tissue, harvested at the time of LVAD implant ("pre-LVAD") or heart transplant ("post-LVAD"), were studied by quantitative proteomics and metabolomics as well as being probed for T-tubule structure and connexin-43 distribution. Moreover, pre-LVAD proteome profiles of ICM context were bioinformatically compared to pre-LVAD proteome profiles of dilated cardiac myopathy (DCM). More than 2120 proteins were reliably identified and quantified in paired patient samples. LVAD therapy led to proteomic remodeling, including reduced levels of α-1-antichymotrypsin together with an overall decrease of immune response proteins and an increase of proteins involved in membrane biology. Metabolomics highlighted increased glucose and glucose-6-phosphate levels in the left ventricle upon LVAD therapy. Wheat germ agglutinin staining demonstrated improved T-tubule structure. Connexin-43 displayed a trend for more pronounced intercalated disc localization. In comparing pre-LVAD proteome profiles of ICM context with pre-LVAD proteome profiles of dilated cardiac myopathy (DCM), we noticed an overrepresentation in ICM of proteins associated with humoral immune response. Our findings underline an impact of LVAD therapy on left ventricular biology in ICM. The proteomic, metabolomic, and structural alterations described here are typically associated with cardiac recovery. On the molecular level, our findings indicate the possibility of cardiac remodeling under LVAD therapy in ICM.
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Ventrículos do Coração/metabolismo , Coração Auxiliar , Metaboloma , Isquemia Miocárdica/terapia , Proteoma/metabolismo , Idoso , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Conexina 43/análise , Conexina 43/metabolismo , Feminino , Glucose/análise , Glucose/metabolismo , Ventrículos do Coração/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Proteoma/análiseRESUMO
INTRODUCTION: Nephronophthisis (NPH) is an inherited form of cystic kidney disease with various extrarenal manifestations accounting for the largest amount of endstage renal disease in childhood. Patient mutations of Anks6 have also been found to cause NPH like phenotypes in animal models. However, little is known about functionality of Anks6. OBJECTIVES/METHODS: We investigated the impact of Anks6 depletion on cellular metabolism of inner medullary collecting duct cells by GC-MS profiling and targeted LC-MS/MS analysis using two different shRNA cell lines for tetracycline-inducible Anks6 downregulation, namely mIMCD3 krab shANKS6 i52 and mIMCD3 krab shANKS6 i12. RESULTS: In combination, we could successfully identify 158 metabolites of which 20 compounds showed similar alterations in both knockdown systems. Especially, large neutral amino acids, such as phenylalanine, where found to be significantly downregulated indicating disturbances in amino acid metabolism. Arginine, lysine and spermidine, which play an important role in cell survival and proliferation, were found to be downregulated. Accordingly, cell growth was diminished in tet treated mIMCD3 krab shANKS6 i52 knockdown cells. Deoxynucleosides were significantly downregulated in both knockdown systems. Hence, PARP1 levels were increased in tet treated mIMCD3 krab shANKS6 i52 cells, but not in tet treated mIMCD3 krab shANKS6 i12 cells. However, yH2AX was found to be increased in the latter. CONCLUSION: In combination, we hypothesise that Anks6 affects DNA damage responses and proliferation and plays a crucial role in physiological amino acid and purine/pyrimidine metabolism.
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Proteínas de Transporte/metabolismo , Metabolômica , Animais , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Cromatografia Líquida , Cromatografia Gasosa-Espectrometria de Massas , Doenças Renais Císticas/metabolismo , Doenças Renais Císticas/patologia , Camundongos , Camundongos KnockoutRESUMO
Ion mobility coupling to mass spectrometry facilitates enhanced identification certitude. Further coupling to liquid chromatography results in multi-dimensional analytical methods, especially suitable for complex matrices with structurally similar compounds. Modified nucleosides represent a large group of very similar members linked to aberrant proliferation. Besides basal production under physiological conditions, they are increasingly excreted by transformed cells and subsequently discussed as putative biomarkers for various cancer types. Here, we report a method for modified nucleosides covering 37 species. We determined collisional cross-sections with high reproducibility from pure analytical standards. For sample purification, we applied an optimized phenylboronic acid solid-phase extraction on media obtained from four different pancreatic cancer cell lines. Our analysis could discriminate different subtypes of pancreatic cancer cell lines. Importantly, they could clearly be separated from a pancreatic control cell line as well as blank medium. m1A, m27G, and Asm were the most important features discriminating cancer cell lines derived from well-differentiated and poorly differentiated cancers. Eventually, we suggest the analytical method reported here for future tumor-marker identification studies. Graphical abstract.
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Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodos , Neoplasias Pancreáticas/metabolismo , RNA Neoplásico/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Pancreáticas/patologiaRESUMO
PURPOSE: Expression profile alterations of nine breast cancer (BC)-associated secreted microRNAs (miRs) were determined under microenvironmental alterations occurring in tumour progression, metastasis or specific oncological treatment modalities. Thereto, the potential influence of the exogenic stimuli hypoxia, acidosis and hyperthermia was investigated in vitro. MATERIAL AND METHODS: Four established BC cell lines were applied as in vitro BC model systems. Quantitative analyses of secreted microRNA specimens were performed by RNA isolation from cell culture supernatant and subsequent real-time PCR in cells under physiological versus hypoxic, acidic or hyperthermia conditions. RESULTS: The in vitro application of exogenic stimuli hypoxia, extracellular acidosis and hyperthermia caused heterogeneous expression alterations for the investigated secreted miRNA phenotypes. The majority of relevant exogenic stimuli-dependent microRNA expression alterations were restricted to single events displaying distinct cell type and stimulus dependent correlations only. Most remarkably, hyperthermia triggered a uniform significant down-regulatory effect on the expression levels of the three secreted microRNAs miR-10b, miR-15b and miR-139, respectively. The marked decrease in miR-10b and miR-15b levels was detectable in all four, while miR-139 was found significantly reduced in three out of four BC cell lines. CONCLUSION: Hyperthermia-dependent down-regulatory influence on three distinct BC-related microRNAs in vitro generates translational aspects for clinical BC treatment, since the identified microRNAs miR-10b, miR-15b and miR-139 are known to have oncogenic as well as tumour suppressor functions in BC. However, an evaluation regarding the potential impact of microRNA-related hyperthermia-dependent alterations for innovative BC treatment approaches demands further analysis including in vivo data.
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Neoplasias da Mama/genética , Hipertermia Induzida/efeitos adversos , MicroRNAs/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , HumanosRESUMO
XD14 is a 4-acyl pyrrole derivative, which was discovered by a high-throughput virtual screening experiment. XD14 inhibits bromodomain and extra-terminal domain (BET) proteins (BRD2, BRD3, BRD4 and BRDT) and consequently suppresses cell proliferation. In this study, metabolic profiling reveals the molecular effects in the human breast cancer cell line MCF-7 (Michigan Cancer Foundation-7) treated by XD14. A three-day time series experiment with two concentrations of XD14 was performed. Gas chromatography-mass spectrometry (GC-MS) was applied for untargeted profiling of treated and non-treated MCF-7 cells. The gained data sets were evaluated by several statistical methods: analysis of variance (ANOVA), clustering analysis, principle component analysis (PCA), and partial least squares discriminant analysis (PLS-DA). Cell proliferation was strongly inhibited by treatment with 50 µM XD14. Samples could be discriminated by time and XD14 concentration using PLS-DA. From the 117 identified metabolites, 67 were significantly altered after XD14 treatment. These metabolites include amino acids, fatty acids, Krebs cycle and glycolysis intermediates, as well as compounds of purine and pyrimidine metabolism. This massive intervention in energy metabolism and the lack of available nucleotides could explain the decreased proliferation rate of the cancer cells.
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Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Metaboloma/efeitos dos fármacos , Pirróis/farmacologia , Antineoplásicos/química , Mama/efeitos dos fármacos , Mama/metabolismo , Análise Discriminante , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Análise dos Mínimos Quadrados , Células MCF-7 , Redes e Vias Metabólicas/efeitos dos fármacos , Metabolômica , Análise de Componente Principal , Pirróis/químicaRESUMO
Sirtuin 3 (SIRT3) is a mitochondrial NAD(+)-dependent deacetylase that regulates energy metabolic enzymes by reversible protein lysine acetylation in various extracardiac tissues. The role of SIRT3 in myocardial energetics and in the development of mitochondrial dysfunction in cardiac pathologies, such as the failing heart, remains to be elucidated. To investigate the role of SIRT3 in the regulation of myocardial energetics and function SIRT3(-/-) mice developed progressive age-related deterioration of cardiac function, as evidenced by a decrease in ejection fraction and an increase in enddiastolic volume at 24 but not 8 weeks of age using echocardiography. Four weeks following transverse aortic constriction, ejection fraction was further decreased in SIRT3(-/-) mice compared to WT mice, accompanied by a greater degree of cardiac hypertrophy and fibrosis. In isolated working hearts, a decrease in cardiac function in SIRT3(-/-) mice was accompanied by a decrease in palmitate oxidation, glucose oxidation, and oxygen consumption, whereas rates of glycolysis were increased. Respiratory capacity and ATP synthesis were decreased in cardiac mitochondria of SIRT3(-/-) mice. HPLC measurements revealed a decrease of the myocardial ATP/AMP ratio and of myocardial energy charge. Using LC-MS/MS, we identified increased acetylation of 84 mitochondrial proteins, including 6 enzymes of fatty acid import and oxidation, 50 subunits of the electron transport chain, and 3 enzymes of the tricarboxylic acid cycle. Lack of SIRT3 impairs mitochondrial and contractile function in the heart, likely due to increased acetylation of various energy metabolic proteins and subsequent myocardial energy depletion.
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Mitocôndrias Cardíacas/fisiologia , Contração Miocárdica , Sirtuína 3/fisiologia , Animais , Ciclo do Ácido Cítrico , Metabolismo Energético , Masculino , Camundongos , Camundongos Knockout , Fosforilação OxidativaRESUMO
Modified nucleosides derived from the RNA metabolism constitute an important chemical class, which are discussed as potential biomarkers in the detection of mammalian breast cancer. Not only the variability of modifications, but also the complexity of biological matrices such as urinary samples poses challenges in the analysis of modified nucleosides. In the present work, a comprehensive two-dimensional liquid chromatography mass spectrometry (2D-LC-MS) approach for the analysis of modified nucleosides in biological samples was established. For prepurification of urinary samples and cell culture supernatants, we performed a cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. In order to establish a 2D-LC method, we tested numerous column combinations and chromatographic conditions. In order to determine the target compounds, we coupled the 2D-LC setup to a triple quadrupole mass spectrometer performing full scans, neutral loss scans, and multiple reaction monitoring (MRM). The combination of a Zorbax Eclipse Plus C18 column with a Zorbax Bonus-RP column was found to deliver a high degree of orthogonality and adequate separation. By application of 2D-LC-MS approaches, we were able to detect 28 target compounds from RNA metabolism and crosslinked pathways in urinary samples and 26 target compounds in cell culture supernatants, respectively. This is the first demonstration of the applicability and benefit of 2D-LC-MS for the targeted metabolome analysis of modified nucleosides and compounds from crosslinked pathways in different biological matrices.
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Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Metaboloma/fisiologia , Nucleosídeos/análise , Nucleosídeos/metabolismo , RNA/metabolismo , Células MCF-7 , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
RATIONALE: A rapid and precise analytical method for the investigation of natural products is required for pathway monitoring of the biosynthesis of secondary metabolites. Phenalinolactones, used in antibiotic research, are produced by Streptomyces sp. Tü6071. For the analysis of those compounds, prior to mass spectrometric analysis, an efficient separation technique is required. METHODS: For the identification of phenalinolactones from liquid cultures of Streptomyces sp. Tü6071, a new method comprising the combination of solid-phase extraction (SPE) prior to liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was established. MS/MS product ion scans were applied for phenalinolactone detection and structure elucidation, performed in negative mode and optimized for sensitivity and specificity. For the discovery of new intermediates, a MS/MS precursor ion scan was applied. RESULTS: Analysis of the extracts revealed that the Oasis® MAX cartridge, containing a quaternary amine functionality, is the most efficient SPE material for purification of phenalinolactones, since it allowed sufficient enrichment and detection of intermediates from the biosynthetic pathway by LC/ESI-MS/MS. Using the precursor ion scan technique, two new secondary metabolites, PL IM1 with m/z 672.6 and PL IM2 with m/z 433.3, have been detected. The structures of the new intermediates are postulated and arranged into the biosynthetic pathway of phenalinolactones. CONCLUSIONS: A precise analytical method was established for the identification of phenalinolactones by combining purification from Streptomyces using SPE prior to LC/ESI-MS/MS. By optimising LC/ESI-MS/MS settings, this method has been successfully applied for pathway monitoring of secondary metabolites. Application of a precursor ion scan allowed for the identification of unknown intermediates in biosynthetic pathways.
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Cromatografia Líquida/métodos , Diterpenos/metabolismo , Glicosídeos/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Streptomyces/metabolismo , Diterpenos/análise , Glicosídeos/análise , Íons/análise , Íons/química , Redes e Vias Metabólicas , Modelos Moleculares , Espectrometria de Massas em TandemRESUMO
Numerous natural substances have anti-cancer properties. Especially indigenous people use aqueous plant extracts for tea or ointments including Dioscorea sansibarensis Pax to treat various diseases. The aim of this study was to evaluate the cytotoxic and radiosensitizing potential of aqueous extracts from Dioscorea sansibarensis Pax collected from Kenya in a panel of HPV-negative and -positive head and neck squamous cell carcinoma (HNSCC) cells grown in three-dimensional laminin-rich extracellular matrix (3D lrECM). The results show cytotoxicity, radiosensitization and increased levels of residual double strand breaks (DBS) by Dioscorea sansibarensis Pax extracts in HPV-negative and -positive HNSCC models in a concentration- and cell model-dependent manner. Application of ROS scavengers indicated an association between ROS-induced DSB and radiosensitization through Dioscorea sansibarensis Pax pretreatment. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based characterization of Dioscorea sansibarensis Pax identified the main components of the extract including camptothecin. Overall, Dioscorea sansibarensis Pax aqueous extracts alone and in combination with X-ray irradiation showed effective anticancer properties, which are worthy of further mechanistic investigation.