RESUMO
Cancer cells communicate with the whole organism via extracellular vesicles (EVs), which propagate molecular information in support of the malignant phenotype. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was employed for protein profiling of EVs derived from CCL-228 as the primary colon tumor, the lymph node metastasis CCL-227, and subclones resistant to 5, 25, and 125 µM 5-fluorouracil (FU). EVs were harvested from cell culture supernatant by ultracentrifugation to serve as a model for circulating cancer cell-derived biomarker carriers from body fluids (i.e., liquid biopsy). Protein mass spectra were recorded using standard MALDI matrixes (e.g., CHCA, sinapinic acid) in the range m/ z 2000-20000 on different MALDI-TOF-MS systems and subjected to multivariate data analysis . By using hierarchical clustering, PCA and PLS-DA, discriminatory protein patterns of the EVs from the different cell populations were obtained. Peaks in the range m/ z 2000-6500 and m/ z 5500-15500 were found to be unique to EVs and the cells, respectively. This clearly demonstrates the differential expression of proteins in EVs as the result of an increasing chemoresistance of their parent cells. The sensitivity of the MALDI-MS based assay was in the low µg/mL (≈1.2-5 × 1010 particles/mL) range. Consequently, our MALDI-MS protein profiling approach shows the potential to serve as novel tool for minimally invasive cancer diagnostics and chemotherapy monitoring in the future, e.g., for early detection of therapy resistance without biopsy.
Assuntos
Biomarcadores Tumorais/análise , Resistencia a Medicamentos Antineoplásicos/fisiologia , Vesículas Extracelulares/química , Proteínas de Neoplasias/análise , Proteômica/métodos , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Humanos , Limite de Detecção , Proteínas de Neoplasias/metabolismo , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
We have reported recently that inactivation of the essential autophagy-related gene 7 (Atg7) in keratinocytes has little or no impact on morphology and function of the epidermal barrier in experimental animals. When these mice aged, mutant males, (Atg7 ΔKC), developed an oily coat. As the keratin 14 promoter driven cre/LoxP system inactivates floxed Atg7 in all keratin 14 (K14) expressing cells, including sebocytes, we investigated whether the oily hair phenotype was the consequence of changes in function of the skin sebaceous glands. Using an antibody to the GFP-LC3 fusion protein, autophagosomes were detected at the border of sebocyte disintegration in control but not in mutant animals, suggesting that autophagy was (a) active in normal sebaceous glands and (b) was inactivated in the mutant mice. Detailed analysis established that dorsal sebaceous glands were about twice as large in all Atg7 ΔKC mice compared to those of controls (Atg7 F/F), and their rate of sebocyte proliferation was increased. In addition, male mutant mice yielded twice as much lipid per unit hair as age-matched controls. Analysis of sebum lipids by thin layer chromatography revealed a 40% reduction in the proportion of free fatty acids (FFA) and cholesterol, and a 5-fold increase in the proportion of fatty acid methyl esters (FAME). In addition, the most common diester wax species (58-60 carbon atoms) were increased, while shorter species (54-55 carbon atoms) were under-represented in mutant sebum. Our data show that autophagy contributes to sebaceous gland function and to the control of sebum composition.
Assuntos
Proteína 7 Relacionada à Autofagia/genética , Autofagia/genética , Glândulas Sebáceas/patologia , Glândulas Sebáceas/fisiopatologia , Sebo/química , Animais , Autofagossomos , Proliferação de Células/genética , Colesterol/análise , Ácidos Graxos não Esterificados/análise , Cabelo , Masculino , Camundongos , Fenótipo , Ceras/análiseRESUMO
Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP2) that binds to ion channels, thereby controlling their gating. Channel properties are also attributed to serotonin transporters (SERTs); however, SERT regulation by PIP2 has not been reported. SERTs control neurotransmission by removing serotonin from the extracellular space. An increase in extracellular serotonin results from transporter-mediated efflux triggered by amphetamine-like psychostimulants. Herein, we altered the abundance of PIP2 by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP2-synthesis. We tested the effects of the verified scarcity of PIP2 on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP2 in pull-down assays. On decreased PIP2 availability, amphetamine-evoked currents were markedly reduced compared with controls, as was amphetamine-induced efflux. Signaling downstream of PLC was excluded as a cause for these effects. A reduction of substrate efflux due to PLC activation was also found with recombinant noradrenaline transporters and in rat hippocampal slices. Transmitter uptake was not affected by PIP2 reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP2. Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP2-dependent effects. Substrate uptake and surface expression were comparable between mutant and WT SERTs. These findings demonstrate that PIP2 binding to monoamine transporters is a prerequisite for amphetamine actions without being a requirement for neurotransmitter uptake. These results open the way to target amphetamine-induced SERT-dependent actions independently of normal SERT function and thus to treat psychostimulant addiction.
Assuntos
Anfetamina/farmacologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Células HEK293 , Humanos , Sistemas do Segundo Mensageiro , Proteínas da Membrana Plasmática de Transporte de Serotonina/genéticaRESUMO
Ovarian cancer (OC) is caused by genetic aberrations in networks that control growth and survival. Importantly, aberrant cancer metabolism interacts with oncogenic signaling providing additional drug targets. Tumors overexpress the lipogenic enzyme fatty acid synthase (FASN) and are inhibited by FASN blockers, whereas normal cells are FASN-negative and FASN-inhibitor-resistant. Here, we demonstrate that this holds true when ovarian/oviductal cells reside in their autochthonous tissues, whereas in culture they express FASN and are FASN-inhibitor-sensitive. Upon subculture, nonmalignant cells cease growth, express senescence-associated ß-galactosidase, lose FASN and become FASN-inhibitor-resistant. Immortalized ovarian/oviductal epithelial cell linesalthough resisting senescencereveal distinct growth activities, which correlate with FASN levels and FASN drug sensitivities. Accordingly, ectopic FASN stimulates growth in these cells. Moreover, FASN levels and lipogenic activities affect cellular lipid composition as demonstrated by thin-layer chromatography. Correlation between proliferation and FASN levels was finally evaluated in cancer cells such as HOC-7, which contain subclones with variable differentiation/senescence and corresponding FASN expression/FASN drug sensitivity. Interestingly, senescent phenotypes can be induced in parental HOC-7 by differentiating agents. In OC cells, FASN drugs induce cell cycle blockade in S and/or G2/M and stimulate apoptosis, whereas in normal cells they only cause cell cycle deceleration without apoptosis. Thus, normal cells, although growth-inhibited, may survive and recover from FASN blockade, whereas malignant cells get extinguished. FASN expression and FASN drug sensitivity are directly linked to cell growth and correlate with transformation/differentiation/senescence only indirectly. FASN is therefore a metabolic marker of cell proliferation rather than a marker of malignancy and is a useful target for future drug development.
Assuntos
Biomarcadores Tumorais/genética , Proliferação de Células/genética , Ácido Graxo Sintase Tipo I/genética , Neoplasias Ovarianas/genética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológicoRESUMO
In this paper we present a pioneering approach exploiting nanoparticles (NPs) for the "on-probe" (i.e., directly from the NP-surface) monitoring of OxPLs by MALDI-MS (i.e., the Nano-MALDI approach). The "electrophilic interaction" with either metal oxide (e.g., ZrO2) or surface-functionalized Fe3O4 core-shell superparamagnetic NPs (100 nm diameter) was exploited for the direct enrichment of short-chain carboxylic (CARBO)-OxPLs, whereas detection of aldehydic (ALDO)-OxPLs was enabled by prior derivatization with bifunctional carbonyl-reactive reagents containing a negatively charged moiety (e.g., 4-AA) followed by NP-binding. Polyetheramine (PEA)-NPs were found best suited in terms of solvent stability, binding efficiency and compatibility with MALDI-MS analysis. For quantitative analysis of the OxPLs a recently introduced MALDI-QIT-TOF-MS/MS platform (Stübiger et al. Atherosclerosis 2012, 224, 177-186) was employed and cross-validated by LC-ESI-SRM-MS/MS. The sensitivity was found in the sub-nanomolar range (LOD ~200 pM), which is 1-4 orders of magnitude higher than necessary for detection of individual OxPLs under normal and diseased conditions in vivo (e.g., in mouse plasma or human lipoproteins). Consequently, the Nano-MALDI approach shows the potential to serve as novel platform for the screening of OxPLs in biological samples and the development of clinical diagnostic tests in the future.
Assuntos
Nanopartículas/química , Fosfolipídeos/sangue , Fosfolipídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Humanos , Nanopartículas de Magnetita/química , Camundongos , Oxirredução , Zircônio/químicaRESUMO
Monitoring bioactive oxidized phospholipids (OxPLs), such as 1-palmitoyl-2-(5'-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine (PONPC), is of major interest as they play a crucial role in a variety of age related diseases, e.g., in the development and progression of atherosclerosis. Since they are in low abundance in samples like oxidized low-density lipoproteins (OxLDL) and human plasma, respectively, their analysis as risk biomarkers requires the combination of an efficient selective sample preparation with highly sensitive detection methods, such as liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). In this study, a nanoparticle-based strategy for successful trapping and enrichment of aldehyde-containing oxidized phospholipids is presented. The concept involves a derivatization step with a bifunctional reagent containing both a hydrazide group for hydrazone formation with carbonyl-containing PLs and a thiol moiety for subsequent trapping on GNPs. After washing, the trapped analytes are quantitatively released from the nanoparticles' surface by transimination with hydroxylamine. The released oxime-derivatives of the carbonylated-OxPLs are subsequently analyzed by LC-ESI-MS/MS in the selected reaction monitoring scan mode. Several parameters of this workflow were optimized. With the optimized nanoparticle-based extraction and enrichment step, very clean extracts of these biomarkers can be obtained and the detection limits can be significantly decreased from 2.76 and 2.65 nM for PONPC and POVPC, respectively, to 0.17 and 0.44 nM. The applicability of this nanoparticle-based sample preparation concept was demonstrated by successful extraction of oxidized phospholipids from biological samples, such as human plasma, MDA-modified LDL and Cu(2+)-oxidized LDL.
Assuntos
Epitopos/sangue , Lipoproteínas LDL/sangue , Fosfatidilcolinas/sangue , Éteres Fosfolipídicos/sangue , Biomarcadores/sangue , Biomarcadores/química , Epitopos/química , Humanos , Hidroxilamina/química , Limite de Detecção , Lipoproteínas LDL/química , Nanopartículas/química , Oxirredução , Fosfatidilcolinas/química , Éteres Fosfolipídicos/química , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Oxidized low-density lipoproteins (OxLDLs), in particular, oxidized phosphatidylcholines (OxPCs), are known to be involved in pathophysiological processes such as cardiovascular diseases and are described as potential biomarkers, for example, for atherosclerosis. In our study, we used the specific affinity of anti-OxLDL antibodies (Abs) conjugated to gold nanoparticles (GNPs) for extraction and enrichment of OxPCs via selective trapping of OxLDLs from plasma combined with the sensitive detection by liquid chromatography/tandem-mass spectrometry (LC-MS/MS). Successful bioconjugation chemistry of Abs via a bifunctional polyethylene glycol (PEG) spacer and protein G linkage, respectively, was controlled by measuring the surface plasmon resonance (SPR) spectra, size, and zeta potentials. Furthermore, the amount of Ab immobilized onto GNP via the PEG linker was determined. With the optimized immobilization chemistry, the ability and potential of the GNP-based extraction procedure was used for the determination of the dissociation constant, K(d), of the OxLDL binding to the GNP-Ab conjugate. Moreover, apparent K(d)'s were determined for individual PCs and their oxidation products using the compound-specific selected reaction monitoring mode, which allows the characterization of the Ab affinity and, thus, assessment of the potential antigenicity of (Ox)PCs bound to OxLDLs. In summary, the application of GNP-based bioanalysis for selective targeting of OxLDLs and the fast and sensitive detection by LC-MS/MS offers new possibilities for targeted lipidomics in lipoproteins as well as for oxidative stress lipid biomarker screening.
Assuntos
Ouro/metabolismo , Lipoproteínas LDL/metabolismo , Nanopartículas Metálicas , Estresse Oxidativo/fisiologia , Espectrometria de Massas em Tandem/métodos , Biomarcadores/metabolismo , Ouro/química , Lipoproteínas LDL/análise , Nanopartículas Metálicas/química , Ligação Proteica/fisiologiaRESUMO
6-Aza-2-thiothymine (ATT) is introduced as novel matrix system for the analysis of oxidized phospholipids (OxPLs) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). A systematic evaluation comparing different established and novel matrix substances, especially 2,4,6-THAP matrix (Stubiger, G.; Belgacem O. Anal. Chem. 2007, 79, 3206-3213) as reference compound for phospholipid analysis, and specific matrix additives was performed. Thereby, ATT turned out to be the reagent of choice for MALDI analysis of major biologically relevant OxPL classes (e.g., OxPC, OxPE, and OxPS) in positive and negative ionization mode. ATT used together with specific chaotropic reagents at low concentration (0.5-2 mM) acting as OxPL ionization enhancers revealed an excellent comatrix system for application with MALDI instrument types employing UV- and Nd:YAG laser systems (337 and 355 nm). Moreover, disposable MALDI targets surfaces with specific physicochemical properties (e.g., metallized glass or polymeric substrates) were revealed as superior over stainless steel in terms of reduced chemical background noise ( approximately 10-fold better S/N ratios), increased mass spectral reproducibility, and enhanced sensitivity (LOD approximately 250-500 fg on target). The combination of these parameters offers a significant advantage for highly sensitive OxPL profiling by MALDI-MS of biological samples (e.g., human plasma) at trace levels.
Assuntos
Fosfolipídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Timina/análogos & derivados , Humanos , Lasers de Estado Sólido , Oxirredução , Fosfolipídeos/sangue , Fosfolipídeos/isolamento & purificação , Extração em Fase Sólida , Timina/química , TriazinasRESUMO
Fatty-acid(FA)-synthase(FASN) is a druggable lipogenic oncoprotein whose blockade causes metabolic disruption. Whether drug-induced metabolic perturbation is essential for anticancer drug-action, or is just a secondary-maybe even a defence response-is still unclear. To address this, SKOV3 and OVCAR3 ovarian cancer(OC) cell lines with clear cell and serous histology, two main OC subtypes, were exposed to FASN-inhibitor G28UCM. Growth-inhibition was compared with treatment-induced cell-metabolomes, lipidomes, proteomes and kinomes. SKOV3 and OVCAR3 were equally sensitive to low-dose G28UCM, but SKOV3 was more resistant than OVCAR3 to higher concentrations. Metabolite levels generally decreased upon treatment, but individual acylcarnitines, glycerophospholipids, sphingolipids, amino-acids, biogenic amines, and monosaccharides reacted differently. Drug-induced effects on central-carbon-metabolism and oxidative-phosphorylation (OXPHOS) were essentially different in the two cell lines, since drug-naïve SKOV3 are known to prefer glycolysis, while OVCAR3 favour OXPHOS. Moreover, drug-dependent increase of desaturases and polyunsaturated-fatty-acids (PUFAs) were more pronounced in SKOV3 and appear to correlate with G28UCM-tolerance. In contrast, expression and phosphorylation of proteins that control apoptosis, FA synthesis and membrane-related processes (beta-oxidation, membrane-maintenance, transport, translation, signalling and stress-response) were concordantly affected. Overall, membrane-disruption and second-messenger-silencing were crucial for anticancer drug-action, while metabolic-rewiring was only secondary and may support high-dose-FASN-inhibitor-tolerance. These findings may guide future anti-metabolic cancer intervention.
Assuntos
Membrana Celular/efeitos dos fármacos , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Ácido Gálico/análogos & derivados , Lipidômica/métodos , Naftalenos/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Proteoma/metabolismo , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Ácido Graxo Sintase Tipo I/metabolismo , Inibidores da Síntese de Ácidos Graxos/farmacologia , Feminino , Ácido Gálico/farmacologia , Humanos , Metaboloma , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Transdução de SinaisRESUMO
An improved analytical strategy for the analysis of complex lipid mixtures using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in combination with high-performance thin-layer chromatography (HPTLC) is reported. Positive ion MALDI RTOF MS was applied as a rapid screening tool for the various neutral (e.g. triacylglycerols) and polar (e.g. glycerophospholipids and -sphingolipids) lipid classes derived from crude lipid extracts of e.g. human plasma as well as soybean lecithin. Finally, MALDI seamless post-source decay (PSD) product ion analysis was performed in order to obtain further structural information (head- and acyl-group identification) of selected lipid species and structure verification. A Coomassie Brilliant Blue R-250 staining protocol for lipids on HPTLC plates was evaluated and was found to be fully compatible with subsequent MALDI-MS. Lipids were analyzed after elution from the HPTLC phase material of the selected band (corresponding to certain lipid classes) by using the proper organic solvent mixture or in few cases directly from the HPTLC plates (a type of on-line HPTLC/MALDI-MS coupling). More than 70 distinct lipid species from seven different lipid classes in the range between m/z 500 and 1500 could be identified from the lipid extracts of human plasma and soybean lecithin, respectively. The general high sensitivity of MALDI-MS detection allowed the analysis of even minor lipid classes from only very small volumes of human plasma (50 microL). The combination of HPTLC, Coomassie staining and positive ion MALDI curved field RTOF-MS represents a straightforward strategy during lipidomics studies of food and clinically relevant human lipid samples.
Assuntos
Cromatografia em Camada Fina/métodos , Glycine max/química , Lecitinas/química , Lipídeos/sangue , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , HumanosRESUMO
Receptor-PI3K-mTORC1 signaling and fatty acid synthase (FASN)-regulated lipid biosynthesis harbor numerous drug targets and are molecularly connected. We hypothesize that unraveling the mechanisms of pathway cross-talk will be useful for designing novel co-targeting strategies for ovarian cancer (OC). The impact of receptor-PI3K-mTORC1 onto FASN is already well-characterized. However, reverse actions-from FASN towards receptor-PI3K-mTORC1-are still elusive. We show that FASN-blockade impairs receptor-PI3K-mTORC1 signaling at multiple levels. Thin-layer chromatography and MALDI-MS/MS reveals that FASN-inhibitors (C75, G28UCM) augment polyunsaturated fatty acids and diminish signaling lipids diacylglycerol (DAG) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) in OC cells (SKOV3, OVCAR-3, A2780, HOC-7). Western blotting and micropatterning demonstrate that FASN-blockers impair phosphorylation/expression of EGF-receptor/ERBB/HER and decrease GRB2-EGF-receptor recruitment leading to PI3K-AKT suppression. FASN-inhibitors activate stress response-genes HIF-1α-REDD1 (RTP801/DIG2/DDIT4) and AMPKα causing mTORC1- and S6-repression. We conclude that FASN-inhibitor-mediated blockade of receptor-PI3K-mTORC1 occurs due to a number of distinct but cooperating processes. Moreover, decrease of PI3K-mTORC1 abolishes cross-repression of MEK-ERK causing ERK activation. Consequently, the MEK-inhibitor selumetinib/AZD6244, in contrast to the PI3K/mTOR-inhibitor dactolisib/NVP-BEZ235, increases growth inhibition when given together with a FASN-blocker. We are the first to provide deep insight on how FASN-inhibition blocks ERBB-PI3K-mTORC1 activity at multiple molecular levels. Moreover, our data encourage therapeutic approaches using FASN-antagonists together with MEK-ERK-inhibitors.
Assuntos
Inibidores Enzimáticos/farmacologia , Ácido Graxo Sintases/antagonistas & inibidores , Complexos Multiproteicos/antagonistas & inibidores , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Serina-Treonina Quinases TOR/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Ácido Graxo Sintases/metabolismo , Feminino , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/metabolismo , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) becomes the method of choice for the rapid identification of microorganisms (i.e. protein biotyping). Although bacterial identification is already quite advanced, biotyping of other microbes including yeasts and fungi are still under development. In this context, lipids (e.g. membrane phospholipids) represent a very important group of molecules with essential functions for cell survival and adaptation to specific environments and habitats of the microorganisms. Therefore, lipids show the potential to serve as additional molecular parameters to be used for biotyping purposes. In this paper we present a molecular characterisation of yeasts and filamentous fungi based on the analysis of lipid composition by MALDI-MS (i.e. MALDI lipid phenotyping). Using a combination of Principal Component Analysis (PCA) and Hierarchical Clustering we could demonstrate that this approach allowed a classification and differentiation of several groups of yeasts (e.g. Saccharomyces) and filamentous fungi (e.g. Aspergillus, Penicillium, Trichoderma) at the species/strain level. By analysing the MALDI lipid profiles we were able to differentiate 26 closely related yeast strains, for which discrimination via genotypic methods like AFLP in this case are relatively more elaborate. Moreover, employing statistical analysis we could identify those lipid parameters (e.g. PCs and LPCs), which were responsible for the differentiation of the strains, thus providing insights into the molecular basis of our results. In summary, MALDI lipid phenotyping represents a suitable method for fungal characterization and shows the potential to be used as companion tool to genotyping and/or protein biotyping for the characterization and identification of yeasts and fungi in diverse areas (e.g. environmental, pharmaceutical, clinical applications, etc.).
Assuntos
Fungos/isolamento & purificação , Lipídeos/isolamento & purificação , Técnicas de Tipagem Micológica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Leveduras/isolamento & purificação , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados/métodos , Técnicas de Cultura de Células , Análise por Conglomerados , DNA Fúngico , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Fungos/química , Fungos/classificação , Fungos/genética , Genótipo , Lipídeos/química , Análise Multivariada , Fenótipo , Fosfolipídeos/química , Fosfolipídeos/isolamento & purificação , Especificidade da Espécie , Leveduras/química , Leveduras/classificação , Leveduras/genéticaRESUMO
Export of macromolecules via extracellular membrane-derived vesicles (MVs) plays an important role in the biology of Gram-negative bacteria. Gram-positive bacteria have also recently been reported to produce MVs; however, the composition and mechanisms governing vesiculogenesis in Gram-positive bacteria remain undefined. Here, we describe MV production in the Gram-positive human pathogen group A streptococcus (GAS), the etiological agent of necrotizing fasciitis and streptococcal toxic shock syndrome. M1 serotype GAS isolates in culture exhibit MV structures both on the cell wall surface and in the near vicinity of bacterial cells. A comprehensive analysis of MV proteins identified both virulence-associated protein substrates of the general secretory pathway in addition to "anchorless surface proteins." Characteristic differences in the contents, distributions, and fatty acid compositions of specific lipids between MVs and GAS cell membrane were also observed. Furthermore, deep RNA sequencing of vesicular RNAs revealed that GAS MVs contained differentially abundant RNA species relative to bacterial cellular RNA. MV production by GAS strains varied in a manner dependent on an intact two-component system, CovRS, with MV production negatively regulated by the system. Modulation of MV production through CovRS was found to be independent of both GAS cysteine protease SpeB and capsule biosynthesis. Our data provide an explanation for GAS secretion of macromolecules, including RNAs, lipids, and proteins, and illustrate a regulatory mechanism coordinating this secretory response. IMPORTANCE: Group A streptococcus (GAS) is a Gram-positive bacterial pathogen responsible for more than 500,000 deaths annually. Establishment of GAS infection is dependent on a suite of proteins exported via the general secretory pathway. Here, we show that GAS naturally produces extracellular vesicles with a unique lipid composition that are laden with proteins and RNAs. Interestingly, both virulence-associated proteins and RNA species were found to be differentially abundant in vesicles relative to the bacteria. Furthermore, we show that genetic disruption of the virulence-associated two-component regulator CovRS leads to an increase in vesicle production. This study comprehensively describes the protein, RNA, and lipid composition of GAS-secreted MVs and alludes to a regulatory system impacting this process.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Repressoras/metabolismo , Vesículas Secretórias/metabolismo , Streptococcus pyogenes/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Histidina Quinase , Lipídeos/análise , Proteínas de Membrana/análise , RNA/análise , RNA/genética , Vesículas Secretórias/química , Fatores de Virulência/análiseRESUMO
BACKGROUND: Splenectomy is a clinical risk factor for complicated thrombosis. We hypothesized that the loss of the mechanical filtering function of the spleen may enrich for thrombogenic phospholipids in the circulation, thereby affecting the vascular remodeling of thrombosis. METHODS AND RESULTS: We investigated the effects of splenectomy both in chronic thromboembolic pulmonary hypertension (CTEPH), a human model disease for thrombus nonresolution, and in a mouse model of stagnant flow venous thrombosis mimicking deep vein thrombosis. Surgically excised thrombi from rare cases of CTEPH patients who had undergone previous splenectomy were enriched for anionic phospholipids like phosphatidylserine. Similar to human thrombi, phosphatidylserine accumulated in thrombi after splenectomy in the mouse model. A postsplenectomy state was associated with larger and more persistent thrombi. Higher counts of procoagulant platelet microparticles and increased leukocyte-platelet aggregates were observed in mice after splenectomy. Histological inspection revealed a decreased number of thrombus vessels. Phosphatidylserine-enriched phospholipids specifically inhibited endothelial proliferation and sprouting. CONCLUSIONS: After splenectomy, an increase in circulating microparticles and negatively charged phospholipids is enhanced by experimental thrombus induction. The initial increase in thrombus volume after splenectomy is due to platelet activation, and the subsequent delay of thrombus resolution is due to inhibition of thrombus angiogenesis. The data illustrate a potential mechanism of disease in CTEPH.
Assuntos
Hipertensão Pulmonar/etiologia , Embolia Pulmonar/etiologia , Esplenectomia/efeitos adversos , Trombose Venosa/etiologia , Idoso , Animais , Coagulação Sanguínea , Estudos de Casos e Controles , Proliferação de Células , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Endarterectomia , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hipertensão Pulmonar/sangue , Hipertensão Pulmonar/diagnóstico , Hipertensão Pulmonar/cirurgia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Neovascularização Patológica , Fosfatidilserinas/sangue , Ativação Plaquetária , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Embolia Pulmonar/sangue , Embolia Pulmonar/diagnóstico , Embolia Pulmonar/cirurgia , Fatores de Risco , Fatores de Tempo , Veia Cava Inferior/metabolismo , Veia Cava Inferior/patologia , Trombose Venosa/sangue , Trombose Venosa/diagnósticoRESUMO
OBJECTIVES: Phospholipids (PLs) are increasingly recognized as key molecules with potential diagnostic value in acute inflammation, CVD and atherosclerosis. We introduce a pioneer mass spectrometry (MS)-based approach aiming to investigate the relationship of specific plasma PL-subsets with atherogenic blood parameters in young patients with familial hyperlipidemia representing high-CVD-risk groups. METHODS: Plasma of carefully phenotyped FH and FCH patients as well as normolipidemic subjects (age 13 ± 5 years, n = 20) was used. Clinical parameters were assessed using standard laboratory techniques and lipids were subjected to a direct targeted monitoring using LC-ESI-SRM- and MALDI-QIT-TOF-MS/MS, respectively. Statistical analysis was performed to evaluate correlations between PL data and the clinical parameters. RESULTS: Most characteristically significant differences of SM/PC and PC/LPC ratios and positive correlations between SM vs. LDL-C (r = 0.946; p = 0.004) and LPC vs. VLDL-C (r = 0.669; p = 0.218) were observed in FH in contrast to the other study groups. OxPC levels were found in the range of â¼2-20 µmol/L with predominance of short-chain aldehydic species (e.g. SOVPC). A positive correlation of OxPCs with IMT (r = 0.952; p = 0.052) and HDL-C (r = 0.893; p = 0.016) but negative correlation with OxLDL (r = -0.910; p = 0.096) was observed. CONCLUSIONS: Our study was a first attempt to use a MALDI-QIT-TOF-MS/MS based clinical lipidomics approach to investigate atherogenic dyslipidemia in young patients with familial hyperlipidemia. This technique represents a promising platform for clinical screening of lipid biomarkers in the future.
Assuntos
Hiperlipidemia Familiar Combinada/sangue , Hiperlipoproteinemia Tipo II/sangue , Lipoproteínas/sangue , Fosfolipídeos/sangue , Adolescente , Aterosclerose/sangue , Biomarcadores/sangue , Criança , LDL-Colesterol , Feminino , Humanos , Lisofosfatidilcolinas/sangue , Masculino , Fosfatidilcolinas/sangue , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Esfingomielinas/sangue , Espectrometria de Massas em Tandem , Adulto JovemRESUMO
In the present study a direct detection approach combining size-exclusion chromatography (SEC) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem-mass spectrometry (MALDI-QIT-TOF-MS/MS) was applied to investigate the influence of HSA and IgG on LDL oxidation in vitro. SEC analysis showed an increase of protein aggregation during LDL-oxidation that could be essentially suppressed in the presence of HSA. In parallel, lipid peroxidation measured by TBARS assay over 24h was inhibited by 95-100% in the presence of HSA but only 0-34% by IgG, respectively. MALDI phospholipid profiles showed considerable decrease of signals from PCs containing sn-2 PUFAs (18:2 or 20:4) accompanied by increase of sn-2 LPCs indicating for specific breakdown of PUFA-containing PLs during LDL-oxidation. These effects were nearly 100% inhibited in the presence of HSA but not by IgG, respectively. Among known pro-atherogenic PL species present in human plasma sphingomyelin (SM16:0) was bound in significant amounts to HSA but not IgG after incubation with oxLDL. Moreover, our investigation showed that LPCs containing SAFAs (16:0 or 18:0) were specifically bound to HSA, while those containing PUFAs (18:2 and 18:3) were preferentially associated with IgG. In summary, the presented methodology provides a promising platform for studying lipid-protein interactions in vivo.
Assuntos
Cromatografia em Gel/métodos , Imunoglobulina G/farmacologia , Lipoproteínas LDL/metabolismo , Fosfolipídeos/metabolismo , Albumina Sérica/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Adulto , Aterosclerose , Ácidos Graxos Insaturados/metabolismo , Feminino , Humanos , Imunoglobulina G/isolamento & purificação , Lisofosfatidilcolinas/metabolismo , Masculino , Pessoa de Meia-Idade , Oxirredução/efeitos dos fármacos , Albumina Sérica/isolamento & purificação , Esfingomielinas/metabolismoRESUMO
In this paper, we present the results of a detailed study using MALDI seamless postsource decay (sPSD) fragment ion analysis of all major glycerophospholipid (GPL) classes, cardiolipin (bisphosphatidylglycerol), and sphingomyelin, respectively. The matrix compound 2,4,6-trihydroxyacetophenon recently introduced for lipid analysis (Stübiger, G.; Belgacem O. Anal. Chem. 2007, 79, 3206-3213) was applied in conjunction with a high-resolution curved field reflectron analyzer allowing detection of the fragment ions without stepping the reflectron voltages (seamless PSD). This instrumental feature helps to define in a fast way the polar headgroups of the different GPL classes and gives information about the constituent fatty acid residues dependent on the type of adduct ion used. Our experiments demonstrate that fragment ions specifying the fatty acid composition of the lipid molecules could only be generated from cationized molecular ions (sodiated or lithiated). Additionally, information about the stereospecificity of the fatty acid residues on the glycerol backbone (sn-1, and -2 position) of particular GPLs could be obtained during sPSD analysis. In the case of phosphatidylcholine, significant fragmentation related to the loss of the acyl groups could only be observed from [M + Li](+) ions. Generally, alkali adduction (sodium and lithium) enhanced fragmentation of most lipid classes, particularly favoring fragment ions associated with the polar headgroups.
Assuntos
Lítio/química , Fosfolipídeos/química , Sódio/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Estrutura MolecularRESUMO
Lipids exhibit a broad range of chemical properties that make their analysis quite demanding. Today, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) represents a versatile tool in the field of lipid analysis, also offering the possibility for molecular structural identification using novel MALDI tandem time-of-flight (TOF/TOF) instrumentation. In this study, we evaluated 2,4,6-trihydroxyacetophenone (THAP) for the analysis of various lipid classes including neutral storage lipids (triacylglycerols), polar membrane lipids (glycerophospho- and sphingolipids), and glycosphingolipids. THAP proved to be a versatile matrix for the routine analysis of various lipids from biological samples ("lipidomics"). A sample preparation methodology was established using selective alkali salt doping for subsequent MS/MS experiments. Sodiated and lithiated molecules provided superior structural information on lipids (i.e., acyl group identification); thus, following this approach, both selective peak detection with high sensitivity and more reliable structural information were obtained simultaneously.
Assuntos
Acetofenonas/química , Lipídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Cerebrosídeos/análise , Cerebrosídeos/química , Lipídeos/químicaRESUMO
Human serum albumin (HSA) is the most abundant protein in plasma. It is known to transport drugs as well as endogenous ligands, like free fatty acids (FFA). A mass spectrometry based method was applied to analyze the albumin bound lipid ligands. HSA was isolated from a human plasma pool by cold ethanol fractionation and ion exchange chromatography. HSA was defatted using a solvent extraction method to release the copurified lipids bound to the protein. The extracts were then analyzed by matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Using this method, phospholipids and acylglycerols were detected. The phospholipids were identified to be lyso-phosphatidylcholine (lyso-PC) with distribution of different fatty acids (palmitic, stearic, oleic, and linoleic acids). An abundant species in the HSA lipid extract was found to be a diacylglycerol, composed of two linoleic and/or oleic acid chains. The identified motifs reflect structures that are known to be present in plasma. The binding of lysophospholipids has already been described but it is the first ever-reported evidence of native diacylglycerol ligands bound to HSA. Besides the native ligands from plasma a triacylglycerol was detected that has been added during the albumin preparation steps.
Assuntos
Ácidos Graxos/isolamento & purificação , Albumina Sérica/química , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Ácidos Graxos/química , Humanos , Albumina Sérica/isolamento & purificação , Soluções , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Our experiments show that it is possible to detect different types of recombinant human erythropoietins (rhEPOs), EPO-alpha, EPO-beta and novel erythropoesis stimulating protein (NESP), based on exact molecular weight (MW) determination by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) applying a high-resolution time-of-flight (TOF) mass analyser in the linear mode. Detection limits for the highly purified, intact glycoproteins were achievable in the low fmol range (25-50 fmol) using a sample preparation method applying a hydrophobic sample support (DropStop) as MALDI target surface. These results are very promising for the development of highly sensitive detection methods for a direct identification of rhEPO after enrichment from human body fluids. During our investigation we were able to differentiate EPO-alpha, EPO-beta and NESP based on distinct molecular substructures at the protein level by specific enzymatic reactions. MW determination of the intact molecules by high resolving one-dimensional sodium dodecyl sulfate /polyacrylamide gel electrophoresis (1D SDS-PAGE) and isoform separation by planar isoelectric focusing (IEF) was compared with MALDI-MS data. Migration differences between the rhEPOs were observed from gel electrophoresis, whereby MWs of 38 kDa in the case of EPO-alpha/beta and 49 kDa for NESP could be estimated. In contrast, an exact MW determination by MALDI-MS based on internal calibration revealed average MWs of 29.8 +/- 0.3 kDa for EPO-alpha/beta and 36.8 +/- 0.4 kDa for NESP. IEF separation of the intact rhEPOs revealed the presence of four to eight distinct isoforms in EPO-alpha and EPO-beta, while four isoforms, which appeared in the more acidic area of the gels, were detected by immunostaining in NESP. A direct detection of the different N- or O-glycoform pattern from rhEPOs using MALDI-MS was possible by de-sialylation of the glycan structures and after de-N-glycosylation of the intact molecules. Thereby, the main glycoforms of EPO-alpha, EPO-beta and NESP could be characterised based on their N-glycan composition. A microheterogeneity of the molecules based on the degree of sialylation of the O-glycan was observable directly from the de-N-glycosylated protein.