RESUMO
Rifampicin is an effective drug for treating tuberculosis (TB) but is not used to treat Mycobacterium abscessus infections due to poor in vitro activity. While rifabutin, another rifamycin, has better anti-M. abscessus activity, its activity is far from the nanomolar potencies of rifamycins against Mycobacterium tuberculosis. Here, we asked (i) why is rifabutin more active against M. abscessus than rifampicin, and (ii) why is rifabutin's anti-M. abscessus activity poorer than its anti-TB activity? Comparative analysis of naphthoquinone- versus naphthohydroquinone-containing rifamycins suggested that the improved activity of rifabutin over rifampicin is linked to its less readily oxidizable naphthoquinone core. Although rifabutin is resistant to bacterial oxidation, metabolite and genetic analyses showed that this rifamycin is metabolized by the ADP-ribosyltransferase ArrMab like rifampicin, preventing it from achieving the nanomolar activity that it displays against M. tuberculosis. Based on the identified dual mechanism of intrinsic rifamycin resistance, we hypothesized that rifamycins more potent than rifabutin should contain the molecule's naphthoquinone core plus a modification that blocks ADP-ribosylation at its C-23. To test these predictions, we performed a blinded screen of a diverse collection of 189 rifamycins and identified two molecules more potent than rifabutin. As predicted, these compounds contained both a more oxidatively resistant naphthoquinone core and C-25 modifications that blocked ADP-ribosylation. Together, this work revealed dual bacterial metabolism as the mechanism of intrinsic resistance of M. abscessus to rifamycins and provides proof of concept for the repositioning of rifamycins for M. abscessus disease by developing derivatives that resist both bacterial oxidation and ADP-ribosylation.
Assuntos
Mycobacterium abscessus , Rifamicinas , ADP-Ribosilação , Testes de Sensibilidade Microbiana , Rifabutina/farmacologia , Rifamicinas/farmacologiaRESUMO
Desmosine (Des) and isodesmosine (Isodes), cross-linking amino acids in the biomolecule elastin, may be used as biomarkers for various pathological conditions associated with elastin degradation. The current study presents a novel approach to quantify Des and Isodes using matrix-assisted laser desorption ionization (MALDI)-tandem mass spectrometry (MS2) in a linear ion trap coupled to a vacuum MALDI source. MALDI-MS2 analyses of Des and Isodes are performed using stable-isotope-labeled desmosine d4 (labeled-Des) as an internal standard in different biological fluids, such as urine and serum. The method demonstrated linearity over two orders of magnitude with a detection limit of 0.02 ng/µL in both urine and serum without enrichment prior to mass spectrometry, and relative standard deviation of < 5%. The method is used to evaluate the time-dependent degradation of Des upon UV irradiation (254 nm) and found to be consistent with quantification by 1H NMR. This is the first characterized MALDI-MS2 method for quantification of Des and Isodes and illustrates the potential of MALDI-ion trap MS2 for effective quantification of biomolecules. The reported method represents improvement over current liquid chromatography-based methods with respect to analysis time and solvent consumption, while maintaining similar analytical characteristics. Graphical abstract á .
Assuntos
Desmosina/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas em Tandem/métodos , Desmosina/sangue , Desmosina/química , Desmosina/urina , Humanos , Limite de Detecção , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Intra-abdominal candidiasis (IAC) is a prominent invasive fungal infection associated with high mortality. Prompt antifungal therapy and source control are crucial for successful treatment. Echinocandin antifungal drugs are first-line agents; however, their clinical effectiveness is highly variable, with known potential for breakthrough resistance, and little is known about drug exposure at the site of infection. Using matrix-assisted desorption ionization mass spectrometry imaging technology, we investigated the spatial and quantitative distribution in tissue lesions for two echinocandin drugs, micafungin and CD101, in a clinically relevant IAC mouse model. Drug accumulation within lesions was observed with both drugs at their humanized therapeutic doses. CD101, but not micafungin, accumulated in lesions at levels above the mutant prevention concentration of the infecting strain. These findings indicate that current echinocandin drugs are limited by penetration at the site of infection and have implications for clinical outcomes and emergence of resistance in patients with IAC.
Assuntos
Abscesso Abdominal/tratamento farmacológico , Antifúngicos/uso terapêutico , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Equinocandinas/farmacocinética , Lipopeptídeos/farmacocinética , Animais , Modelos Animais de Doenças , Farmacorresistência Fúngica/fisiologia , Equinocandinas/uso terapêutico , Feminino , Lipopeptídeos/uso terapêutico , Micafungina , Camundongos , Testes de Sensibilidade Microbiana , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Mutations in HTRA2/Omi/PARK13 have been implicated in Parkinson disease (PD). PARK13 is a neuroprotective serine protease; however, little is known about how PARK13 confers stress protection and which protein targets are directly affected by PARK13. We have reported that Arabidopsis thaliana represents a complementary PD model, and here we demonstrate that AtPARK13, similar to human PARK13 (hPARK13), is a mitochondrial protease. We show that the expression/accumulation of AtPARK13 transcripts are induced by heat stress but not by other stress conditions, including oxidative stress and metals. Our data show that elevated levels of AtPARK13 confer thermotolerance in A. thaliana. Increased temperatures accelerate protein unfolding, and we demonstrate that although AtPARK13 can act on native protein substrates, unfolded proteins represent better AtPARK13 substrates. The results further show that AtPARK13 and hPARK13 can degrade the PD proteins α-synuclein (SNCA) and DJ-1/PARK7 directly, without autophagy involvement, and that misfolded SNCA and DJ-1 represent better substrates than their native counterparts. Comparative proteomic profiling revealed AtPARK13-mediated proteome changes, and we identified four proteins that show altered abundance in response to AtPARK13 overexpression and elevated temperatures. Our study not only suggests that AtPARK13 confers thermotolerance by degrading misfolded protein targets, but it also provides new insight into possible roles of this protease in neurodegeneration.
Assuntos
Adaptação Fisiológica/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Temperatura Alta , Serina Proteases/genética , Sequência de Aminoácidos , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Western Blotting , Clonagem Molecular , Perfilação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Proteínas Oncogênicas/química , Proteínas Oncogênicas/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Plantas Geneticamente Modificadas , Proteína Desglicase DJ-1 , Desdobramento de Proteína , Proteólise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Serina Proteases/metabolismo , Especificidade por Substrato , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMO
RATIONALE: Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry combined with isotope labeling methods are effective for protein and peptide quantification, but limited in their multiplexing capacity, cost-effectiveness and dynamic range. This study investigates MALDI-MS-based quantification of peptide phosphorylation without labeling, and aims to overcome the shot-to-shot variability of MALDI using a mathematical transformation and extended data acquisition times. METHODS: A linear relationship between the reciprocal of phosphopeptide mole fraction and the reciprocal of phosphorylated-to-unphosphorylated signal ratio is derived, and evaluated experimentally using three separate phosphopeptide systems containing phosphorylated serine, threonine and tyrosine residues: mixtures of phosphopeptide and its des-phospho-analog with known stoichiometry measured by vacuum MALDI-linear ion trap mass spectrometry and fit to the linear model. The model is validated for quantifying in vitro phosphorylation assays with inhibition studies on Cdk2/cyclinA. RESULTS: Dynamic range of picomoles to femtomoles, good accuracy (deviations of 1.5-3.0% from expected values) and reproducibility (relative standard deviation (RSD) = 4.3-6.3%) are achieved. Inhibition of cyclin-dependent kinase phosphorylation by the classical inhibitors olomoucine and r-roscovitine was evaluated and IC50 values found to be in agreement with reported literature values. These results, achieved with single-point calibration, without isotope or chromatography, compare favorably to those arrived at using isotope dilution (p > 0.5 for accuracy). CONCLUSIONS: The mathematical relationship derived here can be applied to a method that we term Double Reciprocal Isotope-free Phosphopeptide Quantification (DRIP-Q), as a strategy for quantification of in vitro phosphorylation assays, the first MALDI-based, isotope- and calibration curve-free method of its type. These results also pave the way for further systematic studies investigating the effect of peptide composition and experimental conditions on quantitative, label-free MALDI.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Fosfopeptídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Calibragem , Ciclina A/antagonistas & inibidores , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/metabolismo , Modelos Lineares , Dados de Sequência Molecular , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Fosforilação , Reprodutibilidade dos TestesRESUMO
Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were applied to characterize drug metabolites. Although these two methods have overcome the identification and structural characterization of metabolites analysis, they remain time-consuming processes. In this study, a novel multiple-stage tandem mass spectrometric method (MS(n) ) was evaluated for identification and characterization of new minor metabolism profiling of penicillin G, one of the ß-lactam antibiotics, in human serum. Seven minor metabolites including five phase I metabolites and two phase II metabolites of penicillin G were identified by using data-dependent LC/MS(n) screening in one chromatographic run. The accuracy masses of seven identified metabolites of penicillin G were also confirmed by mass spectral calibration software (MassWorks™). The proposed data-dependent LC/MS(n) method is a powerful tool to provide large amounts of the necessary structural information to characterize minor metabolite in metabolism profiling.
Assuntos
Penicilina G/sangue , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Humanos , Redes e Vias Metabólicas , Ácido Penicilânico/análogos & derivados , Ácido Penicilânico/sangue , Ácido Penicilânico/química , Ácido Penicilânico/metabolismo , Penicilina G/química , Penicilina G/metabolismoRESUMO
We present the application of Bayesian modeling to identify chemical tools and/or drug discovery entities pertinent to drug-resistant Staphylococcus aureus infections. The quinoline JSF-3151 was predicted by modeling and then empirically demonstrated to be active against in vitro cultured clinical methicillin- and vancomycin-resistant strains while also exhibiting efficacy in a mouse peritonitis model of methicillin-resistant S. aureus infection. We highlight the utility of an intrabacterial drug metabolism (IBDM) approach to probe the mechanism by which JSF-3151 is transformed within the bacteria. We also identify and then validate two mechanisms of resistance in S. aureus: one mechanism involves increased expression of a lipocalin protein, and the other arises from the loss of function of an azoreductase. The computational and experimental approaches, discovery of an antibacterial agent, and elucidated resistance mechanisms collectively hold promise to advance our understanding of therapeutic regimens for drug-resistant S. aureus.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Preparações Farmacêuticas , Infecções Estafilocócicas , Animais , Teorema de Bayes , Camundongos , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureusRESUMO
A simple, economical and very effective method is demonstrated for simultaneous determination of 2,4-dichlorophenol, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol and pentachlorophenol, in aqueous samples, by using purge-assisted headspace solid-phase microextraction (PA/HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS). In the new method, purging the sample enhances the removal of the trace chlorophenols without derivatization from the matrices to the headspace. Extraction parameters including extraction temperature, purge gas flow rate and extraction time were systematically investigated. Under optimal conditions, the relative standard deviations (RSDs) were 4-11% at 50 pg/mL and 5-14% at 5 pg/mL, respectively. The recoveries were in the range of 83-114%. Detection limits were determined at the fg level. These results indicate that PA/HS-SPME provides a significant contribution to highly efficient extraction of semi-volatile CPs, especially for pentachlorophenol, which has the smallest Henry's constant and large octanol-water partitioning coefficient. In addition, the proposed method was successfully applied to the analysis of chlorophenols in landfill leachate. New perspectives are opened for headspace extraction of relatively low vapor pressure compounds in complex matrices.
Assuntos
Clorofenóis/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Microextração em Fase Sólida/métodos , Poluentes Químicos da Água/análise , Reprodutibilidade dos Testes , Microextração em Fase Sólida/instrumentação , IncertezaRESUMO
Parkinson's disease and other synucleinopathies are characterized by the presence of intra-neuronal protein aggregates enriched in the presynaptic protein α-synuclein. α-synuclein is considered an intrinsically disordered 14 kDa monomer, and although poorly understood, its transition to higher-order multimeric species may play central roles in healthy neurons and during Parkinson's disease pathogenesis. In this study, we demonstrate that α-synuclein exists as defined, subcellular-specific species that change characteristics in response to oxidative stress in neuroblastoma cells and in response to Parkinson's disease pathogenesis in human cerebellum and frontal cortex. We further show that the phosphorylation patterns of different α-synuclein species are subcellular specific and dependent on the oxidative environment. Using high-performance liquid chromatography and mass spectrometry, we identify a Parkinson's disease enriched, cytosolic ~36-kDa α-synuclein species which can be recapitulated in Parkinson's disease model neuroblastoma cells. The characterization of subcellular-specific α-synuclein features in neurodegeneration will allow for the identification of neurotoxic α-synuclein species, which represent prime targets to reduce α-synuclein pathogenicity.
Assuntos
Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Degeneração Neural/metabolismo , Doença de Parkinson/metabolismo , Frações Subcelulares/metabolismo , Encéfalo/metabolismo , Linhagem Celular Tumoral , Membrana Celular/química , Núcleo Celular/química , Cromatografia Líquida de Alta Pressão/métodos , Citosol/química , Humanos , Frações Subcelulares/químicaRESUMO
MicroRNAs are key regulators associated with numerous diseases. In HEK293 cells, miR-153-3p and miR-205-5p down-regulate alpha-synuclein (SNCA) and Leucine-rich repeat kinase 2 (LRRK2), two key proteins involved in Parkinson's disease (PD). We have used two-dimensional gel electrophoresis (2D-PAGE) coupled to mass spectrometry (MS) to identify a spectrum of miR-153-3p and miR-205-5p targets in neuronal SH-SY5Y cells. We overexpressed and inhibited both microRNAs in SH-SY5Y cells and through comparative proteomics profiling we quantified ~240 protein spots from each analysis. Combined, thirty-three protein spots were identified showing significant (p-value < 0.05) changes in abundance. Modulation of miR-153-3p resulted in seven up-regulated proteins and eight down-regulated proteins. miR-205 modulation resulted in twelve up-regulated proteins and six down-regulated proteins. Several of the proteins are associated with neuronal processes, including peroxiredoxin-2 and -4, cofilin-1, prefoldin 2, alpha-enolase, human nucleoside diphosphate kinase B (Nm23) and 14-3-3 protein epsilon. Many of the differentially expressed proteins are involved in diverse pathways including metabolism, neurotrophin signaling, actin cytoskeletal regulation, HIF-1 signaling and the proteasome indicating that miR-153-3p and miR-205-5p are involved in the regulation of a wide variety of biological processes in neuroblastoma cells.
Assuntos
MicroRNAs/metabolismo , Proteínas de Neoplasias/metabolismo , Neuroblastoma/genética , Proteômica , Ciclo Celular , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Regulação Neoplásica da Expressão Gênica/genética , Humanos , MicroRNAs/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Transcrição Gênica/genéticaRESUMO
HIV-1 reverse transcription represents the predominant target for pharmacological inhibition of viral replication, but cell-intrinsic mechanisms that can block HIV-1 reverse transcription in a clinically significant way are poorly defined. We find that effective HIV-1 reverse transcription depends on the phosphorylation of viral reverse transcriptase by host cyclin-dependent kinase (CDK) 2 at a highly conserved Threonine residue. CDK2-dependent phosphorylation increased the efficacy and stability of viral reverse transcriptase and enhanced viral fitness. Interestingly, p21, a cell-intrinsic CDK inhibitor that is upregulated in CD4(+) T cells from "elite controllers," potently inhibited CDK2-dependent phosphorylation of HIV-1 reverse transcriptase and significantly reduced the efficacy of viral reverse transcription. These data suggest that p21 can indirectly block HIV-1 reverse transcription by inhibiting host cofactors supporting HIV-1 replication and identify sites of viral vulnerability that are effectively targeted in persons with natural control of HIV-1 replication.
Assuntos
Linfócitos T CD4-Positivos/virologia , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Transcriptase Reversa do HIV/metabolismo , Sequência de Aminoácidos , Estudos de Casos e Controles , Quinase 2 Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Estabilidade Enzimática , Transcriptase Reversa do HIV/genética , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Dados de Sequência Molecular , Fosforilação , Transcrição Reversa , Regulação para Cima , Replicação ViralRESUMO
This study describes a new procedure, namely, purge-assisted headspace solid phase microextraction combined with gas chromatography/negative ion chemical ionization mass spectrometry (PA/HS-SPME-GC/NICI-MS), which is used to determine seven nitrated polycyclic aromatic hydrocarbons (NPAHs) in aqueous samples. High extraction efficiency was obtained with PA/HS-SPME with polydimethylsiloxane (PDMS) fiber coating. A programmable temperature vaporizing (PTV) inlet was used in the desorption process. Selected ion monitoring (SIM) was used for quantitative and qualitative purposes. The linear range of detection of the proposed method was 5-5000 pg/mL with coefficients of determination between 0.995 and 0.999. Limits of detection (LODs) for seven NPAHs were 0.01-0.06 pg/mL. The relative standard deviation was below 12.7% at a concentration of 50 pg/mL. Compared with headspace-solid phase microextraction (HS-SPME), the purge procedure enhanced the extraction efficiency for high boiling point analytes, such as 7-nitrobenz[a]anthracene (7-NBA) and 6-nitrochrysene (6-NC). The proposed method provides a sensitive method for NPAH analysis at the pg/mL level. The application of the proposed method for the determination of trace NPAHs in real samples was investigated by analyzing aqueous samples from rivers. The concentrations of NPAHs detected from the samples ranged from 5.2 to 7.5 pg/mL. This method was applied successfully in the analysis of trace NPAHs in river samples.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Compostos Policíclicos/análise , Microextração em Fase Sólida/métodos , Poluentes Químicos da Água/análise , Concentração de Íons de Hidrogênio , Limite de Detecção , Reprodutibilidade dos TestesRESUMO
Moderately thermophilic bacterial strain CC-HSB-11(T) (Muricauda lutaonensis), which was described recently from a coastal hot spring of Green Island, Taiwan, has been identified to produce zeaxanthin as a predominant xanthophyll by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cell culture in bioreactor produced 3.12 ± 0.18 mg zeaxanthin L(-1) of culture. Micronization of zeaxanthin was achieved through supercritical carbon dioxide antisolvent precipitation method. Yield of zeaxanthin after the process was 53.4%. Dynamic light scattering assay determined the polydisperse existence of micronized particles of size 3 nm to 2 µm. Field emission scanning electron microscopy revealed distinct morphology and size distribution heterogeneity of particles. Integrity of zeaxanthin after the antisolvent process was assessed by LC-MS/MS. The technique capitalizes on the inherent ability of CC-HSB-11(T) to synthesize zeaxanthin and the work demonstrated feasibility of antisolvent precipitation method to produce microparticles exploiting a bacterial strain.