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1.
PLoS Pathog ; 19(9): e1011650, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37747938

RESUMEN

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, poses a great threat to human health. With the emergence of drug resistant Mtb strains, new therapeutics are desperately needed. As iron is critical to the growth and survival of Mtb, mechanisms through which Mtb acquires host iron represent attractive therapeutic targets. Mtb scavenges host iron via Mtb siderophore-dependent and heme iron uptake pathways. While multiple studies describe the import of heme and ferric-siderophores and the export of apo-siderophores across the inner membrane, little is known about their transport across the periplasm and cell-wall environments. Mtb FecB and FecB2 are predicted periplasmic binding proteins implicated in host iron acquisition; however, their precise roles are not well understood. This study sought to differentiate the roles FecB and FecB2 play in Mtb iron acquisition. The crystallographic structures of Mtb FecB and FecB2 were determined to 2.0 Å and 2.2 Å resolution, respectively, and show distinct ligand binding pockets. In vitro ligand binding experiments for FecB and FecB2 were performed with heme and bacterial siderophores from Mtb and other species, revealing that both FecB and FecB2 bind heme, while only FecB binds the Mtb sideophore ferric-carboxymycobactin (Fe-cMB). Subsequent structure-guided mutagenesis of FecB identified a single glutamate residue-Glu339-that significantly contributes to Fe-cMB binding. A role for FecB in the Mtb siderophore-mediated iron acquisition pathway was corroborated by Mycobacterium smegmatis and Mtb pull-down assays, which revealed interactions between FecB and members of the mycobacterial siderophore export and import machinery. Similarly, pull-down assays with FecB2 confirms its role in heme uptake revealing interactions with a potential inner membrane heme importer. Due to ligand preference and protein partners, our data suggest that Mtb FecB plays a role in siderophore-dependent iron and heme acquisition pathways; in addition, we confirm that Mtb FecB2 is involved in heme uptake.


Asunto(s)
Hierro , Mycobacterium tuberculosis , Humanos , Hierro/metabolismo , Sideróforos/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Ligandos , Proteínas Bacterianas/metabolismo , Hemo/metabolismo
2.
Environ Sci Technol ; 58(5): 2468-2478, 2024 Feb 06.
Artículo en Inglés | MEDLINE | ID: mdl-38252456

RESUMEN

Wastewater is a source for many contaminants of emerging concern (CECs), and surface waters receiving wastewater discharge often serve as source water for downstream drinking water treatment plants. Nontargeted analysis and suspect screening methods were used to characterize chemicals in residence-time-weighted grab samples and companion polar organic chemical integrative samplers (POCIS) collected on three separate hydrologic sampling events along a surface water flow path representative of de facto water reuse. The goal of this work was to examine the fate of CECs along the study flow path as water is transported from wastewater effluent through drinking water treatment. Grab and POCIS samples provided a comparison between residence-time-weighted single-point and integrative sample results. This unique and rigorous study design, coupled with advanced analytical chemistry tools, provided important insights into chemicals found in drinking water and their potential sources, which can be used to help prioritize chemicals for further study. K-means clustering analysis was used to identify patterns in chemical occurrences across both sampling sites and sampling events. Chemical features that occurred frequently or survived drinking water treatment were prioritized for identification, resulting in the probable identification of over 100 CECs in the watershed and 28 CECs in treated drinking water.


Asunto(s)
Agua Potable , Contaminantes Químicos del Agua , Purificación del Agua , Aguas Residuales , Agua Potable/análisis , Monitoreo del Ambiente/métodos , Contaminantes Químicos del Agua/análisis , Compuestos Orgánicos/análisis
3.
Environ Sci Technol ; 58(4): 1802-1812, 2024 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-38217501

RESUMEN

Humans interact with thousands of chemicals. This study aims to identify substances of emerging concern and in need of human health risk evaluations. Sixteen pooled human serum samples were constructed from 25 individual samples each from the National Institute of Environmental Health Sciences' Clinical Research Unit. Samples were analyzed using gas chromatography (GC) × GC/time-of-flight (TOF)-mass spectrometry (MS) in a suspect screening analysis, with follow-up confirmation analysis of 19 substances. A standard reference material blood sample was also analyzed through the confirmation process for comparison. The pools were stratified by sex (female and male) and by age (≤45 and >45). Publicly available information on potential exposure sources was aggregated to annotate presence in serum as either endogenous, food/nutrient, drug, commerce, or contaminant. Of the 544 unique substances tentatively identified by spectral matching, 472 were identified in females, while only 271 were identified in males. Surprisingly, 273 of the identified substances were found only in females. It is known that behavior and near-field environments can drive exposures, and this work demonstrates the existence of exposure sources uniquely relevant to females.


Asunto(s)
Cromatografía de Gases y Espectrometría de Masas , Pruebas Hematológicas , Femenino , Humanos , Masculino , Cromatografía de Gases y Espectrometría de Masas/métodos , Pruebas Hematológicas/métodos , Adulto , Persona de Mediana Edad
4.
Environ Sci Technol ; 57(8): 3075-3084, 2023 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-36796018

RESUMEN

Several thousand intentional and unintentional chemical releases occur annually in the U.S., with the contents of almost 30% being of unknown composition. When targeted methods are unable to identify the chemicals present, alternative approaches, including non-targeted analysis (NTA) methods, can be used to identify unknown analytes. With new and efficient data processing workflows, it is becoming possible to achieve confident chemical identifications via NTA in a timescale useful for rapid response (typically 24-72 h after sample receipt). To demonstrate the potential usefulness of NTA in rapid response situations, we have designed three mock scenarios that mimic real-world events, including a chemical warfare agent attack, the contamination of a home with illicit drugs, and an accidental industrial spill. Using a novel, focused NTA method that utilizes both existing and new data processing/analysis methods, we have identified the most important chemicals of interest in each of these designed mock scenarios in a rapid manner, correctly assigning structures to more than half of the 17 total features investigated. We have also identified four metrics (speed, confidence, hazard information, and transferability) that successful rapid response analytical methods should address and have discussed our performance for each metric. The results reveal the usefulness of NTA in rapid response scenarios, especially when unknown stressors need timely and confident identification.

5.
Anal Bioanal Chem ; 414(17): 4919-4933, 2022 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-35699740

RESUMEN

Non-targeted analysis (NTA) methods are widely used for chemical discovery but seldom employed for quantitation due to a lack of robust methods to estimate chemical concentrations with confidence limits. Herein, we present and evaluate new statistical methods for quantitative NTA (qNTA) using high-resolution mass spectrometry (HRMS) data from EPA's Non-Targeted Analysis Collaborative Trial (ENTACT). Experimental intensities of ENTACT analytes were observed at multiple concentrations using a semi-automated NTA workflow. Chemical concentrations and corresponding confidence limits were first estimated using traditional calibration curves. Two qNTA estimation methods were then implemented using experimental response factor (RF) data (where RF = intensity/concentration). The bounded response factor method used a non-parametric bootstrap procedure to estimate select quantiles of training set RF distributions. Quantile estimates then were applied to test set HRMS intensities to inversely estimate concentrations with confidence limits. The ionization efficiency estimation method restricted the distribution of likely RFs for each analyte using ionization efficiency predictions. Given the intended future use for chemical risk characterization, predicted upper confidence limits (protective values) were compared to known chemical concentrations. Using traditional calibration curves, 95% of upper confidence limits were within ~tenfold of the true concentrations. The error increased to ~60-fold (ESI+) and ~120-fold (ESI-) for the ionization efficiency estimation method and to ~150-fold (ESI+) and ~130-fold (ESI-) for the bounded response factor method. This work demonstrates successful implementation of confidence limit estimation strategies to support qNTA studies and marks a crucial step towards translating NTA data in a risk-based context.


Asunto(s)
Incertidumbre , Calibración , Espectrometría de Masas/métodos
6.
Anal Chem ; 93(33): 11601-11611, 2021 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-34382770

RESUMEN

There is an increasing need for comparable and harmonized retention times (tR) in liquid chromatography (LC) among different laboratories, to provide supplementary evidence for the identity of compounds in high-resolution mass spectrometry (HRMS)-based suspect and nontarget screening investigations. In this study, a rigorously tested, flexible, and less system-dependent unified retention time index (RTI) approach for LC is presented, based on the calibration of the elution pattern. Two sets of 18 calibrants were selected for each of ESI+ and ESI-based on the maximum overlap with the retention times and chemical similarity indices from a total set of 2123 compounds. The resulting calibration set, with RTI set to range between 1 and 1000, was proposed as the most appropriate RTI system after rigorous evaluation, coordinated by the NORMAN network. The validation of the proposed RTI system was done externally on different instrumentation and LC conditions. The RTI can also be used to check the reproducibility and quality of LC conditions. Two quantitative structure-retention relationship (QSRR)-based models were built based on the developed RTI systems, which assist in the removal of false-positive annotations. The applicability domains of the QSRR models allowed completing the identification process with higher confidence for substances within the domain, while indicating those substances for which results should be treated with caution. The proposed RTI system was used to improve confidence in suspect and nontarget screening and increase the comparability between laboratories as demonstrated for two examples. All RTI-related calculations can be performed online at http://rti.chem.uoa.gr/.


Asunto(s)
Reproducibilidad de los Resultados , Calibración , Cromatografía Liquida , Espectrometría de Masas
7.
Anal Chem ; 93(49): 16289-16296, 2021 12 14.
Artículo en Inglés | MEDLINE | ID: mdl-34842413

RESUMEN

Non-targeted analysis (NTA) encompasses a rapidly evolving set of mass spectrometry techniques aimed at characterizing the chemical composition of complex samples, identifying unknown compounds, and/or classifying samples, without prior knowledge regarding the chemical content of the samples. Recent advances in NTA are the result of improved and more accessible instrumentation for data generation and analysis tools for data evaluation and interpretation. As researchers continue to develop NTA approaches in various scientific fields, there is a growing need to identify, disseminate, and adopt community-wide method reporting guidelines. In 2018, NTA researchers formed the Benchmarking and Publications for Non-Targeted Analysis Working Group (BP4NTA) to address this need. Consisting of participants from around the world and representing fields ranging from environmental science and food chemistry to 'omics and toxicology, BP4NTA provides resources addressing a variety of challenges associated with NTA. Thus far, BP4NTA group members have aimed to establish a consensus on NTA-related terms and concepts and to create consistency in reporting practices by providing resources on a public Web site, including consensus definitions, reference content, and lists of available tools. Moving forward, BP4NTA will provide a setting for NTA researchers to continue discussing emerging challenges and contribute to additional harmonization efforts.


Asunto(s)
Benchmarking , Humanos
8.
Chem Rev ; 119(2): 1193-1220, 2019 01 23.
Artículo en Inglés | MEDLINE | ID: mdl-30474981

RESUMEN

The highly contagious disease tuberculosis (TB) is caused by the bacterium Mycobacterium tuberculosis (Mtb), which has been evolving drug resistance at an alarming rate. Like all human pathogens, Mtb requires iron for growth and virulence. Consequently, Mtb iron transport is an emerging drug target. However, the development of anti-TB drugs aimed at these metabolic pathways has been restricted by the dearth of information on Mtb iron acquisition. In this Review, we describe the multiple strategies utilized by Mtb to acquire ferric iron and heme iron. Mtb iron uptake is a complex process, requiring biosynthesis and subsequent export of Mtb siderophores, followed by ferric iron scavenging and ferric-siderophore import into Mtb. Additionally, Mtb possesses two possible heme uptake pathways and an Mtb-specific mechanism of heme degradation that yields iron and novel heme-degradation products. We conclude with perspectives for potential therapeutics that could directly target Mtb heme and iron uptake machineries. We also highlight how hijacking Mtb heme and iron acquisition pathways for drug import may facilitate drug transport through the notoriously impregnable Mtb cell wall.


Asunto(s)
Hierro/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculosis/microbiología , Proteínas Bacterianas/metabolismo , Transporte Biológico , Hemo/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Humanos , Hierro/química , Mycobacterium tuberculosis/patogenicidad , Sideróforos/química , Sideróforos/metabolismo , Tuberculosis/tratamiento farmacológico , Virulencia
9.
Anal Bioanal Chem ; 413(30): 7495-7508, 2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-34648052

RESUMEN

With the increasing availability of high-resolution mass spectrometers, suspect screening and non-targeted analysis are becoming popular compound identification tools for environmental researchers. Samples of interest often contain a large (unknown) number of chemicals spanning the detectable mass range of the instrument. In an effort to separate these chemicals prior to injection into the mass spectrometer, a chromatography method is often utilized. There are numerous types of gas and liquid chromatographs that can be coupled to commercially available mass spectrometers. Depending on the type of instrument used for analysis, the researcher is likely to observe a different subset of compounds based on the amenability of those chemicals to the selected experimental techniques and equipment. It would be advantageous if this subset of chemicals could be predicted prior to conducting the experiment, in order to minimize potential false-positive and false-negative identifications. In this work, we utilize experimental datasets to predict the amenability of chemical compounds to detection with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The assembled dataset totals 5517 unique chemicals either explicitly detected or not detected with LC-ESI-MS. The resulting detected/not-detected matrix has been modeled using specific molecular descriptors to predict which chemicals are amenable to LC-ESI-MS, and to which form(s) of ionization. Random forest models, including a measure of the applicability domain of the model for both positive and negative modes of the electrospray ionization source, were successfully developed. The outcome of this work will help to inform future suspect screening and non-targeted analyses of chemicals by better defining the potential LC-ESI-MS detectable chemical landscape of interest.

10.
Anal Bioanal Chem ; 412(18): 4221-4233, 2020 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-32335688

RESUMEN

Non-targeted analysis (NTA) methods are being increasingly used to aid in the identification of unknown compounds in the environment, a problem that has challenged environmental chemists for decades. Despite its increased use, quality assurance practices for NTA have not been well established. Furthermore, capabilities and limitations of certain NTA methods have not been thoroughly evaluated. Standard reference material dust (SRM 2585) was used here to evaluate the ability of NTA to identify previously reported compounds, as well as a suite of 365 chemicals that were spiked at various stages of the analytical procedure. Analysis of the unaltered SRM 2585 extracts revealed that several previously reported compounds can be identified by NTA, and that correct identification was dependent on concentration. A manual inspection of unknown features in SRM 2585 revealed the presence of two chlorinated and fluorinated compounds in high abundance, likely precursors to perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonate (PFHxS). A retrospective analysis of data from the American Healthy Homes Survey revealed that these compounds were present in 42% of sampled homes. Spiking the dust at various stages of sample preparation revealed losses from extraction, cleanup, and instrumental analysis; the log Kow for individual compounds influenced the overall recovery levels but no pattern could be discerned from the various degrees of interference that the matrix had on the ionization efficiency of the spiked chemicals. Analysis of the matrix-free chemical mixture at low, medium, and high concentrations led to more correct identifications than analysis at one, very high concentration. Varying the spiked amount and identifying reported compounds at known concentrations allowed an estimation of the lower limits of identification (LOIs) for NTA, analogous to limits of detection in targeted analysis. The LOIs were much lower than levels in dust that would be likely to cause bioactivity in humans, indicating that NTA is useful for identifying and monitoring compounds that may be of toxicological concern. Graphical abstract.

11.
Anal Bioanal Chem ; 412(20): 4931-4939, 2020 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-32494915

RESUMEN

Non-targeted analysis (NTA) is a rapidly evolving analytical technique with numerous opportunities to improve and expand instrumental and data analysis methods. In this work, NTA was performed on eight synthetic mixtures containing 1264 unique chemical substances from the U.S. Environmental Protection Agency's Non-Targeted Analysis Collaborative Trial (ENTACT). These mixtures were analyzed by atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) using both positive and negative polarities for a total of four modes. Out of the 1264 ENTACT chemical substances, 1116 were detected in at least one ionization mode, 185 chemicals were detected using all four ionization modes, whereas 148 were not detected. Forty-four chemicals were detected only by APCI, and 181 were detected only by ESI. Molecular descriptors and physicochemical properties were used to assess which ionization type was preferred for a given compound. One ToxPrint substructure (naphthalene group) was found to be enriched in compounds only detected using APCI, and eight ToxPrints (e.g., several alcohol moieties) were enriched in compounds only detected using ESI. Examination of physicochemical parameters for ENTACT chemicals suggests that those with higher aqueous solubility preferentially ionized by ESI-. While ESI typically detects a larger number of compounds, APCI offers chromatograms with less background, fewer co-elutions, and additional chemical space coverage, suggesting both should be considered for broader coverage in future NTA research. Graphical abstract.

12.
Anal Bioanal Chem ; 412(6): 1303-1315, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31965249

RESUMEN

High-resolution mass spectrometry (HRMS) enables rapid chemical annotation via accurate mass measurements and matching of experimentally derived spectra with reference spectra. Reference libraries are generated from chemical standards and are therefore limited in size relative to known chemical space. To address this limitation, in silico spectra (i.e., MS/MS or MS2 spectra), predicted via Competitive Fragmentation Modeling-ID (CFM-ID) algorithms, were generated for compounds within the U.S. Environmental Protection Agency's (EPA) Distributed Structure-Searchable Toxicity (DSSTox) database (totaling, at the time of analysis, ~ 765,000 substances). Experimental spectra from EPA's Non-Targeted Analysis Collaborative Trial (ENTACT) mixtures (n = 10) were then used to evaluate the performance of the in silico spectra. Overall, MS2 spectra were acquired for 377 unique compounds from the ENTACT mixtures. Approximately 53% of these compounds were correctly identified using a commercial reference library, whereas up to 50% were correctly identified as the top hit using the in silico library. Together, the reference and in silico libraries were able to correctly identify 73% of the 377 ENTACT substances. When using the in silico spectra for candidate filtering, an examination of binary classifiers showed a true positive rate (TPR) of 0.90 associated with false positive rates (FPRs) of 0.10 to 0.85, depending on the sample and method of candidate filtering. Taken together, these findings show the abilities of in silico spectra to correctly identify true positives in complex samples (at rates comparable to those observed with reference spectra), and efficiently filter large numbers of potential false positives from further consideration. Graphical abstract.

13.
Biochemistry ; 58(6): 489-492, 2019 02 12.
Artículo en Inglés | MEDLINE | ID: mdl-30605595

RESUMEN

Mycobacterium tuberculosis heme-degrading protein MhuD degrades heme to mycobilin isomers and iron, while its closest homologues from Staphylococcus aureus, IsdG and IsdI, degrade heme to staphylobilin isomers, formaldehyde, and iron. Superposition of the structures of the heme-bound complexes reveals that the heme molecule in the MhuD active site is rotated ∼90° about the tetrapyrrole plane with respect to IsdG and IsdI active site heme molecules. Therefore, the variation in IsdG/IsdI and MhuD chromophore products may be attributed to the different heme orientations. In MhuD, two arginines, Arg22 and Arg26, stabilize the heme propionates and may account for the heme orientation. Herein, we demonstrate that the MhuD-R26S variant alters the resulting chromophore product from mycobilin to biliverdin IXα (α-BV), whereas the R22S variant does not. Surprisingly, unlike canonical heme oxygenase (HO) that also degrades heme to α-BV, the MhuD-R26S variant produces the C1 product formaldehyde rather than carbon monoxide as observed for HO. The MhuD-R26S variant is an important tool for further probing the mechanism of action of MhuD and for studying the fate of the MhuD product in mycobacterium.


Asunto(s)
Proteínas Bacterianas/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo/metabolismo , Mutación , Mycobacterium tuberculosis/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biliverdina/metabolismo , Monóxido de Carbono/metabolismo , Formaldehído/metabolismo , Hemo/química , Hemo Oxigenasa (Desciclizante)/química , Hemo Oxigenasa (Desciclizante)/genética , Modelos Moleculares , Conformación Proteica
14.
Biochemistry ; 58(46): 4610-4620, 2019 11 19.
Artículo en Inglés | MEDLINE | ID: mdl-31638374

RESUMEN

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, requires iron for survival. In Mtb, MhuD is the cytosolic protein that degrades imported heme. MhuD is distinct, in both sequence and structure, from canonical heme oxygenases (HOs) but homologous with IsdG-type proteins. Canonical HO is found mainly in eukaryotes, while IsdG-type proteins are predominantly found in prokaryotes, including pathogens. While there are several published structures of MhuD and other IsdG-type proteins in complex with the heme substrate, no structures of IsdG-type proteins in complex with a product have been reported, unlike the case for HOs. We recently showed that the Mtb variant MhuD-R26S produces biliverdin IXα (αBV) rather than the wild-type mycobilin isomers. Given that mycobilin and other IsdG-type protein products like staphylobilin are difficult to isolate in quantities sufficient for structure determination, here we use the MhuD-R26S variant and its product αBV as a proxy to study the IsdG-type protein-product complex. First, we show that αBV has a nanomolar affinity for MhuD and the R26S variant. Second, we determined the MhuD-R26S-αBV complex structure to 2.5 Å, which reveals two notable features: (1) two αBV molecules bound per active site and (2) a novel α-helix (α3) that was not observed in previous MhuD-heme structures. Finally, through molecular dynamics simulations, we show that α3 is stable with the proximal αBV alone. MhuD's high affinity for the product and the observed structural and electrostatic changes that accompany substrate turnover suggest that there may be an unidentified class of proteins that are responsible for the extraction of products from MhuD and other IsdG-type proteins.


Asunto(s)
Proteínas Bacterianas/química , Biliverdina/metabolismo , Hemo/metabolismo , Oxigenasas de Función Mixta/química , Mycobacterium tuberculosis/metabolismo , Proteínas Bacterianas/metabolismo , Biliverdina/química , Cristalografía por Rayos X , Hemo/química , Humanos , Oxigenasas de Función Mixta/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Mutación Puntual , Conformación Proteica , Especificidad por Sustrato , Tuberculosis/microbiología
15.
Anal Bioanal Chem ; 411(4): 835-851, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30612177

RESUMEN

Non-targeted analysis (NTA) methods are increasingly used to discover contaminants of emerging concern (CECs), but the extent to which these methods can support exposure and health studies remains to be determined. EPA's Non-Targeted Analysis Collaborative Trial (ENTACT) was launched in 2016 to address this need. As part of ENTACT, 1269 unique substances from EPA's ToxCast library were combined to make ten synthetic mixtures, with each mixture containing between 95 and 365 substances. As a participant in the trial, we first performed blinded NTA on each mixture using liquid chromatography (LC) coupled with high-resolution mass spectrometry (HRMS). We then performed an unblinded evaluation to identify limitations of our NTA method. Overall, at least 60% of spiked substances could be observed using selected methods. Discounting spiked isomers, true positive rates from the blinded and unblinded analyses reached a maximum of 46% and 65%, respectively. An overall reproducibility rate of 75% was observed for substances spiked into more than one mixture and observed at least once. Considerable discordance in substance identification was observed when comparing a subset of our results derived from two separate reversed-phase chromatography methods. We conclude that a single NTA method, even when optimized, can likely characterize only a subset of ToxCast substances (and, by extension, other CECs). Rigorous quality control and self-evaluation practices should be required of labs generating NTA data to support exposure and health studies. Accurate and transparent communication of performance results will best enable meaningful interpretations and defensible use of NTA data. Graphical abstract ᅟ.


Asunto(s)
Cromatografía Liquida/métodos , Cromatografía de Fase Inversa/métodos , Mezclas Complejas , Monitoreo del Ambiente/métodos , Contaminantes Ambientales/análisis , Espectrometría de Masas/métodos , Contaminantes Ambientales/toxicidad , Trazadores Radiactivos , Estándares de Referencia , Reproducibilidad de los Resultados
16.
Anal Chem ; 89(2): 1194-1201, 2017 01 17.
Artículo en Inglés | MEDLINE | ID: mdl-27991763

RESUMEN

The recent increase in extensively drug-resistant bacterial pathogens and the associated increase of morbidity and mortality demonstrate the immediate need for new antibiotic backbones with novel mechanisms of action. Here, we report the development of the PepSAVI-MS pipeline for bioactive peptide discovery. This highly versatile platform employs mass spectrometry and statistics to identify bioactive peptide targets from complex biological samples. We validate the use of this platform through the successful identification of known bioactive peptides from a botanical species, Viola odorata. Using this pipeline, we have widened the known antimicrobial spectrum for V. odorata cyclotides, including antibacterial activity of cycloviolacin O2 against A. baumannii. We further demonstrate the broad applicability of the platform through the identification of novel anticancer activities for cycloviolacins by their cytotoxicity against ovarian, breast, and prostate cancer cell lines.


Asunto(s)
Antibacterianos/química , Antineoplásicos Fitogénicos/química , Productos Biológicos/química , Ciclotidas/química , Descubrimiento de Drogas , Viola/química , Infecciones por Acinetobacter/tratamiento farmacológico , Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/farmacología , Antineoplásicos Fitogénicos/farmacología , Productos Biológicos/farmacología , Línea Celular Tumoral , Ciclotidas/farmacología , Humanos , Neoplasias/tratamiento farmacológico , Biblioteca de Péptidos
18.
Electrophoresis ; 35(24): 3441-51, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24825726

RESUMEN

As primarily sessile organisms, photosynthetic species survive in dynamic environments by using elegant signaling pathways to manifest molecular responses to extracellular cues. These pathways exploit phosphorylation of specific amino acids (e.g. serine, threonine, tyrosine), which impact protein structure, function, and localization. Despite substantial progress in implementation of phosphoproteomics to understand photosynthetic organisms, researchers still struggle to translate a biological question into an experimental strategy and vice versa. This review evaluates the current status of phosphoproteomics in photosynthetic organisms and concludes with recommendations based on current knowledge.


Asunto(s)
Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fotosíntesis , Proteómica/métodos , Fosfoproteínas/análisis , Procesamiento Proteico-Postraduccional
19.
Inorg Chem ; 53(12): 5931-40, 2014 Jun 16.
Artículo en Inglés | MEDLINE | ID: mdl-24901029

RESUMEN

Mycobacterium heme utilization degrader (MhuD) is a heme-degrading protein from Mycobacterium tuberculosis responsible for extracting the essential nutrient iron from host-derived heme. MhuD has been previously shown to produce unique organic products compared to those of canonical heme oxygenases (HOs) as well as those of the IsdG/I heme-degrading enzymes from Staphylococcus aureus. Here, we report the X-ray crystal structure of cyanide-inhibited MhuD (MhuD-heme-CN) as well as detailed (1)H nuclear magnetic resonance (NMR), UV/vis absorption, and magnetic circular dichroism (MCD) spectroscopic characterization of this species. There is no evidence for an ordered network of water molecules on the distal side of the heme substrate in the X-ray crystal structure, as was previously reported for canonical HOs. The degree of heme ruffling in the crystal structure of MhuD is greater than that observed for HO and less than that observed for IsdI. As a consequence, the Fe 3dxz-, 3dyz-, and 3dxy-based MOs are very close in energy, and the room-temperature (1)H NMR spectrum of MhuD-heme-CN is consistent with population of both a (2)Eg electronic state with a (dxy)(2)(dxz,dyz)(3) electron configuration, similar to the ground state of canonical HOs, and a (2)B2g state with a (dxz,dyz)(4)(dxy)(1) electron configuration, similar to the ground state of cyanide-inhibited IsdI. Variable temperature, variable field MCD saturation magnetization data establishes that MhuD-heme-CN has a (2)B2g electronic ground state with a low-lying (2)Eg excited state. Our crystallographic and spectroscopic data suggest that there are both structural and electronic contributions to the α-meso regioselectivity of MhuD-catalyzed heme cleavage. The structural distortion of the heme substrate observed in the X-ray crystal structure of MhuD-heme-CN is likely to favor cleavage at the α- and γ-meso carbons, whereas the spin density distribution may favor selective oxygenation of the α-meso carbon.


Asunto(s)
Cianuros/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo/metabolismo , Mycobacterium tuberculosis/enzimología , Cristalografía por Rayos X , Cianuros/química , Hemo/química , Hemo Oxigenasa (Desciclizante)/química , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Conformación Proteica , Tuberculosis/microbiología
20.
Cell Death Discov ; 10(1): 161, 2024 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-38565596

RESUMEN

Chemokinostatin-1 (CKS1) is a 24-mer peptide originally discovered as an anti-angiogenic peptide derived from the CXCL1 chemokine. Here, we demonstrate that CKS1 acts not only as an anti-angiogenic peptide but also as an oncolytic peptide due to its structural and physical properties. CKS1 induced both necrotic and apoptotic cell death specifically in cancer cells while showing minimal toxicity in non-cancerous cells. Mechanistically, CKS1 disrupted the cell membrane of cancer cells quickly after treatment and activated the apoptotic pathway at later time points. Furthermore, immunogenic molecules were released from CKS1-treated cells, indicating that CKS1 induces immunogenic cell death. CKS1 effectively suppressed tumor growth in vivo. Collectively, these data demonstrate that CKS1 functions as an oncolytic peptide and has a therapeutic potential to treat cancer.

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