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1.
Nat Commun ; 15(1): 2698, 2024 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-38538595

RESUMO

Toxoplasma gondii is an obligate intracellular parasite of rodents and humans. Interferon-inducible guanylate binding proteins (GBPs) are mediators of T. gondii clearance, however, this mechanism is incomplete. Here, using automated spatially targeted optical micro proteomics we demonstrate that inducible nitric oxide synthetase (iNOS) is highly enriched at GBP2+ parasitophorous vacuoles (PV) in murine macrophages. iNOS expression in macrophages is necessary to limit T. gondii load in vivo and in vitro. Although iNOS activity is dispensable for GBP2 recruitment and PV membrane ruffling; parasites can replicate, egress and shed GBP2 when iNOS is inhibited. T. gondii clearance by iNOS requires nitric oxide, leading to nitration of the PV and collapse of the intravacuolar network of membranes in a chromosome 3 GBP-dependent manner. We conclude that reactive nitrogen species generated by iNOS cooperate with GBPs to target distinct structures in the PV that are necessary for optimal parasite clearance in macrophages.


Assuntos
Toxoplasma , Vacúolos , Animais , Humanos , Camundongos , Interferons/metabolismo , Macrófagos/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Toxoplasma/metabolismo , Vacúolos/metabolismo
2.
J Mol Cell Cardiol ; 186: 1-15, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37951204

RESUMO

Myocardial infarction (MI) results from occlusion of blood supply to the heart muscle causing death of cardiac muscle cells. Following myocardial infarction (MI), extracellular matrix deposition and scar formation mechanically stabilize the injured heart as damaged myocytes undergo necrosis and removal. Fibroblasts and macrophages are key drivers of post-MI scar formation, maturation, and ongoing long-term remodelling; however, their individual contributions are difficult to assess from bulk analyses of infarct scar. Here, we employ state-of-the-art automated spatially targeted optical micro proteomics (autoSTOMP) to photochemically tag and isolate proteomes associated with subpopulations of fibroblasts (SMA+) and macrophages (CD68+) in the context of the native, MI tissue environment. Over a time course of 6-weeks post-MI, we captured dynamic changes in the whole-infarct proteome and determined that some of these protein composition signatures were differentially localized near SMA+ fibroblasts or CD68+ macrophages within the scar region. These results link specific cell populations to within-infarct protein remodelling and illustrate the distinct metabolic and structural processes underlying the observed physiology of each cell type.


Assuntos
Cicatriz , Infarto do Miocárdio , Ratos , Animais , Cicatriz/metabolismo , Proteômica , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Fibroblastos/metabolismo , Miócitos Cardíacos/metabolismo , Macrófagos/metabolismo , Remodelação Ventricular
3.
bioRxiv ; 2023 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-37546987

RESUMO

Toxoplasma gondii is an obligate intracellular, protozoan pathogen of rodents and humans. T. gondii's ability to grow within cells and evade cell-autonomous immunity depends on the integrity of the parasitophorous vacuole (PV). Interferon-inducible guanylate binding proteins (GBPs) are central mediators of T. gondii clearance, however, the precise mechanism linking GBP recruitment to the PV and T. gondii restriction is not clear. This knowledge gap is linked to heterogenous GBP-targeting across a population of vacuoles and the lack of tools to selectively purify the intact PV. To identify mediators of parasite clearance associated with GBP2-positive vacuoles, we employed a novel protein discovery tool automated spatially targeted optical micro proteomics (autoSTOMP). This approach identified inducible nitric oxide synthetase (iNOS) enriched at levels similar to the GBPs in infected bone marrow-derived myeloid cells. iNOS expression on myeloid cells was necessary for mice to control T. gondii growth in vivo and survive acute infection. T. gondii infection of IFNγ-primed macrophage was sufficient to robustly induce iNOS expression. iNOS restricted T. gondii infection through nitric oxide synthesis rather than arginine depletion, leading to robust and selective nitration of the PV. Optimal parasite restriction by iNOS and vacuole nitration depended on the chromosome 3 GBPs. Notably, GBP2 recruitment and ruffling of the PV membrane occurred in iNOS knockouts, however, these vacuoles contained dividing parasites. iNOS activity was necessary for the collapse of the intravacuolar network of nanotubular membranes which connects parasites to each other and the host cytosol. Based on these data we conclude reactive nitrogen species generated by iNOS cooperate with the chromosome 3 GBPs to target distinct biology of the PV that are necessary for optimal parasite clearance in murine myeloid cells.

4.
Allergy ; 78(12): 3193-3203, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37497566

RESUMO

BACKGROUND: Recent studies have shown deposition of immunoglobulin G4 (IgG4) and food proteins in the esophageal mucosa of eosinophilic esophagitis (EoE) patients. Our aims were to assess whether co-localization of IgG4 and major cow's milk proteins (CMPs) was associated with EoE disease activity and to investigate the proteins enriched in proximity to IgG4 deposits. METHODS: This study included adult subjects with EoE (n = 13) and non-EoE controls (n = 5). Esophageal biopsies were immunofluorescence stained for IgG4 and CMPs. Co-localization in paired samples from active disease and remission was assessed and compared to controls. The proteome surrounding IgG4 deposits was evaluated by the novel technique, AutoSTOMP. IgG4-food protein interactions were confirmed with co-immunoprecipitation and mass spectrometry. RESULTS: IgG4-CMP co-localization was higher in the active EoE group compared to paired remission samples (Bos d 4, p = .02; Bos d 5, p = .002; Bos d 8, p = .002). Co-localization was also significantly higher in the active EoE group compared to non-EoE controls (Bos d 4, p = .0013; Bos d 5, p = .0007; Bos d 8, p = .0013). AutoSTOMP identified eosinophil-derived proteins (PRG 2 and 3, EPX, RNASE3) and calpain-14 in IgG4-enriched areas. Co-immunoprecipitation and mass spectrometry confirmed IgG4 binding to multiple food allergens. CONCLUSION: These findings further contribute to the understanding of the interaction of IgG4 with food antigens as it relates to EoE disease activity. These data strongly suggest the immune complex formation of IgG4 and major cow's milk proteins. These immune complexes may have a potential role in the pathophysiology of EoE by contributing to eosinophil activation and disease progression.


Assuntos
Esofagite Eosinofílica , Adulto , Feminino , Animais , Bovinos , Humanos , Esofagite Eosinofílica/patologia , Complexo Antígeno-Anticorpo , Imunoglobulina G , Alérgenos , Proteínas do Leite
5.
J Proteome Res ; 20(9): 4543-4552, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34436902

RESUMO

Tissue microenvironment properties like blood flow, extracellular matrix, or proximity to immune-infiltrate are important regulators of cell biology. However, methods to study regional protein expression in the native tissue environment are limited. To address this need, we developed a novel approach to visualize, purify, and measure proteins in situ using automated spatially targeted optical microproteomics (AutoSTOMP). Here, we report custom codes to specify regions of heterogeneity in a tissue section and UV-biotinylate proteins within those regions. We have developed liquid chromatography-mass spectrometry (LC-MS)/MS-compatible biochemistry to purify those proteins and label-free quantification methodology to determine protein enrichment in target cell types or structures relative to nontarget regions in the same sample. These tools were applied to (a) identify inflammatory proteins expressed by CD68+ macrophages in rat cardiac infarcts and (b) characterize inflammatory proteins enriched in IgG4+ lesions in human esophageal tissues. These data indicate that AutoSTOMP is a flexible approach to determine regional protein expression in situ on a range of primary tissues and clinical biopsies where current tools and sample availability are limited.


Assuntos
Proteínas , Proteômica , Animais , Cromatografia Líquida , Espectrometria de Massas , Ratos
6.
Anal Chem ; 92(2): 2005-2010, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31869197

RESUMO

Spatially targeted optical microproteomics (STOMP) is a method to study region-specific protein complexity in primary cells and tissue samples. STOMP uses a confocal microscope to visualize structures of interest and to tag the proteins within those structures by a photodriven cross-linking reaction so that they can be affinity purified and identified by mass spectrometry (eLife 2015, 4, e09579). However, the use of a custom photo-cross-linker and the requirement for extensive user intervention during sample tagging have posed barriers to the utilization of STOMP. To address these limitations, we built automated STOMP (autoSTOMP) which uses a customizable code in SikuliX to coordinate image capture and cross-linking functions in Zeiss Zen Black with image processing in FIJI. To increase protocol accessibility, we implemented a commercially available biotin-benzophenone photo-cross-linking and purification protocol. Here we demonstrate that autoSTOMP can efficiently label, purify, and identify proteins belonging to 1-2 µm structures in primary human foreskin fibroblasts or mouse bone marrow-derived dendritic cells infected with the protozoan parasite Toxoplasma gondii (Tg). AutoSTOMP can easily be adapted to address a range of research questions using Zeiss Zen Black microscopy systems and LC-MS protocols that are standard in many research cores.


Assuntos
Automação , Proteínas/análise , Proteômica , Animais , Células Dendríticas/química , Fibroblastos/química , Humanos , Camundongos , Estrutura Molecular , Imagem Óptica , Conformação Proteica
7.
Eur J Neurosci ; 47(9): 1096-1109, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29480936

RESUMO

The selective vulnerability of hippocampal area CA1 to ischemia-induced injury is a well-known phenomenon. However, the cellular mechanisms that confer resistance to area CA3 against ischemic damage remain elusive. Here, we show that oxygen-glucose deprivation-reperfusion (OGD-RP), an in vitro model that mimic the pathological conditions of the ischemic stroke, increases the phosphorylation level of tropomyosin receptor kinase B (TrkB) in area CA3. Slices preincubated with brain-derived neurotrophic factor (BDNF) or 7,8-dihydroxyflavone (7,8-DHF) exhibited reduced depression of the electrical activity triggered by OGD-RP. Consistently, blockade of TrkB suppressed the resistance of area CA3 to OGD-RP. The protective effect of TrkB activation was limited to area CA3, as OGD-RP caused permanent suppression of CA1 responses. At the cellular level, TrkB activation leads to phosphorylation of the accessory proteins SHC and Gab as well as the serine/threonine kinase Akt, members of the phosphoinositide 3-kinase/Akt (PI-3-K/Akt) pathway, a cascade involved in cell survival. Hence, acute slices pretreated with the Akt antagonist MK2206 in combination with BDNF lost the capability to resist the damage inflicted with OGD-RP. Consistently, with these results, CA3 pyramidal cells exhibited reduced propidium iodide uptake and caspase-3 activity in slices pretreated with BDNF and exposed to OGD-RP. We propose that PI-3-K/Akt downstream activation mediated by TrkB represents an endogenous mechanism responsible for the resistance of area CA3 to ischemic damage.


Assuntos
Glucose/metabolismo , Hipocampo/metabolismo , Oxigênio/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptor trkB/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
8.
ACS Chem Neurosci ; 9(4): 738-748, 2018 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-29172440

RESUMO

Pyramidal cells and astrocytes have differential susceptibility to oxygen-glucose deprivation and reperfusion (OGD-RP). It is known that excessive reactive oxygen species (ROS) in mitochondria initiates cell death, while glutathione (GSH) is one of the major defenses against ROS. Although it is known that astrocytes contain a higher concentration of GSH than neurons, and that astrocytes can provide neurons with GSH, we are unaware of a detailed and quantitative examination of the dynamic changes in the mitochondrial GSH system in the two cell types during OGD-RP. Here, we determined mitochondrial membrane potential and the degrees of oxidation of the mitochondrially targeted roGFP-based sensors for hydrogen peroxide (OxDP) and GSH (OxDG). We also developed a method to estimate the mitochondrial GSH (mGSH) concentration in single cells in the CA1 region of organotypic hippocampal slice cultures at several time-points during OGD-RP. We find that mitochondrial membrane potential drops in pyramidal cells during OGD while it is relatively stable in astrocytes. In both types of cell, the mitochondrial membrane potential decreases during RP. During OGD-RP, mitochondrial peroxide levels are the same. Astrocytic mGSH is more than four times higher than pyramidal cell mGSH (3.2 vs 0.7 mM). Astrocytic mGSH is drained from mitochondria during OGD, whereas in pyramidal cells it remains fairly constant. OxDGSH prior to and during OGD is lower (less oxidized) in pyramidal cells than in astrocytes, but the two nearly converge during RP. The larger changes of redox status in the GSH system in pyramidal cells than astrocytes is an upstream sign of the higher mortality of the pyramidal cells after facing an insult. The pattern of [mGSH] changes in the two cell types could be recognized as another mechanism by which astrocytes protect neurons from transient, extreme conditions.


Assuntos
Astrócitos/metabolismo , Hipocampo/metabolismo , Oxigênio/metabolismo , Células Piramidais/metabolismo , Animais , Células Cultivadas , Glucose/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Neurônios/metabolismo , Espécies Reativas de Oxigênio/metabolismo
9.
Antioxid Redox Signal ; 27(9): 534-549, 2017 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-28129719

RESUMO

AIMS: The susceptibility of CA1 over CA3 to damage from cerebral ischemia may be related to the differences in reactive oxygen species (ROS) production/removal between the two hippocampal subfields. We aimed to measure CA1/CA3 differences in net ROS production in real time in the first 30 min of reperfusion in pyramidal cells. We aimed to determine the underlying cause of the differential vulnerability of CA1 and CA3. RESULTS: Real-time determinations of mitochondrial H2O2 and, independently, glutathione (GSH) redox status from roGFP-based probes in individual pyramidal cells in organotypic hippocampal cultures during oxygen-glucose deprivation (OGD)-reperfusion (RP) demonstrate a significantly more oxidizing environment during RP in CA1 than CA3 mitochondria. Protein levels (immunohistochemistry and Western blots), roGFP2-based probe measurements during controlled mitochondrial production of ROS, and thioredoxin reductase (TrxR) inhibition by auranofin are consistent with a more effective mitochondrial thioredoxin (Trx) system in CA3. Inhibition of TrxR eliminates the differences in redox status and cell death between the regions. Overexpression of cytosolic Trx1 does not influence mitochondrial H2O2 production. INNOVATION: Real-time changes of mitochondrial H2O2 and GSH in tissue cultures during early RP, and also during controlled production of superoxide and peroxide, reveal significant differences between CA1 and CA3. The mitochondrial Trx system is responsible for the observed differences during RP as well as for delayed cell death 18 h afterward. CONCLUSION: Greater mitochondrial Trx efficacy in CA3 pyramidal cells results in less vulnerability to ischemia/reperfusion because of the less oxidizing environment in CA3 mitochondria during RP. Antioxid. Redox Signal. 27, 534-549.


Assuntos
Isquemia Encefálica/metabolismo , Hipocampo/metabolismo , Mitocôndrias/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Isquemia Encefálica/etiologia , Morte Celular , Glutationa/análise , Hipocampo/citologia , Peróxido de Hidrogênio/análise , Masculino , Técnicas de Cultura de Órgãos , Estresse Oxidativo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/complicações , Tiorredoxina Dissulfeto Redutase/metabolismo
10.
ACS Chem Neurosci ; 6(11): 1838-48, 2015 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-26291433

RESUMO

A redox-sensitive Grx1-roGFP2 fusion protein was introduced by transfection into single pyramidal neurons in the CA1 subfield of organotypic hippocampal slice cultures (OHSCs). We assessed changes in the GSH system in neuronal cytoplasm and mitochondria during oxygen-glucose deprivation and reperfusion (OGD/RP), an in vitro model of stroke. Pyramidal cells in a narrow range of depths below the surface of the OHSC were transfected by gene gun or single-cell electroporation with cyto- or mito-Grx1-roGFP2. To mimic the conditions of acute stroke, we developed an optimized superfusion system with the capability of rapid and reproducible exchange of the solution bathing the OHSCs. Measurements of pO2 as a function of tissue depth show that in the region containing the transfected cells, the pO2 is well-controlled. We also found that the pO2 changes on the same time scale as changes in intracranial pressure, cerebral blood flow, and pO2 during acute stroke. Determining the reduction potential, EGSH, from the ratiometric fluorescence signal requires an absolute intensity measurement during calibration of the Grx1-roGFP2. Using the signal from cotransfected tdTomato as an internal standard during calibration improves quantitative measurements of Grx1-roGFP2 redox status and allows EGSH to be determined. EGSH becomes more reducing during OGD and more oxidizing during RP in mitochondria while changes in cytoplasm are not significant compared with controls.


Assuntos
Região CA1 Hipocampal/metabolismo , Glutationa/metabolismo , Células Piramidais/metabolismo , Análise de Célula Única/métodos , Acidente Vascular Cerebral/metabolismo , Técnicas de Cultura de Tecidos/métodos , Animais , Citoplasma/metabolismo , Eletroporação , Glucose/deficiência , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Hipóxia/metabolismo , Microscopia Confocal/métodos , Mitocôndrias/metabolismo , Modelos Biológicos , Oxirredução , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo
11.
J Phys Chem B ; 119(11): 4194-202, 2015 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-25757155

RESUMO

The conformations and motions of lipid molecules under different membrane curvatures have important implications for transmembrane protein function, binding events, and overall membrane organization. This work reports on the local order parameters of saturated lipid molecules, as measured by (13)C NMR relaxation, under several curvature conditions to probe structural changes as a function of lipid bilayer curvature. Different curvature conditions are created by depositing phosphatidylcholine membranes on spherical beads of various diameters. The findings reveal that the order parameters are not a continuous function of the membrane curvature. While small (30 nm) and large (110 nm) diameter bilayers exhibit similar order parameters, bilayers with curvatures of 60-80 nm diameter show a consistently increased order parameter along the entire lipid molecule, indicating a higher packing density and lateral tension. Order parameters for curvatures between 60 and 80 nm also show molecular evidence for interdigitation.


Assuntos
Bicamadas Lipídicas/química , Conformação Molecular , Microesferas , Dióxido de Silício/química
12.
Colloids Surf B Biointerfaces ; 68(2): 245-9, 2009 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-19058952

RESUMO

Composite microparticle drug delivery systems based on chitosan, alginate and pectin with improved pH sensitivity were developed for oral delivery of protein drugs, using bovine serum albumin (BSA) as a model drug. The composite drug-loaded microparticles with a mean particle size less than 200mum were prepared by a convenient shredding method. Since the microparticles were formed by tripolyphosphate cross-linking, electrostatic complexation by alginate and/or pectin, as well as ionotropic gelation with calcium ions, the microparticles exhibited an improved pH-sensitive drug release property. The in vitro drug release behaviors of the microparticles were studied in simulated gastric (pH 1.2 and pH 5.0), intestinal (pH 7.4) and colonic (pH 6.0 and pH 6.8 with enzyme) media. For the composite microparticles with suitable compositions, the releases of BSA at pH 1.2 and pH 5.0 could be effectively sustained, while the releases at pH 7.4, pH 6.8 and pH 6.0 increased significantly, especially in the presence of pectinase. These results clearly suggested that the microparticles had potential for site-specific protein drug delivery through oral administration.


Assuntos
Alginatos/química , Quitosana/química , Sistemas de Liberação de Medicamentos , Nanocompostos/química , Nanopartículas/química , Pectinas/química , Soroalbumina Bovina/farmacologia , Animais , Bovinos , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Nanopartículas/ultraestrutura , Tamanho da Partícula , Poligalacturonase/farmacologia
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