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1.
PLoS Comput Biol ; 16(9): e1008202, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32925922

RESUMO

Hydrogen peroxide (H2O2) promotes a range of phenotypes depending on its intracellular concentration and dosing kinetics, including cell death. While this qualitative relationship has been well established, the quantitative and mechanistic aspects of H2O2 signaling are still being elucidated. Mitochondria, a putative source of intracellular H2O2, have recently been demonstrated to be particularly vulnerable to localized H2O2 perturbations, eliciting a dramatic cell death response in comparison to similar cytosolic perturbations. We sought to improve our dynamic and mechanistic understanding of the mitochondrial H2O2 reaction network in HeLa cells by creating a kinetic model of this system and using it to explore basal and perturbed conditions. The model uses the most current quantitative proteomic and kinetic data available to predict reaction rates and steady-state concentrations of H2O2 and its reaction partners within individual mitochondria. Time scales ranging from milliseconds to one hour were simulated. We predict that basal, steady-state mitochondrial H2O2 will be in the low nM range (2-4 nM) and will be inversely dependent on the total pool of peroxiredoxin-3 (Prx3). Neglecting efflux of H2O2 to the cytosol, the mitochondrial reaction network is expected to control perturbations well up to H2O2 generation rates ~50 µM/s (0.25 nmol/mg-protein/s), above which point the Prx3 system would be expected to collapse. Comparison of these results with redox Western blots of Prx3 and Prx2 oxidation states demonstrated reasonable trend agreement at short times (≤ 15 min) for a range of experimentally perturbed H2O2 generation rates. At longer times, substantial efflux of H2O2 from the mitochondria to the cytosol was evidenced by peroxiredoxin-2 (Prx2) oxidation, and Prx3 collapse was not observed. A refined model using Monte Carlo parameter sampling was used to explore rates of H2O2 efflux that could reconcile model predictions of Prx3 oxidation states with the experimental observations.


Assuntos
Peróxido de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Neoplasias/metabolismo , Biologia Computacional , Citosol/química , Citosol/metabolismo , Células HeLa , Humanos , Cinética , Mitocôndrias/química , Neoplasias/química , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia
2.
Proc Natl Acad Sci U S A ; 117(28): 16383-16390, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601238

RESUMO

Calcium uptake by the mitochondrial calcium uniporter coordinates cytosolic signaling events with mitochondrial bioenergetics. During the past decade all protein components of the mitochondrial calcium uniporter have been identified, including MCU, the pore-forming subunit. However, the specific lipid requirements, if any, for the function and formation of this channel complex are currently not known. Here we utilize yeast, which lacks the mitochondrial calcium uniporter, as a model system to address this problem. We use heterologous expression to functionally reconstitute human uniporter machinery both in wild-type yeast as well as in mutants defective in the biosynthesis of phosphatidylethanolamine, phosphatidylcholine, or cardiolipin (CL). We uncover a specific requirement of CL for in vivo reconstituted MCU stability and activity. The CL requirement of MCU is evolutionarily conserved with loss of CL triggering rapid turnover of MCU homologs and impaired calcium transport. Furthermore, we observe reduced abundance and activity of endogenous MCU in mammalian cellular models of Barth syndrome, which is characterized by a partial loss of CL. MCU abundance is also decreased in the cardiac tissue of Barth syndrome patients. Our work raises the hypothesis that impaired mitochondrial calcium transport contributes to the pathogenesis of Barth syndrome, and more generally, showcases the utility of yeast phospholipid mutants in dissecting the phospholipid requirements of ion channel complexes.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Síndrome de Barth/genética , Síndrome de Barth/metabolismo , Transporte Biológico , Canais de Cálcio/química , Canais de Cálcio/genética , Cardiolipinas/genética , Cardiolipinas/metabolismo , Humanos , Camundongos , Mitocôndrias/química , Mitocôndrias/genética , Mioblastos/metabolismo , Fosfolipídeos , Estabilidade Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
3.
Nature ; 583(7816): 473-478, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32528179

RESUMO

Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a ß-barrel structure1,2. ß-Barrels are sheets of ß-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane3-5. One subunit of the machines is itself a ß-barrel protein that has a central role in folding other ß-barrels. In Gram-negative bacteria, the ß-barrel assembly machine (BAM) consists of the ß-barrel protein BamA, and four lipoproteins5-8. To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP)9, we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia coli folding BamA itself. The BamA catalyst forms an asymmetric hybrid ß-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies10-12; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Dobramento de Proteína , Proteínas da Membrana Bacteriana Externa/química , Cloroplastos/química , Proteínas de Escherichia coli/química , Bactérias Gram-Negativas/química , Ligação de Hidrogênio , Mitocôndrias/química , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato
4.
Nucleic Acids Res ; 48(W1): W239-W243, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32421834

RESUMO

Recent evidences suggest that the localization of mRNAs near the subcellular compartment of the translated proteins is a more robust cellular tool, which optimizes protein expression, post-transcriptionally. Retention of mRNA in the nucleus can regulate the amount of protein translated from each mRNA, thus allowing a tight temporal regulation of translation or buffering of protein levels from bursty transcription. Besides, mRNA localization performs a variety of additional roles like long-distance signaling, facilitating assembly of protein complexes and coordination of developmental processes. Here, we describe a novel machine-learning based tool, mRNALoc, to predict five sub-cellular locations of eukaryotic mRNAs using cDNA/mRNA sequences. During five fold cross-validations, the maximum overall accuracy was 65.19, 75.36, 67.10, 99.70 and 73.59% for the extracellular region, endoplasmic reticulum, cytoplasm, mitochondria, and nucleus, respectively. Assessment on independent datasets revealed the prediction accuracies of 58.10, 69.23, 64.55, 96.88 and 69.35% for extracellular region, endoplasmic reticulum, cytoplasm, mitochondria, and nucleus, respectively. The corresponding values of AUC were 0.76, 0.75, 0.70, 0.98 and 0.74 for the extracellular region, endoplasmic reticulum, cytoplasm, mitochondria, and nucleus, respectively. The mRNALoc standalone software and web-server are freely available for academic use under GNU GPL at http://proteininformatics.org/mkumar/mrnaloc.


Assuntos
RNA Mensageiro/análise , Software , Máquina de Vetores de Suporte , Núcleo Celular/química , Simulação por Computador , Citoplasma/química , Retículo Endoplasmático/química , Mitocôndrias/química , RNA Mensageiro/química , Análise de Sequência de RNA
5.
Nat Commun ; 11(1): 1916, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32317635

RESUMO

mHsp60-mHsp10 assists the folding of mitochondrial matrix proteins without the negative ATP binding inter-ring cooperativity of GroEL-GroES. Here we report the crystal structure of an ATP (ADP:BeF3-bound) ground-state mimic double-ring mHsp6014-(mHsp107)2 football complex, and the cryo-EM structures of the ADP-bound successor mHsp6014-(mHsp107)2 complex, and a single-ring mHsp607-mHsp107 half-football. The structures explain the nucleotide dependence of mHsp60 ring formation, and reveal an inter-ring nucleotide symmetry consistent with the absence of negative cooperativity. In the ground-state a two-fold symmetric H-bond and a salt bridge stitch the double-rings together, whereas only the H-bond remains as the equatorial gap increases in an ADP football poised to split into half-footballs. Refolding assays demonstrate obligate single- and double-ring mHsp60 variants are active, and complementation analysis in bacteria shows the single-ring variant is as efficient as wild-type mHsp60. Our work provides a structural basis for active single- and double-ring complexes coexisting in the mHsp60-mHsp10 chaperonin reaction cycle.


Assuntos
Chaperonina 10/química , Chaperonina 60/química , Mitocôndrias/química , Proteínas Mitocondriais/química , Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Citosol/química , Humanos , Ligação de Hidrogênio , Hidrólise , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Dobramento de Proteína
6.
Proc Natl Acad Sci U S A ; 117(17): 9329-9337, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32291341

RESUMO

The organization of the mitochondrial electron transport chain proteins into supercomplexes (SCs) is now undisputed; however, their assembly process, or the role of differential expression isoforms, remain to be determined. In Saccharomyces cerevisiae, cytochrome c oxidase (CIV) forms SCs of varying stoichiometry with cytochrome bc 1 (CIII). Recent studies have revealed, in normoxic growth conditions, an interface made exclusively by Cox5A, the only yeast respiratory protein that exists as one of two isoforms depending on oxygen levels. Here we present the cryo-EM structures of the III2-IV1 and III2-IV2 SCs containing the hypoxic isoform Cox5B solved at 3.4 and 2.8 Å, respectively. We show that the change of isoform does not affect SC formation or activity, and that SC stoichiometry is dictated by the level of CIII/CIV biosynthesis. Comparison of the CIV5B- and CIV5A-containing SC structures highlighted few differences, found mainly in the region of Cox5. Additional density was revealed in all SCs, independent of the CIV isoform, in a pocket formed by Cox1, Cox3, Cox12, and Cox13, away from the CIII-CIV interface. In the CIV5B-containing hypoxic SCs, this could be confidently assigned to the hypoxia-induced gene 1 (Hig1) type 2 protein Rcf2. With conserved residues in mammalian Hig1 proteins and Cox3/Cox12/Cox13 orthologs, we propose that Hig1 type 2 proteins are stoichiometric subunits of CIV, at least when within a III-IV SC.


Assuntos
Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Microscopia Crioeletrônica/métodos , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/fisiologia , Hipóxia/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Isoformas de Proteínas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia
7.
Food Chem ; 318: 126358, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32145541

RESUMO

Overdoses of SO2 and its derivatives (SO32-/HSO3-) in food or organisms are harmful to health. To detect SO32-/HSO3-, a novel NIR fluorescent probe 1, based upon the intramolecular charge transfer (ICT) mechanism, was developed. This probe was easily synthesized, and gave noticeable colorimetric and linear fluorescence changes at 690 nm after reaction with sulfite from 3.13 to 200 µM. Moreover, probe 1 displayed high sensitivity (LOD = 0.46 µM), excellent selectivity (among 13 kinds of anions and 3 kinds of biothiols) and quick response (within 30 min) towards SO32-/HSO3-. The SO32-/HSO3- sensing mechanism was confirmed as the Michael addition reaction. Furthermore, the probe showed wide applications for measuring SO32-/HSO3- in real samples, including sugar, tap water, wine and traditional Chinese medicine. The probe could also be used to detect SO32-/HSO3- in mitochondria of HepG2 cells and zebrafish, which suggested potential application for monitoring SO2 derivatives in clinical diagnostics.


Assuntos
Colorimetria/métodos , Corantes Fluorescentes/química , Mitocôndrias/química , Sulfitos/análise , Animais , Carboidratos , Medicamentos de Ervas Chinesas/análise , Água Doce/análise , Células Hep G2 , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Imagem Óptica , Açúcares/análise , Vinho/análise , Peixe-Zebra/metabolismo
8.
Chemistry ; 26(26): 5903-5910, 2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32142179

RESUMO

First-in-class CuII and AuIII metaled phosphorus dendrons were synthesized and showed significant antiproliferative activity against several aggressive breast cancer cell lines. The data suggest that the cytotoxicity increases with reducing length of the alkyl chains, whereas the replacement of CuII with AuIII considerably increases the antiproliferative activity of metaled phosphorus dendrons. Very interestingly, we found that the cell death pathway is related to the nature of the metal complexed by the plain dendrons. CuII metaled dendrons showed a potent caspase-independent cell death pathway, whereas AuIII metaled dendrons displayed a caspase-dependent apoptotic pathway. The complexation of plain dendrons with AuIII increased the cellular lethality versus dendrons with CuII and promoted the translocation of Bax into the mitochondria and the release of Cytochrome C (Cyto C).


Assuntos
Citocromos c/metabolismo , Dendrímeros/metabolismo , Metais/química , Mitocôndrias/química , Fósforo/química , Apoptose , Morte Celular , Citocromos c/química , Dendrímeros/química , Humanos , Células MCF-7 , Metais/metabolismo , Mitocôndrias/metabolismo , Estrutura Molecular , Fósforo/metabolismo
9.
Chemistry ; 26(20): 4489-4495, 2020 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-32073730

RESUMO

It is challenging to design metal catalysts for in situ transformation of endogenous biomolecules with good performance inside living cells. Herein, we report a multifunctional metal catalyst, ruthenium-coordinated oligo(p-phenylenevinylene) (OPV-Ru), for intracellular catalysis of transfer hydrogenation of nicotinamide adenine dinucleotide (NAD+ ) to its reduced format (NADH). Owing to its amphiphilic characteristic, OPV-Ru possesses good self-assembly capability in water to form nanoparticles through hydrophobic interaction and π-π stacking, and numerous positive charges on the surface of nanoparticles displayed a strong electrostatic interaction with negatively charged substrate molecules, creating a local microenvironment for enhancing the catalysis efficiency in comparison to dispersed catalytic center molecule (TOF value was enhanced by about 15 fold). OPV-Ru could selectively accumulate in the mitochondria of living cells. Benefiting from its inherent fluorescence, the dynamic distribution in cells and uptake behavior of OPV-Ru could be visualized under fluorescence microscopy. This work represents the first demonstration of a multifunctional organometallic complex catalyzing natural hydrogenation transformation in specific subcellular compartments of living cells with excellent performance, fluorescent imaging ability, specific mitochondria targeting and good chemoselectivity with high catalysis efficiency.


Assuntos
Complexos de Coordenação/química , Mitocôndrias/química , Polivinil/química , Rutênio/química , Catálise , Hidrogenação , Interações Hidrofóbicas e Hidrofílicas , Mitocôndrias/metabolismo , Nanopartículas , Água
10.
Org Biomol Chem ; 18(7): 1487-1492, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32026925

RESUMO

As an important biothiol in living cells, cysteine is closely related to oxidative damage in living organisms. Sulfite from cysteine metabolism in living cells plays a crucial role in maintaining homeostasis in an organism, and the unbalance of sulfite in vivo would lead to multiple diseases. Thus the development of a new fluorescent probe for cysteine metabolism is needed urgently in mitochondria which are the main place of cysteine metabolism. Herein we construct a novel targeting mitochondria fluorescent probe CP-K based on the FRET mechanism to visualize sulfite in living MCF-7 cells. Probe CP-K displays a large Stokes shift of 150 nm, a low detection limit (26.3 nM) and "naked eye" detection after the addition of HSO3-. Importantly, it is appropriate for imaging the endogenous sulfite from cysteine metabolism in living cells.


Assuntos
Cisteína/análise , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Mitocôndrias/química , Cisteína/metabolismo , Humanos , Células MCF-7 , Mitocôndrias/metabolismo , Estrutura Molecular , Imagem Óptica
11.
BMC Bioinformatics ; 21(1): 1, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31898485

RESUMO

BACKGROUND: The green microalga Dunaliella salina accumulates a high proportion of ß-carotene during abiotic stress conditions. To better understand the intracellular flux distribution leading to carotenoid accumulation, this work aimed at reconstructing a carbon core metabolic network for D. salina CCAP 19/18 based on the recently published nuclear genome and its validation with experimental observations and literature data. RESULTS: The reconstruction resulted in a network model with 221 reactions and 212 metabolites within three compartments: cytosol, chloroplast and mitochondrion. The network was implemented in the MATLAB toolbox CellNetAnalyzer and checked for feasibility. Furthermore, a flux balance analysis was carried out for different light and nutrient uptake rates. The comparison of the experimental knowledge with the model prediction revealed that the results of the stoichiometric network analysis are plausible and in good agreement with the observed behavior. Accordingly, our model provides an excellent tool for investigating the carbon core metabolism of D. salina. CONCLUSIONS: The reconstructed metabolic network of D. salina presented in this work is able to predict the biological behavior under light and nutrient stress and will lead to an improved process understanding for the optimized production of high-value products in microalgae.


Assuntos
Carbono/metabolismo , Clorófitas/metabolismo , Microalgas/metabolismo , Carbono/química , Carotenoides/química , Carotenoides/metabolismo , Clorófitas/química , Clorófitas/efeitos da radiação , Cloroplastos/química , Cloroplastos/metabolismo , Citosol/química , Citosol/metabolismo , Luz , Redes e Vias Metabólicas , Microalgas/química , Microalgas/efeitos da radiação , Mitocôndrias/química , Mitocôndrias/metabolismo , Modelos Biológicos , Estresse Fisiológico
12.
Chemosphere ; 243: 125472, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31995896

RESUMO

Repression of the electron transport in mitochondria can result in an increase of reactive oxygen species (ROS) in plant cells. This study was to clarify inhibition of the mitochondrial respiratory components (Complex I and Complex III) as stimuli to induce oxidative damage in Oryza sativa L. under exogenous SCN- exposure with special emphasis on lipid peroxidation, protein modification, and DNA damage at the biochemical and molecular levels. Our results showed that enzymatic activity and gene expression of cytochrome c reductase (Complex III) in roots and shoots of rice seedlings were significantly repressed by SCN- exposure, where significant inhibition of NADH dehydrogenase (Complex I) was only detected in shoots, suggesting that Complex III was the main target attacked by SCN- ligand in rice roots, and both components were arrested in shoots. ROS analysis in tissues indicated that SCN- exposure caused significant accumulation of H2O2 and O2-•, increased malondialdehyde (MDA) and carbonyl content in rice materials in a dose-dependent manner. Similarly, a remarkable elevation of electrolyte leakage was observed in rice tissue samples. The comet assay indicated a positive correlation between DNA damage and external SCN- exposure. In conclusion, oxidative burst generated from the inhibitions of the electron transport in mitochondria in rice seedlings under SCN- exposure can cause lipid peroxidation, protein modification and DNA damage, eventually decreasing fresh weight of rice seedlings.


Assuntos
Flavoproteínas Transferidoras de Elétrons/antagonistas & inibidores , Mitocôndrias/química , Oryza/metabolismo , Estresse Oxidativo , Tiocianatos/farmacologia , Dano ao DNA , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Complexo II de Transporte de Elétrons/antagonistas & inibidores , Peroxidação de Lipídeos , Mitocôndrias/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Plântula/metabolismo
13.
Nat Commun ; 11(1): 94, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31901080

RESUMO

The rapid development of scientific CMOS (sCMOS) technology has greatly advanced optical microscopy for biomedical research with superior sensitivity, resolution, field-of-view, and frame rates. However, for sCMOS sensors, the parallel charge-voltage conversion and different responsivity at each pixel induces extra readout and pattern noise compared to charge-coupled devices (CCD) and electron-multiplying CCD (EM-CCD) sensors. This can produce artifacts, deteriorate imaging capability, and hinder quantification of fluorescent signals, thereby compromising strategies to reduce photo-damage to live samples. Here, we propose a content-adaptive algorithm for the automatic correction of sCMOS-related noise (ACsN) for fluorescence microscopy. ACsN combines camera physics and layered sparse filtering to significantly reduce the most relevant noise sources in a sCMOS sensor while preserving the fine details of the signal. The method improves the camera performance, enabling fast, low-light and quantitative optical microscopy with video-rate denoising for a broad range of imaging conditions and modalities.


Assuntos
Microscopia de Fluorescência/instrumentação , Algoritmos , Animais , Bovinos , Linhagem Celular , Células HeLa , Humanos , Microscopia de Fluorescência/métodos , Microtúbulos/química , Mitocôndrias/química , Semicondutores , Razão Sinal-Ruído
14.
Anal Chim Acta ; 1097: 230-237, 2020 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-31910964

RESUMO

With this research we presented a ratiometric and mitochondria-target fluorescent probe (Mito-HT) for detection of H2O2 both in vitro and in live cells. Mito-HT was constructed by direct conjugation of aryl boronate to fluorophore with three synthetic steps. The borate group is cleaved from Mito-HT in the presence of H2O2, resulting in the exposure of the hydroxyl group of the electron donating group. Then the ICT mechanism was turned on, and the fluorescence emission of Mito-HT at 493 nm was red-shifted to 562 nm, thereby achieving radiometric detection of H2O2. Mito-HT exhibited a highly selectivity towards H2O2, and this interaction can be completed within 40 min. Mito-HT could be used for quantitative detection of H2O2 (0-200 µM) through ratiometric fluorescence signal readout. And limit of detection (LOD) is approximately 0.33 µM. The relatively high stability and medium fluorescence quantum yield of Mito-HT (0.39) and Mito-HT-OH (0.43) enable clear mitochondria localization and dual-channel fluorescence imaging of H2O2 in live cells with confocal microscopy.


Assuntos
Corantes Fluorescentes/química , Peróxido de Hidrogênio/análise , Mitocôndrias/química , Animais , Células CHO , Sobrevivência Celular , Cricetulus , Corantes Fluorescentes/síntese química , Microscopia de Fluorescência , Estrutura Molecular , Fatores de Tempo
15.
Talanta ; 209: 120580, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31892055

RESUMO

In this study, a mitochondria-specific fluorescent probe for efficient ratiometric detection of Cys was designed and investigated. Probe 1 is composed of a xanthylene skeleton and a benzyl group containing an acryloyl moiety. The probe showed excellent water solubility, good selectivity and sensitivity toward Cys over other analytes, and afforded an extremely low detection limit of 33.7 nM. The possible detection mechanism was ascertained by HRMS analysis. Moreover, probe 1 had excellent mitochondrial-targeting ability (the Pearson's correlation coefficient was 0.96), and was capable of monitoring endogenous Cys in living HeLa cells by dual channel ratiometric bioimaging, demonstrating its significant potential in biological applications.


Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Mitocôndrias/química , Xantenos/química , Células HeLa , Humanos , Limite de Detecção , Microscopia Confocal/métodos , Imagem Óptica/métodos
16.
Proc Natl Acad Sci U S A ; 117(5): 2412-2421, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31964824

RESUMO

Mitochondria have a characteristic ultrastructure with invaginations of the inner membrane called cristae that contain the protein complexes of the oxidative phosphorylation system. How this particular morphology of the respiratory membrane impacts energy conversion is currently unknown. One proposed role of cristae formation is to facilitate the establishment of local proton gradients to fuel ATP synthesis. Here, we determined the local pH values at defined sublocations within mitochondria of respiring yeast cells by fusing a pH-sensitive GFP to proteins residing in different mitochondrial subcompartments. Only a small proton gradient was detected over the inner membrane in wild type or cristae-lacking cells. Conversely, the obtained pH values did barely permit ATP synthesis in a reconstituted system containing purified yeast F1F0 ATP synthase, although, thermodynamically, a sufficiently high driving force was applied. At higher driving forces, where robust ATP synthesis was observed, a P-side pH value of 6 increased the ATP synthesis rate 3-fold compared to pH 7. In contrast, when ATP synthase was coreconstituted with an active proton-translocating cytochrome oxidase, ATP synthesis readily occurred at the measured, physiological pH values. Our study thus reveals that the morphology of the inner membrane does not influence the subcompartmental pH values and is not necessary for robust oxidative phosphorylation in mitochondria. Instead, it is likely that the dense packing of the oxidative phosphorylation complexes in the cristae membranes assists kinetic coupling between proton pumping and ATP synthesis.


Assuntos
Trifosfato de Adenosina/biossíntese , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Prótons , Transporte de Elétrons , Concentração de Íons de Hidrogênio , Cinética , Mitocôndrias/química , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/química , Membranas Mitocondriais/enzimologia , ATPases Mitocondriais Próton-Translocadoras/genética , Fosforilação Oxidativa , Proteolipídeos/metabolismo , Bombas de Próton/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
17.
Anal Chim Acta ; 1096: 148-158, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31883581

RESUMO

Nitric oxide (NO) is a very important signal molecule implicated in numerous physiological and pathological processes, and its detection is the key to understand these processes. For this reason, various fluorescent probes have been developed for detection analysis of NO. However, few rapid-response (<1 min) and ratiometric fluorescent probe are reported for real-time detection of short-time NO in biological systems. In this work, we report a rapid-response (within several seconds) and ratiometric fluorescent probe, RatioTr, which displays selective and sensitive detection of NO in solutions, and detections of exo- and endogenous NO in live RAW 264.7 cells. Unexpectedly, the probe RatioTr and its sensing product (p-Nus) display different cellular localizations, the mitochondria and the nucleus, which were demonstrated by co-stained experiments. The sensing process of RatioTr toward NO from mitochondria to nucleus was observed in live cells by confocal fluorescence images. Furthermore, the subcellular localizations were demonstrated by measurements of pKa and interaction of p-Nus and DNA. In the presence of a natural DNA, calf thymus DNA, RatioTr is more sensitive to NO (LOD = 2.8 nM). Therefore, due to the nucleus localization together with a high fluorescence efficiency in the nucleus, p-Nus is a good candidate of cell-permeant nucleic acid stain or a fluorescent probe for the nucleus.


Assuntos
Nucléolo Celular/química , Corantes Fluorescentes/química , Mitocôndrias/química , Óxido Nítrico/análise , Animais , Limite de Detecção , Camundongos , Microscopia de Fluorescência/economia , Microscopia de Fluorescência/métodos , Modelos Moleculares , Imagem Óptica/economia , Imagem Óptica/métodos , Células RAW 264.7 , Espectrometria de Fluorescência/economia , Espectrometria de Fluorescência/métodos , Fatores de Tempo
18.
Chem Commun (Camb) ; 56(7): 1050-1053, 2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31868186

RESUMO

It has been speculated that both the intracellular viscosity and H2O2 level in Alzheimer's disease (AD) brains are higher than that in healthy brains, but direct evidence from living beings is scarce. Herein, we report a NIR emissive fluorescent probe with a large Stokes shift for the associated detection of mitochondrial viscosity and H2O2 in live rat brains with AD for the first time.


Assuntos
Doença de Alzheimer/metabolismo , Corantes Fluorescentes/química , Peróxido de Hidrogênio/análise , Mitocôndrias/metabolismo , Compostos de Quinolínio/química , Precursor de Proteína beta-Amiloide/genética , Animais , Corantes Fluorescentes/efeitos da radiação , Corantes Fluorescentes/toxicidade , Células HeLa , Humanos , Luz , Masculino , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Mitocôndrias/química , Imagem Óptica/métodos , Presenilina-1/genética , Compostos de Quinolínio/efeitos da radiação , Compostos de Quinolínio/toxicidade , Viscosidade
19.
Spectrochim Acta A Mol Biomol Spectrosc ; 224: 117435, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31400745

RESUMO

A novel two-photon pH probe, 3-benzimidazole-7-hydroxycoumarin (BHC), was designed and synthesized based on the structures of hydroxycoumarin and benzimidazole. BHC showed good linearity in the pH ranges of 3.30-5.40 (pKa = 4.20) and 6.50-8.30 (pKa = 7.20) at a maximum emission wavelength of 480 nm. BHC in acidic and alkaline media could be distinguished by an obvious spectral shift of the maximum absorption wavelength from 390 nm to 420 nm. In addition, BHC was well localized to mitochondria and successfully applied to one-photon and two-photon imaging of pH changes in the mitochondria of HeLa cells. The findings presented herein suggest that BHC can serve as an excellent fluorescent probe for selectively sensing mitochondrial pH changes with remarkable photostability and low cytotoxicity.


Assuntos
Benzimidazóis/química , Cumarínicos/química , Corantes Fluorescentes/química , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Mitocôndrias , Umbeliferonas/química , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Mitocôndrias/química , Mitocôndrias/fisiologia
20.
Spectrochim Acta A Mol Biomol Spectrosc ; 224: 117456, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31419747

RESUMO

Mitochondria are essential organelles in eukaryotic cells and act as the energy powerhouse and biosynthetic compartment. Fluorescent dyes are widely used powerful molecular tools for analytical sensing and optical imaging. Low photostability, short excitation and emission wavelengths, and aggregation-induced quenching effects restrict the application of traditional commercial mitochondrial fluorescent probes for bioimaging. In this study, using rhodamine as the acceptor and phenothiazine as the donor, we synthesized a novel mitochondrial-targeted near infrared (NIR) fluorescent probe, MIT-PZR. Due to low cytotoxicity, great photostability and high specificity for mitochondria targeting, MIT-PZR has enormous potential for cell imaging. Furthermore, with a sizeable Stokes shift (emission peak at 705 nm), MIT-PZR penetrated tissues providing stable red fluorescence for imaging in vivo. The histological assessment of various tissues after treatment with MIT-PZR indicated that it has good biocompatibility. Thus, MIT-PZR is a promising mitochondrial NIR fluorescent probe for future application in clinical diagnosis and modern biological research.


Assuntos
Técnicas Citológicas/métodos , Corantes Fluorescentes , Mitocôndrias , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Sobrevivência Celular/efeitos dos fármacos , Feminino , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/toxicidade , Células HeLa , Humanos , Camundongos , Camundongos Nus , Microscopia Confocal/métodos , Mitocôndrias/química , Mitocôndrias/metabolismo , Distribuição Tecidual
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