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1.
Adv Exp Med Biol ; 1159: 5-31, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31502197

RESUMO

Mitochondria and bacteria share a myriad of properties since it is believed that the powerhouses of the eukaryotic cell have evolved from a prokaryotic origin. Ribosomal RNA sequences, DNA architecture and metabolism are strikingly similar in these two entities. Proteins and nucleic acids have been a hallmark for comparison between mitochondria and prokaryotes. In this chapter, similarities (and differences) between mitochondrial and prokaryotic membranes are addressed with a focus on structure-function relationship of different lipid classes. In order to be suitable for the theme of the book, a special emphasis is reserved to the effects of bioactive sphingolipids, mainly ceramide, on mitochondrial membranes and their roles in initiating programmed cell death.


Assuntos
Evolução Biológica , Membrana Celular/química , Lipídeos/química , Mitocôndrias/química , Células Procarióticas/química , Ceramidas , Esfingolipídeos
2.
Adv Exp Med Biol ; 1159: 33-48, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31502198

RESUMO

Are ceramide molecules capable of self-assembling in biological and phospholipid membranes to form ceramide channels: membrane channels capable to translocating proteins through said membranes? A number of papers have been published which support the conclusion that ceramide forms these large channels in membranes. The evidence is extensive and consisting of: flux studies using isolated mitochondria, liposomes and planar membranes; visualization by electron microscopy; elastic deformation studies; and regulation by Bcl-2 family proteins. The evidence supports a structural model of the channel shown to be stable by molecular dynamic simulations and having structural and mechanical properties consistent with multiple experiments. Yet the novelty of this claim raises legitimate questions. Indeed, a recent report questions the existence of ceramide channels based on liposome experiments. This review presents both a comprehensive description of the major observations supporting the case that ceramide channels do exist and addresses the issues raised in the skeptical report.


Assuntos
Membrana Celular/química , Ceramidas/química , Lipossomos/química , Mitocôndrias/química , Fosfolipídeos , Proteínas Proto-Oncogênicas c-bcl-2
3.
Chem Commun (Camb) ; 55(72): 10740-10743, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31432813

RESUMO

We constructed a two-photon fluorescence ratio probe (CST) for in situ quantitative real-time detection of mitochondrial O2˙-. Fluorescence imaging showed that O2˙- was over-generated from mitochondria and conveyed to the cytoplasm via voltage-dependent anion channels in hepatic ischemia-reperfusion mice, damaging the important functional protein aconitase in the cytoplasm.


Assuntos
Fígado/metabolismo , Mitocôndrias/química , Imagem Óptica , Fótons , Traumatismo por Reperfusão/metabolismo , Superóxidos/química , Animais , Ânions/química , Ânions/metabolismo , Transporte Biológico , Corantes Fluorescentes/química , Camundongos , Mitocôndrias/metabolismo , Estrutura Molecular , Superóxidos/metabolismo
4.
Cell Biochem Funct ; 37(7): 494-503, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31387137

RESUMO

Hibernation requires the intricate regulation of physiological and biochemical adaptations to facilitate the decrease in metabolic rate and activation of prosurvival factors needed for winter survival. Mitochondria play important roles in eliciting these responses and in coordinating the required energy shifts. Herein, we report the presence of a novel mitochondrial peptide, s-humanin, in the hibernating 13-lined ground squirrel, Ictidomys tridecemlineatus. S-humanin was shown to have strong structural and sequence similarities to its human analogue, humanin-a powerful neuroprotective mitochondrial peptide. An assessment of the protein and gene expression levels of this peptide in ground squirrels revealed stark tissue-specific regulatory responses whereby transcript levels increased in brain cortex, skeletal muscle, and adipose tissues during hibernation, suggesting a protective torpor-induced activation. Accompanying peptide measurements found that s-humanin levels were suppressed in liver of torpid squirrels but enhanced in brain cortex. The enhanced transcript and protein levels of s-humanin in brain cortex suggest that it is actively involved in protecting delicate brain tissues and neuronal connections from hibernation-associated stresses. We propose that this squirrel-specific peptide is involved in modulating tissue-specific cytoprotective functions, expanding its role from human-specific neuroprotection to environmental stress protection. SIGNIFICANCE OF THE STUDY: Understanding the molecular mechanisms, which protect against oxidative stress in a model hibernator such as the ground squirrel, could be pivotal to the regulation of cytoprotection. This study expands on our knowledge of metabolic rate depression and could suggest a potential role for humanin therapy in neurodegenerative diseases.


Assuntos
Hibernação/efeitos dos fármacos , Mitocôndrias/química , Fármacos Neuroprotetores/farmacologia , Peptídeos/farmacologia , Animais , Feminino , Masculino , Sciuridae
5.
Adv Exp Med Biol ; 1158: 59-70, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31452135

RESUMO

Mitochondria have a central role in cellular metabolism and reversible post-translational modifications regulate activity of mitochondrial proteins. Thanks to advances in proteomics, lysine acetylation has arisen as an important post-translational modification in the mitochondrion. During acetylation an acetyl group is covalently attached to the epsilon amino group in the side chain of lysine residues using acetyl-CoA as the substrate donor. Therefore the positive charge is neutralized, and this can affect the function of proteins thereby regulating enzyme activity, protein interactions, and protein stability. The major deacetylase in mitochondria is SIRT3 whose activity regulates many mitochondrial enzymes. The method of choice for the analysis of acetylated proteins foresees the combination of mass spectrometry-based proteomics with affinity enrichment techniques. Beyond the identification of lysine-acetylated proteins, many studies are moving towards the characterization of acetylated patterns in different diseases. Indeed, modifications in lysine acetylation status can directly alter mitochondrial function and, therefore, be linked to human diseases such as metabolic diseases, cancer, myocardial injury and neurodegenerative diseases. Despite the progress in the characterization of different lysine acetylation sites, additional studies are needed to differentiate the specific changes with a significant biological relevance.


Assuntos
Lisina , Mitocôndrias , Fenótipo , Acetilação , Humanos , Lisina/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Processamento de Proteína Pós-Traducional
6.
Inorg Chem ; 58(20): 13686-13695, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31436962

RESUMO

Iron-sulfur (Fe-S) clusters are common prosthetic groups that are found within a variety of proteins responsible for functions that include electron transfer, regulation of gene expression, and substrate binding and activation. Acquisition of a [4Fe-4S] cluster is essential for the functionality of many such roles, and dysfunctions in Fe-S cluster synthesis and trafficking often result in human disease, such as multiple mitochondrial dysfunctions syndrome. While the topic of [2Fe-2S] cluster biosynthesis and trafficking has been relatively well studied, the understanding of such processes involving [4Fe-4S] centers is less developed. Herein, we focus on the mechanism of the assembly of [4Fe-4S] clusters on two members of the aconitase family, differing also in organelle placement (mitochondrion and cytosol) and biochemical function. Two mechanistic models are evaluated by a combination of kinetic and spectroscopic models, namely, a consecutive model (I), in which two [2Fe-2S] clusters are sequentially delivered to the target, and a prereaction equilibrium model (II), in which a [4Fe-4S] cluster transiently forms on a donor protein before transfer to the target. The paper also addresses the issue of cluster nuclearity for functionally active forms of ISCU, NFU, and ISCA trafficking proteins, each of which has been postulated to exist in both [2Fe-2S] and [4Fe-4S] bound states. By the application of kinetic assays and electron paramagnetic resonance spectroscopy to examine delivery pathways from a variety of potential [2Fe-2S] donor proteins to eukaryotic forms of both aconitase and iron regulatory protein, we conclude that a consecutive model following the delivery of [2Fe-2S] clusters from NFU1 is the most likely mechanism for these target proteins.


Assuntos
Aconitato Hidratase/metabolismo , Citosol/metabolismo , Eucariotos/metabolismo , Proteínas com Ferro-Enxofre/metabolismo , Mitocôndrias/metabolismo , Aconitato Hidratase/química , Citosol/química , Espectroscopia de Ressonância de Spin Eletrônica , Eucariotos/química , Humanos , Proteínas com Ferro-Enxofre/química , Cinética , Mitocôndrias/química
7.
Chem Commun (Camb) ; 55(64): 9444-9447, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31287465

RESUMO

A new mitochondrion targetable molecular probe for carbon monoxide (CO)-specific detection based on palladium-free mediated opening of spirolactam was designed. The turn-on red fluorescence caused by CO enables a safe and powerful method for unravelling the function of CO in biological systems to be established.


Assuntos
Monóxido de Carbono/análise , Corantes Fluorescentes/química , Mitocôndrias/química , Imagem Óptica/métodos , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Lactamas/química , Espectroscopia de Prótons por Ressonância Magnética , Frações Subcelulares/química
8.
Talanta ; 202: 369-374, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171197

RESUMO

Hypochloric acid (HOCl) plays vital roles in cell signaling and homeostasis which involved in many related diseases. Therefore, the accurate detected level of OCl- for discussing the complex contributions of OCl- to human health is of great significance. In this study, a novel mitochondrion-targeting fluorescent probe DMI bearing a carbon-carbon double bond as an OCl--responsive site has been developed. Probe DMI exhibited specific fluorescence response toward OCl- with fast response (within 4 s) and high sensitivity (detection limit is 0.05 µM), where the obvious color changes could be observed by the naked eye. More importantly, DMI could be applied in the bioimaging of OCl- in living A549 cells successfully benefited from its good sensing properties and low toxicity. Fluorescence co-localization test was further carried out and confirmed mitochondria-targetable ability of DMI which can be used for investigating physiological function of HOCl at organelle levels.


Assuntos
Corantes Fluorescentes/química , Ácido Hipocloroso/análise , Mitocôndrias/química , Imagem Óptica , Células A549 , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacologia , Humanos , Estrutura Molecular , Espectrofotometria
9.
Biophys Chem ; 252: 106208, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31238246

RESUMO

The central aspects of the energy economics of living cells revolve around the synthesis and utilization of molecules of adenosine triphosphate (ATP). Current descriptions of cell metabolism and its regulation in most textbooks of biochemistry assume that enzymes and transporters behave in the same way in isolation and in a cell. Calculations of the mechanistic or maximal P/O ratios in oxidative phosphorylation by mammalian cells generally consider only the supply side of the problem without linking to ATP-demand processes. The purpose of this article is to calculate the mechanistic P/O ratio by integration of the supply and demand sides of ATP reactions. The mechanistic stoichiometry calculated from an integrated approach is compared with that obtained from the standard model that considers only ATP supply. After accounting for leaks, slips, and other losses, the actual or operative P/O calculated by the integrated method is found to be in good agreement with the experimental values of the P/O ratio determined in mitochondria for both succinate and NADH-linked respiratory substrates. The thermodynamic consequences of these results and the biological implications are discussed. An integrated model of oxidative phosphorylation that goes beyond the chemiosmotic theory is presented, and a solution to the longstanding fundamental problem of respiratory control is found.


Assuntos
Trifosfato de Adenosina/metabolismo , Respiração Celular , Fosforilação Oxidativa , Trifosfato de Adenosina/química , Animais , Humanos , Mitocôndrias/química , Mitocôndrias/metabolismo , Termodinâmica
10.
Anal Chim Acta ; 1067: 88-97, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31047153

RESUMO

Nitric oxide (NO), a ubiquitous gasotransmitter which plays critical roles in cardiovascular, nervous, and immune systems related diseases, is closely related in the physiological and pathological processes of mitochondria and lysosomes. Thus, monitoring NO in mitochondria or lysosomes is very meaningful for NO related chemical biology. Herein, we rationally designed four NO probes, BDP-NO, Mito-NO-T, Mito-NO and Lyso-NO, based on BODIPY dye substituted at meso position with 5-amino-2-methoxy-phenyl scaffold. These four probes all showed fast fluorescence off-on response toward NO with excellent selectivity and high sensitivity with the detection limit of BDP-NO to reach 5.7 nM. We introduced triphenylphosphonium and morpholine moieties onto BODIPY scaffold respectively to enable organelle-targetability. MTT and flow cytometry assay demonstrated that the probes exhibited low cytotoxicity, which was beneficial to the biological application in living cells. Confocal fluorescence microscopy experiments confirmed excellent mitochondria targeting for Mito-NO and lysosome-targeting with Lyso-NO for the detection of NO in living cells.


Assuntos
Compostos de Boro/química , Corantes Fluorescentes/química , Lisossomos/química , Mitocôndrias/química , Óxido Nítrico/análise , Apoptose/efeitos dos fármacos , Compostos de Boro/síntese química , Compostos de Boro/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacologia , Células HeLa , Humanos , Lisossomos/metabolismo , Microscopia Confocal , Mitocôndrias/metabolismo , Estrutura Molecular , Óxido Nítrico/metabolismo , Imagem Óptica , Células Tumorais Cultivadas
11.
Spectrochim Acta A Mol Biomol Spectrosc ; 219: 509-516, 2019 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-31078818

RESUMO

Hypochlorite anion (ClO-) has been recognized as host defense destructing incursive bacteria and pathogens, a signal molecule inducing occurrence of apoptosis and a noxious agent when it is overproduced. It is significant to detect ClO- in mitochondria for getting meaningful physiological and pathological information. Compared with the fluorescence probes of emission wavelength in ultraviolet or visible region, those with near-infrared (NIR) fluorescence signal are advantageous due to the deeper tissue penetrability and less photo-bleaching effect. In this work, a new "off-on" NIR ClO--specific fluorescence probe (Mito-NClO) especially located in mitochondria was designed and synthesized by condensation of diaminomaleonitrile with a new fluorophore (Mito-NCHO). A marked "turn-on" NIR fluorescence signal was observed on account of the oxidation of the imine bond by NaClO. Moreover, in the range from 0 to 20 µM, this probe had the capability to quantitatively detect ClO- with a detection limit as low as 90.2 nM. Additionally, the probe exerted other excellent properties, including larger stokes shift (117 nm), better aqueous solubility, high selectivity toward ClO-, rapid response and selective mitochondrial location. Furthermore, the bio-imaging experiments clearly demonstrated that Mito-NClO facilitated the visualization of exogenous and endogenous ClO- in living HeLa cells and zebrafish model. Therefore, we speculate that the probe Mito-NClO can be served as an ideal tool for the monitoring of ClO- in biosystems.


Assuntos
Corantes Fluorescentes/química , Ácido Hipocloroso/análise , Mitocôndrias/química , Animais , Células HeLa , Humanos , Microscopia de Fluorescência , Imagem Óptica , Peixe-Zebra
12.
Int J Mol Sci ; 20(10)2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31108962

RESUMO

In aging and neurodegenerative diseases, loss of distinct type of neurons characterizes disease-specific pathological and clinical features, and mitochondria play a pivotal role in neuronal survival and death. Mitochondria are now considered as the organelle to modulate cellular signal pathways and functions, not only to produce energy and reactive oxygen species. Oxidative stress, deficit of neurotrophic factors, and multiple other factors impair mitochondrial function and induce cell death. Multi-functional plant polyphenols, major groups of phytochemicals, are proposed as one of most promising mitochondria-targeting medicine to preserve the activity and structure of mitochondria and neurons. Polyphenols can scavenge reactive oxygen and nitrogen species and activate redox-responsible transcription factors to regulate expression of genes, coding antioxidants, anti-apoptotic Bcl-2 protein family, and pro-survival neurotrophic factors. In mitochondria, polyphenols can directly regulate the mitochondrial apoptosis system either in preventing or promoting way. Polyphenols also modulate mitochondrial biogenesis, dynamics (fission and fusion), and autophagic degradation to keep the quality and number. This review presents the role of polyphenols in regulation of mitochondrial redox state, death signal system, and homeostasis. The dualistic redox properties of polyphenols are associated with controversial regulation of mitochondrial apoptosis system involved in the neuroprotective and anti-carcinogenic functions. Mitochondria-targeted phytochemical derivatives were synthesized based on the phenolic structure to develop a novel series of neuroprotective and anticancer compounds, which promote the bioavailability and effectiveness. Phytochemicals have shown the multiple beneficial effects in mitochondria, but further investigation is required for the clinical application.


Assuntos
Mitocôndrias/química , Compostos Fitoquímicos/farmacologia , Polifenóis/farmacologia , Animais , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Degradação Mitocondrial , Neuroproteção , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Nat Cell Biol ; 21(6): 768-777, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061466

RESUMO

Controlling cellular processes with light can help elucidate their underlying mechanisms. Here we present zapalog, a small-molecule dimerizer that undergoes photolysis when exposed to blue light. Zapalog dimerizes any two proteins tagged with the FKBP and DHFR domains until exposure to light causes its photolysis. Dimerization can be repeatedly restored with uncleaved zapalog. We implement this method to investigate mitochondrial motility and positioning in cultured neurons. Using zapalog, we tether mitochondria to constitutively active kinesin motors, forcing them down the axon towards microtubule (+) ends until their instantaneous release via blue light, which results in full restoration of their endogenous motility. We find that one-third of stationary mitochondria cannot be pulled away from their position and that these firmly anchored mitochondria preferentially localize to VGLUT1-positive presynapses. Furthermore, inhibition of actin polymerization with latrunculin A reduces this firmly anchored pool. On release from exogenous motors, mitochondria are preferentially recaptured at presynapses.


Assuntos
Axônios/metabolismo , Mitocôndrias/genética , Fotólise , Multimerização Proteica/efeitos da radiação , Actinas/antagonistas & inibidores , Animais , Axônios/química , Axônios/efeitos da radiação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células COS , Cercopithecus aethiops , Cinesina/química , Luz , Microtúbulos/genética , Microtúbulos/efeitos da radiação , Mitocôndrias/química , Mitocôndrias/efeitos da radiação , Neurônios/química , Neurônios/efeitos da radiação , Polimerização/efeitos dos fármacos , Domínios Proteicos/genética , Domínios Proteicos/efeitos da radiação , Multimerização Proteica/genética , Sinapses/química , Sinapses/genética , Sinapses/efeitos da radiação , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/genética , Tiazolidinas/farmacologia , Proteína Vesicular 1 de Transporte de Glutamato/genética
14.
Parasitol Res ; 118(6): 1785-1797, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31062084

RESUMO

We report the complete coding sequences of mitochondrial thioredoxin (TsTrx2) and glutaredoxin (TsGrx1) from the cysticerci of T. solium. The full-length DNA of the TsTrx2 gene shows two introns of 88 and 77 bp and three exons. The TsTrx2 gene contains a single ORF of 423 bp, encoding 140 amino acid residues with an estimated molecular weight of 15,560 Da. A conserved C64NPC67 active site and a 30-amino acid extension at its N-terminus were identified. An insulin reduction reaction was used to determine whether it was a functional recombinant protein. The full-length DNA of the TsGrx1 gene shows one intron of 39 bp and a single ORF of 315 bp, encoding 105 amino acid residues with an estimated molecular weight of 12,582 Da. Sequence analysis revealed a conserved dithiol C34PYC37 active site, GSH-binding motifs (CXXC, Lys and Gln/Arg, TVP, and CXD), and a conserved Gly-Gly motif. The r-TsGrx1 kinetic constants for glutathione (GSH) and 2-hydroxyethyl disulfide (HED) were determined. In addition, cytosolic thioredoxin (TsTrx1), as reported by (Jiménez et al., Biomed Res Int 2015:453469, 2015), was cloned and expressed, and its catalytic constants were obtained along with those of the other two reductases. Rabbit-specific antibodies showed immune cross-reactions between TsTrx1 and TsTrx2 but not with TsGrx1. Both TsTGRs as reported by (Plancarte and Nava, Exp Parasitol 149:65-73, 2015) were biochemically purified to obtain and compare the catalytic constants for their natural substrates, r-TsTrx1, and r-TsTrx2, compared to those for Trx-S2E. coli. In addition, we determined the catalytic differences between the glutaredoxin activity of the TsTGRs compared with r-TsGrx1. These data increase the knowledge of the thioredoxin and GSH systems in T. solium, which is relevant for detoxification and immune evasion.


Assuntos
Citosol/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/isolamento & purificação , Mitocôndrias/metabolismo , Taenia solium/genética , Tiorredoxinas/genética , Tiorredoxinas/isolamento & purificação , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cysticercus/genética , Cysticercus/isolamento & purificação , Cysticercus/metabolismo , Citosol/química , Dissulfetos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Etanol/análogos & derivados , Etanol/metabolismo , Glutarredoxinas/química , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Cinética , Mitocôndrias/química , Mitocôndrias/genética , Fases de Leitura Aberta , Coelhos , Taenia solium/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismo
15.
Methods Mol Biol ; 1978: 355-368, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31119674

RESUMO

Spectrophotometric analysis of metabolic enzyme activity from homogenized tissues is a valuable method for investigating mitochondrial content and capacity. Enzyme activity is normally measured in single cuvette spectrophotometers, requiring a large sample volume and low throughput. Here, we describe microplate assays for high-throughput analysis of mitochondrial enzymes citrate synthase, ß-hydroxyacyl CoA dehydrogenase, aconitase, and mitochondrial electron transport system (ETS) complexes I, II, III, and IV.


Assuntos
Complexo I de Transporte de Elétrons/química , Enzimas/química , Mitocôndrias/enzimologia , Espectrofotometria/métodos , Aconitato Hidratase/química , Transporte de Elétrons/genética , Enzimas/metabolismo , Mitocôndrias/química
16.
Talanta ; 199: 140-146, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952238

RESUMO

Small molecules emitting in the NIR for tracking mitochondrial pH alteration with super-resolution are expected to play an essential role in biomedical applications. Herein, two small molecules based on pyridinium salt (P1 and P2) have been synthesized and systematically investigated. It was found that pyridinium salts P1 and P2 emitted in the NIR (about 610 nm), which could detect pH changes from 2.0 to 11.0 with good linearity and high sensitivity. Importantly, P2 could precisely target cellular mitochondria in a real-time manner under stimulated emission depletion (STED). These results implied a chemical strategy with a potential application in super-resolution imaging and mitochondrial pH determination.


Assuntos
Mitocôndrias/química , Compostos de Piridínio/química , Bibliotecas de Moléculas Pequenas/química , Cristalografia por Raios X , Células Hep G2 , Humanos , Concentração de Íons de Hidrogênio , Raios Infravermelhos , Microscopia de Fluorescência , Modelos Moleculares , Estrutura Molecular , Compostos de Piridínio/síntese química , Teoria Quântica , Bibliotecas de Moléculas Pequenas/síntese química
17.
FEBS Open Bio ; 9(4): 571-581, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30984533

RESUMO

The western painted turtle (Chrysemys picta bellii) can survive extended periods of anoxia via a series of mechanisms that serve to reduce its energetic needs. Central to these mechanisms is the response of mitochondria, which depolarize in response to anoxia in turtle pyramidal neurons due to an influx of K+. It is currently unknown how mitochondrial matrix pH is affected by this response and we hypothesized that matrix pH acidifies during anoxia due to increased K+/H+ exchanger activity. Inhibition of K+/H+ exchange via quinine led to a collapse of mitochondrial membrane potential (Ψm) during oxygenated conditions in turtle cortical neurons, as indicated by rhodamine-123 fluorescence, and this occurred twice as quickly during anoxia which indicates an elevation in K+ conductance. Mitochondrial matrix pH acidified during anoxia, as indicated by SNARF-1 fluorescence imaged via confocal microscopy, and further acidification occurred during anoxia when the F1Fo-ATPase was inhibited with oligomycin-A, indicating that ΔpH collapse is prevented during anoxic conditions. Collectively, these results indicate that the mitochondrial proton electrochemical gradient is actively preserved during anoxia to prevent a collapse of Ψm and ΔpH.


Assuntos
Mitocôndrias/química , ATPases Mitocondriais Próton-Translocadoras/genética , Canais de Potássio/metabolismo , Células Piramidais/fisiologia , Proteínas de Répteis/genética , Tartarugas/fisiologia , Anaerobiose , Animais , Concentração de Íons de Hidrogênio , Potencial da Membrana Mitocondrial/fisiologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Antiportadores de Potássio-Hidrogênio/metabolismo , Proteínas de Répteis/metabolismo
18.
Nano Lett ; 19(5): 2905-2913, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-30935203

RESUMO

Multidrug resistance (MDR) is still a formidable obstacle for the majority of anticancer drugs during chemotherapy. MDR is generally divided into the pump and nonpump resistances, which significantly and simultaneously reduce drug accumulation and potency in various cancer cells. In order to concurrently combat the two pathways to completely overcome MDR, a novel siRNA-containing nanomaterial-coated mitochondria complex was developed, which can overcome the barrier of activity loss and electrostatic repulsion to effectively deliver siRNA and mitochondria into the MDR cells. In this way, the functional siRNA could successfully down-regulate pump resistance-related proteins while the transplanted mitochondria efficaciously played its role to improve apoptotic signal activation and transmissions by means of restoring intracellular metabolism environment. We believe this unique organelle-material complex would hold great promise to reverse overall MDR as a result of high spatial-temporal synchronization of potent synthetic and living species.


Assuntos
Antineoplásicos/química , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Nanoestruturas/química , Neoplasias/tratamento farmacológico , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Mitocôndrias/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacologia , Propriedades de Superfície
19.
Int J Mol Sci ; 20(5)2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30845710

RESUMO

In a previous study on chromate toxicity, an increase in the 2Fe2S electron paramagnetic resonance (EPR) signal from mitochondria was found upon addition of chromate to human bronchial epithelial cells and bovine airway tissue ex vivo. This study was undertaken to show that a chromate-induced increase in the 2Fe2S EPR signal is a general phenomenon that can be used as a low-temperature EPR method to determine the maximum concentration of 2Fe2S centers in mitochondria. First, the low-temperature EPR method to determine the concentration of 2Fe2S clusters in cells and tissues is fully developed for other cells and tissues. The EPR signal for the 2Fe2S clusters N1b in Complex I and/or S1 in Complex II and the 2Fe2S cluster in xanthine oxidoreductase in rat liver tissue do not change in intensity because these clusters are already reduced; however, the EPR signals for N2, the terminal cluster in Complex I, and N4, the cluster preceding the terminal cluster, decrease upon adding chromate. More surprising to us, the EPR signals for N3, the cluster preceding the 2Fe2S cluster in Complex I, also decrease upon adding chromate. Moreover, this method is used to obtain the concentration of the 2Fe2S clusters in white blood cells where the 2Fe2S signal is mostly oxidized before treatment with chromate and becomes reduced and EPR detectable after treatment with chromate. The increase of the g = 1.94 2Fe2S EPR signal upon the addition of chromate can thus be used to obtain the relative steady-state concentration of the 2Fe2S clusters and steady-state concentration of Complex I and/or Complex II in mitochondria.


Assuntos
Brônquios/química , Cromatos/efeitos adversos , Fígado/química , Mitocôndrias/química , Animais , Brônquios/citologia , Brônquios/efeitos dos fármacos , Bovinos , Linhagem Celular Tumoral , Espectroscopia de Ressonância de Spin Eletrônica , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Fígado/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Ratos , Xantina Desidrogenase/metabolismo
20.
Mol Cell ; 74(2): 347-362.e6, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30853401

RESUMO

Selective autophagy recycles damaged organelles and clears intracellular pathogens to prevent their aberrant accumulation. How ULK1 kinase is targeted and activated during selective autophagic events remains to be elucidated. In this study, we used chemically inducible dimerization (CID) assays in tandem with CRISPR KO lines to systematically analyze the molecular basis of selective autophagosome biogenesis. We demonstrate that ectopic placement of NDP52 on mitochondria or peroxisomes is sufficient to initiate selective autophagy by focally localizing and activating the ULK1 complex. The capability of NDP52 to induce mitophagy is dependent on its interaction with the FIP200/ULK1 complex, which is facilitated by TBK1. Ectopically tethering ULK1 to cargo bypasses the requirement for autophagy receptors and TBK1. Focal activation of ULK1 occurs independently of AMPK and mTOR. Our findings provide a parsimonious model of selective autophagy, which highlights the coordination of ULK1 complex localization by autophagy receptors and TBK1 as principal drivers of targeted autophagosome biogenesis.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Autofagia/genética , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células HeLa , Humanos , Mitocôndrias/química , Mitocôndrias/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Peroxissomos/química , Peroxissomos/genética , Fosforilação , Proteínas Quinases/genética , Multimerização Proteica , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética
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