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1.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360637

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by increased activation of fibroblasts/myofibroblasts. Previous reports have shown that IPF fibroblasts are resistant to apoptosis, but the mechanisms remain unclear. Since inhibition of the mitochondrial permeability transition pore (mPTP) has been implicated in the resistance to apoptosis, in this study, we analyzed the role of mitochondrial function and the mPTP on the apoptosis resistance of IPF fibroblasts under basal conditions and after mitomycin C-induced apoptosis. We measured the release of cytochrome c, mPTP opening, mitochondrial calcium release, oxygen consumption, mitochondrial membrane potential, ADP/ATP ratio, ATP concentration, and mitochondrial morphology. We found that IPF fibroblasts were resistant to mitomycin C-induced apoptosis and that calcium, a well-established activator of mPTP, is decreased as well as the release of pro-apoptotic proteins such as cytochrome c. Likewise, IPF fibroblasts showed decreased mitochondrial function, while mPTP was less sensitive to ionomycin-induced opening. Although IPF fibroblasts did not present changes in the mitochondrial membrane potential, we found a fragmented mitochondrial network with scarce, thinned, and disordered mitochondria with reduced ATP levels. Our findings demonstrate that IPF fibroblasts are resistant to mitomycin C-induced apoptosis and that altered mPTP opening contributes to this resistance. In addition, IPF fibroblasts show mitochondrial dysfunction evidenced by a decrease in respiratory parameters.


Assuntos
Apoptose , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Mitocôndrias/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Citocromos c/metabolismo , Fibroblastos/patologia , Humanos , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/patologia , Ionomicina , Mitocôndrias/patologia , Mitomicina , Oxigênio/metabolismo , Cultura Primária de Células
2.
Nutrients ; 13(7)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34371916

RESUMO

White adipose tissue (WAT) is a dynamic endocrine organ that can play a significant role in thermoregulation. WAT has the capacity to adopt structural and functional characteristics of the more metabolically active brown adipose tissue (BAT) and contribute to non-shivering thermogenesis under specific stimuli. Non-shivering thermogenesis was previously thought to be uncoupling protein 1 (UCP1)-dependent however, recent evidence suggests that UCP1-independent mechanisms of thermogenesis exist. Namely, futile creatine cycling has been identified as a contributor to WAT thermogenesis. The purpose of this study was to examine the efficacy of creatine supplementation to alter mitochondrial markers as well as adipocyte size and multilocularity in inguinal (iWAT), gonadal (gWAT), and BAT. Thirty-two male and female Sprague-Dawley rats were treated with varying doses (0 g/L, 2.5 g/L, 5 g/L, and 10 g/L) of creatine monohydrate for 8 weeks. We demonstrate that mitochondrial markers respond in a sex and depot specific manner. In iWAT, female rats displayed significant increases in COXIV, PDH-E1alpha, and cytochrome C protein content. Male rats exhibited gWAT specific increases in COXIV and PDH-E1alpha protein content. This study supports creatine supplementation as a potential method of UCP1-independant thermogenesis and highlights the importance of taking a sex-specific approach when examining the efficacy of browning therapeutics in future research.


Assuntos
Tecido Adiposo Branco/efeitos dos fármacos , Regulação da Temperatura Corporal/efeitos dos fármacos , Creatina/farmacologia , Suplementos Nutricionais , Mitocôndrias/efeitos dos fármacos , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Masculino , Mitocôndrias/metabolismo , Piruvato Desidrogenase (Lipoamida) , Ratos Sprague-Dawley , Fatores Sexuais , Proteína Desacopladora 1/metabolismo
3.
Int J Mol Sci ; 22(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34208159

RESUMO

B-cell lymphoma 2 (Bcl-2) and cytochrome c (Cycs) are two important proteins relevant to cellular apoptosis. In this study, we characterized the functions of the promoter regions of two apoptosis-related genes, Bcl-2 and Cycs, in yellow catfish Pelteobagrus fulvidraco. We obtained a 1989 bp Bcl-2 promoter and an 1830 bp Cycs promoter and predicted several key transcription factor binding sites (TFBSs) on the promoters, such as Kruppel-like factor 4 (KLF4), signal transducer and activator of transcription factor 3 (STAT3), forkhead box O (FOXO), metal-responsive element (MRE) and hepatocyte nuclear factor 1α (HNF-1α). Zinc (Zn) increased the activities of the Bcl-2 promoter but decreased the activities of the Cycs promoter. Metal-responsive transcription factor 1 (MTF-1) and HNF-1α directly bound with Bcl-2 and Cycs promoters, and they positively regulated the activity of the Bcl-2 promoter but negatively regulated the activity of the Cycs promoter. Zn promoted the binding ability of HNF-1α to the Bcl-2 promoter but decreased its binding ability to the Cycs promoter. However, Zn had no significant effect on the binding capability of MTF-1 to the regions of Bcl-2 and Cycs promoters. Zn upregulated the mRNA and total protein expression of Bcl-2 but downregulated the mRNA and total protein expression of Cycs. At the same time, Annexin V-FITC/PI staining showed that Zn significantly reduced the apoptosis of primary hepatocytes. For the first time, our study provides evidence for the MRE and HNF-1α response elements on the Bcl-2 and Cycs promoters, offering new insight into the mechanism by which Zn affects apoptosis in vertebrates.


Assuntos
Apoptose/genética , Peixes-Gato/genética , Citocromos c/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Zinco/farmacologia , Animais , Apoptose/efeitos dos fármacos , Sequência de Bases , Sítios de Ligação , Citocromos c/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de Sequência
4.
Molecules ; 26(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200517

RESUMO

Epoxy-α-lapachone (Lap) and Epoxymethyl-lawsone (Law) are oxiranes derived from Lapachol and have been shown to be promising drugs for Leishmaniases treatment. Although, it is known the action spectrum of both compounds affect the Leishmania spp. multiplication, there are gaps in the molecular binding details of target enzymes related to the parasite's physiology. Molecular docking assays simulations were performed using DockThor server to predict the preferred orientation of both compounds to form stable complexes with key enzymes of metabolic pathway, electron transport chain, and lipids metabolism of Leishmania spp. This study showed the hit rates of both compounds interacting with lanosterol C-14 demethylase (-8.4 kcal/mol to -7.4 kcal/mol), cytochrome c (-10.2 kcal/mol to -8.8 kcal/mol), and glyceraldehyde-3-phosphate dehydrogenase (-8.5 kcal/mol to -7.5 kcal/mol) according to Leishmania spp. and assessed compounds. The set of molecular evidence reinforces the potential of both compounds as multi-target drugs for interrupt the network interactions between parasite enzymes, which can lead to a better efficacy of drugs for the treatment of leishmaniases.


Assuntos
Leishmania/efeitos dos fármacos , Naftoquinonas/farmacologia , Simulação por Computador , Citocromos c/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Compostos de Epóxi/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Leishmaniose/tratamento farmacológico , Leishmaniose/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Simulação de Acoplamento Molecular
5.
Phys Chem Chem Phys ; 23(29): 15557-15563, 2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34259248

RESUMO

The deactivation of singlet oxygen, the lowest electronic excited state of molecular oxygen, by proteins is usually described through the interaction of singlet oxygen with certain amino acids. Changes in accessibility of these amino acids influence the quenching rate and the phosphorescence kinetics of singlet oxygen. In the cellular environment, however, numerous proteins with covalently bound or encapsulated cofactors are present. These cofactors could also influence the deactivation of singlet oxygen, and these have received little attention. To confront this issue, we used cytochrome c (cyt c) and apocytochrome c (apocyt c) to illustrate how the heme prosthetic group influences the rate constant of singlet oxygen deactivation upon acidic pH-induced conformational change of cyt c. Photo-excited flavin mononucleotide (FMN) was used to produce singlet oxygen. Our data show that the heme group has a significant and measurable effect on singlet oxygen quenching when the heme is exposed to solvents and is therefore more accessible to singlet oxygen. The effect of amino acids and heme accessibility on the FMN triplet state deactivation was also investigated.


Assuntos
Citocromos c/química , Mononucleotídeo de Flavina/química , Heme/química , Oxigênio Singlete/química , Sequência de Aminoácidos , Citocromos c/metabolismo , Heme/metabolismo , Cinética , Modelos Moleculares , Oxigênio/química , Fotoquímica , Ligação Proteica , Oxigênio Singlete/metabolismo
6.
J Phys Chem B ; 125(28): 7763-7773, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34235935

RESUMO

Electron transport through aromatic species (especially tryptophan and tyrosine) plays a central role in water splitting, redox signaling, oxidative damage protection, and bioenergetics. The cytochrome c peroxidase (CcP)-cytochrome c (Cc) complex (CcP:Cc) is used widely to study interprotein electron transfer (ET) mechanisms. Tryptophan 191 (Trp191) of CcP supports hole hopping charge recombination in the CcP:Cc complex. Experimental studies find that when Trp191 is substituted by tyrosine, phenylalanine, or redox-active aniline derivatives bound in the W191G cavity, enzymatic activity and charge recombination rates both decrease. Theoretical analysis of these CcP:Cc complexes finds that the ET kinetics depend strongly on the chemistry of the modified Trp site. The computed electronic couplings in the W191F and W191G species are orders of magnitude smaller than in the native protein, due largely to the absence of a hopping intermediate and the large tunneling distance. Small molecules bound in the W191G cavity are weakly coupled electronically to the Cc heme, and the structural disorder of the guest molecule in the binding pocket may contribute further to the lack of enzymatic activity. The couplings in W191Y are not substantially weakened compared to the native species, but the redox potential difference for tyrosine vs tryptophan oxidation accounts for the slower rate in the Tyr mutant. Thus, theoretical analysis explains why only the native Trp supports rapid hole hopping in the CcP:Cc complex. Favorable free energies and electronic couplings are essential for establishing an efficient hole hopping relay in this protein-protein complex.


Assuntos
Citocromo-c Peroxidase , Citocromo-c Peroxidase/genética , Citocromo-c Peroxidase/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Transporte de Elétrons , Cinética , Oxirredução
7.
Biomed Pharmacother ; 139: 111707, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34243615

RESUMO

Invasive ductal carcinoma (IDC) is the most recurrent cancer, accounting for 80% of all breast cancers worldwide. Originating from the milk duct, it eventually invades the fibrous tissue of the breast outside the duct, proliferation takes 1-2 months for each division. Quinacrine (QC), an FDA-approved small molecule, has been shown to have anti-cancer activity in numerous cancerous cell lines through diverse pathways; ultimately leading to cell death. Here, we have investigated the mode of action of QC in MCF7 cells. This study demonstrated the modulation of cellular cytoskeleton, such as the formation of distinct filopodial and lamellipodial structures and spikes, through the regulation of small-GTPases. We also observed that QC induces a signaling cascade by inducing apoptotic cell death by increasing ROS generation and altering HSP70 expression; which presumably involves ERK regulation. Our findings show that QC could be an attractive chemotherapeutic agent having a "shotgun" nature with potential of inducing different signaling pathways leading to apoptotic cell death. This opens new avenues for research on developing QC as an effective therapeutic agent for the treatment of invasive ductal carcinomas.


Assuntos
Carcinoma Ductal/tratamento farmacológico , Carcinoma Ductal/metabolismo , Citocromos c/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Quinacrina/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Células MCF-7 , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
Chem Biol Interact ; 345: 109553, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34126102

RESUMO

A hypoxic environment of rapidly growing tumor cells makes them resistant to antitumor drugs. Mimicking hypoxia with iron chelator deferoxamine, suppressed cell death induced by widely used anticancer drugs doxorubicin or cisplatin. Deferoxamine decreased the number of dead (detached) cells, the size of SubG1 population, the release of cytochrome c, and the processing of caspase-3 in HCT116 colon carcinoma cells treated with cisplatin or doxorubicin. Deferoxamine-mediated suppression of apoptosis correlated with the level of pro-apoptotic Bcl-2 family proteins Bax, Bid, and Puma, which stimulate mitochondrial apoptotic pathway through permeabilization of the outer mitochondrial membrane and cytochrome c release. Here we show that one of the reasons for apoptosis suppression is downregulation of p53 expression under hypoxic conditions, and, as a result, attenuation of the expression of pro-apoptotic Bcl-2 family proteins. Indeed, p53 knock-out did not affect the stabilization of hypoxia-inducible factor but made undetectable the expression of pro-apoptotic proteins.


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Regulação para Baixo , Modelos Biológicos , Hipóxia Tumoral , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Desferroxamina/farmacologia , Regulação para Baixo/efeitos dos fármacos , Células HCT116 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Hipóxia Tumoral/efeitos dos fármacos
9.
ACS Appl Mater Interfaces ; 13(24): 27983-27990, 2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34110765

RESUMO

Nanowire-based optical biosensors with high sensitivity are highly desired for the detection of biological microenvironments and analysis of cellular processes. However, the current nanowire biosensors are mostly fabricated with metal and semiconductor materials, which are not suitable for long-term use in biological environments due to their incompatible and nondegradable properties. Biosensors based on biofriendly materials (e.g., spider silk) often do not have high enough sensitivity due to high losses or micron sizes. Here, polylactic acid (PLA), a polymer with high optical transparency, good biocompatibility, biodegradability, and flexibility, is used to fabricate nanowires using a directly drawing method for the first time. Because of the strong evanescent wave and abundant carboxyl groups on the surface of nanowires, an ultralow concentration sensing of cytochrome c is achieved with a limit of detection of 1.38 × 10-17 M, which is much lower than other detection results using semiconductor/metal-based nanosensors (10-6 to 10-12 M). On this basis, a label-free and real-time monitoring of cell apoptosis is realized. In addition, by doping quantum dots, the functionalized PLA nanowires can also sense a change in pH. These results are suggestive of the potential for PLA nanowires applied in multifunctional biosensing and biodetection, pushing forward the photomedicine field.


Assuntos
Apoptose/fisiologia , Técnicas Biossensoriais/métodos , Citocromos c/análise , Nanofios/química , Poliésteres/química , Citocromos c/metabolismo , Concentração de Íons de Hidrogênio , Limite de Detecção , Pontos Quânticos/química , Leveduras/metabolismo
10.
Ecotoxicol Environ Saf ; 220: 112395, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34102394

RESUMO

Copper (Cu), one of the heavy metals, is far beyond the carrying capacity of the environment with Cu mining, industrial wastewater discharging and the use of Cu-containing pesticides. Intaking excess Cu can cause toxic effects on liver, kidney, heart, but few studies report Cu toxicity on brain tissue. It is noteworthy that most toxicity tests are based on rodent models, but large mammals chosen as animal models has no reported. To explore the relationship of the Cu toxicity and mitochondria-mediated apoptosis on hypothalamus in pigs, the content of Cu, histomorphology, mitochondrial related indicators, apoptosis, and AMPK-mTOR signaling pathway were detected. Results showed that Cu could accumulate in hypothalamus and lead to mitochondrial dysfunction, evidenced by the decrease of ATP production, activities of respiratory chain complex I-IV, and mitochondrial respiratory function in Cu-treated groups. Additionally, the genes and proteins expression of Bax, Caspase-3, Cytc in treatment group were higher than control group. Furthermore, the protein level of p-AMPK was enhanced significantly and p-mTOR was declined, which manifested that AMPK-mTOR signaling pathway was activated in Cu-treated groups. In conclusion, this study illuminated that the accumulation of Cu could cause mitochondrial dysfunction, induce mitochondria-mediated apoptosis and activate AMPK-mTOR pathway in hypothalamus.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Cobre/toxicidade , Hipotálamo/efeitos dos fármacos , Metais Pesados/toxicidade , Mitocôndrias/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose , Caspase 3/metabolismo , Cobre/metabolismo , Citocromos c/metabolismo , Exposição Ambiental , Hipotálamo/metabolismo , Metais Pesados/metabolismo , Mitocôndrias/metabolismo , Modelos Animais , Transdução de Sinais , Suínos , Proteína X Associada a bcl-2/metabolismo
11.
Methods Mol Biol ; 2276: 215-225, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060044

RESUMO

Mitochondria play a key role in various modes of cell death. Analysis of mitochondrial dysfunction and the release of proteins from the intermembrane space of mitochondria represent essential tools in cell death investigation. Here we describe how to evaluate release of intermembrane space proteins during apoptosis, alterations in the mitochondrial membrane potential, and oxygen consumption in apoptotic cells.


Assuntos
Membranas Intracelulares/patologia , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/patologia , Morte Celular/fisiologia , Células Cultivadas , Citocromos c/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Consumo de Oxigênio/fisiologia
12.
J Biol Chem ; 297(1): 100816, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34023389

RESUMO

Mitochondrial tRNA 3'-end metabolism is critical for the formation of functional tRNAs. Deficient mitochondrial tRNA 3'-end metabolism is linked to an array of human diseases, including optic neuropathy, but their pathophysiology remains poorly understood. In this report, we investigated the molecular mechanism underlying the Leber's hereditary optic neuropathy (LHON)-associated tRNAAla 5587A>G mutation, which changes a highly conserved adenosine at position 73 (A73) to guanine (G73) on the 3'-end of the tRNA acceptor stem. The m.5587A>G mutation was identified in three Han Chinese families with suggested maternal inheritance of LHON. We hypothesized that the m.5587A>G mutation altered tRNAAla 3'-end metabolism and mitochondrial function. In vitro processing experiments showed that the m.5587A>G mutation impaired the 3'-end processing of tRNAAla precursors by RNase Z and inhibited the addition of CCA by tRNA nucleotidyltransferase (TRNT1). Northern blot analysis revealed that the m.5587A>G mutation perturbed tRNAAla aminoacylation, as evidenced by decreased efficiency of aminoacylation and faster electrophoretic mobility of mutated tRNAAla in these cells. The impact of m.5587A>G mutation on tRNAAla function was further supported by increased melting temperature, conformational changes, and reduced levels of this tRNA. Failures in tRNAAla metabolism impaired mitochondrial translation, perturbed assembly and activity of oxidative phosphorylation complexes, diminished ATP production and membrane potential, and increased production of reactive oxygen species. These pleiotropic defects elevated apoptotic cell death and promoted mitophagy in cells carrying the m.5587A>G mutation, thereby contributing to visual impairment. Our findings may provide new insights into the pathophysiology of LHON arising from mitochondrial tRNA 3'-end metabolism deficiency.


Assuntos
Mitocôndrias/metabolismo , RNA de Transferência de Alanina/metabolismo , Trifosfato de Adenosina/metabolismo , Apoptose , Sequência de Bases , Citocromos c/metabolismo , Transporte de Elétrons , Humanos , Potencial da Membrana Mitocondrial , Proteínas Mitocondriais/metabolismo , Mitofagia , Mutação/genética , Conformação de Ácido Nucleico , Fosforilação Oxidativa , Processamento Pós-Transcricional do RNA/genética , Estabilidade de RNA/genética , RNA Mitocondrial/genética , RNA de Transferência de Alanina/química , Espécies Reativas de Oxigênio/metabolismo , Aminoacilação de RNA de Transferência
13.
AAPS PharmSciTech ; 22(5): 158, 2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34009603

RESUMO

The present study was aimed to enhance the mitochondrial function in oxidative stress-induced diabetes. To achieve this, Ficus religiosa L. extract loaded solid lipid nanoparticles (ETNPs) were prepared and functionalized by using triphenylphosphonium. Developed nanoparticles demonstrated desired quality attributes with sustained release for up to 24 h and excellent storage stability for up to 180 days at 40 ± 2°C and 75 ± 5% relative humidity. In vitro cytotoxicity assessment showed no toxicity of ETNPs. Interestingly, oral administration of ETNPs to diabetic rats demonstrated improved mitochondrial function by normalizing the mitochondrial morphology, intracellular calcium ion concentration, complexes I, II, IV, and V activity, mitochondrial membrane potential, and antioxidant levels. Further, reduction in apoptotic markers viz. cytochrome-C, caspase-3, and caspase-9 was observed following the ETNP treatment. Moreover, significant reduction in blood glucose and glycosylated hemoglobin while significant improvement in plasma insulin was observed as compared to the diabetic group following the treatment with developed formulation. Furthermore, histopathology studies confirmed the safety of the developed formulation and thus, data in hand collectively suggest that proposed strategy can be effectively used to improve the mitochondrial function in oxidative stress-induced diabetes along with better control over blood glucose and glycosylated hemoglobin.


Assuntos
Antioxidantes/farmacologia , Ficus/química , Nanopartículas , Compostos Organofosforados/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Administração Oral , Animais , Apoptose/efeitos dos fármacos , Glicemia/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Citocromos c/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Compostos Organofosforados/administração & dosagem , Compostos Organofosforados/isolamento & purificação , Extratos Vegetais/administração & dosagem , Ratos , Ratos Wistar
14.
J Mol Biol ; 433(15): 167045, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-33971209

RESUMO

Being essential for oxidative protein folding in the mitochondrial intermembrane space, the mitochondrial disulfide relay relies on the electron transfer (ET) from the sulfhydryl oxidase Erv1 to cytochrome c (Cc). Using solution NMR spectroscopy, we demonstrate that while the yeast Cc-Erv1 system is functionally active, no observable binding of the protein partners takes place. The transient interaction between Erv1 and Cc can be rationalized by molecular modeling, suggesting that a large surface area of Erv1 can sustain a fast ET to Cc via a collision-type mechanism, without the need for a canonical protein complex formation. We suggest that, by preventing the direct ET to molecular oxygen (O2), the collision-type Cc-Erv1 interaction plays a role in protecting the organism against reactive oxygen species.


Assuntos
Citocromos c/química , Citocromos c/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Leveduras/metabolismo , Cristalografia por Raios X , Transporte de Elétrons , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Oxigênio/metabolismo , Ligação Proteica , Conformação Proteica , Leveduras/química
15.
J Theor Biol ; 525: 110765, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-34019850

RESUMO

Apoptosis has been extensively characterized by both experimental approaches and model simulations. However, it is still not fully understood how the regulation occurs, especially in the intrinsic pathway, which can be activated by a great variety of signals. In addition, the conditions in which a point of no return could be reached remain elusive. In this work, we use differential equations models to approach these issues. Our starting point was the model for caspase activation of Legewie et al. (Legewie S, et al., PLoS Computational Biology 2006, 2(9): e120), which exhibits irreversible bistability. We added an activation module to this model, with the main events related to mitochondrial outer membrane permeabilization, which includes cytochrome C release by the mitochondria and its effects on caspase activation and respiratory chain disruption. This "Extended Legewie Model" (ELM) uses BAK as the apoptotic stimulus and active caspase 3 as a measure of apoptosis activation. Unexpectedly, in the extended model, BAK cannot trigger apoptosis activation using physiologically sound initial values of the variables, due to limitations in apoptosome concentration increase. Therefore, the next step was to find a regulatory mechanism, allowing apoptosis activation in the ELM, starting from physiological initial concentrations. For this aim, we performed a sensitivity analysis on the 61 parameters of the system, finding that those producing the most relevant changes in the qualitative behaviour were the rates of synthesis of caspase 3, caspase 9 and XIAP. Based on these results, the transcription factor E2F was included in the ELM because it directly regulates the rate of synthesis of caspase 3 and 9. Depending on the concentration of E2F, the ELM shows different qualitative behaviours. On one hand, for low E2F apoptosis is impossible and for high E2F apoptosis is inevitable. Therefore, if E2F is sufficiently increased, the point of no return is crossed. On the other hand, for intermediate values of E2F there is a bistable region where the fate of the system also depends on the concentration of BAK and other signalling species.


Assuntos
Apoptose , Caspases , Caspases/metabolismo , Citocromos c/metabolismo , Mitocôndrias , Membranas Mitocondriais/metabolismo
16.
J Mol Biol ; 433(15): 167057, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34033821

RESUMO

Cardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis.


Assuntos
Cardiolipinas/metabolismo , Citocromo-c Peroxidase/química , Citocromo-c Peroxidase/metabolismo , Fosfatidilgliceróis/metabolismo , Citocromos c/metabolismo , Regulação da Expressão Gênica , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica
17.
Acta Chim Slov ; 68(1): 151-158, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34057526

RESUMO

Phytoalexins are substances with antimicrobial properties produced by plants after being attacked by microorganisms, especially phytopathogenic fungi and viruses. They are also currently being studied for their antitumor effect. We aimed to study the apoptosis-stimulating effect of homobrassinin and thiazino[6,5-b]indol in human ovarian adenocarcinoma A2780 and A2780cis cells via flow cytometric analysis of annexin V/PI, caspase 3 and 9 activity, cytochrome C release, and smac-diablo accumulation. Using the western blot technique, we also monitored the effect of both indoles on the response of heat shock proteins in these cells. Thiazino[6,5-b]indol showed more pronounced sensitizing and/or pro-apoptotic effect compared to homobrassinin accompanied by increased smac-diablo accumulation at earlier time intervals and pronounced externalization of phosphatidylserine at 72 h in A2780cis compared to A2780 cells. The apoptosis stimulating effect of thiazino[6,5-b]indol in A2780cis cells was associated with significant irreversible downregulation of HSP70 and HSP90 and partly with a decrease of HSP40. On the other hand, cisplatin-induced the apoptosis of sensitive A2780 cells with reversible downregulation of HSP40 and HSP57. In conclusion, the effect of thiazino[6,5-b]indol on resistant A2780cis cells could have a great utility in both the potential prevention and the treatment of other cisplatin-resistant tumor cells.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Indóis/farmacologia , Tiazinas/farmacologia , Tiocarbamatos/farmacologia , Anexina A5/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Citocromos c/metabolismo , Regulação para Baixo/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo
18.
Biochem Biophys Res Commun ; 561: 14-18, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34000512

RESUMO

In spite of numerous studies, many details of SARS-Cov-2 interaction with human cells are still poorly understood. The 674-685 fragment of SARS-Cov-2 spike protein is homologous to the fragment of α-cobratoxin underlying its interaction with α7 nicotinic acetylcholine receptors (nAChRs). The interaction of 674-685 peptide with α7 nAChR has been predicted in silico. In the present paper we confirm this prediction experimentally and investigate the effect of SARS-Cov-2 spike protein peptide on mitochondria, which express α7 nAChRs to regulate apoptosis-related events. We demonstrate that SARS-Cov-2 spike protein peptide 674-685 competes with the antibody against 179-190 fragment of α7 nAChR subunit for the binding to α7-expressing cells and mitochondria and prevents the release of cytochrome c from isolated mitochondria in response to 0.5 mM H2O2 but does not protect intact U373 cells against apoptogenic effect of H2O2. Our data suggest that the α7 nAChR-binding portion of SARS-Cov-2 spike protein prevents mitochondria-driven apoptosis when the virus is uncoated inside the cell and, therefore, supports the infected cell viability before the virus replication cycle is complete.


Assuntos
Apoptose , Citocromos c/metabolismo , Mitocôndrias/metabolismo , Fragmentos de Peptídeos/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Simulação por Computador , Feminino , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/crescimento & desenvolvimento , Receptor Nicotínico de Acetilcolina alfa7/metabolismo
19.
Methods Mol Biol ; 2255: 21-26, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033091

RESUMO

Within the cell, proteins are segregated into different organelles depending on their function and activation status. In response to stimulus, posttranslational modifications or loss of organelle membrane integrity lead to the movement of proteins from one compartment to another. This movement of proteins or protein translocation, exerts a significant effect on protein function. This is clearly demonstrated in the context of apoptosis wherein the cytoplasmic translocation of the mitochondrial resident protein, cytochrome C, initiates the activation of the intrinsic arm of the apoptotic pathway. Experimentally, protein translocation can be demonstrated by subcellular fractionation and subsequent western blot analysis of the isolated fractions. This chapter describes the step-by-step procedure in obtaining mitochondrial and cytoplasmic fractions from cell pellets and determining their purity and integrity.


Assuntos
Apoptose , Caspases/metabolismo , Citocromos c/metabolismo , Citoplasma/metabolismo , Mitocôndrias/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Western Blotting , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Células Tumorais Cultivadas
20.
Proc Natl Acad Sci U S A ; 118(11)2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33836592

RESUMO

Energy conversion in aerobic organisms involves an electron current from low-potential donors, such as NADH and succinate, to dioxygen through the membrane-bound respiratory chain. Electron transfer is coupled to transmembrane proton transport, which maintains the electrochemical proton gradient used to produce ATP and drive other cellular processes. Electrons are transferred from respiratory complexes III to IV (CIII and CIV) by water-soluble cytochrome (cyt.) c In Saccharomyces cerevisiae and some other organisms, these complexes assemble into larger CIII2CIV1/2 supercomplexes, the functional significance of which has remained enigmatic. In this work, we measured the kinetics of the S. cerevisiae supercomplex cyt. c-mediated QH2:O2 oxidoreductase activity under various conditions. The data indicate that the electronic link between CIII and CIV is confined to the surface of the supercomplex. Single-particle electron cryomicroscopy (cryo-EM) structures of the supercomplex with cyt. c show the positively charged cyt. c bound to either CIII or CIV or along a continuum of intermediate positions. Collectively, the structural and kinetic data indicate that cyt. c travels along a negatively charged patch on the supercomplex surface. Thus, rather than enhancing electron transfer rates by decreasing the distance that cyt. c must diffuse in three dimensions, formation of the CIII2CIV1/2 supercomplex facilitates electron transfer by two-dimensional (2D) diffusion of cyt. c This mechanism enables the CIII2CIV1/2 supercomplex to increase QH2:O2 oxidoreductase activity and suggests a possible regulatory role for supercomplex formation in the respiratory chain.


Assuntos
Citocromos c/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Saccharomyces cerevisiae/metabolismo , Microscopia Crioeletrônica , Citocromos c/química , Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Cinética , Mitocôndrias/metabolismo , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
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