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1.
Int J Mol Sci ; 22(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34445210

RESUMO

Ischemic episodes are a leading cause of death worldwide with limited therapeutic interventions. The current study explored mitochondrial phosphate-activated glutaminase (GLS1) activity modulation by PKCßII through GC-MS untargeted metabolomics approach. Mitochondria were used to elucidate the endogenous resistance of hippocampal CA2-4 and dentate gyrus (DG) to transient ischemia and reperfusion in a model of ischemic episode in gerbils. In the present investigation, male gerbils were subjected to bilateral carotids occlusion for 5 min followed by reperfusion (IR). Gerbils were randomly divided into three groups as vehicle-treated sham control, vehicle-treated IR and PKCßII specific inhibitor peptide ßIIV5-3-treated IR. Vehicle or ßIIV5-3 (3 mg/kg, i.v.) were administered at the moment of reperfusion. The gerbils hippocampal tissue were isolated at various time of reperfusion and cell lysates or mitochondria were isolated from CA1 and CA2-4,DG hippocampal regions. Recombinant proteins PKCßII and GLS1 were used in in vitro phosphorylation reaction and organotypic hippocampal cultures (OHC) transiently exposed to NMDA (25 µM) to evaluate the inhibition of GLS1 on neuronal viability. PKCßII co-precipitates with GAC (GLS1 isoform) in CA2-4,DG mitochondria and phosphorylates GLS1 in vitro. Cell death was dose dependently increased when GLS1 was inhibited by BPTA while inhibition of mitochondrial pyruvate carrier (MPC) attenuated cell death in NMDA-challenged OHC. Fumarate and malate were increased after IR 1h in CA2-4,DG and this was reversed by ßIIV5-3 what correlated with GLS1 activity increases and earlier showed elevation of neuronal death (Krupska et al., 2017). The present study illustrates that CA2-4,DG resistance to ischemic episode at least partially rely on glutamine and glutamate utilization in mitochondria as a source of carbon to tricarboxylic acid cycle. This phenomenon depends on modulation of GLS1 activity by PKCßII and remodeling of MPC: all these do not occur in ischemia-vulnerable CA1.


Assuntos
Transtornos Cerebrovasculares/enzimologia , Glutaminase/metabolismo , Hipocampo/enzimologia , Mitocôndrias/enzimologia , Proteína Quinase C beta/metabolismo , Traumatismo por Reperfusão/enzimologia , Animais , Transtornos Cerebrovasculares/patologia , Gerbillinae , Hipocampo/patologia , Mitocôndrias/patologia , Ratos , Ratos Wistar , Traumatismo por Reperfusão/patologia
2.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360775

RESUMO

Coupling glycolysis and mitochondrial tricarboxylic acid cycle, pyruvate dehydrogenase (PDH) complex (PDHC) is highly responsive to cellular demands through multiple mechanisms, including PDH phosphorylation. PDHC also produces acetyl-CoA for protein acetylation involved in circadian regulation of metabolism. Thiamine (vitamin B1) diphosphate (ThDP) is known to activate PDH as both coenzyme and inhibitor of the PDH inactivating kinases. Molecular mechanisms integrating the function of thiamine-dependent PDHC into general redox metabolism, underlie physiological fitness of a cell or an organism. Here, we characterize the daytime- and thiamine-dependent changes in the rat brain PDHC function, expression and phosphorylation, assessing their impact on protein acetylation and metabolic regulation. Morning administration of thiamine significantly downregulates both the PDH phosphorylation at Ser293 and SIRT3 protein level, the effects not observed upon the evening administration. This action of thiamine nullifies the daytime-dependent changes in the brain PDHC activity and mitochondrial acetylation, inducing diurnal difference in the cytosolic acetylation and acetylation of total brain proteins. Screening the daytime dependence of central metabolic enzymes and proteins of thiol/disulfide metabolism reveals that thiamine also cancels daily changes in the malate dehydrogenase activity, opposite to those of the PDHC activity. Correlation analysis indicates that thiamine abrogates the strong positive correlation between the total acetylation of the brain proteins and PDHC function. Simultaneously, thiamine heightens interplay between the expression of PDHC components and total acetylation or SIRT2 protein level. These thiamine effects on the brain acetylation system change metabolic impact of acetylation. The changes are exemplified by the thiamine enhancement of the SIRT2 correlations with metabolic enzymes and proteins of thiol-disulfide metabolism. Thus, we show the daytime- and thiamine-dependent changes in the function and phosphorylation of brain PDHC, contributing to regulation of the brain acetylation system and redox metabolism. The daytime-dependent action of thiamine on PDHC and SIRT3 may be of therapeutic significance in correcting perturbed diurnal regulation.


Assuntos
Encéfalo/metabolismo , Cetona Oxirredutases/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sirtuínas/metabolismo , Tiamina/farmacologia , Acetilação/efeitos dos fármacos , Animais , Ciclo do Ácido Cítrico/efeitos dos fármacos , Masculino , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Fatores de Tempo
3.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360547

RESUMO

Mitochondria are essential in eukaryotes. Besides producing 80% of total cellular ATP, mitochondria are involved in various cellular functions such as apoptosis, inflammation, innate immunity, stress tolerance, and Ca2+ homeostasis. Mitochondria are also the site for many critical metabolic pathways and are integrated into the signaling network to maintain cellular homeostasis under stress. Mitochondria require hundreds of proteins to perform all these functions. Since the mitochondrial genome only encodes a handful of proteins, most mitochondrial proteins are imported from the cytosol via receptor/translocase complexes on the mitochondrial outer and inner membranes known as TOMs and TIMs. Many of the subunits of these protein complexes are essential for cell survival in model yeast and other unicellular eukaryotes. Defects in the mitochondrial import machineries are also associated with various metabolic, developmental, and neurodegenerative disorders in multicellular organisms. In addition to their canonical functions, these protein translocases also help maintain mitochondrial structure and dynamics, lipid metabolism, and stress response. This review focuses on the role of Tim50, the receptor component of one of the TIM complexes, in different cellular functions, with an emphasis on the Tim50 homologue in parasitic protozoan Trypanosoma brucei.


Assuntos
Homeostase , Mitocôndrias/enzimologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Trypanosoma brucei brucei/enzimologia , Animais , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Transporte Proteico
4.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360559

RESUMO

The tremendous therapeutic potential of photobiomodulation therapy in different branches of medicine has been described in the literature. One of the molecular mechanisms for this treatment implicates the mitochondrial enzyme, cytochrome C oxidase. However, the efficacy and consistency of clinical outcomes with photobiomodulation treatments has been fiercely debated. This work was motivated by this need to improve photobiomodulation devices and delivery approaches. We designed a novel hand-piece with a flat-top beam profile of irradiation. We compared the beam profile versus a standard hand-piece and a fibre probe. We utilized isolated mitochondria and performed treatments at various spots within the beam, namely, the centre, left and right edge. We examined mitochondrial activity by assessing ATP synthesis with the luciferin/luciferase chemiluminescent method as a primary endpoint, while mitochondrial damage was assessed as the secondary endpoint. We observed a uniform distribution of the power density with the flat-top prototype compared to a wide Gaussian beam profile with the standard fibre and standard hand-piece. We noted increased production of ATP in the centre of all three beams with respect to the non-treated controls (p < 0.05). Both the fibre and standard hand-piece demonstrated less increase in ATP synthesis at the edges than the centre (p < 0.05). In contrast, ATP synthesis was increased homogenously in the flat-top handpiece, both in the centre and the edges of the beam. Fibre, standard hand-piece and the flat-top hand-piece prototype have discrete beam distribution characteristics. This significantly affected the mitochondrial activity with respect to their position within the treated areas. Flat-top hand-piece enhances the uniformity of photobiomodulation treatments and can improve the rigour and reproducibility of PBM clinical outcomes.


Assuntos
Trifosfato de Adenosina/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Lasers Semicondutores/estatística & dados numéricos , Mitocôndrias/enzimologia , Consumo de Oxigênio , Humanos , Mitocôndrias/efeitos da radiação
5.
Mol Biochem Parasitol ; 244: 111393, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34197864

RESUMO

Mitochondrial protein import depends on heterooligomeric translocases in the outer and inner membranes. Using import substrates consisting of various lengths of the N-terminal part of mitochondrial dihydrolipoamide dehydrogenase (LDH) fused to dihydrofolate reductase we present an in vivo analysis showing that in Trypanosoma brucei at least 96 aa of mature LDH are required to efficiently produce an import intermediate that spans both translocases. This is different to yeast, where around 50 aa are sufficient to achieve the same task and likely reflects the different arrangement and architecture of the trypanosomal mitochondrial translocases. Furthermore, we show that formation of the stuck import intermediate leads to a strong growth inhibition suggesting that, depending on the length of the LDH, the import channels in the translocases are quantitatively blocked.


Assuntos
Di-Hidrolipoamida Desidrogenase/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Sistemas de Translocação de Proteínas/genética , Proteínas de Protozoários/genética , Tetra-Hidrofolato Desidrogenase/genética , Trypanosoma brucei brucei/genética , Sequência de Aminoácidos , Di-Hidrolipoamida Desidrogenase/metabolismo , Regulação da Expressão Gênica , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Sistemas de Translocação de Proteínas/metabolismo , Transporte Proteico , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Especificidade da Espécie , Tetra-Hidrofolato Desidrogenase/metabolismo , Trypanosoma brucei brucei/enzimologia
6.
J Stroke Cerebrovasc Dis ; 30(9): 105957, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34217066

RESUMO

BACKGROUND: 1-trifluoromethoxyphenyl-3-(1- propionylpiperidin-4-yl) urea (TPPU) is a novel soluble epoxide hydrolase inhibitor which can protect against cerebral ischemic injury in middle cerebral artery occlusion rat model. However, the effects and potential mechanisms of TPPU on mitochondrial dysfunction are poorly understood. MATERIALS AND METHODS: In oxygen-glucose deprivation/reperfusion (OGD/R)-induced cortical neurons, the effect of TPPU on cell viability was measured by MTT assay and apoptosis was evaluated using TUNEL assay. Mitochondria were observed by transmission electron microscopy and Mitotracker green staining assay, mitochondrial membrane potential was determined by JC-1 staining assay, activities of mitochondrial respiratory chain complexes (MRCC) I-IV and ATPase were measured by MRCC Activity Assay Kits and spectrophotometer. Western blot was used to investigate the effects of TPPU on apoptosis-related proteins. RESULTS: TPPU treatment demonstrated significant protective effect on the OGD/R-induced cortical neurons by reducing cell death and number of apoptotic cells, stabilizing mitochondrial ultrastructure and morphology, increasing mitochondrial membrane potential and activities of MRCC I-IV and ATPase. Furthermore, TPPU treatment might effectively reverse the upregulation of caspase-3, Bax, p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal protein kinase (JNK), alleviate the inhibition of Bcl-2 in OGD/R-induced cortical neurons. CONCLUSIONS: TPPU exerts a marked neuroprotective effect against mitochondrial dysfunction after cerebral ischemia potentially via suppressing JNK/p38 MAPK-mediated mitochondrial apoptosis signal pathway, it may be a promising neuroprotective agent for cerebral ischemia.


Assuntos
Apoptose/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , AVC Isquêmico/tratamento farmacológico , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Compostos de Fenilureia/farmacologia , Piperidinas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Células Cultivadas , Córtex Cerebral/enzimologia , Córtex Cerebral/ultraestrutura , AVC Isquêmico/enzimologia , AVC Isquêmico/patologia , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Neurônios/enzimologia , Neurônios/ultraestrutura , Fosforilação , Ratos , Transdução de Sinais
7.
Int J Mol Sci ; 22(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199464

RESUMO

The influence of salt stress on gene expression, promoter methylation, and enzymatic activity of the mitochondrial and cytosolic forms of aconitase and fumarase has been investigated in maize (Zea mays L.) seedlings. The incubation of maize seedlings in 150-mM NaCl solution resulted in a several-fold increase of the mitochondrial activities of aconitase and fumarase that peaked at 6 h of NaCl treatment, while the cytosolic activity of aconitase and fumarase decreased. This corresponded to the decrease in promoter methylation of the genes Aco1 and Fum1 encoding the mitochondrial forms of these enzymes and the increase in promoter methylation of the genes Aco2 and Fum2 encoding the cytosolic forms. The pattern of expression of the genes encoding the mitochondrial forms of aconitase and fumarase corresponded to the profile of the increase of the stress marker gene ZmCOI6.1. It is concluded that the mitochondrial and cytosolic forms of aconitase and fumarase are regulated via the epigenetic mechanism of promoter methylation of their genes in the opposite ways in response to salt stress. The role of the mitochondrial isoforms of aconitase and fumarase in the elevation of respiration under salt stress is discussed.


Assuntos
Aconitato Hidratase/genética , Metilação de DNA/genética , Fumarato Hidratase/genética , Estresse Salino/genética , Citosol/enzimologia , Regulação da Expressão Gênica de Plantas/genética , Mitocôndrias/enzimologia , Regiões Promotoras Genéticas/genética , Zea mays/genética , Zea mays/crescimento & desenvolvimento
8.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199774

RESUMO

Over a thousand nucleus-encoded mitochondrial proteins are imported from the cytoplasm; however, mitochondrial (mt) DNA encodes for a small number of critical proteins and the entire suite of mt:tRNAs responsible for translating these proteins. Mitochondrial RNase P (mtRNase P) is a three-protein complex responsible for cleaving and processing the 5'-end of mt:tRNAs. Mutations in any of the three proteins can cause mitochondrial disease, as well as mutations in mitochondrial DNA. Great strides have been made in understanding the enzymology of mtRNase P; however, how the loss of each protein causes mitochondrial dysfunction and abnormal mt:tRNA processing in vivo has not been examined in detail. Here, we used Drosophila genetics to selectively remove each member of the complex in order to assess their specific contributions to mt:tRNA cleavage. Using this powerful model, we find differential effects on cleavage depending on which complex member is lost and which mt:tRNA is being processed. These data revealed in vivo subtleties of mtRNase P function that could improve understanding of human diseases.


Assuntos
Mitocôndrias/enzimologia , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Ribonuclease P/metabolismo , Alelos , Animais , Drosophila melanogaster/genética , Mitocôndrias/patologia , Mutação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo
9.
Methods Mol Biol ; 2276: 325-332, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060052

RESUMO

Mitochondrial fusion depends on proteolytic processing of the dynamin-related GTPase protein, OPA1, which is regulated by the mitochondrial zinc metalloproteinase, OMA1. Last year we published a report describing a novel approach to directly measure the enzymatic activity of OMA1 in whole cell lysates. This fluorescence-based reporter assay utilizes an eight amino acid peptide sequence referred to as the S1 cleavage site where OMA1 cleaves within OPA1 and is flanked by a fluorophore and quencher. In this chapter, we provide additional insight into the OMA1 activity assay.


Assuntos
Ensaios Enzimáticos/métodos , Corantes Fluorescentes/química , GTP Fosfo-Hidrolases/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Peptídeos/química , Células Cultivadas , Humanos , Dinâmica Mitocondrial
10.
Nat Commun ; 12(1): 4020, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188055

RESUMO

PrimPol is a human DNA polymerase-primase that localizes to mitochondria and nucleus and bypasses the major oxidative lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis, in mostly error-free manner. We present structures of PrimPol insertion complexes with a DNA template-primer and correct dCTP or erroneous dATP opposite the lesion, as well as extension complexes with C or A as a 3'-terminal primer base. We show that during the insertion of C and extension from it, the active site is unperturbed, reflecting the readiness of PrimPol to accommodate oxoG(anti). The misinsertion of A opposite oxoG(syn) also does not alter the active site, and is likely less favorable due to lower thermodynamic stability of the oxoG(syn)•A base-pair. During the extension step, oxoG(syn) induces an opening of its base-pair with A or misalignment of the 3'-A primer terminus. Together, the structures show how PrimPol accurately synthesizes DNA opposite oxidatively damaged DNA in human cells.


Assuntos
Pareamento de Bases/genética , Dano ao DNA/genética , DNA Primase/metabolismo , Replicação do DNA/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , Guanina/análogos & derivados , Enzimas Multifuncionais/metabolismo , Guanina/química , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/genética , Estresse Oxidativo/genética , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismo
11.
Theranostics ; 11(15): 7294-7307, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34158851

RESUMO

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by death of motor neurons in the brain and spinal cord. However, so far, there is no effective treatment for ALS. Methods: In this study, R13, a prodrug of 7,8-dihydroxyflavone, selectively activating tyrosine kinase receptor B (TrkB) signaling pathway, was administered prophylactically to 40-day old SOD1G93A mice for 90 days. The motor performance was investigated by rotarod test, climbing-pole test, grip strength test and hanging endurance test. Afterwards, the spinal cord and medulla oblongata of 130-day old mice were harvested, and the proteomics revealed the effect of R13 on mouse protein expression profile. Astrocytes and microglial proliferation were assessed by immunohistochemical analysis. The number of motor neurons in the spinal cord is determined by Nissl staining. The effect of R13 on gastrocnemius morphology was assessed by HE staining. The effect of R13 on the survival rate was accomplished with worms stably expressing G93A SOD1. Results: Behavioral tests showed that R13 significantly attenuated abnormal motor performance of SOD1G93A mice. R13 reduced the advance of spinal motor neuron pathology and gastrocnemius muscle atrophy. The proliferation of microglia and astrocytes was reduced by R13 treatment. Mitochondriomics analysis revealed that R13 modified the mitochondrial protein expression profiles in the medulla oblongata and spinal cord of SOD1G93A mice, particularly promoting the expression of proteins related to oxidative phosphorylation (OXPHOS). Further study found that R13 activated AMPK/PGC-1α/Nrf1/Tfam, promoted mitochondrial biogenesis and ameliorated mitochondrial dysfunction. Lastly, R13 prolonged the survival rate of worms stably expressing G93A SOD1. Conclusions: These findings suggest oral R13 treatment slowed the advance of motor system disease in a reliable animal model of ALS, supporting that R13 might be useful for treating ALS.


Assuntos
Esclerose Amiotrófica Lateral , Sistema Nervoso Central/enzimologia , Flavonas/farmacologia , Mitocôndrias , Atividade Motora , Superóxido Dismutase-1 , Superóxido Dismutase , Esclerose Amiotrófica Lateral/tratamento farmacológico , Esclerose Amiotrófica Lateral/enzimologia , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/fisiopatologia , Animais , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/enzimologia , Mitocôndrias/genética , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo
12.
Methods Mol Biol ; 2310: 247-258, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34096006

RESUMO

We compared the activity of complex 1, complex 2, and the expression of the complex 1 subunit, NDUFA9, in isolated brown adipose tissue mitochondria from wild type and mitochondrial uncoupling protein 1 (UCP1) knockout mice. Direct spectrophotometric measurement revealed that complex 2 activity was similar, but complex 1 activity was greater (~2.7 fold) in isolated mitochondria from wild-type mice compared to UCP1 knockout mice, an observation endorsed by greater complex 1 subunit expression (NDUFA9) in mitochondria of wild-type mice. We also measured reactive oxygen species (ROS) production by isolated brown adipose mitochondria respiring on succinate, without rotenone, thus facilitating reverse electron flow through complex 1. We observed that reverse electron flow in isolated mitochondria from wild-type mice, with UCP1 inhibited, produced significantly greater (~1.6 fold) ROS when compared with isolated brown adipose mitochondria from UCP1 knockout mice. In summary, we demonstrate that ROS production by succinate-driven reverse electron flow can occur in brown adipose tissue mitochondria and is a good index of complex 1 activity.


Assuntos
Adipócitos Marrons/efeitos dos fármacos , Tecido Adiposo Marrom/efeitos dos fármacos , Complexo I de Transporte de Elétrons/metabolismo , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Ácido Succínico/farmacologia , Adipócitos Marrons/enzimologia , Tecido Adiposo Marrom/enzimologia , Animais , Biomarcadores/metabolismo , Western Blotting , Fracionamento Celular , Complexo I de Transporte de Elétrons/genética , Eletroforese em Gel de Poliacrilamida , Fluorometria , Camundongos Knockout , Mitocôndrias/enzimologia , Mitocôndrias/genética , Ratos , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo
13.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070501

RESUMO

Our goal was to analyze postmortem tissues of an adult patient with late-onset thymidine kinase 2 (TK2) deficiency who died of respiratory failure. Compared with control tissues, we found a low mtDNA content in the patient's skeletal muscle, liver, kidney, small intestine, and particularly in the diaphragm, whereas heart and brain tissue showed normal mtDNA levels. mtDNA deletions were present in skeletal muscle and diaphragm. All tissues showed a low content of OXPHOS subunits, and this was especially evident in diaphragm, which also exhibited an abnormal protein profile, expression of non-muscular ß-actin and loss of GAPDH and α-actin. MALDI-TOF/TOF mass spectrometry analysis demonstrated the loss of the enzyme fructose-bisphosphate aldolase, and enrichment for serum albumin in the patient's diaphragm tissue. The TK2-deficient patient's diaphragm showed a more profound loss of OXPHOS proteins, with lower levels of catalase, peroxiredoxin 6, cytosolic superoxide dismutase, p62 and the catalytic subunits of proteasome than diaphragms of ventilated controls. Strong overexpression of TK1 was observed in all tissues of the patient with diaphragm showing the highest levels. TK2 deficiency induces a more profound dysfunction of the diaphragm than of other tissues, which manifests as loss of OXPHOS and glycolytic proteins, sarcomeric components, antioxidants and overactivation of the TK1 salvage pathway that is not attributed to mechanical ventilation.


Assuntos
DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Diafragma/metabolismo , Mitocôndrias/metabolismo , Insuficiência Respiratória/metabolismo , Timidina Quinase/deficiência , Timidina Quinase/genética , Actinas/metabolismo , Adulto , Autopsia , Encéfalo/metabolismo , Catalase/metabolismo , Diafragma/enzimologia , Feminino , Frutose-Bifosfato Aldolase/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Humanos , Intestino Delgado/metabolismo , Rim/metabolismo , Fígado/metabolismo , Espectrometria de Massas , Mitocôndrias/enzimologia , Mitocôndrias/genética , Músculo Esquelético/metabolismo , Fosforilação Oxidativa , Peroxirredoxina VI/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteoma/genética , Proteoma/metabolismo , Insuficiência Respiratória/genética , Insuficiência Respiratória/mortalidade , Superóxido Dismutase/metabolismo , Timidina Quinase/metabolismo , Regulação para Cima
14.
Toxicol Lett ; 349: 115-123, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34089817

RESUMO

Cisplatin, the most widely used platinum-based anticancer drug, often causes progressive and irreversible sensorineural hearing loss in cancer patients. However, the precise mechanism underlying cisplatin-associated ototoxicity is still unclear. Nicotinamide adenine dinucleotide (NAD+), a co-substrate for the sirtuin family and PARPs, has emerged as a potent therapeutic molecular target in various diseases. In our investigates, we observed that NAD+ level was changed in the cochlear explants of mice treated with cisplatin. Supplementation of a specific inhibitor (TES-1025) of α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), a rate-limiting enzyme of NAD+de novo synthesis pathway, promoted SIRT1 activity, increased mtDNA contents and enhanced AMPK expression, thus significantly reducing hair cells loss and deformation. The protection was blocked by EX527, a specific SIRT1 inhibitor. Meanwhile, the use of NMN, a precursor of NAD+ salvage synthesis pathway, had shown beneficial effect on hair cell under cisplatin administration, effectively suppressing PARP1. In vivo experiments confirmed the hair cell protection of NAD+ modulators in cisplatin treated mice and zebrafish. In conclusion, we demonstrated that modulation of NAD+ biosynthesis via the de novo synthesis pathway and the salvage synthesis pathway could both prevent ototoxicity of cisplatin. These results suggested that direct modulation of cellular NAD+ levels could be a promising therapeutic approach for protection of hearing from cisplatin-induced ototoxicity.


Assuntos
Inibidores Enzimáticos/farmacologia , Células Ciliadas Auditivas/efeitos dos fármacos , Perda Auditiva/prevenção & controle , Audição/efeitos dos fármacos , NAD/biossíntese , Ototoxicidade/prevenção & controle , Sirtuína 1/metabolismo , Animais , Animais Geneticamente Modificados , Carboxiliases/antagonistas & inibidores , Carboxiliases/metabolismo , Cisplatino , Modelos Animais de Doenças , Ativação Enzimática , Células Ciliadas Auditivas/enzimologia , Células Ciliadas Auditivas/patologia , Perda Auditiva/induzido quimicamente , Perda Auditiva/enzimologia , Perda Auditiva/fisiopatologia , Sistema da Linha Lateral/efeitos dos fármacos , Sistema da Linha Lateral/enzimologia , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Ototoxicidade/enzimologia , Ototoxicidade/etiologia , Ototoxicidade/fisiopatologia , Peixe-Zebra
15.
Nucleic Acids Res ; 49(10): 5798-5812, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34037799

RESUMO

Mitochondria contain their own translation apparatus which enables them to produce the polypeptides encoded in their genome. The mitochondrially-encoded RNA components of the mitochondrial ribosome require various post-transcriptional processing steps. Additional protein factors are required to facilitate the biogenesis of the functional mitoribosome. We have characterized a mitochondrially-localized protein, YbeY, which interacts with the assembling mitoribosome through the small subunit. Loss of YbeY leads to a severe reduction in mitochondrial translation and a loss of cell viability, associated with less accurate mitochondrial tRNASer(AGY) processing from the primary transcript and a defect in the maturation of the mitoribosomal small subunit. Our results suggest that YbeY performs a dual, likely independent, function in mitochondria being involved in precursor RNA processing and mitoribosome biogenesis. Issue Section: Nucleic Acid Enzymes.


Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/metabolismo , Ribonucleases/metabolismo , Subunidades Ribossômicas Menores/metabolismo , Sequência de Aminoácidos , Sobrevivência Celular/genética , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Imuno-Histoquímica , Espectrometria de Massas , Mitocôndrias/enzimologia , Mitocôndrias/genética , Biossíntese de Proteínas/genética , Alinhamento de Sequência
16.
Toxicol Lett ; 347: 36-44, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-33945864

RESUMO

Aconitine, a highly toxic alkaloid derived from Aconitum L., affects the central nervous system and peripheral nervous system. However, the underlying mechanism of aconitine-induced neurotoxicity remains unclear. This study investigates the effects and mechanism of aconitine on mitochondrial energy metabolism in SH-SY5Y cells. Results demonstrated that aconitine exposure suppressed cell proliferation and led to an increase in reactive oxygen species (ROS) and excessive lactate dehydrogenase (LDH) release. Aconitine (400 µmol/L) induced abnormal mitochondrial energy metabolism that quantified by the significant decrease in ATP production, basal respiration, proton leak, maximal respiration, and succinate dehydrogenase (SDH) activity. Phosphorylation of AMPK was significantly reduced in aconitine-treated SH-SY5Y cells. The AMPK activator AIACR pretreatment effectively promoted ATP production to ameliorate mitochondrial energy metabolism disorder caused by aconitine. Mitochondrial biosynthesis was inhibited after treatment with 400 µmol/L aconitine, which was characterized by mitochondria number, TFAM expression, and mtDNA copy number. Moreover, aconitine prompted the down-regulation of mitochondrial fusion proteins OPA1, Mfn1 and Mfn2, and the up-regulation of mitochondrial fission proteins p-Drp1 and p-Mff. These results suggest that aconitine induces mitochondrial energy metabolism dysfunction in SH-SY5Y cells, which may involve the inhibition of AMPK signaling and abnormal mitochondrial dynamics.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aconitina/toxicidade , Metabolismo Energético/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Síndromes Neurotóxicas/etiologia , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Neurônios/enzimologia , Neurônios/patologia , Síndromes Neurotóxicas/enzimologia , Síndromes Neurotóxicas/patologia , Fosforilação , Transdução de Sinais
17.
Front Immunol ; 12: 625627, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33790896

RESUMO

Melatonin reportedly alleviates sepsis-induced multi-organ injury by inducing autophagy and activating class III deacetylase Sirtuin family members (SIRT1-7). However, whether melatonin attenuates small-intestine injury along with the precise underlying mechanism remain to be elucidated. To investigate this, we employed cecal ligation and puncture (CLP)- or endotoxemia-induced sepsis mouse models and confirmed that melatonin treatment significantly prolonged the survival time of mice and ameliorated multiple-organ injury (lung/liver/kidney/small intestine) following sepsis. Melatonin partially protected the intestinal barrier function and restored SIRT1 and SIRT3 activity/protein expression in the small intestine. Mechanistically, melatonin treatment enhanced NF-κB deacetylation and subsequently reduced the inflammatory response and decreased the TNF-α, IL-6, and IL-10 serum levels; these effects were abolished by SIRT1 inhibition with the selective blocker, Ex527. Correspondingly, melatonin treatment triggered SOD2 deacetylation and increased SOD2 activity and subsequently reduced oxidative stress; this amelioration of oxidative stress by melatonin was blocked by the SIRT3-selective inhibitor, 3-TYP, and was independent of SIRT1. We confirmed this mechanistic effect in a CLP-induced sepsis model of intestinal SIRT3 conditional-knockout mice, and found that melatonin preserved mitochondrial function and induced autophagy of small-intestine epithelial cells; these effects were dependent on SIRT3 activation. This study has shown, to the best of our knowledge, for the first time that melatonin alleviates sepsis-induced small-intestine injury, at least partially, by upregulating SIRT3-mediated oxidative-stress inhibition, mitochondrial-function protection, and autophagy induction.


Assuntos
Antioxidantes/farmacologia , Autofagia/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Melatonina/farmacologia , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Sepse/tratamento farmacológico , Sirtuína 3/metabolismo , Animais , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/enzimologia , Mucosa Intestinal/patologia , Intestino Delgado/enzimologia , Intestino Delgado/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Sepse/enzimologia , Sepse/microbiologia , Sepse/patologia , Sirtuína 1/metabolismo , Sirtuína 3/genética
18.
Biochem Cell Biol ; 99(2): 241-248, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33821667

RESUMO

Many reports have indicated that long non-coding RNAs (lncRNAs) are closely associated with the occurrence and development of various cancers. Musculin antisense RNA 1 (MSC-AS1) is a an lncRNA known to act as an oncogene in several types of human cancers; however, its specific function in lung adenocarcinoma (LUAD) is still unclear. For this study, we designed and conducted experiments to clarify the function of the lncRNA MSC-AS1 in LUAD and its underlying mechanisms. We found that the expression of MSC-AS1 was significantly higher in LUAD tissues and cells than that in normal ones. Through loss-of function assays, we confirmed that the proliferation of LUAD cells was significantly restrained by down-regulation of MSC-AS1 and the rate of cell apoptosis was accelerated. The results from our mechanistic experiments showed that MSC-AS1 interacts with microRNA-33b-5p (miR-33b-5p). Moreover, glycerol-3-phosphate acyltransferase, mitochondrial (GPAM) was found to be a direct target gene of miR-33b-5p, and it has similar functions to MSC-AS1. Further, inhibition of miR-33b-5p or overexpression GPAM reversed the inhibitory effects of MSC-AS1 silencing on LUAD cell growth. In short, MSC-AS1 facilitates LUAD progression through sponging miR-33b-5p to up-regulate GPAM.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Glicerol-3-Fosfato O-Aciltransferase/metabolismo , Neoplasias Pulmonares/metabolismo , Mitocôndrias/enzimologia , RNA Longo não Codificante/metabolismo , Adenocarcinoma de Pulmão/patologia , Células Cultivadas , Glicerol-3-Fosfato O-Aciltransferase/genética , Humanos , Neoplasias Pulmonares/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , Regulação para Cima
19.
Anticancer Res ; 41(4): 1831-1840, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33813388

RESUMO

BACKGROUND/AIM: Peroxiredoxin V (Prx V) plays crucial roles in cellular apoptosis and proliferation in various cancer cells by regulating the cellular reactive oxygen species (ROS) levels. MATERIALS AND METHODS: Here, we examined the possible regulatory effects of Prx V on doxorubicin (DOX)-induced cellular apoptosis and its mechanisms in the human gastric adenocarcinoma cell line (AGS cells). RESULTS: Our findings suggest that Prx V knockdown may significantly increase the DOX-induced apoptosis by aggravating intracellular ROS accumulation. We also found that DOX-induced mitochondrial ROS levels and membrane permeability were significantly higher in short hairpin Prx V cells than in mock cells, and these phenomena were dramatically reversed by ROS scavenger treatment. Prx V knockdown also significantly upregulated the cleaved caspase 9, 3, and B-cell lymphoma 2 (Bcl2)-associated agonist of cell death/Bcl2 protein expression levels, suggesting that Prx V knockdown activates mitochondria-dependent apoptotic signaling pathways. CONCLUSION: Taken together, this study suggests that Prx V may be a strong molecular target for gastric cancer (GC) chemotherapy, and further elucidates the role of Prx V in oxidative stress-induced cell apoptosis.


Assuntos
Adenocarcinoma/tratamento farmacológico , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Doxorrubicina/farmacologia , Inativação Gênica , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Peroxirredoxinas/genética , Transdução de Sinais , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
20.
J Parasitol ; 107(2): 289-294, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33844840

RESUMO

Members of the flea family Pulicidae have been the focus of many studies due to their significance as diseases vectors of medical and veterinary importance and their cosmopolitan distribution. They often exhibit variation in morphological features that can make correct species identification and management challenging. This may also apply to Xenopsylla brasiliensis (Baker, 1904), an important plague vector. In the current study, we aimed to provide genetic tools for reliable species identification using a DNA barcoding approach. A total of 73 flea specimens was collected from a native host (Namaqua rock mouse, Micaelamys namaquensis) in South Africa and identified morphologically. In addition, we took measurements of 7 morphological characteristics. Subsequently, we successfully generated barcodes of the mitochondrial cytochrome c oxidase subunit I (COI) gene for X. brasiliensis. We validated this approach by comparing our data to COI sequences from Rwandan X. brasiliensis. While sequences from both regions suggested a close relationship between the 2 X. brasiliensis populations, both haplotype and nucleotide diversity were substantially larger for the South African specimens. This may be attributed to human-assisted spread, differences in habitat, and/or host species sampled and merits further study in the future.


Assuntos
Insetos Vetores/anatomia & histologia , Insetos Vetores/genética , Peste/transmissão , Xenopsylla/anatomia & histologia , Xenopsylla/genética , Animais , Código de Barras de DNA Taxonômico , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Infestações por Pulgas/parasitologia , Infestações por Pulgas/veterinária , Variação Genética , Haplótipos , Masculino , Mitocôndrias/enzimologia , Murinae/parasitologia , África do Sul
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