RESUMO
Coronavirus nonstructural protein 2 (Nsp2) is regarded as a virulence determinant and plays a critical role in virus replication, and innate immunity. Screening and identifying host cell proteins that interact with viral proteins is an effective way to reveal the functions of viral proteins. In this study, the host proteins that interacted with transmissible gastroenteritis virus (TGEV) Nsp2 were identified using immunoprecipitation combined with LC-MS/MS. 77 host cell proteins were identified as putative Nsp2 interaction host cell proteins and a protein-protein interaction (PPI) was constructed. The identified proteins were found to be associated with various subcellular locations and functional categories through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. It is hypothesized that the host cell proteins interacting with TGEV Nsp2 are mainly involved in the formation of the cytoplasmic translation initiation complex, mRNA binding, ribosomes, and proteasomes. Among these, the ATP5B, a core subunit of the mitochondrial ATP synthase was further studied. The Coimmunoprecipitation (Co-IP) and indirect immunofluorescence (IFA) results confirmed that TGEV Nsp2 interacted with ATP5B. Furthermore, the downregulation of ATP5B expression was found to promote TGEV replication, suggesting that ATP5B might function as a negative regulator of TGEV replication. Collectively, our results offer additional insights into the functions of Nsp2 and provide a novel antiviral target against TGEV.
Assuntos
ATPases Mitocondriais Próton-Translocadoras , Vírus da Gastroenterite Transmissível , Proteínas não Estruturais Virais , Replicação Viral , Vírus da Gastroenterite Transmissível/genética , Animais , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Suínos , ATPases Mitocondriais Próton-Translocadoras/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Humanos , Interações Hospedeiro-Patógeno , Gastroenterite Suína Transmissível/virologia , Gastroenterite Suína Transmissível/genética , Linhagem Celular , Imunoprecipitação , Espectrometria de Massas em TandemRESUMO
Cells depend on a continuous supply of adenosine triphosphate (ATP), the universal energy currency. In mitochondria, ATP is produced by a series of redox reactions, whereby an electrochemical gradient is established across the inner mitochondrial membrane. The ATP synthase harnesses the energy of the gradient to generate ATP from adenosine diphosphate (ADP) and inorganic phosphate. We determined the structure of ATP synthase within mitochondria of the unicellular flagellate Polytomella by electron cryo-tomography and subtomogram averaging at up to 4.2-angstrom resolution, revealing six rotary positions of the central stalk, subclassified into 21 substates of the F1 head. The Polytomella ATP synthase forms helical arrays with multiple adjacent rows defining the cristae ridges. The structure of ATP synthase under native operating conditions in the presence of a membrane potential represents a pivotal step toward the analysis of membrane protein complexes in situ.
Assuntos
Clorofíceas , Mitocôndrias , ATPases Mitocondriais Próton-Translocadoras , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Mitocôndrias/enzimologia , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Rotação , Clorofíceas/enzimologiaRESUMO
BACKGROUND: Male infertility due to spermatogenesis defects affects millions of men worldwide. However, the genetic etiology of the vast majority remains unclear. The present study was undertaken to assess the association of DNAH6 and ATPase6 genes in asthenozoospermia patients in the northern region of India. METHODS: A total of 60 semen samples were collected for the study, of which 30 were from the case group and 30 were from the control group. The semen samples for the case group (asthenozoospermia) and control groups were collected from IVF and Reproductive Biology Centre, Maulana Azad Medical College, New Delhi. Sperm count and motility were classified as per World Health Organization (WHO 2021) protocol. A total genomic DNA was extracted as per the stranded TRIZOL method with little modification. RESULTS: In-vitro molecular characterizations of DNAH6 and ATPase6 genes in both groups were checked by Polymerase Chain Reaction (PCR). The 675 bp and 375 bp amplicons were amplified using PCR for ATPase6 and DNAH6 genes. Our study results showed a significant (P ≤ 0.05) null deletion of DNAH6 and ATPase6 genes in asthenozoospermia patients as compared to the control. We found the significant null deletion of DNAH6 in case 45.0%, and the control group was 11.7%. However, in the case of APTase6, it was 26.7% and 10.0%, respectively. CONCLUSIONS: Our study concluded that the presence of DHAH6 and ATPase6 genes had a significant impact on male infertility.
Assuntos
Astenozoospermia , Humanos , Masculino , Astenozoospermia/genética , Índia , Adulto , ATPases Mitocondriais Próton-Translocadoras/genética , DNA Mitocondrial/genéticaRESUMO
Cardiac troponin I (cTnI) is a key regulator of cardiomyocyte contraction. However, its role in mitochondria is unknown. Here we show that cTnI localized to mitochondria in the heart, inhibited mitochondrial functions when stably expressed in noncardiac cells and increased the opening of the mitochondrial permeability transition pore under oxidative stress. Direct, specific and saturable binding of cTnI to F1FO-ATP synthase was demonstrated in vitro using immune-captured ATP synthase and in cells using proximity ligation assay. cTnI binding doubled ATPase activity, whereas skeletal troponin I and several human pathogenic cTnI variants associated with familial hypertrophic cardiomyopathy did not. A rationally designed peptide, P888, inhibited cTnI binding to ATP synthase, inhibited cTnI-induced increase in ATPase activity in vitro and reduced cardiac injury following transient ischemia in vivo. We suggest that cTnI-bound ATP synthase results in lower ATP levels, and releasing this interaction during cardiac ischemia-reperfusion may increase the reservoir of functional mitochondria to reduce cardiac injury.
Assuntos
Mitocôndrias Cardíacas , ATPases Mitocondriais Próton-Translocadoras , Troponina I , Animais , Humanos , Masculino , Camundongos , Ratos , Trifosfato de Adenosina/metabolismo , Modelos Animais de Doenças , Células HEK293 , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/metabolismo , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Estresse Oxidativo/efeitos dos fármacos , Ligação Proteica , Troponina I/metabolismoRESUMO
Background: Sepsis represents a severe manifestation of infection often accompanied by metabolic disorders and mitochondrial dysfunction. Notably, mitochondrial DNA copy number (mtDNA-CN) and the expression of specific mitochondrial genes have emerged as sensitive indicators of mitochondrial function. To investigate the utility of mitochondrial gene expression in peripheral blood cells for distinguishing severe infections and predicting associated outcomes, we conducted a prospective cohort study. Methods: We established a prospective cohort comprising 74 patients with non-sepsis pneumonia and 67 cases of sepsis induced by respiratory infections, aging from 2 to 6 years old. We documented corresponding clinical data and laboratory information and collected blood samples upon initial hospital admission. Peripheral blood cells were promptly isolated, and both total DNA and RNA were extracted. We utilized absolute quantification PCR to assess mtDNA-CN, as well as the expression levels of mt-CO1, mt-ND1, and mt-ATP6. Subsequently, we extended these comparisons to include survivors and non-survivors among patients with sepsis using univariate and multivariate analyses. Receiver operating characteristic (ROC) curves were constructed to assess the diagnostic potential. Results: The mtDNA-CN in peripheral blood cells was significantly lower in the sepsis group. Univariate analysis revealed a significant reduction in the expression of mt-CO1, mt-ND1, and mt-ATP6 in patients with sepsis. However, multivariate analysis did not support the use of mitochondrial function in peripheral blood cells for sepsis diagnosis. In the comparison between pediatric sepsis survivors and non-survivors, univariate analysis indicated a substantial reduction in the expression of mt-CO1, mt-ND1, and mt-ATP6 among non-survivors. Notably, total bilirubin (TB), mt-CO1, mt-ND1, and mt-ATP6 levels were identified as independent risk factors for sepsis-induced mortality. ROC curves were then established for these independent risk factors, revealing areas under the curve (AUCs) of 0.753 for TB (95% CI 0.596-0.910), 0.870 for mt-CO1 (95% CI 0.775-0.965), 0.987 for mt-ND1 (95% CI 0.964-1.000), and 0.877 for mt-ATP6 (95% CI 0.793-0.962). Conclusion: MtDNA-CN and mitochondrial gene expression are closely linked to the severity and clinical outcomes of infectious diseases. Severe infections lead to impaired mitochondrial function in peripheral blood cells. Notably, when compared to other laboratory parameters, the expression levels of mt-CO1, mt-ND1, and mt-ATP6 demonstrate promising potential for assessing the prognosis of pediatric sepsis.
Assuntos
DNA Mitocondrial , Curva ROC , Sepse , Humanos , Sepse/sangue , Sepse/diagnóstico , Sepse/mortalidade , Pré-Escolar , Feminino , Masculino , DNA Mitocondrial/genética , Estudos Prospectivos , Prognóstico , Criança , Mitocôndrias/genética , Mitocôndrias/metabolismo , NADH Desidrogenase/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Células Sanguíneas/metabolismo , Genes Mitocondriais , Expressão Gênica , Pneumonia/diagnóstico , Pneumonia/sangue , Valor Preditivo dos TestesRESUMO
Disorders of ATP synthase, the key enzyme in mitochondrial energy supply, belong to the most severe metabolic diseases, manifesting as early-onset mitochondrial encephalo-cardiomyopathies. Since ATP synthase subunits are encoded by both mitochondrial and nuclear DNA, pathogenic variants can be found in either genome. In addition, the biogenesis of ATP synthase requires several assembly factors, some of which are also hotspots for pathogenic variants. While variants of MT-ATP6 and TMEM70 represent the most common cases of mitochondrial and nuclear DNA mutations respectively, the advent of next-generation sequencing has revealed new pathogenic variants in a number of structural genes and TMEM70, sometimes with truly peculiar genetics. Here we present a systematic review of the reported cases and discuss biochemical mechanisms, through which they are affecting ATP synthase. We explore how the knowledge of pathophysiology can improve our understanding of enzyme biogenesis and function. Keywords: Mitochondrial diseases o ATP synthase o Nuclear DNA o Mitochondrial DNA o TMEM70.
Assuntos
ATPases Mitocondriais Próton-Translocadoras , Fenótipo , Humanos , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mutação , Doenças Mitocondriais/genética , Doenças Mitocondriais/enzimologia , DNA Mitocondrial/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Animais , Mitocôndrias/enzimologia , Mitocôndrias/genéticaRESUMO
PURPOSE: In developed countries, endometrial cancer (EC) is the most prevalent gynecological cancer and its occurrence is associated with chronic inflammation. ATP5F1D is a subunit of ATP synthase (complex V), as well as the important component of mitochondrial electron transport chain (ETC). ETC play compelling roles in carcinogenesis. To date, little is known about the role of ATP5F1D in EC. METHODS: ATP5F1D expression was identified in EC tissues and EC cell lines. We evaluated the influence of ATP5F1D on clinical features and prognosis based on TCGA database. The effects of ATP5F1D in EC malignant progression by applying loss-of-function assays in KLE and Ishikawa cell lines were detected by EdU, CCK-8, wound healing, Transwell, and flow cytometry assays. Additionally, electron microscope, LDH release, ELISA, mitochondrial ROS measurement, and Immunofluorescence were performed to demonstrate ATP5F1D can affect the pyroptosis of EC. To observe the anti-tumor effect on ATP5F1D silencing, we established an in vivo human endometrial tumor model using nude mice. RESULTS: ATP5F1D expression was significantly upregulated in EC and was associated with favorable prognosis. ATP5F1D knockdown inhibited the proliferation, invasion, and migration of EC cells. Similarly, in nude mice, ATP5F1D knockdown suppressed the growth EC cells. Knocking down ATP5F1D lead to decrease the production of mitochondrial ROS (mtROS) and inhibited pyroptosis of EC cells. CONCLUSION: Downregulation of ATP5F1D as a new therapeutic strategy that could mediate pyroptosis via suppressing mtROS/NLRP3/caspase-1/GSDMD pathway to inhibit EC progression.
Assuntos
Caspase 1 , Neoplasias do Endométrio , Camundongos Nus , ATPases Mitocondriais Próton-Translocadoras , Proteína 3 que Contém Domínio de Pirina da Família NLR , Piroptose , Humanos , Feminino , Neoplasias do Endométrio/patologia , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/genética , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Linhagem Celular Tumoral , Caspase 1/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Camundongos , Regulação para Baixo , Progressão da Doença , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Transdução de Sinais , Espécies Reativas de Oxigênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Gasderminas , Proteínas de Ligação a FosfatoRESUMO
Plastid retrograde signaling plays a key role in coordinating the expression of plastid genes and photosynthesis-associated nuclear genes (PhANGs). Although plastid retrograde signaling can be substantially compromised by mitochondrial dysfunction, it is not yet clear whether specific mitochondrial factors are required to regulate plastid retrograde signaling. Here, we show that mitochondrial ATP synthase beta-subunit mutants with decreased ATP synthase activity are impaired in plastid retrograde signaling in Arabidopsis thaliana. Transcriptome analysis revealed that the expression levels of PhANGs were significantly higher in the mutants affected in the AT5G08670 gene encoding the mitochondrial ATP synthase beta-subunit, compared to wild-type (WT) seedlings when treated with lincomycin (LIN) or norflurazon (NF). Further studies indicated that the expression of nuclear genes involved in chloroplast and mitochondrial retrograde signaling was affected in the AT5G08670 mutant seedlings treated with LIN. These changes might be linked to the modulation of some transcription factors (TFs), such as LHY (Late Elongated Hypocotyl), PIF (Phytochrome-Interacting Factors), MYB, WRKY, and AP2/ERF (Ethylene Responsive Factors). These findings suggest that the activity of mitochondrial ATP synthase significantly influences plastid retrograde signaling.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas , ATPases Mitocondriais Próton-Translocadoras , Plastídeos , Transdução de Sinais , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Plastídeos/metabolismo , Plastídeos/genética , Mitocôndrias/metabolismo , Plântula/genética , Plântula/metabolismo , Mutação , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Lincomicina/farmacologia , Perfilação da Expressão GênicaRESUMO
Bedaquiline (BDQ), a first-in-class diarylquinoline anti-tuberculosis drug, and its analogue, TBAJ-587, prevent the growth and proliferation of Mycobacterium tuberculosis by inhibiting ATP synthase1,2. However, BDQ also inhibits human ATP synthase3. At present, how these compounds interact with either M. tuberculosis ATP synthase or human ATP synthase is unclear. Here we present cryogenic electron microscopy structures of M. tuberculosis ATP synthase with and without BDQ and TBAJ-587 bound, and human ATP synthase bound to BDQ. The two inhibitors interact with subunit a and the c-ring at the leading site, c-only sites and lagging site in M. tuberculosis ATP synthase, showing that BDQ and TBAJ-587 have similar modes of action. The quinolinyl and dimethylamino units of the compounds make extensive contacts with the protein. The structure of human ATP synthase in complex with BDQ reveals that the BDQ-binding site is similar to that observed for the leading site in M. tuberculosis ATP synthase, and that the quinolinyl unit also interacts extensively with the human enzyme. This study will improve researchers' understanding of the similarities and differences between human ATP synthase and M. tuberculosis ATP synthase in terms of the mode of BDQ binding, and will allow the rational design of novel diarylquinolines as anti-tuberculosis drugs.
Assuntos
Antituberculosos , Diarilquinolinas , Imidazóis , ATPases Mitocondriais Próton-Translocadoras , Mycobacterium tuberculosis , Piperidinas , Piridinas , Humanos , Antituberculosos/farmacologia , Antituberculosos/química , Sítios de Ligação , Microscopia Crioeletrônica , Diarilquinolinas/química , Diarilquinolinas/farmacologia , Imidazóis/química , Imidazóis/farmacologia , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , ATPases Mitocondriais Próton-Translocadoras/ultraestrutura , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/efeitos dos fármacos , Piperidinas/química , Piperidinas/farmacologia , Subunidades Proteicas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/antagonistas & inibidores , Piridinas/química , Piridinas/farmacologiaRESUMO
BACKGROUND: Numerous studies have confirmed the involvement of extracellular vesicles (EVs) in various physiological processes, including cellular death and tissue damage. Recently, we reported that EVs derived from ischemia-reperfusion heart exacerbate cardiac injury. However, the role of EVs from healthy heart tissue (heart-derived EVs, or cEVs) on myocardial ischemia-reperfusion (MI/R) injury remains unclear. RESULTS: Here, we demonstrated that intramyocardial administration of cEVs significantly enhanced cardiac function and reduced cardiac damage in murine MI/R injury models. cEVs treatment effectively inhibited ferroptosis and maintained mitochondrial homeostasis in cardiomyocytes subjected to ischemia-reperfusion injury. Further results revealed that cEVs can transfer ATP5a1 into cardiomyocytes, thereby suppressing mitochondrial ROS production, alleviating mitochondrial damage, and inhibiting cardiomyocyte ferroptosis. Knockdown of ATP5a1 abolished the protective effects of cEVs. Furthermore, we found that the majority of cEVs are derived from cardiomyocytes, and ATP5a1 in cEVs primarily originates from cardiomyocytes of the healthy murine heart. Moreover, we demonstrated that adipose-derived stem cells (ADSC)-derived EVs with ATP5a1 overexpression showed much better efficacy on the therapy of MI/R injury compared to control ADSC-derived EVs. CONCLUSIONS: These findings emphasized the protective role of cEVs in cardiac injury and highlighted the therapeutic potential of targeting ATP5a1 as an important approach for managing myocardial damage induced by MI/R injury.
Assuntos
Vesículas Extracelulares , Camundongos Endogâmicos C57BL , ATPases Mitocondriais Próton-Translocadoras , Traumatismo por Reperfusão Miocárdica , Miócitos Cardíacos , Animais , Masculino , Camundongos , Modelos Animais de Doenças , Vesículas Extracelulares/metabolismo , Ferroptose/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Hepatocellular carcinoma (HCC) is one of the most lethal malignant tumors worldwide. Brahma-related gene 1 (BRG1), as a catalytic ATPase, is a major regulator of gene expression and is known to mutate and overexpress in HCC. The purpose of this study was to investigate the mechanism of action of BRG1 in HCC cells. In our study, BRG1 was silenced or overexpressed in human HCC cell lines. Transwell and wound healing assays were used to analyze cell invasiveness and migration. Mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (mPTP) detection were used to evaluate mitochondrial function in HCC cells. Colony formation and cell apoptosis assays were used to evaluate the effect of BRG1/TOMM40/ATP5A1 on HCC cell proliferation and apoptosis/death. Immunocytochemistry (ICC), immunofluorescence (IF) staining and western blot analysis were used to determine the effect of BRG1 on TOMM40, ATP5A1 pathway in HCC cells. As a result, knockdown of BRG1 significantly inhibited cell proliferation and invasion, promoted apoptosis in HCC cells, whereas BRG1 overexpression reversed the above effects. Overexpression of BRG1 can up-regulate MMP level, inhibit mPTP opening and activate TOMM40, ATP5A1 expression. Our results suggest that BRG1, as an oncogene, promotes HCC progression by regulating TOMM40 affecting mitochondrial function and ATP5A1 synthesis. Targeting BRG1 may represent a new and effective way to prevent HCC development.
Assuntos
Apoptose , Carcinoma Hepatocelular , Proliferação de Células , DNA Helicases , Neoplasias Hepáticas , Mitocôndrias , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Nucleares , Fatores de Transcrição , Humanos , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , DNA Helicases/metabolismo , DNA Helicases/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Metástase Neoplásica , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
As a persistent organic pollutant, perfluorooctane sulfonate (PFOS) has a serious detrimental impact on human health. It has been suggested that PFOS is associated with liver inflammation. However, the underlying mechanisms are still unclear. Here, PFOS was found to elevate the oligomerization tendency of voltage-dependent anion channel 1 (VDAC1) in the mice liver and human normal liver cells L-02. Inhibition of VDAC1 oligomerization alleviated PFOS-induced nucleotide-binding domain and leucine-rich repeat protein-3 (NLRP3) inflammasome activation. Cytoplasmic membrane VDAC1 translocated to mitochondria was also observed in response to PFOS. Therefore, the oligomerization of VDAC1 occurred mainly in the mitochondria. VDAC1 was found to interact with the ATP synthase beta subunit (ATP5B) under PFOS treatment. Knockdown of ATP5B or immobilization of ATP5B to the cytoplasmic membrane alleviated the increased VDAC1 oligomerization and NLRP3 inflammasome activation. Therefore, our results suggested that PFOS induced NLRP3 inflammasome activation through VDAC1 oligomerization, a process dependent on ATP5B to transfer VDAC1 from the plasma membrane to the mitochondria. The findings offer novel perspectives on the activation of the NLRP3 inflammasome, the regulatory mode on VDAC1 oligomerization, and the mechanism of PFOS toxicity.
Assuntos
Ácidos Alcanossulfônicos , Fluorocarbonos , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Canal de Ânion 1 Dependente de Voltagem , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Animais , Ácidos Alcanossulfônicos/toxicidade , Inflamassomos/metabolismo , Inflamassomos/efeitos dos fármacos , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Canal de Ânion 1 Dependente de Voltagem/genética , Fluorocarbonos/toxicidade , Humanos , Camundongos , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Linhagem Celular , Camundongos Endogâmicos C57BL , Fígado/efeitos dos fármacos , Fígado/metabolismo , Poluentes Ambientais/toxicidade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismoRESUMO
Abiotic stress tends to induce oxidative damage to enzymes and organelles that in turns hampers the phosphorylation process and decreases the adenosine triphosphate (ATP) productivity. Artificial assemblies can alleviate abiotic stress and simultaneously provide nutrients to diminish the oxidative damage. Here, we have integrated natural acid phosphatase (ACP) and ATP synthase with plasmonic Au clusters in a biomimetic microreactor. ACP immobilized on the Au clusters is harnessed to generate proton influx to drive ATP synthase and concurrently supply phosphate to improve phosphorus availability to combat phosphorus-deficiency stress. In tandem with the reactive oxygen species (ROS) scavenging and the photothermal functionality of Au clusters, such an assembled microreactor exhibits an improved abiotic stress tolerance and achieves plasmon-accelerated ATP synthesis. This innovative approach offers an effective route to enhance the stress resistance of ATP synthase-based energy-generating systems, opening an exciting potential of these systems for biomimicking applications.
Assuntos
Mitocôndrias , Mitocôndrias/metabolismo , Ouro/química , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Fosfatase Ácida/metabolismo , Fosfatase Ácida/química , Espécies Reativas de Oxigênio/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismoRESUMO
OBJECTIVE: To analyze the expression level of ATP5A1 in gastric carcinoma and its influence on the prognosis of the patients and glucose metabolism in the tumor cells. METHODS: We retrospectively analyzed the data of 115 patients undergoing radical resection of gastric carcinoma in our hospital from February, 2013 to November, 2016. ATP5A1 expression in the surgical specimens were detected using immunohistochemistry, and the long-term prognosis of the patients with high (n=58) and low ATP5A1 expression (n=57) were analyzed. In gastric carcinoma MGC803 cells, the effects of lentivirus-mediated ATP5A1 knockdown or overexpression on glucose metabolism were investigated. We also observed the growth and glucose metabolism of xenografts derived from MGC803 cells with ATP5A1 knockdown or overexpression in nude mice. RESULTS: ATP5A1 was significantly overexpressed in gastric carcinoma tissues in close correlation with blood CEA and CA19-9 levels, pathological grade, T stage and N stage (P < 0.05). ATP5A1 overexpression was an independent risk factor for a significantly lowered 5-year survival rate of patients with gastric carcinoma (P < 0.05). ROC curve analysis demonstrated the predictive value of high ATP5A1 expression for the patients'prognosis (P < 0.001). In MGC803 cells, ATP5A1 overexpression significantly upregulated cellular glucose uptake and lactate production and increased the protein levels of HK2, PFK1, and LDHA (P < 0.05), while ATP5A1 knockdown produced the opposite changes (P < 0.05). In the tumor-bearing mice, overexpression of ATP5A1 increased glucose metabolism of the tumor cells and promoted tumor growth (P < 0.05). Overexpression of ATP5A1 promoted the expressions of p-JNK and p-JUN in MGC803 cells (P < 0.05), and the JNK inhibitor SP600125 significantly inhibited the enhancement of cellular glucose metabolism induced by ATP5A1 overexpression (P < 0.05). CONCLUSION: High ATP5A1 expression in gastric cancer is associated a poor long-term prognosis of the patients, and its effect is mediated at least partly by promoting glucose metabolism of the cells through the JNK/JUN pathway.
Assuntos
Glucose , Camundongos Nus , Neoplasias Gástricas , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Neoplasias Gástricas/genética , Humanos , Prognóstico , Animais , Linhagem Celular Tumoral , Camundongos , Estudos Retrospectivos , Glucose/metabolismo , Feminino , Masculino , Taxa de Sobrevida , ATPases Mitocondriais Próton-TranslocadorasRESUMO
A 23-month-old neutered male dog of unknown ancestry presented with a history of progressive neurological signs that included anxiety, cognitive impairment, tremors, seizure activity, ataxia, and pronounced visual impairment. The clinical signs were accompanied by global brain atrophy. Due to progression in the severity of disease signs, the dog was euthanized at 26 months of age. An examination of the tissues collected at necropsy revealed dramatic intracellular accumulations of autofluorescent inclusions in the brain, retina, and cardiac muscle. The inclusions were immunopositive for subunit c of mitochondrial ATP synthase, and their ultrastructural appearances were similar to those of lysosomal storage bodies that accumulate in some neuronal ceroid lipofuscinosis (NCL) diseases. The dog also exhibited widespread neuroinflammation. Based on these findings, the dog was deemed likely to have suffered from a form of NCL. A whole genome sequence analysis of the proband's DNA revealed a homozygous C to T substitution that altered the intron 3-exon 4 splice site of CLN6. Other mutations in CLN6 cause NCL diseases in humans and animals, including dogs. The CLN6 protein was undetectable with immunolabeling in the tissues of the proband. Based on the clinical history, fluorescence and electron-microscopy, immunohistochemistry, and molecular genetic findings, the disorder in this dog was classified as an NCL resulting from the absence of the CLN6 protein. Screening the dog's genome for a panel of breed-specific polymorphisms indicated that its ancestry included numerous breeds, with no single breed predominating. This suggests that the CLN6 disease variant is likely to be present in other mixed-breed dogs and at least some ancestral breeds, although it is likely to be rare since other cases have not been reported to date.
Assuntos
Doenças do Cão , Lipofuscinoses Ceroides Neuronais , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/veterinária , Lipofuscinoses Ceroides Neuronais/patologia , Animais , Cães , Masculino , Doenças do Cão/genética , Doenças do Cão/patologia , Sítios de Splice de RNA/genética , Proteínas de Membrana/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Encéfalo/patologia , Encéfalo/metabolismo , MutaçãoRESUMO
BACKGROUND: The mechanism of cardiac reverse remodeling (CRR) mediated by the left ventricular assist device remains unclear. This study aims to identify the specific cell type responsible for CRR and develop the therapeutic target that promotes CRR. METHODS: The nuclei were extracted from the left ventricular tissue of 4 normal controls, 4 CRR patients, and 4 no cardiac reverse remodeling patients and then subjected to single-nucleus RNA sequencing for identifying key cell types responsible for CRR. Gene overexpression in transverse aortic constriction and dilated cardiomyopathy heart failure mouse model (C57BL/6J background) and pathological staining were performed to validate the results of single-nucleus RNA sequencing. RESULTS: Ten cell types were identified among 126â 156 nuclei. Cardiomyocytes in CRR patients expressed higher levels of ATP5F1A than the other 2 groups. The macrophages in CRR patients expressed more anti-inflammatory genes and functioned in angiogenesis. Endothelial cells that elevated in no cardiac reverse remodeling patients were involved in the inflammatory response. Echocardiography showed that overexpressing ATP5F1A through cardiomyocyte-specific adeno-associated virus 9 demonstrated an ability to improve heart function and morphology. Pathological staining showed that overexpressing ATP5F1A could reduce fibrosis and cardiomyocyte size in the heart failure mouse model. CONCLUSIONS: The present results of single-nucleus RNA sequencing and heart failure mouse model indicated that ATP5F1A could mediate CRR and supported the development of therapeutics for overexpressing ATP5F1A in promoting CRR.
Assuntos
Modelos Animais de Doenças , Insuficiência Cardíaca , Camundongos Endogâmicos C57BL , Miócitos Cardíacos , Remodelação Ventricular , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Animais , Humanos , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Camundongos , Masculino , ATPases Mitocondriais Próton-Translocadoras/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Feminino , Pessoa de Meia-IdadeAssuntos
ATPases Mitocondriais Próton-Translocadoras , Doenças Retinianas , Sequenciamento Completo do Genoma , Humanos , Doenças Retinianas/genética , Doenças Retinianas/diagnóstico , ATPases Mitocondriais Próton-Translocadoras/genética , Doenças Mitocondriais/genética , Doenças Mitocondriais/diagnóstico , Masculino , Mutação , DNA Mitocondrial/genética , Tomografia de Coerência Óptica/métodos , Feminino , Angiofluoresceinografia/métodosRESUMO
BACKGROUND: MT-ATP6 is a mitochondrial gene which encodes for the intramembrane subunit 6 (or A) of the mitochondrial ATP synthase, also known asl complex V, which is involved in the last step of oxidative phosphorylation to produce cellular ATP through aerobic metabolism. Although classically associated with the NARP syndrome, recent evidence highlights an important role of MT-ATP6 pathogenic variants in complicated adult-onset ataxias. METHODS: We describe two unrelated patients with adult-onset cerebellar ataxia associated with severe optic atrophy and mild cognitive impairment. Whole mitochondrial DNA sequencing was performed in both patients. We employed patients' primary fibroblasts and cytoplasmic hybrids (cybrids), generated from patients-derived cells, to assess the activity of respiratory chain complexes, oxygen consumption rate (OCR), ATP production and mitochondrial membrane potential. RESULTS: In both patients, we identified the same novel m.8777 T > C variant in MT-ATP6 with variable heteroplasmy level in different tissues. We identifed an additional heteroplasmic novel variant in MT-ATP6, m.8879G > T, in the patients with the most severe phenotype. A significant reduction in complex V activity, OCR and ATP production was observed in cybrid clones homoplasmic for the m.8777 T > C variant, while no functional defect was detected in m.8879G > T homoplasmic clones. In addition, fibroblasts with high heteroplasmic levelsof m.8777 T > C variant showed hyperpolarization of mitochondrial membranes. CONCLUSIONS: We describe a novel pathogenic mtDNA variant in MT-ATP6 associated with adult-onset ataxia, reinforcing the value of mtDNA screening within the diagnostic workflow of selected patients with late onset ataxias.
Assuntos
Ataxia , ATPases Mitocondriais Próton-Translocadoras , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ataxia/genética , Ataxia/patologia , DNA Mitocondrial/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Itália , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismoRESUMO
Diesel exhaust particle (DEP) exposure induces a variety of toxicological effects through oxidative stress and inflammation responses. This research investigated the mechanisms underlying DEP-induced GC-1spg cells oxidative stress by examining ROS accumulation, antioxidant defense systems activation, mitochondrial dysfunction, and the Nrf2/Keap1/HO-1 pathway response. Subsequently, we further evaluated the ATP levels, ATP5α synthase activity and ATP5α synthase S-sulfhydrated modification in DEP-exposed GC-1 spg cells. The results showed that DEP exposure significantly inhibited cell proliferation and viability, increased intracellular ROS production, decreased MMP, down-regulated antioxidant capacity, activated the Nrf2/Keap1/HO-1 pathway. However, DEP-induced oxidative stress was partially alleviated by GSH and exogenous H2S. In addition, DEP exposure induced ATP depletion and ATP5α synthase inactivity in GC-1 spg cells, accompanied by ATP5α synthase S-sulfhydrated modification. In conclusion, our research showed that DEP may incapacitate mitochondria through oxidative stress injury, leading to GC-1 spg cells oxidative stress. This process may be associated with the reduction of ATP5α1 S-sulfhydrated modification. It provides a new perspective for the research of the mechanism related to male reproductive toxicity due to air pollution.