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1.
PLoS Biol ; 18(6): e3000741, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32520929

RESUMO

Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite's metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro-induced differentiation. Our results provide insights into the mechanisms of the parasite's mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.


Assuntos
Metabolômica , Mitocôndrias/metabolismo , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismo , Trifosfato de Adenosina/biossíntese , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Elétrons , Glucose/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Oxirredução , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Transcriptoma/genética , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/genética
2.
Science ; 368(6497): 1371-1376, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32439659

RESUMO

The effect of immunometabolism on age-associated diseases remains uncertain. In this work, we show that T cells with dysfunctional mitochondria owing to mitochondrial transcription factor A (TFAM) deficiency act as accelerators of senescence. In mice, these cells instigate multiple aging-related features, including metabolic, cognitive, physical, and cardiovascular alterations, which together result in premature death. T cell metabolic failure induces the accumulation of circulating cytokines, which resembles the chronic inflammation that is characteristic of aging ("inflammaging"). This cytokine storm itself acts as a systemic inducer of senescence. Blocking tumor necrosis factor-α signaling or preventing senescence with nicotinamide adenine dinucleotide precursors partially rescues premature aging in mice with Tfam-deficient T cells. Thus, T cells can regulate organismal fitness and life span, which highlights the importance of tight immunometabolic control in both aging and the onset of age-associated diseases.


Assuntos
Senilidade Prematura/imunologia , Proteínas de Ligação a DNA/deficiência , Mitocôndrias/metabolismo , Proteínas Mitocondriais/deficiência , Multimorbidade , Linfócitos T/metabolismo , Fatores de Transcrição/deficiência , Senilidade Prematura/genética , Senilidade Prematura/prevenção & controle , Animais , Síndrome da Liberação de Citocina/imunologia , Proteínas de Ligação a DNA/genética , Feminino , Deleção de Genes , Inflamação/genética , Inflamação/imunologia , Longevidade , Masculino , Camundongos , Camundongos Mutantes , Proteínas Mitocondriais/genética , NAD/administração & dosagem , NAD/farmacologia , Aptidão Física , Linfócitos T/ultraestrutura , Fatores de Transcrição/genética , Fator de Necrose Tumoral alfa/antagonistas & inibidores
3.
Parasitol Res ; 119(6): 1857-1871, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32350589

RESUMO

Antimony is an important drug for the treatment of Leishmania parasite infections. In several countries, the emergence of drug-resistant Leishmania species has reduced the effectiveness of this drug. The mechanism of clinical drug resistance is unclear. The aim of this work was to identify mitochondrial proteome alterations associated with resistance against antimonial. A combination of cell fractionation, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and Label-Free Quantification was used to characterize the mitochondrial protein composition of Leishmania tropica field isolates resistant and sensitive to meglumine antimoniate. LC-MS/MS analysis resulted in the identification of about 1200 proteins of the Leishmania tropica mitochondrial proteome. Various criteria were used to allocate about 40% proteins to mitochondrial proteome. Comparative quantitative proteomic analysis of the sensitive and the resistant strains showed proteins with differential abundance in resistance species are involved in TCA and aerobic respiration enzymes, stress proteins, lipid metabolism enzymes, and translation. These results showed that the mechanism of antimony resistance in Leishmania spp. field isolate may be associated with alteration in enzymes involved in mitochondrial pathways.


Assuntos
Antiprotozoários/farmacologia , Leishmania tropica/efeitos dos fármacos , Antimoniato de Meglumina/farmacologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Linhagem Celular , Cromatografia Líquida , Resistência a Medicamentos , Leishmania tropica/isolamento & purificação , Leishmania tropica/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Testes de Sensibilidade Parasitária , Proteoma , Proteômica , Espectrometria de Massas em Tandem
4.
PLoS One ; 15(5): e0231296, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32407378

RESUMO

Phytophthora sansomeana infects soybean and causes root rot. It was recently separated from the species complex P. megasperma sensu lato. In this study, we sequenced and annotated its complete mitochondrial genome and compared it to that of nine other Phytophthora species. The genome was assembled into a circular molecule of 39,618 bp with a 22.03% G+C content. Forty-two protein coding genes, 25 tRNA genes and two rRNA genes were annotated in this genome. The protein coding genes include 14 genes in the respiratory complexes, four ATP synthase genes, 16 ribosomal proteins genes, a tatC translocase gene, six conserved ORFs and a unique orf402. The tRNA genes encode tRNAs for 19 amino acids. Comparison among mitochondrial genomes of 10 Phytophthora species revealed three inversions, each covering multiple genes. These genomes were conserved in gene content with few exceptions. A 3' truncated atp9 gene was found in P. nicotianae. All 10 Phytophthora species, as well as other oomycetes and stramenopiles, lacked tRNA genes for threonine in their mitochondria. Phylogenomic analysis using the mitochondrial genomes supported or enhanced previous findings of the phylogeny of Phytophthora spp.


Assuntos
DNA Mitocondrial/genética , Evolução Molecular , Genoma Mitocondrial , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Phytophthora/classificação , Phytophthora/genética , Sequência de Aminoácidos , Filogenia , Homologia de Sequência , Especificidade da Espécie
5.
Am J Physiol Heart Circ Physiol ; 318(6): H1516-H1524, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32383992

RESUMO

Engineered heart tissues (EHTs) have emerged as a robust in vitro model to study cardiac physiology. Although biomimetic culture environments have been developed to better approximate in vivo conditions, currently available methods do not permit full recapitulation of the four phases of the cardiac cycle. We have developed a bioreactor which allows EHTs to undergo cyclic loading sequences that mimic in vivo work loops. EHTs cultured under these working conditions exhibited enhanced concentric contractions but similar isometric contractions compared with EHTs cultured isometrically. EHTs that were allowed to shorten cyclically in culture had increased capacity for contractile work when tested acutely. Increased work production was correlated with higher levels of mitochondrial proteins and mitochondrial biogenesis; this effect was eliminated when tissues were cyclically shortened in the presence of a myosin ATPase inhibitor. Leveraging our novel in vitro method to precisely apply mechanical loads in culture, we grew EHTs under two loading regimes prescribing the same work output but with different associated afterloads. These groups showed no difference in mitochondrial protein expression. In loading regimes with the same afterload but different work output, tissues subjected to higher work demand exhibited elevated levels of mitochondrial protein. Our findings suggest that regulation of mitochondrial mass in cultured human EHTs is potently modulated by the mechanical work the tissue is permitted to perform in culture, presumably communicated through ATP demand. Precise application of mechanical loads to engineered heart tissues in culture represents a novel in vitro method for studying physiological and pathological cardiac adaptation.NEW & NOTEWORTHY In this work, we present a novel bioreactor that allows for active length control of engineered heart tissues during extended tissue culture. Specific length transients were designed so that engineered heart tissues generated complete cardiac work loops. Chronic culture with various work loops suggests that mitochondrial mass and biogenesis are directly regulated by work output.


Assuntos
Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Humanos , Engenharia Tecidual
6.
BMC Med Genet ; 21(1): 68, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32234020

RESUMO

BACKGROUND: The TWNK gene encodes the twinkle protein, which is a mitochondrial helicase for DNA replication. The dominant TWNK variants cause progressive external ophthalmoplegia with mitochondrial DNA deletions, autosomal dominant 3, while the recessive variants cause mitochondrial DNA depletion syndrome 7 and Perrault syndrome 5. Perrault syndrome is characterized by sensorineural hearing loss in both males and females and gonadal dysfunction in females. Patients with Perrault syndrome may present early-onset cerebellar ataxia, whereas middle-age-onset cerebellar ataxia caused by TWNK variants is rare. CASE PRESENTATION: A Japanese female born to consanguineous parents presented hearing loss at age 48, a staggering gait at age 53, and numbness in her distal extremities at age 57. Neurological examination revealed sensorineural hearing loss, cerebellar ataxia, decreased deep tendon reflexes, and sensory disturbance in the distal extremities. Laboratory tests showed no abnormal findings other than a moderate elevation of pyruvate concentration levels. Brain magnetic resonance imaging revealed mild cerebellar atrophy. Using exome sequencing, we identified a homozygous TWNK variant (NM_021830: c.1358G>A, p.R453Q). CONCLUSIONS: TWNK variants could cause middle-age-onset cerebellar ataxia. Screening for TWNK variants should be considered in cases of cerebellar ataxia associated with deafness and/or peripheral neuropathy, even if the onset is not early.


Assuntos
Ataxia Cerebelar/genética , DNA Helicases/genética , Proteínas Mitocondriais/genética , Ataxia Cerebelar/complicações , Ataxia Cerebelar/diagnóstico , Consanguinidade , Feminino , Marcha Atáxica/complicações , Marcha Atáxica/diagnóstico , Marcha Atáxica/genética , Disgenesia Gonadal 46 XX/diagnóstico , Disgenesia Gonadal 46 XX/genética , Perda Auditiva/complicações , Perda Auditiva/diagnóstico , Perda Auditiva/genética , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/genética , Homozigoto , Humanos , Japão , Transtornos de Início Tardio/diagnóstico , Transtornos de Início Tardio/genética , Pessoa de Meia-Idade , Mutação , Linhagem
7.
Sci Total Environ ; 725: 138192, 2020 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-32278173

RESUMO

Fluoride, a toxic substance, is widely distributed in the environment and causes serious damage to the body. This study was performed to investigate the effects of fluoride on mitochondrial fission in mouse hepatocytes. A total of 48 mice were equally divided into four groups and admisnistered with NaF in drinking water at fluorine ion concentrations of 0, 25, 50 and 100 mg/L for 70 days. The pathomorphology and ultrastructurre of hepatocytes were then observed. The mitochondrial lesion parameters (number, length, width and vacuolization area) are evaluated. The expression of Drp1, Mff, Fis1, MiD49, MiD51 and Dyn2, which are associated with mitochondrial fission, was determined by quantitative real-time PCR and Western blot analysis. Apoptosis was detected by using TUNEL assay. Results showed that fluoride causes notable changes in the pathological morphology of liver tissues and severely damages the ultrastructure of hepatocytes. Damage manifested as nuclear condensation, nuclear membrane breakdown, mitochondrial vacuolation, increased fragmentation, and mitochondrial fission. Moreover, mRNA and protein expression levels were significantly upregulated in the Drp1/Mff signaling pathway. The mRNA expression levels of Cyt c, caspase 9 and 3 markedly increased in the fluoride treated groups in a dose-dependent manner. The percentage of TUNEL-positive nuclei in the liver remarkably increased after fluoride treatment. Overall, the results indicate that excessive fluoride exposure can increase mitochondrial fission via the Drp1/Mff signaling pathway, severely damage the mitochondrial structure, and lead to apoptosis of hepatocytes.


Assuntos
Fluoretos , Proteínas Mitocondriais , Animais , Apoptose , Dinaminas , Hepatócitos , Camundongos , Mitocôndrias , Transdução de Sinais
8.
Sheng Li Xue Bao ; 72(2): 249-254, 2020 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-32328619

RESUMO

The aim of this study was to investigate the effect of edaravone (Eda) on the balance of mitochondrial fusion and fission in Parkinson's disease (PD) cell model. A cell model of PD was established by treating PC12 cells with 500 µmol/L 1-methyl-4-phenylpyridinium (MPP+). Thiazole blue colorimetry (MTT) was used to detect the effect of different concentrations of Eda on the survival rate of PC12 cells exposed to MPP+. The mitochondrial morphology was determined by laser confocal microscope. Western blot was used to measure the protein expression levels of mitochondrial fusion- and fission-related proteins, including OPA1, MFN2, DRP1 and Fis1. The results showed that pretreatment with different concentrations of Eda antagonized MPP+-induced PC12 cell damage in a dose-dependent manner. The PC12 cells treated with MPP+ showed mitochondrial fragmentation, up-regulated DRP1 and Fis1 protein expression levels, and down-regulated MFN2 and OPA1 protein expression levels. Eda could reverse the above changes in the MPP+-treated PC12 cells, but did not affect Fis1 protein expression. These results suggest that Eda has a protective effect on the mitochondrial fusion disruption induced by MPP+ in PC12 cells. The mechanism may be related to the up-regulation of OPA1/MFN2 and down-regulation of DRP1.


Assuntos
Edaravone/farmacologia , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial , 1-Metil-4-fenilpiridínio , Animais , Dinaminas , GTP Fosfo-Hidrolases , Proteínas Mitocondriais , Células PC12 , Doença de Parkinson , Ratos , Regulação para Cima
9.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(2): 152-156, 2020 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-32314713

RESUMO

Objective To construct nucleotide-binding oligomerization domain-like receptors family member X1 (NLRX1) lentivirus plasmid and stable NLRX1-overexpressing A549 cell line. Methods Full-length cDNA encoding human NLRX1 was amplified from pCMV3-NLRX1 plasmid and then was inserted into pCDH-CMV-MCS-EF1-Puro vector to obtain NLRX1-overexpressing plasmid pCDH-NLRX1. pCDH-NLRX1 lentivirus particles were obtained by co-transfecting pCDH-NLRX1 with packaging plasmids (pMD2 and pAX2) into HEK293T cells. A549 cells were infected with concentrated pCDH-NLRX1 lentivirus particles and then were screened by puromycin to obtain stable NLRX1-overexpressed A549 cell line. The mRNA transcription and protein expression of NLRX1 were detected by real-time quantitative PCR, Western blot analysis and immunofluorescence. Results The lentivirus plasmid pCDH-NLRX1 was successfully constructed. After pCDH-NLRX1 and packaging plasmids were co-transfected into HEK293T cells, we obtained NLRX1 lentivirus particles with the titer of 1×105 TU/mL. After A549 cells were infected with lentivirus particles and screened by puromycin, a stable over-expression cell line of NLRX1 was obtained. NLRX1 was obviously expressed in the lentivirus-infected A549 cells and its mRNA and protein levels significantly increased. Conclusion NLRX1 lentivirus plasmid and stable NLRX1-overexpressing A549 cell line have been successfully constructed.


Assuntos
Vetores Genéticos , Lentivirus , Proteínas Mitocondriais/genética , Plasmídeos , Células A549 , Células HEK293 , Humanos , Transfecção
10.
PLoS One ; 15(4): e0232206, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32330194

RESUMO

Hair growth is the cyclically regulated process that is characterized by growing phase (anagen), regression phase (catagen) and resting phase (telogen). Hair follicle stem cells (HFSCs) play pivotal role in the control of hair growth cycle. It has been notified that stem cells have the distinguished metabolic signature compared to differentiated cells, such as the preference to glycolysis rather than mitochondrial respiration. Crif1 is a mitochondrial protein that regulates the synthesis and insertion of oxidative phosphorylation (OXPHOS) polypeptides to inner membrane of mitochondria. Several studies demonstrate that tissue-specific knockout of Crif1 leads to mitochondrial dysfunction. In this study, we investigated the effect of mitochondrial dysfunction in terms of Crif1 deficiency on the hair growth cycle of adult mice. We created two kinds of inducible conditional knockout (icKO) mice. In epidermal specific icKO mice (Crif1 K14icKO), hair growth cycle was significantly retarded compared to wild type mice. Similarly, HFSC specific icKO mice (Crif1 K15icKO) showed significant retardation of hair growth cycle in depilation-induced anagen model. Interestingly, flow cytometry revealed that HFSC populations were maintained in Crif1 K15icKO mice. These results suggest that mitochondrial function in HFSCs is important for the progression of hair growth cycle, but not for maintenance of HFSCs.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Cabelo/crescimento & desenvolvimento , Cabelo/metabolismo , Animais , Diferenciação Celular/fisiologia , Epiderme/crescimento & desenvolvimento , Epiderme/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Peptídeos/metabolismo , Células-Tronco/metabolismo
11.
PLoS One ; 15(4): e0232366, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32348368

RESUMO

Pentatricopeptide repeat (PPR) proteins, composed of PPR motifs repeated in tandem, are sequence-specific RNA binding proteins. Recent bioinformatic studies have shown that the combination of polar amino acids at positions 5 and last in each PPR motif recognizes RNA bases, and an RNA recognition code for PPR proteins has been proposed. Subsequent studies confirmed that the P (canonical length) and S (short) motifs bind to specific nucleotides according to this code. However, the contribution of L (long) motifs to RNA recognition is mostly controversial, owing to the presence of a nonpolar amino acid at position 5. The PLS-class PPR protein PpPPR_56 is a mitochondrial RNA editing factor in the moss Physcomitrella patens. Here, we performed in vitro RNA binding and in vivo complementation assays with PpPPR_56 and its variants containing mutated L motifs to investigate their contributions to RNA recognition. In vitro RNA binding assay showed that the original combination of amino acids at positions 5 and last in the L motifs of PpPPR_56 is not required for RNA recognition. In addition, an in vivo complementation assay with RNA editing factors PpPPR_56 and PpPPR_78 revealed the importance of nonpolar amino acids at position 5 of C-terminal L motifs for efficient RNA editing. Our findings suggest that L motifs function as non-binding spacers, not as RNA-binding motifs, to facilitate the formation of a complex between PLS-class PPR protein and RNA. As a result, the DYW domain, a putative catalytic deaminase responsible for C-to-U RNA editing, is correctly placed in proximity to C, which is to be edited.


Assuntos
Bryopsida/metabolismo , Proteínas de Plantas/metabolismo , Edição de RNA , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Bryopsida/química , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Proteínas de Plantas/química , RNA Mitocondrial/metabolismo , Proteínas de Ligação a RNA/química
12.
PLoS One ; 15(4): e0231202, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32271823

RESUMO

OBJECTIVE: Monoclonal antibody derivatives are promising drugs for the treatment of various diseases due to their high matrix metalloproteinases (MMP) active site specificity. We studied the effects of a novel antibody, SDS3, which specifically recognizes the mature active site of MMP9/2 during ventricular remodeling progression in a mouse model of chronic volume overload (VO). METHODS: VO was induced by creating an aortocaval fistula (ACF) in 10- to 12-week-old C57BL male mice. The VO-induced mice were treated with either vehicle control (PBS) or with SDS3 twice weekly by intraperitoneal (ip) injection. The relative changes in cardiac parameters between baseline (day 1) and end-point (day 30), were evaluated by echocardiography. The effects of SDS3 treatment on cardiac fibrosis, cardiomyocyte volume, and cardiac inflammation were tested by cardiac staining with Masson's trichrome, wheat Germ Agglutinin (WGA), and CD45, respectively. Serum levels of TNFα and IL-6 with and without SDS3 treatment were tested by ELISA. RESULTS: SDS3 significantly reduced cardiac dilatation, left ventricular (LV) mass, and cardiomyocyte hypertrophy compared to the vehicle treated animals. The antibody also reduced the heart-to-body weight ratio of the ACF animals to values comparable to those of the controls. Interestingly, the SDS3 group underwent significant reduction of cardiac inflammation and pro-inflammatory cytokine production, indicating a regulatory role for MMP9/2 in tissue remodeling, possibly by tumor necrosis factor alpha (TNFα) activation. In addition, significant changes in the expression of proteins related to mitochondrial function were observed in ACF animals, these changes were reversed following treatment with SDS3. CONCLUSION: The data suggest that MMP9/2 blockage with SDS3 attenuates myocardial remodeling associated with chronic VO by three potential pathways: downregulating the extracellular matrix proteolytic cleavage, reducing the cardiac inflammatory responses, and preserving the cardiac mitochondrial structure and function.


Assuntos
Anticorpos Bloqueadores/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Remodelação Ventricular/efeitos dos fármacos , Animais , Doença Crônica , Dilatação Patológica , Gelatinases/metabolismo , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Mediadores da Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Fístula Vascular/patologia , Fístula Vascular/fisiopatologia
13.
PLoS Genet ; 16(4): e1008629, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282858

RESUMO

Analyzing 12,361 all-cause cirrhosis cases and 790,095 controls from eight cohorts, we identify a common missense variant in the Mitochondrial Amidoxime Reducing Component 1 gene (MARC1 p.A165T) that associates with protection from all-cause cirrhosis (OR 0.91, p = 2.3*10-11). This same variant also associates with lower levels of hepatic fat on computed tomographic imaging and lower odds of physician-diagnosed fatty liver as well as lower blood levels of alanine transaminase (-0.025 SD, 3.7*10-43), alkaline phosphatase (-0.025 SD, 1.2*10-37), total cholesterol (-0.030 SD, p = 1.9*10-36) and LDL cholesterol (-0.027 SD, p = 5.1*10-30) levels. We identified a series of additional MARC1 alleles (low-frequency missense p.M187K and rare protein-truncating p.R200Ter) that also associated with lower cholesterol levels, liver enzyme levels and reduced risk of cirrhosis (0 cirrhosis cases for 238 R200Ter carriers versus 17,046 cases of cirrhosis among 759,027 non-carriers, p = 0.04) suggesting that deficiency of the MARC1 enzyme may lower blood cholesterol levels and protect against cirrhosis.


Assuntos
Fígado Gorduroso/genética , Fígado Gorduroso/prevenção & controle , Predisposição Genética para Doença , Cirrose Hepática/genética , Cirrose Hepática/prevenção & controle , Proteínas Mitocondriais/genética , Mutação de Sentido Incorreto/genética , Oxirredutases/genética , Alelos , LDL-Colesterol/sangue , Doença da Artéria Coronariana/genética , Conjuntos de Dados como Assunto , Fígado Gorduroso/sangue , Fígado Gorduroso/enzimologia , Feminino , Homozigoto , Humanos , Fígado/enzimologia , Cirrose Hepática/sangue , Cirrose Hepática/enzimologia , Cirrose Hepática Alcoólica/sangue , Cirrose Hepática Alcoólica/enzimologia , Cirrose Hepática Alcoólica/genética , Cirrose Hepática Alcoólica/prevenção & controle , Mutação com Perda de Função/genética , Masculino , Pessoa de Meia-Idade
14.
Med Sci Monit ; 26: e918216, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32129321

RESUMO

BACKGROUND Chemoresistance is a primary hindrance for current cancer treatments. The influence of abnormal mitochondria in chemotherapy resistance is not well known. To explore the correlation between mitochondria and acquired chemoresistance, this work studied alterations in mitochondrial dynamics, biogenesis, and functions for paclitaxel-resistant cancer cell line A549/Taxol and its parental line A549. MATERIAL AND METHODS Mitochondrial morphology was observed by transmission electron microscopy and confocal microscopy. We measured the mitochondrial mass and mitochondrial membrane potential using fluorescent dyes. The glucose metabolic profile and ATP (adenosine triphosphate) content were determined by bioluminescent cell assays. Seahorse bio-energy analyzer XF24 was used to detect the mitochondrial respiratory function. The expressions of mitochondrial dynamics and biogenesis related genes were quantified using real-time polymerase chain reaction. RESULTS We observed fusion morphology of the mitochondrial network in A549/Taxol cells, with upregulation of fusion genes (Mfn1 and Mfn2) and downregulation of fission gene Fis1. In A549/Taxol cells, mitochondrial mass showed a significant decrease, while the mitochondrial biogenesis pathway was strongly activated. Despite the decreased mitochondrial membrane potential, the capability for mitochondrial respiration was not impaired in A549/Taxol cells. CONCLUSIONS Our study revealed a series changes of mitochondrial characteristics in paclitaxel-resistant cells. Mfn1 and Mfn2 and PGC-1alpha increased, while Fis1 expression and mitochondrial oxidative phosphorylation decreased in A549/Taxol cell lines. These changes to mitochondrial fusion, fission, and biological function contributed to the occurrence of paclitaxel resistance in tumor cells which induced paclitaxel resistance.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Dinâmica Mitocondrial , Biogênese de Organelas , Paclitaxel/farmacologia , Células A549 , GTP Fosfo-Hidrolases/metabolismo , Humanos , Potencial da Membrana Mitocondrial , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo
16.
Nature ; 579(7799): 433-437, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132706

RESUMO

Mitochondrial fidelity is tightly linked to overall cellular homeostasis and is compromised in ageing and various pathologies1-3. Mitochondrial malfunction needs to be relayed to the cytosol, where an integrated stress response is triggered by the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) in mammalian cells4,5. eIF2α phosphorylation is mediated by the four eIF2α kinases GCN2, HRI, PERK and PKR, which are activated by diverse types of cellular stress6. However, the machinery that communicates mitochondrial perturbation to the cytosol to trigger the integrated stress response remains unknown1,2,7. Here we combine genome engineering and haploid genetics to unbiasedly identify genes that affect the induction of C/EBP homologous protein (CHOP), a key factor in the integrated stress response. We show that the mitochondrial protease OMA1 and the poorly characterized protein DELE1, together with HRI, constitute the missing pathway that is triggered by mitochondrial stress. Mechanistically, stress-induced activation of OMA1 causes DELE1 to be cleaved into a short form that accumulates in the cytosol, where it binds to and activates HRI via its C-terminal portion. Obstruction of this pathway can be beneficial or adverse depending on the type of mitochondrial perturbation. In addition to the core pathway components, our comparative genetic screening strategy identifies a suite of additional regulators. Together, these findings could be used to inform future strategies to modulate the cellular response to mitochondrial dysfunction in the context of human disease.


Assuntos
Citosol/metabolismo , Citosol/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Ativação Enzimática , Fator de Iniciação 2 em Eucariotos/metabolismo , Genoma Humano/genética , Humanos , Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Fosforilação , Ligação Proteica , Estresse Fisiológico , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/metabolismo
17.
Nature ; 579(7799): 427-432, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132707

RESUMO

In mammalian cells, mitochondrial dysfunction triggers the integrated stress response, in which the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in the induction of the transcription factor ATF41-3. However, how mitochondrial stress is relayed to ATF4 is unknown. Here we show that HRI is the eIF2α kinase that is necessary and sufficient for this relay. In a genome-wide CRISPR interference screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease; and DELE1, a little-characterized protein that we found was associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1 and leads to the accumulation of DELE1 in the cytosol, where it interacts with HRI and activates the eIF2α kinase activity of HRI. In addition, DELE1 is required for ATF4 translation downstream of eIF2α phosphorylation. Blockade of the OMA1-DELE1-HRI pathway triggers an alternative response in which specific molecular chaperones are induced. The OMA1-DELE1-HRI pathway therefore represents a potential therapeutic target that could enable fine-tuning of the integrated stress response for beneficial outcomes in diseases that involve mitochondrial dysfunction.


Assuntos
Citosol/metabolismo , Metaloendopeptidases/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Estresse Fisiológico , eIF-2 Quinase/metabolismo , Fator 4 Ativador da Transcrição/biossíntese , Fator 4 Ativador da Transcrição/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Citosol/enzimologia , Ativação Enzimática , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Masculino , Proteínas Mitocondriais/química , Chaperonas Moleculares/metabolismo , Fosforilação , Ligação Proteica
18.
Clin Sci (Lond) ; 134(7): 889-905, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32219338

RESUMO

Senescence and mitochondrial stress are mutually reinforcing age-related processes that contribute to idiopathic pulmonary fibrosis (IPF); a lethal disease that manifests primarily in the elderly. Whilst evidence is accumulating that GMP-AMP synthase (cGAS) is crucial in perpetuating senescence by binding damaged DNA released into the cytosol, its role in IPF is not known. The present study examines the contributions of cGAS and self DNA to the senescence of lung fibroblasts from IPF patients (IPF-LFs) and age-matched controls (Ctrl-LFs). cGAS immunoreactivity was observed in regions of fibrosis associated with fibroblasts in lung tissue of IPF patients. Pharmacological inhibition of cGAS or its knockdown by silencing RNA (siRNA) diminished the escalation of IPF-LF senescence in culture over 7 days as measured by decreased p21 and p16 expression, histone 2AXγ phosphorylation and/or IL-6 production (P < 0.05, n = 5-8). The targeting of cGAS also attenuated etoposide-induced senescence in Ctrl-LFs (P < 0.05, n = 5-8). Levels of mitochondrial DNA (mDNA) detected by qPCR in the cytosol and medium of IPF-LFs or senescence-induced Ctrl-LFs were higher than Ctrl-LFs at baseline (P < 0.05, n = 5-7). The addition of DNAse I (100 U/ml) deaccelerated IPF-LF senescence (P < 0.05, n = 5), whereas ectopic mDNA or the induction of endogenous mDNA release augmented Ctrl-LF senescence in a cGAS-dependent manner (P < 0.05, n = 5). In conclusion, we provide evidence that cGAS reinforces lung fibroblast senescence involving damaged self DNA. The targeting of cGAS to supress senescent-like responses may have potential important therapeutic implications in the treatment of IPF.


Assuntos
Proliferação de Células , Senescência Celular , DNA Mitocondrial/metabolismo , Fibroblastos/enzimologia , Fibrose Pulmonar Idiopática/enzimologia , Pulmão/enzimologia , Nucleotidiltransferases/metabolismo , Estudos de Casos e Controles , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Histonas/metabolismo , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Interleucina-6/genética , Interleucina-6/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/genética , Comunicação Parácrina , Fosforilação , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
Biochim Biophys Acta Bioenerg ; 1861(7): 148192, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32194061

RESUMO

Mutations of many PDSS and COQ genes are associated with primary coenzyme Q10 (CoQ10) deficiency, whereas mitochondrial DNA (mtDNA) mutations might cause secondary CoQ10 deficiency. Previously, we found that COQ5 and COQ9 proteins are present in different protein complexes in the mitochondria in human 143B cells and demonstrated that COQ5 and COQ9 knockdown suppresses CoQ10 levels. In the present study, we characterized other PDSS and COQ proteins and examined possible crosstalk among various PDSS and COQ proteins. Specific antibodies and mitochondrial localization of mature proteins for these proteins, except PDSS1 and COQ2, were identified. Multiple isoforms of PDSS2 and COQ3 were observed. Moreover, PDSS1, PDSS2, and COQ3 played more important roles in maintaining the stability of the other proteins. Protein complexes containing PDSS2, COQ3, COQ4, COQ6, or COQ7 protein in the mitochondria were detected. Two distinct PDSS2-containing protein complexes could be identified. Transient knockdown of these genes, except COQ6 and COQ8, decreased CoQ10 levels, but only COQ7 knockdown hampered mitochondrial respiration and caused increased ubiquinol:ubiquinone ratios and accumulation of a putative biosynthetic intermediate with reversible redox property as CoQ10. Furthermore, suppressed levels of PDSS2 and various COQ proteins (except COQ3 and COQ8A) were found in cybrids containing the pathogenic mtDNA A8344G mutation or in FCCP-treated 143B cells, which was similar to our previous findings for COQ5. These novel findings may prompt the elucidation of the putative CoQ synthome in human cells and the understanding of these PDSS and COQ protein under physiological and pathological conditions.


Assuntos
Alquil e Aril Transferases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Ubiquinona/análogos & derivados , Especificidade de Anticorpos , Vias Biossintéticas , Linhagem Celular Tumoral , Transporte de Elétrons , Humanos , Peso Molecular , Estabilidade Proteica , Transporte Proteico , Ubiquinona/metabolismo
20.
Mol Cell ; 77(5): 1107-1123.e10, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32142684

RESUMO

Mitochondria import nearly their entire proteome from the cytoplasm by translocating precursor proteins through the translocase of the outer membrane (TOM) complex. Here, we show dynamic regulation of mitochondrial import by the ubiquitin system. Acute pharmacological inhibition or genetic ablation of the mitochondrial deubiquitinase (DUB) USP30 triggers accumulation of Ub-substrates that are normally localized inside the mitochondria. Mitochondrial import of USP30 substrates is impaired in USP30 knockout (KO) cells, suggesting that deubiquitination promotes efficient import. Upstream of USP30, the E3 ligase March5 ubiquitinates mitochondrial proteins whose eventual import depends on USP30. In USP30 KOs, exogenous March5 expression induces accumulation of unimported translocation intermediates that are degraded by the proteasomes. In USP30 KO mice, TOM subunits have reduced abundance across multiple tissues. Together these data highlight how protein import into a subcellular compartment can be regulated by ubiquitination and deubiquitination by E3 ligase and DUB machinery positioned at the gate.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Tioléster Hidrolases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte/genética , Feminino , Células HEK293 , Células HeLa , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Tioléster Hidrolases/genética , Fatores de Tempo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
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