Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 12 de 12
Filtrar
Mais filtros











Base de dados
Intervalo de ano de publicação
1.
Enzymes ; 54: 205-220, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37945172

RESUMO

Mitochondria are multifunctional organelles that play a central role in a wide range of life-sustaining tasks in eukaryotic cells, including adenosine triphosphate (ATP) production, calcium storage and coenzyme generation pathways such as iron-sulfur cluster biosynthesis. The wide range of mitochondrial functions is carried out by a diverse array of proteins comprising approximately 1500 proteins or polypeptides. Degradation of these proteins is mainly performed by four AAA+ proteases localized in mitochondria. These AAA+ proteases play a quality control role in degrading damaged or misfolded proteins and perform various other functions. This chapter describes previously identified roles for these AAA+ proteases that are localized in the mitochondria of animal cells.


Assuntos
Mitocôndrias , Proteínas Mitocondriais , Animais , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Mitocondriais/metabolismo , Peptídeos/metabolismo
2.
mSphere ; 6(6): e0073421, 2021 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-34817239

RESUMO

Five blaCTX-M-14-positive Klebsiella pneumoniae isolates (KpWEA1, KpWEA2, KpWEA3, KpWEA4-1, and KpWEA4-2) were consecutively obtained from a patient with relapsed acute myeloid leukemia who was continuously administered antimicrobials. Compared with KpWEA1 and KpWEA2, KpWEA3 showed decreased susceptibility to antimicrobials, and KpWEA4-1 and KpWEA4-2 (isolated from a single specimen) showed further-elevated multidrug-resistance (MDR) phenotypes. This study aims to clarify the clonality of the five isolates and their evolutionary processes leading to MDR by comparison of these complete genomes. The genome comparison revealed KpWEA1 was the antecedent of the other four isolates, and KpWEA4-1 and KpWEA4-2 independently emerged from KpWEA3. Increasing levels of MDR were acquired by gradual accumulation of genetic alterations related to outer membrane protein expression: the loss of OmpK35 and upregulation of AcrAB-TolC occurred in KpWEA3 due to ramA overexpression caused by a mutation in ramR; then OmpK36 was lost in KpWEA4-1 and KpWEA4-2 by different mechanisms. KpWEA4-2 further acquired colistin resistance by the deletion of mgrB. In addition, we found that exuR and kdgR, which encode repressors of hexuronate metabolism-related genes, were disrupted in different ways in KpWEA4-1 and KpWEA4-2. The two isolates also possessed different amino acid substitutions in AtpG, which occurred at very close positions. These genetic alterations related to metabolisms may compensate for the deleterious effects of major porin loss. Thus, our present study reveals the evolutionary process of a K. pneumoniae clone leading to MDR and also suggests specific survival strategies in the bacteria that acquired MDR by the genome evolution. IMPORTANCE Within-host evolution is a survival strategy that can occur in many pathogens and is often associated with the emergence of novel antimicrobial-resistant (AMR) bacteria. To analyze this process, suitable sets of clinical isolates are required. Here, we analyzed five Klebsiella pneumoniae isolates which were consecutively isolated from a patient and showed a gradual increase in the AMR level. By genome sequencing and other analyses, we show that the first isolate was the antecedent of the later isolates and that they gained increased levels of antimicrobial resistance leading to multidrug resistance (MDR) by stepwise changes in the expression of outer membrane proteins. The isolates showing higher levels of MDR lost major porins but still colonized the patient's gut, suggesting that the deleterious effects of porin loss were compensated for by the mutations in hexuronate metabolism-related genes and atpG, which were commonly detected in the MDR isolates.


Assuntos
Infecções por Klebsiella , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Clonais/metabolismo , Colistina/farmacologia , Resistência a Múltiplos Medicamentos , Farmacorresistência Bacteriana Múltipla/genética , Feminino , Humanos , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , beta-Lactamases/genética
3.
Commun Biol ; 4(1): 974, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400774

RESUMO

Human ATP-dependent Lon protease (LONP1) forms homohexameric, ring-shaped complexes. Depletion of LONP1 causes aggregation of a broad range of proteins in the mitochondrial matrix and decreases the levels of their soluble forms. The ATP hydrolysis activity, but not protease activity, of LONP1 is critical for its chaperone-like anti-aggregation activity. LONP1 forms a complex with the import machinery and an incoming protein, and protein aggregation is linked with matrix protein import. LONP1 also contributes to the degradation of imported, aberrant, unprocessed proteins using its protease activity. Taken together, our results show that LONP1 functions as a gatekeeper for specific proteins imported into the mitochondrial matrix.


Assuntos
Proteases Dependentes de ATP/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Humanos , Transporte Proteico
4.
Oncogenesis ; 6(11): 397, 2017 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-29184058

RESUMO

Tumor heterogeneity can be traced back to a small subset of cancer stem cells (CSCs), which can be derived from a single stem cell and show chemoresistance. Recent studies showed that CSCs are sensitive to mitochondrial targeting antibiotics such as doxycycline. However, little is known about how cancer cells undergo sphere formation and how antibiotics inhibit CSC proliferation. Here we show that under sphere-forming assay conditions, prostate cancer cells acquired CSC-like properties: promoted mitochondrial respiratory chain activity, expression of characteristic CSC markers and resistance to anticancer agents. Furthermore, those CSC-like properties could reversibly change depending on the culture conditions, suggesting some kinds of CSCs have plasticity in tumor microenvironments. The sphere-forming cells (i.e. cancer stem-like cells) showed increased contact between mitochondria and mitochondrial associated-endoplasmic reticulum (ER) membranes (MAM). Mitochondrial targeting doxycycline induced activating transcription factor 4 (ATF4) mediated expression of ER stress response and led to p53-upregulated modulator of apoptosis (PUMA)-dependent apoptosis only in the cancer stem-like cells. We also found that doxycycline effectively suppressed the sphere formation in vitro and blocked CD44v9-expressing tumor growth in vivo. In summary, these data provide new molecular findings that monolayer cancer cells acquire CSC-like properties in a reversible manner. These findings provide important insights into CSC biology and a potential new treatment of targeting mitochondria dependency.

5.
Neurol Genet ; 3(5): e184, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28913435

RESUMO

OBJECTIVE: To determine the molecular factors contributing to progressive cavitating leukoencephalopathy (PCL) to help resolve the underlying genotype-phenotype associations in the mitochondrial iron-sulfur cluster (ISC) assembly system. METHODS: The subjects were 3 patients from 2 families who showed no inconsistencies in either clinical or brain MRI findings as PCL. We used exome sequencing, immunoblotting, and enzyme activity assays to establish a molecular diagnosis and determine the roles of ISC-associated factors in PCL. RESULTS: We performed genetic analyses on these 3 patients and identified compound heterozygosity for the IBA57 gene, which encodes the mitochondrial iron-sulfur protein assembly factor. Protein expression analysis revealed substantial decreases in IBA57 protein expression in myoblasts and fibroblasts. Immunoblotting revealed substantially reduced expression of SDHB, a subunit of complex II, and lipoic acid synthetase (LIAS). Levels of pyruvate dehydrogenase complex-E2 and α-ketoglutarate dehydrogenase-E2, which use lipoic acid as a cofactor, were also reduced. In activity staining, SDH activity was clearly reduced, but it was ameliorated in mitochondrial fractions from rescued myoblasts. In addition, NFU1 protein expression was also decreased, which is required for the assembly of a subset of iron-sulfur proteins to SDH and LIAS in the mitochondrial ISC assembly system. CONCLUSIONS: Defects in IBA57 essentially regulate NFU1 expression, and aberrant NFU1 ultimately affects SDH activity and LIAS expression in the ISC biogenesis pathway. This study provides new insights into the role of the iron-sulfur protein assembly system in disorders related to mitochondrial energy metabolism associated with leukoencephalopathy with cavities.

6.
Oncotarget ; 7(46): 75221-75234, 2016 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-27655692

RESUMO

Cancer cells rewire their metabolism and mitochondrial oxidative phosphorylation (OXPHOS) to promote proliferation and maintenance. Cancer cells use multiple adaptive mechanisms in response to a hypo-nutrient environment. However, little is known about how cancer mitochondria are involved in the ability of these cells to adapt to a hypo-nutrient environment. Oncogenic HRas leads to suppression of the mitochondrial oxygen consumption rate (OCR), but oxygen consumption is essential for tumorigenesis. We found that in oncogenic HRas transformed cells, serum depletion reversibly increased the OCR and membrane potential. Serum depletion promoted a cancer stem cell (CSC)-like phenotype, indicated by an increase in CSC markers expression and resistance to anticancer agents. We also found that nitric oxide (NO) synthesis was significantly induced after serum depletion and that NO donors modified the OCR. An NOS inhibitor, SEITU, inhibited the OCR and CSC gene expression. It also reduced anchorage-independent growth by promoting apoptosis. In summary, our data provide new molecular findings that serum depletion induces NO synthesis and promotes mitochondrial OXPHOS, leading to tumor progression and a CSC phenotype. These results suggest that mitochondrial OCR inhibitors can be used as therapy against CSC.


Assuntos
Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células-Tronco Neoplásicas/metabolismo , Óxido Nítrico/biossíntese , Proteínas ras/genética , Animais , Apoptose/efeitos dos fármacos , Biomarcadores , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Transformada , Linhagem Celular Tumoral , Respiração Celular/genética , Modelos Animais de Doenças , Expressão Gênica , Metformina/farmacologia , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Biológicos , Mutação , Óxido Nítrico Sintase/antagonistas & inibidores , Fosforilação Oxidativa , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
7.
Hum Mutat ; 36(2): 232-9, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25393721

RESUMO

The human ECHS1 gene encodes the short-chain enoyl coenzyme A hydratase, the enzyme that catalyzes the second step of ß-oxidation of fatty acids in the mitochondrial matrix. We report on a boy with ECHS1 deficiency who was diagnosed with Leigh syndrome at 21 months of age. The patient presented with hypotonia, metabolic acidosis, and developmental delay. A combined respiratory chain deficiency was also observed. Targeted exome sequencing of 776 mitochondria-associated genes encoded by nuclear DNA identified compound heterozygous mutations in ECHS1. ECHS1 protein expression was severely depleted in the patient's skeletal muscle and patient-derived myoblasts; a marked decrease in enzyme activity was also evident in patient-derived myoblasts. Immortalized patient-derived myoblasts that expressed exogenous wild-type ECHS1 exhibited the recovery of the ECHS1 activity, indicating that the gene defect was pathogenic. Mitochondrial respiratory complex activity was also mostly restored in these cells, suggesting that there was an unidentified link between deficiency of ECHS1 and respiratory chain. Here, we describe the patient with ECHS1 deficiency; these findings will advance our understanding not only the pathology of mitochondrial fatty acid ß-oxidation disorders, but also the regulation of mitochondrial metabolism.


Assuntos
Enoil-CoA Hidratase/genética , Doença de Leigh/genética , Sequência de Bases , Linhagem Celular Tumoral , Pré-Escolar , Análise Mutacional de DNA , Enoil-CoA Hidratase/deficiência , Estudos de Associação Genética , Humanos , Masculino
8.
J Med Genet ; 49(12): 777-84, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23188110

RESUMO

BACKGROUND: Autosomal recessive hereditary spastic paraplegias (AR-HSP) constitute a heterogeneous group of neurodegenerative diseases involving pyramidal tracts dysfunction. The genes responsible for many types of AR-HSPs remain unknown. We attempted to identify the gene responsible for AR-HSP with optic atrophy and neuropathy. METHODS: The present study involved two patients in a consanguineous Japanese family. Neurologic examination and DNA analysis were performed for both patients, and a skin biopsy for one. We performed genome-wide linkage analysis involving single nucleotide polymorphism arrays, copy-number variation analysis, and exome sequencing. To clarify the mitochondrial functional alteration resulting from the identified mutation, we performed immunoblot analysis, mitochondrial protein synthesis assaying, blue native polyacrylamide gel electrophoresis (BN-PAGE) analysis, and respiratory enzyme activity assaying of cultured fibroblasts of the patient and a control. RESULTS: We identified a homozygous nonsense mutation (c.394C>T, p.R132X) in C12orf65 in the two patients in this family. This C12orf65 mutation was not found in 74 Japanese AR-HSP index patients without any mutations in previously known HSP genes. This mutation resulted in marked reduction of mitochondrial protein synthesis, followed by functional and structural defects in respiratory complexes I and IV. CONCLUSIONS: This novel nonsense mutation in C12orf65 could cause AR-HSP with optic atrophy and neuropathy, resulting in a premature stop codon. The truncated C12orf65 protein must lead to a defect in mitochondrial protein synthesis and a reduction in the respiratory complex enzyme activity. Thus, dysfunction of mitochondrial translation could be one of the pathogenic mechanisms underlying HSPs.


Assuntos
Homozigoto , Mutação , Atrofia Óptica/genética , Fatores de Terminação de Peptídeos/genética , Doenças do Sistema Nervoso Periférico/genética , Paraplegia Espástica Hereditária/genética , Adulto , Sequência de Bases , Variações do Número de Cópias de DNA , Exoma , Ligação Genética , Humanos , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais , Atrofia Óptica/metabolismo , Linhagem , Doenças do Sistema Nervoso Periférico/metabolismo , Paraplegia Espástica Hereditária/metabolismo
9.
J Biol Chem ; 283(35): 23964-71, 2008 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-18593709

RESUMO

Mitochondrial DNA helicase, also called Twinkle, is essential for mtDNA maintenance. Its helicase domain shares high homology with helicases from superfamily 4. Structural analyses of helicases from this family indicate that carboxyl-terminal residues contribute to NTP hydrolysis required for translocation and DNA unwinding, yet genetic and biochemical information is very limited. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying a series of deletion and alanine substitution mutations in the carboxyl terminus and identify critical residues between amino acids 572 and 596 of the 613 amino acid polypeptide that are essential for mitochondrial DNA helicase function in vivo. Likewise, amino acid substitution mutants K574A, R576A, Y577A, F588A, and F595A show dose-dependent dominant-negative phenotypes. Arg-576 and Phe-588 are analogous to the arginine finger and base stack of other helicases, including the bacteriophage T7 gene 4 protein and bacterial DnaB helicase, respectively. We show here that representative human recombinant proteins that are analogous to the alanine substitution mutants exhibit defects in nucleotide hydrolysis. Our findings may be applicable to understand the role of the carboxyl-terminal region in superfamily 4 DNA helicases in general.


Assuntos
Substituição de Aminoácidos , DNA Helicases/química , Proteínas Mitocondriais/química , Mutação , Animais , Bactérias/enzimologia , Bactérias/genética , Bacteriófago T7/enzimologia , Bacteriófago T7/genética , Linhagem Celular , DNA Helicases/genética , DNA Helicases/metabolismo , Drosophila melanogaster , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Estrutura Terciária de Proteína/genética , Homologia de Sequência de Aminoácidos , Spodoptera
10.
J Biol Chem ; 283(18): 12333-42, 2008 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-18308726

RESUMO

Characterization of the basal transcription machinery of mitochondrial DNA (mtDNA) is critical to understand mitochondrial pathophysiology. In mammalian in vitro systems, mtDNA transcription requires mtRNA polymerase, transcription factor A (TFAM), and either transcription factor B1 (TFB1M) or B2 (TFB2M). We have silenced the expression of TFB2M by RNA interference in Drosophila melanogaster. RNA interference knockdown of TF2BM causes lethality by arrest of larval development. Molecular analysis demonstrates that TF2BM is essential for mtDNA transcription during Drosophila development and is not redundant with TFB1M. The impairment of mtDNA transcription causes a dramatic decrease in oxidative phosphorylation and mitochondrial ATP synthesis in the long-lived larvae, and a metabolic shift to glycolysis, which partially restores ATP levels and elicits a compensatory response at the nuclear level that increases mitochondrial mass. At the cellular level, the mitochondrial dysfunction induced by TFB2M knockdown causes a severe reduction in cell proliferation without affecting cell growth, and increases the level of apoptosis. In contrast, cell differentiation and morphogenesis are largely unaffected. Our data demonstrate the essential role of TFB2M in mtDNA transcription in a multicellular organism, and reveal the complex cellular, biochemical, and molecular responses induced by impairment of oxidative phosphorylation during Drosophila development.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Mitocôndrias/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Apoptose , Padronização Corporal , Peso Corporal , Proliferação de Células , DNA Mitocondrial/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Metabolismo Energético , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Glicólise , Larva/citologia , Larva/crescimento & desenvolvimento , Longevidade , Fosforilação Oxidativa , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Asas de Animais/citologia
11.
J Biol Chem ; 282(13): 9436-9444, 2007 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-17272269

RESUMO

We report the cloning and molecular analysis of Drosophila mitochondrial DNA helicase (d-mtDNA helicase) homologous to human TWINKLE, which encodes one of the genes responsible for autosomal dominant progressive external ophthalmoplegia. An RNA interference construct was designed that reduces expression of d-mtDNA helicase to an undetectable level in Schneider cells. RNA interference knockdown of d-mtDNA helicase decreases the copy number of mitochondrial DNA (mtDNA) approximately 5-fold. In a corollary manner, overexpression of d-mtDNA helicase increases mtDNA levels 1.4-fold. Overexpression of helicase active site mutants K388A and D483A results in a severe depletion of mtDNA and a dominant negative lethal phenotype. Overexpression of mutants analogous to human autosomal dominant progressive external ophthalmoplegia mutations shows differential effects. Overexpression of I334T and A442P mutants yields a dominant negative effect as for the active site mutants. In contrast, overexpression of A326T, R341Q, and W441C mutants results in increased mtDNA copy number, as observed with wild-type overexpression. Our dominant negative analysis of d-mtDNA helicase in cultured cells provides a tractable model for understanding human autosomal dominant progressive external ophthalmoplegia mutations.


Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Genes Dominantes , Mitocôndrias/enzimologia , Oftalmoplegia Externa Progressiva Crônica/genética , Fenótipo , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Sítios de Ligação/genética , Linhagem Celular Tumoral , DNA Helicases/biossíntese , DNA Mitocondrial/biossíntese , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Humanos , Mitocôndrias/genética , Dados de Sequência Molecular , Oftalmoplegia Externa Progressiva Crônica/enzimologia , Interferência de RNA
12.
J Biol Chem ; 278(33): 31149-58, 2003 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-12759347

RESUMO

Nuclear and mitochondrial (mt) forms of chicken mt transcription factor A (c-TFAM) generated by alternative splicing of a gene (c-tfam) were cloned. c-tfam mapped at 6q1.1-q1.2 has similar exon/intron organization as mouse tfam except that the first exons encoding the nuclear and the mt form-specific sequences were positioned oppositely. When cDNA encoding the nuclear form was transiently expressed in chicken lymphoma DT40 cells after tagging at the C terminus with c-Myc, the product was localized into nucleus, whereas the only endogenous mt form of DT40 cells was immunostained exclusively within mitochondria. c-TFAM is most similar to Xenopus (xl-) TFAM in having extended C-terminal regions in addition to two high mobility group (HMG) boxes, a linker region between them, and a C-terminal tail, also found in human and mouse TFAM. Similarities between c- and xl-TFAM are higher in linker and C-terminal regions than in HMG boxes. Disruption of both tfam alleles in DT40 cells prevented proliferation. The tfam+/tfam- cells showed a 50 and 40-60% reduction of mtDNA and its transcripts, respectively. Expression of exogenous wild type c-tfam cDNA in the tfam+/tfam- cells increased mtDNA up to 4-fold in a dose-dependent manner, whereas its transcripts increased only marginally. A deletion mutant lacking the first HMG box lost this activity, whereas only marginal reduction of the activity was observed in a deletion mutant at the second HMG box. Despite the essential role of the C-terminal tail in mtDNA transcription demonstrated in vitro, deletion of c-TFAM at this region reduced the activity of maintenance of the mtDNA level only by 50%. A series of deletion mutant at the tail region suggested stimulatory and suppressive sequences in this region for the maintenance of mtDNA level.


Assuntos
DNA Mitocondrial/genética , Proteínas de Ligação a DNA , Dosagem de Genes , Proteínas de Grupo de Alta Mobilidade , Proteínas Mitocondriais , Proteínas Nucleares , Transativadores , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteínas de Xenopus , Sequência de Aminoácidos , Animais , Galinhas , Clonagem Molecular , DNA Complementar , Expressão Gênica , Humanos , Linfoma , Camundongos , Dados de Sequência Molecular , Mutagênese , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Células Tumorais Cultivadas , Xenopus laevis
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA