RESUMO
The human ATP synthase is an assembly of 29 subunits of 18 different types, of which only two (a and 8) are encoded in the mitochondrial genome. Subunit a, together with an oligomeric ring of c-subunit (c-ring), forms the proton pathway responsible for the transport of protons through the mitochondrial inner membrane, coupled to rotation of the c-ring and ATP synthesis. Neuromuscular diseases have been associated to a number of mutations in the gene encoding subunit a, ATP6. The most common, m.8993 T > G, leads to replacement of a strictly conserved leucine residue with arginine (aL156R). We previously showed that the equivalent mutation (aL173R) dramatically compromises respiratory growth of Saccharomyces cerevisiae and causes a 90% drop in the rate of mitochondrial ATP synthesis. Here, we isolated revertants from the aL173R strain that show improved respiratory growth. Four first-site reversions at codon 173 (aL173M, aL173S, aL173K and aL173W) and five second-site reversions at another codon (aR169M, aR169S, aA170P, aA170G and aI216S) were identified. Based on the atomic structures of yeast ATP synthase and the biochemical properties of the revertant strains, we propose that the aL173R mutation is responsible for unfavorable electrostatic interactions that prevent the release of protons from the c-ring into a channel from which protons move from the c-ring to the mitochondrial matrix. The results provide further evidence that yeast aL173 (and thus human aL156) optimizes the exit of protons from ATP synthase, but is not essential despite its strict evolutionary conservation.
Assuntos
Mitocôndrias/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Subunidades Proteicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Complexos de ATP Sintetase/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , DNA Mitocondrial , Genes Mitocondriais , Humanos , Modelos Moleculares , Mutação , Domínios Proteicos , Subunidades Proteicas/metabolismo , PrótonsRESUMO
The m.8993T>G mutation of the mitochondrial MT-ATP6 gene has been associated with numerous cases of neuropathy, ataxia and retinitis pigmentosa and maternally inherited Leigh syndrome, which are diseases known to result from abnormalities affecting mitochondrial energy transduction. We previously reported that an equivalent point mutation severely compromised proton transport through the ATP synthase membrane domain (FO) in Saccharomyces cerevisiae and reduced the content of cytochrome c oxidase (Complex IV or COX) by 80%. Herein, we report that overexpression of the mitochondrial oxodicarboxylate carrier (Odc1p) considerably increases Complex IV abundance and tricarboxylic acid-mediated substrate-level phosphorylation of ADP coupled to conversion of α-ketoglutarate into succinate in m.8993T>G yeast. Consistently in m.8993T>G yeast cells, the retrograde signaling pathway was found to be strongly induced in order to preserve α-ketoglutarate production; when Odc1p was overexpressed, this stress pathway returned to an almost basal activity. Similar beneficial effects were induced by a partial uncoupling of the mitochondrial membrane with the proton ionophore, cyanide m-chlorophenyl hydrazone. This chemical considerably improved the glutamine-based, respiration-dependent growth of human cytoplasmic hybrid cells that are homoplasmic for the m.8993T>G mutation. These findings shed light on the interdependence between ATP synthase and Complex IV biogenesis, which could lay the groundwork for the creation of nutritional or metabolic interventions for attenuating the effects of mtDNA mutations.
Assuntos
Mitocôndrias/metabolismo , Miopatias Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Retinose Pigmentar/metabolismo , Trifosfato de Adenosina/metabolismo , Ataxia/genética , Deficiência de Citocromo-c Oxidase/genética , DNA Mitocondrial/genética , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ácidos Dicarboxílicos/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Transporte de Íons , Doença de Leigh , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mutação , Retinose Pigmentar/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Probing the pathogenicity and functional consequences of mitochondrial DNA (mtDNA) mutations from patient's cells and tissues is difficult due to genetic heteroplasmy (co-existence of wild type and mutated mtDNA in cells), occurrence of numerous mtDNA polymorphisms, and absence of methods for genetically transforming human mitochondria. Owing to its good fermenting capacity that enables survival to loss-of-function mtDNA mutations, its amenability to mitochondrial genome manipulation, and lack of heteroplasmy, Saccharomyces cerevisiae is an excellent model for studying and resolving the molecular bases of human diseases linked to mtDNA in a controlled genetic background. Using this model, we previously showed that a pathogenic mutation in mitochondrial ATP6 gene (m.9191T>C), that converts a highly conserved leucine residue into proline in human ATP synthase subunit a (aL222P), severely compromises the assembly of yeast ATP synthase and reduces by 90% the rate of mitochondrial ATP synthesis. Herein, we report the isolation of intragenic suppressors of this mutation. In light of recently described high resolution structures of ATP synthase, the results indicate that the m.9191T>C mutation disrupts a four α-helix bundle in subunit a and that the leucine residue it targets indirectly optimizes proton conduction through the membrane domain of ATP synthase.
Assuntos
DNA Mitocondrial/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Potencial da Membrana Mitocondrial/genética , ATPases Mitocondriais Próton-Translocadoras/química , Modelos Químicos , Mutação , Fosforilação Oxidativa , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Sudden unexpected death in infancy occurs in apparently healthy infants and remains largely unexplained despite thorough investigation. The vast majority of cases are sporadic. Here we report seven individuals from three families affected by sudden and unexpected cardiac arrest between 4 and 20 months of age. Whole-exome sequencing revealed compound heterozygous missense mutations in PPA2 in affected infants of each family. PPA2 encodes the mitochondrial pyrophosphatase, which hydrolyzes inorganic pyrophosphate into two phosphates. This is an essential activity for many biosynthetic reactions and for energy metabolism of the cell. We show that deletion of the orthologous gene in yeast (ppa2Δ) compromises cell viability due to the loss of mitochondria. Expression of wild-type human PPA2, but not PPA2 containing the mutations identified in affected individuals, preserves mitochondrial function in ppa2Δ yeast. Using a regulatable (doxycycline-repressible) gene expression system, we found that the pathogenic PPA2 mutations rapidly inactivate the mitochondrial energy transducing system and prevent the maintenance of a sufficient electrical potential across the inner membrane, which explains the subsequent disappearance of mitochondria from the mutant yeast cells. Altogether these data demonstrate that PPA2 is an essential gene in yeast and that biallelic mutations in PPA2 cause a mitochondrial disease leading to sudden cardiac arrest in infants.
Assuntos
Alelos , Morte Súbita Cardíaca/etiologia , Pirofosfatase Inorgânica/genética , Proteínas Mitocondriais/genética , Mutação/genética , Morte Súbita Cardíaca/patologia , Difosfatos , Exoma/genética , Feminino , Deleção de Genes , Genes Essenciais/genética , Teste de Complementação Genética , Heterozigoto , Humanos , Lactente , Pirofosfatase Inorgânica/metabolismo , Masculino , Potencial da Membrana Mitocondrial/genética , Viabilidade Microbiana , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Mutação de Sentido Incorreto , Bombas de Próton/deficiência , Bombas de Próton/genética , Bombas de Próton/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The ATP synthase which provides aerobic eukaryotes with ATP, organizes into a membrane-extrinsic catalytic domain, where ATP is generated, and a membrane-embedded FO domain that shuttles protons across the membrane. We previously identified a mutation in the mitochondrial MT-ATP6 gene (m.8969G>A) in a 14-year-old Chinese female who developed an isolated nephropathy followed by brain and muscle problems. This mutation replaces a highly conserved serine residue into asparagine at amino acid position 148 of the membrane-embedded subunit a of ATP synthase. We showed that an equivalent of this mutation in yeast (aS175N) prevents FO-mediated proton translocation. Herein we identified four first-site intragenic suppressors (aN175D, aN175K, aN175I, and aN175T), which, in light of a recently published atomic structure of yeast FO indicates that the detrimental consequences of the original mutation result from the establishment of hydrogen bonds between aN175 and a nearby glutamate residue (aE172) that was proposed to be critical for the exit of protons from the ATP synthase towards the mitochondrial matrix. Interestingly also, we found that the aS175N mutation can be suppressed by second-site suppressors (aP12S, aI171F, aI171N, aI239F, and aI200M), of which some are very distantly located (by 20-30â¯Å) from the original mutation. The possibility to compensate through long-range effects the aS175N mutation is an interesting observation that holds promise for the development of therapeutic molecules.
Assuntos
DNA Mitocondrial/genética , Mitocôndrias/patologia , Doenças Mitocondriais/patologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mutação , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Mitocôndrias/metabolismo , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/genética , Conformação Proteica , Subunidades Proteicas , Prótons , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de SequênciaRESUMO
Only a few genes remain in the mitochondrial genome retained by every eukaryotic organism that carry out essential functions and are implicated in severe diseases. Experimentally relocating these few genes to the nucleus therefore has both therapeutic and evolutionary implications. Numerous unproductive attempts have been made to do so, with a total of only 5 successes across all organisms. We have taken a novel approach to relocating mitochondrial genes that utilizes naturally nuclear versions from other organisms. We demonstrate this approach on subunit 9/c of ATP synthase, successfully relocating this gene for the first time in any organism by expressing the ATP9 genes from Podospora anserina in Saccharomyces cerevisiae. This study substantiates the role of protein structure in mitochondrial gene transfer: expression of chimeric constructs reveals that the P. anserina proteins can be correctly imported into mitochondria due to reduced hydrophobicity of the first transmembrane segment. Nuclear expression of ATP9, while permitting almost fully functional oxidative phosphorylation, perturbs many cellular properties, including cellular morphology, and activates the heat shock response. Altogether, our study establishes a novel strategy for allotopic expression of mitochondrial genes, demonstrates the complex adaptations required to relocate ATP9, and indicates a reason that this gene was only transferred to the nucleus during the evolution of multicellular organisms.
Assuntos
Núcleo Celular/genética , Proteínas Fúngicas/genética , Mitocôndrias/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Podospora/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Evolução Biológica , Núcleo Celular/enzimologia , Proteínas Fúngicas/metabolismo , Deleção de Genes , Genes Mitocondriais , Genoma Mitocondrial , Mitocôndrias/enzimologia , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fosforilação Oxidativa , Podospora/enzimologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , TransgenesRESUMO
ABSTRACT: Diffuse midline glioma, H3K27-altered, is a relatively new entity, characterized by H3K27M histone mutation. This rare pediatric disease with severe prognosis has recently been identified as a new subtype of diffuse astrocytoma due to major breakthrough in histopathological and molecular characterization of gliomas. We report a case of H3K27-altered diffuse midline glioma in a 30-year-old woman.
Assuntos
Fluordesoxiglucose F18 , Glioma , Histonas , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Medula Espinal , Humanos , Feminino , Adulto , Glioma/diagnóstico por imagem , Glioma/patologia , Medula Espinal/diagnóstico por imagem , Medula Espinal/patologia , Histonas/metabolismo , Tomografia Computadorizada por Raios X , Imagem MultimodalRESUMO
The purpose of this study was to determine whether texture analysis features present on pretreatment unenhanced computed tomography (CT) images, derived from 18F-fluorodeoxyglucose positron emission/computed tomography (18-FDG PET/CT), can predict progression-free survival (PFS), progression-free survival at 24 months (PFS 24), time to next treatment (TTNT), and overall survival in patients with high-tumor-burden follicular lymphoma treated with immunochemotherapy and rituximab maintenance. Seventy-two patients with follicular lymphoma were retrospectively included. Texture analysis was performed on unenhanced CT images extracted from 18-FDG PET/CT examinations that were obtained within one month before treatment. Skewness at a fine texture scale (SSF = 2) was an independent predictor of PFS (hazard ratio = 3.72 (95% CI: 1.15, 12.11), p = 0.028), PFS 24 (hazard ratio = 13.38; 95% CI: 1.29, 138.13; p = 0.029), and TTNT (hazard ratio = 5.11; 95% CI: 1.18, 22.13; p = 0.029). Skewness values above -0.015 at SSF = 2 were significantly associated with lower PFS, PFS 24, and TTNT. Kurtosis without filtration was an independent predictor of PFS (SSF = 0; HR = 1.22 (95% CI: 1.04, 1.44), p = 0.013), and TTNT (SSF = 0; hazard ratio = 1.23; 95% CI: 1.04, 1.46; p = 0.013). This study shows that pretreatment unenhanced CT texture analysis-derived tumor skewness and kurtosis may be used as predictive biomarkers of PFS and TTNT in patients with high-tumor-burden follicular lymphoma treated with immunochemotherapy and rituximab maintenance.
RESUMO
Patients (pts) with asymptomatic low-burden follicular lymphoma (FL) are usually observed at diagnosis. Time to lymphoma treatment (TLT) initiation can however be very heterogeneous and risk factors of progression are poorly studied. Our study evaluated 201 pts with grade 1-3a low-tumor burden FL diagnosed in four French centers between 2010 and 2020 and managed by a watch and wait strategy in real-life settings. After a median follow-up of 4.8 years, the median TLT was 4.2 years (95% confidence interval, 3.1-5.5). On multivariate analysis, elevated lactate dehydrogenase (hazard ratio [HR] = 2.2; P = 0.02), more than 4 nodal areas involved (HR = 1.7; P = 0.02) and more than 1 extranodal involvement (HR = 2.7; P = 0.01) were identified as independent predictors of TLT. The median TLT was 5.8 years for pts with no risk factor, 2.4 years for 1 risk factor, and 1.3 years for >1 risk factors (P < 0.01). In a subanalysis of 75 pts staged with positron emission tomography-computed tomography (PET-CT), total metabolic tumor volume (TMTV) ≥14 cm3 and standardized Dmax (reflecting tumor dissemination) >0.32 m-1 were also associated with shorter TLT (HR = 3.4; P = 0.004 and HR = 2.4; P = 0.007, respectively). In multivariate models combining PET-CT parameters and clinical variables, TMTV remained independent predictor of shorter TLT. These simple parameters could help to identify FL patients initially observed at higher risk of early progression. The role of PET-CT (extranodal sites and PET metrics) in low-burden FL appears promising and warrants further assessment in large cohorts.
RESUMO
In yeast, the two main F(O) proton-translocating subunits of the ATP synthase (subunits 6/a and 9/c) are encoded by mitochondrial DNA (mtDNA). Unfortunately, mutations that inactivate the F(O) typically result in loss of mtDNA under the form of ρ(-)/ρ(0) cells. Thus, we have designed a novel genetic strategy to circumvent this problem. It exploits previous findings that a null mutation in the nuclear ATP16 gene encoding ATP synthase subunit δ results in massive and lethal F(O)-mediated protons leaks across the inner mitochondrial membrane. Mutations that inactivate the F(O) can thus, in these conditions, be selected positively as cell viability rescuing events. A first set of seven mutants was analyzed and all showed, as expected, very severe F(O) deficiencies. Two mutants carried nuclear mutations in known genes (AEP1, AEP2) required for subunit c expression. The five other mutations were located in mtDNA. Of these, three affect synthesis or stability of subunit a transcripts and the two last consisted in a single amino acid replacement in subunit c. One of the subunit c mutations is particularly interesting. It consists in an alanine to valine change at position 60 of subunit c adjacent to the essential glutamate of subunit c (at position 59) that interacts with the essential arginine 186 of subunit a. The properties of this mutant suggest that the contact zone between subunit a and the ten subunits c-ring structure only involves critical transient interactions confined to the region where protons are exchanged between the subunit a and the c-ring.
Assuntos
Mutação , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Domínio Catalítico , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , ATPases Translocadoras de Prótons/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
ABSTRACT: The purpose of this study was to investigate the value of the "cerebellum/ liver index for prognosis" (CLIP) as a new prognostic marker in pretherapeutic 18F-Fluorodeoxyglucose positron emission tomography (18F-FDG PET) in patients with follicular lymphoma treated by immunochemotherapy and rituximab maintenance, focusing on progression-free survival (PFS).Clinicobiological and imaging data from patients with follicular lymphoma between March 2010 and September 2015 were retrospectively collected and 5-year PFS was determined. The conventional PET parameters (maximum standardized uptake value and total metabolic tumor volume) and the CLIP, corresponding to the ratio of the cerebellum maximum standardized uptake value over the liver SUVmean, were extracted from the pretherapeutic 18F-FDG PET.Forty-six patients were included. Eighteen patients (39%) progressed within the 5âyears after treatment initiation. Five-year PFS was 78.6% when CLIP was >4.0 and 42.0% when CLIP was <4.0 (Pâ=â.04). CLIP was a significant predictor of PFS on univariate analysis (hazard ratio 3.1, Pâ=â.049) and was near-significant on multivariate analysis (hazard ratio 2.8, Pâ=â.07) with ECOG PS as a cofactor.The CLIP derived from pretherapeutic 18F-FDG PET seems to be an interesting predictive marker of PFS in follicular lymphoma treated by immunochemotherapy and rituximab maintenance. These results should be evaluated prospectively in a larger cohort.
Assuntos
Cerebelo , Imunoterapia , Fígado , Linfoma Folicular , Rituximab , Biomarcadores , Cerebelo/diagnóstico por imagem , Fluordesoxiglucose F18 , Humanos , Fígado/diagnóstico por imagem , Linfoma Folicular/diagnóstico por imagem , Linfoma Folicular/tratamento farmacológico , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Prognóstico , Intervalo Livre de Progressão , Compostos Radiofarmacêuticos , Estudos Retrospectivos , Rituximab/uso terapêuticoRESUMO
Erdheim-Chester disease (ECD) is a rare condition with underestimated neurological involvement. Mild psychiatric symptoms such as mood swings have been rarely described in the clinical spectrum of neuro-ECD. We here describe the first patient with psychiatric manifestations of delirium revealing ECD with neurological involvement with favorable evolution under interferon followed by BRAF inhibitor monotherapy. An 81-year-old woman was referred to the hospital because of delirium and severe cognitive impairment associated with a cerebellar syndrome. Brain magnetic resonance imaging showed "FLAIR-changes" lesions in the pons and upper cerebellum peduncles. Blood and cerebrospinal fluid (CSF) analyses showed normal results except for an elevated neopterin level in the CSF. Whole-body CT scan (18FDG-PET) showed peri-nephric fat infiltration and aorta adventitia sheathing with radiotracer uptake in the pons, vessels, peri-nephric fat, and bone lesions, which was characteristic of ECD. The diagnosis was confirmed on perirenal tissue biopsy, which also showed a BRAFV600E mutation. Treatment with interferon resulted in the resolution of delirium, and treatment with BRAF inhibitor subsequently resulted in a partial remission of all active sites. This case highlights that delirium can be the first manifestation of neurodegenerative ECD. ECD should be screened in unexplained psychiatric features as interferon and targeted therapy appear to be effective in this situation.
Assuntos
Delírio , Doença de Erdheim-Chester , Idoso de 80 Anos ou mais , Antivirais/uso terapêutico , Doença de Erdheim-Chester/diagnóstico , Doença de Erdheim-Chester/tratamento farmacológico , Doença de Erdheim-Chester/genética , Feminino , Humanos , Interferons/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética , Vemurafenib/uso terapêuticoRESUMO
Numerous diseases in humans have been associated with mutations of the mitochondrial genome (mtDNA). This genome encodes 13 protein subunits of complexes involved in oxidative phosphorylation (OXPHOS), a process that provides aerobic eukaryotes with the energy-rich adenosine triphosphate molecule (ATP). Mutations of the mtDNA may therefore have dramatic consequences especially in tissues and organs with high energy demand. Evaluating the pathogenicity of these mutations may be difficult because they often affect only a fraction of the numerous copies of the mitochondrial genome (up to several thousands in a single cell), which is referred to as heteroplasmy. Furthermore, due to its exposure to reactive oxygen species (ROS) produced in mitochondria, the mtDNA is prone to mutations, and some may be simply neutral polymorphisms with no detrimental consequences on human health. Another difficulty is the absence of methods for genetically transforming human mitochondria. Face to these complexities, the yeast Saccharomyces cerevisiae provides a convenient model for investigating the consequences of human mtDNA mutations in a defined genetic background. Owing to its good fermentation capacity, it can survive the loss of OXPHOS, its mitochondrial genome can be manipulated, and genetic heterogeneity in its mitochondria is unstable. Taking advantage of these unique attributes, we herein describe a method we have developed for creating yeast models of mitochondrial ATP6 gene mutations detected in patients, to determine how they impact OXPHOS. Additionally, we describe how these models can be used to discover molecules with therapeutic potential.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Genes Mitocondriais , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , VirulênciaRESUMO
The yeast mitochondrial ATP synthase is an assembly of 28 subunits of 17 types of which 3 (subunits 6, 8, and 9) are encoded by mitochondrial genes, while the 14 others have a nuclear genetic origin. Within the membrane domain (FO) of this enzyme, the subunit 6 and a ring of 10 identical subunits 9 transport protons across the mitochondrial inner membrane coupled to ATP synthesis in the extra-membrane structure (F1) of ATP synthase. As a result of their dual genetic origin, the ATP synthase subunits are synthesized in the cytosol and inside the mitochondrion. How they are produced in the proper stoichiometry from two different cellular compartments is still poorly understood. The experiments herein reported show that the rate of translation of the subunits 9 and 6 is enhanced in strains with mutations leading to specific defects in the assembly of these proteins. These translation modifications involve assembly intermediates interacting with subunits 6 and 9 within the final enzyme and cis-regulatory sequences that control gene expression in the organelle. In addition to enabling a balanced output of the ATP synthase subunits, these assembly-dependent feedback loops are presumably important to limit the accumulation of harmful assembly intermediates that have the potential to dissipate the mitochondrial membrane electrical potential and the main source of chemical energy of the cell.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Trifosfato de Adenosina/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
MADS-domain transcription factors play pivotal roles in various developmental processes. The lack of simple loss-of-function phenotypes provides impediments to understand the biological function of some of the MADS-box transcription factors. Here we have characterized the potential role of the Arabidopsis thaliana AGAMOUS-LIKE6 (AGL6) gene by fusing full-length coding sequence with transcriptional activator and repressor domains and suggest a role for AGL6 in lateral organ development and flowering. Upon photoperiodic induction of flowering, AGL6 becomes expressed in abaxial and proximal regions of cauline leaf primordia, as well as the cryptic bracts subtending flowers. In developing flowers, AGL6 is detected in the proximal regions of all floral organs and in developing ovules. Converting AGL6 into a strong activator through fusion to the VP16 domain triggers bract outgrowth, implicating AGL6 in the development of bractless flowers in Arabidopsis. In addition, ectopic reproductive structures form on both bracts and flowers in gAGL6::VP16 transgenic plants, which is dependent on B and C class homeotic genes, but independent of LEAFY. Overexpression of both AGL6 and its transcriptional repressor form, AGL6::EAR, causes precocious flowering and terminal flower formation, suggesting that AGL6 suppresses the function of a floral repressor.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Flores/crescimento & desenvolvimento , Proteínas de Domínio MADS/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Domínio MADS/genética , Microscopia Eletrônica de Varredura , Óvulo Vegetal/crescimento & desenvolvimento , Óvulo Vegetal/ultraestrutura , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , RNA de Plantas/genéticaRESUMO
ABSTRACT: We report the case of a 21-year-old man referred to our institution for the initial staging of an osteoblastic osteosarcoma of the right femur. An 18F-NaF PET/CT demonstrated millimetric pleuroparenchymal metastases, later confirmed on follow-up. These lesions were not reported on both dedicated chest CT and 18F-FDG PET/CT.
Assuntos
Neoplasias Ósseas/diagnóstico por imagem , Osteossarcoma/diagnóstico por imagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Radiografia Torácica , Fluoreto de Sódio , Neoplasias Ósseas/patologia , Radioisótopos de Flúor , Humanos , Masculino , Metástase Neoplásica , Osteossarcoma/patologia , Adulto JovemRESUMO
Demand for high-performance biocomposites is increasing due to their ease of processing, low environmental impact, and in-service performance. This study investigated the effect of boric acid modification of wood flour on polycarbonate (PC) wood composites' thermal stability, fire retardancy, water absorption, and creep behavior. The composites' fire retardancy increased with increasing wood flour content, and their char residue increased by 102.3% compared to that of pure PC. However, the water absorption of the resulting composites increased due to the hydroxyl groups of the wood flour. Wood flour also improved the composites' anti-creep properties. The excellent fire retardancy and anti-creep properties of wood-PC composites expand their use in the construction sector.
RESUMO
OBJECTIVE: 18F-FDG PET/CT provides valuable informations regarding the prognosis of DLBCL. The aim of this study is to test a novel index based on cerebellar uptake to predict progression free survival in DLBCL patients. METHODS: Data from patients with de novo DLBCL between January 2011 and December 2018 were retrospectively collected and PFS was determined. The conventional PET parameters (SUVmax and total metabolic tumor volume) and the CLIP, corresponding to the ratio of the cerebellum SUVmax over the liver SUVmean, were extracted from baseline 18F-FDG PET. RESULTS: Ninety-five patients were included. When using a threshold of 3.24, CLIP was a significant predictor of PFS on univariate analysis (HR 3.4, p < 0.001) with different 5-year survival rates: 68% (CLIP ≥ 3.24) versus 32% (CLIP < 3.24). Multivariate analysis confirmed the prognostic value of CLIP, as it is one of the two factors remaining significant with ß2-microglobulin (HR 2.1 and 2.5 respectively, p = 0.04 and p = 0.03). A score associating ß2-microglobulin and CLIP allowed to separate the population into three groups of different outcome in terms of 5-year PFS: low risk (80%), intermediate risk (42%) and high risk (17%). CONCLUSIONS: The CLIP derived from pre-therapeutic 18F-FDG PET seems to be an interesting predictive marker of PFS in DLBCL treated by immunochemotherapy.
Assuntos
Linfoma Difuso de Grandes Células B , Adulto , Fluordesoxiglucose F18 , Humanos , Masculino , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Intervalo Livre de ProgressãoRESUMO
We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc(1) complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036-36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8(m)). Subsequently replacing ARG8(m) with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit(-)). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.
Assuntos
Citocromos b/genética , Citocromos b/metabolismo , Mutagênese , Mutação/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Meios de Cultura , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fermentação , Genes Fúngicos , Vetores Genéticos , Íntrons/genética , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transaminases/metabolismoRESUMO
Protons are transported from the mitochondrial matrix to the intermembrane space of mitochondria during the transfer of electrons to oxygen and shuttled back to the matrix by the a subunit and a ring of identical c subunits across the membrane domain (FO) of ATP synthase, which is coupled to ATP synthesis. A mutation (m.9176â¯Tâ¯>â¯G) of the mitochondrial ATP6 gene that replaces an universally conserved leucine residue into arginine at amino acid position 217 of human subunit a (aL217R) has been associated to NARP (Neuropathy, Ataxia and Retinitis Pigmentosa) and MILS (Maternally Inherited Leigh's Syndrome) diseases. We previously showed that an equivalent thereof in Saccharomyces cerevisiae (aL237R) severely impairs subunit a assembly/stability and decreases by >90% the rate of mitochondrial ATP synthesis. Herein we identified three spontaneous first-site intragenic suppressors (aR237M, aR237T and aR237S) that fully restore ATP synthase assembly. However, mitochondrial ATP synthesis rate was only partially recovered (40-50% vs wild type yeast). In light of recently described high-resolution yeast ATP synthase structures, the detrimental consequences of the aL237R change can be explained by steric and electrostatic hindrance with the universally conserved subunit a arginine residue (aR176) that is essential to FO activity. aL237 together with three other nearby hydrophobic residues have been proposed to prevent ion shortage between two physically separated hydrophilic pockets within the FO. Our results suggest that aL237 favors subunit c-ring rotation by optimizing electrostatic interaction between aR176 and an acidic residue in subunit c (cE59) known to be essential also to the activity of FO.