RESUMEN
The human mitochondrial DNA polymerase gamma is a holoenzyme, involved in mitochondrial DNA (mtDNA) replication and maintenance, composed of a catalytic subunit (POLG) and a dimeric accessory subunit (POLG2) conferring processivity. Mutations in POLG or POLG2 cause POLG-related diseases in humans, leading to a subset of Mendelian-inherited mitochondrial disorders characterized by mtDNA depletion (MDD) or accumulation of multiple deletions, presenting multi-organ defects and often leading to premature death at a young age. Considering the paucity of POLG2 models, we have generated a stable zebrafish polg2 mutant line (polg2ia304) by CRISPR/Cas9 technology, carrying a 10-nucleotide deletion with frameshift mutation and premature stop codon. Zebrafish polg2 homozygous mutants present slower development and decreased viability compared to wild type siblings, dying before the juvenile stage. Mutants display a set of POLG-related phenotypes comparable to the symptoms of human patients affected by POLG-related diseases, including remarkable MDD, altered mitochondrial network and dynamics, and reduced mitochondrial respiration. Histological analyses detected morphological alterations in high-energy demanding tissues, along with a significant disorganization of skeletal muscle fibres. Consistent with the last finding, locomotor assays highlighted a decreased larval motility. Of note, treatment with the Clofilium tosylate drug, previously shown to be effective in POLG models, could partially rescue MDD in Polg2 mutant animals. Altogether, our results point at zebrafish as an effective model to study the etiopathology of human POLG-related disorders linked to POLG2, and a suitable platform to screen the efficacy of POLG-directed drugs in POLG2-associated forms.
Asunto(s)
ADN Polimerasa Dirigida por ADN , Enfermedades Mitocondriales , Animales , Humanos , ADN Polimerasa Dirigida por ADN/genética , Pez Cebra/genética , ADN Polimerasa gamma/genética , ADN Mitocondrial/genética , Mitocondrias/genética , Mitocondrias/patología , Mutación/genética , Enfermedades Mitocondriales/tratamiento farmacológico , Enfermedades Mitocondriales/genéticaRESUMEN
Mitochondrial DNA depletion syndromes (MDS) are clinically heterogenous and often severe diseases, characterized by a reduction of the number of copies of mitochondrial DNA (mtDNA) in affected tissues. In the context of MDS, yeast has proved to be both an excellent model for the study of the mechanisms underlying mitochondrial pathologies and for the discovery of new therapies via high-throughput assays. Among the several genes involved in MDS, it has been shown that recessive mutations in MPV17 cause a hepatocerebral form of MDS and Navajo neurohepatopathy. MPV17 encodes a non selective channel in the inner mitochondrial membrane, but its physiological role and the nature of its cargo remains elusive. In this study we identify ten drugs active against MPV17 disorder, modelled in yeast using the homologous gene SYM1. All ten of the identified molecules cause a concomitant increase of both the mitochondrial deoxyribonucleoside triphosphate (mtdNTP) pool and mtDNA stability, which suggests that the reduced availability of DNA synthesis precursors is the cause for the mtDNA deletion and depletion associated with Sym1 deficiency. We finally evaluated the effect of these molecules on mtDNA stability in two other MDS yeast models, extending the potential use of these drugs to a wider range of MDS patients.
Asunto(s)
ADN de Hongos , ADN Mitocondrial , Trastornos Heredodegenerativos del Sistema Nervioso , Hepatopatías , Proteínas de la Membrana , Mitocondrias , Enfermedades Mitocondriales , Proteínas Mitocondriales , Enfermedades del Sistema Nervioso Periférico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , ADN de Hongos/genética , ADN de Hongos/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Trastornos Heredodegenerativos del Sistema Nervioso/genética , Trastornos Heredodegenerativos del Sistema Nervioso/metabolismo , Trastornos Heredodegenerativos del Sistema Nervioso/terapia , Humanos , Hepatopatías/genética , Hepatopatías/metabolismo , Hepatopatías/terapia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/terapia , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Nucleótidos/genética , Nucleótidos/metabolismo , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/metabolismo , Enfermedades del Sistema Nervioso Periférico/terapia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , SíndromeRESUMEN
Mitochondrial diseases result from inherited or spontaneous mutations in mitochondrial or nuclear DNA, leading to an impairment of the oxidative phosphorylation responsible for the synthesis of ATP. To date, there are no effective pharmacological therapies for these pathologies. We performed a yeast-based screening to search for therapeutic drugs to be used for treating mitochondrial diseases associated with dominant mutations in the nuclear ANT1 gene, which encodes for the mitochondrial ADP/ATP carrier. Dominant ANT1 mutations are involved in several degenerative mitochondrial pathologies characterized by the presence of multiple deletions or depletion of mitochondrial DNA in tissues of affected patients. Thanks to the presence in yeast of the AAC2 gene, orthologue of human ANT1, a yeast mutant strain carrying the M114P substitution equivalent to adPEO-associated L98P mutation was created. Five molecules were identified for their ability to suppress the defective respiratory growth phenotype of the haploid aac2M114P. Furthermore, these molecules rescued the mtDNA mutability in the heteroallelic AAC2/aac2M114P strain, which mimics the human heterozygous condition of adPEO patients. The drugs were effective in reducing mtDNA instability also in the heteroallelic strain carrying the R96H mutation equivalent to the more severe de novo dominant missense mutation R80H, suggesting a general therapeutic effect on diseases associated with dominant ANT1 mutations.
Asunto(s)
Translocador 1 del Nucleótido Adenina/genética , Ensayos Analíticos de Alto Rendimiento/métodos , Translocasas Mitocondriales de ADP y ATP/genética , Enfermedades Mitocondriales/tratamiento farmacológico , Mutación , Preparaciones Farmacéuticas/administración & dosificación , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , ADN Mitocondrial/genética , Genes Dominantes , Humanos , Enfermedades Mitocondriales/genética , Oftalmoplejía/tratamiento farmacológico , Oftalmoplejía/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genéticaRESUMEN
The increasing application of next generation sequencing approaches to the analysis of human exome and whole genome data has enabled the identification of novel variants and new genes involved in mitochondrial diseases. The ability of surviving in the absence of oxidative phosphorylation (OXPHOS) and mitochondrial genome makes the yeast Saccharomyces cerevisiae an excellent model system for investigating the role of these new variants in mitochondrial-related conditions and dissecting the molecular mechanisms associated with these diseases. The aim of this review was to highlight the main advantages offered by this model for the study of mitochondrial diseases, from the validation and characterisation of novel mutations to the dissection of the role played by genes in mitochondrial functionality and the discovery of potential therapeutic molecules. The review also provides a summary of the main contributions to the understanding of mitochondrial diseases emerged from the study of this simple eukaryotic organism.
Asunto(s)
Predisposición Genética a la Enfermedad , Enfermedades Mitocondriales/genética , Modelos Biológicos , Mutación , Levaduras/genética , Núcleo Celular/genética , ADN Mitocondrial , Perfilación de la Expresión Génica , Genes Mitocondriales , Variación Genética , Humanos , Mitocondrias/genética , Enfermedades Mitocondriales/metabolismo , Fosforilación Oxidativa , Levaduras/metabolismoRESUMEN
The DNA polymerase gamma (Polg) is a nuclear-encoded enzyme involved in DNA replication in animal mitochondria. In humans, mutations in the POLG gene underlie a set of mitochondrial diseases characterized by mitochondrial DNA (mtDNA) depletion or deletion and multiorgan defects, named POLG disorders, for which an effective therapy is still needed. By applying antisense strategies, ENU- and CRISPR/Cas9-based mutagenesis, we have generated embryonic, larval-lethal and adult-viable zebrafish Polg models. Morphological and functional characterizations detected a set of phenotypes remarkably associated to POLG disorders, including cardiac, skeletal muscle, hepatic and gonadal defects, as well as mitochondrial dysfunctions and, notably, a perturbed mitochondria-to-nucleus retrograde signaling (CREB and Hypoxia pathways). Next, taking advantage of preliminary evidence on the candidate molecule Clofilium tosylate (CLO), we tested CLO toxicity and then its efficacy in our zebrafish lines. Interestingly, at well tolerated doses, the CLO drug could successfully rescue mtDNA and Complex I respiratory activity to normal levels, even in mutant phenotypes worsened by treatment with Ethidium Bromide. In addition, the CLO drug could efficiently restore cardio-skeletal parameters and mitochondrial mass back to normal values. Altogether, these evidences point to zebrafish as a valuable vertebrate organism to faithfully phenocopy multiple defects detected in POLG patients. Moreover, this model represents an excellent platform to screen, at the whole-animal level, candidate molecules with therapeutic effects in POLG disorders.
Asunto(s)
Enfermedades Mitocondriales/genética , Compuestos de Amonio Cuaternario/metabolismo , Animales , Modelos Animales de Enfermedad , Fenotipo , Pez CebraRESUMEN
OPA1 mutations are the major cause of dominant optic atrophy (DOA) and the syndromic form DOA plus, pathologies for which there is no established cure. We used a 'drug repurposing' approach to identify FDA-approved molecules able to rescue the mitochondrial dysfunctions induced by OPA1 mutations. We screened two different chemical libraries by using two yeast strains carrying the mgm1I322M and the chim3P646L mutations, identifying 26 drugs able to rescue their oxidative growth phenotype. Six of them, able to reduce the mitochondrial DNA instability in yeast, have been then tested in Opa1 deleted mouse embryonic fibroblasts expressing the human OPA1 isoform 1 bearing the R445H and D603H mutations. Some of these molecules were able to ameliorate the energetic functions and/or the mitochondrial network morphology, depending on the type of OPA1 mutation. The final validation has been performed in patients' fibroblasts, allowing to select the most effective molecules. Our current results are instrumental to rapidly translating the findings of this drug repurposing approach into clinical trial for DOA and other neurodegenerations caused by OPA1 mutations.
Asunto(s)
Reposicionamiento de Medicamentos , GTP Fosfohidrolasas/genética , Enfermedades Neurodegenerativas/tratamiento farmacológico , Atrofia Óptica Autosómica Dominante/tratamiento farmacológico , Animales , ADN Mitocondrial/efectos de los fármacos , Fibroblastos/efectos de los fármacos , GTP Fosfohidrolasas/antagonistas & inhibidores , Humanos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mutación/efectos de los fármacos , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Atrofia Óptica Autosómica Dominante/genética , Atrofia Óptica Autosómica Dominante/patología , Linaje , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genéticaRESUMEN
Thiosemicarbazones (TSC) and their metal complexes display diverse biological activities and are active against multiple pathological conditions ranging from microbial infections to abnormal cell proliferation. Ribonucleotide reductase (RNR) is considered one of the main targets of TSCs, yet, the existence of additional targets, differently responsible for the multifaceted activities of TSCs and their metal complexes has been proposed. To set the basis for a more comprehensive delineation of their mode of action, we chemogenomically profiled the cellular effects of bis(citronellalthiosemicarbazonato)nickel(II) [Ni(S-tcitr)2] using the unicellular eukaryote Saccharomyces cerevisiae as a model organism. Two complementary genomic phenotyping screens led to the identification of 269 sensitive and 56 tolerant deletion mutant strains and of 14 genes that when overexpressed make yeast cells resistant to an otherwise lethal concentration of Ni(S-tcitr)2. Chromatin remodeling, cytoskeleton organization, mitochondrial function and iron metabolism were identified as lead cellular processes responsible for Ni(S-tcitr)2 toxicity. The latter process, and particularly glutaredoxin-mediated iron loading of RNR, was found to be affected by Ni(S-tcitr)2. Given the multiple pathways regulated by glutaredoxins, targeting of these proteins by Ni(S-tcitr)2 can negatively affect various core cellular processes that may critically contribute to Ni(S-tcitr)2 cytotoxicity.
Asunto(s)
Complejos de Coordinación/farmacología , Níquel , Tiosemicarbazonas/farmacología , Línea Celular Tumoral , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Humanos , Hierro/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Saccharomyces cerevisiaeRESUMEN
BACKGROUND: Mutations in human gene encoding the mitochondrial DNA polymerase γ (HsPolγ) are associated with a broad range of mitochondrial diseases. Here we studied the impact on DNA replication by disease variants clustered around residue HsPolγ-K1191, a residue that in several family-A DNA polymerases interacts with the 3' end of the primer. METHODS: Specifically, we examined the effect of HsPolγ carrying pathogenic variants in residues D1184, I1185, C1188, K1191, D1196, and a stop codon at residue T1199, using as a model the yeast mitochondrial DNA polymerase protein, Mip1p. RESULTS: The introduction of pathogenic variants C1188R (yV945R), and of a stop codon at residue T1199 (yT956X) abolished both polymerization and exonucleolysis in vitro. HsPolγ substitutions in residues D1184 (yD941), I1185 (yI942), K1191 (yK948) and D1196 (yD953) shifted the balance between polymerization and exonucleolysis in favor of exonucleolysis. HsPolγ pathogenic variants at residue K1191 (yK948) and D1184 (yD941) were capable of nucleotide incorporation albeit with reduced processivity. Structural analysis of mitochondrial DNAPs showed that residue HsPolγ-N864 is placed in an optimal distance to interact with the 3' end of the primer and the phosphate backbone previous to the 3' end. Amino acid changes in residue HsPolγ-N864 to Ala, Ser or Asp result in enzymes that did not decrease their polymerization activity on short templates but exhibited a substantial decrease for processive DNA synthesis. CONCLUSION: Our data suggest that in mitochondrial DNA polymerases multiple amino acids are involved in the primer-stand stabilization.
Asunto(s)
ADN Polimerasa gamma/genética , ADN Mitocondrial/metabolismo , Enfermedades Mitocondriales/metabolismo , ADN Polimerasa gamma/química , ADN Polimerasa gamma/metabolismo , Replicación del ADN/genética , ADN Mitocondrial/química , Humanos , Modelos Moleculares , MutaciónRESUMEN
Human and yeast mitochondrial DNA polymerases (DNAPs), POLG and Mip1, are related by evolution to bacteriophage DNAPs. However, mitochondrial DNAPs contain unique amino and carboxyl-terminal extensions that physically interact. Here we describe that N-terminal deletions in Mip1 polymerases abolish polymerization and decrease exonucleolytic degradation, whereas moderate C-terminal deletions reduce polymerization. Similarly, to the N-terminal deletions, an extended C-terminal deletion of 298 amino acids is deficient in nucleotide addition and exonucleolytic degradation of double and single-stranded DNA. The latter observation suggests that the physical interaction between the amino and carboxyl-terminal regions of Mip1 may be related to the spread of pathogenic POLG mutant along its primary sequence.
Asunto(s)
ADN Polimerasa I/metabolismo , ADN de Hongos/biosíntesis , ADN Mitocondrial/biosíntesis , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Dominio Catalítico , ADN Polimerasa I/genética , ADN Polimerasa gamma/genética , ADN Polimerasa gamma/metabolismo , ADN de Hongos/genética , ADN Mitocondrial/genética , Humanos , Proteínas Mitocondriales/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Amongst the various approaches to contain aflatoxin contamination of feed and food commodities, the use of inhibitors of fungal growth and/or toxin biosynthesis is showing great promise for the implementation or the replacement of conventional pesticide-based strategies. Several inhibition mechanisms were found taking place at different levels in the biology of the aflatoxin-producing fungal species such as Aspergillus flavus: compounds that influence aflatoxin production may block the biosynthetic pathway through the direct control of genes belonging to the aflatoxin gene cluster, or interfere with one or more of the several steps involved in the aflatoxin metabolism upstream. Recent findings pointed to mitochondrial functionality as one of the potential targets of some aflatoxin inhibitors. Additionally, we have recently reported that the effect of a compound belonging to the class of thiosemicarbazones might be related to the energy generation/carbon flow and redox homeostasis control by the fungal cell. Here, we report our investigation about a putative molecular target of the 3-isopropylbenzaldehyde thiosemicarbazone (mHtcum), using the yeast Saccharomyces cerevisiae as model system, to demonstrate how the compound can actually interfere with the mitochondrial respiratory chain.
Asunto(s)
Aflatoxinas/antagonistas & inhibidores , Antifúngicos/farmacología , Regulación Fúngica de la Expresión Génica , Mitocondrias/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , Tiosemicarbazonas/farmacología , Aflatoxinas/biosíntesis , Antifúngicos/química , Aspergillus flavus/efectos de los fármacos , Aspergillus flavus/enzimología , Aspergillus flavus/genética , Sitios de Unión , Transporte de Electrón/efectos de los fármacos , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Complejo III de Transporte de Electrones/química , Complejo III de Transporte de Electrones/genética , Complejo III de Transporte de Electrones/metabolismo , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mitocondrias/metabolismo , Modelos Biológicos , Simulación del Acoplamiento Molecular , Familia de Multigenes , Unión Proteica , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Tiosemicarbazonas/químicaRESUMEN
The cysteine-rich PLAC8 domain of unknown function occurs in proteins found in most Eukaryotes. PLAC8-proteins play important yet diverse roles in different organisms, such as control of cell proliferation in animals and plants or heavy metal resistance in plants and fungi. Mammalian Onzin can be either pro-proliferative or pro-apoptotic, depending on the cell type, whereas fungal FCR1 confers cadmium tolerance. Despite their different role in different organisms, we hypothesized common ancestral functions linked to the PLAC8 domain. To address this hypothesis, and to investigate the molecular function of the PLAC8 domain, murine Onzin and fungal FCR1 were expressed in the PLAC8-free yeast Saccharomyces cerevisiae. The two PLAC8-proteins localized in the nucleus and induced almost identical phenotypes and transcriptional changes when exposed to cadmium stress. Like FCR1, Onzin also reduced DNA damage and increased cadmium tolerance by a DUN1-dependent pathway. Both proteins activated transcription of ancient mitochondrial pathways such as leucine and Fe-S cluster biosynthesis, known to regulate cell proliferation and DNA repair in yeast. These results strongly suggest a common ancestral function of PLAC8 proteins and open new perspectives to understand the role of the PLAC8 domain in the cellular biology of Eukaryotes.
Asunto(s)
Cadmio/toxicidad , Núcleo Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteínas Oncogénicas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Animales , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Daño del ADN/genética , Reparación del ADN/genética , Ratones , Proteínas Oncogénicas/genética , Proteínas/genética , Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
OPA1 is the major gene responsible for Dominant Optic Atrophy (DOA) and the syndromic form DOA "plus". Over 370 OPA1 mutations have been identified so far, although their pathogenicity is not always clear. We have analyzed one novel and a set of known OPA1 mutations to investigate their impact on protein functions in primary skin fibroblasts and in two "ad hoc" generated cell systems: the MGM1/OPA1 chimera yeast model and the Opa1-/- MEFs model expressing the mutated human OPA1 isoform 1. The yeast model allowed us to confirm the deleterious effects of these mutations and to gain information on their dominance/recessivity. The MEFs model enhanced the phenotypic alteration caused by mutations, nicely correlating with the clinical severity observed in patients, and suggested that the DOA "plus" phenotype could be induced by the combinatorial effect of mitochondrial network fragmentation with variable degrees of mtDNA depletion. Overall, the two models proved to be valuable tools to functionally assess and define the deleterious mechanism and the pathogenicity of novel OPA1 mutations, and useful to testing new therapeutic interventions.
Asunto(s)
Fibroblastos/citología , GTP Fosfohidrolasas/genética , Proteínas de Unión al GTP/genética , Proteínas Mitocondriales/genética , Mutación , Atrofia Óptica Autosómica Dominante/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/inmunología , Adulto , Animales , Células Cultivadas , Femenino , Fibroblastos/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Masculino , Ratones , Persona de Mediana Edad , Proteínas Mitocondriales/metabolismo , Modelos Biológicos , Atrofia Óptica Autosómica Dominante/patología , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Defects in nuclear-encoded proteins of the mitochondrial translation machinery cause early-onset and tissue-specific deficiency of one or more OXPHOS complexes. Here, we report a 7-year-old Italian boy with childhood-onset rapidly progressive encephalomyopathy and stroke-like episodes. Multiple OXPHOS defects and decreased mtDNA copy number (40%) were detected in muscle homogenate. Clinical features combined with low level of plasma citrulline were highly suggestive of mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, however, the common m.3243 A > G mutation was excluded. Targeted exome sequencing of genes encoding the mitochondrial proteome identified a damaging mutation, c.567 G > A, affecting a highly conserved amino acid residue (p.Gly189Arg) of the MRM2 protein. MRM2 has never before been linked to a human disease and encodes an enzyme responsible for 2'-O-methyl modification at position U1369 in the human mitochondrial 16S rRNA. We generated a knockout yeast model for the orthologous gene that showed a defect in respiration and the reduction of the 2'-O-methyl modification at the equivalent position (U2791) in the yeast mitochondrial 21S rRNA. Complementation with the mrm2 allele carrying the equivalent yeast mutation failed to rescue the respiratory phenotype, which was instead completely rescued by expressing the wild-type allele. Our findings establish that defective MRM2 causes a MELAS-like phenotype, and suggests the genetic screening of the MRM2 gene in patients with a m.3243 A > G negative MELAS-like presentation.
Asunto(s)
Síndrome MELAS/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Niño , ADN Mitocondrial/genética , Humanos , Síndrome MELAS/diagnóstico , Masculino , Mitocondrias/genética , Encefalomiopatías Mitocondriales/genética , Encefalomiopatías Mitocondriales/metabolismo , Mutación , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
The mitochondrial ADP/ATP carrier is a nuclear encoded protein, which catalyzes the exchange of ATP generated in mitochondria with ADP produced in the cytosol. In humans, mutations in the major ADP/ATP carrier gene, ANT1, are involved in several degenerative mitochondrial pathologies, leading to instability of mitochondrial DNA. Recessive mutations have been associated with mitochondrial myopathy and cardiomyopathy whereas dominant mutations have been associated with autosomal dominant Progressive External Ophtalmoplegia (adPEO). Recently, two de novo dominant mutations, R80H and R235G, leading to extremely severe symptoms, have been identified. In order to evaluate if the dominance is due to haploinsufficiency or to a gain of function, the two mutations have been introduced in the equivalent positions of the AAC2 gene, the yeast orthologue of human ANT1, and their dominant effect has been studied in heteroallelic strains, containing both one copy of wild type AAC2 and one copy of mutant aac2 allele. Through phenotypic characterization of these yeast models we showed that the OXPHOS phenotypes in the heteroallelic strains were more affected than in the hemiallelic strain indicating that the dominant trait of the two mutations is due to gain of function.
Asunto(s)
Translocador 1 del Nucleótido Adenina/genética , ADN Mitocondrial/genética , Translocasas Mitocondriales de ADP y ATP/genética , Miopatías Mitocondriales/genética , Mutación Puntual , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Alelos , HumanosRESUMEN
Aspergillus flavus is an opportunistic mold that represents a serious threat for human and animal health due to its ability to synthesize and release, on food and feed commodities, different toxic secondary metabolites. Among them, aflatoxin B1 is one of the most dangerous since it is provided with a strong cancerogenic and mutagenic activity. Controlling fungal contamination on the different crops that may host A. flavus is considered a priority by sanitary authorities of an increasing number of countries due also to the fact that, owing to global temperature increase, the geographic areas that are expected to be prone to experience sudden A. flavus outbreaks are widening. Among the different pre- and post-harvest strategies that may be put forward in order to prevent fungal and/or mycotoxin contamination, fungicides are still considered a prominent weapon. We have here analyzed different structural modifications of a natural-derived compound (cuminaldehyde thiosemicarbazone) for their fungistatic and anti-aflatoxigenic activity. In particular, we have focused our attention on one of the compound that presented a prominent anti-aflatoxin specificity, and performed a set of physiological and molecular analyses, taking also advantage of yeast (Saccharomyces cerevisiae) cell as an experimental model.
Asunto(s)
Aflatoxina B1/biosíntesis , Aspergillus flavus/metabolismo , Benzaldehídos/química , Tiosemicarbazonas/química , Aspergillus flavus/genética , Productos Agrícolas , Cimenos , Regulación Fúngica de la Expresión Génica , Proteómica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
PURPOSE: Addressing the problem of proctologic sequelae after Scopinaro's classical BPD, we elongated the common limb from 50 to 200 cm at the expense of the alimentary limb and simultaneously, with the aim of avoiding weight regain, reduced the gastric pouch from 500 to 40 ml. After increased experience with the new procedure, we observed a favourable tendency towards further weight loss. Thus, we subsequently extended the indication to the procedure to patients with unsatisfactory weight loss after Scopinaro's classical BPD (SBPD). METHODS: We retrospectively reviewed our clinical experience with the new procedure. RESULTS: From March 2008 to December 2014, 38 patients were submitted to the revisional procedure. The indication to surgical revision was proctologic in 26 patients and unsatisfactory weight loss in 12. After the revisional procedure, a significant reduction in bowel movements per day was observed, together with a significant reduction in body weight (from preoperative 87.1 ± 21 to 69.2 ± 13.5 kg at post-operative year 1 and 68.1 ± 11.9 kg at year 5; p < 0.001) and a parallel reduction in BMI (from preoperative 33.03 ± 7.6 to 26.8 ± 4.1 at post-operative year 1 and 26.9 ± 2.8 at year 5; p < 0.001). Mean excess BMI percent loss was 49.5 ± 94.6% at post-operative month 3, 76.51 ± 74.9% at year 1 and 76.2 ± 31.3% at year 5. Nutritional and metabolic parameters remained stable. Similar results were observed, analysing separately both groups of patients. CONCLUSIONS: Our preliminary data suggest that the proposed procedure could represent a safe and effective revisional tool to treat invalidating proctologic sequelae after SBPD, or when weight loss may be deemed unsatisfactory.
Asunto(s)
Desviación Biliopancreática/efectos adversos , Derivación Gástrica/métodos , Obesidad Mórbida/cirugía , Adulto , Anciano , Desviación Biliopancreática/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Obesidad Mórbida/fisiopatología , Periodo Posoperatorio , Reoperación/métodos , Estudios Retrospectivos , Insuficiencia del Tratamiento , Pérdida de PesoRESUMEN
BACKGROUND: Biliopancreatic diversion (BPD) is a bariatric technique burdened, in some instances, by clinical evidence of malabsorption and malnutrition, and by intractable diarrhea. OBJECTIVE: The objective of this study was to assess metabolic and nutritional effects on patients undergoing BPD and BPD plus revisional surgery because of side effects. METHODS: Thirty-five consecutive BPD patients underwent revisional surgery (elongation of the common limb from 50 to 200 cm and reduction of the gastric pouch from 500 to 40 ml) after a median 48-month period [48.3 ± 9.17 months (mean ± SD)] and were observed for a total period of 116.2 ± 6.21 months; 88 patients only undergoing BPD (controls) were observed for 120 months. RESULTS: Revisional surgery significantly improved side effects of BPD, with resolution of clinical symptoms in most instances. After revisional surgery, patients had a further decrease of body weight. The effect on disappearance of diabetes mellitus (DM) was remarkable, with no difference between revisional surgery and BPD. Triglycerides and transaminases decreased in a similar way, while cholesterol levels differed significantly. Estimated glomerular filtration rate improved. Nutritional parameters were similarly affected. CONCLUSION: This study suggests that it is possible to maintain the clinical and metabolic effects of BPD after a revisional procedure that leads to lesser malabsorption and to a greater restriction of the stomach. In particular, the positive effects on DM still persist after revisional surgery. This approach should be kept in mind in the presence of significant side effects due, inter alia, to excessive malabsorption.
Asunto(s)
Desviación Biliopancreática/efectos adversos , Trastornos Nutricionales/etiología , Obesidad Mórbida/cirugía , Adulto , Desviación Biliopancreática/métodos , Femenino , Estudios de Seguimiento , Humanos , Italia , Masculino , Persona de Mediana Edad , Estado Nutricional , Obesidad Mórbida/mortalidad , Complicaciones Posoperatorias , Reoperación , Pérdida de PesoRESUMEN
Mutations in SLC25A4 encoding the mitochondrial ADP/ATP carrier AAC1 are well-recognized causes of mitochondrial disease. Several heterozygous SLC25A4 mutations cause adult-onset autosomal-dominant progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions, whereas recessive SLC25A4 mutations cause childhood-onset mitochondrial myopathy and cardiomyopathy. Here, we describe the identification by whole-exome sequencing of seven probands harboring dominant, de novo SLC25A4 mutations. All affected individuals presented at birth, were ventilator dependent and, where tested, revealed severe combined mitochondrial respiratory chain deficiencies associated with a marked loss of mitochondrial DNA copy number in skeletal muscle. Strikingly, an identical c.239G>A (p.Arg80His) mutation was present in four of the seven subjects, and the other three case subjects harbored the same c.703C>G (p.Arg235Gly) mutation. Analysis of skeletal muscle revealed a marked decrease of AAC1 protein levels and loss of respiratory chain complexes containing mitochondrial DNA-encoded subunits. We show that both recombinant AAC1 mutant proteins are severely impaired in ADP/ATP transport, affecting most likely the substrate binding and mechanics of the carrier, respectively. This highly reduced capacity for transport probably affects mitochondrial DNA maintenance and in turn respiration, causing a severe energy crisis. The confirmation of the pathogenicity of these de novo SLC25A4 mutations highlights a third distinct clinical phenotype associated with mutation of this gene and demonstrates that early-onset mitochondrial disease can be caused by recurrent de novo mutations, which has significant implications for the application and analysis of whole-exome sequencing data in mitochondrial disease.