RESUMEN
Mitochondria play a key role in cancer and their involvement is not limited to the production of ATP only. Mitochondria also produce reactive oxygen species and building blocks to sustain rapid cell proliferation; thus, the deregulation of mitochondrial function is associated with cancer disease development and progression. In cancer cells, a metabolic reprogramming takes place through a different modulation of the mitochondrial metabolic pathways, including oxidative phosphorylation, fatty acid oxidation, the Krebs cycle, glutamine and heme metabolism. Alterations of mitochondrial homeostasis, in particular, of mitochondrial biogenesis, mitophagy, dynamics, redox balance, and protein homeostasis, were also observed in cancer cells. The use of drugs acting on mitochondrial destabilization may represent a promising therapeutic approach in tumors in which mitochondrial respiration is the predominant energy source. In this review, we summarize the main mitochondrial features and metabolic pathways altered in cancer cells, moreover, we present the best known drugs that, by acting on mitochondrial homeostasis and metabolic pathways, may induce mitochondrial alterations and cancer cell death. In addition, new strategies that induce mitochondrial damage, such as photodynamic, photothermal and chemodynamic therapies, and the development of nanoformulations that specifically target drugs in mitochondria are also described. Thus, mitochondria-targeted drugs may open new frontiers to a tailored and personalized cancer therapy.
Asunto(s)
Mitocondrias , Neoplasias , Humanos , Mitocondrias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Fosforilación Oxidativa , Ciclo del Ácido Cítrico , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismoRESUMEN
ADCA-DN and HSN-IE are rare neurodegenerative syndromes caused by dominant mutations in the replication foci targeting sequence (RFTS) of the DNA methyltransferase 1 (DNMT1) gene. Both phenotypes resemble mitochondrial disorders, and mitochondrial dysfunction was first observed in ADCA-DN. To explore mitochondrial involvement, we studied the effects of DNMT1 mutations in fibroblasts from four ADCA-DN and two HSN-IE patients. We documented impaired activity of purified DNMT1 mutant proteins, which in fibroblasts results in increased DNMT1 amount. We demonstrated that DNMT1 is not localized within mitochondria, but it is associated with the mitochondrial outer membrane. Concordantly, mitochondrial DNA failed to show meaningful CpG methylation. Strikingly, we found activated mitobiogenesis and OXPHOS with significant increase of H2O2, sharply contrasting with a reduced ATP content. Metabolomics profiling of mutant cells highlighted purine, arginine/urea cycle and glutamate metabolisms as the most consistently altered pathways, similar to primary mitochondrial diseases. The most severe mutations showed activation of energy shortage AMPK-dependent sensing, leading to mTORC1 inhibition. We propose that DNMT1 RFTS mutations deregulate metabolism lowering ATP levels, as a result of increased purine catabolism and urea cycle pathways. This is associated with a paradoxical mitochondrial hyper-function and increased oxidative stress, possibly resulting in neurodegeneration in non-dividing cells.
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ADN (Citosina-5-)-Metiltransferasa 1/genética , Predisposición Genética a la Enfermedad , Neuropatías Hereditarias Sensoriales y Autónomas/genética , Degeneración Nerviosa/genética , Ataxias Espinocerebelosas/genética , Metilación de ADN/genética , Sordera/genética , Sordera/fisiopatología , Femenino , Fibroblastos/metabolismo , Neuropatías Hereditarias Sensoriales y Autónomas/fisiopatología , Humanos , Masculino , Mitocondrias/genética , Mitocondrias/metabolismo , Mutación/genética , Narcolepsia/genética , Narcolepsia/fisiopatología , Degeneración Nerviosa/fisiopatología , Fosforilación Oxidativa , Fenotipo , Procesamiento Proteico-Postraduccional/genética , Ataxias Espinocerebelosas/fisiopatologíaRESUMEN
Biogenesis of mammalian mitochondrial ribosomes requires a concerted maturation of both the small (SSU) and large subunit (LSU). We demonstrate here that the m(5)C methyltransferase NSUN4, which forms a complex with MTERF4, is essential in mitochondrial ribosomal biogenesis as mitochondrial translation is abolished in conditional Nsun4 mouse knockouts. Deep sequencing of bisulfite-treated RNA shows that NSUN4 methylates cytosine 911 in 12S rRNA (m5C911) of the SSU. Surprisingly, NSUN4 does not need MTERF4 to generate this modification. Instead, the NSUN4/MTERF4 complex is required to assemble the SSU and LSU to form a monosome. NSUN4 is thus a dual function protein, which on the one hand is needed for 12S rRNA methylation and, on the other hand interacts with MTERF4 to facilitate monosome assembly. The presented data suggest that NSUN4 has a key role in controlling a final step in ribosome biogenesis to ensure that only the mature SSU and LSU are assembled.
Asunto(s)
Proteínas Portadoras/genética , Metiltransferasas/genética , Mitocondrias/genética , ARN Ribosómico/genética , Ribosomas/genética , Animales , Proteínas Portadoras/metabolismo , Metilación de ADN/genética , Metiltransferasas/metabolismo , Ratones , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Unión Proteica , ARN Ribosómico/biosíntesis , Ribosomas/ultraestructura , Factores de Transcripción/metabolismoRESUMEN
Leber's hereditary optic neuropathy is a maternally inherited blinding disease caused as a result of homoplasmic point mutations in complex I subunit genes of mitochondrial DNA. It is characterized by incomplete penetrance, as only some mutation carriers become affected. Thus, the mitochondrial DNA mutation is necessary but not sufficient to cause optic neuropathy. Environmental triggers and genetic modifying factors have been considered to explain its variable penetrance. We measured the mitochondrial DNA copy number and mitochondrial mass indicators in blood cells from affected and carrier individuals, screening three large pedigrees and 39 independently collected smaller families with Leber's hereditary optic neuropathy, as well as muscle biopsies and cells isolated by laser capturing from post-mortem specimens of retina and optic nerves, the latter being the disease targets. We show that unaffected mutation carriers have a significantly higher mitochondrial DNA copy number and mitochondrial mass compared with their affected relatives and control individuals. Comparative studies of fibroblasts from affected, carriers and controls, under different paradigms of metabolic demand, show that carriers display the highest capacity for activating mitochondrial biogenesis. Therefore we postulate that the increased mitochondrial biogenesis in carriers may overcome some of the pathogenic effect of mitochondrial DNA mutations. Screening of a few selected genetic variants in candidate genes involved in mitochondrial biogenesis failed to reveal any significant association. Our study provides a valuable mechanism to explain variability of penetrance in Leber's hereditary optic neuropathy and clues for high throughput genetic screening to identify the nuclear modifying gene(s), opening an avenue to develop predictive genetic tests on disease risk and therapeutic strategies.
Asunto(s)
ADN Mitocondrial/genética , Recambio Mitocondrial/genética , Atrofia Óptica Hereditaria de Leber/diagnóstico , Atrofia Óptica Hereditaria de Leber/genética , Penetrancia , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Linaje , Adulto JovenRESUMEN
In mammalian mitochondria, the processing of primary RNA transcripts involves a coordinated series of cleavage and modification events, leading to the formation of processing intermediates and mature mt-RNAs. RNA19 is an unusually stable unprocessed precursor, physiologically polyadenylated, which includes the 16S mt-rRNA, the mt-tRNALeuUUR and the mt-ND1 mRNA. These peculiarities, together with the alteration of its steady-state levels in cellular models with defects in mitochondrial function, make RNA19 a potentially important molecule for the physiological regulation of mitochondrial molecular processes as well as for the pathogenesis of mitochondrial diseases. In this work, we quantitatively and qualitatively examined RNA19 in MELAS trans-mitochondrial cybrids carrying the mtDNA 3243A>G transition and displaying a profound mitochondrial translation defect. Through a combination of isokinetic sucrose gradient and RT-qPCR experiments, we found that RNA19 accumulated and co-sedimented with the mitoribosomal large subunit (mt-LSU) in mutant cells. Intriguingly, exogenous expression of the isolated LARS2 C-terminal domain (Cterm), which was shown to rescue defective translation in MELAS cybrids, decreased the levels of mt-LSU-associated RNA19 by relegating it to the pool of free unbound RNAs. Overall, the data reported here support a regulatory role for RNA19 in mitochondrial physiopathological processes, designating this RNA precursor as a possible molecular target in view of therapeutic strategy development.
RESUMEN
The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. However many GST-tagged proteins are insoluble, and the existing procedures, which employ a mixture of detergents to solubilize the molecules, frequently compromise their functional activity. A further limitation is that large proteins (>80 kDa) are poorly isolated by the current methods and are contaminated by truncated forms. To overcome these problems, we provide here an improved method for efficient purification of active large GST-tagged enzymes such as the 180-kDa GST-fused mitochondrial RNA polymerase.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Glutatión Transferasa/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , ARN Polimerasas Dirigidas por ADN/biosíntesis , ARN Polimerasas Dirigidas por ADN/genética , Electroforesis en Gel de Poliacrilamida , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/genética , Humanos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The m.3243A>G mutation within the mitochondrial mt-tRNALeu(UUR) gene is the most prevalent variant linked to mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome. This pathogenic mutation causes severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA, such as reduced aminoacylation and a lack of post-transcriptional modification. In transmitochondrial cybrids, overexpression of human mitochondrial leucyl-tRNA synthetase (LARS2) has proven effective in rescuing the phenotype associated with m.3243A>G substitution. The rescuing activity resides in the carboxy-terminal domain (Cterm) of the enzyme; however, the precise molecular mechanisms underlying this process have not been fully elucidated. To deepen our knowledge on the rescuing mechanisms, we demonstrated the interactions of the Cterm with mutated mt-tRNALeu(UUR) and its precursor in MELAS cybrids. Further, the effect of Cterm expression on mitochondrial functions was evaluated. We found that Cterm ameliorates de novo mitochondrial protein synthesis, whilst it has no effect on mt-tRNALeu(UUR) steady-state levels and aminoacylation. Despite the complete recovery of cell viability and the increase in mitochondrial translation, Cterm-overexpressing cybrids were not able to recover bioenergetic competence. These data suggest that, in our MELAS cell model, the beneficial effect of Cterm may be mediated by factors that are independent of the mitochondrial bioenergetics.
RESUMEN
The beneficial effects of Cyclooxygenases (COX) inhibitors on human health have been known for thousands of years. Nevertheless, COXs, particularly COX-1, have been linked to a plethora of human diseases such as cancer, heart failure, neurological and neurodegenerative diseases only recently. COXs catalyze the first step in the biosynthesis of prostaglandins (PGs) and are among the most important mediators of inflammation. All published structural work on COX-1 deals with the ovine isoenzyme, which is easier to produce in milligram-quantities than the human enzyme and crystallizes readily. Here, we report the long-sought structure of the human cyclooxygenase-1 (hCOX-1) that we refined to an R/Rfree of 20.82/26.37, at 3.36 Å resolution. hCOX-1 structure provides a detailed picture of the enzyme active site and the residues crucial for inhibitor/substrate binding and catalytic activity. We compared hCOX-1 crystal structure with the ovine COX-1 and human COX-2 structures by using metrics based on Cartesian coordinates, backbone dihedral angles, and solvent accessibility coupled with multivariate methods. Differences and similarities among structures are discussed, with emphasis on the motifs responsible for the diversification of the various enzymes (primary structure, stability, catalytic activity, and specificity). The structure of hCOX-1 represents an essential step towards the development of new and more selective COX-1 inhibitors of enhanced therapeutic potential.
Asunto(s)
Ciclooxigenasa 1/química , Modelos Moleculares , Conformación Proteica , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catálisis , Ciclooxigenasa 1/metabolismo , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Estabilidad de Enzimas , Glicosilación , Humanos , Estructura Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes , Ovinos , Solventes , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The respiratory complexes are organized in supramolecular assemblies called supercomplexes thought to optimize cellular metabolism under physiological and pathological conditions. In this study, we used genetically and biochemically well characterized cells bearing the pathogenic microdeletion m.15,649-15,666 (ΔI300-P305) in MT-CYB gene, to investigate the effects of an assembly-hampered CIII on the re-organization of supercomplexes. First, we found that this mutation also affects the stability of both CI and CIV, and evidences the occurrence of a preferential structural interaction between CI and CIII2, yielding a small amount of active CI+CIII2 supercomplex. Indeed, a residual CI+CIII combined redox activity, and a low but detectable ATP synthesis driven by CI substrates are detectable, suggesting that the assembly of CIII into the CI+CIII2 supercomplex mitigates the detrimental effects of MT-CYB deletion. Second, measurements of oxygen consumption and ATP synthesis driven by NADH-linked and FADH2-linked substrates alone, or in combination, indicate a common ubiquinone pool for the two respiratory pathways. Finally, we report that prolonged incubation with rotenone enhances the amount of CI and CIII2, but reduces CIV assembly. Conversely, the antioxidant N-acetylcysteine increases CIII2 and CIV2 and partially restores respirasome formation. Accordingly, after NAC treatment, the rate of ATP synthesis increases by two-fold compared with untreated cell, while the succinate level, which is enhanced by the homoplasmic mutation, markedly decreases. Overall, our findings show that fine-tuning the supercomplexes stability improves the energetic efficiency of cells with the MT-CYB microdeletion.
Asunto(s)
Adenosina Trifosfato/metabolismo , Complejo III de Transporte de Electrones/deficiencia , Mitocondrias/enzimología , Membranas Mitocondriales/enzimología , Consumo de Oxígeno , Animales , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Eliminación de Gen , Mitocondrias/genética , Oxidación-Reducción , Rotenona/farmacologíaRESUMEN
The MTERF-family is a wide family of proteins identified in Metazoa and plants which includes the known mitochondrial transcription termination factors. With the aim to shed light on the function of MTERF-family members in Drosophila, we performed the cloning and characterization of D-MTERF3, a component of the most conserved group of this family. D-MTERF3 is a mitochondrial protein of 323 amino acids. Sequence analysis in seven different organisms showed that the protein contains five conserved "mTERF-motifs", three of which include a leucine zipper-like domain. D-MTERF3 knock-down, obtained by RNAi in D.Mel-2 cells, did not affect mitochondrial replication and transcription. On the contrary, it decreased to a variable extent the rate of labelling of about half of the mitochondrial polypeptides, with ND1 being the most affected by D-MTERF3 depletion. These results indicate that D-MTERF3 is involved in mitochondrial translation. This role, likely based on protein-protein interactions, may be exerted either through a direct interaction with the translation machinery or by bridging the mitochondrial transcription and translation apparatus.
Asunto(s)
Secuencia Conservada , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Mitocondriales/metabolismo , Biosíntesis de Proteínas/genética , Secuencia de Aminoácidos , Animales , Proteínas de Drosophila/química , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Regulación de la Expresión Génica , Proteínas Mitocondriales/química , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , ARN/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Mitocondrial , Alineación de SecuenciaRESUMEN
Mitochondrial transcription factor A (TFAM) is a key component for the protection and transcription of the mitochondrial genome. TFAM belongs to the high mobility group (HMG) box family of DNA binding proteins that are able to bind to and bend DNA. Human TFAM (huTFAM) contains two HMG box domains separated by a linker region, and a 26 amino acid C-terminal tail distal to the second HMG box. Previous studies on huTFAM have shown that requisites for proper DNA bending and specific binding to the mitochondrial genome are specific intercalating residues and the C-terminal tail. We have characterized TFAM from the sea urchin Paracentrotus lividus (suTFAM). Differently from human, suTFAM contains a short 9 amino acid C-terminal tail, yet it still has the ability to specifically bind to mtDNA. To provide information on the mode of binding of the protein we used fluorescence resonance energy transfer (FRET) assays and found that, in spite of the absence of a canonical C-terminal tail, suTFAM distorts DNA at a great extent and recognizes specific target with high affinity. Site directed mutagenesis showed that the two Phe residues placed in corresponding position of the two intercalating Leu of huTFAM are responsible for the strong bending and the great binding affinity of suTFAM.
Asunto(s)
ADN Mitocondrial/metabolismo , Proteínas Mitocondriales/metabolismo , Erizos de Mar/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Análisis Mutacional de ADN , Transferencia Resonante de Energía de Fluorescencia , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Factores de Transcripción/genéticaRESUMEN
The cloning of the cDNA for two mitochondrial proteins involved in sea urchin mtDNA replication and transcription is reported here. The cDNA for the mitochondrial D-loop binding protein (mtDBP) from the sea urchin Strongylocentrotus purpuratus has been cloned by a polymerase chain reaction-based approach. The protein displays a very high similarity with the Paracentrotus lividus homologue as it contains also the two leucine zipper-like domains which are thought to be involved in intramolecular interactions needed to expose the two DNA binding domains in the correct position for contacting DNA. The cDNA for the mitochondrial single-stranded DNA-binding protein (mtSSB) from P. lividus has been also cloned by a similar approach. The precursor protein is 146 amino acids long with a presequence of 16 residues. The deduced amino acid sequence shows the highest homology with the Xenopus laevis protein and the lowest with the Drosophila mtSSB. The computer modeling of the tertiary structure of P. lividus mtSSB shows a structure very similar to that experimentally determined for human mtSSB, with the conservation of the main residues involved in protein tetramerization and in DNA binding.
Asunto(s)
Replicación del ADN/genética , ADN Mitocondrial/genética , Proteínas de Unión al ADN/genética , Erizos de Mar/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Transcripción Genética/genéticaRESUMEN
Sea urchin mtDNA is transcribed via a different mechanism compared to vertebrates. To gain information on the apparatus of sea urchin mitochondrial transcription we have characterized the DNA binding properties of the mitochondrial transcription factor A (TFAM). The protein contains two HMG box domains but, differently from vertebrates, displays a very short C-terminal tail. Phylogenetic analysis showed that the distribution of tail length is mixed in the different lineages, indicating that it is a trait that undergoes rapid changes during evolution. Homology modeling suggests that the protein adopts the same configuration of the human counterpart and possibly a similar mode of binding to DNA. DNase I footprinting showed that TFAM specifically contacts mtDNA at a fixed distance from three AT-rich consensus sequences that were supposed to act as transcriptional initiation sites. Bound sequences are homologous and contain an inverted repeat motif, which resembles that involved in the intercalation of human TFAM in LSP DNA. The here reported data indicate that sea urchin TFAM specifically binds mtDNA. The protein could intercalate residues at the DNA inverted motif and, despite its short tail, might have a role in mitochondrial transcription.
Asunto(s)
ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Erizos de Mar/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Huella de ADN , Proteínas de Unión al ADN/química , Variación Genética , Proteínas Mitocondriales/química , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Factores de Transcripción/químicaRESUMEN
The MTERF protein family comprises members from Metazoans and plants. All the Metazoan MTERF proteins characterized to date, including the mitochondrial transcription termination factors, play a key role in mitochondrial gene expression. In this study we report the characterization of Drosophila MTERF5 (D-MTERF5), a mitochondrial protein existing only in insects, probably originated from a duplication event of the transcription termination factor DmTTF. D-MTERF5 knock-down in D.Mel-2 cells alters transcript levels with an opposite pattern to that produced by DmTTF knock-down. D-MTERF5 is able to interact with mtDNA at the same sites contacted by DmTTF, but only in the presence of the termination factor. We propose that the two proteins participate in the transcription termination process, with D-MTERF5 engaged in relieving the block exerted by DmTTF. This hypothesis is supported also by D-MTERF5 homology modeling, which suggests that this protein contains protein-protein interaction domains. Co-regulation by DREF (DNA Replication-related Element binding Factor) of D-MTERF5 and DmTTF implies that expression of the two factors needs to be co-ordinated to ensure fine modulation of Drosophila mitochondrial transcription.