RESUMEN

Ischemic stroke is a leading cause of disability worldwide. There is no simple treatment to alleviate ischemic brain injury, as thrombolytic therapy is applicable within a narrow time window. During the last years, the ketogenic diet (KD) and the exogenous administration of the ketone body ß-hydroxybutyrate (BHB) have been proposed as therapeutic tools for acute neurological disorders and both can reduce ischemic brain injury. However, the mechanisms involved are not completely clear. We have previously shown that the D enantiomer of BHB stimulates the autophagic flux in cultured neurons exposed to glucose deprivation (GD) and in the brain of hypoglycemic rats. Here, we have investigated the effect of the systemic administration of D-BHB, followed by its continuous infusion after middle cerebral artery occlusion (MCAO), on the autophagy-lysosomal pathway and the activation of the unfolded protein response (UPR). Results show for the first time that the protective effect of BHB against MCAO injury is enantiomer selective as only D-BHB, the physiologic enantiomer of BHB, significantly reduced brain injury. D-BHB treatment prevented the cleavage of the lysosomal membrane protein LAMP2 and stimulated the autophagic flux in the ischemic core and the penumbra. In addition, D-BHB notably reduced the activation of the PERK/eIF2α/ATF4 pathway of the UPR and inhibited IRE1α phosphorylation. L-BHB showed no significant effect relative to ischemic animals. In cortical cultures under GD, D-BHB prevented LAMP2 cleavage and decreased lysosomal number. It also abated the activation of the PERK/eIF2α/ATF4 pathway, partially sustained protein synthesis, and reduced pIRE1α. In contrast, L-BHB showed no significant effects. Results suggest that protection elicited by D-BHB treatment post-ischemia prevents lysosomal rupture allowing functional autophagy, preventing the loss of proteostasis and UPR activation.

Asunto(s)

Lesiones Encefálicas , Accidente Cerebrovascular , Ratas , Animales , Cuerpos Cetónicos/farmacología , Cuerpos Cetónicos/metabolismo , Endorribonucleasas/farmacología , Proteínas Serina-Treonina Quinasas , Estrés del Retículo Endoplásmico , Ácido 3-Hidroxibutírico/metabolismo , Ácido 3-Hidroxibutírico/farmacología , Glucosa/metabolismo , Autofagia , Infarto de la Arteria Cerebral Media , Modelos Teóricos , Accidente Cerebrovascular/tratamiento farmacológico

RESUMEN

Mitochondrial translation is an intricate process involving both general and mRNA-specific factors. In addition, in the yeast Saccharomyces cerevisiae, translation of mitochondrial mRNAs is coupled to assembly of nascent polypeptides into the membrane. ARG8m is a reporter gene widely used to study the mechanisms of yeast mitochondrial translation. This reporter is a recodified gene that uses the mitochondrial genetic code and is inserted at the desired locus in the mitochondrial genome. After deletion of the endogenous nuclear gene, this reporter produces Arg8, an enzyme necessary for arginine biosynthesis. Since Arg8 is a soluble protein with no relation to oxidative phosphorylation, it is a reliable reporter to study mitochondrial mRNAs translation and dissect translation form assembly processes. In this chapter, we explain how to insert the ARG8m reporter in the desired spot in the mitochondrial DNA, how to analyze Arg8 synthesis inside mitochondria, and how to follow steady-state levels of the protein. We also explain how to use it to find spontaneous suppressors of translation defects.

Asunto(s)

Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Biosíntesis de Proteínas , ADN Mitocondrial/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo

RESUMEN

Deletion of the yeast mitochondrial gene COX2, encoding subunit 2 (mtCox2) of cytochrome c oxidase (CcO), results in a respiratory-incompetent Δcox2 strain. For a cytosol-synthesized Cox2 to restore respiratory growth, it must carry the W56R mutation (cCox2W56R). Nevertheless, only a fraction of cCox2W56R is matured in mitochondria, allowing ∼60% steady-state accumulation of CcO. This can be attributed either to the point mutation or to an inefficient biogenesis of cCox2W56R. We generated a strain expressing the mutant protein mtCox2W56R inside mitochondria which should follow the canonical biogenesis of mitochondria-encoded Cox2. This strain exhibited growth rates, CcO steady-state levels, and CcO activity similar to those of the wild type; therefore, the efficiency of Cox2 biogenesis is the limiting step for successful allotopic expression. Upon coexpression of cCox2W56R and mtCox2, each protein assembled into CcO independently from its genetic origin, resulting in a mixed population of CcO with most complexes containing the mtCox2 version. Notably, the presence of the mtCox2 enhances cCox2W56R incorporation. We provide proof of principle that an allotopically expressed Cox2 may complement a phenotype due to a mutant mitochondrial COX2 gene. These results are relevant to developing a rational design of genes for allotopic expression intended to treat human mitochondrial diseases.

RESUMEN

Cytochrome c oxidase (CcO) is the last electron acceptor in the respiratory chain. The CcO core is formed by mitochondrial DNA-encoded Cox1, Cox2, and Cox3 subunits. Cox1 synthesis is highly regulated; for example, if CcO assembly is blocked, Cox1 synthesis decreases. Mss51 activates translation of COX1 mRNA and interacts with Cox1 protein in high-molecular-weight complexes (COA complexes) to form the Cox1 intermediary assembly module. Thus, Mss51 coordinates both Cox1 synthesis and assembly. We previously reported that the last 15 residues of the Cox1 C terminus regulate Cox1 synthesis by modulating an interaction of Mss51 with Cox14, another component of the COA complexes. Here, using site-directed mutagenesis of the mitochondrial COX1 gene from Saccharomyces cerevisiae, we demonstrate that mutations P521A/P522A and V524E disrupt the regulatory role of the Cox1 C terminus. These mutations, as well as C terminus deletion (Cox1ΔC15), reduced binding of Mss51 and Cox14 to COA complexes. Mss51 was enriched in a translationally active form that maintains full Cox1 synthesis even if CcO assembly is blocked in these mutants. Moreover, Cox1ΔC15, but not Cox1-P521A/P522A and Cox1-V524E, promoted formation of aberrant supercomplexes in CcO assembly mutants lacking Cox2 or Cox4 subunits. The aberrant supercomplex formation depended on the presence of cytochrome b and Cox3, supporting the idea that supercomplex assembly factors associate with Cox3 and demonstrating that supercomplexes can be formed even if CcO is inactive and not fully assembled. Our results indicate that the Cox1 C-terminal end is a key regulator of CcO biogenesis and that it is important for supercomplex formation/stability.

Asunto(s)

Complejo IV de Transporte de Electrones/metabolismo , Mitocondrias/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Sustitución de Aminoácidos , Complejo IV de Transporte de Electrones/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación Missense , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Cytochrome c oxidase assembly requires the synthesis of the mitochondria-encoded core subunits, Cox1, Cox2, and Cox3. In yeast, Pet54 protein is required to activate translation of the COX3 mRNA and to process the aI5ß intron on the COX1 transcript. Here we report a third, novel function of Pet54 on Cox1 synthesis. We observed that Pet54 is necessary to achieve an efficient Cox1 synthesis. Translation of the COX1 mRNA is coupled to the assembly of cytochrome c oxidase by a mechanism that involves Mss51. This protein activates translation of the COX1 mRNA by acting on the COX1 5'-UTR, and, in addition, it interacts with the newly synthesized Cox1 protein in high molecular weight complexes that include the factors Coa3 and Cox14. Deletion of Pet54 decreased Cox1 synthesis, and, in contrast to what is commonly observed for other assembly mutants, double deletion of cox14 or coa3 did not recover Cox1 synthesis. Our results show that Pet54 is a positive regulator of Cox1 synthesis that renders Mss51 competent as a translational activator of the COX1 mRNA and that this role is independent of the assembly feedback regulatory loop of Cox1 synthesis. Pet54 may play a role in Mss51 hemylation/conformational change necessary for translational activity. Moreover, Pet54 physically interacts with the COX1 mRNA, and this binding was independent of the presence of Mss51.

Asunto(s)

Complejo IV de Transporte de Electrones/biosíntesis , Proteínas Mitocondriales/biosíntesis , Biosíntesis de Proteínas/fisiología , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Regiones no Traducidas 5'/fisiología , Complejo IV de Transporte de Electrones/genética , Proteínas Mitocondriales/genética , ARN de Hongos/genética , ARN de Hongos/metabolismo , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Deletion of the yeast mitochondrial gene COX2 encoding subunit 2 (Cox2) of cytochrome c oxidase (CcO) results in loss of respiration (Δcox2 strain). Supekova et al. (2010) [1] transformed a Δcox2 strain with a vector expressing Cox2 with a mitochondrial targeting sequence (MTS) and the point mutation W56R (Cox2(W56R)), restoring respiratory growth. Here, the CcO carrying the allotopically-expressed Cox2(W56R) was characterized. Yeast mitochondria from the wild-type (WT) and the Δcox2+Cox2(W56R) strains were subjected to Blue Native electrophoresis. In-gel activity of CcO and spectroscopic quantitation of cytochromes revealed that only 60% of CcO is present in the complemented strain, and that less CcO is found associated in supercomplexes as compared to WT. CcOs from the WT and the mutant exhibited similar subunit composition, although activity was 20-25% lower in the enzyme containing Cox2(W56R) than in the one with Cox2(WT). Tandem mass spectrometry confirmed that W(56) was substituted by R(56) in Cox2(W56R). In addition, Cox2(W56R) exhibited the same N-terminus than Cox2(WT), indicating that the MTS of Oxa1 and the leader sequence of 15 residues were removed from Cox2(W56R) during maturation. Thus, Cox2(W56R) is identical to Cox2(WT) except for the point mutation W56R. Mitochondrial Cox1 synthesis is strongly reduced in Δcox2 mutants, but the Cox2(W56R) complemented strain led to full restoration of Cox1 synthesis. We conclude that the cytosol-synthesized Cox2(W56R) follows a rate-limiting process of import, maturation or assembly that yields lower steady-state levels of CcO. Still, the allotopically-expressed Cox2(W56R) restores CcO activity and allows mitochondrial Cox1 synthesis to advance at WT levels.

Asunto(s)

Citoplasma/enzimología , Complejo IV de Transporte de Electrones/metabolismo , Oxígeno/metabolismo , Mutación Puntual/genética , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Respiración de la Célula/fisiología , Complejo IV de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/genética , Inmunoensayo , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Datos de Secuencia Molecular , Electroforesis en Gel de Poliacrilamida Nativa , Conformación Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometría de Masas en Tándem

RESUMEN

The mitochondrial permeability transition (PT) involves the opening of a mitochondrial unselective channel (MUC) resulting in membrane depolarization and increased permeability to ions. PT has been observed in many, but not all eukaryotic species. In some species, PT has been linked to cell death, although other functions, such as matrix ion detoxification or regulation of the rate of oxygen consumption have been considered. The identification of the proteins constituting MUC would help understand the biochemistry and physiology of this channel. It has been suggested that the mitochondrial phosphate carrier is a structural component of MUC and we decided to test this in yeast mitochondria. Mersalyl inhibits the phosphate carrier and it has been reported that it also triggers PT. Mersalyl induced opening of the decavanadate-sensitive Yeast Mitochondrial Unselective Channel (YMUC). In isolated yeast mitochondria from a phosphate carrier-null strain the sensitivity to both phosphate and mersalyl was lost, although the permeability transition was still evoked by ATP in a decavanadate-sensitive fashion. Polyethylene glycol (PEG)-induced mitochondrial contraction results indicated that in mitochondria lacking the phosphate carrier the YMUC is smaller: complete contraction for mitochondria from the wild type and the mutant strains was achieved with 1.45 and 1.1 kDa PEGs, respectively. Also, as expected for a smaller channel titration with 1.1 kDa PEG evidenced a higher sensitivity in mitochondria from the mutant strain. The above data suggest that the phosphate carrier is the phosphate sensor in YMUC and contributes to the structure of this channel.

Asunto(s)

Proteínas de Transporte de Fosfato/metabolismo , Canales de Potasio/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Mersalil/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Dilatación Mitocondrial/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Proteínas de Transporte de Fosfato/antagonistas & inhibidores , Fosfatos/metabolismo , Polietilenglicoles/farmacología , Canales de Potasio/química , Canales de Potasio/deficiencia , Canales de Potasio/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de Secuencia , Vanadatos/farmacología , Canales Aniónicos Dependientes del Voltaje/metabolismo

RESUMEN

Pet309 is a protein essential for respiratory growth. It is involved in translation of the yeast mitochondrial COX1 gene, which encodes subunit I of the cytochrome c oxidase. Pet309 is also involved in stabilization of the COX1 mRNA. Mutations in a similar human protein, Lrp130, are associated with Leigh syndrome, where cytochrome c oxidase activity is affected. The sequence of Pet309 reveals the presence of at least seven pentatricopeptide repeats (PPRs) located in tandem in the central portion of the protein. Proteins containing PPR motifs are present in mitochondria and chloroplasts and are in general involved in RNA metabolism. Despite the increasing number of proteins from this family found to play essential roles in mitochondria and chloroplasts, little is understood about the mechanism of action of the PPR domains present in these proteins. In a series of in vivo analyses we constructed a pet309 mutant lacking the PPR motifs. Although the stability of the COX1 mRNA was not affected, synthesis of Cox1 was abolished. The deletion of one PPR motif at a time showed that all the PPR motifs are required for COX1 mRNA translation and respiratory growth. Mutations of basic residues in PPR3 caused reduced respiratory growth. According to a molecular model, these residues are facing a central cavity that could be involved in mRNA-binding activity, forming a possible path for this molecule on Pet309. Our results show that the RNA metabolism function of Pet309 is found in at least two separate domains of the protein.

Asunto(s)

Complejo IV de Transporte de Electrones/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Biosíntesis de Proteínas , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimología , Secuencias de Aminoácidos , Aminoácidos , Complejo IV de Transporte de Electrones/biosíntesis , Regulación Fúngica de la Expresión Génica , Mitocondrias/metabolismo , Proteínas Mitocondriales , Modelos Moleculares , Mutagénesis , Factores de Iniciación de Péptidos , Estructura Terciaria de Proteína , Transporte de Proteínas , ARN de Hongos/metabolismo , ARN Mitocondrial , Secuencias Repetitivas de Aminoácido , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-Actividad

RESUMEN

Mitochondrial diseases display great diversity in clinical symptoms and biochemical characteristics. Although mtDNA mutations have been identified in many patients, there are currently no effective treatments. A number of human diseases result from mutations in mtDNA-encoded proteins, a group of proteins that are hydrophobic and have multiple membrane-spanning regions. One method that has great potential for overcoming the pathogenic consequences of these mutations is to place a wild-type copy of the affected gene in the nucleus, and target the expressed protein to the mitochondrion to function in place of the defective protein. Several respiratory chain subunit genes, which are typically mtDNA encoded, are nucleus encoded in the chlorophyte algae Chlamydomonas reinhardtii and Polytomella sp. Analysis of these genes has revealed adaptations that facilitated their expression from the nucleus. The nucleus-encoded proteins exhibited diminished physical constraints for import as compared to their mtDNA-encoded homologues. The hydrophobicity of the nucleus-encoded proteins is diminished in those regions that are not involved in subunit-subunit interactions or that contain amino acids critical for enzymatic reactions of the proteins. In addition, these proteins have unusually large mitochondrial targeting sequences. Information derived from these studies should be applicable toward the development of genetic therapies for human diseases resulting from mutations in mtDNA-encoded polypeptides.

Asunto(s)

Núcleo Celular/metabolismo , ADN Mitocondrial/metabolismo , Eucariontes/genética , Eucariontes/metabolismo , Genoma , Animales , Chlamydomonas reinhardtii/metabolismo , Terapia Genética , Humanos , Mitocondrias/metabolismo , Modelos Biológicos , Mutación , Péptidos/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína

RESUMEN

Several chlorophyte algae do not have the cox3 gene, encoding subunit III of cytochrome c oxidase, in their mitochondrial genomes. The cox3 gene is nuclear-encoded in the photosynthetic alga Chlamydomonas reinhardtii and in the colorless alga Polytomella sp. In this work, the genomic sequences of the cox3 genes of these two closely related algae are reported. The cox3 genes of both C. reinhardtii and Polytomella sp. contain four introns in the region encoding the putative mitochondrial-targeting sequences. These four introns show low sequence identities, but their locations are conserved between these species. The cox3 gene of C. reinhardtii has five additional introns in the region encoding the mature subunit III of cytochrome c oxidase. Sequence analysis of intron 6 of the cox3 gene of C. reinhardtii revealed similarity with two sequence elements present in introns of several other nuclear genes from this green alga. In the majority of the genes, these conserved sequences are located either near the 3' end or near the 5' end of the introns. Based on these data, we propose that the colorless genus Polytomella separated from C. reinhardtii after the cox3 gene was transferred to the nucleus. The data also support the evolutionary hypothesis of a recent acquisition of introns in C. reinhardtii.

Asunto(s)

Núcleo Celular/genética , Chlamydomonas reinhardtii/genética , Chlorophyta/genética , Complejo IV de Transporte de Electrones/genética , Intrones/genética , Proteínas de la Membrana/genética , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Complejo IV de Transporte de Electrones/química , Evolución Molecular , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Sitios de Empalme de ARN/genética , Proteínas de Saccharomyces cerevisiae

RESUMEN

The atp6 gene, encoding the ATP6 subunit of F(1)F(0)-ATP synthase, has thus far been found only as an mtDNA-encoded gene. However, atp6 is absent from mtDNAs of some species, including that of Chlamydomonas reinhardtii. Analysis of C. reinhardtii expressed sequence tags revealed three overlapping sequences that encoded a protein with similarity to ATP6 proteins. PCR and 5'- and 3'-RACE were used to obtain the complete cDNA and genomic sequences of C. reinhardtii atp6. The atp6 gene exhibited characteristics of a nucleus-encoded gene: Southern hybridization signals consistent with nuclear localization, the presence of introns, and a codon usage and a polyadenylation signal typical of nuclear genes. The corresponding ATP6 protein was confirmed as a subunit of the mitochondrial F(1)F(0)-ATP synthase from C. reinhardtii by N-terminal sequencing. The predicted ATP6 polypeptide has a 107-amino acid cleavable mitochondrial targeting sequence. The mean hydrophobicity of the protein is decreased in those transmembrane regions that are predicted not to participate directly in proton translocation or in intersubunit contacts with the multimeric ring of c subunits. This is the first example of a mitochondrial protein with more than two transmembrane stretches, directly involved in proton translocation, that is nucleus-encoded.

Asunto(s)

Adenosina Trifosfatasas/genética , Núcleo Celular/genética , Chlamydomonas reinhardtii/genética , ADN Mitocondrial/genética , ATPasas de Translocación de Protón/genética , Secuencia de Aminoácidos , Animales , Membrana Celular/enzimología , Chlamydomonas reinhardtii/enzimología , Clonación Molecular , Etiquetas de Secuencia Expresada , ATPasas de Translocación de Protón Mitocondriales , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido