RESUMEN

CHCHD2 and CHCHD10, linked to Parkinson's disease and amyotrophic lateral sclerosis-frontotemporal dementia (ALS), respectively, are mitochondrial intermembrane proteins that form a heterodimer. This study aimed to investigate the impact of the CHCHD2 P14L variant, implicated in ALS, on mitochondrial function and its subsequent effects on cellular homeostasis. The missense variant of CHCHD2, P14L, found in a cohort of patients with ALS, mislocalized CHCHD2 to the cytoplasm, leaving CHCHD10 in the mitochondria. Drosophila lacking the CHCHD2 ortholog exhibited mitochondrial degeneration. In contrast, human CHCHD2 P14L, but not wild-type human CHCHD2, failed to suppress this degeneration, suggesting that P14L is a pathogenic variant. The mitochondrial Ca2+ buffering capacity was reduced in Drosophila neurons expressing human CHCHD2 P14L. The altered Ca2+-buffering phenotype was also observed in cultured human neuroblastoma SH-SY5Y cells expressing CHCHD2 P14L. In these cells, transient elevation of cytoplasmic Ca2+ facilitated the activation of calpain and caspase-3, accompanied by the processing and insolubilization of TDP-43. These observations suggest that CHCHD2 P14L causes abnormal Ca2+ dynamics and TDP-43 aggregation, reflecting the pathophysiology of ALS.

RESUMEN

The transcriptional coactivator PGC-1α has been implicated in the regulation of multiple metabolic processes. However, the previously reported metabolic phenotypes of mice deficient in PGC-1α have been inconsistent. PGC-1α exists as multiple isoforms, including variants transcribed from an alternative first exon. We show here that alternative PGC-1α variants are the main entity that increases PGC-1α during exercise. These variants, unlike the canonical isoform of PGC-1α, are robustly upregulated in human skeletal muscle after exercise. Furthermore, the extent of this upregulation correlates with oxygen consumption. Mice lacking these variants manifest impaired energy expenditure during exercise, leading to the development of obesity and hyperinsulinemia. The alternative variants are also upregulated in brown adipose tissue in response to cold exposure, and mice lacking these variants are intolerant of a cold environment. Our findings thus indicate that an increase in PGC-1α expression, attributable mostly to upregulation of alternative variants, is pivotal for adaptive enhancement of energy expenditure and heat production and thereby essential for the regulation of whole-body energy metabolism.

Asunto(s)

Tejido Adiposo Pardo , Empalme Alternativo , Metabolismo Energético , Músculo Esquelético , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Metabolismo Energético/genética , Animales , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Humanos , Ratones , Empalme Alternativo/genética , Masculino , Músculo Esquelético/metabolismo , Tejido Adiposo Pardo/metabolismo , Ratones Endogámicos C57BL , Condicionamiento Físico Animal , Obesidad/metabolismo , Obesidad/genética , Termogénesis/genética , Consumo de Oxígeno , Ejercicio Físico , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Adulto , Ratones Noqueados

RESUMEN

Abnormal mitochondrial fragmentation by dynamin-related protein1 (Drp1) is associated with the progression of aging-associated heart diseases, including heart failure and myocardial infarction (MI). Here, we report a protective role of outer mitochondrial membrane (OMM)-localized E3 ubiquitin ligase MITOL/MARCH5 against cardiac senescence and MI, partly through Drp1 clearance by OMM-associated degradation (OMMAD). Persistent Drp1 accumulation in cardiomyocyte-specific MITOL conditional-knockout mice induced mitochondrial fragmentation and dysfunction, including reduced ATP production and increased ROS generation, ultimately leading to myocardial senescence and chronic heart failure. Furthermore, ischemic stress-induced acute downregulation of MITOL, which permitted mitochondrial accumulation of Drp1, resulted in mitochondrial fragmentation. Adeno-associated virus-mediated delivery of the MITOL gene to cardiomyocytes ameliorated cardiac dysfunction induced by MI. Our findings suggest that OMMAD activation by MITOL can be a therapeutic target for aging-associated heart diseases, including heart failure and MI.

RESUMEN

While it has been hypothesized that brown adipocytes responsible for mammalian thermogenesis are absent in birds, the existence of beige fat has yet to be studied directly. The present study tests the hypothesis that beige fat emerges in birds as a mechanism of physiological adaptation to cold environments. Subcutaneous neck adipose tissue from cold-acclimated or triiodothyronine (T3)-treated chickens exhibited increases in the expression of avian uncoupling protein (avUCP, an ortholog of mammalian UCP2 and UCP3) gene and some known mammalian beige adipocyte-specific markers. Morphological characteristics of white adipose tissues of treated chickens showed increased numbers of both small and larger clusters of multilocular fat cells within the tissues. Increases in protein levels of avUCP and mitochondrial marker protein, voltage-dependent anion channel, and immunohistochemical analysis for subcutaneous neck fat revealed the presence of potentially thermogenic mitochondria-rich cells. This is the first evidence that the capacity for thermogenesis may be acquired by differentiating adipose tissue into beige-like fat for maintaining temperature homeostasis in the subcutaneous fat 'neck warmer' in chickens exposed to a cold environment.

Asunto(s)

Aclimatación/fisiología , Pollos/fisiología , Grasa Subcutánea/metabolismo , Grasa Abdominal/citología , Grasa Abdominal/metabolismo , Adipocitos Beige/metabolismo , Tejido Adiposo/metabolismo , Animales , Peso Corporal , Frío , Ingestión de Alimentos , Mitocondrias/metabolismo , Cuello/fisiología , Grasa Subcutánea/citología , Grasa Subcutánea/efectos de los fármacos , Termogénesis/efectos de los fármacos , Triyodotironina/farmacología , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismo

RESUMEN

Inactivation of constitutive autophagy results in the formation of cytoplasmic inclusions in neurones, but the relationship between impaired autophagy and Lewy bodies (LBs) remains unknown. α-Synuclein and p62, components of LBs, are the defining characteristic of Parkinson's disease (PD). Until now, we have analyzed mice models and demonstrated p62 aggregates derived from an autophagic defect might serve as 'seeds' and can potentially be a cause of LB formation. P62 may be the key molecule for aggregate formation. To understand the mechanisms of LBs, we analyzed p62 homeostasis and inclusion formation using PD model mice. In PARK22-linked PD, intrinsically disordered mutant CHCHD2 initiates Lewy pathology. To determine the function of CHCHD2 for inclusions formation, we generated Chchd2-knockout (KO) mice and characterized the age-related pathological and motor phenotypes. Chchd2 KO mice exhibited p62 inclusion formation and dopaminergic neuronal loss in an age-dependent manner. These changes were associated with a reduction in mitochondria complex activity and abrogation of inner mitochondria structure. In particular, the OPA1 proteins, which regulate fusion of mitochondrial inner membranes, were immature in the mitochondria of CHCHD2-deficient mice. CHCHD2 regulates mitochondrial morphology and p62 homeostasis by controlling the level of OPA1. Our findings highlight the unexpected role of the homeostatic level of p62, which is regulated by a non-autophagic system, in controlling intracellular inclusion body formation, and indicate that the pathologic processes associated with the mitochondrial proteolytic system are crucial for loss of DA neurones.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Homeostasis , Cuerpos de Inclusión/patología , Cuerpos de Lewy/patología , Mitocondrias/patología , Enfermedad de Parkinson/patología , Proteína Sequestosoma-1/metabolismo , Factores de Transcripción/fisiología , Animales , Autofagia , Modelos Animales de Enfermedad , Cuerpos de Inclusión/metabolismo , Cuerpos de Lewy/genética , Cuerpos de Lewy/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Proteína Sequestosoma-1/genética

RESUMEN

1-Methyl-4-phenylpyridinium (MPP+)-treated human neuroblastoma SH-SY5Y cells have been generally accepted as a cellular model for Parkinson's disease. This article contains metabolic analysis data of not only cell lysate but also culture supernatants to understand comprehensive metabolic disturbances in this model. Metabolic analysis employed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Data obtained by CE-TOFMS were processed to extract peak information including m/z, peak area, and migration time. The data provided in this manuscript have been analyzed and discussed in the research article entitled "Metabolomic analysis revealed mitochondrial dysfunction and aberrant choline metabolism in MPP+-exposed SH-SY5Y cells" [1].

RESUMEN

The effect of adding ethylammonium nitrate (EAN), which is an ionic liquid (IL), on the aggregate formation of α-synuclein (α-Syn) in aqueous solution has been investigated. FTIR and Raman spectroscopy were used to investigate changes in the secondary structure of α-Syn and in the states of water molecules and EAN. The results presented here show that the addition of EAN to α-Syn causes the formation of an intermolecular ß-sheet structure in the following manner: native disordered state → polyproline II (PPII)-helix → intermolecular ß-sheet (α-Syn amyloid-like aggregates: α-SynA). Although cations and anions of EAN play roles in masking the charged side chains and PPII-helix-forming ability involved in the formation of α-SynA, water molecules are not directly related to its formation. We conclude that EAN-induced α-Syn amyloid-like aggregates form at hydrophobic associations in the middle of the molecules after masking the charged side chains at the N- and C-terminals of α-Syn.

Asunto(s)

Agregado de Proteínas , Compuestos de Amonio Cuaternario/química , alfa-Sinucleína/química , Amiloide/síntesis química , Amiloide/química , Precipitación Química/efectos de los fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Agregado de Proteínas/efectos de los fármacos , Conformación Proteica en Lámina beta/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Estructura Secundaria de Proteína , Compuestos de Amonio Cuaternario/farmacología , Espectroscopía Infrarroja por Transformada de Fourier , Espectrometría Raman , Agua/química

RESUMEN

1-Methyl-4-phenylpyridinium (MPP+)-treated human neuroblastoma SH-SY5Y cells have been generally accepted as a cellular model for Parkinson's disease. To understand comprehensive metabolic disturbances in this model, both cell lysates and culture supernatants were subjected to metabolomic analysis. As expected from the fact that MPP+ inhibits mitochondrial complex I, a metabolic shift from mitochondrial oxidative phosphorylation to glycolysis was indicated by an increase in extracellular lactic acid and a parallel depletion of pyruvic acid. In cell lysates, the metabolic shift was supported by consistent decreases in TCA cycle intermediates. Metabolomic analysis also revealed aberrant choline metabolism. Choline in the culture supernatant was elevated 8.5- and 17-fold by 30 and 300 µM MPP+ exposure, respectively; therefore, extracellular choline might be a metabolic biomarker for Parkinson's disease.

Asunto(s)

1-Metil-4-fenilpiridinio/farmacología , Colina/antagonistas & inhibidores , Metabolómica , Mitocondrias/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colina/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Relación Estructura-Actividad , Células Tumorales Cultivadas

RESUMEN

Iron is an essential nutrient for mitochondrial metabolic processes, including mitochondrial respiration. Ferritin complexes store excess iron and protect cells from iron toxicity. Therefore, iron stored in the ferritin complex might be utilized under iron-depleted conditions. In this study, we show that the inhibition of lysosome-dependent protein degradation by bafilomycin A1 and the knockdown of NCOA4, an autophagic receptor for ferritin, reduced mitochondrial respiration, respiratory chain complex assembly, and membrane potential under iron-sufficient conditions. However, autophagy did not contribute to degradation of the ferritin complex under iron-sufficient conditions. Knockout of the ferritin light chain, a subunit of the ferritin complex, inhibited ferritin degradation by decreasing interactions with NCOA4. However, ferritin light chain knockout did not affect mitochondrial functions under iron-sufficient conditions, and ferritin light chain knockout cells showed a rapid reduction of mitochondrial functions compared with wild-type cells under iron-depleted conditions. These results indicate that the constitutive degradation of the ferritin complex contributes to the maintenance of mitochondrial functions.

Asunto(s)

Ferritinas/química , Hierro/metabolismo , Lisosomas/metabolismo , Mitocondrias/metabolismo , Coactivadores de Receptor Nuclear/metabolismo , Autofagia , Respiración de la Célula/efectos de los fármacos , Ferritinas/genética , Ferritinas/metabolismo , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Macrólidos/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Coactivadores de Receptor Nuclear/genética , Proteolisis/efectos de los fármacos

RESUMEN

Mitochondrial content is regarded a useful feature to distinguish muscle-fiber types in terms of energy metabolism in skeletal muscles. Increasing evidence suggests that specific mitochondrial bioenergetic phenotypes exist in metabolically different muscle fibers. A few studies have examined the energetic properties of skeletal muscle in domestic fowls; however, no information on muscle bioenergetics in broiler chickens selectively bred for faster growth is available. In this study, we aimed to characterize the mitochondrial contents and functions of chicken skeletal muscle consisting entirely of type I (oxidative) (M. pubo-ischio-femoralis pars medialis), type IIA (glycolytic/oxidative) (M. pubo-ischio-femoralis pars lateralis), and type IIB (glycolytic) (M. pectoralis) muscle fibers. Citrate synthase (CS) activity was the highest in type IIA muscle tissues and isolated mitochondria, among the muscle tissues tested. Although no difference was registered in mitochondrial CS activity between type IIB and type I muscles, tissue CS activity was significantly higher in the latter. Histochemical staining for NADH tetrazolium reductase and the ratio of muscle-tissue to mitochondrial CS activity indicated that type I, type IIA, and type IIB muscle-fiber types showed decreasing mitochondrial content. Mitochondria from type I muscle exhibited a higher coupled respiration rate induced by pyruvate/malate, palmitoyl-CoA/malate, and palmitoyl-carnitine, as respiratory substrates, than type IIB-muscle mitochondria, while the response of mitochondria from type IIA muscle to those substrates was comparable to that of mitochondria from type I muscle. Type IIA-muscle mitochondria exhibited the highest carnitine palmitoyltransferase-2 level among all tissues tested, which may contribute to the higher fatty acid oxidation in these mitochondria. The results suggest that mitochondrial abundance is one of the features differentiating metabolic characteristics of different chicken skeletal muscle types. Moreover, the study demonstrated that type IIA-muscle mitochondria may have distinct metabolic capacities.

RESUMEN

Increasing evidence shows that metabolic abnormalities in body fluids are distinguishing features of the pathophysiology of Parkinson's disease. However, a non-invasive approach has not been established in the earliest or pre-symptomatic phases. Here, we report comprehensive double-cohort analyses of the metabolome using capillary electrophoresis/liquid chromatography mass-spectrometry. The plasma analyses identified 18 Parkinson's disease-specific metabolites and revealed decreased levels of seven long-chain acylcarnitines in two Parkinson's disease cohorts (n = 109, 145) compared with controls (n = 32, 45), respectively. Furthermore, statistically significant decreases in five long-chain acylcarnitines were detected in Hoehn and Yahr stage I. Likewise, decreased levels of acylcarnitine(16:0), a decreased ratio of acylcarnitine(16:0) to fatty acid(16:0), and an increased index of carnitine palmitoyltransferase 1 were identified in Hoehn and Yahr stage I of both cohorts, suggesting of initial ß-oxidation suppression. Receiver operating characteristic curves produced using 12-14 long-chain acylcarnitines provided a large area of under the curve, high specificity and moderate sensitivity for diagnosing Parkinson's disease. Our data demonstrate that a primary decrement of mitochondrial ß-oxidation and that 12-14 long-chain acylcarnitines decreases would be promising diagnostic biomarkers for Parkinson's disease.

Asunto(s)

Carnitina/análogos & derivados , Oxidación-Reducción , Enfermedad de Parkinson/diagnóstico , Enfermedad de Parkinson/metabolismo , Anciano , Biomarcadores , Carnitina/metabolismo , Estudios de Cohortes , Ácidos Grasos/metabolismo , Femenino , Humanos , Masculino , Metaboloma , Metabolómica/métodos , Persona de Mediana Edad , Mitocondrias/metabolismo , Modelos Biológicos , Músculo Esquelético/metabolismo , Curva ROC , Índice de Severidad de la Enfermedad

RESUMEN

A number of alternations in mitochondrial DNA (mtDNA) have been reported in different types of cancers, and the role of mtDNA in cancer has been attracting increasing interest. In order to investigate the relationship between mtDNA alternations and chemosensitivity, we constructed cybrid (trans-mitochondrial hybrid) cell lines carrying a HeLa nucleus and the mtDNA of healthy individuals because of the presence of somatic alternations in the mtDNA of many cancer cells. After a treatment with 1.0 µg/mL cisplatin for 10 days, we isolated 100 cisplatin-resistant clones, 70 of which carried the shorter mtDNA OriB variant (16184-16193 poly-cytosine tract), which was located in the control region of mtDNA. Whole mtDNA sequencing of 10 clones revealed no additional alternations. Re-construction of the HeLa nucleus and mtDNA from cisplatin-resistant cells showed that cisplatin resistance was only acquired by mtDNA alternations in the control region, and not by possible alternation(s) in the nuclear genome.

Asunto(s)

Cisplatino/farmacología , ADN Mitocondrial/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Variación Genética , Antineoplásicos/farmacología , Secuencia de Bases , Separación Celular , Supervivencia Celular/efectos de los fármacos , Citosina/metabolismo , Fluorouracilo/farmacología , Células HeLa , Humanos , Células Híbridas/efectos de los fármacos , Células Híbridas/metabolismo , Oxidorreductasas/metabolismo , Análisis de Secuencia de ADN

RESUMEN

BACKGROUND: Identification of causative genes in mendelian forms of Parkinson's disease is valuable for understanding the cause of the disease. We did genetic studies in a Japanese family with autosomal dominant Parkinson's disease to identify novel causative genes. METHODS: We did a genome-wide linkage analysis on eight affected and five unaffected individuals from a family with autosomal dominant Parkinson's disease (family A). Subsequently, we did exome sequencing on three patients and whole-genome sequencing on one patient in family A. Variants were validated by Sanger sequencing in samples from patients with autosomal dominant Parkinson's disease, patients with sporadic Parkinson's disease, and controls. Participants were identified from the DNA bank of the Comprehensive Genetic Study on Parkinson's Disease and Related Disorders (Juntendo University School of Medicine, Tokyo, Japan) and were classified according to clinical information obtained by neurologists. Splicing abnormalities of CHCHD2 mutants were analysed in SH-SY5Y cells. We used the Fisher's exact test to calculate the significance of allele frequencies between patients with sporadic Parkinson's disease and unaffected controls, and we calculated odds ratios and 95% CIs of minor alleles. FINDINGS: We identified a missense mutation (CHCHD2, 182C>T, Thr61Ile) in family A by next-generation sequencing. We obtained samples from a further 340 index patients with autosomal dominant Parkinson's disease, 517 patients with sporadic Parkinson's disease, and 559 controls. Three CHCHD2 mutations in four of 341 index cases from independent families with autosomal dominant Parkinson's disease were detected by CHCHD2 mutation screening: 182C>T (Thr61Ile), 434G>A (Arg145Gln), and 300+5G>A. Two single nucleotide variants (-9T>G and 5C>T) in CHCHD2 were confirmed to have different frequencies between sporadic Parkinson's disease and controls, with odds ratios of 2·51 (95% CI 1·48-4·24; p=0·0004) and 4·69 (1·59-13·83, p=0·0025), respectively. One single nucleotide polymorphism (rs816411) was found in CHCHD2 from a previously reported genome-wide association study; however, there was no significant difference in its frequency between patients with Parkinson's disease and controls in a previously reported genome-wide association study (odds ratio 1·17, 95% CI 0·96-1·19; p=0·22). In SH-SY5Y cells, the 300+5G>A mutation but not the other two mutations caused exon 2 skipping. INTERPRETATION: CHCHD2 mutations are associated with, and might be a cause of, autosomal dominant Parkinson's disease. Further genetic studies in other populations are needed to confirm the pathogenicity of CHCHD2 mutations in autosomal dominant Parkinson's disease and susceptibility for sporadic Parkinson's disease, and further functional studies are needed to understand how mutant CHCHD2 might play a part in the pathophysiology of Parkinson's disease. FUNDING: Japan Society for the Promotion of Science; Japanese Ministry of Education, Culture, Sports, Science and Technology; Japanese Ministry of Health, Labour and Welfare; Takeda Scientific Foundation; Cell Science Research Foundation; and Nakajima Foundation.

Asunto(s)

Ligamiento Genético/genética , Estudio de Asociación del Genoma Completo/métodos , Proteínas Mitocondriales/genética , Mutación Missense/genética , Trastornos Parkinsonianos/genética , Análisis de Secuencia de ADN/métodos , Factores de Transcripción/genética , Edad de Inicio , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Proteínas de Unión al ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Trastornos Parkinsonianos/diagnóstico , Linaje

RESUMEN

Mutations in PTEN-induced putative kinase 1 (PINK1) cause recessive forms of Parkinson's disease (PD). PINK1 acts upstream of parkin, regulating mitochondrial elimination (mitophagy) in cultured cells treated with mitochondrial uncouplers that cause mitochondrial depolarization. PINK1 loss-of-function decreases mitochondrial membrane potential, resulting in mitochondrial dysfunction, although the exact function of PINK1 in mitochondria has not been fully elucidated. We have previously found that PINK1 deficiency causes a decrease in mitochondrial membrane potential, which is not due to a proton leak, but to respiratory chain defects. Here, we examine mitochondrial respiratory chain defects in PINK1-deficient cells, and find both complex I and complex III are defective. These results suggest that mitochondrial respiratory chain defects may be associated with PD pathogenesis caused by mutations in the PINK1 gene.

Asunto(s)

Mitocondrias/metabolismo , Proteínas Quinasas/metabolismo , Animales , Células Cultivadas , Transporte de Electrón , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Fibroblastos/metabolismo , Ratones Noqueados , Canales Aniónicos Dependientes del Voltaje/metabolismo

RESUMEN

The mitochondrial ubiquitin ligase MITOL regulates mitochondrial dynamics. We report here that MITOL regulates mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) domain formation through mitofusin2 (Mfn2). MITOL interacts with and ubiquitinates mitochondrial Mfn2, but not ER-associated Mfn2. Mutation analysis identified a specific interaction between MITOL C-terminal domain and Mfn2 HR1 domain. MITOL mediated lysine-63-linked polyubiquitin chain addition to Mfn2, but not its proteasomal degradation. MITOL knockdown inhibited Mfn2 complex formation and caused Mfn2 mislocalization and MAM dysfunction. Sucrose-density gradient centrifugation and blue native PAGE retardation assay demonstrated that MITOL is required for GTP-dependent Mfn2 oligomerization. MITOL knockdown reduced Mfn2 GTP binding, resulting in reduced GTP hydrolysis. We identified K192 in the GTPase domain of Mfn2 as a major ubiquitination site for MITOL. A K192R mutation blocked oligomerization even in the presence of GTP. Taken together, these results suggested that MITOL regulates ER tethering to mitochondria by activating Mfn2 via K192 ubiquitination.

Asunto(s)

Retículo Endoplásmico/metabolismo , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/fisiología , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Animales , GTP Fosfohidrolasas/análisis , Células HeLa , Humanos , Proteínas de la Membrana , Ratones , Proteínas Mitocondriales/análisis , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación

RESUMEN

RATIONALE: Caloric restriction (CR) confers cardioprotection against ischemia/reperfusion injury. However, the exact mechanism(s) underlying CR-induced cardioprotection remain(s) unknown. Recent evidence indicates that Sirtuins, NAD(+)-dependent deacetylases, regulate various favorable aspects of the CR response. Thus, we hypothesized that deacetylation of specific mitochondrial proteins during CR preserves mitochondrial function and attenuates production of reactive oxygen species during ischemia/reperfusion. OBJECTIVE: The objectives of the present study were (1) to investigate the effect of CR on mitochondrial function and mitochondrial proteome and (2) to investigate what molecular mechanisms mediate CR-induced cardioprotection. METHODS AND RESULTS: Male 26-week-old Fischer344 rats were randomly divided into ad libitum-fed and CR (40% reduction) groups for 6 months. No change was observed in basal mitochondrial function, but CR preserved postischemic mitochondrial respiration and attenuated postischemic mitochondrial H(2)O(2) production. CR decreased the level of acetylated mitochondrial proteins that were associated with enhanced Sirtuin activity in the mitochondrial fraction. We confirmed a significant decrease in the acetylated forms of NDUFS1 and cytochrome bc1 complex Rieske subunit in the CR heart. Low-dose resveratrol treatment mimicked the effect of CR on deacetylating them and attenuated reactive oxygen species production during anoxia/reoxygenation in cultured cardiomyocytes without changing the expression levels of manganese superoxide dismutase. Treatment with nicotinamide completely abrogated the effect of low-dose resveratrol. CONCLUSIONS: These results strongly suggest that CR primes mitochondria for stress resistance by deacetylating specific mitochondrial proteins of the electron transport chain. Targeted deacetylation of NDUFS1 and/or Rieske subunit might have potential as a novel therapeutic approach for cardioprotection against ischemia/reperfusion.

Asunto(s)

Restricción Calórica , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Mitocondrias Cardíacas/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/metabolismo , Estrés Oxidativo , Sirtuinas/metabolismo , Acetilación , Animales , Antioxidantes/farmacología , Western Blotting , Células Cultivadas , Modelos Animales de Enfermedad , Complejo III de Transporte de Electrones/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Mitocondrias Cardíacas/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Daño por Reperfusión Miocárdica/metabolismo , Miocitos Cardíacos/efectos de los fármacos , NAD/metabolismo , NADH Deshidrogenasa/metabolismo , Niacinamida/farmacología , Estrés Oxidativo/efectos de los fármacos , Proteómica , Ratas , Ratas Endogámicas F344 , Resveratrol , Estilbenos/farmacología

RESUMEN

Somatic mutations of mitochondrial DNA (mtDNA) have been reported in different types of cancers and are suggested to play roles in metastasis, cancer development and response to anticancer agents. To predict potential roles of mtDNA alterations in colorectal cancer, we determined the entire mtDNA sequence of eleven human-derived colorectal cancer cell lines and compared with the revised Cambridge Reference Sequence to identify nucleotide alterations. Four homoplasmic and six heteroplasmic alterations were found to be novel. Among them, homoplasmic G6709A (MT-CO1) and G14804A (MT-CYB) alterations cause amino acid changes in the highly conserved residues. Heteroplasmic G1576A (MT-RNR1) and G2975A (MT-RNR2) alterations are expected to make the stem structure of mitochondrial ribosomal RNAs unstable. These nucleotide alterations are candidates that could play important roles in cancer.

Asunto(s)

ADN Mitocondrial/genética , Proteínas Mitocondriales/genética , Mutación , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Citocromos b/genética , ADN Mitocondrial/química , Complejo IV de Transporte de Electrones/genética , Humanos , Datos de Secuencia Molecular , Mutación Puntual , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido

RESUMEN

Meat-type chickens show high feed efficiency and have a very rapid growth rate compared with laying-type chickens. To clarify whether the type-specific difference in feed conversion efficiency is involved in mitochondrial bioenergetics, modular kinetic analysis was applied to oxidative phosphorylation in skeletal muscle mitochondria of both type chickens. Mitochondria from skeletal muscle of meat-type chickens showed greater substrate oxidation and phosphorylating activities, and less proton leak than those of the laying-type, resulting in a higher efficiency of oxidative phosphorylation. Gene expression and protein content of uncoupling protein (avUCP) but not adenine nucleotide translocase (avANT) gene expression were lower in skeletal muscle mitochondria of meat-type chickens than the laying-type. The current results regarding a higher efficiency of oxidative phosphorylation and UCP content may partially support the high feed efficiency of meat-type chickens.

Asunto(s)

Pollos/metabolismo , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Fosforilación Oxidativa , Animales , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Peso Corporal , Pollos/clasificación , Pollos/genética , Conducta Alimentaria , Expresión Génica , Immunoblotting , Cinética , Masculino , Carne , Potencial de la Membrana Mitocondrial , Translocasas Mitocondriales de ADP y ATP/genética , Translocasas Mitocondriales de ADP y ATP/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas Desacopladoras Mitocondriales , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Mutations in PTEN-induced putative kinase 1 (PINK1) cause a recessive form of Parkinson's disease (PD). PINK1 is associated with mitochondrial quality control and its partial knock-down induces mitochondrial dysfunction including decreased membrane potential and increased vulnerability against mitochondrial toxins, but the exact function of PINK1 in mitochondria has not been investigated using cells with null expression of PINK1. Here, we show that loss of PINK1 caused mitochondrial dysfunction. In PINK1-deficient (PINK1(-/-)) mouse embryonic fibroblasts (MEFs), mitochondrial membrane potential and cellular ATP levels were decreased compared with those in littermate wild-type MEFs. However, mitochondrial proton leak, which reduces membrane potential in the absence of ATP synthesis, was not altered by loss of PINK1. Instead, activity of the respiratory chain, which produces the membrane potential by oxidizing substrates using oxygen, declined. H(2)O(2) production rate by PINK1(-/-) mitochondria was lower than PINK1(+/+) mitochondria as a consequence of decreased oxygen consumption rate, while the proportion (H(2)O(2) production rate per oxygen consumption rate) was higher. These results suggest that mitochondrial dysfunctions in PD pathogenesis are caused not by proton leak, but by respiratory chain defects.

Asunto(s)

Respiración de la Célula , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , Proteínas Quinasas/deficiencia , Protones , Animales , Respiración de la Célula/genética , Células Cultivadas , Fibroblastos/metabolismo , Potencial de la Membrana Mitocondrial/genética , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/patología , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/patología , Neuronas/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Proteínas Quinasas/genética

RESUMEN

We previously showed that heat stress stimulates reactive oxygen species (ROS) production in skeletal muscle mitochondria of birds, probably via an elevation in mitochondrial membrane potential (DeltaPsi). To clarify the mechanism underlying the elevation of DeltaPsi, modular kinetic analysis was applied to oxidative phosphorylation in skeletal muscle mitochondria of heat-stressed birds (34 degrees C for 12h). In the birds exposed to heat stress, 'substrate oxidation' (a DeltaPsi-producer) was increased compared to control (24 degrees C) birds, although there was little difference in 'proton leak' (a DeltaPsi-consumer), suggesting that an elevation in the DeltaPsi at state 4 may be due to enhanced substrate oxidation. It thus appears that enhanced substrate oxidation plays a crucial role in the overproduction of ROS for heat-stressed birds, probably via elevated DeltaPsi.

Asunto(s)

Trastornos de Estrés por Calor/metabolismo , Calor , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Fosforilación Oxidativa , Animales , Aves/metabolismo , Cinética , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Oxidación-Reducción , Protones , Especies Reactivas de Oxígeno/metabolismo