RESUMEN
Dengue virus (DENV) infection is known to affect host cell metabolism, but the molecular players involved are still poorly known. Using a proteomics approach, we identified six DENV proteins associated with mitochondria isolated from infected hepatocytes, and most of the peptides identified were from NS3. We also found an at least twofold decrease of several electron transport system (ETS) host proteins. Thus, we investigated whether NS3 could modulate the ETS function by incubating recombinant DENV NS3 constructs in mitochondria isolated from mouse liver. We found that NS3pro (NS3 protease domain), but not the correspondent catalytically inactive mutant (NS3proS135A), impairs complex I (CI)-dependent NADH:ubiquinone oxidoreductase activity, but not the activities of complexes II, III, IV, or V. Accordingly, using high-resolution respirometry, we found that both NS3pro and full-length NS3 decrease the respiratory rates associated with malate/pyruvate oxidation in mitochondria. The NS3-induced impairment in mitochondrial respiration occurs without altering either leak respiration or mitochondria's capacity to maintain membrane potential, suggesting that NS3 does not deeply affect mitochondrial integrity. Remarkably, CI activity is also inhibited in DENV-infected cells, supporting that the NS3 effects observed in isolated mitochondria may be relevant in the context of the infection. Finally, in silico analyses revealed the presence of potential NS3 cleavage sites in 17 subunits of mouse CI and 16 subunits of human CI, most of them located on the CI surface, suggesting that CI is prone to undergo proteolysis by NS3. Our findings suggest that DENV NS3 can modulate mitochondrial bioenergetics by directly affecting CI function. IMPORTANCE: Dengue virus (DENV) infection is a major public health problem worldwide, affecting about 400 million people yearly. Despite its importance, many molecular aspects of dengue pathogenesis remain poorly known. For several years, our group has been investigating DENV-induced metabolic alterations in the host cells, focusing on the bioenergetics of mitochondrial respiration. The results of the present study reveal that the DENV non-structural protein 3 (NS3) is found in the mitochondria of infected cells, impairing mitochondrial respiration by directly targeting one of the components of the electron transport system, the respiratory complex I (CI). NS3 acts as the viral protease during the DENV replication cycle, and its proteolytic activity seems necessary for inhibiting CI function. Our findings uncover new nuances of DENV-induced metabolic alterations, highlighting NS3 as an important player in the modulation of mitochondria function during infection.
Asunto(s)
Virus del Dengue , Complejo I de Transporte de Electrón , Mitocondrias , Proteínas no Estructurales Virales , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Animales , Virus del Dengue/fisiología , Virus del Dengue/genética , Ratones , Complejo I de Transporte de Electrón/metabolismo , Complejo I de Transporte de Electrón/genética , Humanos , Mitocondrias/metabolismo , Hepatocitos/virología , Hepatocitos/metabolismo , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Dengue/virología , Dengue/metabolismo , Respiración de la Célula , Proteómica , Proteasas ViralesRESUMEN
The respiratory chain alternative enzymes (AEs) NDX and AOX from the tunicate Ciona intestinalis (Ascidiacea) have been xenotopically expressed and characterized in human cells in culture and in the model organisms Drosophila melanogaster and mouse, with the purpose of developing bypass therapies to combat mitochondrial diseases in human patients with defective complexes I and III/IV, respectively. The fact that the genes coding for NDX and AOX have been lost from genomes of evolutionarily successful animal groups, such as vertebrates and insects, led us to investigate if the composition of the respiratory chain of Ciona and other tunicates differs significantly from that of humans and Drosophila, to accommodate the natural presence of AEs. We have failed to identify in tunicate genomes fifteen orthologous genes that code for subunits of the respiratory chain complexes; all of these putatively missing subunits are peripheral to complexes I, III and IV in mammals, and many are important for complex-complex interaction in supercomplexes (SCs), such as NDUFA11, UQCR11 and COX7A. Modeling of all respiratory chain subunit polypeptides of Ciona indicates significant structural divergence that is consistent with the lack of these fifteen clear orthologous subunits. We also provide evidence using Ciona AOX expressed in Drosophila that this AE cannot access the coenzyme Q pool reduced by complex I, but it is readily available to oxidize coenzyme Q molecules reduced by glycerophosphate oxidase, a mitochondrial inner membrane-bound dehydrogenase that is not involved in SCs. Altogether, our results suggest that Ciona AEs might have evolved in a mitochondrial inner membrane environment much different from that of mammals and insects, possibly without SCs; this correlates with the preferential functional interaction between these AEs and non-SC dehydrogenases in heterologous mammalian and insect systems. We discuss the implications of these findings for the applicability of Ciona AEs in human bypass therapies and for our understanding of the evolution of animal respiratory chain.
Asunto(s)
Ciona intestinalis , Proteínas Mitocondriales , Fosforilación Oxidativa , Animales , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Ciona intestinalis/genética , Ciona intestinalis/enzimología , Humanos , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Subunidades de Proteína/metabolismo , Subunidades de Proteína/genética , Drosophila melanogaster/genética , Drosophila melanogaster/enzimología , Urocordados/genética , Urocordados/enzimología , Transporte de Electrón , Complejo I de Transporte de Electrón/metabolismo , Complejo I de Transporte de Electrón/genética , Filogenia , Proteínas de PlantasRESUMEN
More than 100 years since the first description of Chagas Disease and with over 29,000 new cases annually due to vector transmission (in 2010), American Trypanosomiasis remains a Neglected Tropical Disease (NTD). This study presents the most comprehensive Trypanosoma cruzi sampling in terms of geographic locations and triatomine species analyzed to date and includes both nuclear and mitochondrial genomes. This addresses the gap of information from North and Central America. We incorporate new and previously published DNA sequence data from two mitochondrial genes, Cytochrome oxidase II (COII) and NADH dehydrogenase subunit 1 (ND1). These T. cruzi samples were collected over a broad geographic range including 111 parasite DNA samples extracted from triatomines newly collected across North and Central America, all of which were infected with T. cruzi in their natural environment. In addition, we present parasite reduced representation (Restriction site Associated DNA markers, RAD-tag) genomic nuclear data combined with the mitochondrial gene sequences for a subset of the triatomines (27 specimens) collected from Guatemala and El Salvador. Our mitochondrial phylogenetic reconstruction revealed two of the major mitochondrial lineages circulating across North and Central America, as well as the first ever mitochondrial data for TcBat from a triatomine collected in Central America. Our data also show that within mtTcIII, North and Central America represent an independent, distinct clade from South America, named here as mtTcIIINA-CA, geographically restricted to North and Central America. Lastly, the most frequent lineage detected across North and Central America, mtTcI, was also an independent, distinct clade from South America, noted as mtTcINA-CA. Furthermore, nuclear genome data based on Single Nucleotide Polymorphism (SNP) showed genetic structure of lineage TcI from specimens collected in Guatemala and El Salvador supporting the hypothesis that genetic diversity at a local scale has a geographical component. Our multiscale analysis contributes to the understanding of the independent and distinct evolution of T. cruzi lineages in North and Central America regions.
Asunto(s)
Enfermedad de Chagas/parasitología , Mitocondrias/genética , Trypanosoma cruzi/clasificación , Trypanosoma cruzi/aislamiento & purificación , América Central , 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 , Humanos , Mitocondrias/metabolismo , Filogenia , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , América del Sur , Trypanosoma cruzi/genéticaRESUMEN
The impairment of the CFTR channel activity, a cAMP-activated chloride (Cl-) channel responsible for cystic fibrosis (CF), has been associated with a variety of mitochondrial alterations such as modified gene expression, impairment in oxidative phosphorylation, increased reactive oxygen species (ROS), and a disbalance in calcium homeostasis. The mechanisms by which these processes occur in CF are not fully understood. Previously, we demonstrated a reduced MTND4 expression and a failure in the mitochondrial complex I (mCx-I) activity in CF cells. Here we hypothesized that the activity of CFTR might modulate the mitochondrial fission/fusion balance, explaining the decreased mCx-I. The mitochondrial morphology and the levels of mitochondrial dynamic proteins MFN1 and DRP1 were analysed in IB3-1 CF cells, and S9 (IB3-1 expressing wt-CFTR), and C38 (IB3-1 expressing a truncated functional CFTR) cells. The mitochondrial morphology of IB3-1 cells compared to S9 and C38 cells showed that the impaired CFTR activity induced a fragmented mitochondrial network with increased rounded mitochondria and shorter branches. Similar results were obtained by using the CFTR pharmacological inhibitors CFTR(inh)-172 and GlyH101 on C38 cells. These morphological changes were accompanied by modifications in the levels of the mitochondrial dynamic proteins MFN1, DRP1, and p(616)-DRP1. IB3-1 CF cells treated with Mdivi-1, an inhibitor of mitochondrial fission, restored the mCx-I activity to values similar to those seen in S9 and C38 cells. These results suggest that the mitochondrial fission/fusion balance is regulated by the CFTR activity and might be a potential target to treat the impaired mCx-I activity in CF.
Asunto(s)
Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Células Epiteliales/patología , Mitocondrias/patología , Dinámicas Mitocondriales , Mutación , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Células Epiteliales/metabolismo , Humanos , Transporte Iónico , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fosforilación Oxidativa , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: Mitochondria play a significant role in plant cytoplasmic male sterility (CMS). In our previous study, mitochondrial complex I genes, nad4, nad5, and nad7 showed polymorphisms between the transgenic CMS line M2BS and its wild type M2B. The sterility mechanism of the M2BS at cytological, physiological, biochemical, and molecular level is not clear. RESULTS: Cytological observation showed that the anthers were light yellow, fissured, invalid in KI-I2, and full of irregularly typical abortion pollen grains in M2BS. Transmission electron microscopic (TEM) observation revealed no nucleus and degraded mitochondria with obscure cristae in anther cells of M2BS. The results of staining for H2O2 presented a large number of electron dense precipitates (edp) in intercellular space of anther cells of M2BS at anthesis. Moreover, the anther respiration rate and complex I activity of M2BS were significantly lower than those of wild type M2B during pollen development. Furthermore, RNA editing results showed only nad7 presented partially edited at 534th nucleotides. The expression of nad5 and nad7 revealed significant differences between M2B and M2BS. CONCLUSIONS: Our data demonstrated that mitochondrial structural degradation and complex I deficiency might be associated with transgenic CMS of rice.
Asunto(s)
Complejo I de Transporte de Electrón/genética , Mitocondrias/patología , Oryza , Infertilidad Vegetal , Regulación de la Expresión Génica de las Plantas , Peróxido de Hidrógeno , Mitocondrias/ultraestructura , Oryza/genética , Plantas Modificadas GenéticamenteRESUMEN
Diatoms are unicellular organisms containing red algal-derived plastids that probably originated as result of serial endosymbioses between an ancestral heterotrophic organism and a red alga or cryptophyta algae from which has only the chloroplast left. Diatom mitochondria are thus believed to derive from the exosymbiont. Unlike animals and fungi, diatoms seem to contain ancestral respiratory chains. In support of this, genes encoding gamma type carbonic anhydrases (CAs) whose products were shown to be intrinsic complex I subunits in plants, Euglena and Acanthamoeba were found in diatoms, a representative of Stramenopiles. In this work, we experimentally show that mitochondrial complex I in diatoms is a large complex containing gamma type CA subunits, supporting an ancestral origin. By using a bioinformatic approach, a complex I integrated CA domain with heterotrimeric subunit composition is proposed.
Asunto(s)
Anhidrasas Carbónicas/metabolismo , Diatomeas/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Mitocondrias/metabolismo , Secuencia de Aminoácidos , Anhidrasas Carbónicas/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Diatomeas/genética , Complejo I de Transporte de Electrón/genética , Evolución Molecular , Mitocondrias/genética , Filogenia , RNA-Seq , Rhodophyta/genética , Alineación de Secuencia , Simbiosis/genéticaRESUMEN
BACKGROUND: Mitochondria play a significant role in plant cytoplasmic male sterility (CMS). In our previous study, mitochondrial complex I genes, nad4, nad5, and nad7 showed polymorphisms between the transgenic CMS line M2BS and its wild type M2B. The sterility mechanism of the M2BS at cytological, physiological, biochemical, and molecular level is not clear. RESULTS: Cytological observation showed that the anthers were light yellow, fissured, invalid in KI-I2, and full of irregularly typical abortion pollen grains in M2BS. Transmission electron microscopic (TEM) observation revealed no nucleus and degraded mitochondria with obscure cristae in anther cells of M2BS. The results of staining for H2O2 presented a large number of electron dense precipitates (edp) in intercellular space of anther cells of M2BS at anthesis. Moreover, the anther respiration rate and complex I activity of M2BS were significantly lower than those of wild type M2B during pollen development. Furthermore, RNA editing results showed only nad7 presented partially edited at 534th nucleotides. The expression of nad5 and nad7 revealed significant differences between M2B and M2BS. CONCLUSIONS: Our data demonstrated that mitochondrial structural degradation and complex I deficiency might be associated with transgenic CMS of rice.
Asunto(s)
Oryza/genética , Complejo I de Transporte de Electrón/genética , Infertilidad Vegetal , Mitocondrias/patología , Plantas Modificadas Genéticamente , Regulación de la Expresión Génica de las Plantas , Peróxido de Hidrógeno , Mitocondrias/ultraestructuraRESUMEN
BACKGROUND: Kidney cancer is one of the most common cancers in the world. It is necessary to clarify its underlying mechanism and find its prognostic biomarkers. Current studies showed that SHMT2 may be participated in several kinds of cancer. METHODS: Our studies investigated the expression of SHMT2 in kidney cancer by Oncomine, Human Protein Atlas database and ULCAN database. Meanwhile, we found its co-expression gene by cBioPortal online tool and validated their relationship in A498 and ACHN cells by cell transfection, western blot and qRT-PCR. Besides these, we also explored their prognostic values via the Kaplan-Meier plotter database in different types of kidney cancer patients. RESULTS: SHMT2 was found to be increased in 7 kidney cancer datasets, compared to normal renal tissues. For the cancer stages, ages and races, there existed significant difference in the expression of SHMT2 among different groups by mining of the UALCAN database. High SHMT2 expression is associated with poor overall survival in patients with kidney cancer. Among all co-expressed genes, NDUFA4L2 and SHMT2 had a high co-expression efficient. SHMT2 overexpression led to the increased expression of NDUFA4L2 at both mRNA and protein levels. Like SHMT2, overexpressed NDUFA4L2 also was associated with worse overall survival in patients with kidney cancer. CONCLUSION: Based on above results, overexpressed SHMT2 and its co-expressed gene NDUFA4L2 were all correlated with the prognosis in kidney cancer. The present study might be benefit for better understanding the clinical significance of SHMT2 and provided a potential therapeutic target for kidney cancer in future.
Asunto(s)
Complejo I de Transporte de Electrón/genética , Glicina Hidroximetiltransferasa/genética , Neoplasias Renales , Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Renales/genética , Neoplasias Renales/patología , Estadificación de Neoplasias , ARN MensajeroRESUMEN
Several studies suggest that the assembly of mitochondrial respiratory complexes into structures known as supercomplexes (SCs) may increase the efficiency of the electron transport chain, reducing the rate of production of reactive oxygen species. Therefore, the study of the (dis)assembly of SCs may be relevant for the understanding of mitochondrial dysfunction reported in brain aging and major neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD). Here we briefly reviewed the biogenesis and structural properties of SCs, the impact of mtDNA mutations and mitochondrial dynamics on SCs assembly, the role of lipids on stabilization of SCs and the methodological limitations for the study of SCs. More specifically, we summarized what is known about mitochondrial dysfunction and SCs organization and activity in aging, AD and PD. We focused on the critical variables to take into account when postmortem tissues are used to study the (dis)assembly of SCs. Since few works have been performed to study SCs in AD and PD, the impact of SCs dysfunction on the alteration of brain energetics in these diseases remains poorly understood. The convergence of future progress in the study of SCs structure at high resolution and the refinement of animal models of AD and PD, as well as the use of iPSC-based and somatic cell-derived neurons, will be critical in understanding the biological relevance of the structural remodeling of SCs.
Asunto(s)
Encéfalo/metabolismo , Metabolismo Energético/fisiología , Mitocondrias/metabolismo , Dinámicas Mitocondriales/fisiología , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Encéfalo/patología , 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 , Humanos , Mitocondrias/patología , Enfermedades Neurodegenerativas/patología , Neuronas/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Varroa destructor, a parasitic mite of the western honey bee, Apis mellifera L., is a serious threat to colonies and beekeeping worldwide. Population genetics studies of the mite have provided information on two mitochondrial haplotypes infecting honey bee colonies, named K and J (after Korea and Japan, respectively, where they were originally identified). On the American continent, the K haplotype is much more prevalent, with the J haplotype only detected in some areas of Brazil. The aims of the present study were to assess the genetic diversity of V. destructor populations in the major beekeeping region of Argentina and to evaluate the presence of heteroplasmy at the nucleotide level. Phoretic mites were collected from managed A. mellifera colonies in ten localities, and four mitochondrial DNA (mtDNA) regions (COXI, ND4, ND4L, and ND5) were analyzed. Based on cytochrome oxidase subunit I (COXI) sequencing, exclusively the K haplotype of V. destructor was detected. Furthermore, two sub-haplotypes (KArg-N1 and KArg-N2) were identified from a variation in ND4 sequences and the frequency of these sub-haplotypes was found to significantly correlate with geographical latitude. The occurrence of site heteroplasmy was also evident for this gene. Therefore, ND4 appears to be a sensitive marker for detecting genetic variability in mite populations. Site heteroplasmy emerges as a phenomenon that could be relatively frequent in V. destructor.
Asunto(s)
Abejas/parasitología , ADN Mitocondrial/genética , Variación Genética/genética , Proteínas Mitocondriales/genética , Varroidae/genética , Animales , Argentina , Apicultura , Brasil , Complejo I de Transporte de Electrón/genética , Complejo IV de Transporte de Electrones/genética , Haplotipos , Japón , NADH Deshidrogenasa/genética , República de CoreaRESUMEN
BACKGROUND: Kidney cancer is one of the most common cancers in the world. It is necessary to clarify its underlying mechanism and find its prognostic biomarkers. Current studies showed that SHMT2 may be participated in several kinds of cancer. METHODS: Our studies investigated the expression of SHMT2 in kidney cancer by Oncomine, Human Protein Atlas database and ULCAN database. Meanwhile, we found its co-expression gene by cBioPortal online tool and validated their relationship in A498 and ACHN cells by cell transfection, western blot and qRT-PCR. Besides these, we also explored their prognostic values via the Kaplan-Meier plotter database in different types of kidney cancer patients. RESULTS: SHMT2 was found to be increased in 7 kidney cancer datasets, compared to normal renal tissues. For the cancer stages, ages and races, there existed significant difference in the expression of SHMT2 among different groups by mining of the UALCAN database. High SHMT2 expression is associated with poor overall survival in patients with kidney cancer. Among all co-expressed genes, NDUFA4L2 and SHMT2 had a high co-expression efficient. SHMT2 overexpression led to the increased expression of NDUFA4L2 at both mRNA and protein levels. Like SHMT2, overexpressed NDUFA4L2 also was associated with worse overall survival in patients with kidney cancer. CONCLUSION: Based on above results, overexpressed SHMT2 and its co-expressed gene NDUFA4L2 were all correlated with the prognosis in kidney cancer. The present study might be benefit for better understanding the clinical significance of SHMT2 and provided a potential therapeutic target for kidney cancer in future.
Asunto(s)
Humanos , Glicina Hidroximetiltransferasa/genética , Complejo I de Transporte de Electrón/genética , Neoplasias Renales/genética , Neoplasias Renales/patología , ARN Mensajero , Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Estadificación de NeoplasiasRESUMEN
Cytosolic calcium (cCa2+) entry into mitochondria is facilitated by the mitochondrial membrane potential (ΔΨm), an electrochemical gradient generated by the electron transport chain (ETC). Is has been assumed that as long as mutations that affect the ETC do not affect the ΔΨm, the mitochondrial Ca2+ (mCa2+) homeostasis remains normal. We show that knockdown of NDUFAF3 and SDHB reduce ETC activity altering mCa2+ efflux and influx rates while ΔΨm remains intact. Shifting the equilibrium toward lower [Ca2+]m accumulation renders cells resistant to death. Our findings reveal an unexpected relationship between complex I and II with the mCa2+ homeostasis independent of ΔΨm.
Asunto(s)
Calcio/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Homeostasis , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Succinato Deshidrogenasa/metabolismo , Complejo I de Transporte de Electrón/genética , Humanos , Células MCF-7 , Potencial de la Membrana Mitocondrial/genética , Mitocondrias/genética , Proteínas Mitocondriales/genética , Succinato Deshidrogenasa/genéticaRESUMEN
HIG2A promotes cell survival under hypoxia and mediates the assembly of complex III and complex IV into respiratory chain supercomplexes. In the present study, we show that human HIGD2A and mouse Higd2a gene expressions are regulated by hypoxia, glucose, and the cell cycle-related transcription factor E2F1. The latter was found to bind the promoter region of HIGD2A. Differential expression of the HIGD2A gene was found in C57BL/6 mice in relation to tissue and age. Besides, the silencing of HIGD2A evidenced the modulation of mitochondrial dynamics proteins namely, OPA1 as a fusion protein increases, while FIS1, a fission protein, decreases. Besides, the mitochondrial membrane potential (ΔΨm) increased. The protein HIG2A is localized in the mitochondria and nucleus. Moreover, we observed that the HIG2A protein interacts with OPA1. Changes in oxygen concentration, glucose availability, and cell cycle regulate HIGD2A expression. Alterations in HIGD2A expression are associated with changes in mitochondrial physiology.
Asunto(s)
Ciclo Celular/fisiología , Potencial de la Membrana Mitocondrial/fisiología , Dinámicas Mitocondriales/fisiología , Membranas Mitocondriales/metabolismo , Animales , Complejo I de Transporte de Electrón/genética , Humanos , Ratones Endogámicos C57BL , Proteínas Mitocondriales/metabolismo , Proteínas de Neoplasias/metabolismoRESUMEN
Electron leakage from dysfunctional respiratory chain and consequent superoxide formation leads to mitochondrial and cell injury during ischemia and reperfusion (IR). In this work we evaluate if the supramolecular assembly of the respiratory complexes into supercomplexes (SCs) is associated with preserved energy efficiency and diminished oxidative stress in post-ischemic hearts treated with the antioxidant N-acetylcysteine (NAC) and the cardioprotective maneuver of Postconditioning (PostC). Hemodynamic variables, infarct size, oxidative stress markers, oxygen consumption and the activity/stability of SCs were compared between groups. We found that mitochondrial oxygen consumption and the activity of respiratory complexes are preserved in mitochondria from reperfused hearts treated with both NAC and PostC. Both treatments contribute to recover the activity of individual complexes. NAC reduced oxidative stress and maintained SCs assemblies containing Complex I, Complex III, Complex IV and the adapter protein SCAFI more effectively than PostC. On the other hand, the activities of CI, CIII and CIV associated to SCs assemblies were preserved by this maneuver, suggesting that the activation of other cardioprotective mechanisms besides oxidative stress contention might participate in maintaining the activity of the mitochondrial respiratory complexes in such superstructures. We conclude that both the monomeric and the SCs assembly of the respiratory chain contribute to the in vivo functionality of the mitochondria. However, although the ROS-induced damage and the consequent increased production of ROS affect the assembly of SCs, other levels of regulation as those induced by PostC, might participate in maintaining the activity of the respiratory complexes in such superstructures.
Asunto(s)
Acetilcisteína/farmacología , Antioxidantes/farmacología , Cardiotónicos/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Daño por Reperfusión Miocárdica/prevención & control , Animales , Transporte de Electrón/efectos de los fármacos , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Complejo III de Transporte de Electrones/genética , Complejo III de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Regulación de la Expresión Génica , Poscondicionamiento Isquémico/métodos , Mitocondrias Cardíacas/enzimología , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/enzimología , Daño por Reperfusión Miocárdica/enzimología , Daño por Reperfusión Miocárdica/genética , Daño por Reperfusión Miocárdica/patología , Miocardio/enzimología , Miocardio/patología , Fosforilación Oxidativa/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Variations in phenotypic expression of feed efficiency could be associated with differences or inefficiencies in mitochondria function due to its impact on energy expenditure. The aim of this study was to determine hepatic mitochondrial density and function in terms of respiration, gene and protein expression, and enzyme activity of mitochondrial respiratory complex proteins, in steers of divergent residual feed intake (RFI) phenotypes. Hereford steers (n = 111 and n = 122 for year 1 and 2, respectively) were evaluated in postweaning 70 d standard test for RFI. Forty-six steers exhibiting the greatest (n = 9 and 16 for year 1 and 2; high-RFI) and the lowest (n = 9 and 12 for year 1 and 2; low-RFI) RFI values were selected for this study. After the test, steers were managed together until slaughter under grazing conditions until they reached the slaughter body weight. At slaughter, hepatic samples (biopsies) were obtained. Tissue respiration was evaluated using high-resolution respirometry methods. Data were analyzed using a mixed model that included RFI group as fixed effect and slaughter date and year as a random effect using PROC MIXED of SAS. RFI and dry matter intake were different (P < 0.001) between low and high-RFI groups of year 1 and year 2. Basal respiration and maximum respiratory rate were greater (P ≤ 0.04) for low than high-RFI steers when complex II substrates (succinate) were supplied. However, when Complex I substrates (glutamate/malate) were used maximum respiratory capacity tended to be greater (P < 0.09) for low vs. high-RFI steers. Low-RFI steers presented greater mitochondria density markers (greater (P < 0.05) citrate synthase (CS) activity and tended (P ≤ 0.08) to have greater CS mRNA and mtDNA:nDNA ratio) than high-RFI steers. Hepatic expression SDHA, UQCRC1, and CYC1 mRNA was greater (P ≤ 0.02) and expression of NDUFA4, NDUFA13, SDHD, UQCRH, and ATP5E mRNA tended (P ≤ 0.10) to be greater in low than high-RFI steers. Hepatic SDHA protein expression tended (P < 0.08) to be greater while succinate dehydrogenase activity was greater (P = 0.04) and NADH dehydrogenase activity was greater (P = 0.03) for low than high-RFI steers. High-efficiency steers (low-RFI) probably had greater efficiency in hepatic nutrient metabolism, which was strongly associated with greater hepatic mitochondrial density and functioning, mainly of mitochondrial complex II.
Asunto(s)
Alimentación Animal/análisis , Bovinos/fisiología , Ingestión de Alimentos , Complejo I de Transporte de Electrón/metabolismo , Metabolismo Energético , Mitocondrias/enzimología , Animales , Peso Corporal , Bovinos/genética , Citrato (si)-Sintasa/genética , Citrato (si)-Sintasa/metabolismo , ADN Mitocondrial/genética , Complejo I de Transporte de Electrón/genética , Hígado/enzimología , Masculino , Mitocondrias/genética , Oxígeno/metabolismo , Fenotipo , ARN Mensajero/genéticaRESUMEN
The aim of this work was to develop a cryopreservation method of small liver biopsies for in situ mitochondrial function assessment. Herein we describe a detailed protocol for tissue collection, cryopreservation, high-resolution respirometry using complex I and II substrates, calculation and interpretation of respiratory parameters. Liver biopsies from cow and rat were sequentially frozen in a medium containing dimethylsulfoxide as cryoprotectant and stored for up to 3 months at -80⯰C. Oxygen consumption rate studies of fresh and cryopreserved samples revealed that most respiratory parameters remained unchanged. Additionally, outer mitochondrial membrane integrity was assessed adding cytochrome c, proving that our cryopreservation method does not harm mitochondrial structure. In sum, we present a reliable way to cryopreserve small liver biopsies without affecting mitochondrial function. Our protocol will enable the transport and storage of samples, extending and facilitating mitochondrial function analysis of liver biopsies.
Asunto(s)
Criopreservación , Hígado/metabolismo , Mitocondrias Hepáticas/genética , Consumo de Oxígeno/genética , Animales , Biopsia , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/fisiología , Hígado/fisiología , Mitocondrias Hepáticas/fisiología , Membranas Mitocondriales/metabolismo , Consumo de Oxígeno/fisiología , RatasRESUMEN
AMP-activated protein kinase (AMPK) regulates many different metabolic pathways in eukaryote cells including mitochondria biogenesis and energy homeostasis. Here we identify a patient with hypotonia, weakness, delayed milestones and neurological impairment since birth harbouring a novel homozygous mutation in the AMPK catalytic α-subunit 1, encoded by the PRKAA1 gene. The homozygous mutation p.S487L in isoform 1 present in the patient is in a cryptic residue for AMPK activity. In the present study, we performed the characterization of mitochondrial respiratory properties of the patient, in comparison to healthy controls, through the culture of skin fibroblasts in order to understand some of the cellular consequences of the PRKAA1 mutation. In these assays, mitochondrial respiratory complex I showed lower activity, which was followed by a decrement in the mtDNA copy number, which is a probable consequence of the lower expression of PGC-1α and PRKAA1 itself as measured in our quantitative PCRs experiments. Confirming the effect of the patient mutation in respiration, transfection of patient fibroblasts with wild type PRKAA1 partially restore complex I level. The preliminary clinic evaluations of the patient suggested a metabolic defect related to the mitochondrial respiratory function, therefore treatment with CoQ10 supplementation dose started four years ago and a clear improvement in motor skills and strength has been achieved with this treatment.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Fibroblastos , Homocigoto , Mitocondrias , Mutación Missense , Consumo de Oxígeno , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Sustitución de Aminoácidos , Preescolar , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Masculino , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismoRESUMEN
We demonstrate that a heterozygous nuclear variant in the gene encoding mitochondrial complex I subunit NDUFV1 aggravates the cellular phenotype in the presence of a mitochondrial DNA variant in complex I subunit ND1. Our findings suggest that heterozygous variants could be more significant in inherited mitochondrial diseases than hitherto assumed.
Asunto(s)
Complejo I de Transporte de Electrón/deficiencia , Enfermedades Mitocondriales/genética , NADH Deshidrogenasa/genética , Niño , ADN Mitocondrial/genética , Complejo I de Transporte de Electrón/genética , Femenino , Pruebas Genéticas/métodos , Heterocigoto , Humanos , Recién Nacido , Masculino , Enfermedades Mitocondriales/diagnóstico , Mutación , FenotipoRESUMEN
Due to the fact that mitochondrial defects and oxidative stress have been related with obesity and breast cancer is more aggressive in women with obesity, we investigated if postmenopausal Mexican-Mestizo women with breast cancer presented somatic mutations in the sequence of the ATP6 and/or ND3 genes. Twenty one postmenopausal Mexican-Mestizo women with breast cancer who underwent mastectomy or breast conserving surgery were studied. Height and weight were used to calculate body mass index. DNA from tumor tissue samples and blood leukocytes was amplified by polymerase chain reaction and sequenced the ATP6 and ND3 mitochondrial genes. Ages ranged from 46 to 82. According to World Health Organization criteria among the 21 women, 7 had a normal BMI, 7 were overweight and 7 had obesity. In regard to the molecular study, after sequencing the coding region of ATP6 and ND3 genes of the DNA obtained from both leukocytes and tumor tissue, we did not find somatic mutations. All of the changes that we found in both genes were polymorphisms: in ATP6, we identified in ten patients 3 non-synonymous nucleotide changes and in ND3 we observed that six patients presented polymorphisms, three of them were synonymous and two non-synonymous. To our knowledge, this constitutes the first report where the complete sequence of the ATP6 and ND3 genes has been analyzed in postmenopausal Mexican-Mestizo women with breast cancer and diverse BMI. Our results differ with those reported in Caucasian and Asian populations, possibly due to ethnic differences.