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1.
Pathol Res Pract ; 214(10): 1613-1620, 2018 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-30115536

RESUMEN

Ubiquinol-cytochrome c reductase complex core protein 2 (UQCRC2) is an important subunit of mitochondrial respiratory complex III. However, its role in tumorigenesis and tumor progression remains unknown, especially with regards to colorectal cancer (CRC). In this research, we measured the expression of UQCRC2 protein by immunohistochemistry assay in 89 paired paraffin-embedded tumor tissues and corresponding adjacent normal tissues from patients with colorectal adenocarcinoma and investigated possible correlations of UQCRC2 expression with clinicopathological parameters and prognosis. We found that UQCRC2 was significantly upregulated in CRC tissues compared with adjacent normal tissues, and immunohistochemical UQCRC2 status was correlated to the depth of invasion (T), lymph node metastasis (N), advanced TNM stage. Multivariate analysis indicated that UQCRC2 remained an independent prognostic factor for poorer overall survival. Furthermore, we determined the role of UQCRC2-knockdown in CRC cells (RKO and HCT116) using lentivirus-mediated small hairpin RNAs (shRNAs). The effects of UQCRC2 knockdown on CRC cells (RKO and HCT116) proliferation were analyzed by cell proliferation and colony formation assay, and cell cycle and apoptosis were assessed by flow cytometry. We found that silencing UQCRC2 suppressed cell proliferation and colony formation in RKO and HCT116 cells, led to a cell cycle arrest and induced cell apoptosis in vitro. These results provided novel insights into the potential role of UQCRC2 in the tumorigenesis and progression of CRC, and revealed that UQCRC2 may serve as a new prognostic and therapeutic target in CRC.


Asunto(s)
Adenocarcinoma/patología , Biomarcadores de Tumor/análisis , Neoplasias Colorrectales/patología , Complejo III de Transporte de Electrones/biosíntesis , Adenocarcinoma/mortalidad , Adulto , Anciano , Apoptosis/fisiología , Proliferación Celular/fisiología , Neoplasias Colorrectales/mortalidad , Progresión de la Enfermedad , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Proteínas Mitocondriales , Pronóstico , Modelos de Riesgos Proporcionales , Regulación hacia Arriba
2.
Biomed Res Int ; 2017: 1314297, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28676853

RESUMEN

In several recent studies, proteomics analyses suggest that increase of ubiquinol-cytochrome c reductase core protein 1 (UQCRC1) is cardio-protective. However, direct evidence for this effect has not yet been obtained. Thus, the current study aimed to determine this effect and the mechanism underlying this effect. The results showed that overexpression of UQCRC1 protected H9c2 cardiac cells against in vitro simulated ischemia-reperfusion by maintaining mitochondrial membrane potential and suppressing the expression of caspase-3. These protective effects were significantly enhanced by exogenous Zn2+ but completely abolished by Zn2+-selective chelator TPEN. Furthermore, the upregulation of UQCRC1 reduced the concentration of free Zn2+ in mitochondria, whereas the downregulation of UQCRC1 increased the concentration of free Zn2+ in mitochondria. In conclusion, the overexpression of UQCRC1 can protect H9c2 cardiac cells against simulated ischemia/reperfusion, and this cardio-protective effect is likely mediated by zinc binding.


Asunto(s)
Complejo III de Transporte de Electrones/biosíntesis , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Mitocondrias Cardíacas/enzimología , Daño por Reperfusión Miocárdica/enzimología , Zinc/farmacología , Animales , Caspasa 3/biosíntesis , Línea Celular , Supervivencia Celular/efectos de los fármacos , Complejo III de Transporte de Electrones/genética , Miocitos Cardíacos , Ratas
3.
Oncotarget ; 6(31): 30628-39, 2015 Oct 13.
Artículo en Inglés | MEDLINE | ID: mdl-26415228

RESUMEN

High energy ionizing radiation can cause DNA damage and cell death. During clinical radiation therapy, the radiation dose could range from 15 to 60 Gy depending on targets. While 2 Gy radiation has been shown to cause cancer cell death, studies also suggest a protective potential by low dose radiation. In this study, we examined the effect of 0.2-2 Gy radiation on hippocampal neurons. Low dose 0.2 Gy radiation treatment increased the levels of MTT. Since hippocampal neurons are post-mitotic, this result reveals a possibility that 0.2 Gy irradiation may increase mitochondrial activity to cope with stimuli. Maintaining neural plasticity is an energy-demanding process that requires high efficient mitochondrial function. We thus hypothesized that low dose radiation may regulate mitochondrial dynamics and function to ensure survival of neurons. Our results showed that five days after 0.2 Gy irradiation, no obvious changes on neuronal survival, neuronal synapses, membrane potential of mitochondria, reactive oxygen species levels, and mitochondrial DNA copy numbers. Interestingly, 0.2 Gy irradiation promoted the mitochondria fusion, resulting in part from the increased level of a mitochondrial fusion protein, Mfn2, and inhibition of Drp1 fission protein trafficking to the mitochondria. Accompanying with the increased mitochondrial fusion, the expressions of complexes I and III of the electron transport chain were also increased. These findings suggest that, hippocampal neurons undergo increased mitochondrial fusion to modulate cellular activity as an adaptive mechanism in response to low dose radiation.


Asunto(s)
Complejo III de Transporte de Electrones/biosíntesis , Complejo I de Transporte de Electrón/biosíntesis , Hipocampo/efectos de la radiación , Mitocondrias/metabolismo , Dinámicas Mitocondriales/efectos de la radiación , Neuronas/efectos de la radiación , Radiación Ionizante , Animales , Plasticidad de la Célula/fisiología , Supervivencia Celular/efectos de la radiación , Células Cultivadas , ADN Mitocondrial/genética , Dinaminas/metabolismo , GTP Fosfohidrolasas , Dosificación de Gen/genética , Potencial de la Membrana Mitocondrial/fisiología , Potencial de la Membrana Mitocondrial/efectos de la radiación , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Oxidación-Reducción/efectos de la radiación , Transporte de Proteínas/efectos de la radiación , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo
4.
J Affect Disord ; 184: 160-3, 2015 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-26093828

RESUMEN

BACKGROUND: Studies that have focused on the mitochondrial electron transport chain indicate that bipolar disorder (BD) is associated with pathology in mitochondrial function. These pathological processes occur in the brain circuits that regulate affective functions, emotions, and motor behaviors. The present study aimed to determine the relationship between mitochondrial complex dysfunction and BD. METHODS: The BD group included 32 male patients diagnosed with first-episode manic BD. The control group included 35 sociodemographically matched healthy males. Messenger ribonucleic acid (mRNA) was isolated from peripheral blood samples obtained from the patients and control group, and the mRNA levels of the NDUFV1, NDUFV2, and NDUFS1 genes of mitochondrial complex I and the UQCR10 gene of mitochondrial complex III were investigated. RESULTS: Significant differences were identified in complex I gene mRNA levels between the BD group (n = 32) and the control group (n = 35) for the following genes: NDUFV1 (P = 0.01), NDUFV2 (P < 0.01), and NDUFS1 (P = 0.02). The UQCR10 gene (complex III) mRNA level did not differ between the groups (P = 0.1). The mRNA levels of the four genes studied were lower at the 3-month follow-up; however, these differences were not significant (P > 0.05). LIMITATIONS: All of the BD patients were in manic episodes; thus, we were unable to separately compare these levels with those during depressive and euthymic episodes. CONCLUSIONS: The mRNA levels of all of the genes representing the subunits of mitochondrial complex I (NDUFV1, NDUFV2, and NDUFS1) were significantly higher in the present study's BD patients during manic episodes than in the controls. With the data obtained from further research, biomarkers that could be used for the diagnosis and follow-up of neuropsychiatric disorders may be discovered.


Asunto(s)
Trastorno Bipolar/metabolismo , Complejo III de Transporte de Electrones/biosíntesis , Complejo I de Transporte de Electrón/biosíntesis , ARN Mensajero/biosíntesis , Adulto , Biomarcadores , Trastorno Bipolar/genética , Trastorno Bipolar/psicología , Complejo I de Transporte de Electrón/genética , Complejo III de Transporte de Electrones/genética , Femenino , Estudios de Seguimiento , Humanos , Masculino , NADH Deshidrogenasa/biosíntesis , NADH Deshidrogenasa/genética , ARN Mensajero/genética , Factores Socioeconómicos , Adulto Joven
5.
Placenta ; 33(10): 824-9, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22858023

RESUMEN

MiR-155 is known to participate in various cellular processes by targeting gene expression. We previously revealed a link between miR-155 and perturbation of trophoblast invasion and differentiation. This study aimed to investigate the target molecule(s) of miR-155 on the influence on the proliferation and migration of trophoblast cells. Bioinformatics analysis showed that, at the 3' untranslated region (UTR) of cyclin D1, six bases are complementary to the seed region of miR-155. Luciferase assays and cyclin D1 3'UTR transfection assays validated that cyclin D1 3'UTR was the target of miR-155 in HTR-8/SVneo cells. Overexpression of miR-155 in HTR-8/SVneo cells reduced the level of cyclin D1 protein, decreased cell proliferation and invasion, and increased cell number at the G1 stage. Furthermore, the increased expression of miR-155 also regulated the protein levels of kinase inhibitory protein p27 and phosphorylated cytoskeletal protein filamin A. In conclusion, we found that cyclin D1 may be a target of miR-155 in HTR-8/SVneo cells, and demonstrated a negative regulatory role of miR-155 involved in cyclin D1/p27 pathway in proliferation and migration of the cells.


Asunto(s)
Movimiento Celular/efectos de los fármacos , Ciclina D1/metabolismo , MicroARNs/farmacología , Trofoblastos/fisiología , Regiones no Traducidas 3' , Adulto , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas Contráctiles/metabolismo , Ciclina D1/genética , Regulación hacia Abajo , Complejo I de Transporte de Electrón/biosíntesis , Complejo III de Transporte de Electrones/biosíntesis , Complejo IV de Transporte de Electrones/biosíntesis , Femenino , Filaminas , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , MicroARNs/antagonistas & inhibidores , Proteínas de Microfilamentos/metabolismo , Preeclampsia/fisiopatología , Embarazo , Trofoblastos/efectos de los fármacos , Trofoblastos/metabolismo
6.
Mol Cell Biol ; 32(21): 4400-9, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22927643

RESUMEN

The final step in the assembly of the ubiquinol-cytochrome c reductase or bc(1) complex involves the insertion of the Rieske Fe/S cluster protein, Rip1. Maturation of Rip1 occurs within the mitochondrial matrix prior to its translocation across the inner membrane (IM) in a process mediated by the Bcs1 ATPase and subsequent insertion into the bc(1) complex. Here we show that the matrix protein Mzm1 functions as a Rip1 chaperone, stabilizing Rip1 prior to the translocation step. In the absence of Mzm1, Rip1 is prone to either proteolytic degradation or temperature-induced aggregation. A series of Rip1 truncations were engineered to probe motifs necessary for Mzm1 interaction and Bcs1-mediated translocation of Rip1. The Mzm1 interaction with Rip1 persists in Rip1 variants lacking its transmembrane domain or containing only its C-terminal globular Fe/S domain. Replacement of the globular domain of Rip1 with that of the heterologous folded protein Grx3 abrogated Mzm1 interaction; however, appending the C-terminal 30 residues of Rip1 to the Rip1-Grx3 chimera restored Mzm1 interaction. The Rip1-Grx3 chimera and a Rip1 truncation containing only the N-terminal 92 residues each induced stabilization of the bc(1):cytochrome oxidase supercomplex in a Bcs1-dependent manner. However, the Rip1 variants were not stably associated with the supercomplex. The induced supercomplex stabilization by the Rip1 N terminus was independent of Mzm1.


Asunto(s)
Complejo III de Transporte de Electrones/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Complejo III de Transporte de Electrones/biosíntesis , Mitocondrias/metabolismo , Oxidorreductasas/metabolismo , Pliegue de Proteína , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Transporte de Proteínas
7.
Biochim Biophys Acta ; 1817(6): 872-82, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22564912

RESUMEN

The cytochrome bc(1) complex is an essential component of the electron transport chain in most prokaryotes and in eukaryotic mitochondria. The catalytic subunits of the complex that are responsible for its redox functions are largely conserved across kingdoms. In eukarya, the bc(1) complex contains supernumerary subunits in addition to the catalytic core, and the biogenesis of the functional bc(1) complex occurs as a modular assembly pathway. Individual steps of this biogenesis have been recently investigated and are discussed in this review with an emphasis on the assembly of the bc(1) complex in the model eukaryote Saccharomyces cerevisiae. Additionally, a number of assembly factors have been recently identified. Their roles in bc(1) complex biogenesis are described, with special emphasis on the maturation and topogenesis of the yeast Rieske iron-sulfur protein and its role in completing the assembly of functional bc(1) complex. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.


Asunto(s)
Complejo III de Transporte de Electrones/biosíntesis , Complejo III de Transporte de Electrones/metabolismo , Multimerización de Proteína/fisiología , Animales , Humanos
8.
Biochim Biophys Acta ; 1817(2): 276-86, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22138626

RESUMEN

The cytochrome bc(1) complex is an essential component of the electron transport chain in most prokaryotes and in eukaryotic mitochondria. The catalytic subunits of the complex that are responsible for its redox functions are largely conserved across kingdoms. In eukarya, the bc(1) complex contains supernumerary subunits in addition to the catalytic core, and the biogenesis of the functional bc(1) complex occurs as a modular assembly pathway. Individual steps of this biogenesis have been recently investigated and are discussed in this review with an emphasis on the assembly of the bc(1) complex in the model eukaryote Saccharomyces cerevisiae. Additionally, a number of assembly factors have been recently identified. Their roles in bc(1) complex biogenesis are described, with special emphasis on the maturation and topogenesis of the yeast Rieske iron-sulfur protein and its role in completing the assembly of functional bc(1) complex. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.


Asunto(s)
Complejo III de Transporte de Electrones/biosíntesis , Complejo III de Transporte de Electrones/metabolismo , Multimerización de Proteína/fisiología , Animales , Complejo III de Transporte de Electrones/química , Complejo III de Transporte de Electrones/fisiología , Células Eucariotas/metabolismo , Células Eucariotas/fisiología , Humanos , Modelos Biológicos , Modelos Moleculares , Células Procariotas/metabolismo , Células Procariotas/fisiología , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología
9.
Mitochondrion ; 9(5): 299-305, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19389488

RESUMEN

Mutations in the assembly chaperone BCS1L constitute a major cause of mitochondrial complex III deficiency. We studied the presence of BCS1L mutations in a complex III-deficient patient with metabolic acidosis, liver failure, and tubulopathy. A previously reported mutation, p.R56X, was identified in one BCS1L allele, and two novel heterozygous mutations, g.1181A>G and g.1164C>G, were detected in the second allele. The g.1181A>G mutation generated an alternative splicing site in the BCS1L transcript, causing a 19-nucleotides deletion in its 5'UTR region. Decreased BCS1L mRNA and protein levels, and a respiratory chain complex III assembly impairment, determine a pathogenic role for the novel BCS1L mutations.


Asunto(s)
Regiones no Traducidas 5'/genética , Complejo III de Transporte de Electrones/deficiencia , Mutación Puntual , ARN Mensajero/genética , ATPasas Asociadas con Actividades Celulares Diversas , Secuencia de Bases , Complejo III de Transporte de Electrones/biosíntesis , Complejo III de Transporte de Electrones/genética , Femenino , Humanos , Recién Nacido , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Embarazo , Análisis de Secuencia de ADN
10.
Hum Mol Genet ; 17(24): 4001-11, 2008 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-18806273

RESUMEN

Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disorder, is mostly due to three mitochondrial DNA (mtDNA) mutations in respiratory chain complex I subunit genes: 3460/ND1, 11778/ND4 and 14484/ND6. Despite considerable clinical evidences, a genetic modifying role of the mtDNA haplogroup background in the clinical expression of LHON remains experimentally unproven. We investigated the effect of mtDNA haplogroups on the assembly of oxidative phosphorylation (OXPHOS) complexes in transmitochondrial hybrids (cybrids) harboring the three common LHON mutations. The steady-state levels of respiratory chain complexes appeared normal in mutant cybrids. However, an accumulation of low molecular weight subcomplexes suggested a complex I assembly/stability defect, which was further demonstrated by reversibly inhibiting mitochondrial protein translation with doxycycline. Our results showed differentially delayed assembly rates of respiratory chain complexes I, III and IV amongst mutants belonging to different mtDNA haplogroups, revealing that specific mtDNA polymorphisms may modify the pathogenic potential of LHON mutations by affecting the overall assembly kinetics of OXPHOS complexes.


Asunto(s)
ADN Mitocondrial/genética , Mitocondrias/genética , Modelos Genéticos , Atrofia Óptica Hereditaria de Leber/genética , Fosforilación Oxidativa , Línea Celular , Línea Celular Tumoral , ADN Mitocondrial/efectos de los fármacos , ADN Mitocondrial/metabolismo , Doxiciclina/farmacología , Transporte de Electrón/efectos de los fármacos , Transporte de Electrón/genética , Complejo I de Transporte de Electrón/biosíntesis , Complejo I de Transporte de Electrón/genética , Complejo III de Transporte de Electrones/biosíntesis , Complejo III de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/biosíntesis , Complejo IV de Transporte de Electrones/genética , Estabilidad de Enzimas/efectos de los fármacos , Estabilidad de Enzimas/genética , Humanos , Cinética , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Datos de Secuencia Molecular , NADH Deshidrogenasa/genética , Atrofia Óptica Hereditaria de Leber/metabolismo , Atrofia Óptica Hereditaria de Leber/patología , Fosforilación Oxidativa/efectos de los fármacos , Subunidades de Proteína/genética , Análisis de Secuencia de ADN
11.
Biochem J ; 416(1): 15-26, 2008 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-18729827

RESUMEN

Mice homozygous for a defect in the PTCD2 (pentatricopeptide repeat domain protein 2) gene were generated in order to study the role of this protein in mitochondrial RNA metabolism. These mice displayed specific but variable reduction of ubiquinol-cytochrome c reductase complex activity in mitochondria of heart, liver and skeletal muscle due to a decrease in the expression of mitochondrial DNA-encoded cytochrome b, the catalytic core of the complex. This reduction in mitochondrial function has a profound effect on the myocardium, with replacement of ventricular cardiomyocytes by fibro-fatty tissue. Northern blotting showed a reduction in the mRNA for the mitochondrial DNA encoded proteins cytochrome b (cytb) and ND5 (NADH dehydrogenase subunit 5) and an elevation in a combined pre-processed ND5-CYTB transcript. This suggests that the PTCD2 protein is involved in processing RNA transcripts involving cytochrome b derived from mitochondrial DNA. This defines the site for PTCD2 action in mammalian mitochondria and suggests a possible role for dysfunction of this protein in the aetiology of heart failure.


Asunto(s)
Citocromos b/biosíntesis , Complejo III de Transporte de Electrones/biosíntesis , Genes Mitocondriales/fisiología , Mitocondrias Cardíacas/enzimología , Proteínas Mitocondriales/genética , Proteínas de Unión al ARN/genética , Animales , Regulación Enzimológica de la Expresión Génica , Células HeLa , Humanos , Ratones , Microscopía Electrónica , Mitocondrias Cardíacas/ultraestructura , Mitocondrias Hepáticas/enzimología , Mitocondrias Musculares/enzimología , Proteínas Mitocondriales/fisiología , ARN/metabolismo , ARN Mitocondrial , Proteínas de Unión al ARN/fisiología
12.
Biochim Biophys Acta ; 1777(4): 388-96, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18258177

RESUMEN

In the green alga Chlamydomonas reinhardtii, a mutant deprived of complex I enzyme activity presents a 1T deletion in the mitochondrial nd5 gene. The loss of the ND5 subunit prevents the assembly of the 950 kDa whole complex I. Instead, a low abundant 700 kDa subcomplex, loosely associated to the inner mitochondrial membrane, is assembled. The resolution of the subcomplex by SDS-PAGE gave rise to 19 individual spots, sixteen having been identified by mass spectrometry analysis. Eleven, mainly associated to the hydrophilic part of the complex, are homologs to subunits of the bovine enzyme whereas five (including gamma-type carbonic anhydrase subunits) are specific to green plants or to plants and fungi. None of the subunits typical of the beta membrane domain of complex I enzyme has been identified in the mutant. This allows us to propose that the truncated enzyme misses the membrane distal domain of complex I but retains the proximal domain associated to the matrix arm of the enzyme. A complex I topology model is presented in the light of our results. Finally, a supercomplex most probably corresponding to complex I-complex III association, was identified in mutant mitochondria, indicating that the missing part of the enzyme is not required for the formation of the supercomplex.


Asunto(s)
Chlamydomonas reinhardtii/química , Complejo I de Transporte de Electrón/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Arabidopsis/química , Proteínas Bacterianas/química , Chlamydomonas reinhardtii/genética , Proteínas de Drosophila/química , Complejo I de Transporte de Electrón/genética , Complejo III de Transporte de Electrones/biosíntesis , Magnetospirillum/química , Subunidades de Proteína , Alineación de Secuencia , Espectrometría de Masas en Tándem
13.
Can J Physiol Pharmacol ; 85(10): 986-96, 2007 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18066099

RESUMEN

Women and men do not respond identically to cardiac insults; premenopausal women are somewhat protected from cardiovascular disease. Our objective was to isolate and characterize hormone-responsive genes in the heart. Differential display identified an estrogen-inducible fragment that was found to encode the ubiquinol-cytochrome-c reductase (UCCR) 7.2 kDa protein of the mitochondrial respiratory complex III. We found UCCR7.2 mRNA to be highly expressed in the heart, and this expression increased in hearts of 4-, 10-, and 28-week-old spontaneously hypertensive rats (SHR) compared with normotensive Wistar-Kyoto rats. Oral hydralazine treatment to reduce hypertension reduced SHR UCCR7.2 expression. Cardiac UCCR7.2 mRNA expression was also increased significantly after a 5/6 nephrectomy compared with mock surgery. Cardiac expression after ovariectomy was 50% that of intact rats. Supplementation of ovariectomized rats with estrogen had no effect, whereas progesterone increased cardiac expression, although not to intact levels. No change in cardiac UCCR7.2 expression was found when intact rats were treated with either tamoxifen or ICI 182780. Thus, UCCR7.2 expression is reduced in the absence of ovarian hormones, but is not directly regulated by estrogen in the heart. We conclude that UCCR7.2 is a steroid hormone-responsive gene in the heart, with expression increased in cardiac hypertrophy and in response to hypertension.


Asunto(s)
Cardiomegalia/enzimología , Complejo III de Transporte de Electrones/biosíntesis , Hormonas Esteroides Gonadales/farmacología , Hipertensión/enzimología , Mitocondrias Cardíacas/enzimología , Animales , Northern Blotting , Complejo III de Transporte de Electrones/genética , Estradiol/farmacología , Femenino , Mitocondrias Cardíacas/efectos de los fármacos , Ovariectomía , Progesterona/farmacología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Ratas Sprague-Dawley
14.
Biochim Biophys Acta ; 1764(4): 735-42, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16458619

RESUMEN

The Rieske 2Fe-2S protein is a central component of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for expression in Escherichia coli of full-length and truncated Spinacia oleracea Rieske (PetC) proteins fused to the MalE, maltose binding protein. The expressed Rieske fusion proteins were found predominantly in soluble form in the E. coli cytoplasm. These proteins could be readily purified for further experimentation. In vitro reconstitution of the characteristic, "Rieske-type" 2Fe-2S cluster into these fused proteins was accomplished by a chemical method employing reduced iron and sulfide. Cluster incorporation was monitored by electron paramagnetic resonance and optical circular dichroism (CD) spectroscopy. CD spectral analysis in the ultraviolet region suggests that the spinach Rieske apoprotein must be in a partially folded conformation to incorporate an appropriate iron-sulfur cluster. These data further suggest that upon cluster integration, further folding occurs, allowing the Rieske protein to attain a final, native structure. The data presented here are the first to demonstrate successful chemical reconstitution of the 2Fe-2S cluster into a Rieske apoprotein from higher plant chloroplasts.


Asunto(s)
Apoproteínas/química , Complejo III de Transporte de Electrones/química , Proteínas Hierro-Azufre/química , Pliegue de Proteína , Cloroplastos/química , Dicroismo Circular , Complejo III de Transporte de Electrones/biosíntesis , Escherichia coli/metabolismo , Proteínas Hierro-Azufre/biosíntesis , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Spinacia oleracea/química
15.
Biochemistry ; 44(18): 7048-58, 2005 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-15865449

RESUMEN

Rieske [2Fe-2S] clusters have reduction potentials which vary by over 500 mV, and which are pH dependent. In the cytochrome bc(1) complex, the high-potential and low-pK values of the cluster may be important in the mechanism of quinol oxidation. Hydrogen bonds, from both side-chain and mainchain groups, are crucial for these properties, but solvent accessibility and a disulfide bond (present in only high-potential Rieske proteins) have been suggested to be important determinants also. Previous studies have addressed the hydrogen bonds, disulfide bond, and a leucine residue which may restrict solvent access, by mutations in the cytochrome bc(1) complex. However, influences on the complex (disruption of quinol binding and displacement of the Rieske domain) are difficult to deconvolute from intrinsic effects on the Rieske cluster. Here, the effects of similar mutations on cluster potential, pK values, and stability are characterized comprehensively in the isolated Rieske domain of the bovine protein. Hydrogen bonds from Ser163 and Tyr165 are important in increasing the reduction potential and decreasing the pK values. The disulfide has a limited effect on the redox properties, but is crucial for cluster stability, particularly in the oxidized state. Mutations of Leu142 had little effect on cluster potential, pK values, or stability, in contrast to the significant effects which were observed in the complex. The sum of the effects of all the mutated residues accounts for most of the differences between high- and low-potential Rieske proteins.


Asunto(s)
Disulfuros/química , Complejo III de Transporte de Electrones/química , Proteínas Hierro-Azufre/química , Termodinámica , Sustitución de Aminoácidos/genética , Animales , Bovinos , Electroquímica , Complejo III de Transporte de Electrones/biosíntesis , Complejo III de Transporte de Electrones/genética , Complejo III de Transporte de Electrones/metabolismo , Estabilidad de Enzimas/genética , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Proteínas Hierro-Azufre/biosíntesis , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Leucina/genética , Modelos Químicos , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Serina/genética , Relación Estructura-Actividad , Tirosina/genética
16.
EMBO J ; 23(23): 4679-89, 2004 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-15526035

RESUMEN

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, after apoptosis induction, translocates to the nucleus where it participates in apoptotic chromatinolysis. Here, we show that human or mouse cells lacking AIF as a result of homologous recombination or small interfering RNA exhibit high lactate production and enhanced dependency on glycolytic ATP generation, due to severe reduction of respiratory chain complex I activity. Although AIF itself is not a part of complex I, AIF-deficient cells exhibit a reduced content of complex I and of its components, pointing to a role of AIF in the biogenesis and/or maintenance of this polyprotein complex. Harlequin mice with reduced AIF expression due to a retroviral insertion into the AIF gene also manifest a reduced oxidative phosphorylation (OXPHOS) in the retina and in the brain, correlating with reduced expression of complex I subunits, retinal degeneration, and neuronal defects. Altogether, these data point to a role of AIF in OXPHOS and emphasize the dual role of AIF in life and death.


Asunto(s)
Proteínas de la Membrana/deficiencia , Adenosina Trifosfato/biosíntesis , Animales , Apoptosis , Factor Inductor de la Apoptosis , Encéfalo/metabolismo , Células Cultivadas , Complejo I de Transporte de Electrón/biosíntesis , Complejo III de Transporte de Electrones/biosíntesis , Flavoproteínas/genética , Flavoproteínas/metabolismo , Glucosa/metabolismo , Humanos , Ácido Láctico/biosíntesis , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Miocardio/metabolismo , Especificidad de Órganos , Fosforilación Oxidativa , Filogenia , ARN Interferente Pequeño/metabolismo , Retina/metabolismo , Levaduras/genética , Levaduras/crecimiento & desarrollo , Levaduras/metabolismo
17.
J Mol Biol ; 309(1): 121-38, 2001 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-11491283

RESUMEN

Recently, we demonstrated that the RegB/RegA two-component regulatory system from Rhodobacter capsulatus functions as a global regulator of metabolic processes that either generate or consume reducing equivalents. For example, the RegB/RegA system controls expression of such energy generating processes as photosynthesis and hydrogen utilization. In addition, RegB/RegA also control nitrogen and carbon fixation pathways that utilize reducing equivalents. Here, we use a combination of DNase I protection and plasmid-based reporter expression studies to demonstrate that RegA directly controls synthesis of cytochrome cbb3 and ubiquinol oxidases that function as terminal electron acceptors in a branched respiratory chain. We also demonstrate that RegA controls expression of cytochromes c2, c(y) and the cytochrome bc1 complex that are involved in both photosynthetic and respiratory electron transfer events. These data provide evidence that the RegB/RegA two-component system has a major role in controlling the synthesis of numerous processes that affect reducing equivalents in Rhodobacter capsulatus.


Asunto(s)
Proteínas Bacterianas/metabolismo , Transporte de Electrón/genética , Regulación Bacteriana de la Expresión Génica , Fotosíntesis/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Proteínas Quinasas , Rhodobacter capsulatus/genética , Secuencia de Bases , Sitios de Unión , Grupo Citocromo c/biosíntesis , Grupo Citocromo c/genética , Citocromos c2 , Huella de ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Complejo III de Transporte de Electrones/biosíntesis , Complejo III de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/biosíntesis , Complejo IV de Transporte de Electrones/genética , Inducción Enzimática , Genes Bacterianos/genética , Genes Reporteros/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Quinona Reductasas/biosíntesis , Quinona Reductasas/genética , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Rhodobacter capsulatus/enzimología , Rhodobacter capsulatus/metabolismo , Alineación de Secuencia , Factores de Transcripción/metabolismo
18.
Genetics ; 156(4): 1727-36, 2000 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11102369

RESUMEN

A P-element insertion in the oxen gene, ox(1), has been isolated in a search for modifiers of white gene expression. The mutation preferentially exerts a negative dosage effect upon the expression of three genes encoding ABC transporters involved in pigment precursor transport, white, brown, and scarlet. A precise excision of the P element reverts the mutant phenotype. Five different transcription units were identified around the insertion site. To distinguish a transcript responsible for the mutant phenotype, a set of deletions within the oxen region was generated. Analysis of gene expression within the oxen region in the case of deletions as well as generation of transgenic flies allowed us to identify the transcript responsible for oxen function. It encodes a 6.6-kD homolog of mitochondrial ubiquinol cytochrome c oxidoreductase (QCR9), subunit 9 of the bc(1) complex in yeast. In addition to white, brown, and scarlet, oxen regulates the expression of three of seven tested genes. Thus, our data provide additional evidence for a cellular response to changes in mitochondrial function. The oxen mutation provides a model for the genetic analysis in multicellular organisms of the effect of mitochondrial activity on nuclear gene expression.


Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas de Drosophila , Drosophila melanogaster/genética , Complejo III de Transporte de Electrones/genética , Proteínas Fúngicas/genética , Regulación de la Expresión Génica/genética , Genes de Insecto , Proteínas de Insectos/genética , Mitocondrias/enzimología , Complejos Multienzimáticos/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Drosophila melanogaster/enzimología , Drosophila melanogaster/crecimiento & desarrollo , Complejo III de Transporte de Electrones/biosíntesis , Complejo III de Transporte de Electrones/fisiología , Color del Ojo/genética , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/genética , Regulación Fúngica de la Expresión Génica , Genotipo , Proteínas de Insectos/biosíntesis , Datos de Secuencia Molecular , Fenotipo , Subunidades de Proteína , Pupa , ARN Mensajero/biosíntesis , Saccharomyces cerevisiae/enzimología , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Transcripción Genética
19.
J Bacteriol ; 181(17): 5365-72, 1999 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-10464208

RESUMEN

The ubihydroquinone-cytochrome c oxidoreductase (or the cytochrome bc1 complex) from Rhodobacter capsulatus is composed of the Fe-S protein, cytochrome b, and cytochrome c1 subunits encoded by petA(fbcF), petB(fbcB), and petC(fbcC) genes organized as an operon. In the work reported here, petB(fbcB) was split genetically into two cistrons, petB6 and petBIV, which encoded two polypeptides corresponding to the four amino-terminal and four carboxyl-terminal transmembrane helices of cytochrome b, respectively. These polypeptides resembled the cytochrome b6 and su IV subunits of chloroplast cytochrome b6f complexes, and together with the unmodified subunits of the cytochrome bc1 complex, they formed a novel enzyme, named cytochrome b6c1 complex. This membrane-bound multisubunit complex was functional, and despite its smaller amount, it was able to support the photosynthetic growth of R. capsulatus. Upon further mutagenesis, a mutant overproducing it, due to a C-to-T transition at the second base of the second codon of petBIV, was obtained. Biochemical analyses, including electron paramagnetic spectroscopy, with this mutant revealed that the properties of the cytochrome b6c1 complex were similar to those of the cytochrome bc1 complex. In particular, it was highly sensitive to inhibitors of the cytochrome bc1 complex, including antimycin A, and the redox properties of its b- and c-type heme prosthetic groups were unchanged. However, the optical absorption spectrum of its cytochrome bL heme was modified in a way reminiscent of that of a cytochrome b6f complex. Based on the work described here and that with Rhodobacter sphaeroides (R. Kuras, M. Guergova-Kuras, and A. R. Crofts, Biochemistry 37:16280-16288, 1998), it appears that neither the inhibitor resistance nor the redox potential differences observed between the bacterial (or mitochondrial) cytochrome bc1 complexes and the chloroplast cytochrome b6f complexes are direct consequences of splitting cytochrome b into two separate polypeptides. The overall findings also illustrate the possible evolutionary relationships among various cytochrome bc oxidoreductases.


Asunto(s)
Grupo Citocromo b/biosíntesis , Complejo III de Transporte de Electrones/biosíntesis , Rhodobacter capsulatus/crecimiento & desarrollo , Grupo Citocromo b/química , Grupo Citocromo b/genética , Complejo de Citocromo b6f , Citocromos c1/biosíntesis , Citocromos c1/genética , Complejo III de Transporte de Electrones/química , Complejo III de Transporte de Electrones/genética , Ingeniería Genética , Mutagénesis , Fotosíntesis , Rhodobacter capsulatus/metabolismo
20.
FEBS Lett ; 395(2-3): 199-203, 1996 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-8898095

RESUMEN

The aromatic character of the region 66(YWYWW)70 of the 11-kDa subunit VIII of ubiquinol-cytochrome c oxidoreductase (bc1 complex) of the yeast Saccharomyces cerevisiae has previously been demonstrated to be important for assembly of a functional complex [Hemrika et al. (1994) FEBS Lett. 344, 15-19]. Especially the aromatic nature of residue 66 appeared to be relevant, as the very low level (5%) of bc1 complex in the mutant 66(SASAA)70 was restored to nearly 70% of the wild-type level in a phenotypic revertant with the sequence 66(FASAA)70. In the present study, three other site-directed mutants (66(SAYAA)70, 66(SASAW)70 and 66(SWYWW)70) were constructed and analysed. The data indicate that the presence of one aromatic residue is enough for a substantial level of assembly and that its position modulates the level of both assembly and electron transfer activity. The results also confirm the relevance of this region of subunit VIII for the formation of the Q(out) reaction domain, as reported by Hemrika et al. [(1993) Eur. J. Biochem. 215, 601-609]. It is further shown that the lowered specific activity of the mutant described by these authors is not due to the introduction of a cysteine in the sequence of subunit VIII.


Asunto(s)
Complejo III de Transporte de Electrones/química , Complejo III de Transporte de Electrones/metabolismo , Saccharomyces cerevisiae/enzimología , Secuencia de Aminoácidos , Antifúngicos/farmacología , Antimicina A/análogos & derivados , Antimicina A/farmacología , Secuencia de Bases , Secuencia de Consenso , Complejo III de Transporte de Electrones/biosíntesis , Hongos/enzimología , Humanos , Cinética , Sustancias Macromoleculares , Metacrilatos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plantas/enzimología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Tiazoles/farmacología
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