RESUMO
NADH-ubiquinone (UQ) oxidoreductase (complex I) couples electron transfer from NADH to UQ with proton translocation in its membrane part. The UQ reduction step is key to triggering proton translocation. Structural studies have identified a long, narrow, tunnel-like cavity within complex I, through which UQ may access a deep reaction site. To elucidate the physiological relevance of this UQ-accessing tunnel, we previously investigated whether a series of oversized UQs (OS-UQs), whose tail moiety is too large to enter and transit the narrow tunnel, can be catalytically reduced by complex I using the native enzyme in bovine heart submitochondrial particles (SMPs) and the isolated enzyme reconstituted into liposomes. Nevertheless, the physiological relevance remained unclear because some amphiphilic OS-UQs were reduced in SMPs but not in proteoliposomes, and investigation of extremely hydrophobic OS-UQs was not possible in SMPs. To uniformly assess the electron transfer activities of all OS-UQs with the native complex I, here we present a new assay system using SMPs, which were fused with liposomes incorporating OS-UQ and supplemented with a parasitic quinol oxidase to recycle reduced OS-UQ. In this system, all OS-UQs tested were reduced by the native enzyme, and the reduction was coupled with proton translocation. This finding does not support the canonical tunnel model. We propose that the UQ reaction cavity is flexibly open in the native enzyme to allow OS-UQs to access the reaction site, but their access is obstructed in the isolated enzyme as the cavity is altered by detergent-solubilizing from the mitochondrial membrane.
Assuntos
Complexo I de Transporte de Elétrons , Ubiquinona , Animais , Bovinos , Ubiquinona/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Membranas Mitocondriais/metabolismo , NAD/metabolismo , Prótons , LipossomosRESUMO
Cytochrome c oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a proton pump across the membrane. In all crystals reported to date, bovine CcO exists as a dimer with the same intermonomer contacts, whereas CcOs and related enzymes from prokaryotes exist as monomers. Recent structural analyses of the mitochondrial respiratory supercomplex revealed that CcO monomer associates with complex I and complex III, indicating that the monomeric state is functionally important. In this study, we prepared monomeric and dimeric bovine CcO, stabilized using amphipol, and showed that the monomer had high activity. In addition, using a newly synthesized detergent, we determined the oxidized and reduced structures of monomer with resolutions of 1.85 and 1.95 Å, respectively. Structural comparison of the monomer and dimer revealed that a hydrogen bond network of water molecules is formed at the entry surface of the proton transfer pathway, termed the K-pathway, in monomeric CcO, whereas this network is altered in dimeric CcO. Based on these results, we propose that the monomer is the activated form, whereas the dimer can be regarded as a physiological standby form in the mitochondrial membrane. We also determined phospholipid structures based on electron density together with the anomalous scattering effect of phosphorus atoms. Two cardiolipins are found at the interface region of the supercomplex. We discuss formation of the monomeric CcO, dimeric CcO, and supercomplex, as well as their role in regulation of CcO activity.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/química , Mitocôndrias Cardíacas/enzimologia , Animais , Cardiolipinas/química , Bovinos , Cristalografia por Raios X , Digitonina/química , Transporte de Elétrons , Complexo I de Transporte de Elétrons/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Membranas Mitocondriais/enzimologia , Conformação Molecular , Oxirredução , Oxigênio/química , Fosfolipídeos/química , Fósforo/química , Ligação Proteica , Conformação Proteica , Multimerização ProteicaRESUMO
BACKGROUND AND AIM: We aimed to clarify the influences of aldehyde dehydrogenase 2 (ALDH2), alcohol dehydrogenase 1B (ADH1B) polymorphisms, and ethanol consumption profile to hepatocellular carcinoma (HCC) development in alcoholic liver cirrhosis without chronic hepatitis B and C virus infection (non-B non-C). METHODS: Of 236 freshly diagnosed non-B non-C alcoholic liver cirrhosis patients, 67 were diagnosed as HCC and the remaining 169 as not having HCC. The relationship between the genetic polymorphisms and development to HCC were evaluated in well-matched patients with HCC (HCC group, n = 67) and without HCC (non-HCC group, n = 67) using propensity scores in age, sex, and prevalence of diabetes mellitus. RESULTS: Daily amount of ethanol consumption was significantly lower (P = 0.005), and consumptive period was significantly longer (P = 0.003) in HCC group than non-HCC group. Of 134 well-matched patients, 113 (84.3%) had ALDH2*1/*1 genotype and 21 (15.7%) had ALDH2*1/*2 genotype. In HCC development, consumptive long period (P = 0.007) and carrying ALDH2*1/*2 genotype (P = 0.026) were identified as significant factors independently participated, while there was no relation to ADH1B polymorphism. In addition, consumptive period was significantly longer in HCC group than non-HCC group in ALDH2*1/*1 genotype patients (P = 0.0005), while there was no difference in profile of ethanol consumption in ALDH2*1/*2 genotype patients. Among HCC group, daily (P = 3.78 × 10(-6) ) and cumulative amount (P = 4.89 × 10(-6) ) of ethanol consumption were significantly higher in ALDH2*1/*1 genotype patients than ALDH2*1/*2 genotype patients. CONCLUSION: In alcoholic liver cirrhosis, investigations of ALDH2 polymorphism and ethanol consumption profile are useful for prediction of HCC development.
Assuntos
Consumo de Bebidas Alcoólicas/genética , Aldeído Desidrogenase/genética , Carcinoma Hepatocelular/genética , Cirrose Hepática Alcoólica/genética , Neoplasias Hepáticas/genética , Polimorfismo Genético/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Álcool Desidrogenase/genética , Consumo de Bebidas Alcoólicas/efeitos adversos , Aldeído-Desidrogenase Mitocondrial , Povo Asiático , Carcinoma Hepatocelular/epidemiologia , Carcinoma Hepatocelular/etiologia , Medicamentos de Ervas Chinesas , Eleutherococcus , Ásia Oriental/epidemiologia , Feminino , Previsões , Humanos , Cirrose Hepática Alcoólica/etiologia , Neoplasias Hepáticas/epidemiologia , Neoplasias Hepáticas/etiologia , Masculino , Pessoa de Meia-IdadeRESUMO
Prenatal exposure to low-doses of bisphenol A (BPA) has been shown to affect murine neocortical development by accelerating neuronal differentiation/migration through disrupting thyroid hormone function. We therefore studied whether prenatal exposure to low-doses of BPA affected organization of adult neocortical structures. Pregnant mice were injected with 20 microg/kg of BPA daily from embryonic day 0.5 (E0.5) and bromodeoxyuridine (BrdU) was injected at E12.5, E14.5 and at E16.5, and the fetal brains were analyzed after birth. The BrdU-positive cells labeled at E14.5 were significantly increased in the Vth and VIth cortical layers of BPA-treated mice at postnatal 3 weeks (P3W), whereas they were confined to the IVth layer of control mice, though such differences disappeared at P12W. The thalamocortical projections demonstrated by DiI-labeling were abnormal at P3W and P12W in BPA-treated mice. These results indicate that BPA might affect not only neocortical development but also thalamocortical connections.