Dataset Information

Intracellular gene transfer: reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase.

ABSTRACT: Subunit 2 of cytochrome c oxidase (Cox2) in legumes offers a rare opportunity to investigate factors necessary for successful gene transfer of a hydrophobic protein that is usually mitochondrial-encoded. We found that changes in local hydrophobicity were necessary to allow import of this nuclear-encoded protein into mitochondria. All legume species containing both a mitochondrial and nuclear encoded Cox2 displayed a similar pattern, with a large decrease in hydrophobicity evident in the first transmembrane region of the nuclear encoded protein compared with the organelle-encoded protein. Mitochondrial-encoded Cox2 could not be imported into mitochondria under the direction of the mitochondrial targeting sequence that readily supports the import of nuclear encoded Cox2. Removal of the first transmembrane region promotes import ability of the mitochondrial-encoded Cox2. Changing just two amino acids in the first transmembrane region of mitochondrial-encoded Cox2 to the corresponding amino acids in the nuclear encoded Cox2 also promotes import ability, whereas changing the same two amino acids in the nuclear encoded Cox2 to what they are in the mitochondrial-encoded copy prevents import. Therefore, changes in amino acids in the mature protein were necessary and sufficient for gene transfer to allow import under the direction of an appropriate signal to achieve the functional topology of Cox2.

SUBMITTER: Daley DO

PROVIDER: S-EPMC124958 | biostudies-literature | 2002 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Intracellular gene transfer: reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase.

Daley Daniel O DO Clifton Rachel R Whelan James J

Proceedings of the National Academy of Sciences of the United States of America 20020725 16

Subunit 2 of cytochrome c oxidase (Cox2) in legumes offers a rare opportunity to investigate factors necessary for successful gene transfer of a hydrophobic protein that is usually mitochondrial-encoded. We found that changes in local hydrophobicity were necessary to allow import of this nuclear-encoded protein into mitochondria. All legume species containing both a mitochondrial and nuclear encoded Cox2 displayed a similar pattern, with a large decrease in hydrophobicity evident in the first tr ...[more]

PMID: 12142462

Similar Datasets

Project description:BACKGROUND:Treatment of heart failure remains one of the most challenging task for intensive care medicine, cardiology and cardiac surgery. New options and better indicators are always required. Understanding the basic mechanisms underlying heart failure promote the development of adjusted therapy e.g. assist devices and monitoring of recovery. If cardiac failure is related to compromised cellular respiration of the heart, remains unclear. Myocardial respiration depends on Cytochrome c- Oxidase (CytOx) activity representing the rate limiting step for the mitochondrial respiratory chain. The enzymatic activity as well as mRNA expression of enzyme's mitochondrial encoded catalytic subunit 2, nuclear encoded regulatory subunit 4 and protein contents were studied in biopsies of cardiac patients suffering from myocardial insufficiency and dilated cardiomyopathy (DCM). METHODS:Fifty-four patients were enrolled in the study and underwent coronary angiography. Thirty male patients (mean age: 45 +/-?15 yrs.) had a reduced ejection fraction (EF) 35?±?12% below 45% and a left ventricular end diastolic diameter (LVEDD) of 71?±?10 mm bigger than 56 mm. They were diagnosed as having idiopathic dilated cardiomyopathy (DCM) without coronary heart disease and NYHA-class 3 and 4. Additionally, 24 male patients (mean age: 52 +/-?11 yrs.) after exclusion of secondary cardiomyopathies, coronary artery or valve disease, served as control (EF: 68?±?7, LVEDD: 51?±?7 mm). Total RNA was extracted from two biopsies of each person. Real-time PCR analysis was performed with specific primers followed by a melt curve analysis. Corresponding protein expression in the tissue was studied with immune-histochemistry while enzymatic activity was evaluated by spectroscopy. RESULTS:Gene and protein expression analysis of patients showed a significant decrease of subunit 4 (1.1 vs. 0.6, p?<?0.001; 7.7?±?3.1% vs. 2.8?±?1.4%, p?<?0.0001) but no differences in subunit 2. Correlations were found between reduced subunit 2 expression, low EF (r?=?0.766, p?<?0.00045) and increased LVEDD (r?=?0.492, p?<?0.0068). In case of DCM less subunit 4 expression and reduced shortening fraction (r?=?0.524, p?<?0.017) was found, but enzymatic activity was higher (0.08?±?0.06 vs. 0.26?±?0.08 U/mg, p?<?0.001) although myocardial oxygen consumption continued to the same extent. CONCLUSION:In case of myocardial insufficiency and DCM, decreased expression of COX 4 results in an impaired CytOx activity. Higher enzymatic activity but equal oxygen consumption contribute to the pathophysiology of the myocardial insufficiency and appears as an indicator of oxidative stress. This kind of dysregulation should be in the focus for the development of diagnostic and therapy procedures.

Project description:Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung-specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2-knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2-knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2-knockout mice, lung COX activity and cellular ATP levels were significantly reduced (-50 and -29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced P(enh) and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot-Leyden crystals. In addition, there was an interesting sex-specific phenotype, in which the knockout females showed reduced lean mass (-12%), reduced total oxygen consumption rate (-8%), improved glucose tolerance, and reduced grip force (-14%) compared to wild-type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction and thus reduced airway responsiveness; long-term lung pathology develops in the knockout mice due to impairment of energy-costly lung maintenance processes; and therefore, we propose mitochondrial oxidative phosphorylation as a novel target for the treatment of respiratory diseases, such as asthma.

Project description:BackgroundAccurate identification of mosquito species is essential for the development and optimization of strategies to control mosquitoes and mosquito-borne diseases. Problems with the morphological identification of mosquito species have led to the use of molecular identification techniques, in particular the Folmer cytochrome c oxidase subunit I (COI) PCR system (FCOS), originally designed to identify a range of other invertebrates.MethodsAs there can be difficulties identifying mosquitoes using FCOS, we re-evaluated the FCOS primers and developed a new COI-based SYBR PCR (the Auburn COI system-AUCOS) to improve the molecular identification of mosquitoes. Sequence data in GenBank for 33 species from 10 genera of mosquitoes were used to develop our AUCOS primers. Two molecular assays (AUCOS, FCOS) and morphological identification were carried out on mosquitoes collected from the field in Auburn, Alabama (USA) and on Saint Kitts.ResultsWith a convenience sample of individual mosquitoes comprising 19 species from six genera in Saint Kitts (n = 77) and Auburn (n = 48), our AUCOS provided higher-quality sequence data than FCOS. It also proved more sensitive than FCOS, successfully amplifying 67.5% (85/126) as opposed to 16.7% (21/126) of the samples. The species determined by morphology, or genus with damaged samples, matched that as determined by AUCOS for 84.9% (62/73) of the samples. Morphological classification was confirmed by FCOS with 81.0% (17/21) of samples producing utilizable sequences. While both FCOS and AUCOS correctly identified all the Aedes, Anopheles, Deinocerites, and Uranotaenia species in the study, identification of Culex species was less successful with both methods: 50.0% (3/6) by FCOS and 35.7% (5/14) by AUCOS.ConclusionsThe AUCOS DNA barcoding system for mosquito species described in this study is superior to the existing FCOS for the identification of mosquito species. As AUCOS and FCOS amplify the same variable region of the COI, the large amount of existing data on GenBank can be used to identify mosquito species with sequences produced by either PCR.

Dataset Information

Intracellular gene transfer: reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase.

Publications

Intracellular gene transfer: reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets