Subunit VIIa Research Articles

MicroRNA-10a negatively controls regulatory T cell suppression of colitis through inhibition of mitochondrial oxidation and Blimp1 expression

Abstract MicroRNAs play critical roles in maintaining intestinal homeostasis, and regulatory T cells (Tregs) restrict intestinal inflammation. However, how Tregs is regulated is still not completely understood. In this study, we investigate the mechanisms by which miR-10a controls Tregs’ suppressive function, thereby regulating intestinal inflammation. We found that miR-10a deficient Tregs expressed higher function markers in the intestine and were more potent in suppressing T cell proliferation and colitis induced by CD4+ CD45Rbhi T cells. Moreover, miR-10a did not affect Treg stability, which was evidenced by no differences in Foxp3 expression and methylation in Regulatory T-Cell-Specific Demethylated Region (TSDR). RNA sequencing data indicated that miR-10a regulates Tregs’ oxidative phosphorylation, which was verified by a higher level of oxygen consumption and mitochondrial ROS in miR-10a deficient Tregs. Pretreatment with a low dose of oligomycin, the ATP inhibitor which reduces mitochondrial respiration, impaired Tregs function in suppressing naïve T cell proliferation and colitis induced by CD4+ CD45Rbhi T cells in Rag−/− mice. Mechanically, miR-10a directly binds to uqcrq, the subunit VII of mitochondrial respiratory chain complex III, and Prdm1, which encodes Blimp1, a transcription factor that regulates Tregs’ functions. Furthermore, Blimp1-deficient Tregs showed decreased expression of function markers with the lower level of OCR and failed to restrict CD45Rbhi T cell-induced colitis. Collectively, miR-10a negatively regulates Tregs’ suppressive function by altering the mitochondrial metabolic process and inhibiting Blimp1, thereby controlling intestinal inflammation.

Regulation of β-mercuryl alcohol metabolic flow in Saccharomyces cerevisiae cells

In this study, citrate synthase gene(CIT2), and malate synthase gene(MLS1) were successfully knocked out in β-amyrin-producing yeast cells by using CRISPR/CAS9. The promoter of phosphoglucose isomerase gene(PGI1) was replaced by that of cytochrome c oxidase subunit Ⅶa(Cox9)to weaken its expression, aiming to channel more carbon flux into the NADPH-producing pathway. The fermentation results showed that CIT2 deletion had no effect on the β-amyrin production. Compared with the control strain, the production of β-amyrin was increased by 1.85 times after deleting MLS1, reaching into 3.3 mg·L~(-1). By replacing the promoter of PGI1, the β-amyrin yield was 3.75 times higher than that of the control strain, reaching up to 6.7 mg·L~(-1). This study successfully knocked out the CITT2 and MLS1 genes and weakened the PGI1 gene by using CRISPR/CAS9, which directly influenced the production of β-amyrin and provided some reference for the the metabolic engineering of triterpernoid producing strain.

Cartilage rings contribute to the proper embryonic tracheal epithelial differentiation, metabolism, and expression of inflammatory genes.

The signaling cross talk between the tracheal mesenchyme and epithelium has not been researched extensively, leaving a substantial gap of knowledge in the mechanisms dictating embryonic development of the proximal airways by the adjacent mesenchyme. Recently, we reported that embryos lacking mesenchymal expression of Sox9 did not develop tracheal cartilage rings and showed aberrant differentiation of the tracheal epithelium. Here, we propose that tracheal cartilage provides local inductive signals responsible for the proper differentiation, metabolism, and inflammatory status regulation of the tracheal epithelium. The tracheal epithelium of mesenchyme-specific Sox9Δ/Δ mutant embryos showed altered mRNA expression of various epithelial markers such as Pb1fa1, surfactant protein B (Sftpb), secretoglobulin, family 1A, member 1 (Scgb1a1), and trefoil factor 1 (Tff1). In vitro tracheal epithelial cell cultures confirmed that tracheal chondrocytes secrete factors that inhibit club cell differentiation. Whole gene expression profiling and ingenuity pathway analysis showed that the tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and transforming growth factor-β (TGF-β) signaling pathways were significantly altered in the Sox9 mutant trachea. TNF-α and IFN-γ interfered with the differentiation of tracheal epithelial progenitor cells into mature epithelial cell types in vitro. Mesenchymal knockout of Tgf-β1 in vivo resulted in altered differentiation of the tracheal epithelium. Finally, mitochondrial enzymes involved in fat and glycogen metabolism, cytochrome c oxidase subunit VIIIb (Cox8b) and cytochrome c oxidase subunit VIIa polypeptide 1 (Cox7a1), were strongly upregulated in the Sox9 mutant trachea, resulting in increases in the number and size of glycogen storage vacuoles. Our results support a role for tracheal cartilage in modulation of the differentiation and metabolism and the expression of inflammatory-related genes in the tracheal epithelium by feeding into the TNF-α, IFN-γ, and TGF-β signaling pathways.

COX7AR is a Stress-inducible Mitochondrial COX Subunit that Promotes Breast Cancer Malignancy.

Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, plays a key role in regulating mitochondrial energy production and cell survival. COX subunit VIIa polypeptide 2-like protein (COX7AR) is a novel COX subunit that was recently found to be involved in mitochondrial supercomplex assembly and mitochondrial respiration activity. Here, we report that COX7AR is expressed in high energy-demanding tissues, such as brain, heart, liver, and aggressive forms of human breast cancer cells. Under cellular stress that stimulates energy metabolism, COX7AR is induced and incorporated into the mitochondrial COX complex. Functionally, COX7AR promotes cellular energy production in human mammary epithelial cells. Gain- and loss-of-function analysis demonstrates that COX7AR is required for human breast cancer cells to maintain higher rates of proliferation, clone formation, and invasion. In summary, our study revealed that COX7AR is a stress-inducible mitochondrial COX subunit that facilitates human breast cancer malignancy. These findings have important implications in the understanding and treatment of human breast cancer and the diseases associated with mitochondrial energy metabolism.

Prediction of genetic risk factors of atherosclerosis using various bioinformatic tools.

The aim of this study was to identify potential markers of atherosclerosis development in familial hypercholesterolemia (FH) patients. GSE13985 microarray data, generated using blood samples from 5 FH patients and 5 matched controls, was downloaded from the Gene Expression Omnibus. Differentially expressed genes (DEGs) between FH and controls were identified and a protein-protein interaction (PPI) network was constructed. Module and hub proteins were screened in this network. The module genes were subjected to a gene ontology (GO) analysis, and a Kyoto Encyclopedia of Genes and Genomes enrichment analysis was also performed. A total of 394 genes, including 125 up- and 269 down-regulated genes, were differentially expressed. Ribosomal proteins L9 (RPL9), L35 (RPL35), and S7 (RPS7) were designated as hub nodes in the PPI network. The DEGs were found to be significantly enriched in ribosomal and oxidative phosphorylation pathways. Ribosomal protein genes were found to be involved in the ribosomal pathway. The cytochrome-c oxidase (COX) genes COX subunit VIIa polypeptide 2 (COX7A2), COX subunit VIIb (COX7B), COX subunit VIIc (COX7C), and COX subunit VIc (COX6C) were enriched in the oxidative phosphorylation pathway. Module analysis and GO enrichment analysis identified ribosomal proteins as important regulators of FH. Ribosomal and oxidative phosphorylation pathways may be closely associated with atherosclerosis development. Ribosomal protein genes and cytochrome-coxidase genes may be potential therapeutic targets for atherosclerosis.

Destabilization of the Quaternary Structure of Bovine Heart Cytochrome c Oxidase upon Removal of Tightly Bound Cardiolipin

The quaternary structural stability of cardiolipin-containing (CcO(CL+)) versus CL-free cytochrome c oxidase (CcO(CL-)) was compared using structural perturbants as probes. Exposure to increasing concentrations of urea or guanidinium chloride causes sequential dissociation of five subunits from CcO(CL+) in the order VIa and VIb, followed by III and VIIa, and ultimately Vb. Removal of CL from CcO destabilizes the association of each of these five subunits with the core of CcO. Subunits VIa and VIb spontaneously dissociate from CcO(CL-) even in the absence of denaturant and are no longer present after purification of the CL-free 11-subunit complex by ion exchange chromatography. The other 11 subunits remain associated in a partially active complex, but the association of subunits III, VIIa, and Vb is weakened; i.e., the midpoints for the subunit dissociation curves are each shifted to a lower perturbant concentration (lower by 1.1-1.7 M urea; lower by 0.3-0.4 M GdmCl). This corresponds to a decrease of ∼9 kJ in the Gibbs free association energy for each of these subunits when CL is removed from CcO. With either CcO(CL+) or CcO(CL-), loss of enzymatic activity occurs coincident with dissociation of subunits III and VIIa. The loss of activity is irreversible, and reactivation of CcO(CL-) by exogenous CL occurs only if both subunits remain associated with the core of CcO. Inclusion of sulfate anions stabilizes the association of VIIa more than III, resulting in a slight separation of the urea-induced dissociation curves. In this case, activity loss correlates much better with dissociation of subunit VIIa than III. We conclude that (1) bound cardiolipin is an important stabilizing factor in the quaternary structure of CcO and (2) association of subunit VIIa (possibly together with subunit III) is critical for functional CL binding and full electron-transfer activity of CcO.

Self-Administrationof Oxycodone by Adolescent and Adult Mice Differentially Affects Hypothalamic Mitochondrial Metabolism Gene Expression

Objective: Illicit prescription drug use among adolescents is a pressing public health concern: 12% of high school students report using prescription opioids with many progressing to use heroin. However, little is known about the effects of these drugs on the adolescent brain compared to the adult brain. This study examined the effect of adolescent oxycodone self-administration on gene expression specifically related to mitochondrial energy metabolism in the hypothalamus as the hypothalamus is involved in the regulation of feeding, reproduction and stress-induced drug seeking behavior. Methods: Adolescent and adult mice self-administered oxycodone (0.25mg/kg/infusion) or served as yoked saline controls 2 hours daily for 14 days. The hypothalamic mRNAs were analyzed with qPCR using a commercially available “mitochondrial energy metabolism” PCR array containing 84 genes. Results: mRNA levels of the ubiquinol-cytochrome c reductase, complex III subunit VII (Uqcrq) gene showed an experiment-wise significant increase in adolescents that self-administered oxycodone compared with controls. This effect was not found in adult mice. We also found that mRNA levels of the oxidase assembly 1-like (Oxa1l) gene showed a point-wise significant decrease in adult mice that had self-administered oxycodone. Additionally, twentyseven genes had increased expression in adolescents that self-administered oxycodone. Conversely, adults that self-administered oxycodone had eight genes with lower expression; none showed higher expression. Conclusion: These findings demonstrate that prescription opioid use caused significant changes of gene expression related to mitochondrial metabolism. The differences between adolescents and adults demonstrate the importance of studying adolescents in order to develop effective treatments.

Decreased Cytochrome c Oxidase Subunit VIIa in Aged Rat Heart Mitochondria: Immunocytochemistry

Aging decreases oxidative phosphorylation through cytochrome oxidase (COX) in cardiac interfibrillar mitochondria (IFM) in 24-month old (aged) rats compared to 6-month old adult Fischer 344 rats, whereas subsarcolemmal mitochondria (SSM) located beneath the plasma membrane remain unaffected. Immunoelectron microscopy (IEM) reveals in aged rats a 25% reduction in cardiac COX subunit VIIa in cardiac IFM, but not in SSM. In contrast, the content of subunit IV remains unchanged in both SSM and IFM, irrespective of age. These subunits are localized mainly on cristae membranes. In contrast, semi-quantitative immunoblotting, which detects denatured protein, indicates that the content of COX VIIa is similar in IFM and SSM from both aged and adult hearts. IEM provides a sensitive method for precise localizing and quantifying specific mitochondrial proteins. The lack of immunoreaction of COX VIIa subunit by IEM in aged IFM is not explained by a reduction in protein, but rather by a masking phenomenon or by an in situ change in protein structure affecting COX activity.

Miltefosine Induces Apoptosis-Like Cell Death in Yeast via Cox9p in CytochromecOxidase

Miltefosine has antifungal properties and potential for development as a therapeutic for invasive fungal infections. However, its mode of action in fungi is poorly understood. We demonstrate that miltefosine is rapidly incorporated into yeast, where it penetrates the mitochondrial inner membrane, disrupting mitochondrial membrane potential and leading to an apoptosis-like cell death. COX9, which encodes subunit VIIa of the cytochrome c oxidase (COX) complex in the electron transport chain of the mitochondrial membrane, was identified as a potential target of miltefosine from a genomic library screen of the model yeast Saccharomyces cerevisiae. When overexpressed in S. cerevisiae, COX9, but not COX7 or COX8, led to a miltefosine-resistant phenotype. The effect of miltefosine on COX activity was assessed in cells expressing different levels of COX9. Miltefosine inhibited COX activity in a dose-dependent manner in Cox9p-positive cells. This inhibition most likely contributed to the miltefosine-induced apoptosis-like cell death.

Two common genetic variants near nuclear-encoded OXPHOS genes are associated with insulin secretion in vivo

ContextMitochondrial ATP production is important in the regulation of glucose-stimulated insulin secretion. Genetic factors may modulate the capacity of the β-cells to secrete insulin and thereby contribute to the risk of type 2 diabetes.ObjectiveThe aim of this study was to identify genetic loci in or adjacent to nuclear-encoded genes of the oxidative phosphorylation (OXPHOS) pathway that are associated with insulin secretion in vivo.Design and methodsTo find polymorphisms associated with glucose-stimulated insulin secretion, data from a genome-wide association study (GWAS) of 1467 non-diabetic individuals, including the Diabetes Genetic Initiative (DGI), was examined. A total of 413 single nucleotide polymorphisms with a minor allele frequency ≥0.05 located in or adjacent to 76 OXPHOS genes were included in the DGI GWAS. A more extensive population-based study of 4323 non-diabetics, the PPP-Botnia, was used as a replication cohort. Insulinogenic index during an oral glucose tolerance test was used as a surrogate marker of glucose-stimulated insulin secretion. Multivariate linear regression analyses were used to test genotype–phenotype associations.ResultsTwo common variants were identified in the DGI, where the major C-allele of rs606164, adjacent to NADH dehydrogenase (ubiquinone) 1 subunit C2 (NDUFC2), and the minor G-allele of rs1323070, adjacent to cytochrome c oxidase subunit VIIa polypeptide 2 (COX7A2), showed nominal associations with decreased glucose-stimulated insulin secretion (P=0.0009, respective P=0.003). These associations were replicated in PPP-Botnia (P=0.002 and P=0.05).ConclusionOur study shows that genetic variation near genes involved in OXPHOS may influence glucose-stimulated insulin secretion in vivo.

Isolation and Characterization of a Silkworm cDNA Encoding a Protein Homologous to the 14kDa Protein of Bovine Ubiquinol-cytochrome C Reductase

In this study, we characterized the small subunit of ubiquinol-cytochrome C reductase (Bmuccr) of the silkwormBombyx mori, a model insect of Lepidopteron species. The Bmuccr gene covers a 1.4 kb genome region and contains 3exons. The ORF contained 354bp and encoded 117 amino acid residues, which shares 69% overall amino acid sequenceidentities with the subunit VII of ubiquinol-cytochrome C reductase from bovine. Phylogenetic tree showed Bmuccr hadhigh homology with T. castaneum homologous. The multiple sequence alignment of 16 subunit VII homologues showsthat Bmuccr is very hydrophilic, has a characteristic charge distribution, and has a high helical content. Expressionanalysis indicated that Bmuccr was highly expressed in larva stage and was down-regulated in embryos stage and adultstage of silkworm. The tissue-specific expression indicated Bmuccr had high-expression level in tissues that consumeoxygen. The analysis of domain structure of this protein suggested that it might be involved in correct assembly of thecytochrome bcl complex. Definition of the homologous of bovine subunit VII of ubiquinol-cytochrome C reductaseshould facilitate further analysis of structure/function relationships of silkworm cytochrome bcl complex.

Mitochondrial Complex III Deficiency Associated with a Homozygous Mutation in UQCRQ

A consanguineous Israeli Bedouin kindred presented with an autosomal-recessive nonlethal phenotype of severe psychomotor retardation and extrapyramidal signs, dystonia, athetosis and ataxia, mild axial hypotonia, and marked global dementia with defects in verbal and expressive communication skills. Metabolic workup was normal except for mildly elevated blood lactate levels. Brain magnetic resonance imaging (MRI) showed increased density in the putamen, with decreased density and size of the caudate and lentiform nuclei. Reduced activity specifically of mitochondrial complex III and variable decrease in complex I activity were evident in muscle biopsies. Homozygosity of affected individuals to UQCRB and to BCSIL, previously associated with isolated complex III deficiency, was ruled out. Genome-wide linkage analysis identified a homozygosity locus of approximately 9 cM on chromosome 5q31 that was further narrowed down to 2.14 cM, harboring 30 genes (logarithm of the odds [LOD] score 8.82 at theta = 0). All 30 genes were sequenced, revealing a single missense (p.Ser45Phe) mutation in UQCRQ (encoding ubiquinol-cytochrome c reductase, complex III subunit VII, 9.5 kDa), one of the ten nuclear genes encoding proteins of mitochondrial complex III.

ANESTHETIC PRECONDITIONING CONFERS ACUTE CARDIOPROTECTION VIA UP-REGULATION OF MANGANESE SUPEROXIDE DISMUTASE AND PRESERVATION OF MITOCHONDRIAL RESPIRATORY ENZYME ACTIVITY

The cellular and molecular mechanisms that underlie cardioprotection against I/R by anesthetic-induced preconditioning (APC) require further elucidation. Using isoflurane as a representative anesthetic, we evaluated the hypothesis that APC induces myocardial protection against I/R by attenuation of excessive reactive oxygen species and restoration of mitochondrial bioenergetics through postischemic up-regulation of manganese superoxide dismutase (MnSOD) expression and preservation of respiratory enzyme activity. Pentobarbital anesthetized open-chest Sprague-Dawley rats were subject to 30-min left coronary artery occlusion, followed by 120-min reperfusion. Before ischemia, rats were randomly assigned to receive 0.9% saline, two cycles of brief coronary artery occlusion and reperfusion, or a 30-min exposure to 1.0 minimum alveolar concentration isoflurane in the absence or presence of a specific mitochondrial adenosine triphosphate-sensitive potassium (KATP) channel blocker, 5-hydroxydecanoate; a membrane-permeable superoxide scavenger, 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl; or a NOS inhibitor, N(G)-nitro-L-arginine methyl ester. Isoflurane exposure induced an initial increase in myocardial superoxide (O2-), but not NO level. It also significantly decreased infarct size and restored mitochondrial respiratory enzyme activity or ATP production in I/R rat hearts, along with suppression of the O2- surge at reperfusion and increase in MnSOD expression or enzyme activity. These protective effects were abrogated by 5-hydroxydecanoate or 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl, but not by N(G)-nitro-L-arginine methyl ester pretreatment. These results suggest that opening of mitochondrial KATP channel, followed by O2- signaling, induces postischemic augmentation of MnSOD and preservation of mitochondrial respiratory enzyme activities, leading to attenuated cardiac O2- surge and restored ATP production during reperfusion, and underlie APC-induced cardioprotection.

Aup1p, a Yeast Mitochondrial Protein Phosphatase Homolog, Is Required for Efficient Stationary Phase Mitophagy and Cell Survival

Autophagy is a catabolic membrane-trafficking process that occurs in all eukaryotic cells and leads to the hydrolytic degradation of cytosolic material in the vacuolar or lysosomal lumen. Mitophagy, a selective form of autophagy targeting mitochondria, is poorly understood at present. Several recent reports suggest that mitophagy is a selective process that targets damaged mitochondria, whereas other studies imply a role for mitophagy in cell death processes. In a screen for protein phosphatase homologs that functionally interact with the autophagy-dedicated protein kinase Atg1p in yeast, we have identified Aup1p, encoded by Saccharomyces cerevisiae reading frame YCR079w. Aup1p is highly similar to a family of protein phosphatase homologs in animal cells that are predicted to localize to mitochondria based on sequence analysis. Interestingly, we found that Aup1p localizes to the mitochondrial intermembrane space and is required for efficient mitophagy in stationary phase cells. Viability studies demonstrate that Aup1p is required for efficient survival of cells in prolonged stationary phase cultures, implying a pro-survival role for mitophagy under our working conditions. Our data suggest that Aup1p may be part of a signal transduction mechanism that marks mitochondria for sequestration into autophagosomes.

Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast

The plasma membrane potential is mainly considered as the driving force for ion and nutrient translocation. Using the yeast Saccharomyces cerevisiae as a model organism, we have discovered a novel role of the membrane potential in the organization of the plasma membrane. Within the yeast plasma membrane, two non-overlapping sub-compartments can be visualized. The first one, represented by a network-like structure, is occupied by the proton ATPase, Pma1, and the second one, forming 300-nm patches, houses a number of proton symporters (Can1, Fur4, Tat2 and HUP1) and Sur7, a component of the recently described eisosomes. Evidence is presented that sterols, the main lipid constituent of the plasma membrane, also accumulate within the patchy compartment. It is documented that this compartmentation is highly dependent on the energization of the membrane. Plasma membrane depolarization causes reversible dispersion of the H(+)-symporters, not however of the Sur7 protein. Mitochondrial mutants, affected in plasma membrane energization, show a significantly lower degree of membrane protein segregation. In accordance with these observations, depolarized membranes also considerably change their physical properties (detergent sensitivity).

Photolabeling of Cardiolipin Binding Subunits within Bovine Heart CytochromecOxidase

Subunits located near the cardiolipin binding sites of bovine heart cytochrome c oxidase (CcO) were identified by photolabeling with arylazido-cardiolipin analogues and detecting labeled subunits by reversed-phase HPLC and HPLC-electrospray ionization mass spectrometry. Two arylazido-containing cardiolipin analogues were synthesized: (1) 2-SAND-gly-CL with a nitrophenylazido group attached to the polar headgroup of cardiolipin (CL) via a linker containing a cleavable disulfide; (2) 2',2''-bis-(AzC12)-CL with two of the four fatty acid tails of cardiolipin replaced by 12-(N-4-azido-2-nitrophenyl) aminododecanoic acid. Both arylazido-CL derivatives were used to map the cardiolipin binding sites within two types of detergent-solubilized CcO: (1) intact 13-subunit CL-containing CcO (three to four molecules of endogenous CL remain bound per CcO monomer); (2) 11-subunit CL-free CcO (subunits VIa and VIb are missing because they dissociate during CL removal). Upon the basis of these photolabeling studies, we conclude that (1) subunits VIIa, VIIc, and possibly VIII are located near the two high-affinity cardiolipin binding sites, which are present in either form of CcO, and (2) subunit VIa is located adjacent to the lower affinity cardiolipin binding site, which is only present in the 13-subunit form of CcO. These data are consistent with the recent CcO crystal structure in which one cardiolipin is located near subunit VIIa and a second is located near subunit VIa (PDB ID code referenced in Tomitake, T. et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 15304-15309). However, we propose that a third cardiolipin is bound between subunits VIIa and VIIc near the entrance to the D-channel. Cardiolipin bound at this location could potentially function as a proton antenna to facilitate proton entry into the D-channel. If true, it would explain the CcO requirement of bound cardiolipin for full electron transport activity.

Compromised liver mitochondrial function and complex activity in low feed efficient broilers are associated with higher oxidative stress and differential protein expression

Variations in broiler growth and efficiency have been explained in part by differences in mitochondrial function and biochemistry in broilers. To further our knowledge in this regard, 2 experiments were carried out to determine the relationships of a) mitochondrial function and activities of various electron transport chain (ETC) complexes; b) production of H2O2, a reactive oxygen species (ROS), and its association with protein oxidation; and c) mitochondrial protein expression in liver of a single line male broilers with low or high feed efficiency (FE, n = 5 to 8 per group). Mitochondrial function and complex activities were measured polarographically and spectrophotometrically, respectively. H2O2 was measured fluorimetrically, whereas oxidized protein (carbonyls) and specific mitochondrial proteins were analyzed using Western blots. Mitochondrial function (ETC coupling) and activities of ETC complexes (I, II, III, and IV) were higher in high FE compared with low FE broilers. H2O2 and protein carbonyls were higher in the livers of low FE broilers than in high FE broilers. Whereas the expression of 4 immunoreactive proteins [NAD3 (complex I), subunit VII (complex III), cytochrome c oxidase subunits (COX) II, and COX IVb (complex IV)] were higher in low FE liver mitochondria and 2 proteins [subunit 70 (complex II) and a-ATP synthase (complex V)] were higher in high FE birds, there were no differences between groups in the expression of 18 other mitochondrial proteins. In conclusion, increases in oxidative stress in low FE broilers were caused by or may contribute to differences in mitochondrial function (ETC coupling and complex activities) or the differential expression of steady-state levels of some mitochondrial proteins in the liver. Understanding the role of oxidative stress in Low FE broilers will provide clues in understanding the cellular basis of feed efficiency.

A Role for Pet100p in the Assembly of Yeast Cytochrome c Oxidase: INTERACTION WITH A SUBASSEMBLY THAT ACCUMULATES IN A pet100 MUTANT

The biogenesis of multimeric protein complexes of the inner mitochondrial membrane in yeast requires a number of nuclear-coded ancillary proteins. One of these, Pet100p, is required for cytochrome c oxidase. Previous studies have shown that Pet100p is not required for the synthesis, processing, or targeting of cytochrome c oxidase subunits to the mitochondrion nor for heme A biosynthesis. Here, we report that Pet100p does not affect the localization of cytochrome c oxidase subunit polypeptides to the inner mitochondrial membrane but instead functions after they have arrived at the inner membrane. We have also localized Pet100p to the inner mitochondrial membrane in wild type cells, where it is present in a subassembly (Complex A) with cytochrome c oxidase subunits VII, VIIa, and VIII. Pet100p does not interact with the same subunits after they have been assembled into the holoenzyme. In addition, we have identified two subassemblies that are present in pet100 null mutant cells: one subassembly (Complex A') is composed of subunits VII, VIIa, and VIII but not Pet100p, and another subassembly (Complex B) is composed of subunits Va and VI. Because pet100 null mutant cells lack assembled cytochrome c oxidase but accumulate Complexes A' and B it appears likely that these subassemblies of cytochrome c oxidase subunits are intermediates along an assembly pathway for holocytochrome c oxidase and that Pet100p functions in this pathway to facilitate the interaction(s) between Complex A' and other cytochrome c oxidase subassemblies and subunits.

Specific modification of two tryptophans within the nuclear-encoded subunits of bovine cytochrome c oxidase by hydrogen peroxide.

Hydrogen peroxide does more than react with the binuclear center of oxidized bovine cytochrome c oxidase and generate the well-characterized "peroxy" and "ferryl" forms. Hydrogen peroxide also inactivates detergent-solubilized cytochrome c oxidase in a time- and concentration-dependent manner. There is a 70-80% decrease of electron-transport activity, peroxidation of bound cardiolipin, modification of two nuclear-encoded subunits (IV and VIIc), and dissociation of approximately 60% of subunits VIa and VIIa. Modification of subunit VIIc and dissociation of subunit VIIa are coupled events that probably are responsible for the inactivation of cytochrome c oxidase. When cytochrome c oxidase is exposed to 500 microM hydrogen peroxide for 30 min at pH 7.4 and room temperature, subunits IV (modified up to 20%) and VIIc (modified up to 70%) each have an increased mass of 16 Da as detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization mass spectrometry. In each case, the increased mass is caused by oxidation of a tryptophan (Trp19 within subunit VIIc and Trp48 within subunit IV), almost certainly due to formation of hydroxytryptophan. We conclude that hydrogen peroxide-induced oxidation of tryptophan and cardiolipin proceeds via the binuclear center since both modifications are prevented if the binuclear center is first blocked with cyanide. Bound cardiolipin and oxidized tryptophans are localized relatively far from the binuclear center (30-60 A); therefore, oxidation probably occurs by migration of a free radical generated at the binuclear center to these distal reaction sites.

Retention of a duplicate gene through changes in subcellular targeting: an electron transport protein homologue localizes to the golgi.

Cytochrome c oxidase (COX), the terminal enzyme complex of the electron transport chain, contains 13 subunits, 3 encoded by mitochondrial DNA and 10 by nuclear. Several of the nuclear subunits, including subunit VIIa, are known to have two tissue- and development-specific isoforms in mammals. A recently identified third member of the gene family, COX7AR, encodes a protein previously thought to function in mitochondria. However, observation of fluorescent pCOX7AR C-terminal fusion proteins in HeLa cells showed that pCOX7AR is localized to the Golgi apparatus. Sequence analyses indicate that the duplication of COX7AR occurred prior to the origin of the Euteleostomi (bony vertebrates) and that pCOX7AR is more highly conserved than the two other isoforms. These results indicate that, after gene duplication and modification of the mitochondrial targeting signal, pCOX7AR was evolutionarily altered to a new and apparently important function in the Golgi. These results also suggest that predictions of function from homology can be misleading and show that specialization and modification of subcellular localization are similar to cis-element subfunctionalization. In cis-element subfunctionalization, complementary null mutations occur to the cis-elements of the descendents of a gene duplication, causing both descendent genes to be obligate. In the process described in this paper, which could be termed subcellular subfunctionalization, complementary null mutations can occur to the subcellular localization signals of the descendants of a gene duplication, causing both descendent genes to be similarly obligate. Noncomplementary null mutations could also uncover an alternate localization, which is the more likely case for pCOX7AR.