Evidence for cytochrome oxidase subunit I and a cytochrome c--subunit II fused protein in the cytochrome 'c1aa3' of Thermus thermophilus. How old is cytochrome oxidase?

The terminal cytochrome c1aa3 of the respiratory chain of Thermus thermophilus has been isolated and purified to homogeneity by a novel procedure. The two subunit proteins (55 and 33 kDa) have been characterized chemically. Computer searches with partial amino acid sequences obtained from both subunits show that the larger subunit belongs to the cytochrome oxidase subunit I protein family while the smaller covalently heme-binding subunit is not a cytochrome c1 but appears to be a fused protein between cytochrome c and cytochrome oxidase subunit II. With respect to the 16-S rRNA-derived phylogeny of procaryotes, the results show that the genetic information for an O2-reacting cytochrome oxidase (EC 1.9.3.1) existed already in early eubacteria.     

TGF-β sensitivity is determined by N-linked glycosylation of the type II TGF-β receptor

N-linked glycosylation is a critical determinant of protein structure and function, regulating processes such as protein folding, stability and localization, ligand-receptor binding and intracellular signalling. TβRII [type II TGF-β (transforming growth factor β) receptor] plays a crucial role in the TGF-β signalling pathway. Although N-linked glycosylation of TβRII was first demonstrated over a decade ago, it was unclear how this modification influenced TβRII biology. In the present study, we show that inhibiting the N-linked glycosylation process successfully hinders binding of TGF-β1 to TβRII and subsequently renders cells resistant to TGF-β signalling. The lung cancer cell line A549, the gastric carcinoma cell line MKN1 and the immortal cell line HEK (human embryonic kidney)-293 exhibit reduced TGF-β signalling when either treated with two inhibitors, including tunicamycin (a potent N-linked glycosylation inhibitor) and kifunensine [an inhibitor of ER (endoplasmic reticulum) and Golgi mannosidase I family members], or introduced with a non-glycosylated mutant version of TβRII. We demonstrate that defective N-linked glycosylation prevents TβRII proteins from being transported to the cell surface. Moreover, we clearly show that not only the complex type, but also a high-mannose type, of TβRII can be localized on the cell surface. Collectively, these findings demonstrate that N-linked glycosylation is essentially required for the successful cell surface transportation of TβRII, suggesting a novel mechanism by which the TGF-β sensitivity can be regulated by N-linked glycosylation levels of TβRII.     

Molecular evolution of cytochrome c oxidase subunit I in primates: is there coevolution between mitochondrial and nuclear genomes?

Phylogenetic analyses carried out on cytochrome c oxidase (COX) subunit I mitochondrial genes from 14 primates representing the major branches of the order and four outgroup nonprimate eutherians revealed that transversions and amino acid replacements (i.e., the more slowly occurring sequence changes) contained lower levels of homoplasy and thus provided more accurate information on cladistic relationships than transitions (i.e., the more rapidly occurring sequence changes). Several amino acids, each with a high likelihood of functionality involving the binding of cytochrome c or interaction with COX VIII, have changed in Anthropoidea, the primate suborder grouping New World monkey, Old World monkey, ape, and human lineages. They are conserved in other mammalian lineages and in nonanthropoid primates. Maximum-likelihood ancestral COX I nucleotide sequences were determined utilizing a near most parsimonious branching arrangement for the primate sequences that was consistent with previously hypothesized primate cladistic relationships based on larger and more diverse data sets. Relative rate tests of COX I mitochondrial sequences showed an elevated nonsynonymous (N) substitution rate for anthropoid-nonanthropoid comparisons. This finding for the largest mitochondrial (mt) DNA-encoded subunit is consistent with previous observations of elevated nonsynonymous substitution/synonymous substitution (S) rates in primates for mt-encoded COX II and for the nuclear-encoded COX IV and COX VIIa-H. Other COX-related proteins, including cytochrome c and cytochrome b, also show elevated amino acid replacement rates or N/S during similar time frames, suggesting that this group of interacting genes is likely to have coevolved during primate evolution.     

Comparison of DNA extraction methods for PCR amplification of mitochondrial cytochrome c oxidase subunit II (COII) DNA from primate fecal samples

Mitochondrial COII DNA was amplified by PCR from total DNA extracted from field collected primate fecal samples (n=24) which had been stored without refrigeration for over 30 days. High molecular weight DNA total DNA was obtained from samples stored in 70% (v/v) ethanol, SDS lysis buffer (LB) and guanidine isothiocyanate buffer (GTB) than from samples stored in 10% formalin. Fecal DNA quality and COII amplification varied according to storage solution (formalin, ethanol, LB and GTB), extraction method (LB-based and GTB-based) and primate species (chimpanzee, baboon, human). It is recommended that fecal samples be collected in LB for DNA analysis. However, GTB-based protocols are suitable when total RNA is needed for epidemiological studies of viral diseases or gene expression analysis.     

Searching interaction partners of protein kinase CK2β subunit by two-hybrid screening

To date, the intracellular regulation of protein kinase CK2 is unknown. However it was observed that the enzyme associates with several intracellular proteins and the formation of such molecular complexes may represent a mechanism for the control of CK2 activity. Using the Interaction Trap system in yeast, with the CK2 as a bait, we looked for CK2 partners. We present the identification of new potential partners of CK2 and it is hoped that their classification will help in understanding the physiological roles and the regulation of CK2 in the cell.     

The binding interface of cytochrome c and cytochrome c₁ in the bc₁ complex: rationalizing the role of key residues

The interaction of cytochrome c with ubiquinol-cytochrome c oxidoreductase (bc₁ complex) has been studied for >30 years, yet many aspects remain unclear or controversial. We report the first molecular dynamic simulations of the cyt c-bc₁ complex interaction. Contrary to the results of crystallographic studies, our results show that there are multiple dynamic hydrogen bonds and salt bridges in the cyt c-c₁ interface. These include most of the basic cyt c residues previously implicated in chemical modification studies. We suggest that the static nature of x-ray structures can obscure the quantitative significance of electrostatic interactions between highly mobile residues. This provides a clear resolution of the discrepancy between the structural data and functional studies. It also suggests a general need to consider dynamic interactions of charged residues in protein-protein interfaces. In addition, a novel structural change in cyt c is reported, involving residues 21-25, which may be responsible for cyt c destabilization upon binding. We also propose a mechanism of interaction between cyt c₁ monomers responsible for limiting the binding of cyt c to only one molecule per bc₁ dimer by altering the affinity of the cytochrome c binding site on the second cyt c₁ monomer.     

Regulation of TGF-β receptor hetero-oligomerization and signaling by endoglin

Complex formation among transforming growth factor-β (TGF-β) receptors and its modulation by coreceptors represent an important level of regulation for TGF-β signaling. Oligomerization of ALK5 and the type II TGF-β receptor (TβRII) has been thoroughly investigated, both in vitro and in intact cells. However, such studies, especially in live cells, are missing for the endothelial cell coreceptor endoglin and for the ALK1 type I receptor, which enables endothelial cells to respond to TGF-β by activation of both Smad2/3 and Smad1/5/8. Here we combined immunoglobulin G–mediated immobilization of one cell-surface receptor with lateral mobility studies of a coexpressed receptor by fluorescence recovery after photobleaching (FRAP) to demonstrate that endoglin forms stable homodimers that function as a scaffold for binding TβRII, ALK5, and ALK1. ALK1 and ALK5 bind to endoglin with differential dependence on TβRII, which plays a major role in recruiting ALK5 to the complex. Signaling data indicate a role for the quaternary receptor complex in regulating the balance between TGF-β signaling to Smad1/5/8 and to Smad2/3.     

The CO adduct of yeast cytochrome c oxidase. M?ssbauer and photolysis studies

M?ssbauer spectra of 57Fe-enriched NADH-reduced yeast cytochrome c oxidase reveal two quadrupole doublets of unequal intensity; one (approximately 33%) is typical of high-spin ferrous heme with histidine coordination and is assigned to heme a3, while the other (approximately 67%) is typical of low-spin heme with two nitrogeneous axial ligands as expected from heme a. The excess intensity (approximately 17%) of the low-spin doublet must therefore be assigned to heme a3 in a modified environment. The M?ssbauer spectra of the same sample exposed to CO show that 50% of the heme iron forms a CO adduct, consistent with heme a3 being inhibited by CO. While low-spin hem a has the same M?ssbauer parameters as in the reduced sample, its intensity has dropped to 35%. A distinctly new high-spin species (approximately 15%) is observed and assigned to heme a in a modified environment. The comparable size of the unexpected high-spin heme a fraction in the CO adduct and the low-spin heme a3 fraction in the reduced enzyme suggest that they arise from the same material. This material is likely to be the inactive fraction that has been found in all preparations of resting yeast cytochrome c oxidase (Siedow, J.N., Miller, S., and Palmer, G. (1981) J. Bioenerg. Biomembr. 14, 171-179). The kinetics of CO recombination following photolysis of the CO complex further confirms the coexistence of two distinct fractions associated with active and inactive protein. The majority (approximately 74%), presumably active protein, recombines exponentially from 160 to 270 K following an Arrhenius law. The large activation enthalpy, delta H approximately 35 kJ/mol, is comparable to that found in the beef heart enzyme, suggesting that the flashed-off CO is bound by the nearby CuB as in the mammalian system (Fiamingo, F.G., Altschuld, R.A., Moh, P.P., and Alben, J.O. (1982) J. Biol. Chem. 250, 1639-1650). In the minority, presumably inactive, fraction the CO recombination has fast nonexponential kinetics with a distribution of activation enthalpies peaking near delta Hp = 13 kJ/mol reminiscent of CO binding to myoglobin. In this inactive fraction CuB is apparently not accessible to the flashed-off CO.     

Shedding of epithin/PRSS14 is induced by TGF-β and mediated by tumor necrosis factor-α converting enzyme

Epithin/PRSS14, a type II transmembrane serine protease, plays critical roles in cancer metastasis. Previously, we have reported that epithin/PRSS14 undergoes ectodomain shedding in response to phorbol myristate acetate (PMA) stimulation. In this study, we show that transforming growth factor-β (TGF-β) induces rapid epithin/PRSS14 shedding through receptor mediated pathway in 427.1.86 thymoma cells. Tumor necrosis factor-α converting enzyme (TACE) is responsible for this shedding. Amino acid sequence encompassing the putative shedding cleavage site of epithin/PRSS14 exhibit strong homology to the cleavage site of l-selectin, a known TACE substrate. TACE inhibitor, TAPI-0 and TACE siRNA greatly reduced TGF-β-induced epithin/PRSS14 shedding. TGF-β treatment induces translocation of intracellular pool of TACE to the membrane where epithin/PRSS14 resides. These findings suggest that TGF-β induces epithin/PRSS14 shedding by mediating translocation of epithin/PRSS14 sheddase, TACE, to the membrane.     

Mutations in the C-terminal domain of topoisomerase II affect meiotic function and interaction with the casein kinase 2β subunit

Topoisomerase II is a major target of the protein kinase casein kinase 2 (PK CK2) in vivo. All major phosphorylation acceptor sites in the yeast enzyme are found in the C-terminal 350aa. The acceptor sites are generally clustered such that there is more than one modified Ser or Thr within a short peptide. Mutagenesis of the predicted acceptor sites have confirmed that five of the eight predicted sites are targeted in vitro and in vivo by PK CK2. Mutation to nonphosphorylatable, neutral residues provokes at most a 10% increase in mitotic doubling time. Truncation of the enzyme leaves the enzyme catalytically active, but slightly lengthens the doubling time during mitotic growth and impedes progress through meiosis. Since this could reflect the loss of interaction with an important ligand, we have examined whether the C-terminal domain of the yeast enzyme mediates interaction with the regulatory subunit of PK CK2, which was previously reported to bind topoisomerase II. We find that point mutation of the phospho-acceptor sites does not abrogate the interaction with a small region of PK CK2 , while truncation at aal276 or aal236 does. The site of interaction within PK CK2 does not coincide with the highly negatively charged spermine binding site.     

Regulation of TGF-β signal transduction by mono- and deubiquitylation of Smads

Polyubiquitylation leading to proteasomal degradation is a well-established mechanism for regulating TGF-β signal transduction components such as receptors and Smads. Recently, an equally important role was suggested for monoubiquitylation of both Smad4 and receptor-associated Smads that regulates their function without protein degradation. Monoubiquitylation of Smads was discovered following the identification of deubiquitylases required for TGF-β signaling, suggesting that continuous cycles of Smad mono- and deubiquitylation are required for proper TGF-β signal transduction. Here we summarize and discuss recent work on Smad mono- and deubiquitylation.     

The role of TGF-β and epithelial-to mesenchymal transition in diabetic nephropathy

Transforming Growth Factor-beta (TGF-β) is a pro-sclerotic cytokine widely associated with the development of fibrosis in diabetic nephropathy. Central to the underlying pathology of tubulointerstitial fibrosis is epithelial-to-mesenchymal transition (EMT), or the trans-differentiation of tubular epithelial cells into myofibroblasts. This process is accompanied by a number of key morphological and phenotypic changes culminating in detachment of cells from the tubular basement membrane and migration into the interstitium. Ultimately these cells reside as activated myofibroblasts and further exacerbate the state of fibrosis. A large body of evidence supports a role for TGF-β and downstream Smad signalling in the development and progression of renal fibrosis. Here we discuss a role for TGF-β as the principle effector in the development of renal fibrosis in diabetic nephropathy, focusing on the role of the TGF-β1 isoform and its downstream signalling intermediates, the Smad proteins. Specifically we review evidence for TGF-β1 induced EMT in both the proximal and distal regions of the nephron and describe potential therapeutic strategies that may target TGF-β1 activity.     

Removal of H₂O₂ and generation of superoxide radical: role of cytochrome c and NADH

In cells, mitochondria, endoplasmic reticulum, and peroxisomes are the major sources of reactive oxygen species (ROS) under physiological and pathophysiological conditions. Cytochrome c (cyt c) is known to participate in mitochondrial electron transport and has antioxidant and peroxidase activities. Under oxidative or nitrative stress, the peroxidase activity of Fe³⁺cyt c is increased. The level of NADH is also increased under pathophysiological conditions such as ischemia and diabetes and a concurrent increase in hydrogen peroxide (H₂O₂) production occurs. Studies were performed to understand the related mechanisms of radical generation and NADH oxidation by Fe³⁺cyt c in the presence of H₂O₂. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with NADH, Fe³⁺cyt c, and H₂O₂ in the presence of methyl-β-cyclodextrin. An EPR spectrum corresponding to the superoxide radical adduct of DMPO encapsulated in methyl-β-cyclodextrin was obtained. This EPR signal was quenched by the addition of the superoxide scavenging enzyme Cu,Zn-superoxide dismutase (SOD1). The amount of superoxide radical adduct formed from the oxidation of NADH by the peroxidase activity of Fe³⁺cyt c increased with NADH and H₂O₂ concentration. From these results, we propose a mechanism in which the peroxidase activity of Fe³⁺cyt c oxidizes NADH to NAD^•, which in turn donates an electron to O₂, resulting in superoxide radical formation. A UV-visible spectroscopic study shows that Fe³⁺cyt c is reduced in the presence of both NADH and H₂O₂. Our results suggest that Fe³⁺cyt c could have a novel role in the deleterious effects of ischemia/reperfusion and diabetes due to increased production of superoxide radical. In addition, Fe³⁺cyt c may play a key role in the mitochondrial “ROS-induced ROS-release” signaling and in mitochondrial and cellular injury/death. The increased oxidation of NADH and generation of superoxide radical by this mechanism may have implications for the regulation of apoptotic cell death, endothelial dysfunction, and neurological diseases. We also propose an alternative electron transfer pathway, which may protect mitochondria and mitochondrial proteins from oxidative damage.