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51.
52.
Quinol:fumarate reductase (QFR) is the terminal enzyme of anaerobic fumarate respiration. This membrane protein complex couples the oxidation of menaquinol to menaquinone to the reduction of fumarate to succinate. Although the diheme-containing QFR from Wolinella succinogenes is known to catalyze an electroneutral process, its three-dimensional structure at 2.2 A resolution and the structural and functional characterization of variant enzymes revealed locations of the active sites that indicated electrogenic catalysis. A solution to this apparent controversy was proposed with the so-called "E-pathway hypothesis". According to this, transmembrane electron transfer via the heme groups is strictly coupled to a parallel, compensatory transfer of protons via a transiently established pathway, which is inactive in the oxidized state of the enzyme. Proposed constituents of the E-pathway are the side chain of Glu C180 and the ring C propionate of the distal heme. Previous experimental evidence strongly supports such a role of the former constituent. Here, we investigate a possible heme-propionate involvement in redox-coupled proton transfer by a combination of specific (13)C-heme propionate labeling and Fourier transform infrared (FTIR) difference spectroscopy. The labeling was achieved by creating a W. succinogenes mutant that was auxotrophic for the heme-precursor 5-aminolevulinate and by providing [1-(13)C]-5-aminolevulinate to the medium. FTIR difference spectroscopy revealed a variation on characteristic heme propionate vibrations in the mid-infrared range upon redox changes of the distal heme. These results support a functional role of the distal heme ring C propionate in the context of the proposed E-pathway hypothesis of coupled transmembrane electron and proton transfer. 相似文献
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54.
An HJ Miyamoto S Lancaster KS Kirmiz C Li B Lam KS Leiserowitz GS Lebrilla CB 《Journal of proteome research》2006,5(7):1626-1635
A glycomic approach is developed to identify oligosaccharide markers for ovarian cancer by rapidly profiling globally released oligosaccharides. Glycoproteins shed by cancer cells are found in the supernatant (or conditioned media) of cultured cells. In this approach, shed glycoproteins are profiled for their oligosaccharide content using beta-elimination conditions. Changes in glycosylation are monitored by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Because shed glycoproteins would also be found in serum, similar glycan profiling was performed to observe potential oligosaccharide markers. Oligosaccharide profiling data on a limited set of patient and normal serum samples were studied to determine potential glycan markers in ovarian cancer. We were able to demonstrate the presence of at least 15 unique serum glycan markers in all patients but absent in normal individuals. To determine the structure of the glycan biomarkers, a number of the ions were isolated and further analyzed using infrared multiphoton dissociation (IRMPD). One major advantage of this approach is that glycans are examined directly from patient sera without the need to obtain cancer biopsy specimens or to purify glycosylated proteins from these specimens. 相似文献
55.
Membrane protein complexes can support both the generation and utilisation of a transmembrane electrochemical proton potential ('proton-motive force'), either by transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by transmembrane proton transfer. Here we provide the first evidence that both of these mechanisms are combined in the case of a specific respiratory membrane protein complex, the dihaem-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes, so as to facilitate transmembrane electron transfer by transmembrane proton transfer. We also demonstrate the non-functionality of this novel transmembrane proton transfer pathway ('E-pathway') in a variant QFR where a key glutamate residue has been replaced. The 'E-pathway', discussed on the basis of the 1.78-Angstrom-resolution crystal structure of QFR, can be concluded to be essential also for the viability of pathogenic varepsilon-proteobacteria such as Helicobacter pylori and is possibly relevant to proton transfer in other dihaem-containing membrane proteins, performing very different physiological functions. 相似文献
56.
Reconciliation of apparently contradictory experimental results obtained on the quinol: fumarate reductase (QFR), a dihaem-containing respiratory membrane protein complex from Wolinella succinogenes, was previously obtained by the proposal of the so-called E-pathway hypothesis. According to this hypothesis, transmembrane electron transfer via the haem groups is strictly coupled to co-transfer of protons via a transiently established, novel pathway, proposed to contain the side chain of residue Glu-C180 and the distal haem ring-C propionate as the most prominent components. This hypothesis has recently been supported by both theoretical and experimental results. Multiconformation continuum electrostatics calculations predict Glu-C180 to undergo a combination of proton uptake and conformational change upon haem reduction. Strong experimental support for the proposed role of Glu-C180 in the context of the “E-pathway hypothesis” is provided by the effects of replacing Glu-C180 with Gln or Ile by site-directed mutagenesis, the consequences of these mutations for the viability of the resulting mutants, together with the structural and functional characterisation of the corresponding variant enzymes, and the comparison of redox-induced Fourier-transform infrared (FTIR) difference spectra for the wild type and Glu-C180 → Gln variant. A possible haem propionate involvement has recently been supported by combining 13C-haem propionate labelling with redox-induced FTIR difference spectroscopy. 相似文献
57.
Potti A Dressman HK Bild A Riedel RF Chan G Sayer R Cragun J Cottrill H Kelley MJ Petersen R Harpole D Marks J Berchuck A Ginsburg GS Febbo P Lancaster J Nevins JR 《Nature medicine》2006,12(11):1294-1300
Using in vitro drug sensitivity data coupled with Affymetrix microarray data, we developed gene expression signatures that predict sensitivity to individual chemotherapeutic drugs. Each signature was validated with response data from an independent set of cell line studies. We further show that many of these signatures can accurately predict clinical response in individuals treated with these drugs. Notably, signatures developed to predict response to individual agents, when combined, could also predict response to multidrug regimens. Finally, we integrated the chemotherapy response signatures with signatures of oncogenic pathway deregulation to identify new therapeutic strategies that make use of all available drugs. The development of gene expression profiles that can predict response to commonly used cytotoxic agents provides opportunities to better use these drugs, including using them in combination with existing targeted therapies. 相似文献
58.
Cenacchi L Busch M Schleidt PG Müller FG Stumpp TV Mäntele W Trost P Lancaster CR 《Biochimica et biophysica acta》2012,1818(3):679-688
Cytochrome (cyt) b(561) proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b(561)-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b(561) paralogs from Arabidopsis thaliana (Acytb(561)-A, Acytb(561)-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb(561)-A resembles the best characterised member of the CYBASC family, the cytochrome b(561) from adrenomedullary chromaffin vesicles, and that Acytb(561)-B is atypical compared to other CYBASC proteins. Haem oxidation-reduction midpoint potential (E(M)) values were found to be fully consistent with ascorbate oxidation activities and Fe(3+)-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b(561) from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E(M) values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E(M) values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe(3+)-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E(M) values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b(561) paralogs exist as homodimers. 相似文献
59.
Lucia Cenacchi Manuela Busch Philipp G. Schleidt Florian G. Müller Tina V.M. Stumpp Werner Mäntele Paolo Trost C. Roy D. Lancaster 《生物化学与生物物理学报:生物膜》2012,1818(3):679-688
Cytochrome (cyt) b561 proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b561-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b561 paralogs from Arabidopsis thaliana (Acytb561-A, Acytb561-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb561-A resembles the best characterised member of the CYBASC family, the cytochrome b561 from adrenomedullary chromaffin vesicles, and that Acytb561-B is atypical compared to other CYBASC proteins. Haem oxidation–reduction midpoint potential (EM) values were found to be fully consistent with ascorbate oxidation activities and Fe3 +-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b561 from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem EM values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem EM values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe3 +-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem EM values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b561 paralogs exist as homodimers. 相似文献
60.
Lancaster GI Kowalski GM Estevez E Kraakman MJ Grigoriadis G Febbraio MA Gerondakis S Banerjee A 《PloS one》2012,7(6):e39100
Obesity is associated with a state of chronic low grade inflammation that plays an important role in the development of insulin resistance. Tumor progression locus 2 (Tpl2) is a serine/threonine mitogen activated protein kinase kinase kinase (MAP3K) involved in regulating responses to specific inflammatory stimuli. Here we have used mice lacking Tpl2 to examine its role in obesity-associated insulin resistance. Wild type (wt) and tpl2(-/-) mice accumulated comparable amounts of fat and lean mass when fed either a standard chow diet or two different high fat (HF) diets containing either 42% or 59% of energy content derived from fat. No differences in glucose tolerance were observed between wt and tpl2(-/-) mice on any of these diets. Insulin tolerance was similar on both standard chow and 42% HF diets, but was slightly impaired in tpl2(-/-) mice fed the 59% HFD. While gene expression markers of macrophage recruitment and inflammation were increased in the white adipose tissue of HF fed mice compared with standard chow fed mice, no differences were observed between wt and tpl2(-/-) mice. Finally, a HF diet did not increase Tpl2 expression nor did it activate Extracellular Signal-Regulated Kinase 1/2 (ERK1/2), the MAPK downstream of Tpl2. These findings argue that Tpl2 does not play a non-redundant role in obesity-associated metabolic dysfunction. 相似文献