171,146 research outputs found
A cooperative model for proton pumping in cytochrome c oxidase
No full textIn this paper, the mechanism of proton pumping in cytochrome c oxidase is examined. Data on cooperative linkage of vectorial proton
translocation to oxido-reduction of CuA and heme a in the CO-inhibited, liposome-reconstituted bovine cytochrome c oxidase are reviewed.
Results on proton translocation associated to single-turnover oxido-reduction of the four metal centers in the unliganded, membrane reconstituted
oxidase are also presented. On the basis of these results, X-ray crystallographic structures and spectrometric data for a proton
pumping model in cytochrome c oxidase is proposed.
This model, which is specifically derived from data available for the bovine cytochrome c oxidase, is intended to illustrate the essential
features of cooperative coupling of proton translocation at the low potential redox site. Variants will have to be introduced for those members
of the heme copper oxidase family which differ in the redox components of the low potential site and in the amino acid network connected to
this site.
The model we present describes in detail steps of cooperative coupling of proton pumping at the low potential CuA-heme a site in the
bovine enzyme. It is then outlined how this cooperative proton transfer can be thermodynamically and kinetically coupled to the chemistry of
oxygen reduction to water at the high potential CuB-heme a3 center, so as to result in proton pumping, in the turning-over enzyme, against a
transmembrane electrochemical proton gradient of some 250 mV
Replication Data for: Astrocytic ATM deficiency triggers complement C3a-mediated synaptic dysfunction and depressive-like behaviors
No full textThis dataset includes LC-MS/MS data of differentially expressed proteins in the astrocyte proteome and secretome generated in the study entitled “Astrocytic ATM deficiency triggers complement C3a–mediated synaptic dysfunction and depressive-like behaviors” by Sabrina Briguglio, Anna Selimi, Clara Cambria, Ramona Stringhi, Manuela Moriggi, Mario Federici, Maria Vittoria Zavaglia, Elena Borroni, Francesco Rusconi, Elena Battaglioli, Elena Marcello, Laura Musazzi, Daniele Capitanio, and Flavia Antonucci.
Astrocytes were gently scraped, collected in PBS and pelletted. Astrocytes were then suspended in lysis buffer (2% SDS, 100 mM Tris-HCl pH 7.6, 0.1 M DTT, and 1% phenylmethanesulfonylfluoride) and sonicated on ice until completely dissolved. After incubation at 95 °C for 3 min, lysates were clarified by centrifugation at 12 000 g for 20 min at 4 °C. Protein quantitation with 2-D Quant-kit protein assay (Cytiva) was then performed. Culture media (15 ml) were centrifuged in 30 KDa MWCO filters (ThermoFisher scientific) to a final volume of 50 µl. Lysis buffer (50 µl) was then added, and each medium was processed as described for cells. Protein extracts (50 µg for each sample) were processed following the filter-aided sample preparation (FASP) protocol. Peptide samples were concentrated and then separated on a Dionex UltiMate 3000 HPLC System with an Easy Spray PepMap RSLC C18 column (250 mm, internal diameter of 75 µm) (ThermoFisher Scientific), and finally electrosprayed into an Orbitrap Fusion Tribrid (ThermoFisher Scientific) mass spectrometer. Three technical replicates for each sample were acquired.
Mass spectra were analysed using MaxQuant software (Max Planck Institute of Biochemistry, Munich, Germany, version 1.6.17.0). The maximum allowed mass deviation was set to 6 ppm for monoisotopic precursor ions and 0.5 Da for MS/MS peaks. Enzyme specificity was set to trypsin/P, and a maximum of two missed cleavages was allowed. Carbamidomethylation was set as a fixed modification, while N-terminal acetylation and methionine oxidation were set as variable modifications. Spectra were searched by the Andromeda search engine against the Mus musculus Uniprot UP000000589 sequence database (54707 proteins, March 2024). Protein identification required at least one unique or razor peptide per protein group. Quantification in MaxQuant was performed using the built-in extracted ion chromatogram (XIC)-based label-free quantification (LFQ) algorithm using fast LFQ. The required FDR was set to 1% at the peptide, 1% at the protein, and 1% at the site-modification levels, and the minimum required peptide length was set to 7 amino acids. Statistical analyses were performed using Perseus software (v.1.6.15.0, Max Planck Institute of Biochemistry, Martinsried, Germany). For each experimental group, proteins identified in at least 80% of samples were considered. False positives were reduced utilizing the Benjamini–Hochberg false discovery rate test
Inhibition of proton pumping in membrane reconstituted bovine heart cytochrome c oxidase by zinc binding at the inner matrix side
No full textA study is presented on the effect of zinc binding at the matrix side, on the proton pump of purified liposome
reconstituted bovine heart cytochrome c oxidase (COV). Internally trapped Zn2+ resulted in 50% decoupling
of the proton pump at level flow. Analysis of the pH dependence of inhibition by internal Zn2+ of proton
release in the oxidative and reductive phases of the catalytic cycle of cytochrome c oxidase indicates that Zn2+
suppresses two of the four proton pumping steps in the cycle, those taking place when the 2 OH− produced in
the reduction of O2 at the binuclear center are protonated to 2 H2O. This decoupling effect could be associated
with Zn2+ induced conformational alteration of an acid/base cluster linked to heme a3
Protonmotive cooperativity in cytochrome c oxidase
No full textCooperative linkage of solute binding at separate binding sites in allosteric proteins is an important functional attribute of soluble and membrane bound hemoproteins. Analysis of proton/electron coupling at the four redox centers, i.e. CuA, heme a, heme a3 and CuB, in the purified bovine cytochrome c oxidase in the unliganded, CO-liganded and CN-liganded states is presented. These studies are based on direct measurement of scalar proton translocation associated with oxido-reduction of the metal centers and pH dependence of the midpoint potential of the redox centers. Heme a (and CuA) exhibits a cooperative proton/electron linkage (Bohr effect). Bohr effect seems also to be associated with the oxygenreduction chemistry at the heme a3–CuB binuclear center. Data on electron transfer in cytochrome c oxidase are also presented, which, together with structural data, provide evidence showing the occurrence of direct electron transfer from CuA to the binuclear center in addition to electron transfer via heme a. A survey of structural and functional data showing the essential role of cooperative proton/electron linkage at heme a in the proton pump of cytochrome c oxidase is presented. On the basis of this and related functional and structural information, variants for cooperative mechanisms in the proton pump of the oxidase are examined
Replication Data for: PROTEOME ANALYSIS REVEALS COMMON PLAYERS BETWEEN THE PHYSIOLOGICAL NEURODEGENERATION OF THE ASCIDIAN CIONA INTESTINALIS AND THE PATHOLOGICAL NEURODEGENERATION IN HUMANS
No full textThis dataset includes LC-MS/MS data of differentially expressed proteins in the Ciona intestinalis proteome generated in the study entitled “PROTEOME ANALYSIS REVEALS COMMON PLAYERS BETWEEN THE PHYSIOLOGICAL NEURODEGENERATION OF THE ASCIDIAN CIONA INTESTINALIS AND THE PATHOLOGICAL NEURODEGENERATION IN HUMANS", by Daniele Capitanio, Silvia Mercurio, Ettore Mosca, Cristina Battaglia, Marco Venturin, and Roberta Pennati.
Tunicates, including ascidians, are recognized as the true “sister group” of vertebrates and are emerging as models to study the development and degeneration of central nervous system (CNS). Ascidian larvae have the typical chordate body plan that includes a dorsal neural tube. During their metamorphosis, a deep tissue reorganization takes place, with some tissues that degenerate while others develop to become functional during the adult life. The larval CNS also degenerates and most neurons disappear, making room to the formation of adult CNS. The genome of the ascidian Ciona intestinalis has been sequenced and annotated, with several CNS specific genes that have been characterized, revealing specification mechanisms shared with humans. These features make ascidian metamorphosis a good model to study the mechanisms underlying physiological CNS degeneration and to compare them to the pathological condition typical of neurodegenerative diseases.
In order to shed light on the molecular determinants of C. intestinalis metamorphosis and neurodegeneration, we analyzed its proteome at three stages of development: swimming larva (SwL, Hotta stage 28), settled larva (SetL, Hotta stage 32) and metamorphosing larva (MetL, Hotta stage 34).
A total of 9 samples were collected from three stages of larval development of Ciona (3 biological replicates for each stage) and their proteins were extracted in lysis buffer (0.1 M Tris/HCl pH 7.6, 4% SDS, 0.1 M DTT and 0.001 M PMSF) through sonication. Proteins were denatured at 95°C for 3 min and lysates clarified by centrifugation at 16000 g for 15 min. Protein concentrations were determined using the 2D-Quant Kit (Cytiva) and 120 ug of each extract were processed following the Filter aided sample preparation (FASP) method. Proteins were identified to match with Ciona intestinalis UNIPROT reference proteome (UP000008144) and their peaks were quantified with MaxQuant software (version 2.7.0.0, Max Planck Institute of Biochemistry, Germany) (Cox and Mann 2008). Detection of differentially expressed proteins (DEPs) was performed with Perseus software (Tyanova et al. 2016) (version 2.0.11, Max Planck Institute of Biochemistry). ANOVA and Tukey post-hoc test (p-value < 0.05) were used to obtain DEPs with their corresponding log fold change (LFC) values for the pairwise comparisons between the three developmental stages. Proteins with less than 6 valid values in at least 1 group were filtered out. False positives were excluded utilizing the Benjamini–Hochberg false discovery rate test. STRING (Version 12.0, https://string-db.org/) annotation was also used to further characterize DE proteins.
405 modulated proteins were identified by mass spectrometry, 156 of which are also regulated at transcript level. Enrichment analysis showed the involvement of several processes/pathways, including authophagy, proteasome and mTOR pathway among the most significant terms.
This work will contribute to give a comprehensive picture of the molecular pathways and mechanisms underlying ascidian metamorphosis, also pointing to new possible actors involved in the neurodegeneration process in humans
Intorno a San Ferdinando
No full textIl testo ripercorre i contributi recenti relativi alla chiesa di san Ferdinando di Livorno, concludendo con alcune novità sul suo patrimonio di argenterie sacre
Vectorial nature of redox Bohr effects in bovine heart cytochrome c oxidase
No full textThe vectorial nature of redox Bohr effects (redox-linked pK shifts) in cytochrome c oxidase from bovine heart incorporated in liposomes has been analyzed. The Bohr effects linked to oxide-reduction of heme a and CUB display membrane vectorial asymmetry. This provides evidence for involvement of redox Bohr effects in the proton pump of the oxidase
Redox Bohr effects and the role of heme a in the proton pump of bovine heart cytochrome c oxidase
No full textStructural and functional observations are reviewed which provide evidence for a central role of redox Bohr
effect linked to the low-spin heme a in the proton pump of bovine heart cytochrome c oxidase. Data on the
membrane sidedness of Bohr protons linked to anaerobic oxido-reduction of the individual metal centers in
the liposome reconstituted oxidase are analysed. Redox Bohr protons coupled to anaerobic oxido-reduction of
heme a (and CuA) and CuB exhibit membrane vectoriality, i.e. protons are taken up from the inner space upon
reduction of these centers and released in the outer space upon their oxidation. Redox Bohr protons coupled
to anaerobic oxido-reduction of heme a3 do not, on the contrary, exhibit vectorial nature: protons are
exchanged only with the outer space. A model of the proton pump of the oxidase, in which redox Bohr protons
linked to the low-spin heme a play a central role, is described. This article is part of a Special Issue entitled:
Allosteric cooperativity in respiratory proteins
Italia
No full textIl sistema di controllo della produzione delle argenterie negli stati preunitari e nell'Italia unita
PH DEPENDENCE OF THE VARIOUS PHASES OF THE PROTON PUMP OF CYTOCHROME C OXIDASE
No full textCytochrome c oxidase(COX)catalyses the reduction of dioxygen to water by ferrocytochrome c.This reaction is coupled to the translocation of up to 1H+/e-
4H+/O2 across the coupling membrane from the inner to the outer aqueous phase (1,2).
The catalytic cycle of COX can be divided into two phases: a reductive phase and an oxidative phase.A study is presented on the pHdependence of proton transfer of COX reconstituted in vesicles,associated with the oxidation phase,the reduction phase and the oxidation-rereduction rapid transitio
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