Oxaloacetate decarboxylase of Vibrio cholerae: purification, characterization, and expression of the genes in Escherichia coli

The oxaloacetate decarboxylase (OAD) Na+ pump consists of subunits α, β, and γ, which are expressed from an oadGAB gene cluster present in various anaerobic bacteria. Vibrio cholerae has two copies of oad genes, which are termed oad-1 and oad-2. The oad-2 genes are part of the citrate fermentation operon, while the oad-1 genes are flanked by genes encoding products not involved in a catabolic pathway. The gene sequences of oad-1 and oad-2 of V. cholerae strain O395-N1 were determined. The apparent frameshift in the published sequence of the oadA-2 gene from V. cholerae El Tor N16961 was not present in strain O395-N1. Upon anaerobic growth of V. cholerae on citrate, exclusively the oad-2 genes are expressed. OAD was isolated from these cells by monomeric avidin-Sepharose affinity chromatography. The enzyme was of higher specific activity than that from Klebsiella pneumoniae and was significantly more stable. Decarboxylase activity was Na+ dependent, and the activation profile showed strong cooperativity with a Hill coefficient nH=1.8. Oxalate and oxomalonate inhibited the enzyme with half-maximal concentrations of 10μM and 200μM, respectively. After reconstitution into proteoliposomes, the enzyme acted as a Na+ pump. With size-exclusion chromatography, the enzyme eluted in a symmetrical peak at a retention volume corresponding to an apparent molecular mass of approximately 570kDa, suggesting a tetrameric structure for OAD-2. The two oad gene clusters were heterologously expressed in Escherichia coli, and the decarboxylases were isolated from the host cell

Caspase-8 binding to cardiolipin in giant unilamellar vesicles provides a functional docking platform for bid

Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding. Biophysical approaches, including Laurdan fluorescence and rupture/tension measurements, were used to determine the ability of these three components (cardiolipin, caspase-8 and Bid) to fulfil the minimal requirements for the formation and function of the platform at the mitochondrial membrane. Our results shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria

Structure-Function Relations in Oxaloacetate Decarboxylase Complex. Fluorescence and Infrared Approaches to Monitor Oxomalonate and Na+ Binding Effect

ions across the membrane, which drives endergonic membrane reactions such as ATP synthesis, transport and motility. OAD is a membrane-bound enzyme composed of α, β and γ subunits. The α subunit contains the carboxyltransferase catalytic site. characteristic of a high content of α helix structures. Addition of oxomalonate induced a shift of the amide-I band of OAD toward higher wavenumbers, interpreted as a slight decrease of β sheet structures and a concomitant increase of α helix structures. Oxomalonate binding to αγand α subunits also provoked secondary structure variations, but these effects were negligible compared to OAD complex. alters the tryptophan environment of the β subunit, consistent with the function of these subunits within the enzyme complex. Formation of a complex between OAD and its substrates elicits structural changes in the α-helical as well as β-strand secondary structure elements

Population dynamics and genetic connectivity in recent chimpanzee history

The European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 864203) (to T.M.-B.). BFU2017-86471-P (MINECO/FEDER, UE) (to T.M.-B.). “Unidad de Excelencia María de Maeztu”, funded by the AEI (CEX2018-000792-M) (to T.M.-B.). Howard Hughes International Early Career (to T.M.-B.). NIH 1R01HG010898-01A1 (to T.M.-B.). Secretaria d’Universitats i Recerca and CERCA Program del Departament d’Economia i Coneixement de la Generalitat de Catalunya (GRC 2017 SGR 880) (to T.M.-B.). UCL’s Wellcome Trust ISSF3 award 204841/Z/16/Z (to A.M.A. and J.M.S.). Generalitat de Catalunya (2017 SGR-1040) (to M. Llorente). Wellcome Trust Investigator Award 202802/Z/16/Z (to D.A.H.). The Pan African Program: The Cultured Chimpanzee (PanAf) is generously funded by the Max Planck Society, the Max Planck Society Innovation Fund, and the Heinz L. Krekeler Foundation.Knowledge on the population history of endangered species is critical for conservation, but whole-genome data on chimpanzees (Pan troglodytes) is geographically sparse. Here, we produced the first non-invasive geolocalized catalog of genomic diversity by capturing chromosome 21 from 828 non-invasive samples collected at 48 sampling sites across Africa. The four recognized subspecies show clear genetic differentiation correlating with known barriers, while previously undescribed genetic exchange suggests that these have been permeable on a local scale. We obtained a detailed reconstruction of population stratification and fine-scale patterns of isolation, migration, and connectivity, including a comprehensive picture of admixture with bonobos (Pan paniscus). Unlike humans, chimpanzees did not experience extended episodes of long-distance migrations, which might have limited cultural transmission. Finally, based on local rare variation, we implement a fine-grained geolocalization approach demonstrating improved precision in determining the origin of confiscated chimpanzees.Publisher PDFPeer reviewe

Quantitative estimates of glacial refugia for chimpanzees (Pan troglodytes) since the Last Interglacial (120,000 BP).

Paleoclimate reconstructions have enhanced our understanding of how past climates have shaped present-day biodiversity. We hypothesize that the geographic extent of Pleistocene forest refugia and suitable habitat fluctuated significantly in time during the late Quaternary for chimpanzees (Pan troglodytes). Using bioclimatic variables representing monthly temperature and precipitation estimates, past human population density data, and an extensive database of georeferenced presence points, we built a model of changing habitat suitability for chimpanzees at fine spatio-temporal scales dating back to the Last Interglacial (120,000 BP). Our models cover a spatial resolution of 0.0467° (approximately 5.19 km2 grid cells) and a temporal resolution of between 1000 and 4000 years. Using our model, we mapped habitat stability over time using three approaches, comparing our modeled stability estimates to existing knowledge of Afrotropical refugia, as well as contemporary patterns of major keystone tropical food resources used by chimpanzees, figs (Moraceae), and palms (Arecacae). Results show habitat stability congruent with known glacial refugia across Africa, suggesting their extents may have been underestimated for chimpanzees, with potentially up to approximately 60,000 km2 of previously unrecognized glacial refugia. The refugia we highlight coincide with higher species richness for figs and palms. Our results provide spatio-temporally explicit insights into the role of refugia across the chimpanzee range, forming the empirical foundation for developing and testing hypotheses about behavioral, ecological, and genetic diversity with additional data. This methodology can be applied to other species and geographic areas when sufficient data are available

The bii4africa dataset of faunal and floral population intactness estimates across Africa’s major land uses

Sub-Saharan Africa is under-represented in global biodiversity datasets, particularly regarding the impact of land use on species’ population abundances. Drawing on recent advances in expert elicitation to ensure data consistency, 200 experts were convened using a modified-Delphi process to estimate ‘intactness scores’: the remaining proportion of an ‘intact’ reference population of a species group in a particular land use, on a scale from 0 (no remaining individuals) to 1 (same abundance as the reference) and, in rare cases, to 2 (populations that thrive in human-modified landscapes). The resulting bii4africa dataset contains intactness scores representing terrestrial vertebrates (tetrapods: ±5,400 amphibians, reptiles, birds, mammals) and vascular plants (±45,000 forbs, graminoids, trees, shrubs) in sub-Saharan Africa across the region’s major land uses (urban, cropland, rangeland, plantation, protected, etc.) and intensities (e.g., large-scale vs smallholder cropland). This dataset was co-produced as part of the Biodiversity Intactness Index for Africa Project. Additional uses include assessing ecosystem condition; rectifying geographic/ taxonomic biases in global biodiversity indicators and maps; and informing the Red List of Ecosystems

La créatine kinase mitochondriale - organisatrice de la membrane mitochondriale ? (ségrégation de la cardiolipine sur des monocouches phospholipidiques)

En utilisant comme modèle membranaire les monocouches de Langmuir à l interface air-tampon et des techniques de caractérisation spécifiques, nous avons obtenu des nouvelles informations sur l interaction entre la créatine kinase mitochondriale et la membrane interne de la mitochondrie. Par microscopie à l angle de Brewster nous avons pu visualiser la formation de complexes spécifiques cardiolipine-CKmt, avec ségrégation de la cardiolipine dans la membrane biomimétique. La fixation de la CKmt a lieu avec insertion de domaines protéiques parmi les lipides et abouti à une stabilisation globale du film interfacial, comme indiqué par des mesures de capacité différentielle d une électrode de mercure. Une réorientation des domaines protéiques, principalement des hélices a, a également pu être mise en évidence par spectroscopie infrarouge de réflexion-absorption par modulation de la polarisation (PM-IRRAS). L effet de la CKmt sur la morphologie de la monocouche dépend du degré d insaturation des chaînes acyles. Ceci est en relation avec la forte proportion d acide linoléique (C18:2), caractéristique des molécules de cardiolipine dans les cellules musculaires. Le phénomène de ségrégation induit par la CKmt, pourrait contribuer à la modulation de la distribution de cardiolipine et avoir des conséquences sur la fixation d autres molécules comme le cytochrome c ou les protéines de la famille Bcl-2. La membrane mitochondriale étant siège des nombreux phénomènes de peroxydation, de part sa structure et sa localisation, la CKmt est une cible des molécules oxydantes générées. Parmi ces molécules, le 4-hydroxynonénal, inactive l enzyme, modifie sa structure et induit l accumulation de complexes protéiques agrégés au niveau de la membrane. L interaction de la CKmt avec la membrane biomimétique est fortement perturbée, aussi bien en monocouche que sur les liposomesaThe interaction between the mitochondrial isoform of creatine kinase (mtCK) and a mitochondrial inner membrane biomimetic model was analysed using phospholipid monolayers at the air-buffer interface. New insight on the structural and morphological consequences both on protein and membrane was obtained. As visualised by Brewster angle microscopy, mtCK induces formation of specific protein cardiolipin complexes, leading to the segregation of cardiolipin molecules. MtCK binding occurs with partial insertion between lipids and results in an overall stabilisation of the membrane, as indicated by differential capacity measurements. A reorientation of protein -helical structures was also observed by polarisation modulation infrared reflexion-absorption spectroscopy (PM-IRRAS). Formation of mtCK cardiolipin complexes was found to be highly dependable of acyl chain unsaturation. This is of physiological relevance as one of the main characteristics of cardiolipin in muscle mitochondria is the high percentage of linoleic acid (C18:2). MtCK-induced segregation may contribute to membrane organisation by modulating cardiolipin distribution, and consequently membrane binding of other molecules such as cytochrome c or Bcl-2 family proteins. Mitochondrial membranes are the site of numerous peroxydation processes. MtCK structure and localisation in the vicinity of the membrane make it a target for oxidative molecules produced during such processes. Among these molecules, 4-hydroxynonenal, induces enzyme inactivation, modifies its structure and induces accumulation of aggregates at membranes. MtCK binding to model membranes, liposomes or monolayers, is thus disturbed. All these complex perturbation were analysed and debated under a physiological point of view along this thesis workLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Structural comparison of highly similar nucleoside diphosphate kinases: molecular explanation of distinct membrane binding behavior

International audienceNDPK-A, NDPK-B and NDPK-D are three enzymes which belong to the NDPK group I isoforms and are not only involved in metabolism process but also in transcriptional regulation, DNA cleavage, histidine protein kinase activity and metastasis development. Those enzymes were reported to bind to membranes either in mitochondria where NDPK-D influences cardiolipin lateral organization and is thought to be involved in apoptotic pathway or in cytosol where NDPK-A and NDPK-B membrane association was shown to influence several cellular processes like endocytosis, cellular adhesion, ion transport, etc. However, despite numerous studies, the role of NDPK-membrane association and the molecular details of the binding process are still elusive. In the present work, a comparative study of the three NDPK isoforms allowed us to show that although membrane binding is a common feature of these enzymes, mechanisms differ at the molecular scale. NDPK-A was not able to bind to model membranes mimicking the inner leaflet of plasma membrane, suggesting that its in vivo membrane association is mediated by a non-lipidic partner or other partners than the studied phospholipids. On the contrary, NDPK-B and NDPK-D were shown to bind efficiently to liposomes mimicking plasma membrane and mitochondrial inner membrane respectively but details of the binding mechanism differ between the two enzymes as NDPK-B binding necessarily involved an anionic phospholipid partner while NDPK-D can bind either zwitterionic or anionic phospholipids. Although sharing similar secondary structure and homohexameric quaternary arrangement, tryptophan fluorescence revealed fine disparities in NDPK tertiary structures. Interfacial behavior as well as ANS fluorescence showed further dissimilarities between NDPK isoforms, notably the presence of distinct accessible hydrophobic areas as well as different capacity to form Gibbs monolayers related to their surface activity properties. Those distinct features may contribute to explain the differences in the protein behavior towards membrane binding