Biomedical subjects

Daphné Seigneurin-Berny

Publications and source records attributed to Daphné Seigneurin-Berny.

7 recordsLinked to original sources

PubMed2006

A versatile method for deciphering plant membrane proteomes.

Proteomics is a very powerful approach to link the information contained in sequenced genomes, like that of Arabidopsis, to the functional knowledge provided by studies of plant cell compartments. This article summarizes the different steps of a versatile strategy that has been developed to decipher plant membrane proteomes. Initiated with envelope membranes from spinach chloroplasts, this strategy has been adapted to thylakoids, and further extended to a series of membranes from the model plant Arabidopsis: chloroplast envelope membranes, plasma membrane, and mitochondrial membranes. The first step is the preparation of highly purified membrane fractions from plant tissues. The second step in the strategy is the fractionation of membrane proteins on the basis of their physico-chemical properties. Chloroform/methanol extraction and washing of membranes with NaOH, NaCl or any other agent led to the simplification of the protein content of the fraction to be analysed. The next step is the genuine proteomic step, i.e. the separation of proteins by 1D-gel electrophoresis followed by in-gel proteolytic digestion of the polypeptides, analysis of the proteolytic peptides using mass spectrometry, and protein identification by searching through databases. The last step is the validation of the procedure by checking the subcellular location. The results obtained by using this strategy demonstrate that a combination of different proteomics approaches, together with bioinformatics, indeed provide a better understanding of the biochemical machinery of the different plant membranes at the molecular level.

Alkalies↗

PubMed2005

HMA1, a new Cu-ATPase of the chloroplast envelope, is essential for growth under adverse light conditions.

Although ions play important roles in the cell and chloroplast metabolism, little is known about ion transport across the chloroplast envelope. Using a proteomic approach specifically targeted to the Arabidopsis chloroplast envelope, we have identified HMA1, which belongs to the metal-transporting P1B-type ATPases family. HMA1 is mainly expressed in green tissues, and we validated its chloroplast envelope localization. Yeast expression experiments demonstrated that HMA1 is involved in copper homeostasis and that deletion of its N-terminal His-domain partially affects the metal transport. Characterization of hma1 Arabidopsis mutants revealed a lower chloroplast copper content and a diminution of the total chloroplast superoxide dismutase activity. No effect was observed on the plastocyanin content in these lines. The hma1 insertional mutants grew like WT plants in standard condition but presented a photosensitivity phenotype under high light. Finally, direct biochemical ATPase assays performed on purified chloroplast envelope membranes showed that the ATPase activity of HMA1 is specifically stimulated by copper. Our results demonstrate that HMA1 offers an additional way to the previously characterized chloroplast envelope Cu-ATPase PAA1 to import copper in the chloroplast.

Adenosine Triphosphatases↗

PubMed2004

The hydrophobic proteome of mitochondrial membranes from Arabidopsis cell suspensions.

The development of mitochondria and the integration of their function within a plant cell rely on the presence of a complex biochemical machinery located within their limiting membranes. The aim of the present work was: (1) to enhance our understanding of the biochemical machinery of mitochondrial membranes and (2) to test the versatility of the procedure developed for the identification of the hydrophobic proteome of the chloroplast envelope [Molecular and Cellular Proteomics 2 (2003) 325-345]. A proteomic analysis was performed, to provide the most exhaustive view of the protein repertoire of these membranes. For this purpose, highly purified mitochondria were prepared from Arabidopsis cultured cells and membrane proteins were extracted. To get a more exhaustive array of membrane proteins from Arabidopsis mitochondria, from the most to the less hydrophobic ones, various extraction procedures (chloroform/methanol extraction, alkaline or saline treatments) were applied. LC-MS/MS analyses were then performed on each membrane subfraction, leading to the identification of more than 110 proteins. The identification of these proteins is discussed with respect to their mitochondrial localization, their physicochemical properties and their implications in the metabolism of mitochondria. In order to provide a new overview of the biochemical machinery of the plant mitochondria, proteins identified during this work were compared to the lists of proteins identified during previous proteomic analyses performed on plant and algae mitochondria (Arabidopsis, pea, Chlamydomonas, rice, etc.). A total of 502 proteins are listed. About 40% of the 114 proteins identified during this work were not identified during previous proteomic studies performed on mitochondria.

Arabidopsis↗

PubMed2003

HDAC6-induced premature chromatin compaction in mouse oocytes and fertilised eggs.

Chromatin remodelling in the fertilised mouse egg is intimately linked to protein synthesis and degradation, to protamine by histone replacement and to specific histone modifications. The involvement of histone deacetylases (HDACs) in the beginning of development is poorly understood. HDACs are essential for cell proliferation and in the control of gene expression in a wide variety of mammalian systems. Here we focus on mHDAC6, a recently identified class II histone deacetylase, and we analyse its expression and localisation in oocytes and pronuclear zygotes. By indirect immunofluorescence we show that mHDAC6 is detected in the cytoplasm of germinal vesicle (GV) stage oocytes and 1-cell embryos. Ectopic expression of this enzyme after injection into germinal vesicles and pronuclei alters the nuclear structure and causes premature compaction of the chromatin. Our data suggest that the effect of condensation is linked to the ubiquitin-binding activity of mHDAC6, rather than to its function as a deacetylase.

Animals↗

PubMed2003

Proteomics of chloroplast envelope membranes.

Proteomics is a very powerful approach to link the information contained in sequenced genomes, like Arabidopsis, to the functional knowledge provided by studies of plant cell compartments, such as chloroplast envelope membranes. This review summarizes the present state of proteomic analyses of highly purified spinach and Arabidopsis envelope membranes. Methods targeted towards the hydrophobic core of the envelope allow identifying new proteins, and especially new transport systems. Common features were identified among the known and newly identified putative envelope inner membrane transporters and were used to mine the complete Arabidopsis genome to establish a virtual plastid envelope integral protein database. Arabidopsis envelope membrane proteins were extracted using different methods, that is, chloroform/methanol extraction, alkaline or saline treatments, in order to retrieve as many proteins as possible, from the most to the less hydrophobic ones. Mass spectrometry analyses lead to the identification of more than 100 proteins. More than 50% of the identified proteins have functions known or very likely to be associated with the chloroplast envelope. These proteins are (a) involved in ion and metabolite transport, (b) components of the protein import machinery and (c) involved in chloroplast lipid metabolism. Some soluble proteins, like proteases, proteins involved in carbon metabolism or in responses to oxidative stress, were associated with envelope membranes. Almost one third of the newly identified proteins have no known function. The present stage of the work demonstrates that a combination of different proteomics approaches together with bioinformatics and the use of different biological models indeed provide a better understanding of chloroplast envelope biochemical machinery at the molecular level.

Journal Article↗

PubMed2003

HSP27 is a ubiquitin-binding protein involved in I-kappaBalpha proteasomal degradation.

HSP27 is an ATP-independent chaperone that confers protection against apoptosis through various mechanisms, including a direct interaction with cytochrome c. Here we show that HSP27 overexpression in various cell types enhances the degradation of ubiquitinated proteins by the 26S proteasome in response to stressful stimuli, such as etoposide or tumor necrosis factor alpha (TNF-alpha). We demonstrate that HSP27 binds to polyubiquitin chains and to the 26S proteasome in vitro and in vivo. The ubiquitin-proteasome pathway is involved in the activation of transcription factor NF-kappaB by degrading its main inhibitor, I-kappaBalpha. HSP27 overexpression increases NF-kappaB nuclear relocalization, DNA binding, and transcriptional activity induced by etoposide, TNF-alpha, and interleukin 1beta. HSP27 does not affect I-kappaBalpha phosphorylation but enhances the degradation of phosphorylated I-kappaBalpha by the proteasome. The interaction of HSP27 with the 26S proteasome is required to activate the proteasome and the degradation of phosphorylated I-kappaBalpha. A protein complex that includes HSP27, phosphorylated I-kappaBalpha, and the 26S proteasome is formed. Based on these observations, we propose that HSP27, under stress conditions, favors the degradation of ubiquitinated proteins, such as phosphorylated I-kappaBalpha. This novel function of HSP27 would account for its antiapoptotic properties through the enhancement of NF-kappaB activity.

Animals↗

PubMed2002

In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation.

Trichostatin A (TSA) inhibits all histone deacetylases (HDACs) of both class I and II, whereas trapoxin (TPX) cannot inhibit HDAC6, a cytoplasmic member of class II HDACs. We took advantage of this differential sensitivity of HDAC6 to TSA and TPX to identify its substrates. Using this approach, alpha-tubulin was identified as an HDAC6 substrate. HDAC6 deacetylated alpha-tubulin both in vivo and in vitro. Our investigations suggest that HDAC6 controls the stability of a dynamic pool of microtubules. Indeed, we found that highly acetylated microtubules observed after TSA treatment exhibited delayed drug-induced depolymerization and that HDAC6 overexpression prompted their induced depolymerization. Depolymerized tubulin was rapidly deacetylated in vivo, whereas tubulin acetylation occurred only after polymerization. We therefore suggest that acetylation and deacetylation are coupled to the microtubule turnover and that HDAC6 plays a key regulatory role in the stability of the dynamic microtubules.

Acetylation↗