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Thierry Rabilloud

Publications and source records attributed to Thierry Rabilloud.

25 records · Page 2Linked to original sources
PubMed2003

Physical-chemical features of non-detergent sulfobetaines active as protein-folding helpers.

Some non-detergent sulfobetaines had been shown to prevent aggregation and improve the yield of active proteins when added to the buffer during in vitro protein renaturation. With the aim of designing more efficient folding helpers, a series of non-detergent sulfobetaines have been synthesized and their efficiency in improving the renaturation of a variety of proteins (E. coli tryptophan synthase and beta-D-galactosidase, hen lysozyme, bovine serum albumin, a monoclonal antibody) have been investigated. Attempts to correlate the structure of each sulfobetaines with its effect on folding revealed some molecular features that appear important in helping renaturation. This enabled us to design and synthesize new non-detergent sulfobetaines that act as potent folding helpers.

Animals↗
PubMed2002

A method for detection of overoxidation of cysteines: peroxiredoxins are oxidized in vivo at the active-site cysteine during oxidative stress.

Peroxiredoxins are often encountered as double spots when analysed by two-dimensional electrophoresis. The quantitative balance between these two spots depends on the physiological conditions, and is altered in favour of the acidic variant by oxidative stress for all the peroxiredoxins we could analyse. Using HeLa cells as a model system, we have further analysed the two protein isoforms represented by the two spots for each peroxiredoxin. The use of selected enzyme digestion and MS demonstrated that the acidic variant of all the peroxiredoxins analysed is irreversibly oxidized at the active-site cysteine into cysteine sulphinic or sulphonic acid. Thus, this acidic variant represents an inactivation form of the peroxiredoxins, and provides a useful marker of oxidative damage to the cells.

Amino Acid Sequence↗
PubMed2002

Proteomics analysis of cellular response to oxidative stress. Evidence for in vivo overoxidation of peroxiredoxins at their active site.

The proteomics analysis reported here shows that a major cellular response to oxidative stress is the modification of several peroxiredoxins. An acidic form of the peroxiredoxins appeared to be systematically increased under oxidative stress conditions. Peroxiredoxins are enzymes catalyzing the destruction of peroxides. In doing so, a reactive cysteine in the peroxiredoxin active site is weakly oxidized (disulfide or sulfenic acid) by the destroyed peroxides. Cellular thiols (e.g. thioredoxin) are used to regenerate the peroxiredoxins to their active state. Tandem mass spectrometry was carried out to characterize the modified form of the protein produced in vivo by oxidative stress. The cysteine present in the active site was shown to be oxidized into cysteic acid, leading to an inactivated form of peroxiredoxin. This strongly suggested that peroxiredoxins behave as a dam upon oxidative stress, being both important peroxide-destroying enzymes and peroxide targets. Results obtained in a primary culture of Leydig cells challenged with tumor necrosis factor alpha suggested that this oxidized/native balance of peroxiredoxin 2 may play an active role in resistance or susceptibility to tumor necrosis factor alpha-induced apoptosis.

Animals↗
PubMed2002

Comparative proteomics as a new tool for exploring human mitochondrial tRNA disorders.

More than 70 different point mutations in human mitochondrial tRNA genes are correlated with severe disorders, including fatal cardiopathies, encephalopathies, myopathies, and others. So far, investigation of the molecular impact(s) of mutations has focused on the affected tRNA itself by seeking structural and/or functional perturbations capable of interfering with synthesis of the 13 mitochondrion-encoded subunits of respiratory chain complexes. Here, a proteomic approach was used to investigate whether such mutations would affect the pattern of mitochondrial proteins at a broader level. Analysis of several hundred mitochondrial proteins from sibling cybrid cell lines by two-dimensional electrophoresis, an approach that takes into account all regulatory steps of mitochondrial and nuclear gene expression, indeed reveals a number of up- and downregulated proteins when healthy and single-point-mutation-carrying mitochondria representative of either MELAS or MERRF syndrome were compared. Assignment by mass spectrometry of the two proteins which exhibit obvious large quantitative decreases in the levels of both pathologic mitochondria identified nuclear-encoded subunits of cytochrome c oxidase, a respiratory chain complex. This clearly shows a linkage between the effects of mutations in mitochondrial tRNA genes and the steady-state level of nuclear-encoded proteins in mitochondria. It opens new routes toward a large-scale exploration of potential proteic partners involved in the genotype-phenotype correlation of mitochondrial disorders.

Amino Acid Sequence↗
PubMed2002

A survey of the plant mitochondrial proteome in relation to development.

To expand the functional analysis of plant mitochondria, we have undertaken the building of the proteome of pea mitochondria purified from leaves (green and etiolated), roots and seeds. In the first stage, we focused our proteomic exploration on the soluble protein complement of the green leaf mitochondria. We used traditional two-dimensional polyacrylamide gel electrophoresis, in combination with size exclusion chromatography as a third dimension, to identify the major proteins and further resolve their macromolecular complexity. The two-dimensional map of soluble proteins of green leaf mitochondria revealed 433 spots (with Coomassie blue staining) and around 73% of the proteins (in mass) were identified using three different approaches: Edman degradation, matrix-assisted laser desorption/ionization mass spectrometry and electrospray ionization tandem mass spectrometry. Quite a lot of the polypeptides were present in multiforms which indicated the presence of isoforms or the occurrence of post-translational modifications. Among these proteins, we uncovered an abundant family that was identified as aldehyde dehydrogenases, representing approximately 7.5% of the soluble proteins. The comparative analysis of soluble mitochondrial proteomes led to the identification of a number of proteins which were specifically present in root or in seed mitochondria, thus revealing the impact of tissue differentiation at the mitochondrial level.

Cell Fractionation↗
PubMed2002

Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains.

Proteomics has traditionally used the separating power of two-dimensional electrophoresis for the quantitative analysis of protein amounts in complex extracts. However, the limitations of this approach in terms of throughput and analyzable protein range have elicited the development of other proteomics approaches, based either on peptide separations instead of protein separations, or based on direct protein recognition and selection on dedicated arrays (protein chips). These recent methods seem very promising, and probably look more promising than they will ultimately be, just because their weaknesses are not fully characterized yet. The purpose of this paper is thus to highlight the strengths and weaknesses of all the proteomics approaches proposed to date and to try to deduce the respective niches in proteomics that these approaches will have in the future.

Electrophoresis, Gel, Two-Dimensional↗