Biomedical subjects

Frédéric Colland

Publications and source records attributed to Frédéric Colland.

5 recordsLinked to original sources

PubMed2006

FOXO4 transcriptional activity is regulated by monoubiquitination and USP7/HAUSP.

FOXO (Forkhead box O) transcription factors are important regulators of cellular metabolism, cell-cycle progression and cell death. FOXO activity is regulated by multiple post-translational modifications, including phosphorylation, acetylation and polyubiquitination. Here, we show that FOXO becomes monoubiquitinated in response to increased cellular oxidative stress, resulting in its re-localization to the nucleus and an increase in its transcriptional activity. Deubiquitination of FOXO requires the deubiquitinating enzyme USP7/HAUSP (herpesvirus-associated ubiquitin-specific protease), which interacts with and deubiquitinates FOXO in response to oxidative stress. Oxidative stress-induced ubiquitination and deubiquitination by USP7 do not influence FOXO protein half-life. However, USP7 does negatively regulate FOXO transcriptional activity towards endogenous promoters. Our results demonstrate a novel mechanism of FOXO regulation and indicate that USP7 has an important role in regulating FOXO-mediated stress responses.

Animals↗

PubMed2005

ARHGAP10 is necessary for alpha-catenin recruitment at adherens junctions and for Listeria invasion.

E-cadherin mediates the formation of adherens junctions between epithelial cells. It serves as a receptor for Listeria monocytogenes, a bacterial pathogen that enters epithelial cells. The L. monocytogenes surface protein, InlA, interacts with the extracellular domain of E-cadherin. In adherens junctions, this ectodomain is involved in homophilic interactions whereas the cytoplasmic domain binds beta-catenin, which then recruits alpha-catenin. alpha-catenin binds to actin directly, or indirectly, thus linking E-cadherin to the actin cytoskeleton. Entry of L. monocytogenes into cells and adherens junction formation are dynamic events that involve actin and membrane rearrangements. To understand these processes better, we searched for new ligands of alpha-catenin. Using a two-hybrid screen, we identified a new partner of alpha-catenin: ARHGAP10. This protein colocalized with alpha-catenin at cell-cell junctions and was recruited at L. monocytogenes entry sites. In ARHGAP10-knockdown cells, L. monocytogenes entry and alpha-catenin recruitment at cell-cell contacts were impaired. The GAP domain of ARHGAP10 has GAP activity for RhoA and Cdc42. Its overexpression disrupted actin cables, enhanced alpha-catenin and cortical actin levels at cell-cell junctions and inhibited L. monocytogenes entry. Altogether, our results show that ARHGAP10 is a new component of cell-cell junctions that controls alpha-catenin recruitment and has a key role during L. monocytogenes uptake.

Actins↗

PubMed2004

Functional proteomics mapping of a human signaling pathway.

Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.

Adaptor Proteins, Signal Transducing↗

PubMed2002

The interaction between sigmaS, the stationary phase sigma factor, and the core enzyme of Escherichia coli RNA polymerase.

BACKGROUND: The RNA polymerase holoenzyme of Escherichia coli is composed of a core enzyme (subunit structure alpha2betabeta') associated with one of the sigma subunits, required for promoter recognition. Different sigma factors compete for core binding. Among the seven sigma factors present in E. coli, sigma70 controls gene transcription during the exponential phase, whereas sigmaS regulates the transcription of genes in the stationary phase or in response to different stresses. Using labelled sigmaS and sigma70, we compared the affinities of both sigma factors for core binding and investigated the structural changes in the different subunits involved in the formation of the holoenzymes. RESULTS: Using native polyacrylamide gel electrophoresis, we demonstrate that sigmaS binds to the core enzyme with fivefold reduced affinity compared to sigma70. Using iron chelate protein footprinting, we show that the core enzyme significantly reduces polypeptide backbone solvent accessibility in regions 1.1, 2.5, 3.1 and 3.2 of sigmaS, while increasing the accessibility in region 4.1 of sigmaS. We have also analysed the positioning of sigmaS on the holoenzyme by the proximity-dependent protein cleavage method using sigmaS derivatives in which FeBABE was tethered to single cysteine residues at nine different positions. Protein cutting patterns are observed on the beta and beta' subunits, but not alpha. Regions 2.5, 3.1 and 3.2 of sigmaS are close to both beta and beta' subunits, in agreement with iron chelate protein footprinting data. CONCLUSIONS: A comparison between these results using sigmaS and previous data from sigma70 indicates similar contact patterns on the core subunits and similar characteristic changes associated with holoenzyme formation, despite striking differences in the accessibility of regions 4.1 and 4.2.

Bacterial Proteins↗

PubMedDate not supplied

Integrating a functional proteomic approach into the target discovery process.

Functional proteomics is a promising technique for the rational identification of novel therapeutic targets by elucidation of the function of newly identified proteins in disease-relevant cellular pathways. Of the recently described high-throughput approaches for analyzing protein-protein interactions, the yeast two-hybrid (Y2H) system has turned out to be one of the most suitable for genome-wide analysis. However, this system presents a challenging technical problem: the high prevalence of false positives and false negatives in datasets due to intrinsic limitations of the technology and the use of a high-throughput, genetic assay. We discuss here the different experimental strategies applied to Y2H assays, their general limitations and advantages. We also address the issue of the contribution of protein interaction mapping to functional biology, especially when combined with complementary genomic and proteomic analyses. Finally, we illustrate how the combination of protein interaction maps with relevant functional assays can provide biological support to large-scale protein interaction datasets and contribute to the identification and validation of potential therapeutic targets.

Animals↗