Biomedical subjects

Nathalie Rouleau

Publications and source records attributed to Nathalie Rouleau.

4 recordsLinked to original sources

PubMed2006

Homogeneous and nonradioactive high-throughput screening platform for the characterization of kinase inhibitors in cell lysates.

Protein kinases are directly implicated in many human diseases; therefore, kinase inhibitors show great promises as new therapeutic drugs. In an effort to facilitate the screening and the characterization of kinase inhibitors, a novel application of the AlphaScreen technology was developed to monitor JNK activity from (1) purified kinase preparations and (2) endogenous kinase from cell lysates preactivated with different cytokines. The authors confirmed that both adenosine triphosphate (ATP) competitive as well as peptide-based JNK inhibitors were able to block the activity of both recombinant and HepG2 endogenous JNK activity. Using the same luminescence technique adapted for binding studies, the authors characterized peptide inhibitor mechanisms by measuring the binding affinity of the inhibitors for JNK. Because of the versatility of the technology, this cell-based JNK kinase assay could be adapted to other kinases and would represent a powerful tool to evaluate endogenous kinase activity and test a large number of potential inhibitors in a more physiologically relevant environment.

Binding Sites↗

PubMed2005

A high throughput screen to identify substrates for the ubiquitin ligase Rsp5.

Ubiquitin-protein ligases (E3s) are implicated in various human disorders and are attractive targets for therapeutic intervention. Although most cellular proteins are ubiquitinated, ubiquitination cannot be linked directly to a specific E3 for a large fraction of these proteins, and the substrates of most E3 enzymes are unknown. We have developed a luminescent assay to detect ubiquitination in vitro, which is more quantitative, effective, and sensitive than conventional ubiquitination assays. By taking advantage of the abundance of purified proteins made available by genomic efforts, we screened hundreds of purified yeast proteins for ubiquitination, and we identified previously reported and novel substrates of the yeast E3 ligase Rsp5. The relevance of these substrates was confirmed in vivo by showing that a number of them interact genetically with Rsp5, and some were ubiquitinated by Rsp5 in vivo. The combination of this sensitive assay and the availability of purified substrates will enable the identification of substrates for any purified E3 enzyme.

Endosomal Sorting Complexes Required for Transport↗

PubMed2003

Development of a versatile platform for nuclear receptor screening using AlphaScreen.

The interaction between nuclear receptors (NRs) and their coactivators, a key step in transcription regulation, requires a short consensus sequence called the LXXLL motif found in the coactivators' structure. Using the AlphaScreen technology, the authors have taken advantage of this receptor-coactivator interaction to develop a highly sensitive assay to identify and characterize compounds modulating NR activity. Estrogen and retinoic acid receptors were chosen as models to demonstrate the versatility of the AlphaScreen technology: (1) the assay can be designed using different antibodies to capture either full-length receptors or receptor domains that have been tagged, (2) the assay can differentiate between ligands that act as agonists or antagonists because only agonists will allow recruitment of the coactivator sequence-derived peptide, and (3) the assay gives the opportunity to screen for antagonists targeting the ligand-binding site or the dimerization interface between the receptor and the coactivator. Titration of the receptor and biotinylated peptide indicates that AlphaScreen is highly sensitive, requiring nanomolar concentration of reagents. Competition isotherms performed with known receptor antagonists demonstrate that the assay is a useful tool to rank the antagonists according to their order of potency. Overall, the results presented here indicate that the versatility, sensitivity, robustness, and ease of execution of the AlphaScreen NR assay will allow for efficient screening of NR modulators.

Antibodies↗

PubMed2002

Novel ATPase of SNF2-like protein family interacts with androgen receptor and modulates androgen-dependent transcription.

Nuclear receptors, including the androgen receptor (AR), regulate target cell transcription through interaction with auxiliary proteins to modify chromatin structure. We describe herein a novel AR-interacting protein, termed ARIP4, that has structural features typical of the SNF2-like protein family. With regard to the Snf2 domain, the closest homolog of ARIP4 is the ATRX protein. ARIP4 is a nuclear protein and comprises 1466 amino acids. It interacts with AR in vitro and in cultured yeast and mammalian cells. ARIP4 can be labeled with 8-azido-[gamma-32P]ATP and exhibits DNA-dependent ATPase activity. Like several ATP-dependent chromatin remodeling proteins, ARIP4 generates superhelical torsion within linear DNA fragments in an ATP-dependent manner. With a stably integrated target promoter, ARIP4 elicits a modest enhancement of AR-dependent transactivation. In transient cotransfection assays, ARIP4 modulates AR function in a promoter-dependent manner; it enhances receptor activity on minimal promoters, but does not activate more complex promoters. ARIP4 mutants devoid of ATPase activity fail to alter DNA topology and behave as trans-dominant negative regulators of AR function in transient assays.

Adenosine Triphosphatases↗