Dr Eric Chevet is a team leader at the French National Institute for Health Research (INSERM). His laboratory is located within an INSERM laboratory at the University of Bordeaux, Bordeaux, France. The lab research interests revolve around the study of endoplasmic reticulum (ER) functions and more particularly on ER signalling pathways.
Dr Chevet holds a PhD in molecular and cellular biology from Paris XI University (France). After his PhD he moved to Montreal for a post-doc at McGill University in the department of Anatomy and Cell biology under the supervision of John JM Bergeron. He started his own research group at McGill University, then moved back to France in 2007 to accept his current position at INSERM in Bordeaux.
One research focus of the Chevet lab is the study of ER stress signalling pathways. These stress signalling pathways are activated upon accumulation of improperly folded in the ER [1]. This situation is observed under numerous physiological and pathological contexts such as diabetes, cancer, or neurodegenerative disorders. Dr. Chevet’s group has been using Alpha technologies for their research since 2002. In particular, his group has developed assays to monitor the activity of small GTPases of the Ras superfamily [2, 3], to evaluate the activation of ER stress signalling (using AlphaLISA-based assays) or to identify new artificial regulators of ER stress signalling.
In a typical GTP binding assay using AlphaScreen® technology (Figure 1), Biotinylated GTPgS and GST-GTPase are incubated in the presence of streptavidin-coated Donor and anti-GST-coated Acceptor beads as well as increasing concentrations of GTP (or any other nucleotides).
Figure 1: AlphaScreen GTPase/GTP binding assay schematic.
Figure 2. Typical competition assay to determine GTPase affinity for GTP (ATP and GST are used as internal controls).
Figure 3. GTPases are classified based on their affinity for GTP .
The results of a typical competition assay are shown in Figure 2 using C. elegans Cdc42 as an example. In this case GTP and ATP are used as competitors.
The IC50 values deduced from these competition assays were calculated for the entire family of C. elegans Rho GTPases (Figure 3). The variation in the results reflects the variability between these highly homologous proteins.
In conjunction with the use of FlashPlate® assays, these AlphaScreen GTP binding assays provided an integrated assay platform to measure GTPase affinity for GTP, GTP hydrolysis activity, and GDP/GTP exchange activity [3]. This allowed us to propose a novel classification of small GTPases of the Rho sub-family based on biochemical data and not only on amino-acid sequences.
For higher throughput, the assay was automated using a JANUS® Mini liquid handler with an MDT dispensing head is in line with a stacker and a JANUS robot containing an 8 pin head and a gripper. This liquid handling tool is in line with an Envision® Multidetection Plate Reader.
Figure 4. Automated AlphaScreen® assay platform as of November 2009.
The National Institute for Health and Medical Research (INSERM) is a public scientific and technological organization. By performing research in the diverse fields of fundamental biology, cognitive and applied medicine, and public health; INSERM meets its overriding objectives of to improve our understanding of human diseases and to ensure that patients and the medical community benefit rapidly from the latest research findings.
To find more about INSERM please visit http://www.inserm.fr/
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References
- Taouji S, Dahan S, Bossé R, Chevet E. Current Screens Based on the AlphaScreen Technology for Deciphering Cell Signalling Pathways. Curr Genomics. 2009 Apr;10(2):93-101
- Caruso ME, Jenna S, Beaulne S, Lee EH, Bergeron A, Chauve C, Roby P, Rual JF, Hill DE, Vidal M, Bossé R and Chevet E. Biochemical clustering of monomeric GTPases of the Ras superfamily. Mol. Cell. Proteomics, 2005 4(7): 936-944
- Caruso ME, Sarah Jenna, Marion Bouchecareilh, David Baillie, Daniel Boismenu, Dalia Halawani, Martin Latterich and Eric Chevet. GTPase-mediated regulation of the Unfolded Protein Response in C. elegans is dependent on the AAA+ ATPase CDC-48. Mol. Cell. Biol., 2008, 28(13):4261-74