ayout 1 - EMBL Grenoble

EMBL Grenoble
High-throughput protein technologies
Previous and current research
Our group develops new molecular biology methods and uses them to work on difficult biological
problems. Combinatorial methods (e.g. directed evolution, phage display) are used to address
problems that are too complex for rational design approaches. Large random libraries of variants
are constructed and screened to identify rare hits with the desired property. In our ESPRIT process,
for example, all truncations of a target protein are generated and screened using advanced picking
and arraying robotics. With such technologies in hand, we are able to study certain biological
questions with advantages over classical approaches. The proteins we study are generally enzymes
of biological and medicinal interest:
Influenza RNA polymerase: There is worldwide concern that currently circulating avian influenza
viruses will cross the species barrier and become highly pathogenic, human transmissible strains with
pandemic potential. This could result from residue changes in several influenza proteins, either by
point mutations or through shuffling of the segmented avian and mammalian viral genomes. We are
now characterising the interactions of these mutants with host cell factors using both structural and
biophysical methods with the aim of understanding mechanisms of influenza host specificity.
Human Kinases: Cells have intricate mechanisms of sensing and responding to environmental
changes. Upon a stimulus detected by a cellular receptor the complex system of signal transduction is activated that results in changes in gene
expression. Protein kinases play a crucial role in cellular stress responses as mediators between the upstream receptor and downstream gene
regulation and are key components in coping with changes in the intra-/extracellular environment.
When these mechanisms malfunction, diseases such as excessive inflammation, autoimmune
disorders and cancer can occur. Kinases therefore represent important
pharmaceutical targets for drug design. The multidomain nature of many kinases reflects the
need to regulate the activity of the catalytic activity. We are screening for stable constructs that
extend beyond the conserved regions of the catalytic domain, and well-expressed internal domains
presumably implicated in complex formation or regulation.
Histone Deacetylases (HDACs): Using our construct screening technology, we have identified
well-expressing, catalytically active constructs of an HDAC involved in cholesterol homeostasis.
Using these proteins, we are investigating how new inhibitors bind using X-ray crystallography
and enzymatic inhibition assays. Secondly, using a library-format protein interaction screen, we
are trying to identify HDAC-interacting domains of cellular proteins. If identified, disruption of
such protein-protein interactions suggests a new route towards specific HDAC inhibition.
Future projects and goals
Difficult biological projects require advanced new tools. We will continue to develop
expression methods to handle protein complexes, targets that require eukaryotic
expression for correct folding, and possibly aspects of membrane proteins.
Each project uses ‘real’ targets of interest and the aim is use method advancements
to yield previously unobtainable biological knowledge. For example, we are testing
permutations of influenza-influenza and influenza-host
proteins with the aim of defining expressible, crystallisable
protein complexes that should provide insights into
virus host cell interactions.
Darren Hart
PhD 1996, Oxford University.
Postdoctoral research at
Cambridge University.
Group leader at Sense
Proteomic Ltd., Cambridge.
Team leader at EMBL
Grenoble since 2003.
Figure 1: Screening tens of thousands of
expression constructs of a target gene.
Constructs are made as a random library
and printed on membranes for soluble
expression analysis by hybridisation of
fluorescent antibodies.
Figure 2: A previously unsuspected domain from influenza polymerase, identified by
high throughput expression screening of tens of thousands of random DNA
constructs, and structurally characterised by X-ray crystallography. A single mutation
to lysine at residue 627 (A) can be responsible for the evolution of human influenza
viruses from wild-type avian viruses that have a glutamic acid at this position (B). The
mutation of residue 627 reinforces or disrupts a striking basic surface patch and we
are seeking to understand how this affects polymerase function.
Selected references
Angelini, A., Tosi, T., Mas, P., Acajjaoui, S., Zanotti, G., Terradot, L.
& Hart, D.J. (2009). Expression of Helicobacter pylori CagA domains
by library-based construct screening. FEBS J., 276, 816-82
Guilligay, D., Tarendeau, F., Resa-Infante, P., Coloma, R., Crepin, T.,
Sehr, P., Lewis, J., Ruigrok, R.W., Ortin, J., Hart, D.J. & Cusack, S.
(2008). The structural basis for cap binding by influenza virus
polymerase subunit PB2. Nat. Struct. Mol. Biol., 15, 500-506
Tarendeau, F., Crepin, T., Guilligay, D., Ruigrok, R.W., Cusack, S. &
Hart, D.J. (2008). Host determinant residue lysine 627 lies on the
surface of a discrete, folded domain of influenza virus polymerase
PB2 subunit. PLoS Pathog., , e1000136
Tarendeau, F., Boudet, J., Guilligay, D., Mas, P.J., Bougault, C.M.,
Boulo, S., Baudin, F., Ruigrok, R.W., Daigle, N., Ellenberg, J.,
Cusack, S., Simorre, J.P. & Hart, D.J. (2007). Structure and nuclear
import function of the C-terminal domain of influenza virus
polymerase PB2 subunit. Nat. Struct. Mol. Biol., 1, 229-233
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