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Session XIV : Transcriptional and translational control Poster XIV, 76
Blocking NF-kB activation by engineering proteins targeted to the minimal
oligomerization domain of NEMO
Emanuel Wyler*, Monika Kaminska, Andreas Plückthun, Michel Veron, and Fabrice
Agou
The Unit of Enzymatic Regulation of Cell Activities, Pasteur Institut, Paris, France,
*Present address: Institut of Biochemistry, ETH, Zurich, Switzerland
wyler@bc.biol.ethz.ch,kaminska@pasteur.fr,plueckthun@bioc.unizh.ch
mveron@pasteur.fr, fagou@pasteur.fr
The link between NF-kB signal transduction pathway and cancer is now well established.
Thus, inhibiting this pathway is a promising clinical approach through a pro-apoptotic effect
in the treatment of certains cancers. It has already proved to be efficient in therapy, in
combination with chemotherapy and radiation for a variety of cancers. The IKK complex is a
priviledged target for designing inhibitors due to its central role of the pathway. We
previously defined a minimal oligomerization domain of NEMO and showed that
oligomerization was necessary for NEMO function. This allowed us to develop new
inhibitory peptides of the NF-kB pathway targeting NEMO oligomerization (1).
Ankyrins constitute an attractive class of stable and small repeat proteins that provide variable
and modular binding surfaces to a target protein. We used the ribosom display method to
select ankyrins binding to the NEMO minimal oligomerization domain from a native ankyrin
library. After four rounds of selection, several ankyrins with affinity in the low nanomolar
range were isolated.When expressed in 293T cells, the selected ankyrins strongly inhibit
TNF-a-mediated NF-kB activation while having no effect on the basal activity. Controls with
native ankyrin or null plasmid were without effect. Furthermore, we could show that this NFkB
inhibition occurs through a specific interaction between ankyrin binders and the
endogenous NEMO, resulting in the IKK inhibition. Our findings indicate that high-affinity
binders selected from peptide or chemical libraries against the minimal oligomerization
domain of NEMO can be a promising strategy to search for specific NF-kB inhibitors. Our
binders might also be used for structural studies for both NEMO and the minimal
oligomerization domain.
F. Agou et al. (2004) J.Biol.Chem. 279, 54248-54257
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