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Inhibition of NF-kappaB activation with designed ankyrin-repeat proteins targeting the ubiquitin-binding/oligomerization domain of NEMO.

ABSTRACT: The link between the NF-kappaB signal transduction pathway and cancer is now well established. Inhibiting this pathway is therefore a promising approach in the treatment of certain cancers through a pro-apoptotic effect in malignant cells. Owing to its central role in the pathway, the IkappaB kinase (IKK) complex is a privileged target for designing inhibitors. Previously, we showed that oligomerization of NEMO is necessary for IKK activation and defined a minimal oligomerization domain (CC2-LZ) for NEMO, and we developed NEMO peptides inhibiting NF-kappaB activation at the level of the IKK complex. To improve the low-affinity inhibitors, we used ribosome display to select small and stable proteins with high affinity against the individual CC2-LZ because the entire NEMO protein is poorly soluble. Several binders with affinities in the low nanomolar range were obtained. When expressed in human cells, some of the selected molecules, despite their partial degradation, inhibited TNF-alpha-mediated NF-kappaB activation while having no effect on the basal activity. Controls with a naive library member or null plasmid had no effect. Furthermore, we could show that this NF-kappaB inhibition occurs through a specific interaction between the binders and the endogenous NEMO, resulting in decreased IKK activation. These results indicate that in vitro selections with the NEMO subdomain alone as a target may be sufficient to lead to interesting compounds that are able to inhibit NF-kappaB activation.

SUBMITTER: Wyler E

PROVIDER: S-EPMC2206981 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Inhibition of NF-kappaB activation with designed ankyrin-repeat proteins targeting the ubiquitin-binding/oligomerization domain of NEMO.

Wyler Emanuel E Kaminska Monika M Coïc Yves-Marie YM Baleux Françoise F Véron Michel M Agou Fabrice F

Protein science : a publication of the Protein Society 20070901 9

The link between the NF-kappaB signal transduction pathway and cancer is now well established. Inhibiting this pathway is therefore a promising approach in the treatment of certain cancers through a pro-apoptotic effect in malignant cells. Owing to its central role in the pathway, the IkappaB kinase (IKK) complex is a privileged target for designing inhibitors. Previously, we showed that oligomerization of NEMO is necessary for IKK activation and defined a minimal oligomerization domain (CC2-LZ) ...[more]

PMID: 17766391

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Project description:Total internal reflection fluorescence-based single-molecule Förster resonance energy transfer (FRET) measurements were previously carried out on the ankyrin repeat domain (ARD) of I?B?, the temporally regulated inhibitor of canonical NF?B signaling. Under native conditions, most of the I?B? molecules showed stable, high FRET signals consistent with distances between the fluorophores estimated from the crystal structures of the NF?B(RelA/p50)-I?B? complex. Similar high FRET efficiencies were found when the I?B? molecules were either free or in complex with NF?B(RelA/p50), and were interpreted as being consistent with the crystallographically observed ARD structure. An exception to this was observed when the donor and acceptor fluorophores were attached in AR3 (residue 166) and AR6 (residue 262). Surprisingly, the FRET efficiency was lower for the bound I?B? molecules (0.67) than for the free I?B? molecules (0.74), apparently indicating that binding of NF?B(RelA/p50) stretches the ARD of I?B?. Here, we conducted confocal-based single-molecule FRET studies to investigate this phenomenon in greater detail. The results not only recapitulated the apparent stretching of the ARD but also showed that the effect was more pronounced when the N-terminal domains (NTDs) of both RelA and p50 were present, even though the interface between NF?B(RelA/p50) and I?B? encompasses only the dimerization domains. We also performed mass spectrometry-detected amide hydrogen/deuterium exchange (HDXMS) experiments on I?B? as well as I?B? bound to dimerization-domain-only constructs or full-length NF?B(RelA/p50). Although we expected the stretched I?B? to have regions with increased exchange, instead the HDXMS experiments showed decreases in exchange in AR3 and AR6 that were more pronounced when the NF?B NTDs were present. Simulations of the interaction recapitulated the increased distance between residues 166 and 262, and also provide a plausible mechanism for a twisting of the I?B? ARD induced by interactions of the I?B? proline-glutamate-serine-threonine-rich sequence with positively charged residues in the RelA NTD.

Dataset Information

Inhibition of NF-kappaB activation with designed ankyrin-repeat proteins targeting the ubiquitin-binding/oligomerization domain of NEMO.

Publications

Inhibition of NF-kappaB activation with designed ankyrin-repeat proteins targeting the ubiquitin-binding/oligomerization domain of NEMO.

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