Sandomenico, Annamaria (2008) Peptides as modulators of protein-protein interactions. [Tesi di dottorato] (Inedito)

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Abstract

One of the major challenges for the comprehension of physiological and pathological functions regulating the cellular processes, is the elucidation of molecular mechanisms underlining protein–protein interactions. Protein–protein interactions indeed play a key role in a variety of biological processes and are therefore important targets for the design of novel therapeutics. Unlike the design of enzyme inhibitors, the disruption of protein–protein interactions is far more challenging, due to large interfacial areas involved in protein recognition and to the relatively flat topologies of these surfaces. Nevertheless, in spite of such difficulties, there has been considerable progress in the recent years. The purpose of this PhD thesis is the identification of small peptides acting as modulators of protein–protein interactions playing a crucial role in pathological processes, such as allergy and cancer. Different strategies have been employed: in one case, starting from a crystallographic structure, the rational design of short polypeptides mimicking the receptor binding surface has been achieved. In a second case, potent inhibitors of a protein have been identified following an approach of screening of peptide pools generated from the protein itself. In the first part of the study, we have dealt with de novo designed inhibitors of the IgE-FcεRI interaction, one of the main target for blocking the IgE-mediated allergic reactions. We have selected several short peptides interacting with IgE with a dissociation constant in the low micromolar range but with a very high selectivity for this immunoglobulin subclass. We have evaluated their kinetic and thermodynamic properties and assessed the capacity to block the interaction of IgE with a recombinant variant of the receptor second domain. Despite the relatively low affinity, these peptides have a potential applicability as modulators of the IgE-FcεRI interaction and therefore as new molecules to be developed for contrasting allergy. In a second study, we have studied a homotypic protein-protein interaction mediated by specific α-helical domains, known as Capase Recruitment Domains (CARD). We have studied the CARD of the protein BCL10, a main regulator of the innate immune response that is a component of the CBM (CARMA1-BCL10-MALT1) protein complex, by which it promotes the activation of the NF-B transcription factor. In this case, we have studied a very short peptide derived from the protein structure, which is able to block the protein self-interaction. The peptide has been selected by functionally ranking CARD-CARD antagonists by a binding competition assay between differently tagged recombinant domains. The antagonists have been prepared by enzymatically digesting the CARD itself and fractionating the resulting peptides. Using synthetic peptides, we have been able to identify single residues involved in CARD-CARD contacts. The peptide appears to mimic the protein binding interface and is a useful and effective inhibitor of the BCL10 activity in cellular assays.

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