INTERACTIONS BETWEEN PROTEINS AND IDENTIFICATION OF HIGH AFFINITY ANTAGONISTS

tornatore, laura (2008) INTERACTIONS BETWEEN PROTEINS AND IDENTIFICATION OF HIGH AFFINITY ANTAGONISTS. [Tesi di dottorato] (Inedito)

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Abstract

Interactions between proteins and identification of high affinity antagonists The relationship between the pathological processes of infection, inflammation and cancer is correlated to the role of the transcription factor NF-κB in cellular homeostasis. Therefore studying NF-κB target genes makes it is possible to understand the molecular mechanisms of inflammation, of tumor growth and progression. Gadd45β is one of the anti-apoptotic regulators controlled by NF-κB in response to pro-inflammatory stimuli and genotoxic stress exerting its functions by interacting with several partners such as MAP kinases and DNA clamp protein. The interaction between Gadd45β and MKK7 has been described as a molecular link between the NF-kB cytoprotective effects and the suppression of the signaling of JNK, one of the main MKK7 substrates that strongly promotes apoptosis. Understanding this complex network of interactions and finding effective antagonists is of preeminent importance for elucidating the balance between cell death and cell survival and to modulate cell homeostasis for therapeutic applications. For this reason, this PhD project has been focused on the structural and functional characterization of the Gadd45β-MKK7 complex and on the identification of compounds able to disrupt this interaction. In fact, developing molecules able to block the pro-survival action of NF-κB without significantly compromising the innate activation of the immune system is an effective way to suppress the proliferative effects induced by Gadd45β expression. Biochemical analyses confirmed that Gadd45β exists in solution prevalently as a non covalent dimer in a 1:1 stoichiometry. The self-association dissociation constant was estimated to be about 100 nM and by limited proteolysis we identified the regions of Gadd45β involved in the self-association as corresponding to the predicted helix 1 (H1) and helix 5 (H5) of the protein. The biochemical analysis of Gadd45β-MKK7 complex has been investigated and provided a new context for dissecting the structure-activity relationship of this interaction which exhibited a KD of about 13 nM. To select antagonists of the Gadd45β-MKK7 interaction, a combinatorial chemistry approach has been followed. By this process, Lead Compounds 1 and 2 have been selected from deconvolution of two generations of tetrapeptide libraries. These novel antagonists can disrupt very efficiently the interaction between Gadd45β and MKK7 at concentrations in the low nanomolar range. Further investigations by co-immunoprecipitation and kinase assays have confirmed that the measured antagonistic activity matches perfectly with the IC50 values from ELISA data. Additionally, the results suggest that the selected peptides disrupt the interaction between Gadd45β and MKK7 without directly interfering with the kinase activity. Indeed several kinase assays have confirmed that both Lead Compounds do not block JNK phosphorylation, therefore they do not enter the kinase ATP binding site. These results suggest that Lead Peptides 1 and 2 have the potential of becoming very useful new drug candidates, since the selective inhibition of NF-kB downstream targets, such as Gadd45β, could be a way to convert inflammation-driven tumor growth into inflammation–induced tumor suppression.

Tipologia di documento:Tesi di dottorato
Parole chiave:Gadd45b; MKK7; oligomerization ; protein-protein interaction; combinatorial chemistry and drug discovery
Settori scientifico-disciplinari MIUR:Area 05 Scienze biologiche > BIO/10 BIOCHIMICA
Coordinatori della Scuola di dottorato:
Coordinatore del Corso di dottoratoe-mail (se nota)
Prof. Sannia, Giovannigiovanni.sannia@unina.it
Tutor della Scuola di dottorato:
Tutor del Corso di dottoratoe-mail (se nota)
Prof. Benedetti, ettoreettore.benedetti@unina.it
Dott.ssa Monti, simona Mariamarmonti@unina.it
Stato del full text:Accessibile
Data:2008
Numero di pagine:125
Istituzione:Universita' degli studi di Napoli Federico II
Dipartimento o Struttura:Dipartimento di Biostrutture e Bioimmagini
Tipo di tesi:Dottorato
Stato dell'Eprint:Inedito
Scuola di dottorato:Dottorato in Scienze Biotecnologiche –Università di Napoli Federico II
Denominazione del dottorato:Dottorato in Scienze Biotecnologiche – XXI- cicloIndirizzo Biotecnologie molecolari
Ciclo di dottorato: XXI ciclo
Numero di sistema:3485
Depositato il:19 Novembre 2009 10:23
Ultima modifica:19 Novembre 2009 10:23

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