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Tesi etd-02262009-190704


Thesis type
Tesi di dottorato di ricerca
Author
BARILARI, MANUELA
URN
etd-02262009-190704
Title
Dissecting the interaction network of a multi-PDZ protein
Settore scientifico disciplinare
BIO/11
Corso di studi
BIOTECNOLOGIE MOLECOLARI
Commissione
Relatore Prof. Dente, Luciana
Parole chiave
  • PDZ
  • IRSp53
  • Cypin
  • CIPP
Data inizio appello
17/03/2009;
Consultabilità
parziale
Data di rilascio
17/03/2049
Riassunto analitico
Multimodular scaffolds, containing multiple copies of binding domains, such as multi-PDZ proteins, play a crucial role in the formation and localization of complex multicomponent assemblies, which are fundamental for many activities of the cell. Unravelling the components of the networks, mediated by such multivalent binding proteins, is essential for understanding how signal transduction and intracellular pathways are organized. During my doctorate research, I analyzed the network mediated by the protein CIPP, “channel-interacting PDZ protein”, a typical example of multi-PDZ protein, engaged in the interaction with several channels and receptors, at membrane level. <br>CIPP belongs to a large family of proteins, composed of several isoforms, whose tissue specific expression I have analyzed by RT-PCR. In particular, I concentrated my study on an isoform containing four PDZ domains and I analyzed its brain-specific expression, using in situ hybridization assays. In order to identify the precise components of the protein interaction complex mediated by CIPP PDZ domains, I characterized two novel cytoplasmic CIPP interactors: the proteins Cypin and IRSp53, both widely involved in mechanisms of cytoskeletal remodelling. Their direct and specific interaction with CIPP-PDZ1 and CIPP-PDZ2, respectively, was verified using multiple biochemical binding assays. The binding occurrence was also proved with the full length CIPP recombinant protein and, by generating mutant constructs, I showed the importance of an intact canonical PDZ binding motif at the carboxyl termini of the ligands. Immunolocalization analysis on cultured cells confirmed the reciprocal interactions and, moreover, showed that IRSp53, following PDZ-mediated interaction with CIPP, was able to induce the formation of punctate cytoplasmic clusters. These CIPP-containing puncta were not associated with endocytic vesicles, but they were simple protein assemblies, containing CIPP, IRSp53 and Cypin. Finally, I established that IRSp53 and Cypin were able to directly interact one with each other, through specific regions, independently from the PDZ-mediated binding to CIPP. Interestingly, their clustering in cytoplasmic puncta occurred only when also CIPP was present. <br>All the information derived from these studies suggest that CIPP-PDZ domains are responsible for the formation of a tripartite CIPP-IRSp53-Cypin complex, which could contact membrane channels or other receptors, such as NMDA receptors, and mediate signal transduction inside the cell to the final cytoskeletal targets.<br>
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