Abstract
Proteomics is a new discipline that studies in a systematic way all the proteins produced in organisms, whose genome sequencing is underway or has already been completed. In the past, in order to analyze the differential expression of proteins in specific conditions or biological states, methods of two-dimensional gel electrophoresis were mainly used.
Following the emergence of new powerful analytical technologies, such as mass spectrometry and the development of bioinformatics methods for analysing genomic sequences and for deducing the aminoacidic composition of gene products, proteomics has extended its action field to “functional genomics”, including studies on the identification, localization, expression and interaction among proteins.
In particular, the creation of a protein-protein interaction map of the cell could be of great value for the understanding of the biology of the cell itself. The recognition and the binding between proteins is at the basis of the formation of multiprotein complexes, which are fundamental for many activities of the cell, such as development, signal trasduction, adherence and communication with other cells. A great number of interactions are mediated by families of protein-protein interaction domains, that are portions of proteins, autonomously folded from the rest of the molecule, including about 40-200 amino acids. They are non catalytic domains that bind specifically and reversibly to a more or less extended region of the target molecule, through a cavity specialised for recognition.
For this dissertation, I concentrated my study on a particular family of protein interaction domains that are very frequent in proteins and take part in the formation of
Abstract V
several supramolecular complexes. The protein modules “PDZ” were originally described as structural elements contained in three proteins, PSD95 (component of a complex under the post-sinaptic membrane), DLG (a tumor suppressor) e ZO-1 (present in mammalian tight junctions), afterwards they have been identified in organisms as diverse as bacteria, plants, invertebrates and vertebrates, mainly at cellular membranes level, where they play the role of “adapters” or of “scaffolds”.
In order to characterize the modalities PDZ domains use in order to recognize and bind their targets, I isolated the coding regions for some PDZ modules present in proteins of different organisms: man, mouse, zebrafish. I then used an integrated proteomic approach in order to study: 1) the specificity of recognition of the various domains, 2) the target proteins they can interact with, 3) the components of the possible multiprotein complexes they are part of. Therefore I analysed ranges of peptides with random sequences, displayed on a phage capsid (“peptide phage-display”) or phage libraries of cDNA products (“cDNA-phage display”), identifying peptides that are bound preferably by each PDZ. Moreover in the framework of a collaboration project, starting from protein extracts from mouse brains, I selected, by means of affinity chromatography and mass spectrometry analysis, proteins that keep connected to PDZ in vitro. I confirmed one of these interactions also in a cultured cell system.
Finally, the use of different approaches enabled to understand advantages and limits of the various technologies, highlighting the need to compare and integrate different results, in order to have understand how these domains modulate proteins activities within cells.
