PROTEIN-PROTEIN INTERACTIONS: CHARACTERIZATION AND NEW STRATEGY FOR INDUSTRIAL APPLICATIONS

Biological systems are made up of very large numbers of different components interacting at various scales. Most genes, proteins and other cell components carry out their functions within a complex network of interactions and a single component can affect a wide range of other components. Interactions involved in biological processes have been previously characterized individually, but this ‘‘reductionist’’ approach suffers from a lack of information about time, space, and context in which the interactions occur in vivo. A global, integrative, approach has been developed for several years, focusing on the building of protein-protein interaction maps (interactomes). These interaction networks are complex systems, where new properties arise. This is part of an emergent field, called systems biology which is ‘‘the study of an organism, viewed as an integrated and interacting network of genes, proteins and biochemical reactions which give rise to life’’ (http://www.systemsbiology.org/). This interdisciplinary approach, involving techniques from the mathematical, computational, physical and engineering sciences is required to understand complex networks. Detailed knowledge of protein interactions and protein complex structures is therefore fundamental to understand how individual proteins function within a complex and how the complex functions as a whole. This PhD thesis targeted the characterization by structural and functional proteomics approaches of selected protein complexes involved in relevant molecular processes and can have an impact on their industrial applications. The following systems of biotechnological interest have been analysed in this study: 1) Phenol Hydroxylase (PH) from Pseudomonas sp. OX1. It is a bacterium endowed with the ability to grow on a wide spectrum of hydroxylated and non-hydroxylated aromatic compounds for this reason it can be use in bioremediation of contaminated environments. In this context the research has been aimed at characterizing the endogenously expressed Phenol Hydroxylase (PH) complex from Pseudomonas sp. OX1, using biochemical approaches integrated to mass spectrometry analysis. Moreover, in order to define the protein-protein interaction networks of this complex a functional proteomic approach was carried out. 2) Characterization of the complex between pLG72 and human D-amino acid oxidase (hDAAO), linked to the onset of schizophrenia. The molecular basis of schizophrenia is still elusive and current treatments focus on eliminating the symptoms of the disease. The clarification of the role of this specific complex and the identification of other physiological or conditional partners in this biochemical process will be useful in developing molecules that can be effective in the disease treatment. Moreover, as a parallel goal, and strictly connected with the former issue, I worked at 3) the development of an innovative structural analysis approach based on the use of a femtosecond UV laser as a novel zero length protein-protein cross-linker. Such photo-physical approach could obviate many of the problems associated with standard chemical cross-linking reagents, and put cross-linking in a proteome-wide position for the characterizations of protein-protein interactions in vivo in intact cells, the study of the transient interactions among proteins, mapping molecular interfaces in protein complexes, providing information on the dynamics of the contacts within a multi-protein complex