Content:
The study and the characterization of protein-protein interactions (PPIs) are essential to define the molecular networks that contribute to cellular physiological processes or to the development of a disease. To this purpose, my research project was focused on the development of an innovative methodology aimed at identifying protein-protein interaction networks in living cells by inducing UV laser cross-linking between two interacting bio-partners in close molecular contact without altering proteins and their natural environment. In particular, we used a femtosecond UV laser to irradiate human living cells, i.e. HeLa cells, in order to cross-link proteins in vivo. We focused our attention on glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein implicated in many cellular activities, which is a homo-tetrameric enzyme, structurally and functionally well characterized. Our experiments allowed us to demonstrate that the exposure of HeLa cells to UV laser induces the formation of covalent adducts of GAPDH inside the cells and that the phenomenon can be modulated, since the amount of cross-linked products was found to be a linear function of the total irradiation energy. By this way, we were able to setting up the optimal parameters to maximize UV laser protein-protein cross-linking yield without damaging cells. Immunoprecipitation experiments and mass spectrometry analyses allowed us to isolate and identify, among the cross-linked products, stable dimers of GAPDH, as also observed upon in vitro UV laser irradiation of the pure protein. Since aromatic residues are supposed to be involved in the cross-linking reaction, we performed GAPDH structural and spectroscopic analyses to add knowledge to the molecular bases of UV laser cross-linking. Interestingly, a detailed structure analysis of the distances between aromatic residues showed the presence of a defined aromatic patch at the C2 symmetric R-interface of GAPDH natural tetramer, so that a dimer is the sole product expected to be generated by light excitation. Furthermore, spectroscopic analyses, performed on rabbit muscle GAPDH irradiated in vitro, suggested that a cross-coupled aromatic moiety is produced upon laser irradiation. Taken together, collected data confirmed that UV laser acts as an agent able to photo-fix protein surfaces that are in direct contact. Due to the ultra-short time scale of UV laser induced cross-linking, this technique may represent a powerful tool to weld transient protein interactions (at zero length) in their native context. Since this novel methodology needs to be validated and deeply characterized, in the period I spent in the laboratories of Professor C. Zurzolo in Paris and Professor M. Tramier in Rennes, I set up a suitable system based on FLIM-FRET microscopy, which will be helpful to validate by an independent approach data obtained by UV laser cross-linking in living cells and which will allow the spatio-temporal characterization of the dynamics of interactions photo-fixed" by UV laser irradiation. The impact of the UV laser technique will be relevant both in the generation of scientific knowledge and insight, as well as in practical applications. Our results pave the way to understand the basic mechanisms of interaction between biomolecules in a native context at a molecular level, what represents a consistent boost in physics, chemistry, and so-called "omics" sciences, such as genomics, epigenomics and proteomics."
Note:
Dissertation 2016
Language:
Italian
