RESEARCH ACTIVITY
FONTANA-POLVERINO DE LAURETO-SPOLAORE
Folding and Misfolding. The research activity of the Laboratory of Protein Chemistry is focused on the study of the mechanisms of protein folding and misfolding. A main interest is devoted to partly folded states of proteins, which are considered intermediates in the process of protein folding and that are involved in misfolding and aggregation of several proteins, responsible of several human diseases, known as amyloidosis (Parkinson's disease, Alzheimer's disease). Our research aims at clarifying at the molecular level the mechanisms of formation of protein amyloid aggregates by using several model proteins and peptides. These studies are conducted using different biophysical techniques (circular dichroism, fluorescence, infrared spectroscopy), as well as biochemical methods (e.g., limited proteolysis). Moreover, the morphology of protein aggregates is analyzed by electron microscopy. The proteins under study are human and bovine alpha-lactabumin, human and chicken lysozyme and myoglobin. These small globular proteins are excellent protein models for structural and folding studies, as well as for an understanding of the mechanism of protein aggregation, since they are able to form in vitro fibrils that are indistinguishable from those extracted from patients affected by amyloidosis. Another protein of interest is human alpha-synuclein that is involved in Parkinson's disease. Since this protein is abundant in the brain and in particular in neurons, we are particularly interested in the interaction of this protein with membranes and brain lipids.
Transglutaminase. Transglutaminase (TGase) catalyses the formation of an isopeptide bond between a protein-bound glutamine residue and a variety of primary amines, thus enabling the covalent binding of numerous chemical entities to proteins. The TGase-mediated enzymatic reaction can be used to develop a variety of strategies of protein modification for the purposes of basic studies, as well as for numerous biotechnological applications. We are using microbial TGase for the enzymatic modification of proteins and our results have clearly demonstrated that the TGase-mediated reactions can be site-specific. We have concluded that a reactive glutamine residue should be located in flexible or locally unfolded region of the protein chain in order to act as TGase's substrate, as deduced from TGase reactions conducted on apomyoglobin as model protein substrate. We are using microbial TGase for the site-specific modification of several proteins of pharmaceutical interest, such as interferon, human growth hormone, granulocyte colony-stimulating factor and erythropoietin. In particular, we are interested in devising novel approaches for the TGase-mediated modification of these protein drugs with polymers as polythethylene glycol (PEG) and with metal-chelating agents, including radio-nuclides useful for PET diagnosis and radio-therapy.
Mass spectrometry. A general aim of our research is the development and optimization of techniques for the purification and characterization of proteins and of methodologies for the analysis of proteins by mass spectrometry (MS), in particular for the detection of chemical and post-translational modifications of proteins. The variety of MS techniques and approaches nowadays used in modern proteomics research are being implemented. Recently, these methods are used to detect allergenic proteins contained in minute amounts in foods.