Aggregating Proteins and their Interactions with Chemical Chaperones

Our interest is focused on the investigation of protein misfolding and aggregating processes, that are known to follow a multidegree polymerization pattern, from dimers, to small oligomer, further to protofibrils and fibril complexes, found in amyloid fibrils and also associated with other Loss Of Function (LOS) or Gain Of Function (GOF) diseases [1]. In this context, we pursuit the evaluation of the capability of small natural molecules to interfere with protein misfolding and with the subsequent aggregation process. Therefore we developed a reliable model of protein misfolding, using different proteins in physiological conditions mimicking buffers, such as ?- and ?-crystallin from pig crystalline lens, as their aggregation is involved in cataract pathogenesis; recombinant human or hen egg white lysozyme, as lysozyme and its mutated proteins are associated with hereditary non-neuropathic systemic amyloidosis; or VVTL1 protein purified from white wine, as it is a pathogen-related protein that withstands the vinification and persists in wine after bottling and may cause haze formation in wine. We proceeded by treating them with various physical or chemical methods to induce misfolding and aggregation (e.g., increasing temperature, UV radiation as oxidative stress inducers, high ions concentration, as for example Ca2+) and then we monitored the aggregation process by spectrophotometric techniques such as fluorescence, UV, CD, also taking advantage of dedicated probes. We profited of UV radiation to give rise to protein aggregation because we already have demonstrated [2] that the UV light is able to generate reactive oxygen species in aqueous solution, made observable for example by using the fluorescent probe DHR, which emission signal intensity is proportional to the concentration of ROS generated, as indeed is prevented in presence of 1mM of ascorbic acid, already known to be a radical scavenger. The ROS generation rate is affected by the actual buffer composition, moreover, the presence of proteins, and in general of aromatic compounds, influences the efficiency of the UV-light induced ROS production. With regard to this phenomena, by synchrotron radiation photo-denaturation assays we observed a significant decrease of secondary structure upon light irradiation using intense far-UV radiations in structured proteins. ROS are so supposed to be the leading cause of the protein denaturation experienced as a consequence of the 25 repeated scans of the SR-CD photon flux at the B23 beamline of the Diamond Light Source synchrotron. As well as thermal denaturation, UV photo-denaturation varies from protein to protein, and also in the presence of ligands.