ANALYTICAL STRATEGIES FOR THE CHARACTERIZATION OF PROTEIN AGGREGATION AND INHIBITION IN HUMAN DISEASES

Protein misfolding and aggregation has been related to several human disorders, generally termed protein aggregation diseases. These diseases include neurodegenerative disorders such as Alzheimer’s, Parkinson’s and Huntington’s diseases and peripheral disorders such as systemic amyloidosis and type 2 diabetes. The complexity of the aggregation process and the intertwined events account for the fact that no effective disease-modifying treatment is currently available for these disorders. Nevertheless, in-depth research into the aggregation processes has recently yielded major insights into some key mechanisms of aggregation-mediated cell toxicity, offering new targets for drug development. A multiple-methodological study including atomic force microscopy (AFM), mass spectrometry techniques (MALDI-TOF, ESI-IT, ESI-Q-TOF), direct Thioflavin T fuorescence spectroscopy, transmission electron microscopy (TEM), fluorescence microscopy (FLM), is here presented, which enables the characterization of the morphology, dimension and molecular weight of aggregates in the self-assembly kinetics. This approach is useful to elucidate in detail the diverse assembly species formed in amyloid peptides folding process. Different orthogonal techniques resulted suitable to obtain technique-independent outcomes. Moreover, MALDI–TOF mass spectrometry allowed the detection and characterization of the very early stages and nucleating species of the self-assembly process. This multi-methodological approach to the characterization of the mechanism of aggregation inhibition was validated highlighting inhibitors mechanism of action and elucidating which are the species whose formation is impeded.