Expression and purification of potential amyloidogenic proteins: AB peptides and hSOD1 to investigate the mechanism of fibrils formation.

One of the major paradigms in structural biology, according to which a rigid well-folded 3D structure is required for protein function, has clearly changed over the last decades. Recent studies show that some proteins despite the absence of a stable secondary or tertiary structure, play important roles in a number of biological processes, such as differentiation, transcription regulation, DNA condensation, mRNA processing, and apoptosis. Such proteins are known as intrinsically disordered proteins (IDPs) and are characterized by high flexibility and plasticity, that facilitate their interactions with a broad range of binding partners, such as proteins, membranes, nucleic acids and other molecules of biological relevance. (Uversky & Dunker, 2010). Intrinsically disordered proteins are also prone to misfolding and tend to evade normal clearance pathways. In turn, the combination of misfolding and lack of clearing mechanisms can result in aberrant processes, often associated with the onset of pathologies. In these cases, it is common to observe progressive protein aggregation into intracellular and/or extracellular deposits. The consequence is a diverse group of neurodegenerative disorders, each of which entails the aggregation of particular proteins in characteristic patterns and locations (Jucker & Walker, 2013). Under some particular conditions (e.g. environmental change or mutation) even native folded proteins might lose theirs stable, biochemically functional forms, and for this reason they can be investigated by the same methodologies used for IDPs. Examples of such proteins are beta amyloid peptide (Aβ) (intrinsically disordered in the monomeric form) associated to the Alzheimer’s disease (AD), and superoxide dismutase (SOD1) (which is intrinsically disordered in the reduced apo form) related to amyotrophic lateral sclerosis (ALS). These proteins are the main topics of this thesis. In this work we will present detailed structural characterization of Aβ prefibrillar and fibrillar assemblies by Solid State Nuclear Magnetic Resonance (SSNMR) that is crucial for understanding Aβ precise mechanism aggregation pathways and identifying toxic Aβ species involved in Alzheimer’s disease. This thesis will demonstrate also attempt of reverting side effects caused by cisplatin, that is effectie drug strongly inhibiting process of SOD1 oligomerization. That could open a way toward a novel therapeutic strategies in amyotrophic lateral sclerosis (ALS), since the clinical use of cisplatin is still limited cause of the development of neurotoxicity and others undesirable effects.