|Tipo di tesi||Tesi di dottorato di ricerca|
|Autore||MASCHIO, MARIA CELESTE|
|Titolo||Modellizzazione di una proteina amiloidogenica in soluzione e su superfici|
|Titolo in inglese||Modeling an amyloidogenic protein in solution and on surfaces|
|Settore scientifico disciplinare||FIS/03 - FISICA DELLA MATERIA|
|Corso di studi||PHYSICS AND NANO SCIENCES|
|Data inizio appello||2019-08-29|
|Disponibilità||Accessibile via web (tutti i file della tesi sono accessibili)|
L’aggregazione di proteine in fibrille insolubili è all’origine di particolari patologie, chiamate amiloidosi. Le malattie neurodegenerative i cui depositi di amiloidi si trovano nel cervello (e.g. Alzheimer e Parkinson) ed i danni ad altri tessuti ad opera delle amiloidosi sistemiche trovano trattamento adeguato solo nella sintomatologia. La scoperta dei meccanismi di misfolding e aggregazione di una proteina si pone pertanto come un importante obiettivo da raggiungere.
The aggregation of soluble proteins into insoluble fibrillar amyloids is at the origin of pathological conditions, named amyloid disorders. Ranging from neurodegenerative disorders that are characterized by amyloid deposits in the brain (e.g. Alzheimer’s and Parkinson’s disease) to systemic amyloidoses affecting other tissues in the human body, such diseases do not have proper medical treatments except for symptoms: the comprehension of protein behavior during misfolding and aggregation is then one of the most important goals to reach. The so-called Dialysis Related Amyloidosis (DRA), very common in patients undergoing hemodialysis, is driven by the pathological aggregation of the protein β2-microglobulin (β2-m), targeting mostly bones, ligaments and joint structures. Recent studies have highlighted the role of many events as triggering factors for fibril formation, varying from the external environment that surrounds the protein to its internal rearrangements. In this work, we decided to focus on three of them: (i) the dimerization as a first step of aggregation, characterizing the protein-protein interaction for β2-m in physiological pH conditions; (ii) the cis-to-trans isomerisation of a particular aminoacid, the proline P32, causing severe conformational changes that drive β2-m unfolding towards fibrils; (iii) the presence of graphitic surfaces, which may alter the aggregation mechanism of β2-m through competitive adsorption. In the present PhD thesis we investigate the behavior of β2-m and its variants in relation to the aforementioned triggering factors for fibrillogenesis, using state-of-the-art computer simulations. (i) Docking, molecular dynamics and replica exchange methods are employed to study the mechanisms underlying dimer stabilization in the case of the β2-m wild type and its amyloidogenic variants D76N and ΔN6. Our analyses on the number and the strength of the intermolecular salt bridges involved in dimer interface reveal a correlation with the degree of amyloidogenicity of each individual species. (ii) Metadynamics, a powerful enhanced sampling method to accelerate rare events, is used to evaluate the proline cis-to-trans isomerization free energy landscape of the native β2-m and D76N variant. Simulations describe how the isomerization of P32 changes the protein conformation and disrupts fundamental hydrogen bond networks. (iii) The effects of graphitic surfaces on the misfolding of native β2-m and D76N variant are studied by means of replica exchange, complementing in vitro experiments. Our simulations show that adsorption of amyloidogenic proteins on graphite is fast and accompanied by a partial unfolding, consistently with previous experimental findings. The results obtained in this PhD thesis contribute to the fundamental understanding of the amyloidogenic β2-m behavior by helping to unravel the variety of triggering factors contributing to the fibrillogenical pathways.