Amyloidoses are diseases caused by the misfolding of proteins and their aggregation into amyloid fibrils. In AL amyloidosis, the proteins responsible are the light chains of immunoglobulins and their deposition can affect various organs causing anatomical and functional damage. The mechanisms underlying the formation of fibrils by light chains are still poorly understood, and fragments derived from the proteolytic digestion of light chains could have a role in initiating the process at the tissue level. Understanding the aggregation mechanisms and developing an in vitro model of fibrillogenesis in physiological conditions are two fundamental steps to be able to develop targeted therapies as well as a platform for in vitro evaluation of novel pharmaceutical molecules. This thesis project was conducted at the Biochemistry Laboratory of the Department of Molecular Medicine. Starting from data previously acquired by the team on the biochemical characteristics of amyloid deposits in patients, I worked on the production of fragments of natural amyloidogenic antibody light chains (consistent with those present in vivo) and on the characterization of their amyloidogenic potential. For this purpose, I employed a combination of molecular biology techniques regarding heterologous expression of recombinant proteins, their purification by chromatography and their subsequent characterization by gel electrophoresis and circular dichroism. Four different length proteoforms of a IGLV6-57 germline light chain were produced. The kinetics of fibrillogenesis were monitored with specific probes for amyloid conformation and the results confirmed with microscopy. The aggregation kinetics were evaluated both for the proteoforms taken individually and in mixture. We confirmed the importance of the presence of proteolyzed forms (already found in ex vivo samples) in the molecular mechanisms associated with the aggregation of the protein in question.
Le amiloidosi sono patologie causate dal misfolding di proteine e dalla loro aggregazione in fibrille amiloidi. Nell'amiloidosi AL, le proteine responsabili sono le catene leggere delle immunoglobuline e la deposizione può interessare diversi organi causandone un danno anatomico-funzionale. I meccanismi alla base della formazione di fibrille da parte di catene leggere sono ancora poco noti, e frammenti derivati dalla digestione proteolitica di catene leggere potrebbero avere un ruolo nell'iniziare il processo a livello tissutale. Capire i meccanismi di aggregazione e sviluppare un modello in vitro di fibrillogenesi in condizioni fisiologiche sono due passaggi fondamentali per poter sviluppare terapie mirate e disporre di una piattaforma per la valutazione di nuovi farmaci in vitro. Questo progetto di tesi è stato svolto presso il Laboratorio di Biochimica del Dipartimento di Medicina Molecolare. Partendo da informazioni precedentemente acquisite dal gruppo sulle caratteristiche biochimiche dei depositi amiloidi nei pazienti ho lavorato alla produzione di frammenti di catene leggere amiloidogeniche naturali (consistenti con quelli presenti in vivo) e alla caratterizzazione del loro potenziale amiloidogenico. A tale scopo ho utilizzato una combinazione di approcci di biologia molecolare per l'espressione eterologa di proteine ricombinanti, purificazione con tecniche cromatografiche, caratterizzazione con elettroforesi su gel e dicroismo circolare. Sono state prodotte quatto proteoforme di una catena leggera di germline IGLV6-57 di diversa lunghezza. Le cinetiche di fibrillogenesi sono state studiate mediante l'uso di sonde specifiche per la conformazione amiloide e tecniche di microscopia. La cinetica di aggregazione è stata valutata sia analizzando le proteoforme prese singolarmente che in miscela. Abbiamo confermato l'importanza della presenza di forme proteolizzate, già riscontrate in campioni ex vivo, nei meccanismi molecolari associati all'aggregazione della proteina in questione.
Creazione e caratterizzazione di un nuovo modello di amiloidogenesi in vitro di catene leggere immunoglobuliniche
LAMPIS, ALESSIO
2021/2022
Abstract
Amyloidoses are diseases caused by the misfolding of proteins and their aggregation into amyloid fibrils. In AL amyloidosis, the proteins responsible are the light chains of immunoglobulins and their deposition can affect various organs causing anatomical and functional damage. The mechanisms underlying the formation of fibrils by light chains are still poorly understood, and fragments derived from the proteolytic digestion of light chains could have a role in initiating the process at the tissue level. Understanding the aggregation mechanisms and developing an in vitro model of fibrillogenesis in physiological conditions are two fundamental steps to be able to develop targeted therapies as well as a platform for in vitro evaluation of novel pharmaceutical molecules. This thesis project was conducted at the Biochemistry Laboratory of the Department of Molecular Medicine. Starting from data previously acquired by the team on the biochemical characteristics of amyloid deposits in patients, I worked on the production of fragments of natural amyloidogenic antibody light chains (consistent with those present in vivo) and on the characterization of their amyloidogenic potential. For this purpose, I employed a combination of molecular biology techniques regarding heterologous expression of recombinant proteins, their purification by chromatography and their subsequent characterization by gel electrophoresis and circular dichroism. Four different length proteoforms of a IGLV6-57 germline light chain were produced. The kinetics of fibrillogenesis were monitored with specific probes for amyloid conformation and the results confirmed with microscopy. The aggregation kinetics were evaluated both for the proteoforms taken individually and in mixture. We confirmed the importance of the presence of proteolyzed forms (already found in ex vivo samples) in the molecular mechanisms associated with the aggregation of the protein in question.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/15246