Optical tweezers have evolved as an exemplary Single Molecule Force Spectroscopy (SMFS) technique over the past three decades. A distinct and bio medically relevant application of Optical Tweezers is their ability to observe directly at single molecule level the folding, misfolding and aggregation of protein molecules. Additionally the dynamic approach of Optical Tweezer setup also allows for the isolated study of interactions between two or more biomolecules, such as chaperone-protein interactions, in real time. The medical relevance of such studies stems from the fact that misfolding and aggregation of proteins are deleterious processes and have been linked to many neurodegenerative disorders. While molecular chaperones have evolved as an evolutionarily conserved sword and shield mechanism against such deleterious processes, wherein their holdase action acts as a shield preventing further aggregation of misfolded protein species and their foldase action acts as a sword and actively assists misfolded structure to regains their natively folded state. The dysfunction of this chaperone activity is also cytotoxic and can lead to loss of proteostasis. The present thesis dwells deeper in this specific application of Optical tweezer. The thesis will elaborate upon how optical tweezers can extract the mechanistic details of the folding and misfolding of protein molecules by reviewing the experiments performed on NCS-1 (Neuronal Calcium Sensor 1). It will also discuss the experimental approach taken by SMFS techniques like Optical Tweezers and AFM (Atomic Force Microscopy) to study the structural and functional dynamics of molecular chaperones. Furthermore, the thesis will explore the recent developments in Optical Tweezers and their biological applications. Finally, I describe the results of experiments we have carried out on the maltose binding protein to elucidate the mechanism of action of the chaperone HSPB8. We have mechanically denatured homotetramers of MBP as well as single MBP molecules and analyzed their folding and aggregation processes in the presence and absence of wild-type HSPB8 and its mutant form HSPB8-K141E/N. Our results reveal a strong holdase activity of wild type HSPB8, which either prevents completely the aggregation of denatured MBP molecules or allows the substrate to form only small and mechanically weak aggregates while this holdase activity is significantly suppressed in the mutant. Moreover, and importantly, a careful analysis of the data also discloses an unexpected foldase activity of both wild type and mutated forms of HSPB8, which guides the folding process of denatured MBP molecules into their native states. Our findings highlight new mechanisms of interaction between HSPB8 and its substrates and suggest a more complex physiological role for this chaperone than previously assumed.

Negli ultimi decenni le pinze ottiche si sono rivelate una tecnica sperimentale estremamente efficace per eseguire studi di spettroscopia di forza a livello di singola molecola. In particolare, un’applicazione delle pinze ottiche che sta avendo una rilevanza biomedica sempre più importante è quella relativa allo studio dei processi di ripiegamento corretto (folding), non corretto (misfolding) e dell’aggregazione di proteine. Di forte rilevanza biomedica è anche la possibilità offerta dalle pinze ottiche di caratterizzare in grande dettaglio i meccanismi molecolari che mediano le interazioni tra due o più biomolecole, come ad esempio tra uno chaperone molecolare e il suo substrato. La rilevanza medica di questi studi deriva dal fatto che l'errato ripiegamento e l'aggregazione delle proteine sono processi deleteri, spesso associati a neurodegenerazione. Gli chaperoni molecolari si sono evoluti come strumento molecolare per combattere sia il misfolding che l’aggregazione proteica. Un funzionamento non corretto degli chaperoni molecolari spesso causa perdita di proteostasi e l’insorgenza di varie patologie umane. Il lavoro descritto in questa tesi spiega in maniera dettagliata l’approccio sperimentale utilizzato per utilizzare le pinze ottiche per lo studio del folding, misfolding e aggregazione di proteine. In particolare in questa tesi vengono descritti: i) i risultati di esperimenti mirati alla elucidazione del processo di ripiegamento corretto e non del sensore al calcio NCS-1 (Neuronal Calcium Sensor 1; ii) l'approccio sperimentale adottato per descrivere la dinamica strutturale e funzionale di vari chaperoni molecolari utilizzando le pinze ottiche e la microscopia a forza atomica; iii) recenti sviluppi tecnici che hanno ampliato le possibili applicazioni delle pinze ottiche in campo biologico; iv) i risultati di esperimenti mirati a far luce sui meccanismi molecolari che mediano l’attività chaperonica dello chaperone molecolare HSPB8. In quest’ultimi esperimenti abbiamo manipolato meccanicamente monomeri e tetrameri della Maltose Binding Protein (MBP) e analizzato i loro processi di folding, misfolding e aggregazione in presenza e assenza del HSPB8 wild-type e del suo mutante HSPB8-K141E. I nostri risultati dimostrano una forte attività antiaggregante (holdase activity) della HSPB8 che riduce significativamente l'aggregazione delle molecole di MBP e un’attività antiaggregante molto ridotta del mutante HSPB8-K141E. Inoltre, i nostri studi rivelano una inaspettata attività pro-folding (foldase activity) sia della forma mutata che di quella wild-type della HSPB8. Questi dati sperimentali evidenziano nuovi meccanismi di interazione tra HSPB8 e i suoi substrati e suggeriscono un ruolo fisiologico più complesso per questo chaperone molecolare di quanto precedentemente ipotizzato.

Studio a livello di singola molecola del folding, misfolding e aggregazione di proteine e dell’attività chaperonica della HSPB8 / Dhawal Choudhary , 2020 Jan 13. 32. ciclo, Anno Accademico 2018/2019.

Studio a livello di singola molecola del folding, misfolding e aggregazione di proteine e dell’attività chaperonica della HSPB8

CHOUDHARY, DHAWAL
2020

Abstract

Optical tweezers have evolved as an exemplary Single Molecule Force Spectroscopy (SMFS) technique over the past three decades. A distinct and bio medically relevant application of Optical Tweezers is their ability to observe directly at single molecule level the folding, misfolding and aggregation of protein molecules. Additionally the dynamic approach of Optical Tweezer setup also allows for the isolated study of interactions between two or more biomolecules, such as chaperone-protein interactions, in real time. The medical relevance of such studies stems from the fact that misfolding and aggregation of proteins are deleterious processes and have been linked to many neurodegenerative disorders. While molecular chaperones have evolved as an evolutionarily conserved sword and shield mechanism against such deleterious processes, wherein their holdase action acts as a shield preventing further aggregation of misfolded protein species and their foldase action acts as a sword and actively assists misfolded structure to regains their natively folded state. The dysfunction of this chaperone activity is also cytotoxic and can lead to loss of proteostasis. The present thesis dwells deeper in this specific application of Optical tweezer. The thesis will elaborate upon how optical tweezers can extract the mechanistic details of the folding and misfolding of protein molecules by reviewing the experiments performed on NCS-1 (Neuronal Calcium Sensor 1). It will also discuss the experimental approach taken by SMFS techniques like Optical Tweezers and AFM (Atomic Force Microscopy) to study the structural and functional dynamics of molecular chaperones. Furthermore, the thesis will explore the recent developments in Optical Tweezers and their biological applications. Finally, I describe the results of experiments we have carried out on the maltose binding protein to elucidate the mechanism of action of the chaperone HSPB8. We have mechanically denatured homotetramers of MBP as well as single MBP molecules and analyzed their folding and aggregation processes in the presence and absence of wild-type HSPB8 and its mutant form HSPB8-K141E/N. Our results reveal a strong holdase activity of wild type HSPB8, which either prevents completely the aggregation of denatured MBP molecules or allows the substrate to form only small and mechanically weak aggregates while this holdase activity is significantly suppressed in the mutant. Moreover, and importantly, a careful analysis of the data also discloses an unexpected foldase activity of both wild type and mutated forms of HSPB8, which guides the folding process of denatured MBP molecules into their native states. Our findings highlight new mechanisms of interaction between HSPB8 and its substrates and suggest a more complex physiological role for this chaperone than previously assumed.
Single molecule studies of folding, misfolding and aggregation of proteins and chaperone activity of HSPB8
13-gen-2020
CECCONI, CIRO
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/1199862
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