Transthyretin (TTR) is an extracellular protein able to deposit into well-defined protein aggregates called amyloid, in pathological conditions known as senile systemic amyloidosis, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and leptomeningeal amyloidosis. At least three distinct partially folded states have been described for TTR, including the widely studied amyloidogenic state at mildly acidic pH. In this study, I have used fluorescence resonance energy transfer (FRET) experiments in a monomeric variant of TTR (M-TTR) and in its W41F and W79F mutants, taking advantage of the presence of a unique, solvent-exposed, cysteine residue at position 10, that I have labelled with a coumarin derivative (DACM, acceptor), and of the two natural tryptophan residues at positions 41 and 79 (donors). Trp41 is located in an ideal position as it is one of the residues of -strand C, whose degree of unfolding is debated. I found that the amyloidogenic state at low pH has the same FRET efficiency as the folded state at neutral pH in both M-TTR and W79F-M-TTR, indicating an unmodified Cys10-Trp41 distance. The partially folded state populated at low denaturant concentrations also has a similar FRET efficiency, but other spectroscopic probes indicate that it is distinct from the amyloidogenic state at acidic pH. By contrast, the off-pathway state accumulating transiently during refolding has a higher FRET efficiency, indicating nonnative interactions that reduce the Cys10-Trp41 spatial distance, revealing a third distinct conformational state. Overall, these results clarify a negligible degree of unfolding of -strand C in the formation of the amyloidogenic state and establish the concept that TTR is a highly plastic protein able to populate at least three distinct conformational states. TTR is also a protein able to inhibit amyloid sibril formation of A, the peptide associate with alzheimer diseases, and to suppres the toxcicity of performed oligomers of Aβ. Pervious analyses of the in vitro interaction between human TTR and Aβ, have shown that TTR binds to all forms of Aβ: monomers, oligomers and fibrils. The binding occurs with higher affinity for A oligomers, aggregates and fibrils with respect to A monomers. Previous data do not offer any insight into the mechanism by which TTR inhibits A amyloid fibril formation, oligomer toxicity and on the TTR form responsible for such an effect. In this thesis, the interaction between different variants of TTR (WT-TTR, M-TTR and W79F M-TTR) and the A peptide in both the process of aggregation, as well as on pre-formed toxic oligomers called amyloid-derived diffusible ligands (ADDLs), was studied by FRET technique. The time course of amyloid fibril formation was studied under conditions close to physiological and starting from a monomeric state of the A40 peptide, in the presence of WT-TTR, M-TTR and W97F M-TTR at different concentrations. This study showed inhibition of aggregation of Aβ40 by transthyretin molecules. I also studied the conformational change of all three TTR variants following the interaction with Aβ40 during aggregation at a molar ratio of 1:3 (TTR:Aβ40). In particular, the FRET E of TTRs during aggregation of Aβ40 was monitored and the results showed a change in FRET E of all TTR molecules as Aβ40 aggregation proceeds, indicating a conformational conversion upon Aβ40/TTR interaction. The far-UV CD spectra of WT-TTR, M-TTR and W79F M-TTR in the absence and presence of Aβ40 undergoing aggregation were compared, showing that there are no changes in the secondary structure of TTRs after binding to Aβ40 while it converts from monomers to oligomers. The fluorescence spectra recorded for both M-TTR and DACM-M-TTR in the presence of A42 ADDLs decreased in intensity with time, indicating a progressive interaction with the ADDLs. The FRET E value of M-TTR measured during incubation with A42 ADDLs was found to decrease progressively, indicating that a conformational change occurs for M-TTR following the interaction with A42 ADDLs. This analysis was repeated for the W79F mutant of M-TTR leading to very similar results. This confirms that a conformational change occurs also for W79F M-TTR following the interaction with A42 ADDLs and indicates an increased spatial distance between the DACM moiety attached to Cys10 and the two tryptophan residues, particularly Trp41.

Study of the conformational changes occurring in human transthyretin that are necessary for amyloid fibril formation in disease and for its role as a detoxifier / Seyyed Abolghasem Ghadami. - (2017).

Study of the conformational changes occurring in human transthyretin that are necessary for amyloid fibril formation in disease and for its role as a detoxifier.

GHADAMI, SEYYED ABOLGHASEM
2017

Abstract

Transthyretin (TTR) is an extracellular protein able to deposit into well-defined protein aggregates called amyloid, in pathological conditions known as senile systemic amyloidosis, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and leptomeningeal amyloidosis. At least three distinct partially folded states have been described for TTR, including the widely studied amyloidogenic state at mildly acidic pH. In this study, I have used fluorescence resonance energy transfer (FRET) experiments in a monomeric variant of TTR (M-TTR) and in its W41F and W79F mutants, taking advantage of the presence of a unique, solvent-exposed, cysteine residue at position 10, that I have labelled with a coumarin derivative (DACM, acceptor), and of the two natural tryptophan residues at positions 41 and 79 (donors). Trp41 is located in an ideal position as it is one of the residues of -strand C, whose degree of unfolding is debated. I found that the amyloidogenic state at low pH has the same FRET efficiency as the folded state at neutral pH in both M-TTR and W79F-M-TTR, indicating an unmodified Cys10-Trp41 distance. The partially folded state populated at low denaturant concentrations also has a similar FRET efficiency, but other spectroscopic probes indicate that it is distinct from the amyloidogenic state at acidic pH. By contrast, the off-pathway state accumulating transiently during refolding has a higher FRET efficiency, indicating nonnative interactions that reduce the Cys10-Trp41 spatial distance, revealing a third distinct conformational state. Overall, these results clarify a negligible degree of unfolding of -strand C in the formation of the amyloidogenic state and establish the concept that TTR is a highly plastic protein able to populate at least three distinct conformational states. TTR is also a protein able to inhibit amyloid sibril formation of A, the peptide associate with alzheimer diseases, and to suppres the toxcicity of performed oligomers of Aβ. Pervious analyses of the in vitro interaction between human TTR and Aβ, have shown that TTR binds to all forms of Aβ: monomers, oligomers and fibrils. The binding occurs with higher affinity for A oligomers, aggregates and fibrils with respect to A monomers. Previous data do not offer any insight into the mechanism by which TTR inhibits A amyloid fibril formation, oligomer toxicity and on the TTR form responsible for such an effect. In this thesis, the interaction between different variants of TTR (WT-TTR, M-TTR and W79F M-TTR) and the A peptide in both the process of aggregation, as well as on pre-formed toxic oligomers called amyloid-derived diffusible ligands (ADDLs), was studied by FRET technique. The time course of amyloid fibril formation was studied under conditions close to physiological and starting from a monomeric state of the A40 peptide, in the presence of WT-TTR, M-TTR and W97F M-TTR at different concentrations. This study showed inhibition of aggregation of Aβ40 by transthyretin molecules. I also studied the conformational change of all three TTR variants following the interaction with Aβ40 during aggregation at a molar ratio of 1:3 (TTR:Aβ40). In particular, the FRET E of TTRs during aggregation of Aβ40 was monitored and the results showed a change in FRET E of all TTR molecules as Aβ40 aggregation proceeds, indicating a conformational conversion upon Aβ40/TTR interaction. The far-UV CD spectra of WT-TTR, M-TTR and W79F M-TTR in the absence and presence of Aβ40 undergoing aggregation were compared, showing that there are no changes in the secondary structure of TTRs after binding to Aβ40 while it converts from monomers to oligomers. The fluorescence spectra recorded for both M-TTR and DACM-M-TTR in the presence of A42 ADDLs decreased in intensity with time, indicating a progressive interaction with the ADDLs. The FRET E value of M-TTR measured during incubation with A42 ADDLs was found to decrease progressively, indicating that a conformational change occurs for M-TTR following the interaction with A42 ADDLs. This analysis was repeated for the W79F mutant of M-TTR leading to very similar results. This confirms that a conformational change occurs also for W79F M-TTR following the interaction with A42 ADDLs and indicates an increased spatial distance between the DACM moiety attached to Cys10 and the two tryptophan residues, particularly Trp41.
2017
Fabrizio Chiti
Seyyed Abolghasem Ghadami
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/1076898
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