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A New Folding Kinetic Mechanism for Human Transthyretin and the Influence of the Amyloidogenic V30M Mutation

View Article: PubMed Central - PubMed

ABSTRACT

Protein aggregation into insoluble amyloid fibrils is the hallmark of several neurodegenerative diseases, chief among them Alzheimer’s and Parkinson’s. Although caused by different proteins, these pathologies share some basic molecular mechanisms with familial amyloidotic polyneuropathy (FAP), a rare hereditary neuropathy caused by amyloid formation and deposition by transthyretin (TTR) in the peripheral and autonomic nervous systems. Among the amyloidogenic TTR mutations known, V30M-TTR is the most common in FAP. TTR amyloidogenesis (ATTR) is triggered by tetramer dissociation, followed by partial unfolding and aggregation of the low conformational stability monomers formed. Thus, tetramer dissociation kinetics, monomer conformational stability and competition between refolding and aggregation pathways do play a critical role in ATTR. Here, we propose a new model to analyze the refolding kinetics of WT-TTR and V30M-TTR, showing that at pH and protein concentrations close to physiological, a two-step mechanism with a unimolecular first step followed by a second-order second step adjusts well to the experimental data. Interestingly, although sharing the same kinetic mechanism, V30M-TTR refolds at a much slower rate than WT-TTR, a feature that may favor the formation of transient species leading to kinetic partition into amyloidogenic pathways and, thus, significantly increasing the probability of amyloid formation in vivo.

No MeSH data available.


Simulation of molar fractions of TTR protein species. The three protein species (U, unfolded monomer (dashed lines); I, intermediate (solid lines); T, tetramer (dotted lines)) participate in the two-step refolding process (U → I → T) of TTR over time, as predicted by the rate constants (k1 and k2) obtained by extrapolation to 0.0 M urea (Equation (7); Table 1). Although the initial rate of conversion of urea-denatured TTR monomers into the corresponding intermediate is fast for both proteins, while unfolded monomers of WT-TTR are completely consumed within less than 5 min, the same process takes longer than 15 min for V30M-TTR. Additionally, whereas in the case of WT-TTR, the intermediate is the major species between 45 s and 4.6 min of reaction time, in the case of V30M-TTR, this occurs between 2.7 and 19.6 min.
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ijms-17-01428-f007: Simulation of molar fractions of TTR protein species. The three protein species (U, unfolded monomer (dashed lines); I, intermediate (solid lines); T, tetramer (dotted lines)) participate in the two-step refolding process (U → I → T) of TTR over time, as predicted by the rate constants (k1 and k2) obtained by extrapolation to 0.0 M urea (Equation (7); Table 1). Although the initial rate of conversion of urea-denatured TTR monomers into the corresponding intermediate is fast for both proteins, while unfolded monomers of WT-TTR are completely consumed within less than 5 min, the same process takes longer than 15 min for V30M-TTR. Additionally, whereas in the case of WT-TTR, the intermediate is the major species between 45 s and 4.6 min of reaction time, in the case of V30M-TTR, this occurs between 2.7 and 19.6 min.

Mentions: Results indicate that the refolding mechanism that better fits our experimental data, as concerns WT- and V30M-TTR, involves two steps and the presence of an intermediate. The data also show that the amyloidogenic variant V30M-TTR refolds about five-times slower than WT-TTR (Table 1). Thus, the refolding process from unfolded monomers to the corresponding native homotetramer is kinetically more favorable for WT-TTR than for V30M-TTR. The rate constants emerging from the best fits were used to simulate the molar fractions of all protein species (U, I and T) over time, extrapolated to a 0.0 M urea concentration, for WT- and V30M-TTR (Figure 7). Analysis of Figure 7 shows that the refolding process of WT-TTR occurs within a 2 h period, whereas for the amyloidogenic variant V30M-TTR (Figure 7), the refolding process is only complete after more than 10 h. More importantly, the time the intermediate species persist in solution is significantly longer for V30M-TTR. While for WT-TTR, the intermediate appears and is reduced to less than 10% in less than 30 min, for V30M-TTR after 2 h, the intermediate is still above 10% (Figure 7).


A New Folding Kinetic Mechanism for Human Transthyretin and the Influence of the Amyloidogenic V30M Mutation
Simulation of molar fractions of TTR protein species. The three protein species (U, unfolded monomer (dashed lines); I, intermediate (solid lines); T, tetramer (dotted lines)) participate in the two-step refolding process (U → I → T) of TTR over time, as predicted by the rate constants (k1 and k2) obtained by extrapolation to 0.0 M urea (Equation (7); Table 1). Although the initial rate of conversion of urea-denatured TTR monomers into the corresponding intermediate is fast for both proteins, while unfolded monomers of WT-TTR are completely consumed within less than 5 min, the same process takes longer than 15 min for V30M-TTR. Additionally, whereas in the case of WT-TTR, the intermediate is the major species between 45 s and 4.6 min of reaction time, in the case of V30M-TTR, this occurs between 2.7 and 19.6 min.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037707&req=5

ijms-17-01428-f007: Simulation of molar fractions of TTR protein species. The three protein species (U, unfolded monomer (dashed lines); I, intermediate (solid lines); T, tetramer (dotted lines)) participate in the two-step refolding process (U → I → T) of TTR over time, as predicted by the rate constants (k1 and k2) obtained by extrapolation to 0.0 M urea (Equation (7); Table 1). Although the initial rate of conversion of urea-denatured TTR monomers into the corresponding intermediate is fast for both proteins, while unfolded monomers of WT-TTR are completely consumed within less than 5 min, the same process takes longer than 15 min for V30M-TTR. Additionally, whereas in the case of WT-TTR, the intermediate is the major species between 45 s and 4.6 min of reaction time, in the case of V30M-TTR, this occurs between 2.7 and 19.6 min.
Mentions: Results indicate that the refolding mechanism that better fits our experimental data, as concerns WT- and V30M-TTR, involves two steps and the presence of an intermediate. The data also show that the amyloidogenic variant V30M-TTR refolds about five-times slower than WT-TTR (Table 1). Thus, the refolding process from unfolded monomers to the corresponding native homotetramer is kinetically more favorable for WT-TTR than for V30M-TTR. The rate constants emerging from the best fits were used to simulate the molar fractions of all protein species (U, I and T) over time, extrapolated to a 0.0 M urea concentration, for WT- and V30M-TTR (Figure 7). Analysis of Figure 7 shows that the refolding process of WT-TTR occurs within a 2 h period, whereas for the amyloidogenic variant V30M-TTR (Figure 7), the refolding process is only complete after more than 10 h. More importantly, the time the intermediate species persist in solution is significantly longer for V30M-TTR. While for WT-TTR, the intermediate appears and is reduced to less than 10% in less than 30 min, for V30M-TTR after 2 h, the intermediate is still above 10% (Figure 7).

View Article: PubMed Central - PubMed

ABSTRACT

Protein aggregation into insoluble amyloid fibrils is the hallmark of several neurodegenerative diseases, chief among them Alzheimer’s and Parkinson’s. Although caused by different proteins, these pathologies share some basic molecular mechanisms with familial amyloidotic polyneuropathy (FAP), a rare hereditary neuropathy caused by amyloid formation and deposition by transthyretin (TTR) in the peripheral and autonomic nervous systems. Among the amyloidogenic TTR mutations known, V30M-TTR is the most common in FAP. TTR amyloidogenesis (ATTR) is triggered by tetramer dissociation, followed by partial unfolding and aggregation of the low conformational stability monomers formed. Thus, tetramer dissociation kinetics, monomer conformational stability and competition between refolding and aggregation pathways do play a critical role in ATTR. Here, we propose a new model to analyze the refolding kinetics of WT-TTR and V30M-TTR, showing that at pH and protein concentrations close to physiological, a two-step mechanism with a unimolecular first step followed by a second-order second step adjusts well to the experimental data. Interestingly, although sharing the same kinetic mechanism, V30M-TTR refolds at a much slower rate than WT-TTR, a feature that may favor the formation of transient species leading to kinetic partition into amyloidogenic pathways and, thus, significantly increasing the probability of amyloid formation in vivo.

No MeSH data available.