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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.

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Related in: MedlinePlus

Urea dependence of the WT-TTR and V30M-TTR apparent refolding rate constants, measured by intrinsic fluorescence at 380 nm. Plots of the apparent refolding rate constants (kapp) of WT- and V30M-TTR against urea concentration, obtained at 1.0 µM protein concentration, pH 7.0 and 25 °C. The plotted rate constants and standard deviation (SD) error bars are an average of at least three independent experiments. Symbols (●) and (■) represent the values of kapp1 and kapp2 at each urea concentration, respectively. Dashed lines are the linear least-squares fits for k1 and k2 using Equation (7). * Although values for kapp1 at 0.4 M urea for V30M-TTR and 1.8 M urea for WT-TTR have also been determined, they were not taken into account for the extrapolation of k1, since at these concentrations of urea, the initial phase of the exponential decays was too fast to be measured accurately.
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ijms-17-01428-f006: Urea dependence of the WT-TTR and V30M-TTR apparent refolding rate constants, measured by intrinsic fluorescence at 380 nm. Plots of the apparent refolding rate constants (kapp) of WT- and V30M-TTR against urea concentration, obtained at 1.0 µM protein concentration, pH 7.0 and 25 °C. The plotted rate constants and standard deviation (SD) error bars are an average of at least three independent experiments. Symbols (●) and (■) represent the values of kapp1 and kapp2 at each urea concentration, respectively. Dashed lines are the linear least-squares fits for k1 and k2 using Equation (7). * Although values for kapp1 at 0.4 M urea for V30M-TTR and 1.8 M urea for WT-TTR have also been determined, they were not taken into account for the extrapolation of k1, since at these concentrations of urea, the initial phase of the exponential decays was too fast to be measured accurately.

Mentions: In order to obtain kinetic constants independent of protein concentration, a two-step kinetic mechanism based on a first-order reaction followed by a second-order reaction had to be postulated (Figure S4B). As shown in Figure 4, the weighted residuals of the fits, using equations representing such a mechanism, exhibit small dispersion, which indicates that the chosen kinetic model is appropriate to describe the refolding mechanism of the TTR variants under analysis. The apparent refolding rate constants for WT- and V30M-TTR, at different final urea concentrations, are shown in Table 1. The extrapolation of the experimentally-determined apparent rate constants (kapp) to conditions of absence of urea, allows the direct comparison of the folding rates of WT- and V30M-TTR. Plots of the apparent rate constants kapp1 and kapp2 as a function of urea concentration are shown in Figure 6. Each data point is an average of at least three independent measurements, and a linear least-square fit of Equation (7) to the data yields kinetic constants in the absence of urea (Table 1). Rate constants for V30M-TTR refolding in the absence of urea are lower than those for WT-TTR: k1 is four-fold lower, and k2 is five-fold smaller. In addition, there is no experimental evidence to suggest that the TTR refolding pathway may include rate-limiting processes, such as isomerization [45], because in general, these kinetic steps present weak or even no denaturant concentration dependence.


A New Folding Kinetic Mechanism for Human Transthyretin and the Influence of the Amyloidogenic V30M Mutation
Urea dependence of the WT-TTR and V30M-TTR apparent refolding rate constants, measured by intrinsic fluorescence at 380 nm. Plots of the apparent refolding rate constants (kapp) of WT- and V30M-TTR against urea concentration, obtained at 1.0 µM protein concentration, pH 7.0 and 25 °C. The plotted rate constants and standard deviation (SD) error bars are an average of at least three independent experiments. Symbols (●) and (■) represent the values of kapp1 and kapp2 at each urea concentration, respectively. Dashed lines are the linear least-squares fits for k1 and k2 using Equation (7). * Although values for kapp1 at 0.4 M urea for V30M-TTR and 1.8 M urea for WT-TTR have also been determined, they were not taken into account for the extrapolation of k1, since at these concentrations of urea, the initial phase of the exponential decays was too fast to be measured accurately.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-17-01428-f006: Urea dependence of the WT-TTR and V30M-TTR apparent refolding rate constants, measured by intrinsic fluorescence at 380 nm. Plots of the apparent refolding rate constants (kapp) of WT- and V30M-TTR against urea concentration, obtained at 1.0 µM protein concentration, pH 7.0 and 25 °C. The plotted rate constants and standard deviation (SD) error bars are an average of at least three independent experiments. Symbols (●) and (■) represent the values of kapp1 and kapp2 at each urea concentration, respectively. Dashed lines are the linear least-squares fits for k1 and k2 using Equation (7). * Although values for kapp1 at 0.4 M urea for V30M-TTR and 1.8 M urea for WT-TTR have also been determined, they were not taken into account for the extrapolation of k1, since at these concentrations of urea, the initial phase of the exponential decays was too fast to be measured accurately.
Mentions: In order to obtain kinetic constants independent of protein concentration, a two-step kinetic mechanism based on a first-order reaction followed by a second-order reaction had to be postulated (Figure S4B). As shown in Figure 4, the weighted residuals of the fits, using equations representing such a mechanism, exhibit small dispersion, which indicates that the chosen kinetic model is appropriate to describe the refolding mechanism of the TTR variants under analysis. The apparent refolding rate constants for WT- and V30M-TTR, at different final urea concentrations, are shown in Table 1. The extrapolation of the experimentally-determined apparent rate constants (kapp) to conditions of absence of urea, allows the direct comparison of the folding rates of WT- and V30M-TTR. Plots of the apparent rate constants kapp1 and kapp2 as a function of urea concentration are shown in Figure 6. Each data point is an average of at least three independent measurements, and a linear least-square fit of Equation (7) to the data yields kinetic constants in the absence of urea (Table 1). Rate constants for V30M-TTR refolding in the absence of urea are lower than those for WT-TTR: k1 is four-fold lower, and k2 is five-fold smaller. In addition, there is no experimental evidence to suggest that the TTR refolding pathway may include rate-limiting processes, such as isomerization [45], because in general, these kinetic steps present weak or even no denaturant concentration dependence.

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.


Related in: MedlinePlus