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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.


Related in: MedlinePlus

Refolding kinetics of WT-TTR and V30M-TTR monitored by intrinsic fluorescence emission. Fluorescence intensity decays (upper panels) for WT- and V30M-TTR monitored at different urea concentrations, pH 7.0 and 25 °C. The best fitting curves (red lines) to the experimental data points were obtained using Equations (3) to (6). Refolding assays were performed at constant protein concentrations (1.0 µM). Intrinsic fluorescence was monitored at 380 nm with an excitation wavelength of 290 nm. Lower panels show weighted residuals.
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ijms-17-01428-f004: Refolding kinetics of WT-TTR and V30M-TTR monitored by intrinsic fluorescence emission. Fluorescence intensity decays (upper panels) for WT- and V30M-TTR monitored at different urea concentrations, pH 7.0 and 25 °C. The best fitting curves (red lines) to the experimental data points were obtained using Equations (3) to (6). Refolding assays were performed at constant protein concentrations (1.0 µM). Intrinsic fluorescence was monitored at 380 nm with an excitation wavelength of 290 nm. Lower panels show weighted residuals.

Mentions: TTR refolding was initiated by the dilution of solutions of unfolded TTR, containing 6.0 M urea, to the desired final urea and protein concentrations. Figure 4 shows two examples of fluorescence intensity decays obtained for WT- and V30M-TTR refolding. In order to fit the data, several kinetic models were tested, among them kinetic models based on a single-step (U → T) and on two-step (U → I → T) refolding mechanisms. Data were initially fit to kinetic models based on a single-step mechanism (simulating first-order and second-order reactions), but the fits were of poor quality and were discarded (Figure S3). Consequently, more complex mechanisms, based on a two-step model (U → I → T), were attempted with more success. Good fits to the data were obtained with a two-step mechanism with two consecutive first-order reactions, but the rate constants obtained showed dependence on protein concentration (Figure S4A), indicating that higher order reactions have to be considered. Additionally, although previous fluorescence experiments have led us to propose a two-step mechanism of two consecutive bimolecular second-order steps [44], taking into account the monomeric nature of the intermediate species identified by SEC and DGGE (see below), a two-step model (U → I → T) involving a first-order reaction followed by a second-order reaction was tried. Fits of the experimental data were very good, with reduced weighted residuals and with no protein concentration dependencies of the rate constants (Figure S4B).


A New Folding Kinetic Mechanism for Human Transthyretin and the Influence of the Amyloidogenic V30M Mutation
Refolding kinetics of WT-TTR and V30M-TTR monitored by intrinsic fluorescence emission. Fluorescence intensity decays (upper panels) for WT- and V30M-TTR monitored at different urea concentrations, pH 7.0 and 25 °C. The best fitting curves (red lines) to the experimental data points were obtained using Equations (3) to (6). Refolding assays were performed at constant protein concentrations (1.0 µM). Intrinsic fluorescence was monitored at 380 nm with an excitation wavelength of 290 nm. Lower panels show weighted residuals.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037707&req=5

ijms-17-01428-f004: Refolding kinetics of WT-TTR and V30M-TTR monitored by intrinsic fluorescence emission. Fluorescence intensity decays (upper panels) for WT- and V30M-TTR monitored at different urea concentrations, pH 7.0 and 25 °C. The best fitting curves (red lines) to the experimental data points were obtained using Equations (3) to (6). Refolding assays were performed at constant protein concentrations (1.0 µM). Intrinsic fluorescence was monitored at 380 nm with an excitation wavelength of 290 nm. Lower panels show weighted residuals.
Mentions: TTR refolding was initiated by the dilution of solutions of unfolded TTR, containing 6.0 M urea, to the desired final urea and protein concentrations. Figure 4 shows two examples of fluorescence intensity decays obtained for WT- and V30M-TTR refolding. In order to fit the data, several kinetic models were tested, among them kinetic models based on a single-step (U → T) and on two-step (U → I → T) refolding mechanisms. Data were initially fit to kinetic models based on a single-step mechanism (simulating first-order and second-order reactions), but the fits were of poor quality and were discarded (Figure S3). Consequently, more complex mechanisms, based on a two-step model (U → I → T), were attempted with more success. Good fits to the data were obtained with a two-step mechanism with two consecutive first-order reactions, but the rate constants obtained showed dependence on protein concentration (Figure S4A), indicating that higher order reactions have to be considered. Additionally, although previous fluorescence experiments have led us to propose a two-step mechanism of two consecutive bimolecular second-order steps [44], taking into account the monomeric nature of the intermediate species identified by SEC and DGGE (see below), a two-step model (U → I → T) involving a first-order reaction followed by a second-order reaction was tried. Fits of the experimental data were very good, with reduced weighted residuals and with no protein concentration dependencies of the rate constants (Figure S4B).

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