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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.


Characterization of the TTR refolded species. (A,C) Size-exclusion chromatograms of WT-TTR (A) and V30M-TTR (C) after complete protein refolding. The size-exclusion chromatography (SEC) experiments were run at a flow rate of 0.4 mL/min, in 20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.0, at 25 °C; (B,D) Binding isotherm curves of thyroxine to refolded WT-TTR (B) and to refolded V30M-TTR (D) monitored by the variation of the intrinsic protein fluorescence emission at 350 nm. Protein concentrations were 1.0 μM in 20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.0. Graph insets show the emission spectra of the protein samples in the presence of increasing concentrations of thyroxine.
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ijms-17-01428-f003: Characterization of the TTR refolded species. (A,C) Size-exclusion chromatograms of WT-TTR (A) and V30M-TTR (C) after complete protein refolding. The size-exclusion chromatography (SEC) experiments were run at a flow rate of 0.4 mL/min, in 20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.0, at 25 °C; (B,D) Binding isotherm curves of thyroxine to refolded WT-TTR (B) and to refolded V30M-TTR (D) monitored by the variation of the intrinsic protein fluorescence emission at 350 nm. Protein concentrations were 1.0 μM in 20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.0. Graph insets show the emission spectra of the protein samples in the presence of increasing concentrations of thyroxine.

Mentions: In addition to fluorescence and CD, refolded species of WT- and V30M-TTR were characterized by size-exclusion chromatography (SEC) (Figure 3A,C) and thyroxine binding assays (Figure 3B,D). SEC chromatograms show the presence of one major peak (>95%), with an elution volume of approximately 22 mL and an apparent molecular mass of 55 kDa, clearly demonstrating the tetrameric nature of the refolded species of WT- and V30M-TTR. The elution volume of these refolded species is exactly the same as the one observed for native tetrameric TTR (Figure S1). Furthermore, the absence in the chromatogram of other molecular species, in any significant amount, indicates that both TTR variants refold to the tetrameric form with a very high yield. Thyroxine binding assays were performed to assess if the refolded tetramers of WT- and V30M-TTR displayed native-like binding properties. Figure 3B,D shows the isothermal binding curves of thyroxine to WT- and V30M-TTR after the refolding reaction, obtained by plotting the variation of TTR intrinsic fluorescence intensity as a function of thyroxine concentration. The fluorescence spectra of the refolded tetramers of WT- and V30M-TTR (inset graphics in Figure 3B,D) show quenching in fluorescence upon thyroxine addition. This decrease in fluorescence intensity is most likely due to the deactivation of the TTR fluorophores’ excited state by the iodine atoms of thyroxine. Several synthetic TTR ligands lacking halogen atoms do not quench the TTR fluorescence upon binding, which evidences the role of the iodine atoms in the mechanism of fluorescence quenching [34]. Because no shifts in the fluorescence emission maxima are observed, no major conformational changes are expected to occur in TTR upon thyroxine binding, which is also evident from the analysis of the crystal structures of TTR in the absence and presence of thyroxine [43].


A New Folding Kinetic Mechanism for Human Transthyretin and the Influence of the Amyloidogenic V30M Mutation
Characterization of the TTR refolded species. (A,C) Size-exclusion chromatograms of WT-TTR (A) and V30M-TTR (C) after complete protein refolding. The size-exclusion chromatography (SEC) experiments were run at a flow rate of 0.4 mL/min, in 20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.0, at 25 °C; (B,D) Binding isotherm curves of thyroxine to refolded WT-TTR (B) and to refolded V30M-TTR (D) monitored by the variation of the intrinsic protein fluorescence emission at 350 nm. Protein concentrations were 1.0 μM in 20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.0. Graph insets show the emission spectra of the protein samples in the presence of increasing concentrations of thyroxine.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-17-01428-f003: Characterization of the TTR refolded species. (A,C) Size-exclusion chromatograms of WT-TTR (A) and V30M-TTR (C) after complete protein refolding. The size-exclusion chromatography (SEC) experiments were run at a flow rate of 0.4 mL/min, in 20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.0, at 25 °C; (B,D) Binding isotherm curves of thyroxine to refolded WT-TTR (B) and to refolded V30M-TTR (D) monitored by the variation of the intrinsic protein fluorescence emission at 350 nm. Protein concentrations were 1.0 μM in 20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.0. Graph insets show the emission spectra of the protein samples in the presence of increasing concentrations of thyroxine.
Mentions: In addition to fluorescence and CD, refolded species of WT- and V30M-TTR were characterized by size-exclusion chromatography (SEC) (Figure 3A,C) and thyroxine binding assays (Figure 3B,D). SEC chromatograms show the presence of one major peak (>95%), with an elution volume of approximately 22 mL and an apparent molecular mass of 55 kDa, clearly demonstrating the tetrameric nature of the refolded species of WT- and V30M-TTR. The elution volume of these refolded species is exactly the same as the one observed for native tetrameric TTR (Figure S1). Furthermore, the absence in the chromatogram of other molecular species, in any significant amount, indicates that both TTR variants refold to the tetrameric form with a very high yield. Thyroxine binding assays were performed to assess if the refolded tetramers of WT- and V30M-TTR displayed native-like binding properties. Figure 3B,D shows the isothermal binding curves of thyroxine to WT- and V30M-TTR after the refolding reaction, obtained by plotting the variation of TTR intrinsic fluorescence intensity as a function of thyroxine concentration. The fluorescence spectra of the refolded tetramers of WT- and V30M-TTR (inset graphics in Figure 3B,D) show quenching in fluorescence upon thyroxine addition. This decrease in fluorescence intensity is most likely due to the deactivation of the TTR fluorophores’ excited state by the iodine atoms of thyroxine. Several synthetic TTR ligands lacking halogen atoms do not quench the TTR fluorescence upon binding, which evidences the role of the iodine atoms in the mechanism of fluorescence quenching [34]. Because no shifts in the fluorescence emission maxima are observed, no major conformational changes are expected to occur in TTR upon thyroxine binding, which is also evident from the analysis of the crystal structures of TTR in the absence and presence of thyroxine [43].

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.