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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 intermediate species. Size-exclusion chromatograms of WT-TTR (A) and V30M-TTR (B) after dilution-induced protein refolding, in the presence of low concentrations of urea. 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, in the presence of 1.0 M and of 0.4 M urea, for WT- and V30M-TTR, respectively. Prior to SEC, protein samples were submitted to 2.0 M GdmSCN for 12 h and then dialyzed against the chromatography buffer. In both chromatograms, the two main peaks can be assigned to tetramers and monomers with apparent molecular weights differing by about four-fold.
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ijms-17-01428-f005: Characterization of the TTR intermediate species. Size-exclusion chromatograms of WT-TTR (A) and V30M-TTR (B) after dilution-induced protein refolding, in the presence of low concentrations of urea. 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, in the presence of 1.0 M and of 0.4 M urea, for WT- and V30M-TTR, respectively. Prior to SEC, protein samples were submitted to 2.0 M GdmSCN for 12 h and then dialyzed against the chromatography buffer. In both chromatograms, the two main peaks can be assigned to tetramers and monomers with apparent molecular weights differing by about four-fold.

Mentions: In order to investigate the nature of the intermediate species involved in the refolding process of TTR, we conducted SEC experiments, as shown in Figure 5. These experiments were run at a relatively low concentration of denaturant, favoring the occurrence and accumulation of intermediate species in solution upon partial refolding. SEC chromatograms (Figure 5) show only two main populations of protein species in solution at low denaturant concentration. Peaks at earlier elution times are attributed to the refolded forms of tetrameric WT- and V30M-TTR (apparent molecular weights of 50 to 60 kDa), whereas peaks with later elution times are consistent with apparent molecular weights of TTR monomers (11 to 16 kDa). Understandably, while the elution volume registered for tetrameric V30M-TTR remains unchanged (approximately 22 mL) and is equal to the volume observed when a sample of tetrameric WT-TTR is loaded onto the column (Figure S5), the elution volume for WT-TTR in the presence of 1.0 M urea is slightly larger (approximately 27 mL) due to the considerable increase in viscosity of the mobile phase, caused by the presence of urea in the equilibrating buffer (20 mM sodium phosphate buffer, 150 mM sodium chloride, 1.0 M urea, pH 7.0). Likewise, the elution volumes detected for the monomeric species are also slightly different in the two chromatograms, namely 32 mL for V30M-TTR (Figure 5B) and 35 mL for WT-TTR (Figure 5A). Additionally, the broader peaks observed for the monomeric species, in particular for V30M-TTR, may reflect the less compact nature of these molecular species and exchange between multiple monomeric conformations.


A New Folding Kinetic Mechanism for Human Transthyretin and the Influence of the Amyloidogenic V30M Mutation
Characterization of the TTR intermediate species. Size-exclusion chromatograms of WT-TTR (A) and V30M-TTR (B) after dilution-induced protein refolding, in the presence of low concentrations of urea. 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, in the presence of 1.0 M and of 0.4 M urea, for WT- and V30M-TTR, respectively. Prior to SEC, protein samples were submitted to 2.0 M GdmSCN for 12 h and then dialyzed against the chromatography buffer. In both chromatograms, the two main peaks can be assigned to tetramers and monomers with apparent molecular weights differing by about four-fold.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-17-01428-f005: Characterization of the TTR intermediate species. Size-exclusion chromatograms of WT-TTR (A) and V30M-TTR (B) after dilution-induced protein refolding, in the presence of low concentrations of urea. 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, in the presence of 1.0 M and of 0.4 M urea, for WT- and V30M-TTR, respectively. Prior to SEC, protein samples were submitted to 2.0 M GdmSCN for 12 h and then dialyzed against the chromatography buffer. In both chromatograms, the two main peaks can be assigned to tetramers and monomers with apparent molecular weights differing by about four-fold.
Mentions: In order to investigate the nature of the intermediate species involved in the refolding process of TTR, we conducted SEC experiments, as shown in Figure 5. These experiments were run at a relatively low concentration of denaturant, favoring the occurrence and accumulation of intermediate species in solution upon partial refolding. SEC chromatograms (Figure 5) show only two main populations of protein species in solution at low denaturant concentration. Peaks at earlier elution times are attributed to the refolded forms of tetrameric WT- and V30M-TTR (apparent molecular weights of 50 to 60 kDa), whereas peaks with later elution times are consistent with apparent molecular weights of TTR monomers (11 to 16 kDa). Understandably, while the elution volume registered for tetrameric V30M-TTR remains unchanged (approximately 22 mL) and is equal to the volume observed when a sample of tetrameric WT-TTR is loaded onto the column (Figure S5), the elution volume for WT-TTR in the presence of 1.0 M urea is slightly larger (approximately 27 mL) due to the considerable increase in viscosity of the mobile phase, caused by the presence of urea in the equilibrating buffer (20 mM sodium phosphate buffer, 150 mM sodium chloride, 1.0 M urea, pH 7.0). Likewise, the elution volumes detected for the monomeric species are also slightly different in the two chromatograms, namely 32 mL for V30M-TTR (Figure 5B) and 35 mL for WT-TTR (Figure 5A). Additionally, the broader peaks observed for the monomeric species, in particular for V30M-TTR, may reflect the less compact nature of these molecular species and exchange between multiple monomeric conformations.

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.