Limits...
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

Conformational changes of WT-TTR and V30M-TTR upon unfolding and refolding. Fluorescence spectra (A,C) and CD spectra (B,D) of WT-TTR (A,B) and V30M-TTR (C,D) under native conditions (solid lines) (20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.0), after incubation with 2.0 M GdmSCN for 12 h (dashed lines), followed by dialysis against 6.0 M urea for 10 h (dashed dotted lines) and after refolding upon extensive dialysis against sodium phosphate buffer (20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.0) (dotted lines). Protein concentrations were 1.0 µM in the final refolding mixture. Fluorescence spectra were recorded with an excitation wavelength of 290 nm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037707&req=5

ijms-17-01428-f002: Conformational changes of WT-TTR and V30M-TTR upon unfolding and refolding. Fluorescence spectra (A,C) and CD spectra (B,D) of WT-TTR (A,B) and V30M-TTR (C,D) under native conditions (solid lines) (20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.0), after incubation with 2.0 M GdmSCN for 12 h (dashed lines), followed by dialysis against 6.0 M urea for 10 h (dashed dotted lines) and after refolding upon extensive dialysis against sodium phosphate buffer (20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.0) (dotted lines). Protein concentrations were 1.0 µM in the final refolding mixture. Fluorescence spectra were recorded with an excitation wavelength of 290 nm.

Mentions: Analysis of the intrinsic tryptophan fluorescence spectra (Figure 2A,C) revealed that the native tetramers of WT- and V30M-TTR have very similar emission maxima, at approximately 340 nm, characteristic of partially-buried tryptophans. TTR has two tryptophan residues in each of its four identical subunits at positions 41 (Trp 41) and 79 (Trp 79). Trp 79 is located in the single α-helix of the protein positioned between β-strands E and F, while Trp 41 is located in the loop proximal to the beginning of β-strand C [25]. Previous studies showed that in the tetrameric form of the protein, while Trp 41 has a solvent exposure of 34.1%, Trp 79 is almost totally buried in the protein core and presents a solvent exposure of only 1.0% [34]. Moreover, it is known that the intrinsic fluorescence exhibited by TTR at pH 7.0 is mainly due to Trp 41 [33]. This agrees well with our results (Figure 2A,C).


A New Folding Kinetic Mechanism for Human Transthyretin and the Influence of the Amyloidogenic V30M Mutation
Conformational changes of WT-TTR and V30M-TTR upon unfolding and refolding. Fluorescence spectra (A,C) and CD spectra (B,D) of WT-TTR (A,B) and V30M-TTR (C,D) under native conditions (solid lines) (20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.0), after incubation with 2.0 M GdmSCN for 12 h (dashed lines), followed by dialysis against 6.0 M urea for 10 h (dashed dotted lines) and after refolding upon extensive dialysis against sodium phosphate buffer (20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.0) (dotted lines). Protein concentrations were 1.0 µM in the final refolding mixture. Fluorescence spectra were recorded with an excitation wavelength of 290 nm.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-17-01428-f002: Conformational changes of WT-TTR and V30M-TTR upon unfolding and refolding. Fluorescence spectra (A,C) and CD spectra (B,D) of WT-TTR (A,B) and V30M-TTR (C,D) under native conditions (solid lines) (20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.0), after incubation with 2.0 M GdmSCN for 12 h (dashed lines), followed by dialysis against 6.0 M urea for 10 h (dashed dotted lines) and after refolding upon extensive dialysis against sodium phosphate buffer (20 mM sodium phosphate buffer, 150 mM sodium chloride, pH 7.0) (dotted lines). Protein concentrations were 1.0 µM in the final refolding mixture. Fluorescence spectra were recorded with an excitation wavelength of 290 nm.
Mentions: Analysis of the intrinsic tryptophan fluorescence spectra (Figure 2A,C) revealed that the native tetramers of WT- and V30M-TTR have very similar emission maxima, at approximately 340 nm, characteristic of partially-buried tryptophans. TTR has two tryptophan residues in each of its four identical subunits at positions 41 (Trp 41) and 79 (Trp 79). Trp 79 is located in the single α-helix of the protein positioned between β-strands E and F, while Trp 41 is located in the loop proximal to the beginning of β-strand C [25]. Previous studies showed that in the tetrameric form of the protein, while Trp 41 has a solvent exposure of 34.1%, Trp 79 is almost totally buried in the protein core and presents a solvent exposure of only 1.0% [34]. Moreover, it is known that the intrinsic fluorescence exhibited by TTR at pH 7.0 is mainly due to Trp 41 [33]. This agrees well with our results (Figure 2A,C).

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