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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.


Denaturant gradient gel electrophoresis (DGGE) patterns of TTR (0.5 magnification factor). (A) Pattern for WT-TTR when a native sample is loaded onto the gel. Refolding patterns of WT-TTR (B) and V30M-TTR (C) when previously urea-unfolded samples are applied to the gel; TTR samples at approximately 1.0 µM were applied across the top of the gels containing a continuous urea gradient from left to right and submitted to electrophoresis at 20 mA during 4 h (A–C).
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ijms-17-01428-f001: Denaturant gradient gel electrophoresis (DGGE) patterns of TTR (0.5 magnification factor). (A) Pattern for WT-TTR when a native sample is loaded onto the gel. Refolding patterns of WT-TTR (B) and V30M-TTR (C) when previously urea-unfolded samples are applied to the gel; TTR samples at approximately 1.0 µM were applied across the top of the gels containing a continuous urea gradient from left to right and submitted to electrophoresis at 20 mA during 4 h (A–C).

Mentions: Patterns generated in urea gradient gel electrophoresis experiments depend on the thermodynamics and kinetics of urea-induced unfolding/refolding transitions [38,39,40,41]. Within the electrophoresis time scale, rapid transitions are described by continuous protein bands, with a single S-shaped curve and a single inflexion point in the transition zone; whereas, slow conformational interconversions are described by discontinuous bands [38,39,40,41]. Figure 1 shows the DGGE refolding patterns of WT- and V30M-TTR, when previously unfolded TTR is applied to a 0.0 to 5.0 M urea gradient gel. Analysis of Figure 1 allows the identification of two main protein bands in each gel. In an electrophoretic migration, the mobility of the sample is dictated by the charge, hydrodynamic volume, shape and the characteristics of the separation medium [38,42]. In the case of TTR and under the conditions tested, the electrophoretic mobility of the unfolded protein species is lower than the electrophoretic mobility of the folded globular protein. Hence, while upper bands correspond to unfolded TTR monomers, lower bands have been identified as belonging to refolded tetrameric TTR, because they exhibit similar electrophoretic mobility to the bands observed when native WT-TTR is applied onto the gel (Figure 1A). Moreover, in these conditions, TTR tetramers do not fully unfold even at high urea concentrations (9.0 M), on the time scale of the electrophoresis (4 h).


A New Folding Kinetic Mechanism for Human Transthyretin and the Influence of the Amyloidogenic V30M Mutation
Denaturant gradient gel electrophoresis (DGGE) patterns of TTR (0.5 magnification factor). (A) Pattern for WT-TTR when a native sample is loaded onto the gel. Refolding patterns of WT-TTR (B) and V30M-TTR (C) when previously urea-unfolded samples are applied to the gel; TTR samples at approximately 1.0 µM were applied across the top of the gels containing a continuous urea gradient from left to right and submitted to electrophoresis at 20 mA during 4 h (A–C).
© Copyright Policy
Related In: Results  -  Collection

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

ijms-17-01428-f001: Denaturant gradient gel electrophoresis (DGGE) patterns of TTR (0.5 magnification factor). (A) Pattern for WT-TTR when a native sample is loaded onto the gel. Refolding patterns of WT-TTR (B) and V30M-TTR (C) when previously urea-unfolded samples are applied to the gel; TTR samples at approximately 1.0 µM were applied across the top of the gels containing a continuous urea gradient from left to right and submitted to electrophoresis at 20 mA during 4 h (A–C).
Mentions: Patterns generated in urea gradient gel electrophoresis experiments depend on the thermodynamics and kinetics of urea-induced unfolding/refolding transitions [38,39,40,41]. Within the electrophoresis time scale, rapid transitions are described by continuous protein bands, with a single S-shaped curve and a single inflexion point in the transition zone; whereas, slow conformational interconversions are described by discontinuous bands [38,39,40,41]. Figure 1 shows the DGGE refolding patterns of WT- and V30M-TTR, when previously unfolded TTR is applied to a 0.0 to 5.0 M urea gradient gel. Analysis of Figure 1 allows the identification of two main protein bands in each gel. In an electrophoretic migration, the mobility of the sample is dictated by the charge, hydrodynamic volume, shape and the characteristics of the separation medium [38,42]. In the case of TTR and under the conditions tested, the electrophoretic mobility of the unfolded protein species is lower than the electrophoretic mobility of the folded globular protein. Hence, while upper bands correspond to unfolded TTR monomers, lower bands have been identified as belonging to refolded tetrameric TTR, because they exhibit similar electrophoretic mobility to the bands observed when native WT-TTR is applied onto the gel (Figure 1A). Moreover, in these conditions, TTR tetramers do not fully unfold even at high urea concentrations (9.0 M), on the time scale of the electrophoresis (4 h).

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.