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N-alpha-acetylation of α-synuclein increases its helical folding propensity, GM1 binding specificity and resistance to aggregation.

Bartels T, Kim NC, Luth ES, Selkoe DJ - PLoS ONE (2014)

Bottom Line: Interestingly, the folding and lipid binding enhancements with phosphatidylserine in vitro were weak when compared to that of αS with GM1, a lipid enriched in presynaptic membranes.The resultant increase in helical folding propensity of N-acetylated αS enhanced its resistance to aggregation.Our findings demonstrate the significance of the extreme N-terminus for folding nucleation, for relative GM1 specificity of αS-membrane interaction, and for a protective function of N-terminal-acetylation against αS aggregation mediated by GM1.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
A switch in the conformational properties of α-synuclein (αS) is hypothesized to be a key step in the pathogenic mechanism of Parkinson's disease (PD). Whereas the beta-sheet-rich state of αS has long been associated with its pathological aggregation in PD, a partially alpha-helical state was found to be related to physiological lipid binding; this suggests a potential role of the alpha-helical state in controlling synaptic vesicle cycling and resistance to β-sheet rich aggregation. N-terminal acetylation is the predominant post-translational modification of mammalian αS. Using circular dichroism, isothermal titration calorimetry, and fluorescence spectroscopy, we have analyzed the effects of N-terminal acetylation on the propensity of recombinant human αS to form the two conformational states in interaction with lipid membranes. Small unilamellar vesicles of negatively charged lipids served as model membranes. Consistent with previous NMR studies using phosphatidylserine, we found that membrane-induced α-helical folding was enhanced by N-terminal acetylation and that greater exothermic heat could be measured upon vesicle binding of the modified protein. Interestingly, the folding and lipid binding enhancements with phosphatidylserine in vitro were weak when compared to that of αS with GM1, a lipid enriched in presynaptic membranes. The resultant increase in helical folding propensity of N-acetylated αS enhanced its resistance to aggregation. Our findings demonstrate the significance of the extreme N-terminus for folding nucleation, for relative GM1 specificity of αS-membrane interaction, and for a protective function of N-terminal-acetylation against αS aggregation mediated by GM1.

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Variability in the folding of purified recombinant NAA-αS under non-denaturing conditions.A: CD spectroscopy of bacterially expressed and purified NAA-αS and αS under non-denaturing conditions. No variability in the secondary structure was seen with unmodified αS. In contrast, NAA-αS showed batch-to-batch variability in folding of the obtained preps. B: SDS-PAGE Western blot and Coomassie stain of αS and NAA-αS (“expression C”). The samples were not boiled in SDS-sample buffer before gel loading for Western blotting. The NAA-αS banding pattern indicates some SDS-stable tetrameric protein present in the sample, while no higher bands could be detected in non-acetylated αS. For coomassie staining, the samples were boiled in sample buffer, leading to a single stainable band at 14 kDa, indicative of the high purity of the sample.
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pone-0103727-g001: Variability in the folding of purified recombinant NAA-αS under non-denaturing conditions.A: CD spectroscopy of bacterially expressed and purified NAA-αS and αS under non-denaturing conditions. No variability in the secondary structure was seen with unmodified αS. In contrast, NAA-αS showed batch-to-batch variability in folding of the obtained preps. B: SDS-PAGE Western blot and Coomassie stain of αS and NAA-αS (“expression C”). The samples were not boiled in SDS-sample buffer before gel loading for Western blotting. The NAA-αS banding pattern indicates some SDS-stable tetrameric protein present in the sample, while no higher bands could be detected in non-acetylated αS. For coomassie staining, the samples were boiled in sample buffer, leading to a single stainable band at 14 kDa, indicative of the high purity of the sample.

Mentions: We recently discovered the existence of a helically folded oligomeric form of endogenous αS in human cells, an observation that has led to an ongoing controversy in the field. The main origin of this controversy is that the protein had previously been characterized as “natively unfolded”, based principally on studies of recombinantly expressed αS purified from bacterial lysates. This seemed to disagree directly with our characterization of the protein isolated under non-denaturing conditions from human cells. A major difference between αS obtained from these two sources is the presence of N-terminal alpha acetylation (NAA) in the human protein, a post-translational modification found commonly in mammalian proteins. We therefore first examined the effect of NAA on the overall folding of bacterially expressed αS in the absence of structure-inducing detergents or lipids, in comparison with αS isolated from fresh human red blood cells (RBC-αS). To this end, we co-expressed αS in bacteria with the fission yeast complex N-acetyltransferase B (NatB), which is reported to cause targeted NAA of other recombinantly expressed proteins that share with αS the N-terminal amino acid motif Met-Asp (MD) [19]. In order to preserve any structure induced by the NAA, we used a non-denaturing protocol to purify the bacterially expressed recombinant protein. This method generated preparations that were 100% N-α-acetylated αS (NAA-αS), as confirmed by mass spectrometry (data not shown). As shown by circular dichroism (CD) spectra, we obtained some batches of α-helically folded NAA-αS in the absence of detergent (e.g. the BOG used by Trexler et al. [14]) or lipids (Fig. 1A), and such preparations contained minor amounts of SDS-stable oligomeric NAA-αS (Fig. 1B), in agreement with a recent report by Trexler et al. [14] and closely resembling endogenous RBC-αS. However, in other batches ostensibly prepared identically under non-denaturing conditions, we observed mainly unfolded NAA-αS, similar to the NAA-αS preparations reported by Fauvet et al. [20], and yet other preparations were folded in an intermediate state (Fig. 1A). Even the maximally unfolded preparations had a slightly higher α-helical content than comparable preparations of αS obtained without co-expression of NatB (approximately 9% vs 5% helicity measured by CD spectroscopy). This increased helicity could not be prevented by application of heat denaturation (data not shown) and thus appeared to be an intrinsic property of NAA-αS independent of the purification method. It is important to note that no such variability in the amount of helical content was observed for bacterial preparations of αS without N-acetylation under non-denaturing conditions; it was all unfolded with only minor helical content (5%). The cause(s) of the conformational variability in the recombinant batches of NAA-αS prepared under seemingly identical conditions are under investigation but currently remain elusive. None of the variations in the temperature during IPTG induction (25 or 37°C), cell density at harvest (OD600: 0.8–1.5) or the IPTG concentration used for induction (0.1–1 mM) had a clear correlation with helicity. It has to be noted though that preparations with higher helical content typically had lower overall protein yield and that N-terminally acetylated αS seemed to be more resistant to N-terminal degradation during the purification procedure in general. Given the lack of control over batch variability under non-denaturing conditions, we decided to analyze separately the properties of the invariably mostly unfolded NAA-αS obtained under denaturing conditions in regards to its interaction with lipids in order to gain more insight into possible folding mechanisms of this post-translationally modified form of αS. We therefore used a heat denaturation step in our purification protocol for both NAA-αS and non-acetylated αS in all experiments described below to obtain maximally unfolded protein preparations. This denaturation step allowed us to control variability in helical content and assess the impact of N-terminal acetylation on initiation of in vitro folding upon in interaction with lipids.


N-alpha-acetylation of α-synuclein increases its helical folding propensity, GM1 binding specificity and resistance to aggregation.

Bartels T, Kim NC, Luth ES, Selkoe DJ - PLoS ONE (2014)

Variability in the folding of purified recombinant NAA-αS under non-denaturing conditions.A: CD spectroscopy of bacterially expressed and purified NAA-αS and αS under non-denaturing conditions. No variability in the secondary structure was seen with unmodified αS. In contrast, NAA-αS showed batch-to-batch variability in folding of the obtained preps. B: SDS-PAGE Western blot and Coomassie stain of αS and NAA-αS (“expression C”). The samples were not boiled in SDS-sample buffer before gel loading for Western blotting. The NAA-αS banding pattern indicates some SDS-stable tetrameric protein present in the sample, while no higher bands could be detected in non-acetylated αS. For coomassie staining, the samples were boiled in sample buffer, leading to a single stainable band at 14 kDa, indicative of the high purity of the sample.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4116227&req=5

pone-0103727-g001: Variability in the folding of purified recombinant NAA-αS under non-denaturing conditions.A: CD spectroscopy of bacterially expressed and purified NAA-αS and αS under non-denaturing conditions. No variability in the secondary structure was seen with unmodified αS. In contrast, NAA-αS showed batch-to-batch variability in folding of the obtained preps. B: SDS-PAGE Western blot and Coomassie stain of αS and NAA-αS (“expression C”). The samples were not boiled in SDS-sample buffer before gel loading for Western blotting. The NAA-αS banding pattern indicates some SDS-stable tetrameric protein present in the sample, while no higher bands could be detected in non-acetylated αS. For coomassie staining, the samples were boiled in sample buffer, leading to a single stainable band at 14 kDa, indicative of the high purity of the sample.
Mentions: We recently discovered the existence of a helically folded oligomeric form of endogenous αS in human cells, an observation that has led to an ongoing controversy in the field. The main origin of this controversy is that the protein had previously been characterized as “natively unfolded”, based principally on studies of recombinantly expressed αS purified from bacterial lysates. This seemed to disagree directly with our characterization of the protein isolated under non-denaturing conditions from human cells. A major difference between αS obtained from these two sources is the presence of N-terminal alpha acetylation (NAA) in the human protein, a post-translational modification found commonly in mammalian proteins. We therefore first examined the effect of NAA on the overall folding of bacterially expressed αS in the absence of structure-inducing detergents or lipids, in comparison with αS isolated from fresh human red blood cells (RBC-αS). To this end, we co-expressed αS in bacteria with the fission yeast complex N-acetyltransferase B (NatB), which is reported to cause targeted NAA of other recombinantly expressed proteins that share with αS the N-terminal amino acid motif Met-Asp (MD) [19]. In order to preserve any structure induced by the NAA, we used a non-denaturing protocol to purify the bacterially expressed recombinant protein. This method generated preparations that were 100% N-α-acetylated αS (NAA-αS), as confirmed by mass spectrometry (data not shown). As shown by circular dichroism (CD) spectra, we obtained some batches of α-helically folded NAA-αS in the absence of detergent (e.g. the BOG used by Trexler et al. [14]) or lipids (Fig. 1A), and such preparations contained minor amounts of SDS-stable oligomeric NAA-αS (Fig. 1B), in agreement with a recent report by Trexler et al. [14] and closely resembling endogenous RBC-αS. However, in other batches ostensibly prepared identically under non-denaturing conditions, we observed mainly unfolded NAA-αS, similar to the NAA-αS preparations reported by Fauvet et al. [20], and yet other preparations were folded in an intermediate state (Fig. 1A). Even the maximally unfolded preparations had a slightly higher α-helical content than comparable preparations of αS obtained without co-expression of NatB (approximately 9% vs 5% helicity measured by CD spectroscopy). This increased helicity could not be prevented by application of heat denaturation (data not shown) and thus appeared to be an intrinsic property of NAA-αS independent of the purification method. It is important to note that no such variability in the amount of helical content was observed for bacterial preparations of αS without N-acetylation under non-denaturing conditions; it was all unfolded with only minor helical content (5%). The cause(s) of the conformational variability in the recombinant batches of NAA-αS prepared under seemingly identical conditions are under investigation but currently remain elusive. None of the variations in the temperature during IPTG induction (25 or 37°C), cell density at harvest (OD600: 0.8–1.5) or the IPTG concentration used for induction (0.1–1 mM) had a clear correlation with helicity. It has to be noted though that preparations with higher helical content typically had lower overall protein yield and that N-terminally acetylated αS seemed to be more resistant to N-terminal degradation during the purification procedure in general. Given the lack of control over batch variability under non-denaturing conditions, we decided to analyze separately the properties of the invariably mostly unfolded NAA-αS obtained under denaturing conditions in regards to its interaction with lipids in order to gain more insight into possible folding mechanisms of this post-translationally modified form of αS. We therefore used a heat denaturation step in our purification protocol for both NAA-αS and non-acetylated αS in all experiments described below to obtain maximally unfolded protein preparations. This denaturation step allowed us to control variability in helical content and assess the impact of N-terminal acetylation on initiation of in vitro folding upon in interaction with lipids.

Bottom Line: Interestingly, the folding and lipid binding enhancements with phosphatidylserine in vitro were weak when compared to that of αS with GM1, a lipid enriched in presynaptic membranes.The resultant increase in helical folding propensity of N-acetylated αS enhanced its resistance to aggregation.Our findings demonstrate the significance of the extreme N-terminus for folding nucleation, for relative GM1 specificity of αS-membrane interaction, and for a protective function of N-terminal-acetylation against αS aggregation mediated by GM1.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT
A switch in the conformational properties of α-synuclein (αS) is hypothesized to be a key step in the pathogenic mechanism of Parkinson's disease (PD). Whereas the beta-sheet-rich state of αS has long been associated with its pathological aggregation in PD, a partially alpha-helical state was found to be related to physiological lipid binding; this suggests a potential role of the alpha-helical state in controlling synaptic vesicle cycling and resistance to β-sheet rich aggregation. N-terminal acetylation is the predominant post-translational modification of mammalian αS. Using circular dichroism, isothermal titration calorimetry, and fluorescence spectroscopy, we have analyzed the effects of N-terminal acetylation on the propensity of recombinant human αS to form the two conformational states in interaction with lipid membranes. Small unilamellar vesicles of negatively charged lipids served as model membranes. Consistent with previous NMR studies using phosphatidylserine, we found that membrane-induced α-helical folding was enhanced by N-terminal acetylation and that greater exothermic heat could be measured upon vesicle binding of the modified protein. Interestingly, the folding and lipid binding enhancements with phosphatidylserine in vitro were weak when compared to that of αS with GM1, a lipid enriched in presynaptic membranes. The resultant increase in helical folding propensity of N-acetylated αS enhanced its resistance to aggregation. Our findings demonstrate the significance of the extreme N-terminus for folding nucleation, for relative GM1 specificity of αS-membrane interaction, and for a protective function of N-terminal-acetylation against αS aggregation mediated by GM1.

Show MeSH
Related in: MedlinePlus