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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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Characterization of synuclein-GM1 interaction.A. Cumulative reaction enthalpy of αS vs. NAA-αS (each at 5 µM) upon vesicle titration with GM1 vesicles is shown as a function of lipid/protein ratio. Experiments were conducted at 25°C. Note the strongly increased heat release for NAA-αS upon titration with GM1. B. ITC-CD correlation plots showing the different interactions of NAA-αS and αS with GM1 vesicles. The interaction of NAA-αS and αS with GM1 vesicles is shown as correlation of MRE at 222 nm (abscissa) with corresponding cumulative heat release (ordinate). The greater slope obtained for NAA-αS indicates an increased heat release accompanied by similar secondary structure induction.
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pone-0103727-g004: Characterization of synuclein-GM1 interaction.A. Cumulative reaction enthalpy of αS vs. NAA-αS (each at 5 µM) upon vesicle titration with GM1 vesicles is shown as a function of lipid/protein ratio. Experiments were conducted at 25°C. Note the strongly increased heat release for NAA-αS upon titration with GM1. B. ITC-CD correlation plots showing the different interactions of NAA-αS and αS with GM1 vesicles. The interaction of NAA-αS and αS with GM1 vesicles is shown as correlation of MRE at 222 nm (abscissa) with corresponding cumulative heat release (ordinate). The greater slope obtained for NAA-αS indicates an increased heat release accompanied by similar secondary structure induction.

Mentions: The increased heat release upon lipid binding can be easily visualized when the cumulative obtained heat is plotted against the amount of titrated lipid. As seen in Fig. 4A, each single lipid titration leads to an increased exothermic reaction when the N-terminal acetyl group is present. This is surprising, as both forms of the protein have similar lipid binding constants. However, the heat measured by ITC must be treated as a composite quantity that summates interfacial binding and structure formation in the protein. A linear relationship was found between the cumulative heat released by vesicle binding and the MRE at 222 nm, which indicates that the energy transfer is associated with an equivalent increment in secondary structure formation (MRE) (Fig. 4B).


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)

Characterization of synuclein-GM1 interaction.A. Cumulative reaction enthalpy of αS vs. NAA-αS (each at 5 µM) upon vesicle titration with GM1 vesicles is shown as a function of lipid/protein ratio. Experiments were conducted at 25°C. Note the strongly increased heat release for NAA-αS upon titration with GM1. B. ITC-CD correlation plots showing the different interactions of NAA-αS and αS with GM1 vesicles. The interaction of NAA-αS and αS with GM1 vesicles is shown as correlation of MRE at 222 nm (abscissa) with corresponding cumulative heat release (ordinate). The greater slope obtained for NAA-αS indicates an increased heat release accompanied by similar secondary structure induction.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0103727-g004: Characterization of synuclein-GM1 interaction.A. Cumulative reaction enthalpy of αS vs. NAA-αS (each at 5 µM) upon vesicle titration with GM1 vesicles is shown as a function of lipid/protein ratio. Experiments were conducted at 25°C. Note the strongly increased heat release for NAA-αS upon titration with GM1. B. ITC-CD correlation plots showing the different interactions of NAA-αS and αS with GM1 vesicles. The interaction of NAA-αS and αS with GM1 vesicles is shown as correlation of MRE at 222 nm (abscissa) with corresponding cumulative heat release (ordinate). The greater slope obtained for NAA-αS indicates an increased heat release accompanied by similar secondary structure induction.
Mentions: The increased heat release upon lipid binding can be easily visualized when the cumulative obtained heat is plotted against the amount of titrated lipid. As seen in Fig. 4A, each single lipid titration leads to an increased exothermic reaction when the N-terminal acetyl group is present. This is surprising, as both forms of the protein have similar lipid binding constants. However, the heat measured by ITC must be treated as a composite quantity that summates interfacial binding and structure formation in the protein. A linear relationship was found between the cumulative heat released by vesicle binding and the MRE at 222 nm, which indicates that the energy transfer is associated with an equivalent increment in secondary structure formation (MRE) (Fig. 4B).

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