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Differential regulation of wild-type and mutant alpha-synuclein binding to synaptic membranes by cytosolic factors.

Wislet-Gendebien S, Visanji NP, Whitehead SN, Marsilio D, Hou W, Figeys D, Fraser PE, Bennett SA, Tandon A - BMC Neurosci (2008)

Bottom Line: We show that co-incubation with brain cytosol significantly increases the membrane binding of normal and PD-linked mutant alpha-syn.We further show that 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) is one of the principal lipids found in complex with cytosolic proteins and is required to enhance alpha-syn interaction with synaptic membrane.In addition, the impaired membrane binding observed for A30P alpha-syn was significantly mitigated by the presence of protease-sensitive factors in brain cytosol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, M5S 3H2 Canada. s.wislet@ulg.ac.be

ABSTRACT

Background: Alpha-Synuclein (alpha-syn), a 140 amino acid protein associated with presynaptic membranes in brain, is a major constituent of Lewy bodies in Parkinson's disease (PD). Three missense mutations (A30P, A53T and E46K) in the alpha-syn gene are associated with rare autosomal dominant forms of familial PD. However, the regulation of alpha-syn's cellular localization in neurons and the effects of the PD-linked mutations are poorly understood.

Results: In the present study, we analysed the ability of cytosolic factors to regulate alpha-syn binding to synaptic membranes. We show that co-incubation with brain cytosol significantly increases the membrane binding of normal and PD-linked mutant alpha-syn. To characterize cytosolic factor(s) that modulate alpha-syn binding properties, we investigated the ability of proteins, lipids, ATP and calcium to modulate alpha-syn membrane interactions. We report that lipids and ATP are two of the principal cytosolic components that modulate Wt and A53T alpha-syn binding to the synaptic membrane. We further show that 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) is one of the principal lipids found in complex with cytosolic proteins and is required to enhance alpha-syn interaction with synaptic membrane. In addition, the impaired membrane binding observed for A30P alpha-syn was significantly mitigated by the presence of protease-sensitive factors in brain cytosol.

Conclusion: These findings suggest that endogenous brain cytosolic factors regulate Wt and mutant alpha-syn membrane binding, and could represent potential targets to influence alpha-syn solubility in brain.

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Purified E-coli α-syn is monomeric and unstructured. Each recombinant α-syn (Wt, A30P and A53T) was analyzed by size exclusion chromatography to determine the presence of monomeric, dimeric, or other higher order forms. Eluate peaks (fraction 27) were then assessed by circular dichroism spectra to define the secondary structure of the α-syn proteins (Inset). Far-UV circular dichroism spectra were recorded on an Aviv circular dichroism spectrometer model 62DS (Lakewood, NJ, USA) at 25°C using quartz cells with a path length of 0.1 cm. Spectra were obtained from 195 nm to 260 nm, with a 1.0-nm step, 1.0-nm bandwidth, and 4-s collection time per step. The experimental data were expressed as mean residue ellipticity (θ) (deg·cm2·dmol-1). Only monomeric forms of α-syn where identified by size exclusion chromatography, and all α-syn share similar random secondary structure.
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Figure 4: Purified E-coli α-syn is monomeric and unstructured. Each recombinant α-syn (Wt, A30P and A53T) was analyzed by size exclusion chromatography to determine the presence of monomeric, dimeric, or other higher order forms. Eluate peaks (fraction 27) were then assessed by circular dichroism spectra to define the secondary structure of the α-syn proteins (Inset). Far-UV circular dichroism spectra were recorded on an Aviv circular dichroism spectrometer model 62DS (Lakewood, NJ, USA) at 25°C using quartz cells with a path length of 0.1 cm. Spectra were obtained from 195 nm to 260 nm, with a 1.0-nm step, 1.0-nm bandwidth, and 4-s collection time per step. The experimental data were expressed as mean residue ellipticity (θ) (deg·cm2·dmol-1). Only monomeric forms of α-syn where identified by size exclusion chromatography, and all α-syn share similar random secondary structure.

Mentions: Our results above, though consistent with previous reports showing that the A30P mutation impairs membrane binding ability compared to Wt and A53T α-syn, notably indicate that A30P α-syn binding is also significantly enhanced by cytosol, albeit not to the extent of Wt α-syn. Because α-syn is prone to self-aggregation and changes to the secondary structure of α-syn could induce artifactual differences between Wt, A53T and A30P membrane binding, we assessed whether each of the α-syn proteins are structurally similar in their soluble form prior to exposure to membranes, and not dimerized or aggregated which could affect membrane binding ability. All three α-syn proteins eluted in the same fractions as monomers from a size-exclusion column, and their circular dichroism spectra showed the characteristic minima of a randomly structured protein near 200 nm (Figure 4).


Differential regulation of wild-type and mutant alpha-synuclein binding to synaptic membranes by cytosolic factors.

Wislet-Gendebien S, Visanji NP, Whitehead SN, Marsilio D, Hou W, Figeys D, Fraser PE, Bennett SA, Tandon A - BMC Neurosci (2008)

Purified E-coli α-syn is monomeric and unstructured. Each recombinant α-syn (Wt, A30P and A53T) was analyzed by size exclusion chromatography to determine the presence of monomeric, dimeric, or other higher order forms. Eluate peaks (fraction 27) were then assessed by circular dichroism spectra to define the secondary structure of the α-syn proteins (Inset). Far-UV circular dichroism spectra were recorded on an Aviv circular dichroism spectrometer model 62DS (Lakewood, NJ, USA) at 25°C using quartz cells with a path length of 0.1 cm. Spectra were obtained from 195 nm to 260 nm, with a 1.0-nm step, 1.0-nm bandwidth, and 4-s collection time per step. The experimental data were expressed as mean residue ellipticity (θ) (deg·cm2·dmol-1). Only monomeric forms of α-syn where identified by size exclusion chromatography, and all α-syn share similar random secondary structure.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562387&req=5

Figure 4: Purified E-coli α-syn is monomeric and unstructured. Each recombinant α-syn (Wt, A30P and A53T) was analyzed by size exclusion chromatography to determine the presence of monomeric, dimeric, or other higher order forms. Eluate peaks (fraction 27) were then assessed by circular dichroism spectra to define the secondary structure of the α-syn proteins (Inset). Far-UV circular dichroism spectra were recorded on an Aviv circular dichroism spectrometer model 62DS (Lakewood, NJ, USA) at 25°C using quartz cells with a path length of 0.1 cm. Spectra were obtained from 195 nm to 260 nm, with a 1.0-nm step, 1.0-nm bandwidth, and 4-s collection time per step. The experimental data were expressed as mean residue ellipticity (θ) (deg·cm2·dmol-1). Only monomeric forms of α-syn where identified by size exclusion chromatography, and all α-syn share similar random secondary structure.
Mentions: Our results above, though consistent with previous reports showing that the A30P mutation impairs membrane binding ability compared to Wt and A53T α-syn, notably indicate that A30P α-syn binding is also significantly enhanced by cytosol, albeit not to the extent of Wt α-syn. Because α-syn is prone to self-aggregation and changes to the secondary structure of α-syn could induce artifactual differences between Wt, A53T and A30P membrane binding, we assessed whether each of the α-syn proteins are structurally similar in their soluble form prior to exposure to membranes, and not dimerized or aggregated which could affect membrane binding ability. All three α-syn proteins eluted in the same fractions as monomers from a size-exclusion column, and their circular dichroism spectra showed the characteristic minima of a randomly structured protein near 200 nm (Figure 4).

Bottom Line: We show that co-incubation with brain cytosol significantly increases the membrane binding of normal and PD-linked mutant alpha-syn.We further show that 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) is one of the principal lipids found in complex with cytosolic proteins and is required to enhance alpha-syn interaction with synaptic membrane.In addition, the impaired membrane binding observed for A30P alpha-syn was significantly mitigated by the presence of protease-sensitive factors in brain cytosol.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, M5S 3H2 Canada. s.wislet@ulg.ac.be

ABSTRACT

Background: Alpha-Synuclein (alpha-syn), a 140 amino acid protein associated with presynaptic membranes in brain, is a major constituent of Lewy bodies in Parkinson's disease (PD). Three missense mutations (A30P, A53T and E46K) in the alpha-syn gene are associated with rare autosomal dominant forms of familial PD. However, the regulation of alpha-syn's cellular localization in neurons and the effects of the PD-linked mutations are poorly understood.

Results: In the present study, we analysed the ability of cytosolic factors to regulate alpha-syn binding to synaptic membranes. We show that co-incubation with brain cytosol significantly increases the membrane binding of normal and PD-linked mutant alpha-syn. To characterize cytosolic factor(s) that modulate alpha-syn binding properties, we investigated the ability of proteins, lipids, ATP and calcium to modulate alpha-syn membrane interactions. We report that lipids and ATP are two of the principal cytosolic components that modulate Wt and A53T alpha-syn binding to the synaptic membrane. We further show that 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) is one of the principal lipids found in complex with cytosolic proteins and is required to enhance alpha-syn interaction with synaptic membrane. In addition, the impaired membrane binding observed for A30P alpha-syn was significantly mitigated by the presence of protease-sensitive factors in brain cytosol.

Conclusion: These findings suggest that endogenous brain cytosolic factors regulate Wt and mutant alpha-syn membrane binding, and could represent potential targets to influence alpha-syn solubility in brain.

Show MeSH
Related in: MedlinePlus