1. Structural Biology and Molecular Biophysics

N-acetylation of α-synuclein enhances synaptic vesicle clustering mediated by α-synuclein and lysophosphatidylcholine

  1. Chuchu Wang
  2. Chunyu Zhao
  3. Hu Xiao
  4. Jiali Qiang
  5. Zhenying Liu
  6. Jinge Gu
  7. Shengnan Zhang
  8. Dan Li
  9. Yaoyang Zhang
  10. Jacqueline Burré
  11. Jiajia Diao  Is a corresponding author
  12. Cong Liu  Is a corresponding author
  1. Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, China
  2. University of Chinese Academy of Sciences, China
  3. Department of Molecular and Cellular Physiology, Stanford University, United States
  4. Department of Cancer Biology, University of Cincinnati College of Medicine, United States
  5. Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, China
  6. Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, China
  7. Brain and Mind Research Institute & Appel Institute for Alzheimer’s Disease Research, Weill Cornell Medicine, United States
  8. State Key Laboratory of Chemical Biology, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, China
https://doi.org/10.7554/eLife.97228.3
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Cite this article
  1. Chuchu Wang
  2. Chunyu Zhao
  3. Hu Xiao
  4. Jiali Qiang
  5. Zhenying Liu
  6. Jinge Gu
  7. Shengnan Zhang
  8. Dan Li
  9. Yaoyang Zhang
  10. Jacqueline Burré
  11. Jiajia Diao
  12. Cong Liu
(2024)
N-acetylation of α-synuclein enhances synaptic vesicle clustering mediated by α-synuclein and lysophosphatidylcholine
eLife 13:RP97228.
https://doi.org/10.7554/eLife.97228.3
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5 figures, 1 table and 2 additional files

Figures

Figure 1
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N-terminal acetylation enhances synaptic vesicle (SV) clustering induced by α-synuclein (α-syn).

(a) α-syn is N-terminally acetylated in cells. Upper panel: Schematic representation of the delivery of 15N-labeled N-terminally unmodified α-syn (un-α-syn) into mammalian cells through electroporation. Lower panel: Comparisons of 2D 1H-15N HSQC spectra of N-acetylated α-syn (Ac-α-syn) (black) with in-buffer un-α-syn (blue) and in-cell un-α-syn (red). Distinct assignments for nuclear magnetic resonance (NMR) cross-peaks corresponding to amino acids in Ac-α-syn and un-α-syn are enclosed and labeled. (b) Scheme of experimental design for the functional study of Ac-α-syn on SV clustering. SVs isolated from mouse brains were added to Ac-α-syn (Upper) for measuring size distribution by dynamic light scattering (DLS) (middle panel), and the sample was further visualized by using negatively stained transmission electron microscopy (TEM) (lower panel). (c) The influences of N-acetylation of α-syn and (d) α-syn without the N-terminal thirty residues on SV clustering measured by DLS. The X-axis represents the number percent of the single SV and clustered SVs counted by DLS. Error bars are standard deviations from three biological replicates. **p-value <0.01; ***p-value <0.001; analysis by Student’s t-test. (e) Representative negatively stained TEM images of the single SV and clustered SVs in SV samples with no α-syn (gray) and Ac-α-syn (red), respectively. Figure 1—source data 1 (c&d): the DLS numerical data of SV&Ac-α-syn, SV&un-α-syn, SV&Δ30-α-Syn, and SV-only (three biological replicates of each sample).

Figure 1—source data 1

The DLS numerical data.

https://cdn.elifesciences.org/articles/97228/elife-97228-fig1-data1-v1.xlsx
Figure 2
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Lysophosphatidylcholine (LPC) mediates the enhancement of vesicle clustering by N-terminal acetylation of α-synuclein (α-syn).

(a) Scheme of single-vesicle clustering assay for the functional study of Ac-α-syn on vesicle clustering. Vesicles were prepared with different amounts of LPC, and were labeled with DiD or remained unlabeled, respectively. A saturated layer of unlabeled vesicles was immobilized on the imaging surface. Free DiD-vesicles were injected into the system with Ac-α-syn. Red laser illumination imaged the DiD-vesicles that clustered with unlabeled vesicles. The enhancement of LPC (b) and dioleoyl-phosphoserine (DOPS) (c) on single vesicle clustering count by Ac-α-syn and un-α-syn, respectively, was measured. Error bars are standard deviations from six random imaging locations in the same sample channel. *** indicates p-value <0.001, analysis by Student’s t-test.

Figure 3
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N-terminal acetylation increases the α-synuclein (α-syn)–lysophosphatidylcholine (LPC) interaction.

Comparisons of residue-resolved nuclear magnetic resonance (NMR) signal intensity ratios (I/I0) of un-α-syn (upper) and Ac-α-syn (lower) during titration with LPC micelles (a), LPC-containing liposomes (DOPC:LPC = 4:1, mol:mol) (b), and dioleoyl-phosphoserine (DOPS) liposomes (c) at indicated protein/lipid molar ratios. Dashed lines highlight the residue positions 30 and 95. (d) SVs isolated from mouse brains were employed for NMR titration with 15N-Ac-α-syn, approximating the physiological ratio (α-syn:SV = 4000:300, mol:mol). Residue-resolved NMR signal intensity ratios (I/I0) of Ac-α-syn is titrated by synaptic vesicles (SVs) to that in solution. The molar ratios of SV to Ac-α-syn are indicated. LPC titration in the Ac-α-syn/LPC ratio of 1:10 (blue curve) is overlaid on the SV titration. (e) 2D 1H-15N HSQC spectra of NMR for un-α-syn with LPC micelles and Ac-α-syn with LPC micelles, LPC-containing liposomes, and mouse SVs. The NMR cross-peaks of the first 10 residues are highlighted and magnified, as depicted on the right side of each spectrum set (Note: the first and second residues of un-α-syn cannot be assigned). Figure 3—source data 1 (a–d): the NMR titration numerical data of α-syn&LPC, Ac-α-syn&LPC, Ac-α-syn&4PC/LPC, α-syn&DOPS, Ac-α-syn&DOPS, and Ac-α-syn&SV.

Figure 3—source data 1

The NMR titration numerical data.

https://cdn.elifesciences.org/articles/97228/elife-97228-fig3-data1-v1.xlsx
Figure 4
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Ac-α-syn binding on lysophosphatidylcholine (LPC) shows high intermolecular interactions.

(a) The cross-linking patterns of Ac-α-syn in the presence of LPC and dioleoyl-phosphoserine (DOPS) mapped by mass spectrometry (MS) at the protein/lipid molar ratio of 1:50. Lines present the inter-molecular cross-linked residues between two individual 15N-labeled and unlabeled Ac-α-syn. The grayscale of the lines corresponds to the frequency of the cross-linked pairs identified in three individual experiments. Source data are provided in Supplementary file 1a-f. (b) Ac-α-syn binds strongly to LPC through the N-terminal region (red arrow), and leaves more unbound NAC and C-terminal region for intermolecular interaction (green arrow). In contrast, N-terminal acetylation reduces α-syn’s binding to DOPS, and due to the negatively charged headgroup of PS, Ac-α-syn binding on DOPS extends to the NAC region, which limits intermolecular interactions. Supplementary file 1 (a-–c) Three biological replicates of identified cross-linked peptides between 15N-Ac-α-syn and 14N-Ac-α-syn in LPC; (d-e) Three biological replicates of identified cross-linked peptides between 15N-Ac-α-syn and 14N-Ac-α-syn in DOPS; Protein:lipid = 1:50, mol:mol.

Author response image 1
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Representative raw dataset of α-Syn-mediated synaptic vesicle (SV) clustering monitored by dynamic light scattering (DLS).

The gray-colored rows represent small particles (< 5 nm) that contributed zero to the particle number count.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Escherichia coli)BL21(DE3)BioRad156–3003Electrocompetent cells
Cell line (Homo-sapiens)HEK-293T epithelial-like cellsATCCCRL-3216Cell line authentication services (STR profiling) are offered by ATCC, not detected mycoplasma contamination
Transfected construct (human)α-syn, Δ30 α-syn to pET22 vectorThis paper; Zhao et al., 2024Constructs saved in C. Liu lab
Commercial assay or kitNeon transfection system kitInvitrogenMPK5000
Chemical compound, drug16:0 LPC, DOPC, DOPS, POPC, POPE, biotin-DPPE, cholesterolAvanti Polar Lipids855675, 850375, 840035, 850457, 850757, 870277, 70000
Chemical compound, drugDisuccinimidyl suberate (DSS)Thermo Scientific21658
Chemical compound, druguranyl acetateSigma AldrichCDS021290
Chemical compound, drugDiDInvitrogenD307
Software, algorithmpLinkpLinkV1.9For XL-MS data
Software, algorithmSPARKYSPARKYV3.115For NMR data
Software, algorithmNMRpipeNMRpipeBuild2018For NMR data
Software, algorithmsmCameraTJ Ha’s labFor single vesicle data
Software, algorithmDynamicsWyattV7.0For DLS data
OtherC57BL6 miceLingchang Shanghai8 wk old, male

Additional files

Supplementary file 1

Supplementary tables.

https://cdn.elifesciences.org/articles/97228/elife-97228-supp1-v1.docx
MDAR checklist
https://cdn.elifesciences.org/articles/97228/elife-97228-mdarchecklist1-v1.docx

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  1. Chuchu Wang
  2. Chunyu Zhao
  3. Hu Xiao
  4. Jiali Qiang
  5. Zhenying Liu
  6. Jinge Gu
  7. Shengnan Zhang
  8. Dan Li
  9. Yaoyang Zhang
  10. Jacqueline Burré
  11. Jiajia Diao
  12. Cong Liu
(2024)
N-acetylation of α-synuclein enhances synaptic vesicle clustering mediated by α-synuclein and lysophosphatidylcholine
eLife 13:RP97228.
https://doi.org/10.7554/eLife.97228.3