N-terminal acetylation enhances SV clustering induced by α-syn.

(a) α-syn is N-terminally acetylated in cells. Upper: Schematic representation of the delivery of 15N-labeled N-terminally unmodified α-synuclein (un-α-syn) into mammalian cells through electroporation. Lower: 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 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 DLS (middle), and the sample was further visualized by using negatively stained TEM (lower). (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 3 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 (grey) and Ac-α-syn (red), respectively.

LPC mediates the enhancement of vesicle clustering by N-terminal acetylation of 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 labeled with DiD and unlabeled, respectively. A saturated layer of unlabeled vesicles was immobilized on the imaging surface. DiD-vesicles were injected into the system with Ac-α-syn. Red laser illumination imaged Free the DiD-vesicles that clustered with unlabeled vesicles. The enhancement of LPC (b) and DOPS (c) on single vesicle clustering count by Ac-α-syn and un-α-syn, respectively. Error bars are standard deviations from 6 random imaging locations in the same sample channel. *** indicates p-value < 0.001, analysis by Student’s t-test.

N-terminal acetylation increases the α-syn–LPC interaction.

Comparisons of residue-resolved 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 DOPS liposomes (c) at indicated protein/lipid molar ratios. Dash lines highlight the residue positions of 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 titrated by 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).

Ac-α-syn binding on LPC shows high intermolecular interactions.

(a) The cross-linking patterns of Ac-α-syn in the presence of LPC and DOPS mapped by 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 Tables S1-S6. (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.