1. ABOUT THE DATASET -------------------- Title: Residues 2 to 7 of α-synuclein regulate amyloid formation via lipid-dependent and lipid-independent pathways Creator(s):Katherine M. Dewison[1], Benjamin Rowlinson[1], Jonathan M. Machin[1], Joel A. Crossley[1], Dev Thacker[1], Martin Wilkinson[1], Sabine M. Ulamec[1], G. Nasir Khan[1], Neil A. Ranson[1], Patricija van Oosten-Hawle[2], David J. Brockwell[1], Sheena E. Radford[1] Organisation(s): 1. Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom 2. Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, NC, USA Rights-holder(s): Copyright 2024 University of Leeds Publication Year: 2024 Description: The role of residues 2-7 in the N-terminal region of αSyn are investigated in terms of their ability to regulate amyloid fibril formation in vitro and in vivo. Deletion of these residues (αSynΔN7) slows the rate of fibril formation in vitro and reduces the capacity of the protein to be recruited by wild-type (αSynWT) fibril seeds, despite cryo-EM showing a fibril structure consistent with those of full-length αSyn. Strikingly, fibril formation of αSynΔN7 is not induced by liposomes, despite the protein binding to liposomes with similar affinity to αSynWT. A Caenorhabditis elegans model also showed that αSynΔN7::YFP forms few puncta and lacks motility and lifespan defects typified by expression of αSynWT::YFP. Cite as: Dewison K.M., Rowlinson B., Machin J.M., Crossley J.A., Thacker D., Wilkinson M., Ulamec S.M., Khan G.N., Ranson N.A., van Oosten-Hawle P., Brockwell D.J., Radford S.E. (2024): Residues 2-7 of α-synuclein regulate amyloid formation via lipid-dependent and lipid-independent pathways. University of Leeds. https:/doi.org/https://doi.org/10.5518/1422 Related publication: Contact: Sheena E. Radford - S.E.Radford@leeds.ac.uk; David J. Brockwell - D.J.Brockwell@leeds.ac.uk 2. TERMS OF USE --------------- Copyright © 2024 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY) 3. PROJECT AND FUNDING INFORMATION ---------------------------------- Negative-stain EM, CD, and NMR data were collected using facilities in the Astbury Biostructure Laboratory and were funded by the University of Leeds and the Wellcome Trust (094232/Z/10/Z). Confocal microscopy data were collected with instrumentation from the Leeds Bioimaging Facility [Wellcome Trust funded (104918MA)]. K.M.D. and M.W. are funded by MRC (MR/N013840/1 and MR/T011149/1). B.R. and J.A.C. by BBSRC (BB/W007649/1 and BB/T008059/1), J.M.M., D.T., M.W., and S.M.U. by Wellcome (222373/Z/21/Z, 215062/Z/18/Z, and 204963), S.E.R. holds a Royal Society Professorial Research Fellowship (RSRP/R1/211057). 4. CONTENTS ----------- File listing DOI_Figure1.xlsx - ThT fluorescence data, with corresponding T50 and Tlag values and calculated percentage insoluble data used for Figure 1. DOI_Figure2.xlsx - ThT fluorescence data for the seeding experiment using sonicated seeds, with percentage insoluble data for 3 repeats. DOI_Figure3.xlsx - liposome-binding data used for Figure 3. Sheets 1 and 2 contain MRE values for aSynWT and aSynDN7, respectively. Sheet 3 contains the MRE values at 222nm for the 3 CD repeats. Sheets 4 and 5 contain the NMR intensty values for aSynWT and aSynDN7 in the absence and presence of DMPS liposomes and the resulting intensity ratios. DOI_Figure4.xlsx - ThT flurescence data for aSynWT (sheet 1) and aSynDN7 (sheet 2) in the presence of DMPS liposomes. DOI_Figure5.xlsx - Quantification of aggregates per C. elegans (sheet 1) and number of body bends per second for each C. elegans (sheet 2). DOI_SI_Figure1.xlsx - ThT fluorescence data for DP1, DP2 and DD (sheet 1) and percentage insoluble for each aggregation assay (sheet 2). DOI_SI_Figure4.xlsx - ThT fluorescence data for the monomer mixing experiment (sheet 1), with corresponding percentage insoluble values for 3 repeats (sheet 2). Reversed-phase HPLC values for the souble fraction at the end of the ThT assay compared with aSynWT and aSynDN7 monomeric controls (sheet 3). DOI_SI_Figure6.xlsx - T50 values for the sonictaed seeding ThT assay. DOI_SI_Figure7.xlsx - ThT fluorescence values for the seeding assay using unsonicated seeds (sheet 1) with corresponding T50 values (sheet 2) and percentage insoluble values (sheet 3). DOI_SI_Figure9.xlsx - NMR HMQC intensity values for aSynWT (sheet 1) and aSynDN7 (sheet 2) at temperatures of 20, 30, and 40 degC in the absence and presence of liposomes. DOI_SI_Figure10.xlsx - DPH fluorescence anisotropy data for aSynWT, aSynDN7, and aSynDP1 at LPRs of 8:1 and 60:1. DOI_SI_Figure12.xlsx - DP1 liposome experimental data. MRE data for aSynDP1 (sheet 1). MRE values at 222nm for aSynDP1 for three repeats (sheet 2). Liposome ThT fluorescence values for aSynDP1 (sheet 3). NMR HMQC intensity values for aSynDP1 in the absence and presence of DMPS liposomes. DOI_SI_Figure13.xlsx - MRE values for the N7 alanine scan mutants in the absence or presence of liposomes DOI_SI_Figure14.xlsx - ThT fluorescence values for the N7 alanine scan mutants in the presence of liposomes (sheet 1) and the corresponding T50 values (sheet 2). DOI_SI_Figure15.xlsx - Western blot densitometry values for expression levels of aSynWT versus aSynDN7, normalised to levels of tubulin. DOI_SI_Figure16.xlsx - Lifespan data for aSynDN7 C. elegans strain compared with aSynWT and N2. C.elegans images - zipped folder containing all C. elegans confocal images, e.g. "aSynDN7_D1AD_1.czi", where aSynDN7 is the strain name, D1AD refers to the image aquired at Day 1 of Adulthood, and 1 is the repeat number. SDS-PAGE gels - folder containing all SDS-PAGE gels used for densitometry calculations (well information contained in parentheses). aSynDP1DP2DD_ThT_pelletingassay1.tif (ladder, DP1 whole, DP1 soluble, DP2 whole, DP2 soluble, DD whole, DD soluble) aSynDP1DP2DD_ThT_pelletingassay2.tif (ladder, DP1 whole, DP1 soluble , DP2 whole, DP2 soluble, DD whole, DD soluble) aSynDP1DP2DD_ThT_pelletingassay3.tif (ladder, DP1 whole, DP1 soluble, DP2 whole, DP2 soluble, DD whole, DD soluble) aSynWTDN7_monomermix_ThT_pelletingassay_1.tif (ladder, aSynWT 100uM whole, aSynWT 100 uM soluble, aSynWT 50 uM whole, aSynWT 50 uM soluble, DN7 100 uM whole, DN7 100 uM soluble, DN7 50 uM whole, DN7 50 uM soluble, WT + DN7 mix whole, WT + DN7 mix soluble) aSynWTDN7_monomermix_ThT_pelletingassay_2.tif (ladder, aSynWT 100uM whole, aSynWT 100 uM soluble, aSynWT 50 uM whole, aSynWT 50 uM soluble, DN7 100 uM whole, DN7 100 uM soluble, DN7 50 uM whole, DN7 50 uM soluble, WT + DN7 mix whole, WT + DN7 mix soluble) aSynWTDN7_monomermix_ThT_pelletingassay_3.tif (ladder, aSynWT 100uM whole, aSynWT 100 uM soluble, aSynWT 50 uM whole, aSynWT 50 uM soluble, DN7 100 uM whole, DN7 100 uM soluble, DN7 50 uM whole, DN7 50 uM soluble, WT + DN7 mix whole, WT + DN7 mix soluble) aSynWTDN7_soni_seeding_ThT_pelletingassay_1.tif (ladder, WT + WT seeds whole, WT + WT seeds soluble, WT + DN7 seeds whole, WT + DN7 seeds soluble, DN7 + WT seeds whole, DN7 + WT seeds soluble, DN7 + DN7 seeds whole, DN7 + DN7 seeds soluble) aSynWTDN7_soni_seeding_ThT_pelletingassay_2.tif (ladder, WT + WT seeds whole, WT + WT seeds soluble, WT + DN7 seeds whole, WT + DN7 seeds soluble, DN7 + WT seeds whole, DN7 + WT seeds soluble, DN7 + DN7 seeds whole, DN7 + DN7 seeds soluble) aSynWTDN7_soni_seeding_ThT_pelletingassay_3.tif (ladder, WT + WT seeds whole, WT + WT seeds soluble, DN7 + WT seeds whole, DN7 + WT seeds soluble, WT + DN7 seeds whole, WT + DN7 seeds soluble, DN7 + DN7 seeds whole, DN7 + DN7 seeds soluble) aSynWTDN7_ThT_pelletingassay_1.tif (ladder, WT whole, WT soluble, DN7 whole, DN7 soluble) aSynWTDN7_ThT_pelletingassay_2.tif (ladder, WT whole, WT soluble, DN7 whole, DN7 soluble) aSynWTDN7_ThT_pelletingassay_3.tif (ladder, WT whole, WT soluble, lanes 4-9 not used, DN7 whole, DN7 soluble) aSynWTDN7_unsoni_seeding_ThT_pelletingassay_1A.tif (ladder, lane not used, lane not used, WT + WT seeds whole, WT + WT seeds soluble, lane not used, lane not used, DN7 + DN7 seeds whole, DN7 + DN7 seeds soluble) aSynWTDN7_unsoni_seeding_ThT_pelletingassay_1B.tif (ladder, lane not used, lane not used, WT + DN7 seeds whole, WT + DN7 seeds soluble, lane not used, lane not used, DN7 + WT seeds whole, DN7 + WT seeds soluble) aSynWTDN7_unsoni_seeding_ThT_pelletingassay_2.tif (ladder, WT + WT seeds whole, WT + WT seeds soluble, DN7 + WT seeds whole, DN7 + WT seeds soluble, WT + DN7 seeds whole, WT + DN7 seeds soluble, DN7 + DN7 seeds whole, DN7 + DN7 seeds soluble) aSynWTDN7_unsoni_seeding_ThT_pelletingassay_3.tif (ladder, WT + WT seeds whole, WT + WT seeds soluble, DN7 + WT seeds whole, DN7 + WT seeds soluble, WT + DN7 seeds whole, WT + DN7 seeds soluble, DN7 + DN7 seeds whole, DN7 + DN7 seeds soluble) Ce_aSynWTDN7_WB_1_antiGFP.tif (tubulin, N2 lysate, aSynWT lysate, aSynDN7 lysate) Ce_aSynWTDN7_WB_1_antiGFP_ladder.tif (tubulin, N2 lysate, aSynWT lysate, aSynDN7 lysate) Ce_aSynWTDN7_WB_1_antitubulin.tif (tubulin, N2 lysate, aSynWT lysate, aSynDN7 lysate) Ce_aSynWTDN7_WB_1_antitubulin_ladder.tif (tubulin, N2 lysate, aSynWT lysate, aSynDN7 lysate) Ce_aSynWTDN7_WB_2_antiGFP.tif (tubulin, N2 lysate, aSynWT lysate, aSynDN7 lysate) Ce_aSynWTDN7_WB_2_antiGFP_ladder.tif (tubulin, N2 lysate, aSynWT lysate, aSynDN7 lysate) Ce_aSynWTDN7_WB_2_antitubulin.tif (tubulin, N2 lysate, aSynWT lysate, aSynDN7 lysate) Ce_aSynWTDN7_WB_2_antitubulin_ladder.tif (tubulin, N2 lysate, aSynWT lysate, aSynDN7 lysate) Ce_aSynWTDN7_WB_3_antiGFP.tif (tubulin, N2 lysate, aSynWT lysate, aSynDN7 lysate) Ce_aSynWTDN7_WB_3_antiGFP_ladder.tif (tubulin, N2 lysate, aSynWT lysate, aSynDN7 lysate) Ce_aSynWTDN7_WB_3_antitubulin.tif (tubulin, N2 lysate, aSynWT lysate, aSynDN7 lysate) Ce_aSynWTDN7_WB_3_antitubulin_ladder.tif (tubulin, N2 lysate, aSynWT lysate, aSynDN7 lysate) 5. METHODS ---------- For details on the Methods used to collect the data in this dataset, please find information in the Supporting Information document coresponding to this publication.