Title | KTKEGV repeat motifs are key mediators of normal α-synuclein tetramerization: Their mutation causes excess monomers and neurotoxicity. |
Publication Type | Journal Article |
Year of Publication | 2015 |
Authors | Dettmer U, Newman AJ, von Saucken VE, Bartels T, Selkoe D |
Journal | Proc Natl Acad Sci U S A |
Volume | 112 |
Issue | 31 |
Pagination | 9596-601 |
Date Published | 2015 Aug 04 |
ISSN | 1091-6490 |
Keywords | alpha-Synuclein, Amino Acid Motifs, Amino Acid Sequence, Animals, Cell Death, Conserved Sequence, Cross-Linking Reagents, Humans, Inclusion Bodies, Microscopy, Fluorescence, Molecular Sequence Data, Mutant Proteins, Mutation, Neurons, Protein Multimerization, Rats, Sprague-Dawley, Repetitive Sequences, Amino Acid, Sequence Deletion, Structure-Activity Relationship |
Abstract | α-Synuclein (αS) is a highly abundant neuronal protein that aggregates into β-sheet-rich inclusions in Parkinson's disease (PD). αS was long thought to occur as a natively unfolded monomer, but recent work suggests it also occurs normally in α-helix-rich tetramers and related multimers. To elucidate the fundamental relationship between αS multimers and monomers in living neurons, we performed systematic mutagenesis to abolish self-interactions and learn which structural determinants underlie native multimerization. Unexpectedly, tetramers/multimers still formed in cells expressing each of 14 sequential 10-residue deletions across the 140-residue polypeptide. We postulated compensatory effects among the six highly conserved and one to three additional αS repeat motifs (consensus: KTKEGV), consistent with αS and its homologs β- and γ-synuclein all forming tetramers while sharing only the repeats. Upon inserting in-register missense mutations into six or more αS repeats, certain mutations abolished tetramer formation, shown by intact-cell cross-linking and independently by fluorescent-protein complementation. For example, altered repeat motifs KLKEGV, KTKKGV, KTKEIV, or KTKEGW did not support tetramerization, indicating the importance of charged or small residues. When we expressed numerous different in-register repeat mutants in human neural cells, all multimer-abolishing but no multimer-neutral mutants caused frank neurotoxicity akin to the proapoptotic protein Bax. The multimer-abolishing variants became enriched in buffer-insoluble cell fractions and formed round cytoplasmic inclusions in primary cortical neurons. We conclude that the αS repeat motifs mediate physiological tetramerization, and perturbing them causes PD-like neurotoxicity. Moreover, the mutants we describe are valuable tools for studying normal and pathological properties of αS and screening for tetramer-stabilizing therapeutics. |
DOI | 10.1073/pnas.1505953112 |
Alternate Journal | Proc. Natl. Acad. Sci. U.S.A. |
PubMed ID | 26153422 |
PubMed Central ID | PMC4534262 |
Grant List | R01 NS083845 / NS / NINDS NIH HHS / United States R01 NSNS083845 / / PHS HHS / United States |
Submitted by api_import on December 20, 2019 - 1:39pm