Blocking of αSyn tyrosine nitration decreases aggregation and cytotoxicity.

Abstract

<p>(A) Expression of αSyn, A30P, 4(Y/F) and A30P/4(Y/F) αSyn was induced for 12 h in galactose-containing medium and the proteins were enriched by Ni²⁺ pull-down from yeast cell extracts. For <i>in vitro</i> nitration, 1 μl peroxynitrite (PON) was mixed with 15 μg of αSyn extracts in the presence of 1 μl 0.3 M HCl. Western blotting with di-tyrosine antibody reveals a major band at about 36 kDa, corresponding to dimers. Additional bands with lower molecular weights are observed, probably due to intramolecular di-tyrosine crosslinking. The same membrane was stripped and re-probed with αSyn antibody. (B) Western blotting using 3-nitro-tyrosine antibody (3-NT). Phenylalanine codon substitutions eliminate immunoreactivity. The same membrane was stripped and re-probed with αSyn antibody. (C) Spotting analysis of yeast cells expressing <i>GAL1</i>-driven αSyn, A30P, 4(Y/F), A30P/4(Y/F) αSyn and GFP (control). Yeast cells were spotted in 10-fold dilutions on SC-Ura plates containing glucose (αSyn ‘OFF’) or galactose (αSyn ‘ON’). (D) Cell growth analysis of yeast cells expressing αSyn, A30P, 4(Y/F), A30P/4(Y/F) αSyn and GFP (control) in galactose-containing SC-Ura medium for 40 h. Error bars represent standard deviations of three independent experiments. (E) Fluorescence microscopy of yeast cells, expressing indicated αSyn-GFP variants after 6 h of induction in galactose-containing medium. Scale bar: 1 μm. (F) Quantification of the percentage of cells displaying aggregates after 6 h induction in galactose-containing medium. Significance of differences was calculated with t-test (*, <i>p</i> < 0.05, n = 6). (G) Western blotting analysis of protein crude extracts of GFP-tagged αSyn, 4(Y/F), A30P and A30P/4(Y/F) after 6 h induction in galactose-containing medium. GAPDH antibody was used as loading control.</p