Figure 1: S-nitrosylation of UCHL1 in neuroblastoma SH-SY5Y cells and in recombinant UCHL1.
From: S-nitrosylation of UCHL1 induces its structural instability and promotes α-synuclein aggregation
(a) Biotin switch assay was performed after 16 h exposure of rotenone (1 μM) to SH-SY5Y cells. Lower panel represents loading control. For specificity of this assay sodium ascorbate (20 mM) was omitted in right lane. MMTS was used for effective blocking of free thiols to minimize background biotinylation and false signal (b) nNOS was activated in SH-SY5Y cells by NMDA (50 μM) and calcium ionophore A23187 (5 μM) separately and in presence and absence of NOS inhibitor L-NAME. (c) Nitrosylation quantification was done by incubating UCHL1 (10 μM) with GSNO (10 molar excess) for 15 min at 37 °C. S-nitrosylated UCHL1 generated was analyzed by release of NO, causing the conversion of NO to NAT. Values are mean ± s.e.m., n ≥ 3; ***P < 0.0001 by one way analysis of variance with Bonferroni post hoc test. (d) Nitrosylation in recombinant UCHL1. Biotin switch assay was performed after incubating 2 mg/ml UCHL1 with 10 molar excess GSNO. Lower panel represents loading control. UCHL1 protein is detected by anti-UCHL1 antibody after streptavidin pull down. (e–g) Mass spectrometry (MALDI MS) analysis identifies C90, C152 and C220 as nitrosylation site. MS/MS spectra of trypsin digested peptide fragments (e) QTIGNSCGTIGLIHAVANNQDK (f) NEAIQAAHDAVAQEGQCR and (g) FSAVALCK were shown to be modified with HPDP Biotin. (h) Crystal structure of UCHL1 (PDB 2ETL), nitrosylated cysteine shown as yellow spheres and unmodified cysteine as brown spheres. Uncropped western blots were presented in Supplementary Fig. S9.
