Project description:WAC is a known positive regulator of (macro)autophagy. WAC also forms a complex with RNF20/RNF40 to promote H2B monoubiquitination and hence to affect transcriptional regulation. This study addresses whether the WAC/RNF20/RNF40 complex regulates autophagy through effects on gene expression. WAC, RNF20 and RNF40 were knocked-down using pools of siRNAs in HEK293A cells. Each knockdown was in triplicate and the control was RISCfree siRNA. mRNA expression profiles were investigated using an Illumina HT12v4 Bead Array.
Project description:Hydrogen peroxide (H2O2) reacts, directly or indirectly, with cysteines to form cysteine sulfenic acid, also known as S-sulfenylation. This cysteine oxidation steers diverse cellular processes by altering protein interactions, trafficking, conformation, and function. We present a method to identify S-sulfenylated cysteines sites in proteins using a genetic probe based on the yeast AP-1–like (YAP1) transcription factor that specifically reacts with sulfenic acid sites to form mixed disulfides. After a tryptic digest and with the help of an antibody directed against a 7-amino-acid YAP1C-derived peptide that contains the redox-active cysteine, we enriched for disulfide-linked peptides. The mass spectral characteristics for fragment ions of the mixed disulfides made it possible to identify 1,747 S-sulfenylation protein sites in Arabidopsis under H2O2 stress. Furthermore, cross-study comparison shows that 55% of cross-linked sites match with previously reported S-sulfenylated, S-nitrosylated and reversibly oxidized cysteines in Arabidopsis. These include well-characterized redox-sensitive cysteines, such as Cys20 of DEHYDROASCORBATE REDUCTASE 2 (DHAR2) and Cys181 of MAP KINASE 4 (MAPK4). Altogether, we describe a novel approach to identify S-sulfenylated sites in situ using the YAP1C probe, thereby offering a non-invasive manner to study site-specific cysteine oxidation and which can be applied for identification of S-sulfenylated sites at the organellar level.