FAT-switch-based quantitative S-nitrosoproteomics reveals a key role of GSNOR1 in regulating ER functions

Abstract

Reversible protein S-nitrosylation regulates a wide range of biological functions and physiological activities in plants. However, it is challenging to quantitively determine the S-nitrosylation targets and dynamics in vivo. In this study, we develop a highly sensitive and efficient fluorous affinity tag-switch (FAT-switch) chemical proteomics approach for S-nitrosylation peptide enrichment and detection. We quantitatively compare the global S-nitrosylation profiles in wild-type Arabidopsis and gsnor1/hot5/par2 mutant using this approach, and identify 2,121 S-nitrosylation peptides in 1,595 protein groups, including many previously unrevealed S-nitrosylated proteins. These are 408 S-nitrosylated sites in 360 protein groups showing an accumulation in hot5-4 mutant when compared to wild type. Biochemical and genetic validation reveal that S-nitrosylation at Cys337 in ER OXIDOREDUCTASE 1 (ERO1) causes the rearrangement of disulfide, resulting in enhanced ERO1 activity. This study offers a powerful and applicable tool for S-nitrosylation research, which provides valuable resources for studies on S-nitrosylation-regulated ER functions in plants.

Localization of four class I glutaredoxins in the cytosol and the secretory pathway and characterization of their biochemical diversification

“…In the absence of direct interaction, one can speculate about possible posttranslational modification (PTM) and an indirect regulatory function. In this context, it is interesting that C96 has been reported recently to get nitrosylated although no experimental evidence for a distinct function of this PTM is available yet (Qin et al., 2023). Similarly, the homologous C83 in mammalian GLRX1 was recently reported to be a target of glutathionylation and oxidation by H 2 O 2 in vitro , which causes oxidative inactivation of the protein (Corteselli et al., 2023).…”

CysQuant: Simultaneous quantification of cysteine oxidation and protein abundance using data dependent or independent acquisition mass spectrometry

“…One limitation of the current workflow is that TCEP reduction indiscriminately reduces various oxidative modifications, such as S -nitrosylation, S -sulfenylation, S -glutathionylation, and others. However, using more selective reductive agents such as ascorbate ( S -nitrosothiols) [ 53 ], hydroxylamine ( S -acylation) [ 54 ], or arsenite ( S -sulfenylation) [ 55 ] would also be compatible with the CysQuant workflow. Noteworthy, in case that Cys are subject to sulfinic and sulfonic acid modifications (–SO 2 H and –SO 3 H) that cannot be reversed by TCEP, the calculated oxidation degrees would be an underestimation.…”