A substrate trapping approach identifies proteins regulated by reversible S-nitrosylation

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Protein S-nitrosylation is a critical nitric oxide-mediated posttranslational modification impacting cellular processes.
• Limited knowledge exists regarding the S-nitrosoproteome across different cell types and conditions.
• The precise roles of protein S-nitrosylation and denitrosylation require further investigation.

Purpose of the Study:

• To develop and present a novel strategy for identifying reversibly nitrosylated proteins.
• To explore the S-nitrosoproteome in human monocytes and mouse macrophages.
• To investigate the role of thioredoxin in denitrosylation and enzyme activation.

Main Methods:

• Utilized a thioredoxin trap mutant for nitrosothiol capture and enrichment.
• Employed mass spectrometry for protein identification.
• Conducted biochemical and functional experiments in vitro and in cells for validation.

Main Results:

• Identified over 400 putative nitroso-proteins in human monocytes and approximately 200 in mouse macrophages.
• The majority of identified proteins are novel S-nitrosylation targets involved in cellular homeostasis and signaling.
• Validated proteomic findings and suggested thioredoxin's role in denitrosylation and activation of iNOS and MEK1.

Conclusions:

• The novel approach facilitates the identification and exploration of nitroso-proteomes.
• Findings enhance understanding of the macrophage S-nitrosoproteome and nitric oxide signaling.
• Thioredoxin plays a role in denitrosylation and activation of key signaling proteins.