Disulfide Bond–Modified Proteomics Reveals the Effects of Riboflavin on Protein Folding Dynamics

ABSTRACT

Riboflavin is a crucial micronutrient essential for maintaining cellular homeostasis, acting as an important precursor for flavoproteins that utilize flavin mononucleotide and flavin adenine dinucleotide as cofactors. Some flavoproteins play roles in the folding of newly synthesized proteins within the endoplasmic reticulum. However, the specific proteins whose folding is influenced by riboflavin remain unidentified. Disulfide bond–modified proteomics to identify proteins whose folding is influenced by riboflavin. Our findings identified 34 proteins in HepG2 cells, a highly riboflavin‐dependent model, with riboflavin‐dependent disulfide bond modifications. These proteins are primarily involved in metabolic pathways such as endoplasmic reticulum protein processing, antigen processing and presentation, glycolysis/gluconeogenesis, lysosomal function, and amino acid biosynthesis. Furthermore, the accumulation of these unfolded or misfolded proteins in the endoplasmic reticulum enhanced the activity of the CHOP promoter, leading to increased expression of the CHOP gene and protein, thereby triggering the cellular apoptosis pathway. For the first time, we have identified riboflavin‐dependent protein folding substrates using disulfide bond–modified proteomics. These findings may provide new perspectives for future therapies targeting endoplasmic reticulum stress–related diseases.