Illuminating the functions of understudied Fructosamine-3-kinase (FN3K) using a multi-omics approach reveals new links to nicotinamide adenine dinucleotides (NAD)

Fructosamine-3-kinases (FN3Ks) are a conserved family of repair enzymes that phosphorylate reactive sugars attached to lysine residues in peptides and proteins. Although FN3Ks are present across the tree of life and share detectable sequence similarity to eukaryotic protein kinases, the biological processes regulated by these kinases are largely unknown. To address this knowledge gap, we leveraged the FN3K CRISPR Knock-Out (KO) cell line alongside an integrative multi-omics study combining transcriptomics, interactomics, metabolomics, and genomics to place these enzymes in a pathway context. The integrative analyses revealed the enrichment of pathways related to oxidative stress response, lipid biosynthesis (cholesterol and fatty acids), carbon and co-factor metabolism, and protein translational machinery. Moreover, statistically significant enrichment of metallothioneins and nicotinamide adenine dinucleotide (NAD) binding proteins suggest potential links between FN3Ks and NAD-mediated energy metabolism and redox balance. We report specific binding of human FN3K to NAD compounds in a metal and concentration-dependent manner and provide insight into their binding mode using modeling and experimental site-directed mutagenesis. By identifying a potential link between FN3Ks, redox regulation, and NAD-dependent metabolic processes, our studies provide a framework for targeting these understudied kinases in diabetic complications and other metabolic disorders. Overall design: To investigate the pathways related to FN3K we Knocked out FN3K gene in HepG2 cells using Crispr-cas. We then performed gene expression profiling analysis obtained from RNA-seq of 3 different samples from the WT and the KO HepG2 cells