Dehydration/1,6-addition-based Site-specific Bioconjugation Unveils Norepinephrinylation as a Widespread Post-translational Modification in the Cellular Proteome

Norepinephrine (NE) is a key neurotransmitter and hormone involved in diverse physiological and pathological processes. Beyond its canonical non-covalent signaling through adrenergic receptors, NE also induces protein post-translational modifications (PTMs), representing an emerging regulatory mechanism. Two major forms of NE-derived PTMs have been identified: non-enzymatic norepinephrinylation (NEylation) of cysteine residues mediated by NE quinone and transglutaminase 2 (TG2)-catalyzed NEylation of glutamine residues. However, the biochemical basis and pathophysiological roles of NEylation remain poorly understood due to limited detection tools. Here, we report a bioorthogonal reaction for selective labeling and enrichment of the NEylation proteome in cell lines and tissues, which is based on acid-catalyzed dehydration and 1,6-addition to thiol probes. This strategy enables fluorescence imaging and chemical proteomic profiling, revealing NEylation as a widespread PTM that affects enzymatic activities of modified proteins, including protein tyrosine-protein phosphatase non-receptor type 11 (PTPN11). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/731782v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@19dce3aorg.highwire.dtl.DTLVardef@14e7f30org.highwire.dtl.DTLVardef@80354aorg.highwire.dtl.DTLVardef@12ac6f9_HPS_FORMAT_FIGEXP M_FIG C_FIG