Proteomic Cysteine Profiling Links DMI and DMF to Antiviral Activity

Dimethyl fumarate (DMF) and dimethyl itaconate (DMI) are electrophilic metabolite derivatives with potent immunomodulatory properties. Both have been reported to covalently modify cysteine residues in proteins, thereby altering their structure and function. Thus, we hypothesized that DMI and DMF share common cysteine targets that account for their overlapping immunosuppressive effects. Using tandem mass tag activity-based protein profiling (TMT-ABPP) in mouse embryonic fibroblasts (MEFs), we systematically mapped cysteine sites whose activity toward an iodoacetamide-desthiobiotin probe was altered by DMI and DMF in MEF lysates. A subset of these targets responded to both compounds, and the proteins carrying them included proteins directly involved in immune signaling such as RelA (p65/NF-κB) and the cytosolic RNA sensor Ddx58 (RIG-I). Functional analyses demonstrated that both compounds suppressed RIG-I-dependent antiviral responses, leading to inhibition of type I interferon induction as well as impaired NF-κB nuclear translocation and transcriptional activation. Importantly, these effects were observed in nonimmune stromal cells, highlighting a broader regulatory role beyond professional immune cells. Our findings nominate RIG-I as a shared candidate target of DMI and DMF and associate altered cysteine reactivity with the dampening of antiviral and inflammatory signaling. This work provides new mechanistic insights into the immunosuppressive actions of these electrophiles and highlights their potential as chemical probes and therapeutic leads for modulating innate immunity.