EGCG inactivates tumor necrosis factor-alpha (TNFα) by inducing its higher-order assembly

Background Tumor necrosis factor-alpha (TNFα) is an important therapeutic target for treating a range of inflammatory and autoimmune disorders. The TNFα trimer functions by interacting with its receptors (TNFRs) on the immune cell surface and triggers downstream intracellular signaling. (-)-Epigallocatechin-3-gallate (EGCG) is the primary bioactive polyphenol compound in green tea. Our previous study found that EGCG can inhibit TNFα activity; however, the underlying molecular mechanism remains unclear. Purpose In this study, we investigated the interaction between EGCG and TNFα to elucidate the mechanism by which EGCG inactivates TNFα. Methods EGCG-treated TNFα was analyzed using size exclusion chromatography (SEC), multiangle light scattering (MALS), and cryo-electron microscopy (cryo-EM). Mass spectrometry (MS), chemical modifications, and cell-based assays were further conducted to assess the function of the endogenous cysteines. Results EGCG induces assembly of TNFα trimers into higher-order aggregation states, characterized as a non-strictly defined oligomerization. This process involves reorganization of the endogenous disulfide bond (C69-C101) through reduction and subsequent oxidation reactions. The resulting oligomers are incapable of triggering TNFα-TNFR signaling, and capping the free cysteines in TNFα abrogates the inhibitory effect of EGCG. Conclusion These findings reveal the molecular basis for the beneficial effect of EGCG in TNFα-associated inflammatory and autoimmune diseases, giving a new support to the consumption of regular green tea as a dietary therapy. This approach may be particularly relevant in the post-COVID-19 context for managing long COVID symptoms linked to elevated TNFα levels.