Bilirubin directly activates RIPK3 to induce non-classical necroptosis.

Bilirubin, a byproduct of heme metabolism, is traditionally recognized for its antioxidant properties. However, its accumulation under pathological conditions can result in severe clinical manifestations, including neurotoxicity. Here, we report that bilirubin acts as an activator of receptor-interacting protein kinase 3 (RIPK3), inducing necroptosis and contributing to neurotoxic effects. Unlike the canonical pathway, in which RIPK3-mediated necroptosis is dependent on RIPK1, bilirubin directly binds to the D161 and S102 residues within the kinase domain of RIPK3. This binding facilitates RIPK3 oligomerization and autophosphorylation, leading to the activation of MLKL, the primary effector of necroptosis. Notably, bilirubin-induced necroptosis does not require the RHIM domain of RIPK3 or other RHIM-containing proteins, such as ZBP1 and TRIF. Functionally, the depletion of Ripk3 reduces bilirubin-induced neurotoxicity in murine models. These findings reveal a previously unrecognized mechanism of RIPK3-mediated non-canonical necroptosis, establishing it as a key mediator of bilirubin-induced neurotoxicity.