Characterizing The Mechanism By Which Hsp90 Regulates TNF-induced Necroptosis

Programmed cell death plays a crucial role in development and adult tissue homeostasis.There are three major forms of cell death:apoptosis,autophagy and necrosis.For a long time,necrosis has been viewed as a negative,uncontrolled type of cell death.However,in recent years,genetic evidence as well as the discovery of chemical inhibitors of necrosis has shown that necrosis is a new type of regulated cell death or programmed necrosis.TNF induced necrotic cell death is named 'necroptosis'.By far,necroptosis is one of the best understood types of regulated necrosis.Upon stimulation of TNFR1(TNF receptor 1)by TNF,RIP1 kinase interacts with RIP3 through its RHIM domain.RIP3 then recruits its downstream signaling target protein MLKL to form a multiple protein complex as a signaling platform called necrosome.Within the necrosome,RIP3 phosphorylates MLKL at the Thr357 and Ser358.This phosphorylation modification changes the conformation of MLKL which leads to MLKL activation.Upon activation,MLKL forms oligomers and translocates to the plasma membrane to execute necrotic cell death through a mechanism not well understood.MLKL is the key component of TNF-inducced necroptosis and plays a crucial role in necroptosis execution.However,the mechanisms that control MLKL activity are not completely understood.In this work,we demonstrated that Hsp90 associates with MLKL in mammalian cells and is essential for MLKL stability.Moreover,we found that Hsp90 also regulates the stability of the upstream RIP3 kinase.Disruption of Hsp90 function with the 17AAG inhibitor destabilizes MLKL and RIP3,resulting in their degradation by the proteasome pathway.Furthermore,we found Hsp90 is required for TNF-induced necroptosis in HT-29 cells.Interference with Hsp90 function with the 17AAG inhibitor prevents necrosome formation and strongly reduces MLKL phosphorylation and inhibits TNF-induced necroptosis in HT-29 cells.Conversely,coexpression of Hsp90 increases MLKL oligomerization and plasma membrane translocation and enhances MLKL-mediated necroptosis in 293T cells.Taken these together,our findings demonstrate that Hsp90 is a crucial regulator in TNF-induced necroptosis.Hsp90 modulates the stabilities of MLKL and RIP3 and controls the formation of the necrosome.Hence,Hsp90 is required for TNF-induced necroptosis.