Ubiquitin mediated protein degradation is crucial for regulation of cell signaling and protein quality control. Poly(ADP-ribose) (PAR) is a cell-signaling molecule that mediates changes in protein function through binding at PAR binding sites. Here we characterize the PAR binding protein, Iduna, and show that it is a PAR-dependent ubiquitin E3 ligase. Iduna’s E3 ligase activity requires PAR binding because point mutations at Y156A and R157A eliminate Iduna’s PAR binding and Iduna’s E3 ligase activity. Iduna’s E3 ligase activity also requires an intact really interesting new gene (RING) domain because Iduna possessing point mutations at either H54A or C60A is devoid of ubiquitination activity. Tandem affinity purification reveals that Iduna binds to a number of proteins that are either PARsylated or bind PAR including PAR polymerase-1, 2 (PARP1, 2), nucleolin, DNA ligase III, KU70, KU86, XRCC1, and histones. PAR binding to Iduna activates its E3 ligase function, and PAR binding is required for Iduna ubiquitination of PARP1, XRCC1, DNA ligase III, and KU70. Iduna’s PAR-dependent ubiquitination of PARP1 targets it for proteasomal degradation. Via PAR binding and ubiquitin E3 ligase activity, Iduna protects against cell death induced by the DNA damaging agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and rescues cells from G1 arrest and promotes cell survival after γ-irradiation. Moreover, Iduna facilitates DNA repair by reducing apurinic/apyrimidinic (AP) sites after MNNG exposure and facilitates DNA repair following γ-irradiation as assessed by the comet assay. These results define Iduna as a PAR-dependent E3 ligase that regulates cell survival and DNA repair.
Glutamate acting on N-methyl-D-aspartate (NMDA) receptors plays an important role in neurodegenerative diseases and neuronal injury following stroke, through activation of poly(ADP-ribose) polymerase-1 and generation of the death molecule poly(ADP-ribose) (PAR) polymer. Here we identify Iduna, a novel NMDA receptor-induced survival gene that is neuroprotective against glutamate NMDA receptor mediated excitotoxicity both in vitro and in vivo and against stroke through interfering with PAR polymer induced cell death (parthanatos). Iduna’s protective effects are independent and downstream of PARP-1 activity. Iduna is a PAR polymer binding protein and mutations at the PAR polymer binding site abolishes the PAR binding activity of Iduna and attenuates its protective actions. Iduna is protective in vivo against NMDA-induced excitotoxicity and middle cerebral artery occlusion (MCAO)-induced stroke in mice. These results define Iduna as the first endogenous inhibitor of parthanatos. Interfering with PAR polymer signaling offers a new therapeutic strategy for the treatment of neurologic disorders.
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