AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX

Abstract

Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543-559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.

BID regulates AIF-mediated caspase-independent necroptosis by promoting BAX activation

“…This approach showed that MNNG Figure 2D). Overtime, similarly to what has been previously reported for MNNG-treated H2AX À/À MEFs, 14 it seems that the cell is unable to achieve the necroptotic PCD program in the absence of BID. As a result, the 'highly damaged' cell, which is unable to proliferate/divide (Supplementary Figure 3), explodes by an uncontrolled form of necrosis.…”

“…8 Once in mitochondria, BAX provokes a mitochondrial damage that is accompanied by the release of tAIF, the truncated form of AIF, to the cytosol and nucleus (Figures 1d and e). 8,14 In the nucleus, this apoptogenic protein generates TUNELdetectable 3 0 -OH DNA breaks in DNA (Figure 1f). Altogether, these events represent the biochemical hallmarks of MNNG-mediated necroptosis.…”

“…Altogether, these events represent the biochemical hallmarks of MNNG-mediated necroptosis. 8,14 BAX activation is critical in MNNG-induced necroptosis. The presence of tAIF in extramitochondrial compartments is vital in the PCD process triggered by MNNG.…”

“…[5][6][7] This type of cell death, enabled here by the action of PARP-1, is a caspase-independent process with significant alterations detected as soon as 6 h of MNNG treatment ( Figure 1a). 8,14 As in staurosporine (STS)-induced caspase-dependent apoptosis (our positive control), MNNG-treated cells undergo Annexin V-detectable phosphatidylserine (PS) exposure. However, in contrast to STS-induced PCD, MNNG treatment provokes a double Annexin V/PI(propidium iodide)-positive labeling (Figure 1a), which is indeed a hallmark of this type of PCD.…”

“…[9][10][11][12][13] Cytosolic tAIF rapidly redistributes to the nuclear compartment where, assisted by gH2AX and cyclophilin A (CypA), it promotes chromatinolysis and cell-viability loss. 14,15 Among the features of MNNG-induced necroptosis, the implication of BCL-2 family members appears as a milestone. The BCL-2 set of proteins is distributed in three subgroups according to their properties.…”