Poly(ADP-ribose) (PAR) binding to apoptosis-inducing factor is critical for PAR polymerase-1-dependent cell death (parthanatos)

Abstract

The mitochondrial protein apoptosis-inducing factor (AIF) plays a pivotal role in poly(ADP-ribose) polymerase-1 (PARP-1)-mediated cell death (parthanatos), during which it is released from the mitochondria and translocates to the nucleus. We show that AIF is a high-affinity poly(ADP-ribose) (PAR)-binding protein and that PAR binding to AIF is required for parthanatos both in vitro and in vivo. AIF bound PAR at a site distinct from AIF's DNA binding site, and this interaction triggered AIF release from the cytosolic side of the mitochondrial outer membrane. Mutation of the PAR binding site in AIF did not affect its NADH (reduced form of nicotinamide adenine dinucleotide) oxidase activity, its ability to bind FAD (flavin adenine dinucleotide) or DNA, or its ability to induce nuclear condensation. However, this AIF mutant was not released from mitochondria and did not translocate to the nucleus or mediate cell death after PARP-1 activation. These results suggest a mechanism for PARP-1 to initiate AIF-mediated cell death and indicate that AIF's bioenergetic cell survival-promoting functions are separate from its effects as a mitochondrially derived death effector. Interference with the PAR-AIF interaction or PAR signaling may provide notable opportunities for preventing cell death after activation of PARP-1.

Fig. 1. PAR binds to AIF

(A) Overlay assay of mouse AIF and biotin-labeled PAR. H3 and BSA were used as positive and negative controls, respectively. n = 3. (B) EMSA of AIF using ³²P-labeled PAR. Histone H1 was used as a positive control, n = 3. (C) Co-immunoprecipitation of WT-AIF-Flag with PAR in post-nuclear fractions isolated from HeLa cells 2 h after MNNG treatment (50 μM for 15 min). rIgG, rabbit IgG. n = 3. (D) Co-immunoprecipitation of endogenous AIF with PAR in post-nuclear fractions isolated from cortical neurons 2 h after NMDA treatment (500 μM for 5 min). The intensity of AIF signal was quantified and normalized to input (right panel). *** p < 0.001 by Student’s t-test, n = 6. (E) Dot-blot analysis of full length WT-AIF, three AIF peptides, histone H3, and BSA in presence of 100 nM purified ³²P-labeled PAR, n = 4. (F) Overlay assay of His-tagged WT-AIF, AIFm222-244, AIFm441-463, AIFm567-592, AIFΔ567-592, and AIF (K254A, R264A) mutants with ³²P-labeled automodified PARP-1 (equivalent to 100 nM of PAR), purified ³²P-labeled PAR (100 nM), or purified ³²P-labeled PAR with either a 100-fold excess of DNA or cold PAR.

Fig. 2. Mouse AIF structure

(A) Ribbon diagram of the AIF structure showing the three domains (D1 in blue, D2 in cyan and D3 in green) along with the basic helix-loop-helix domain constituting the PAR-binding domain (in red). Side chains of essential basic amino acids are indicated. Modified from Mate et al. (20) (B) Structural details of the PAR-binding domain of AIF. Stabilizing hydrogen bonds are shown in pink. D, aspartic acid; E, glutamic acid; G, glycine; I, isoleucine; Q, glutamine. (C) The surface and electrostatic potential of AIF. DNA binding sites are shown in blue and the PAR binding sites that do not overlap with the DNA binding sites are shown in red.

Fig. 3. Determination of amino acids in AIF responsible for PAR binding

(A) PAR overlay assay of full length WT and mutant AIFs (amino acid 567–592 sequences shown in upper panel). The radioactive signal was quantified and normalized to WT-AIF (lower panel). n = 3. V, valine; W, tryptophan; N, asparagine; F, phenylalanine; M, methionine; P, proline; X, any amino acid; H, hydrophobic amino acid; B, basic amino acid. (B) Dot-blot analysis of WT and mutant AIF peptides (sequences shown in upper panel), and p21, a peptide that binds strongly to PAR, (43) in the presence of 100 nM purified ³²P-labeled PAR, n = 3. (C) EMSA of WT-AIF and Pbm-AIF using ³²P-labeled PAR, n = 3. (D) [³²P]-PAR bound to WT-AIF or Pbm-AIF was analyzed in 20% TBE-PAGE. Values represent ADP-ribose units. The radioactive signal was quantified and normalized to Input.*** p < 0.001 by one-way ANOVA, n = 5. (E) Pulldown assay of WT-AIF and Pbm-AIF using biotin-labeled PAR-conjugated NeutrAvidin beads. W/O, buffer. Beads, NeutrAvidin beads without biotin-labeled PAR polymer, n = 4.

Fig. 4. Characterization of PAR-independent properties of WT-AIF and Pbm-AIF

(A) NADH oxidase activity of His-WT-AIF and His-Pbm-AIF was visualized on native gel by NBT reduction. BSA was used as a negative control. (B) NADH oxidase activity of His-WT-AIF and His-Pbm-AIF was determined by monitoring the changes in absorbance at OD 340 nm. (C) FAD binding of His-WT-AIF and His-Pbm-AIF was determined by spectrophotometric wave length scanning of purified proteins. (D) DNA retardation assay of His-WT-AIF and His-Pbm-AIF by incubating with DNA (200 ng) for 30 min. BSA was used as a negative control. (E) Non-tagged WT-AIF and Pbm-AIF cause chromatin condensation and nuclear shrinkage in isolated HeLa cell nuclei. Scale bar, 20 μm. (F) Quantification of the number of nuclei treated by WT-AIF, Pbm-AIF, and control (CTL) in E. Caspase 3 (Casp3) was applied as a positive control, n = 5.

Figure 5. Pbm-AIF is resistant to release from mitochondria and subsequent nuclear translocation

(A) Co-immunoprecipitation of PAR with WT-AIF-Flag and Pbm-AIF-Flag in post-nuclear fractions isolated from Hq cortical neurons 2 h after NMDA treatment (500 μM for 5 min), rabbit IgG (rIgG). (B) The signal was quantified and normalized to input. n = 5. (C) Subcellular localization of WT-AIF-Flag and Pbm-AIF-Flag in Hq cortical neurons 2 h after NMDA treatment, n = 4. (D) NMDA-induced AIF nuclear translocation in cortical neurons. DAPI, nuclei staining. Scale, 20 μm, n = 3. (E) Mitochondria isolated from cortical neurons were incubated with PAR for 30 min. The levels of AIF were determined in the mitochondria (left) and in the supernatant (right), n = 6. (F & G) WT-AIF-Flag or Pbm-AIF-Flag (500 ng/ml) were incubated with isolated mitochondria (1 mg/ml) in the absence or presence of PAR for 30 min. The supernatant was collected for analyses of AIF, Tom20, and Cyt C. AIF binding to mitochondria in the absence of PAR was regarded as 100% binding. ^##p < 0.01, ^###p < 0.001, compared to control (absence of PAR); * p < 0.05, **p < 0.01, ***p <0.001 by one-way ANOVA, n = 5.

Figure 6. WT-AIF, but not Pbm-AIF, sensitizes cells to parthanatos in vitro

(A) The effect of WT-AIF-Flag or Pbm-AIF-Flag on NMDA-induced cytotoxicity in Hq cortical neurons 24 h after NMDA (500 μM for 5 min). Cells were transduced with lentivirus carrying WT-AIF-Flag or Pbm-AIF-Flag. Non-transduced neurons and GFP-lentivirus infected cells were used as negative controls. Blue indicates Hoechst 33342 staining and red indicates propidium iodide staining. (B) Cytotoxicity in Hq cortical neurons was determined at 24 h, 36 h, and 48 h after NMDA (500 μM for 5 min) treatment. (C) DPQ (30 μM) but not zVAD (100 μM) inhibited NMDA-induced cortical neuron death. *p < 0.05, ***p < 0.001 as compared to its control group treated with CSS. ^###p < 0.001, n = 4. By one-way ANOVA analysis. Neurons prepared from wild type mice were used as a control.

Figure 7. Pbm-AIF, but not WT-AIF, protects cells from parthanatos in vivo

(A & B) Nissl staining and lesion volume of WT and Hq mice 60 h after 20 nmols NMDA injection in striatum. **p < 0.01 by Student’s t-test, n = 6 mice. (C) Time windows for virus injection. (D) Transduction of WT-AIF-Flag, Pbm-AIF-Flag, or GFP in striatum of Hq mouse brains. (E) Nissl staining of WT-AIF-, Pbm-AIF-, and GFP-lentivirus-, and saline-injected mice 60 h after NMDA injection in striatum. A dotted line demarcates the striatum for panels A, D and E. (F) Lesion volumes of WT-AIF-, Pbm-AIF-, GFP-, and saline-injected mice assessed at 48 h, 60 h, and 168 h after injection of NMDA or saline. n (WT-AIF) = 11, n (Pbm-AIF) = 10, n (saline) = 9, n (GFP) =9. (G) Transduction of WT-AIF-Flag, Pbm-AIF-Flag, and GFP in CA1 of Hq mouse brains. (H & I) Nissl staining and lesion volumes of WT-AIF, Pbm-AIF, and GFP AAV2 injected mice 60 h after NMDA injection in CA1. (**p < 0.01, as compared to saline injection. ***p < 0.001, #p < 0.05, by one-way ANOVA, n (WT-AIF) = 4, n (Pbm-AIF) = 3, n (GFP) =3.

Figure 8. Model of PAR-dependent AIF release in parthanatos

The scheme shows that DNA damage induced by MNNG administration (or other alkylating agents, as indicated by the “…”) or NMDA excitotoxicity activates PARP-1, which catalyzes PAR formation. PAR then translocates from the nucleus to the cytosol and mitochondria where it binds to a pool of AIF that is on the cytosolic side of the outer membrane of the mitochondria (25) inducing its release. AIF then translocates to nucleus and causes cell death. In contrast, PAR fails to bind Pbm-AIF and Pbm-AIF is not released during PARP-1 activation and cells survive the toxic stimuli.