Research Article

A nuclease that mediates cell death induced by DNA damage and poly(ADP-ribose) polymerase-1

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Science  07 Oct 2016:
Vol. 354, Issue 6308, aad6872
DOI: 10.1126/science.aad6872

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DNA damage-activated nuclease identified

Cells that experience stresses and accumulate excessive damage to DNA undergo cell death mediated by a nuclear enzyme known as PARP-1. During this process, apoptosis-inducing factor (AIF) translocates to the nucleus and activates one or more nucleases to cleave DNA. Wang et al. found that macrophage migration inhibitory factor (MIF) is an AIF-associated endonuclease that contributes to PARP-1-induced DNA fragmentation (see the Perspective by Jonas). In mouse neurons in culture, loss of MIF protected neurons from cell death caused by excessive stimulation. Targeting MIF could thus provide a therapeutic strategy against diseases in which PARP-1 activation is excessive.

Science, this issue p. 82; see also p. 36

Structured Abstract

INTRODUCTION

Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is a nuclear enzyme responding to oxidative stress and DNA damage. Excessive activation of PARP-1 causes an intrinsic caspase-independent cell death program designated parthanatos, which occurs in many organ systems because of toxic or stressful insults, including ischemia-reperfusion injury after stroke and myocardial infarction, inflammatory injury, reactive oxygen species–induced injury, glutamate excitotoxicity, and neurodegenerative diseases. Inhibition or genetic deletion of PARP-1 is profoundly protective against such cellular injury in models of human disease.

RATIONALE

The molecular mechanisms underlying parthanatos involve release of mitochondrial apoptosis-inducing factor (AIF) and its translocation to the nucleus, which results in chromatinolysis into 20- to 50-kb large DNA fragments—a commitment point for parthanatos. Because AIF itself has no obvious nuclease activity, we propose that AIF recruits a nuclease or a nuclease complex to the nucleus to trigger DNA cleavage and parthanatos. Although the endonuclease G (EndoG) homolog may promote DNA degradation in Caenorhabditis elegans through cooperating with the AIF homolog, our group and others showed that EndoG does not have an essential role in PARP-dependent chromatinolysis and cell death in mammals. Thus, the identity of the nuclease responsible for large DNA fragmentation following AIF entry to the nucleus during parthanatos has been a long-standing mystery.

RESULTS

Using two sequential unbiased screens, including a human protein array and a small interfering RNA screen, we discovered that macrophage migration inhibitory factor (MIF) binds AIF and is required for parthanatos. Three-dimensional modeling of MIF revealed that the MIF trimer has the same core topology structure as PD-D/E(X)K superfamily nucleases. In the presence of Mg2+ or Ca2+, MIF has both 3′ exonuclease and endonuclease activity. It binds to 5′ unpaired bases of single-stranded DNA with stem loop structure and cleaves its 3′ unpaired bases. These nuclease activities allow MIF to cleave genomic DNA into large fragments. Depletion of MIF markedly reduced chromatinolysis and cell death induced by N-methyl-d-aspartate (NMDA) receptor–activated glutamate excitotoxicity in primary neuronal cultures, DNA damage caused by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or focal stroke in mice. Mutating key amino acid residues in the PD-D/E(X)K nuclease domain of MIF eliminated its nuclease activity and prevented parthanatos. Disrupting the AIF and MIF interaction prevented the translocation of MIF from the cytosol to the nucleus and protected against parthanatos. Moreover, depletion of MIF, disruption of AIF and MIF interaction, and eliminating MIF’s nuclease activity has long-lasting histological and behavioral rescue in the focal ischemia model of stroke.

CONCLUSION

We identified MIF as a PARP-1–dependent AIF-associated nuclease that is required for parthanatos. In response to oxidative stress or DNA damage, PARP-1 activation triggers AIF release from the mitochondria. AIF then recruits MIF to the nucleus where MIF cleaves genomic DNA into large fragments and causes cell death. Depletion of MIF, disruption of AIF and MIF interaction, or blocking MIF nuclease activity inhibited chromatinolysis and parthanatos. Targeting MIF nuclease activity may offer an important therapeutic opportunity for a variety of disorders with excessive PARP-1 activation.

Stressors lead to DNA damage, PARP-1 activation, and PAR formation. PAR facilitates the release of AIF from mitochondria where it binds MIF. This complex translocates to the nucleus to bind DNA; the result is DNA fragmentation and cell death. Interference with this cascade by preventing the formation of the AIF-MIF complex or by a nuclease-deficient MIF prevents DNA fragmentation and promotes cell survival.

Abstract

Inhibition or genetic deletion of poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is protective against toxic insults in many organ systems. The molecular mechanisms underlying PARP-1–dependent cell death involve release of mitochondrial apoptosis-inducing factor (AIF) and its translocation to the nucleus, which results in chromatinolysis. We identified macrophage migration inhibitory factor (MIF) as a PARP-1–dependent AIF-associated nuclease (PAAN). AIF was required for recruitment of MIF to the nucleus, where MIF cleaves genomic DNA into large fragments. Depletion of MIF, disruption of the AIF-MIF interaction, or mutation of glutamic acid at position 22 in the catalytic nuclease domain blocked MIF nuclease activity and inhibited chromatinolysis, cell death induced by glutamate excitotoxicity, and focal stroke. Inhibition of MIF’s nuclease activity is a potential therapeutic target for diseases caused by excessive PARP-1 activation.

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