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. 2012 Apr 15;443(2):485-90.
doi: 10.1042/BJ20111057.

3'-5' phosphoadenosine phosphate is an inhibitor of PARP-1 and a potential mediator of the lithium-dependent inhibition of PARP-1 in vivo

Elie Toledano  1 , Vasily Ogryzko, Antoine Danchin, Daniel Ladant, Undine Mechold
Affiliations

3'-5' phosphoadenosine phosphate is an inhibitor of PARP-1 and a potential mediator of the lithium-dependent inhibition of PARP-1 in vivo

Elie Toledano et al. Biochem J. .

Abstract

pAp (3'-5' phosphoadenosine phosphate) is a by-product of sulfur and lipid metabolism and has been shown to have strong inhibitory properties on RNA catabolism. In the present paper we report a new target of pAp, PARP-1 [poly(ADP-ribose) polymerase 1], a key enzyme in the detection of DNA single-strand breaks. We show that pAp can interact with PARP-1 and inhibit its poly(ADP-ribosyl)ation activity. In vitro, inhibition of PARP-1 was detectable at micromolar concentrations of pAp and altered both PARP-1 automodification and heteromodification of histones. Analysis of the kinetic parameters revealed that pAp acted as a mixed inhibitor that modulated both the Km and the Vmax of PARP-1. In addition, we showed that upon treatment with lithium, a very potent inhibitor of the enzyme responsible for pAp recycling, HeLa cells exhibited a reduced level of poly(ADP-ribosyl)ation in response to oxidative stress. From these results, we propose that pAp might be a physiological regulator of PARP-1 activity.

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Figures

Figure 1
Figure 1. Physical interactions between pAp and PARP
(A) SDS/PAGE of pAp-affinity chromatography from HeLa cell nuclear extracts. The gel was stained with Colloidal Blue. Lane 1, pAp-binding fraction; lane 2, binding on control agarose beads; lane 3, molecular mass markers. (B) PARP-1-affinity chromatography with AMP-agarose or pAp-agarose. Partially purified PARP-1 (1 μg) was incubated with AMP-agarose (lanes 1–4) or pAp-agarose (lanes 5–8) in the presence (+) or absence (−) of 3 mM pAp. Detection of PARP-1 in the bound (B) or unbound (U) fractions was performed by Western blot analysis. Lane 9, PARP-1 input.
Figure 2
Figure 2. Inhibition of PARP-1 activity by various adenine derivatives
PARP-1 activity was measured as described in the Materials and methods section, in the presence of 400 μM [32P]NAD+ with an incubation time of 7 min. Inhibition of PARP-1 activity was measured in the presence of 50 μM of the indicated adenine nucleotides. The two assays shown on the right-hand side were performed in the presence of 1 mM ATP. Values are means±S.D. for duplicate experiments, and are expressed as the percentage of activity without adenine nucleotides. 100% corresponds to 14 pmol of incorporated ADP-ribose per s. *P<0.05; **P<0.01; ***P<0.001, as compared with the control (with or without 1 mM ATP respectively) calculated using Student's t test.
Figure 3
Figure 3. Kinetic parameters of pAp inhibition of PARP-1 activity
(A) The kcat, defined as mol of NAD+ incorporated/mol of PARP-1 per s, is expressed as a function of the pAp concentration. PARP-1 activity was measured as described in Figure 2. Inset: PhosphorImager autoradiography of poly(ADP-ribosyl)ated PARP-1 blotted on to a nitrocellulose membrane. (B) Activity of PARP-1, expressed as pmol of NAD+ incorporated/μg of PARP-1 per s, as a function of the NAD+ concentration, and in the presence of the pAp concentrations indicated. For each data point ADP-ribose incorporation was measured at 2, 5, 7 and 10 min. The activity was then determined from the slope of the ADP-ribose incorporated as a function of time. (C) Kinetic parameters of PARP-1 deduced from the results shown in (B).
Figure 4
Figure 4. pAp inhibition of histone poly(ADP-ribosyl)ation
PARP-1 catalysed poly(ADP-ribosyl)ation of histone H1 was measured as described in the Materials and methods section in the presence of the indicated concentrations of pAp. The PARP-1 activity (arbitrary unit) is expressed as a percentage of the activity in the absence of pAp.
Figure 5
Figure 5. Inhibition of poly(ADP-ribosyl)ation by lithium treatment
(A) PARP-1 was detected by Western blot analysis in protein extracts from HeLa cells treated or not with lithium. The left-hand lane presents an extract from cells entering apoptosis as a positive control for PARP-1 cleavage. (B) HeLa cells were treated with the lithium concentrations indicated for 4 days (long) or 30 min (short) and were exposed to 800 μM H2O2 for 6 min. Poly(ADP-ribosyl)ation was then measured by immunofluorescence detection of poly(ADP-ribose) polymers as described in the Materials and methods section.
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