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Poly(ADP-ribosyl)ation of p53 contributes to TPEN-induced neuronal apoptosis.

Kim HL, Ra H, Kim KR, Lee JM, Im H, Kim YH - Mol. Cells (2015)

Bottom Line: Poly(ADP-ribosyl)ation of p53 occurred starting 1 h after TPEN treatment.Consistent with this, the induction of downstream proapoptotic proteins PUMA and NOXA was noticeably reduced by chemical inhibitors or genetic deletion of PARP-1.Taken together, these findings indicate that PARP-1 is essential for TPEN-induced neuronal apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Sejong University, Seoul 143-747, Korea.

ABSTRACT
Depletion of intracellular zinc by N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces p53-mediated protein synthesis-dependent apoptosis of mouse cortical neurons. Here, we examined the requirement for poly(ADP-ribose) polymerase (PARP)-1 as an upstream regulator of p53 in zinc depletion-induced neuronal apoptosis. First, we found that chemical inhibition or genetic deletion of PARP-1 markedly attenuated TPEN-induced apoptosis of cultured mouse cortical neurons. Poly(ADP-ribosyl)ation of p53 occurred starting 1 h after TPEN treatment. Suggesting the critical role of PARP-1, the TPEN-induced increase of stability and activity of p53 as well as poly(ADP-ribosyl)ation of p53 was almost completely blocked by PARP inhibition. Consistent with this, the induction of downstream proapoptotic proteins PUMA and NOXA was noticeably reduced by chemical inhibitors or genetic deletion of PARP-1. TPEN-induced cytochrome C release into the cytosol and caspase-3 activation were also blocked by inhibition of PARP-1. Taken together, these findings indicate that PARP-1 is essential for TPEN-induced neuronal apoptosis.

No MeSH data available.


Related in: MedlinePlus

Post-translational modification of p53 by PARP-1 in TPEN-induced neuronal apoptosis. (A) Immunoprecipitation (IP) and immunoblotting over the time course of p53 PARylation. Protein samples were prepared from nearly pure cortical neuron cultures at the indicated time points after TPEN treatment. Protein extracts were immunoprecipitated with p53 antibody and analyzed by SDS-PAGE and immunoblotting with poly(ADP-ribose) (PAR) antibody. PARylation bands of p53 were detected from 1 hr after TPEN treatment. To show that the same amount of protein extract was used in IP, 30 μl of protein (1 μg/μl) was prepared before IP incubation and analyzed using actin antibody. (B) IP and immunoblotting for p53 PARylation. Protein samples were prepared after 4-h exposure to TPEN with or without NAM or AB. PARylation of p53 was almost completely blocked by PARP inhibitors. (C) PARylation of p53 in PARP-1+/+ or PARP-1−/− mouse cortical neuron cultures. Protein samples were prepared after 4-h exposure to sham wash (CTRL) or TPEN and then immunoprecipitated with p53 antibody. PARylation and accumulation of p53 by TPEN were not induced in PARP-1−/− mouse cortical neuron cultures. (D) Western blot analysis of p53 phosphorylation (upper) and accumulation (lower). Protein samples were prepared after 6-h exposure to TPEN with or without NAM or AB. PARP inhibitors markedly attenuated the TPEN-induced increase in p53 activity and stability. (E) Western blot analysis of p53 phosphorylation (upper) and accumulation (lower) in PARP-1+/+ or PARP-1−/− mouse cortical neuron cultures. Protein samples were prepared after 6-h exposure to sham wash (CTRL) or TPEN. The increase of p53 activity and stability by TPEN was not detected in PARP-1 deficient neuronal cultures.
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f2-molce-38-4-312: Post-translational modification of p53 by PARP-1 in TPEN-induced neuronal apoptosis. (A) Immunoprecipitation (IP) and immunoblotting over the time course of p53 PARylation. Protein samples were prepared from nearly pure cortical neuron cultures at the indicated time points after TPEN treatment. Protein extracts were immunoprecipitated with p53 antibody and analyzed by SDS-PAGE and immunoblotting with poly(ADP-ribose) (PAR) antibody. PARylation bands of p53 were detected from 1 hr after TPEN treatment. To show that the same amount of protein extract was used in IP, 30 μl of protein (1 μg/μl) was prepared before IP incubation and analyzed using actin antibody. (B) IP and immunoblotting for p53 PARylation. Protein samples were prepared after 4-h exposure to TPEN with or without NAM or AB. PARylation of p53 was almost completely blocked by PARP inhibitors. (C) PARylation of p53 in PARP-1+/+ or PARP-1−/− mouse cortical neuron cultures. Protein samples were prepared after 4-h exposure to sham wash (CTRL) or TPEN and then immunoprecipitated with p53 antibody. PARylation and accumulation of p53 by TPEN were not induced in PARP-1−/− mouse cortical neuron cultures. (D) Western blot analysis of p53 phosphorylation (upper) and accumulation (lower). Protein samples were prepared after 6-h exposure to TPEN with or without NAM or AB. PARP inhibitors markedly attenuated the TPEN-induced increase in p53 activity and stability. (E) Western blot analysis of p53 phosphorylation (upper) and accumulation (lower) in PARP-1+/+ or PARP-1−/− mouse cortical neuron cultures. Protein samples were prepared after 6-h exposure to sham wash (CTRL) or TPEN. The increase of p53 activity and stability by TPEN was not detected in PARP-1 deficient neuronal cultures.

Mentions: Next, we investigated the PARylation of p53 in TPEN-induced neuronal apoptosis. Substantial PARylation of p53 was detected 1 h after TPEN treatment (Fig. 2A) and blocked by PARP inhibitors (Fig. 2B). In PARP-1−/− cortical neuron cultures, PARylation by TPEN was not detected (Fig. 2C). Consistent with this observation, TPEN-induced accumulation and phosphorylation of p53 were markedly attenuated by chemical inhibitors (Fig. 2D) or genetic deletion of PARP-1 (Fig. 2E). These results strongly suggest that PARP-1 regulates the stability and activity of p53 via post-translational modification (i.e., PARylation) in TPEN-induced neuronal apoptosis.


Poly(ADP-ribosyl)ation of p53 contributes to TPEN-induced neuronal apoptosis.

Kim HL, Ra H, Kim KR, Lee JM, Im H, Kim YH - Mol. Cells (2015)

Post-translational modification of p53 by PARP-1 in TPEN-induced neuronal apoptosis. (A) Immunoprecipitation (IP) and immunoblotting over the time course of p53 PARylation. Protein samples were prepared from nearly pure cortical neuron cultures at the indicated time points after TPEN treatment. Protein extracts were immunoprecipitated with p53 antibody and analyzed by SDS-PAGE and immunoblotting with poly(ADP-ribose) (PAR) antibody. PARylation bands of p53 were detected from 1 hr after TPEN treatment. To show that the same amount of protein extract was used in IP, 30 μl of protein (1 μg/μl) was prepared before IP incubation and analyzed using actin antibody. (B) IP and immunoblotting for p53 PARylation. Protein samples were prepared after 4-h exposure to TPEN with or without NAM or AB. PARylation of p53 was almost completely blocked by PARP inhibitors. (C) PARylation of p53 in PARP-1+/+ or PARP-1−/− mouse cortical neuron cultures. Protein samples were prepared after 4-h exposure to sham wash (CTRL) or TPEN and then immunoprecipitated with p53 antibody. PARylation and accumulation of p53 by TPEN were not induced in PARP-1−/− mouse cortical neuron cultures. (D) Western blot analysis of p53 phosphorylation (upper) and accumulation (lower). Protein samples were prepared after 6-h exposure to TPEN with or without NAM or AB. PARP inhibitors markedly attenuated the TPEN-induced increase in p53 activity and stability. (E) Western blot analysis of p53 phosphorylation (upper) and accumulation (lower) in PARP-1+/+ or PARP-1−/− mouse cortical neuron cultures. Protein samples were prepared after 6-h exposure to sham wash (CTRL) or TPEN. The increase of p53 activity and stability by TPEN was not detected in PARP-1 deficient neuronal cultures.
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f2-molce-38-4-312: Post-translational modification of p53 by PARP-1 in TPEN-induced neuronal apoptosis. (A) Immunoprecipitation (IP) and immunoblotting over the time course of p53 PARylation. Protein samples were prepared from nearly pure cortical neuron cultures at the indicated time points after TPEN treatment. Protein extracts were immunoprecipitated with p53 antibody and analyzed by SDS-PAGE and immunoblotting with poly(ADP-ribose) (PAR) antibody. PARylation bands of p53 were detected from 1 hr after TPEN treatment. To show that the same amount of protein extract was used in IP, 30 μl of protein (1 μg/μl) was prepared before IP incubation and analyzed using actin antibody. (B) IP and immunoblotting for p53 PARylation. Protein samples were prepared after 4-h exposure to TPEN with or without NAM or AB. PARylation of p53 was almost completely blocked by PARP inhibitors. (C) PARylation of p53 in PARP-1+/+ or PARP-1−/− mouse cortical neuron cultures. Protein samples were prepared after 4-h exposure to sham wash (CTRL) or TPEN and then immunoprecipitated with p53 antibody. PARylation and accumulation of p53 by TPEN were not induced in PARP-1−/− mouse cortical neuron cultures. (D) Western blot analysis of p53 phosphorylation (upper) and accumulation (lower). Protein samples were prepared after 6-h exposure to TPEN with or without NAM or AB. PARP inhibitors markedly attenuated the TPEN-induced increase in p53 activity and stability. (E) Western blot analysis of p53 phosphorylation (upper) and accumulation (lower) in PARP-1+/+ or PARP-1−/− mouse cortical neuron cultures. Protein samples were prepared after 6-h exposure to sham wash (CTRL) or TPEN. The increase of p53 activity and stability by TPEN was not detected in PARP-1 deficient neuronal cultures.
Mentions: Next, we investigated the PARylation of p53 in TPEN-induced neuronal apoptosis. Substantial PARylation of p53 was detected 1 h after TPEN treatment (Fig. 2A) and blocked by PARP inhibitors (Fig. 2B). In PARP-1−/− cortical neuron cultures, PARylation by TPEN was not detected (Fig. 2C). Consistent with this observation, TPEN-induced accumulation and phosphorylation of p53 were markedly attenuated by chemical inhibitors (Fig. 2D) or genetic deletion of PARP-1 (Fig. 2E). These results strongly suggest that PARP-1 regulates the stability and activity of p53 via post-translational modification (i.e., PARylation) in TPEN-induced neuronal apoptosis.

Bottom Line: Poly(ADP-ribosyl)ation of p53 occurred starting 1 h after TPEN treatment.Consistent with this, the induction of downstream proapoptotic proteins PUMA and NOXA was noticeably reduced by chemical inhibitors or genetic deletion of PARP-1.Taken together, these findings indicate that PARP-1 is essential for TPEN-induced neuronal apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Sejong University, Seoul 143-747, Korea.

ABSTRACT
Depletion of intracellular zinc by N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) induces p53-mediated protein synthesis-dependent apoptosis of mouse cortical neurons. Here, we examined the requirement for poly(ADP-ribose) polymerase (PARP)-1 as an upstream regulator of p53 in zinc depletion-induced neuronal apoptosis. First, we found that chemical inhibition or genetic deletion of PARP-1 markedly attenuated TPEN-induced apoptosis of cultured mouse cortical neurons. Poly(ADP-ribosyl)ation of p53 occurred starting 1 h after TPEN treatment. Suggesting the critical role of PARP-1, the TPEN-induced increase of stability and activity of p53 as well as poly(ADP-ribosyl)ation of p53 was almost completely blocked by PARP inhibition. Consistent with this, the induction of downstream proapoptotic proteins PUMA and NOXA was noticeably reduced by chemical inhibitors or genetic deletion of PARP-1. TPEN-induced cytochrome C release into the cytosol and caspase-3 activation were also blocked by inhibition of PARP-1. Taken together, these findings indicate that PARP-1 is essential for TPEN-induced neuronal apoptosis.

No MeSH data available.


Related in: MedlinePlus