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Structure/function analysis of PARP-1 in oxidative and nitrosative stress-induced monomeric ADPR formation.

Buelow B, Uzunparmak B, Paddock M, Scharenberg AM - PLoS ONE (2009)

Bottom Line: Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2(nd) messenger monomeric adenosine diphosphate-ribose (mADPR).To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1's BRCT protein interaction domain.Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2(nd) messenger monomeric adenosine diphosphate-ribose (mADPR). Previous studies have delineated specific roles for several of PARP-1's structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1's BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2(nd) messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.

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The effects of DBD mutations on PARP-dependent O/N STRESS induced NAD degradation and TRPM2-dependent Ca2+ transients.A) PARP-dZF mutant expression levels relative to PARP-WT:Left Panel: 50 µg of cellular protein were loaded into each lane of an 8% SDS-PAGE gel and analyzed by western blotting. Rabbit anti-human PARP-1 polyclonal antibody was used as the primary antibody for immunoblotting of PARP-1 (1∶4000, Alexis Biochemicals), and IR680 conjugated goat anti-rabbit as secondary antibody (1∶3000, Licor Inc). Blots were analyzed on a Licor Odyssey. Middle Panel: Relative transcript abundance of PARP-dZF mutants normalized to PARP-WT, as determined by Q-PCR. Right panel: TRPM2-dependent whole cell currents are similar in PARP-WT and PARP-dZF mutant clones. Average whole cell currents were not statistically different from one another across all cell types. Cells were patched in the whole cell configuration: the pipette solution contained 100 µM mADPR. The I-V relationship and current development across all cell types was characteristic of TRPM2 and identical to that previously shown by our lab [5]. At least 3 whole cell recordings were taken for each cell type. B) Left panel: NAD turnover in PARP-WT and PARP-dZF1 DT40 cells. PARP-dZF1 did not show NAD degradation over the course of 30 minutes following application of 100 µM MNNG. Stars indicate a p-value of p≤.001 from baseline for all subsequent points. Right panel: TRPM2-dependent Ca2+ transients in PARP-WT and PARP-dZF1 DT40 cells after stimulation with 500 µM MNNG, as measured by ratiometric analysis of Indo-1 stained cells by FACS. No transients were seen in PARP-KO or PARP-dZF1 at 100 µM MNNG (data not shown). C) Left panel: NAD turnover in PARP-WT and PARP-dZF2 DT40 cells. PARP-dZF2 cells show NAD degradation over the course of 240 minutes following application of 100 µM MNNG. Stars indicate a p-value of p≤.001 from baseline for all subsequent points. Right panel: TRPM2-dependent Ca2+ transients in PARP-WT and PARP-dZF2 DT40 cells after stimulation with 100 µM MNNG, as measured by ratiometric analysis of Indo-1 stained cells by FACS. No transients were seen in PARP-KO or PARP-dZF2 at 100 µM MNNG (data not shown).
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pone-0006339-g004: The effects of DBD mutations on PARP-dependent O/N STRESS induced NAD degradation and TRPM2-dependent Ca2+ transients.A) PARP-dZF mutant expression levels relative to PARP-WT:Left Panel: 50 µg of cellular protein were loaded into each lane of an 8% SDS-PAGE gel and analyzed by western blotting. Rabbit anti-human PARP-1 polyclonal antibody was used as the primary antibody for immunoblotting of PARP-1 (1∶4000, Alexis Biochemicals), and IR680 conjugated goat anti-rabbit as secondary antibody (1∶3000, Licor Inc). Blots were analyzed on a Licor Odyssey. Middle Panel: Relative transcript abundance of PARP-dZF mutants normalized to PARP-WT, as determined by Q-PCR. Right panel: TRPM2-dependent whole cell currents are similar in PARP-WT and PARP-dZF mutant clones. Average whole cell currents were not statistically different from one another across all cell types. Cells were patched in the whole cell configuration: the pipette solution contained 100 µM mADPR. The I-V relationship and current development across all cell types was characteristic of TRPM2 and identical to that previously shown by our lab [5]. At least 3 whole cell recordings were taken for each cell type. B) Left panel: NAD turnover in PARP-WT and PARP-dZF1 DT40 cells. PARP-dZF1 did not show NAD degradation over the course of 30 minutes following application of 100 µM MNNG. Stars indicate a p-value of p≤.001 from baseline for all subsequent points. Right panel: TRPM2-dependent Ca2+ transients in PARP-WT and PARP-dZF1 DT40 cells after stimulation with 500 µM MNNG, as measured by ratiometric analysis of Indo-1 stained cells by FACS. No transients were seen in PARP-KO or PARP-dZF1 at 100 µM MNNG (data not shown). C) Left panel: NAD turnover in PARP-WT and PARP-dZF2 DT40 cells. PARP-dZF2 cells show NAD degradation over the course of 240 minutes following application of 100 µM MNNG. Stars indicate a p-value of p≤.001 from baseline for all subsequent points. Right panel: TRPM2-dependent Ca2+ transients in PARP-WT and PARP-dZF2 DT40 cells after stimulation with 100 µM MNNG, as measured by ratiometric analysis of Indo-1 stained cells by FACS. No transients were seen in PARP-KO or PARP-dZF2 at 100 µM MNNG (data not shown).

Mentions: Following transfection of TRPM2 expressing PARP-deficient DT40 cells with each mutant construct (Figure 2), panels of clones were selected and TRPM2 and mutant PARP protein expression were compared to the PARP-WT control cell line selected above. For each PARP mutant, a clone closely matched to the PARP-WT cell line for both hPARP-1 by western blot (Figures 3B [left panel], 4A [left panel], and 5A [left panel]) and TRPM2 by whole cell current amplitude (Figure 3B [right panel], 4A [right panel], and 5A [right panel]), was selected for use in subsequent experiments. Importantly, taken together with the data from Figure 3A, these data demonstrate that, absent a significant alteration in function of a mutant protein relative to WT PARP-1, the range of expression levels observed in the clones chosen for analysis would not be expected to significantly impact the rate of O/N-stress induced mADPR accumulation.


Structure/function analysis of PARP-1 in oxidative and nitrosative stress-induced monomeric ADPR formation.

Buelow B, Uzunparmak B, Paddock M, Scharenberg AM - PLoS ONE (2009)

The effects of DBD mutations on PARP-dependent O/N STRESS induced NAD degradation and TRPM2-dependent Ca2+ transients.A) PARP-dZF mutant expression levels relative to PARP-WT:Left Panel: 50 µg of cellular protein were loaded into each lane of an 8% SDS-PAGE gel and analyzed by western blotting. Rabbit anti-human PARP-1 polyclonal antibody was used as the primary antibody for immunoblotting of PARP-1 (1∶4000, Alexis Biochemicals), and IR680 conjugated goat anti-rabbit as secondary antibody (1∶3000, Licor Inc). Blots were analyzed on a Licor Odyssey. Middle Panel: Relative transcript abundance of PARP-dZF mutants normalized to PARP-WT, as determined by Q-PCR. Right panel: TRPM2-dependent whole cell currents are similar in PARP-WT and PARP-dZF mutant clones. Average whole cell currents were not statistically different from one another across all cell types. Cells were patched in the whole cell configuration: the pipette solution contained 100 µM mADPR. The I-V relationship and current development across all cell types was characteristic of TRPM2 and identical to that previously shown by our lab [5]. At least 3 whole cell recordings were taken for each cell type. B) Left panel: NAD turnover in PARP-WT and PARP-dZF1 DT40 cells. PARP-dZF1 did not show NAD degradation over the course of 30 minutes following application of 100 µM MNNG. Stars indicate a p-value of p≤.001 from baseline for all subsequent points. Right panel: TRPM2-dependent Ca2+ transients in PARP-WT and PARP-dZF1 DT40 cells after stimulation with 500 µM MNNG, as measured by ratiometric analysis of Indo-1 stained cells by FACS. No transients were seen in PARP-KO or PARP-dZF1 at 100 µM MNNG (data not shown). C) Left panel: NAD turnover in PARP-WT and PARP-dZF2 DT40 cells. PARP-dZF2 cells show NAD degradation over the course of 240 minutes following application of 100 µM MNNG. Stars indicate a p-value of p≤.001 from baseline for all subsequent points. Right panel: TRPM2-dependent Ca2+ transients in PARP-WT and PARP-dZF2 DT40 cells after stimulation with 100 µM MNNG, as measured by ratiometric analysis of Indo-1 stained cells by FACS. No transients were seen in PARP-KO or PARP-dZF2 at 100 µM MNNG (data not shown).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2713433&req=5

pone-0006339-g004: The effects of DBD mutations on PARP-dependent O/N STRESS induced NAD degradation and TRPM2-dependent Ca2+ transients.A) PARP-dZF mutant expression levels relative to PARP-WT:Left Panel: 50 µg of cellular protein were loaded into each lane of an 8% SDS-PAGE gel and analyzed by western blotting. Rabbit anti-human PARP-1 polyclonal antibody was used as the primary antibody for immunoblotting of PARP-1 (1∶4000, Alexis Biochemicals), and IR680 conjugated goat anti-rabbit as secondary antibody (1∶3000, Licor Inc). Blots were analyzed on a Licor Odyssey. Middle Panel: Relative transcript abundance of PARP-dZF mutants normalized to PARP-WT, as determined by Q-PCR. Right panel: TRPM2-dependent whole cell currents are similar in PARP-WT and PARP-dZF mutant clones. Average whole cell currents were not statistically different from one another across all cell types. Cells were patched in the whole cell configuration: the pipette solution contained 100 µM mADPR. The I-V relationship and current development across all cell types was characteristic of TRPM2 and identical to that previously shown by our lab [5]. At least 3 whole cell recordings were taken for each cell type. B) Left panel: NAD turnover in PARP-WT and PARP-dZF1 DT40 cells. PARP-dZF1 did not show NAD degradation over the course of 30 minutes following application of 100 µM MNNG. Stars indicate a p-value of p≤.001 from baseline for all subsequent points. Right panel: TRPM2-dependent Ca2+ transients in PARP-WT and PARP-dZF1 DT40 cells after stimulation with 500 µM MNNG, as measured by ratiometric analysis of Indo-1 stained cells by FACS. No transients were seen in PARP-KO or PARP-dZF1 at 100 µM MNNG (data not shown). C) Left panel: NAD turnover in PARP-WT and PARP-dZF2 DT40 cells. PARP-dZF2 cells show NAD degradation over the course of 240 minutes following application of 100 µM MNNG. Stars indicate a p-value of p≤.001 from baseline for all subsequent points. Right panel: TRPM2-dependent Ca2+ transients in PARP-WT and PARP-dZF2 DT40 cells after stimulation with 100 µM MNNG, as measured by ratiometric analysis of Indo-1 stained cells by FACS. No transients were seen in PARP-KO or PARP-dZF2 at 100 µM MNNG (data not shown).
Mentions: Following transfection of TRPM2 expressing PARP-deficient DT40 cells with each mutant construct (Figure 2), panels of clones were selected and TRPM2 and mutant PARP protein expression were compared to the PARP-WT control cell line selected above. For each PARP mutant, a clone closely matched to the PARP-WT cell line for both hPARP-1 by western blot (Figures 3B [left panel], 4A [left panel], and 5A [left panel]) and TRPM2 by whole cell current amplitude (Figure 3B [right panel], 4A [right panel], and 5A [right panel]), was selected for use in subsequent experiments. Importantly, taken together with the data from Figure 3A, these data demonstrate that, absent a significant alteration in function of a mutant protein relative to WT PARP-1, the range of expression levels observed in the clones chosen for analysis would not be expected to significantly impact the rate of O/N-stress induced mADPR accumulation.

Bottom Line: Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2(nd) messenger monomeric adenosine diphosphate-ribose (mADPR).To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1's BRCT protein interaction domain.Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2(nd) messenger monomeric adenosine diphosphate-ribose (mADPR). Previous studies have delineated specific roles for several of PARP-1's structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1's BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2(nd) messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.

Show MeSH
Related in: MedlinePlus