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The 193-kD vault protein, VPARP, is a novel poly(ADP-ribose) polymerase.

Kickhoefer VA, Siva AC, Kedersha NL, Inman EM, Ruland C, Streuli M, Rome LH - J. Cell Biol. (1999)

Bottom Line: Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP.Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s).A portion of p193 is nuclear and localizes to the mitotic spindle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of California, Los Angeles School of Medicine, Los Angeles, California 90095-1737, USA. vkick@medne.tucla.edu

ABSTRACT
Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of approximately 350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle.

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The catalytic subunit of p193 is a PARP that ADP-ribosylates itself. The catalytic domain of p193 (255–611) was expressed and purified from E. coli. In vitro assays containing p193 (255–611) were incubated in the presence of [32P]NAD+ and the products separated by SDS-PAGE followed by Coomassie blue staining (left panel) and by autoradiography (right panel). Reactions contained 1 μg of p193 (255–611) and 1.3 μM [32P]NAD+. Preincubation of p193 (255–611) at 65°C for 10 min before the addition of labeled NAD+ inactivated the activity (heat). Reactions were supplemented with either 1 mM unlabeled NAD+ (cold chase) or with 1 mM 3ABA, the PARP inhibitor (inhibitor).
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Figure 4: The catalytic subunit of p193 is a PARP that ADP-ribosylates itself. The catalytic domain of p193 (255–611) was expressed and purified from E. coli. In vitro assays containing p193 (255–611) were incubated in the presence of [32P]NAD+ and the products separated by SDS-PAGE followed by Coomassie blue staining (left panel) and by autoradiography (right panel). Reactions contained 1 μg of p193 (255–611) and 1.3 μM [32P]NAD+. Preincubation of p193 (255–611) at 65°C for 10 min before the addition of labeled NAD+ inactivated the activity (heat). Reactions were supplemented with either 1 mM unlabeled NAD+ (cold chase) or with 1 mM 3ABA, the PARP inhibitor (inhibitor).

Mentions: To determine whether p193 has PARP activity, the catalytic domain of p193, aa 255–611, were expressed in E. coli as a His-tagged fusion protein and purified. An in vitro PARP activity assay, which measures the addition of radiolabeled ADP-ribose to protein acceptors with [32P]NAD+ used as a substrate, was carried out. A Coomassie stain of the gel before exposure to a PhosphorImager screen shows that equal amounts of proteins were used in all of the assays (Fig. 4, left panel). Like PARP, the catalytic domain of p193 contains ADP-ribosylation activity, and it ADP-ribosylates itself (Fig. 4, right panel). This activity is heat inactivatable (Fig. 4, right panel). The addition of unlabeled NAD+ (1 mM) decreased the level of labeled ADP-ribose polymers added to p193 (255–611) about threefold (Fig. 4, right panel). To confirm that the labeling reaction with p193 was analogous to PARP-catalyzed poly(ADP-ribosyl)ation, the PARP-specific inhibitor 3ABA was included in a reaction. Modification of p193 (255–611) was decreased about twofold in the presence of the inhibitor (Fig. 4, right panel). Furthermore, modified p193 (255–611) reacted with a monoclonal anti–poly (ADP-ribose) antibody (data not shown), consistent with it carrying ADP-ribose polymers. These data indicate that p193 (255–611) is a PARP.


The 193-kD vault protein, VPARP, is a novel poly(ADP-ribose) polymerase.

Kickhoefer VA, Siva AC, Kedersha NL, Inman EM, Ruland C, Streuli M, Rome LH - J. Cell Biol. (1999)

The catalytic subunit of p193 is a PARP that ADP-ribosylates itself. The catalytic domain of p193 (255–611) was expressed and purified from E. coli. In vitro assays containing p193 (255–611) were incubated in the presence of [32P]NAD+ and the products separated by SDS-PAGE followed by Coomassie blue staining (left panel) and by autoradiography (right panel). Reactions contained 1 μg of p193 (255–611) and 1.3 μM [32P]NAD+. Preincubation of p193 (255–611) at 65°C for 10 min before the addition of labeled NAD+ inactivated the activity (heat). Reactions were supplemented with either 1 mM unlabeled NAD+ (cold chase) or with 1 mM 3ABA, the PARP inhibitor (inhibitor).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169495&req=5

Figure 4: The catalytic subunit of p193 is a PARP that ADP-ribosylates itself. The catalytic domain of p193 (255–611) was expressed and purified from E. coli. In vitro assays containing p193 (255–611) were incubated in the presence of [32P]NAD+ and the products separated by SDS-PAGE followed by Coomassie blue staining (left panel) and by autoradiography (right panel). Reactions contained 1 μg of p193 (255–611) and 1.3 μM [32P]NAD+. Preincubation of p193 (255–611) at 65°C for 10 min before the addition of labeled NAD+ inactivated the activity (heat). Reactions were supplemented with either 1 mM unlabeled NAD+ (cold chase) or with 1 mM 3ABA, the PARP inhibitor (inhibitor).
Mentions: To determine whether p193 has PARP activity, the catalytic domain of p193, aa 255–611, were expressed in E. coli as a His-tagged fusion protein and purified. An in vitro PARP activity assay, which measures the addition of radiolabeled ADP-ribose to protein acceptors with [32P]NAD+ used as a substrate, was carried out. A Coomassie stain of the gel before exposure to a PhosphorImager screen shows that equal amounts of proteins were used in all of the assays (Fig. 4, left panel). Like PARP, the catalytic domain of p193 contains ADP-ribosylation activity, and it ADP-ribosylates itself (Fig. 4, right panel). This activity is heat inactivatable (Fig. 4, right panel). The addition of unlabeled NAD+ (1 mM) decreased the level of labeled ADP-ribose polymers added to p193 (255–611) about threefold (Fig. 4, right panel). To confirm that the labeling reaction with p193 was analogous to PARP-catalyzed poly(ADP-ribosyl)ation, the PARP-specific inhibitor 3ABA was included in a reaction. Modification of p193 (255–611) was decreased about twofold in the presence of the inhibitor (Fig. 4, right panel). Furthermore, modified p193 (255–611) reacted with a monoclonal anti–poly (ADP-ribose) antibody (data not shown), consistent with it carrying ADP-ribose polymers. These data indicate that p193 (255–611) is a PARP.

Bottom Line: Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP.Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s).A portion of p193 is nuclear and localizes to the mitotic spindle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of California, Los Angeles School of Medicine, Los Angeles, California 90095-1737, USA. vkick@medne.tucla.edu

ABSTRACT
Mammalian vaults are ribonucleoprotein (RNP) complexes, composed of a small ribonucleic acid and three proteins of 100, 193, and 240 kD in size. The 100-kD major vault protein (MVP) accounts for >70% of the particle mass. We have identified the 193-kD vault protein by its interaction with the MVP in a yeast two-hybrid screen and confirmed its identity by peptide sequence analysis. Analysis of the protein sequence revealed a region of approximately 350 amino acids that shares 28% identity with the catalytic domain of poly(ADP-ribose) polymerase (PARP). PARP is a nuclear protein that catalyzes the formation of ADP-ribose polymers in response to DNA damage. The catalytic domain of p193 was expressed and purified from bacterial extracts. Like PARP, this domain is capable of catalyzing a poly(ADP-ribosyl)ation reaction; thus, the 193-kD protein is a new PARP. Purified vaults also contain the poly(ADP-ribosyl)ation activity, indicating that the assembled particle retains enzymatic activity. Furthermore, we show that one substrate for this vault-associated PARP activity is the MVP. Immunofluorescence and biochemical data reveal that p193 protein is not entirely associated with the vault particle, suggesting that it may interact with other protein(s). A portion of p193 is nuclear and localizes to the mitotic spindle.

Show MeSH
Related in: MedlinePlus