Limits...
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.

Show MeSH

Related in: MedlinePlus

Alignment of p193 and PARP catalytic domains. Colons indicate conserved aa. Periods indicate semi-conserved aa. Highlighted aa form the binding pocket of the catalytic domain in the crystal structure of the catalytic fragment of PARP (Ruf et al. 1996, Ruf et al. 1998).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169495&req=5

Figure 3: Alignment of p193 and PARP catalytic domains. Colons indicate conserved aa. Periods indicate semi-conserved aa. Highlighted aa form the binding pocket of the catalytic domain in the crystal structure of the catalytic fragment of PARP (Ruf et al. 1996, Ruf et al. 1998).

Mentions: The second domain, aa 209–563, shares 29% identity with the catalytic subunit of PARP (PARP, sequence data available from EMBL/GenBank/DDBJ under accession no. G130781) (Fig. 3). PARP is a nuclear protein that can be divided into three domains: the NH2-terminal DNA binding domain (containing two zinc fingers), a central automodification domain, and a COOH-terminal catalytic domain (for review see de Murcia et al. 1991). The catalytic subunit binds to NAD+, hydrolyzes the nicotine moiety, and polymerizes the ADP-ribose group in response to DNA damage. Poly(ADP-ribose) is attached mainly to PARP, but also to other substrates including histones H1 and H2B (Simonin et al. 1993b). A number of drugs have been shown to bind to the active site of the catalytic subunit, thus blocking NAD binding (including PD128763 and 3ABA; Ruf et al. 1996, Ruf et al. 1998). The minimum region necessary for PARP to retain catalytic activity is a 40-kD fragment (aa 654–1014; Simonin et al. 1990). The crystal structure of the catalytic fragment of PARP has been determined (Ruf et al. 1996, Ruf et al. 1998). Based on the crystal structure, the residues that form the NAD binding pocket are conserved between PARP and p193 (Fig. 3, shaded residues). These data suggest that this region of the p193 will form a similar binding pocket, which could have catalytic activity.


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)

Alignment of p193 and PARP catalytic domains. Colons indicate conserved aa. Periods indicate semi-conserved aa. Highlighted aa form the binding pocket of the catalytic domain in the crystal structure of the catalytic fragment of PARP (Ruf et al. 1996, Ruf et al. 1998).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169495&req=5

Figure 3: Alignment of p193 and PARP catalytic domains. Colons indicate conserved aa. Periods indicate semi-conserved aa. Highlighted aa form the binding pocket of the catalytic domain in the crystal structure of the catalytic fragment of PARP (Ruf et al. 1996, Ruf et al. 1998).
Mentions: The second domain, aa 209–563, shares 29% identity with the catalytic subunit of PARP (PARP, sequence data available from EMBL/GenBank/DDBJ under accession no. G130781) (Fig. 3). PARP is a nuclear protein that can be divided into three domains: the NH2-terminal DNA binding domain (containing two zinc fingers), a central automodification domain, and a COOH-terminal catalytic domain (for review see de Murcia et al. 1991). The catalytic subunit binds to NAD+, hydrolyzes the nicotine moiety, and polymerizes the ADP-ribose group in response to DNA damage. Poly(ADP-ribose) is attached mainly to PARP, but also to other substrates including histones H1 and H2B (Simonin et al. 1993b). A number of drugs have been shown to bind to the active site of the catalytic subunit, thus blocking NAD binding (including PD128763 and 3ABA; Ruf et al. 1996, Ruf et al. 1998). The minimum region necessary for PARP to retain catalytic activity is a 40-kD fragment (aa 654–1014; Simonin et al. 1990). The crystal structure of the catalytic fragment of PARP has been determined (Ruf et al. 1996, Ruf et al. 1998). Based on the crystal structure, the residues that form the NAD binding pocket are conserved between PARP and p193 (Fig. 3, shaded residues). These data suggest that this region of the p193 will form a similar binding pocket, which could have catalytic activity.

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