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 ∼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.
SummaryFibrin-based biomaterial preparations can be used as provisional growth matrices for cells important in tissue repair during wound healing in vivo. Their efficacy can be enhanced by including bioactive agents that promote specific cellular responses. This study examined the controlled delivery of the angiogenic growth factors bFGF, VEGF165, and VEGF121 using biomatrix preparations prepared from Fibrin Sealant product components. The growth factors were added prior to formation of the Fibrin Sealant clots, and the release kinetics of the proteins from the clots measured. The results indicated that the proteins were released from the clots more slowly in the order bFGF << VEGF165 < VEGF121. The biologic activity of the growth factors delivered from Fibrin Sealant clots was established by assaying growth stimulation of human microvascular endothelial cells (HMVEC) and angiogenesis in the chicken embryo chorioallantoic membrane (CAM) model of neovascularization. In the latter assay, clots containing bFGF, VEGF165, or V EGF121 all displayed angiogenic activity. However, delivery of either bFGF, VEGF165, or VEGF121 alone resulted in a significant percentage of clots becoming filled with blood, indicating that the newly developing vessels invading the clots were leaky and immature. In contrast, this hemorrhaging behavior did not occur with delivery of combinations, e.g., (VEGF165 + VEGF121) or (VEGF165 + bFGF), indicating that the vessels were more mature than those produced in response to single growth factors. Thus, delivering a combination of growth factors constituted an improvement over the delivery of individual growth factors for enhancing neovascularization.Part of this research was presented at the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society held in Munich, Germany.
The major vault protein (MVP) is the predominant constituent of ubiquitous, evolutionarily conserved large cytoplasmic ribonucleoprotein particles of unknown function. Vaults are multimeric protein complexes with several copies of an untranslated RNA. Double labeling employing laser-assisted confocal microscopy and indirect immunofluorescence demonstrates partial colocalization of vaults with cytoskeletal elements in Chinese hamster ovary (CHO) and nerve growth factor (NGF)-treated neuronlike PC12 cells. Transfection of CHO and PC12 cells with a cDNA encoding the rat major vault protein containing a vesicular stomatitis virus glycoprotein epitope tag demonstrates that the recombinant protein is sorted into vault particles and targeted like endogenous MVPs. In neuritic extensions of differentiated PC12 cells, there is an almost complete overlap of the distribution of microtubules and vaults. A pronounced colocalization of vaults with filamentous actin can be seen in the tips of neurites. Moreover, in NGF-treated PC12 cells the location of vaults partially coincides with vesicular markers. Within the terminal tips of neurites vaults are located near secretory organelles. Our observations suggest that the vault particles are transported along cytoskeletal-based cellular tracks.
The major vault protein (MVP) is the predominant member of a large ribonucleoprotein particle, named vault. Vaults are abundant in the cytosol of mammalian cells. Mammalian MVP has previously been reported to be associated with the nucleus, particularly its cytosolic surface on which vaults are thought to dock at or near the nuclear pore complex. To date the presence of vault particles inside the nucleus has been convincingly reported only for sea urchin cells. We have addressed the potential nuclear localization of MVP in mammalian cells by employing confocal laser microscopy and cryo-immunoelectron microscopy. As revealed by immunostaining and by analysis of cells transfected with a construct encoding MVP and green fluorescent protein, MVP is present in both the cytosol and in the nucleus. Cryo-electron microscopy of human astroglioma U373 cells reveals clusters of immunogold particles at nuclear pores and in the nucleoplasm suggesting that nuclear MVP is associated with particulate structures. Quantification of the fluorescence observed in the cytosol and in the nuclei reveals that about 5% of the MVP in U373 cells is localized inside the nucleus. Our results further support the notion that part of the cellular MVP can enter the nucleus.
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