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Query: UNIPROT:P04637 (p53)
 77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To facilitate the purification of wild type p53 protein, we established a recombinant p53 vaccinia viral expression system. Using this efficient eukaryotic expression vector, we found that the expressed p53 proteins retained their specific structural characteristics. A comparison between wild type and mutant p53 proteins showed the conservation of the typical subcellular localization and the expression of specific antigenic determinants. Furthermore, wild type p53 exhibited a typical binding with large T antigen, whereas no binding was detected with mutant p53. Both wild type and mutant p53 proteins were highly stable and constituted 5-7% of total protein expressed in the infected cells. These expression recombinant viruses offer a simple, valuable system for the purification of wild type and mutant p53 proteins that are expressed abundantly in eukaryotic cells.
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PMID:Expression of wild-type and mutant p53 proteins by recombinant vaccinia viruses. 163 Sep 14

The full-length provirus of human T-cell leukemia virus type I (HTLV-I) was isolated from MT-2, a lymphoid cell line producing HTLV-I. In transfected cells, structural proteins of HTLV-I, the gag and env products, were formed and processed in the same manner as observed in MT-2 cells. The nucleotide sequence was determined for a region between the gag and pol genes of the proviral DNA clone containing an open-reading frame. The deduced amino acid sequences show that this open-reading frame encodes a putative HTLV-I protease. The protease gene (pro) of HTLV-I was investigated using a vaccinia virus expression vector. Processing of 53k gag precursor polyprotein into mature p19, p24, and p15 gag structural proteins was detectable with a recombinant plasmid harboring the entire gag- and protease-coding sequence. We demonstrated that the protease processed the gag precursor polyprotein in a trans-action. A change in the sequence Asp(64)-Thr-Gly, the catalytic core sequence among aspartyl proteases, to Gly-Thr-Gly was shown to abolish correct processing, suggesting that HTLV-I protease may belong to the aspartyl protease group. The 76k gag-pro precursor polyprotein was identified, implying that a cis-acting function of HTLV-I protease may be necessary to trigger the initial cleavage event for its own release from a precursor protein, followed by the release of p53 gag precursor protein. The p53 gag precursor protein is then processed by the trans-action of the released protease to form p19, p24, and p15.
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PMID:Identification of HTLV-I gag protease and its sequential processing of the gag gene product. 266 87

A fragment of HTLV-IIIB gag-gene, coding for the first 441 amino acids of the p53 gag-precursor was expressed in the recombinant vaccinia virus, vC5. Two HIV specific proteins were detected by western blot in CV-1 cells infected with vC5. Their relative molecular masses were 50 and 35 Kd, pointing out that the first of the proteins is a full length expression product of the cloned sequence, while the second one is a result of processing or abortive translation. Possibilities of using such a strain as a vaccine or in Western blot conformation test are discussed.
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PMID:[Expression of gag gene of human immunodeficiency virus in recombinant vaccinia virus]. 326 87

Interaction of wild type p53 with specific DNA target sequences, which is dictated by several structural domains, can be modified by blocking the different antigenic epitopes of the protein. Comparison of p53 protein expressed by recombinant bacteria (wtp53-Bac) to that produced in an eukaryotic system by a vaccinia expression vector (wtp53-Vac), indicated that only the later exhibited spontaneous DNA-binding activity. Furthermore, DNA-binding patterns of these wild type p53 proteins were affected differently by their interactions with monoclonal anti-p53 antibodies recognizing individual antigenic epitopes of the molecule. While the vaccinia derived p53 that spontaneously bound DNA is supershifted by the N'-terminal specific antibodies PAb-248, the bacterial derived p53 protein that retains this antigenic epitope but does not bind DNA spontaneously, is not affected. The C'-terminal specific PAb-421 antibodies accelerated binding of the bacterial p53 protein and modified the pattern of the interaction of the vaccinia derived p53 DNA. DNA-binding patterns generated by PAb-421 and PAb-248, suggest that either interaction of wild type p53 is dependent on modification of the p53 protein or that it interacts with cellular factors which their activity can be mimicked by PAb-421. Saturation of both types of wild type p53 with several anti-p53 monoclonal antibodies directed against the wild type p53 specific epitope that maps to the N'-terminal border of the DNA-binding region, blocked specific DNA-binding. The fact that most p53 mutants have lost the wild type p53 conformation specific epitope coupled with the observation that blocking of this site by binding specific antibodies, prevents the interaction of wild type p53 with DNA, suggests that maintaining the correct structural conformation of this site is central for DNA-binding activity. The wild type specific epitope which maps to the N'-terminal border of the DNA-binding region is neighboring the first beta-strand detected by the recent crystallographic analysis.
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PMID:The DNA binding activity of wild type p53 is modulated by blocking its various antigenic epitopes. 753 17

A recombinant vaccinia virus was constructed using the wild-type human p53 gene as an insert. The p 53 protein produced by this recombinant virus was used to investigate p53 binding proteins in seventeen cell lines, including 10 derived from human hepatocellular carcinoma, four from other human cancers, and three from non-human primate tissues. In all 17 cell lines tested, two proteins of 40 kD and 50 kD were identified that bound to wild-type p53 and that may be cellular regulators of p53 function.
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PMID:Identification of two p53-binding proteins using a recombinant vaccinia virus containing the wild-type human p53 gene. 765 64

The Ku autoantigen is a heterodimer of 70 kDa (p70) and -80 kDa (p80) subunits that is the DNA-binding component of a DNA-dependent protein kinase (DNA-PK). The 350 kDa (p350) catalytic subunit of DNA-PK phosphorylates Sp-1, Oct-1, p53 and RNA polymerase II in vitro, but the precise cellular role of DNA-PK remains unclear. In the present studies, the assembly of p70/p80 heterodimers and the interaction of Ku with DNA was investigated using recombinant vaccinia viruses directing the synthesis of human p70 (p70-vacc) and p80 (p80-vacc), and monoclonal antibodies (mAbs). Expression of human Ku antigens in rabbit kidney (RK13) cells could be demonstrated by immunofluorescent staining because this cell line contains little endogenous Ku. A novel mAb designated 162 stained the nuclei of RK13 cells coinfected with p70-vacc and p80-vacc, but not cells that were infected with either virus alone, suggesting that it recognized the p70/p80 heterodimer but not monomeric p70 or p80. In agreement with the immunofluorescence data, 162 immunoprecipitated both p70 and p80 from extracts of coinfected cells, but did not immunoprecipitate either subunit by itself from extracts of cells infected with p70-vacc or p80-vacc, respectively. Conversely, the binding of 162 to Ku isolated from human K562 cells stabilized the p70/p80 heterodimer under conditions that normally dissociate p70 from p80. The nuclei of cells infected with p70-vacc alone could be stained with mAb N3H10 (anti-p70) and cells infected with p80-vacc alone could be stained with mAb 111 (anti-p80), indicating that the formation of p70/p80 heterodimers was not required for nuclear transport. Finally, free recombinant and cellular p70 both bound to DNA efficiently in vitro, suggesting that free p70, like the p70/p80 heterodimer, serves as a DNA-binding factor. Moreover, free human p70 could be released from the nuclei of p70-vacc-infected RK13 cells by deoxyribonuclease I treatment, suggesting that it was associated with chromatin in vivo. The nuclear transport of free p70 and the association of free p70 with chromatin in vivo raise the possibility that newly synthesized cellular p70 might undergo nuclear transport and DNA-binding prior to dimerization with p80 or assembly with p350.
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PMID:Assembly and DNA binding of recombinant Ku (p70/p80) autoantigen defined by a novel monoclonal antibody specific for p70/p80 heterodimers. 769 19

The simian virus 40 large T antigen immortalizes growing primary cells in culture. In addition, this viral oncoprotein cooperates with an activated ras protein to produce dense foci on monolayers of rat embryo fibroblasts (REF). The relationship between independent immortalization and cooperative transformation with ras has not been defined. Previously, two regions of T antigen were shown to contain immortalization activities. An N-terminal fragment consisting of amino acids 1 to 147 immortalizes rodent cells (L. Sompayrac and K. J. Danna, Virology 181:412-415, 1991). Loss-of-function analysis indicated that immortalization depended on integrity of the T-antigen segments containing amino acids 351 to 450 and 533 to 626 (T. D. Kierstead and M. J. Tevethia, J. Virol. 67:1817-1829, 1993). The experiments described here were directed toward determining whether these same T-antigen regions were sufficient for cooperation with ras. Initially, constructs that produce T antigens containing amino acids 176 to 708 (T176-708) or 1 to 147 were tested in a ras cooperation assay. Both polypeptides cooperated with ras to produce dense foci on monolayers of primary REF. These results showed that T antigen contains two separate ras cooperation activities. In order to determine the N-terminal limit of the ras cooperation activity contained within the T176-708 polypeptide, a series of constructs designed to produce fusion proteins containing T-antigen segments beginning at residues 251, 301, 337, 351, 371, 401, 451, 501, 551, 601, and 651 was generated. Each of these constructs was tested for the capacity to cooperate with ras to produce dense foci on REF monolayers. The results indicated that a polypeptide containing T-antigen amino acids 251 to 708 (T251-708) was sufficient to cooperate with ras, whereas the more extensively truncated products were not. The abilities of the N-terminally truncated T antigens to bind p53 were examined in p53-deficient cells infected with a recombinant vaccinia virus expressing a phenotypically wild-type mouse p53. The results showed that polypeptides containing T-antigen amino acids 251 to 708, 301 to 708, 337 to 708, or 351 to 708 retained p53-binding capacity. The introduction into the T251-708 polypeptide of deletions that either prevented p53 binding (dl434-444) or did not prevent p53 binding (dl400) abrogated ras cooperation. These results indicated that although p53 binding may be necessary for ras cooperation, an additional, as-yet-undefined activity contained within the T251-708 polypeptide is needed.
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PMID:Simian virus 40 large T antigen contains two independent activities that cooperate with a ras oncogene to transform rat embryo fibroblasts. 781 61

The immunogenicity of viral oncoproteins has been established beyond doubt. Cytotoxic T lymphocytes directed against viral oncogene products can eradicate large established tumor masses. This stage has not yet been reached for cellular oncogene and tumor suppressor gene products, but T cells have been raised against MHC-binding peptides encoded by both mutant and wild-type alleles of the ras oncogene and the p53 tumor suppressor gene. In addition, T cells specific for joining region peptides of abnormal fusion proteins resulting from chromosome translocation in tumor cells have been generated. Some of these peptides are processed in cells infected with, for example, vaccinia-ras, but direct anti-tumor effects of peptide specific T lymphocytes remain to be demonstrated.
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PMID:Potential immunogenicity of oncogene and tumor suppressor gene products. 824 Jul 32

The observation that wild-type p53 may induce cells to undergo either apoptosis or differentiation raises the question of whether these two events share similar p53-dependent pathways. To evaluate the interrelationship between these two p53-dependent processes, our study focused on the human HL-60, a promyelocytic p53 nonproducer cell line in which p53 expression was introduced and the induction of apoptosis and differentiation was followed under controlled conditions. p53 expression was induced in the HL-60 cell line by infection with the recombinant wild-type p53 (p53WT) vaccinia virus. Viral infection gave rise to cells expressing various levels of wild-type p53 protein. High levels of p53 protein induced cells to undergo rapid apoptosis, whereas lower levels of p53 protein induced cells to undergo cell differentiation at a more moderate rate of kinetics. These results suggest that p53 protein levels may determine whether a given cell should prefer one pathway over the other to exit the cell cycle. Accordingly, we propose that the p53 vaccinia virus may be used as a potential vector for cell therapy leading toward the exit of p53 null human primary hematopoetic tumors from the malignant state in vivo via the apoptotic or cell differentiation pathways.
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PMID:Induction of HL-60 cells to undergo apoptosis is determined by high levels of wild-type p53 protein whereas differentiation of the cells is mediated by lower p53 levels. 878 30

By using vaccinia virus-T7 hybrid expression system, subcellular localization of the NS3 protein of hepatitis C virus was studied. Full-size NS3 (NS3F) and a carboxy-terminally truncated form (NS3 deltaC) were localized in the cytoplasm and the nucleus when expressed alone. However, NS3F and NS3 deltaC, but not amino- and carboxy-terminally truncated form (NS3 deltaN deltaC), were each co-localized with wild-type p53 almost exclusively in the nucleus upon co-expression. The wild-type p53-induced nuclear accumulation of NS3F was inhibited only partially by NS4A. When co-expressed with mutant-type p53, NS3F and NS3 deltaC were each co-localized with it exclusively in the cytoplasm. Taken together, the present results suggest that wild-type p53 enhances nuclear accumulation of NS3F and NS3 deltaC through the involvement of their amino-terminal sequences even in the presence of NS4A, and that mutant-type p53 inhibits their nuclear, and enhances their cytoplasmic, accumulation.
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PMID:Wild-type, but not mutant-type, p53 enhances nuclear accumulation of the NS3 protein of hepatitis C virus. 901 97


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