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1
Estebanell, Eva, Aleix Cases, José López-Pedret, Ricardo Castillo, Antonio Ordinas, Ginés Escolar, and Maribel Diaz-Ricart. "Erythropoietin Improves Signaling through Tyrosine Phosphorylation in Platelets from Uremic Patients." Thrombosis and Haemostasis 82, no. 10 (1999): 1312–17. http://dx.doi.org/10.1055/s-0037-1614382.
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SummaryErythropoietin has shown to be effective in the correction of the hemostatic defect present in uremic patients. We have investigated the possible effect of recombinant human erythropoietin (rHuEPO) on the signaling processes occurring in platelets. Platelet suspensions were obtained from hemodialyzed patients before and after at least one month of initiating treatment with rHuEPO. Aliquots of non-activated or thrombin-activated platelets were treated to obtain platelet lysates or processed to extract platelet cytoskeleton. Samples were resolved by 8% SDS-polyacrylamide gel electrophoresis followed by Western blotting. After thrombin activation, proteins p120, p85, p78, p75, pp62, pp60, p59, p58, p56, p54 and p52 associated with the Triton-insoluble cytoskeletal fraction appeared phosphorylated in control profiles. In profiles from platelets obtained from uremic patients before treatment with rHuEPO, only proteins p58 and p56 appeared clearly and p54 was slightly phosphorylated. However, in platelets from the same patients under rHuEPO treatment, thrombin-induced phosphorylation improved to levels even above those observed in control profiles. Specially, the band at 54KDa appeared consistently more phosphorylated in all the patients under rHuEPO treatment. Although it is accepted that part of the hemostatic effect of erythropoietin is mediated by an increase in hematocrit, our study suggests that it enhances platelet signaling in uremic platelets which may explain the improvement of platelet response to activating stimulus before clinically noticeable elevation of hematocrit. Abbrevations: rHuEPO = recombinant human erythropoietin; SDS-PAGE = sodium dodecyl sulphate-polyacrylamide gel electrophoresis; CPD = citrate/phosphate/dextrose; PRP = platelet-rich plasma; HBSS = Hanks’ balanced salt solution; EGTA = ethylene glycol bis (β-aminoethylether)-N,N,N’,N’-tetraacetic acid; EDTA = ethylenediaminetetraacetic acid, PMSF = phenylmethylsulphonyl fluoride, ECL = enhanced chemiluminiscence
2
Ashcroft, Margaret, Michael H. G. Kubbutat, and Karen H. Vousden. "Regulation of p53 Function and Stability by Phosphorylation." Molecular and Cellular Biology 19, no. 3 (March 1, 1999): 1751–58. http://dx.doi.org/10.1128/mcb.19.3.1751.
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ABSTRACT The p53 tumor suppressor protein can be phosphorylated at several sites within the N- and C-terminal domains, and several protein kinases have been shown to phosphorylate p53 in vitro. In this study, we examined the activity of p53 proteins with combined mutations at all of the reported N-terminal phosphorylation sites (p53N-term), all of the C-terminal phosphorylation sites (p53C-term), or all of the phosphorylation sites together (p53N/C-term). Each of these mutant proteins retained transcriptional transactivation functions, indicating that phosphorylation is not essential for this activity of p53, although a subtle contribution of the C-terminal phosphorylation sites to the activation of expression of the endogenous p21Waf1/Cip1-encoding gene was detected. Mutation of the phosphorylation sites to alanine did not affect the sensitivity of p53 to binding to or degradation by Mdm2, although alteration of residues 15 and 37 to aspartic acid, which could mimic phosphorylation, resulted in a slight resistance to Mdm2-mediated degradation, consistent with recent reports that phosphorylation at these sites inhibits the p53-Mdm2 interaction. However, expression of the phosphorylation site mutant proteins in both wild-type p53-expressing and p53-null lines showed that all of the mutant proteins retained the ability to be stabilized following DNA damage. This indicates that phosphorylation is not essential for DNA damage-induced stabilization of p53, although phosphorylation could clearly contribute to p53 stabilization under some conditions.
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Wienzek, Sandra, Judith Roth, and Matthias Dobbelstein. "E1B 55-Kilodalton Oncoproteins of Adenovirus Types 5 and 12 Inactivate and Relocalize p53, but Not p51 or p73, and Cooperate with E4orf6 Proteins To Destabilize p53." Journal of Virology 74, no. 1 (January 1, 2000): 193–202. http://dx.doi.org/10.1128/jvi.74.1.193-202.2000.
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ABSTRACT The p53 tumor suppressor protein represents a target for viral and cellular oncoproteins, including adenovirus gene products. Recently, it was discovered that several proteins with structural and functional homologies to p53 exist in human cells. Two of them were termed p51 and p73. We have shown previously that the E1B 55-kDa protein (E1B-55 kDa) of adenovirus type 5 (Ad5) binds and inactivates p53 but not p73. Further, p53 is rapidly degraded in the presence of E1B-55 kDa and the E4orf6 protein of this virus. Here, it is demonstrated that p51 does not detectably associate with E1B-55 kDa. While p53 is relocalized to the cytoplasm by E1B-55 kDa, p51's location is unaffected. Finally, p51 retains its full transcriptional activity in the presence of E1B-55 kDa. Apparently, p51 does not represent a target of Ad5 E1B-55 kDa, suggesting that the functions of p51 are distinct from p53-like tumor suppression. E1B-55 kDa from highly oncogenic adenovirus type 12 (Ad12) was previously shown to surpass the oncogenic activity of Ad5 E1B-55 kDa in various assay systems, raising the possibility that Ad12 E1B-55 kDa might target a broader range of p53-like proteins. However, we show here that Ad12 E1B-55 kDa also inhibits p53's transcriptional activity without measurably affecting p73 or p51. Moderate inhibition of p51's transcriptional activity was observed in the presence of the E4orf6 proteins from Ad5 and Ad12. p53 and Ad12-E1B-55 kDa colocalize in the nucleus and also in cytoplasmic clusters when transiently coexpressed. Finally, E1B-55 kDa and E4orf6 of Ad12 mediate rapid degradation of p53 with an efficiency comparable to that of the Ad5 proteins in human and rodent cells. Our results suggest that E1B-55 kDa of either virus type has similar effects on p53 but does not affect p73 and p51.
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Legagneux, V., P. Bouvet, F. Omilli, S. Chevalier, and H. B. Osborne. "Identification of RNA-binding proteins specific to Xenopus Eg maternal mRNAs: association with the portion of Eg2 mRNA that promotes deadenylation in embryos." Development 116, no. 4 (December 1, 1992): 1193–202. http://dx.doi.org/10.1242/dev.116.4.1193.
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Maternal Xenopus Eg mRNAs have been previously identified as transcripts that are specifically deadenylated after fertilization and degraded after the mid blastula transition. Destabilizing cis sequences were previously localised in the 3′ untranslated region of Eg2 mRNA. In order to characterize possible trans-acting factors which are involved in the post-transcriptional regulation of Eg mRNAs, gel-shift and u.v. cross-linking experiments were performed, which allowed the identification of a p53-p55 RNA-binding protein doublet specific for the 3′ untranslated regions of Eg mRNAs. These p53-p55 proteins do not bind to the 3′ untranslated regions of either ornithine decarboxylase or phosphatase 2Ac mRNAs, which remain polyadenylated in embryos. These novel RNA-binding proteins are distinct from the cytoplasmic polyadenylation element-binding protein that controls the polyadenylation of maternal mRNAs in maturing Xenopus oocytes, and from previously identified thermoresistant RNA-binding proteins present in oocyte mRNP storage particles. The p53-p55 bind a portion of the Eg2 mRNA 3′ untranslated region, distinct from the previously identified destabilizing region, that is able to confer the postfertilization deadenylation of CAT-coding chimeric mRNAs. This suggests that the p53-p55 RNA-binding proteins are good candidates for trans-acting factors involved in the deadenylation of Eg mRNAs in Xenopus embryos.
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Porubiaková, Otília, Natália Bohálová, Alberto Inga, Natália Vadovičová, Jan Coufal, Miroslav Fojta, and Václav Brázda. "The Influence of Quadruplex Structure in Proximity to P53 Target Sequences on the Transactivation Potential of P53 Alpha Isoforms." International Journal of Molecular Sciences 21, no. 1 (December 24, 2019): 127. http://dx.doi.org/10.3390/ijms21010127.
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p53 is one of the most studied tumor suppressor proteins that plays an important role in basic biological processes including cell cycle, DNA damage response, apoptosis, and senescence. The human TP53 gene contains alternative promoters that produce N-terminally truncated proteins and can produce several isoforms due to alternative splicing. p53 function is realized by binding to a specific DNA response element (RE), resulting in the transactivation of target genes. Here, we evaluated the influence of quadruplex DNA structure on the transactivation potential of full-length and N-terminal truncated p53α isoforms in a panel of S. cerevisiae luciferase reporter strains. Our results show that a G-quadruplex prone sequence is not sufficient for transcription activation by p53α isoforms, but the presence of this feature in proximity to a p53 RE leads to a significant reduction of transcriptional activity and changes the dynamics between co-expressed p53α isoforms.
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Chen, Jing, Dadong Zhang, Xiaodi Qin, Kouros Owzar, Jennifer J. McCann, and Michael B. Kastan. "DNA-Damage-Induced Alternative Splicing of p53." Cancers 13, no. 2 (January 12, 2021): 251. http://dx.doi.org/10.3390/cancers13020251.
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Cellular responses to DNA damage and other stresses are important determinants of mutagenesis and impact the development of a wide range of human diseases. TP53 is highly mutated in human cancers and plays an essential role in stress responses and cell fate determination. A central dogma of p53 induction after DNA damage has been that the induction results from a transient increase in the half-life of the p53 protein. Our laboratory recently demonstrated that this long-standing paradigm is an incomplete picture of p53 regulation by uncovering a critical role for protein translational regulation in p53 induction after DNA damage. These investigations led to the discovery of a DNA-damage-induced alternative splicing (AS) pathway that affects p53 and other gene products. The damage-induced AS of p53 pre-mRNA generates the beta isoform of p53 (p53β) RNA and protein, which is specifically required for the induction of cellular senescence markers after ionizing irradiation (IR). In an attempt to elucidate the mechanisms behind the differential regulation and apparent functional divergence between full-length (FL) p53 and the p53β isoform (apoptosis versus senescence, respectively), we identified the differential transcriptome and protein interactome between these two proteins that may result from the unique 10-amino-acid tail in p53β protein.
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Roth, Judith, and Matthias Dobbelstein. "Failure of viral oncoproteins to target the p53-homologue p51A." Journal of General Virology 80, no. 12 (December 1, 1999): 3251–55. http://dx.doi.org/10.1099/0022-1317-80-12-3251.
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The p51/p63/KET proteins were identified based on their strong homology to the tumour suppressor p53 and a related set of proteins termed p73. All these protein species were shown to activate transcription from at least some p53-responsive promoters. To evaluate a possible role of the transcriptionally active splicing variant p51A/p63γ in tumour suppression, we determined whether viral oncoproteins that inactivate p53 might also target p51A. Neither the large T-antigen of simian vacuolating virus 40 (SV40) nor the E6 protein from human papillomavirus type 18 were found to inhibit p51A-mediated transcription, whereas they strongly suppress the activity of p53. Further, SV40 T-antigen directly interacts with p53 but not detectably with p51A. Finally, a cytoplasmic mutant (K128A) of SV40 T-antigen relocalizes p53 from the nucleus to the cytoplasm, but p51A remains in the nucleus when coexpressed with cytoplasmic T-antigen. These results strongly suggest that the inhibitory effect of these viral oncoproteins is specific for p53 and does not measurably affect p51A. Thus, unlike p53, p51A does not appear to be a necessary target in virus-induced cell transformation and may not exert a role comparable to p53 in tumour suppression.
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Martinou, I., P. A. Fernandez, M. Missotten, E. White, B. Allet, R. Sadoul, and J. C. Martinou. "Viral proteins E1B19K and p35 protect sympathetic neurons from cell death induced by NGF deprivation." Journal of Cell Biology 128, no. 1 (January 1, 1995): 201–8. http://dx.doi.org/10.1083/jcb.128.1.201.
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To study molecular mechanisms underlying neuronal cell death, we have used sympathetic neurons from superior cervical ganglia which undergo programmed cell death when deprived of nerve growth factor. These neurons have been microinjected with expression vectors containing cDNAs encoding selected proteins to test their regulatory influence over cell death. Using this procedure, we have shown previously that sympathetic neurons can be protected from NGF deprivation by the protooncogene Bcl-2. We now report that the E1B19K protein from adenovirus and the p35 protein from baculovirus also rescue neurons. Other adenoviral proteins, E1A and E1B55K, have no effect on neuronal survival. E1B55K, known to block apoptosis mediated by p53 in proliferative cells, failed to rescue sympathetic neurons suggesting that p53 is not involved in neuronal death induced by NGF deprivation. E1B19K and p35 were also coinjected with Bcl-Xs which blocks Bcl-2 function in lymphoid cells. Although Bcl-Xs blocked the ability of Bcl-2 to rescue neurons, it had no effect on survival that was dependent upon expression of E1B19K or p35.
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Whitesell, Luke, Patrick D. Sutphin, Elizabeth J. Pulcini, Jesse D. Martinez, and Paul H. Cook. "The Physical Association of Multiple Molecular Chaperone Proteins with Mutant p53 Is Altered by Geldanamycin, an hsp90-Binding Agent." Molecular and Cellular Biology 18, no. 3 (March 1, 1998): 1517–24. http://dx.doi.org/10.1128/mcb.18.3.1517.
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ABSTRACT Wild-type p53 is a short-lived protein which turns over very rapidly via selective proteolysis in the ubiquitin-proteasome pathway. Most p53 mutations, however, encode for protein products which display markedly increased intracellular levels and are associated with positive tumor-promoting activity. The mechanism by which mutation leads to impairment of ubiquitination and proteasome-mediated degradation is unknown, but it has been noted that many transforming p53 mutants are found in stable physical association with molecular chaperones of the hsp70 class. To explore a possible role for aberrant chaperone interactions in mediating the altered function of mutant p53 and its intracellular accumulation, we examined the chaperone proteins which physically associate with a temperature-sensitive murine p53 mutant. In lysate prepared from A1-5 cells grown under mutant temperature conditions, hsp70 coprecipitated with p53Val135 as previously reported by others, but in addition, other well-recognized elements of the cellular chaperone machinery, including hsp90, cyclophilin 40, and p23, were detected. Under temperature conditions favoring wild-type p53 conformation, the coprecipitation of chaperone proteins with p53 was lost in conjunction with the restoration of its transcriptional activating activity. Chaperone interactions similar to those demonstrated in A1-5 cells under mutant conditions were also detected in human breast cancer cells expressing two different hot-spot mutations. To examine the effect of directly disrupting chaperone interactions with mutant p53, we made use of geldanamycin (GA), a selective hsp90-binding agent which has been shown to alter the chaperone associations regulating the function of unliganded steroid receptors. GA treatment of cells altered heteroprotein complex formation with several different mutant p53 species. It increased p53 turnover and resulted in nuclear translocation of the protein in A1-5 cells. GA did not, however, appear to restore wild-type transcriptional activating activity to mutant p53 proteins in either A1-5 cells or human breast cancer cell lines.
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Dao, Thi Nguyet Minh, Sung-Hwan Kang, Aurélie Bak, and Svetlana Y. Folimonova. "A Non-Conserved p33 Protein of Citrus Tristeza Virus Interacts with Multiple Viral Partners." Molecular Plant-Microbe Interactions® 33, no. 6 (June 2020): 859–70. http://dx.doi.org/10.1094/mpmi-11-19-0328-fi.
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The RNA genome of citrus tristeza virus (CTV), one of the most damaging viral pathogens of citrus, contains 12 open reading frames resulting in production of at least 19 proteins. Previous studies on the intraviral interactome of CTV revealed self-interaction of the viral RNA-dependent RNA polymerase, the major coat protein (CP), p20, p23, and p33 proteins, while heterologous interactions between the CTV proteins have not been characterized. In this work, we examined interactions between the p33 protein, a nonconserved protein of CTV, which performs multiple functions in the virus infection cycle and is needed for virus ability to infect the extended host range, with other CTV proteins shown to mediate virus interactions with its plant hosts. Using yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays, we demonstrated that p33 interacts with three viral proteins, i.e., CP, p20, and p23, in vivo and in planta. Coexpression of p33, which is an integral membrane protein, resulted in a shift in the localization of the p20 and p23 proteins toward the subcellular crude-membrane fraction. Upon CTV infection, the four proteins colocalized in the CTV replication factories. In addition, three of them, CP, p20, and p23, were found in the p33-formed membranous structures. Using bioinformatic analyses and mutagenesis, we found that the N-terminus of p33 is involved in the interactions with all three protein partners. A potential role of these interactions in virus ability to infect the extended host range is discussed.
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Johnson, J. L., T. G. Beito, C. J. Krco, and D. O. Toft. "Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes." Molecular and Cellular Biology 14, no. 3 (March 1994): 1956–63. http://dx.doi.org/10.1128/mcb.14.3.1956.
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Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60. Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol.
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Johnson, J. L., T. G. Beito, C. J. Krco, and D. O. Toft. "Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes." Molecular and Cellular Biology 14, no. 3 (March 1994): 1956–63. http://dx.doi.org/10.1128/mcb.14.3.1956-1963.1994.
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Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60. Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol.
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Tisdale, Ellen J., Helen Plutner, Jeanne Matteson, and William E. Balch. "p53/58 Binds COPI and Is Required for Selective Transport through the Early Secretory Pathway." Journal of Cell Biology 137, no. 3 (May 5, 1997): 581–93. http://dx.doi.org/10.1083/jcb.137.3.581.
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p53/58 is a transmembrane protein that continuously recycles between the ER and pre-Golgi intermediates composed of vesicular-tubular clusters (VTCs) found in the cell periphery and at the cis face of the Golgi complex. We have generated an antibody that uniquely recognizes the p53/58 cytoplasmic tail. Here we present evidence that this antibody arrests the anterograde transport of vesicular stomatitis virus glycoprotein and leads to the accumulation of p58 in preGolgi intermediates. Consistent with a role for the KKXX retrieval motif found at the cytoplasmic carboxyl terminus of p53/58 in retrograde traffic, inhibition of transport through VTCs correlates with the ability of the antibody to block recruitment of COPI coats to the p53/58 cytoplasmic tail and to p53/58-containing membranes. We suggest that p53/58 function may be required for the coupled exchange of COPII for COPI coats during segregation of anterograde and retrograde transported proteins.
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Iwakura, Ricardo, Antonio Carlos Pereira Martins, Silvio Tucci Jr, Monica T. Pastorello, Haylton Jorge Suaid, Adauto José Cologna, João José Carneiro, and Antônio D. Campos. "Proteína p53 em nefroblastomas." Acta Cirurgica Brasileira 15, suppl 2 (2000): 50–52. http://dx.doi.org/10.1590/s0102-86502000000600015.
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Cui, Yu-Xin, Alan Kerby, Fiona Kate Elizabeth McDuff, Hongtao Ye, and Suzanne Dawn Turner. "NPM-ALK inhibits the p53 tumor suppressor pathway in an MDM2 and JNK-dependent manner." Blood 113, no. 21 (May 21, 2009): 5217–27. http://dx.doi.org/10.1182/blood-2008-06-160168.
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Abstract Anaplastic large cell lymphoma (ALCL) is characterized by the presence of the t(2;5)(p23;q35) generating the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein, a hyperactive kinase with transforming properties. Among these properties is the ability to regulate activity of the p53 tumor suppressor protein. In many human cancers, p53 is inactivated by mutation or other means, in some cases as a result of up-regulation of the negative regulator MDM2. However, the majority of ALK-expressing ALCL carry wild-type p53 and do not over express MDM2. We demonstrate a novel p53-dependent pathogenetic mechanism in ALK-expressing lymphoma. We confirm previously published reports of NPM-ALK–induced activation of the phosphoinositide (PI) 3-kinase and Jun N-terminal kinase (JNK) stress-activated protein (SAP) kinase proteins, but in this study demonstrate a role for these in the regulation of p53 activity in an intricate signaling system. Specifically, constitutive ALK signaling leads to the functional inactivation and/or degradation of p53 in JNK and MDM2 dependent manners. We also show nuclear exclusion of p53 in a PI 3-kinase–dependent manner. Furthermore, we demonstrate that reactivation of p53 in ALK-expressing cells as a result of pharmacologic inhibition of JNK, PI 3-kinase, and/or MDM2 activities results in the induction of apoptosis suggesting a novel therapeutic modality.
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Bernal-Mizrachi, Leon, Sarah K. Edwards, and Lee Ratner. "Accumulation of NFkB1 (p105) and NFkB2 (p100) Is Essential for Apoptosis Induced by Proteasome Inhibition in a Lymphoma Model." Blood 106, no. 11 (November 16, 2005): 2607. http://dx.doi.org/10.1182/blood.v106.11.2607.2607.
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Abstract The proteasome is a novel target in the therapeutic approach against different subtypes of lymphomas due to its role in the degradation of many proteins involved in cell cycle progression and apoptosis. This multi-enzyme complex controls gene expression by degrading transcription factors such as NF-kB, p53, c-Jun, c-Myc, HIFla, and MATa2. Among these, the best characterized is the NFkB family of transcription factors. Mammals express five NFkB proteins including REL-A (p65), c-REL, REL-B, p50 and p52. All NFkB proteins contain a highly conserved REL-homology domain (RHD), which is responsible for DNA binding, dimerization, nuclear translocation, and interaction with the IkB proteins. Active NFkB is present in the nucleus as heterodimers of p65 and p50 or p52 and REL-B subunits. In contrast, inactive NFkB dimers are sequestered in the cytoplasm due to their interaction with inhibitory proteins such as the IkB’s or the large NFkB subunits p105 and p100. Activation of the NFkB pathway is mediated by proteasome processing of p105 and p100 to produce p50 and p52, respectively, and proteasome degradation of the IkB proteins. Therefore, the proteasome plays a central role in the equilibrium between anti-apoptotic signals derived from p50 and p52, and the NFkB inhibitory signals of p105 and p100. However, it is still undetermined whether modulation of the inhibitory signals or the anti-apoptotic signals by proteasome inhibition is more important for the induction of apoptosis in lymphomas. To address this question we tested the apoptotic effect of the proteasome inhibitor PS341 in two lymphoma cell lines (Daudi and SC) after knocking down p105 and/or p100, using lentivirus expressing siRNA’s. After 24 hours of treatment with titration doses of PS341 (5, 10, 50, and 100nM), we measured apoptosis and NFkB inhibition by Annexin V-Alexa fluorescence and an NFkB luciferase reporter assay, respectively. Interestingly, co-expression of siRNA against both p100 and p105 rendered cells resistant to the induction of apoptosis at clinical doses of PS341 (5 and 10nM) compared to the controls (see table). Similar results were obtained after selective knock down of either p105 or p100. Western Blot analysis showed much lower accumulation of p105 and p100 in siRNA expressing cells than in control cells. However, IkB levels remain stable or increase during treatment in the p105 and/or p100 siRNA expressing cells, despite the observed reduction in apoptosis. These results suggest that the accumulation of p105 and p100, rather than IkB, contributes to the induction of apoptosis. In addition, the expression of NFkB anti-apoptotic signals, such as BCL-xL, increased in all cell lines, including controls. Therefore, the presence of anti-apoptotic signals has no impact on the apoptotic effect produced by PS341. In conclusion, the current study in our lymphoma model demonstrates that the accumulation of p105 and p100 is essential for the induction of apoptosis produced by proteasome inhibition. siRNA Against p100 and/or p105 Reduces PS341 Induction of Apoptosis ps314(nM) 5 10 50 100 SC Luciferase siRNA 61.2±3.2 70±3.6 72.5±3.6 79.5±5 Both siRNA’s 23.5±1.5 33±2.9 59.2±3.9 72.1±5.7 p100 siRNA 27.2±0.4 29±0.5 56±2.7 66.3±6.3 p105siRNA 32.2±7.3 45±2.4 55.4±7.7 78.4±4.1 DAUDI Luciferase siRNA 45.6±3.3 49.5±5.1 61.5±7.1 69.9±5 Both siRNA’s 21.4±4.6 29.3±1.7 34.5±2.4 55.3±7.7 p100 siRNA 17.3±4.2 24.4±9.9 44.5±3.3 56.6±4.7 p105 siRNA 19.7±1.6 34.3±3.3 31.3±2.6 46.3±3.4
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Ito, Noriko, Yuai Li, Tsuneo Suzuki, Daniel J. Stechschulte, and Kottarappat N. Dileepan. "Transient degradation of NF-κB proteins in macrophages after interaction with mast cell granules." Mediators of Inflammation 7, no. 6 (1998): 397–407. http://dx.doi.org/10.1080/09629359890776.
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The exposure of the macrophage cell line, J774 to mast cell granules (MCG) led to the form ation of altered nuclear transcription factor proteins (NFκBx), which had faster electrophoretic mobility than the p50 homodimer of NF-κB, but retained comparable DNA binding capacity. Antibodies to N-terminal peptides of p50, p52, p65 or c-Rel supershifted only a fraction of NF-κBx. Western blot analyses revealed that nuclear p65 and c-Rel were progressively degraded after exposure to MCG, whereas nuclear p50 appeared to be unaffected. In contrast, cytoplasmic p50, p65, c-Rel as well as IkBα remained intact after MCG treatment, although p52 was clearly degraded. In comparison to J774 cells, incubation of m ouse peritoneal macrophages with MCG resulted in more extensive alterations to NF-κB proteins. The alterations in NF-κB proteins did not affect the expression of inducible nitric oxide synthase (iNOS) or TNF-α mRNA in J774 cells. These data indicate that exposure of J774 cells to MCG leads to generation of altered nuclear p52, p65 and c-Rel, which retain intact N-terminal peptides, specific oligonucleotide binding and transactivating activity. On the other hand, in peritoneal macrophages, MCG induce more extensive modifications to NF-κB proteins with associated inhibition of iNOS or TNF-α mRNA expression.
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Wang, GuoZhen, and Alan R. Fersht. "Propagation of aggregated p53: Cross-reaction and coaggregation vs. seeding." Proceedings of the National Academy of Sciences 112, no. 8 (February 9, 2015): 2443–48. http://dx.doi.org/10.1073/pnas.1500262112.
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Destabilized mutant p53s coaggregate with WT p53, p63, and p73 in cancer cell lines. We found that stoichiometric amounts of aggregation-prone mutants induced only small amounts of WT p53 to coaggregate, and preformed aggregates did not significantly seed the aggregation of bulk protein. Similarly, p53 mutants trapped only small amounts of p63 and p73 into their p53 aggregates. Tetrameric full-length protein aggregated at similar rates and kinetics to isolated core domains, but there was some induced aggregation of WT by mutants in hetero-tetramers. p53 aggregation thus differs from the usual formation of amyloid fibril or prion aggregates where tiny amounts of preformed aggregate rapidly seed further aggregation. The proposed aggregation mechanism of p53 of rate-determining sequential unfolding and combination of two molecules accounts for the difference. A molecule of fast-unfolding mutant preferentially reacts with another molecule of mutant and only occasionally traps a slower unfolding WT molecule. The mutant population rapidly self-aggregates before much WT protein is depleted. Subsequently, WT protein self-aggregates at its normal rate. However, the continual production of mutant p53 in a cancer cell would gradually trap more and more WT and other proteins, accounting for the observations of coaggregates in vivo. The mechanism corresponds more to trapping by cross-reaction and coaggregation rather than classical seeding and growth.
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Zhang, Jiping, Mary Ann Warren, Suzanne F. Shoemaker, and Margot M. Ip. "NFκB1/p50 Is Not Required for Tumor Necrosis Factor-Stimulated Growth of Primary Mammary Epithelial Cells: Implications for NFκB2/p52 and RelB." Endocrinology 148, no. 1 (January 1, 2007): 268–78. http://dx.doi.org/10.1210/en.2006-0500.
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Nuclear factor κB (NFκB) plays an important role in mammary gland development and breast cancer. We previously demonstrated that TNF stimulates growth of mammary epithelial cells (MEC) in a physiologically relevant three-dimensional primary culture system, accompanied by enhanced DNA-binding of the NFκB p50 homodimer. To further understand the mechanism of TNF-stimulated growth of primary MEC, the requirement for NFκB1/p50, and the role of cyclin D1 in TNF-stimulated growth were examined. TNF induced the formation of DNA-binding complexes of p50 and p52 with their coactivator bcl3 in MEC nuclear extracts. Concomitantly, TNF increased the binding of NFκB proteins to the κB site on the cyclin D1 promoter, and increased expression of cyclin D1 mRNA and protein. Using MEC from p50 null mice, we found that p50 was not required for TNF-induced growth nor for up-regulation of cyclin D1. However, TNF induced a p52/RelB NFκB DNA-binding complex in p50 null MEC nuclear extracts. In addition, we found that in wild-type MEC, TNF stimulated the occupancy of p52 and RelB on the cyclin D1 promoter κB site, whereas p50 was present constitutively. These data suggest that in wild-type MEC, TNF stimulates the interaction of bcl3 with p50 and p52, and the binding of p52, as well as RelB, to cyclin D1 promoter κB sites, and as a consequence, stimulates the growth of MEC. In the absence of p50, p52 and RelB can compensate for p50 in TNF-stimulated growth and cyclin D1 induction in MEC.
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Raycroft, L., J. R. Schmidt, K. Yoas, M. M. Hao, and G. Lozano. "Analysis of p53 mutants for transcriptional activity." Molecular and Cellular Biology 11, no. 12 (December 1991): 6067–74. http://dx.doi.org/10.1128/mcb.11.12.6067.
Full textAbstract:
The wild-type p53 protein functions to suppress transformation, but numerous mutant p53 proteins are transformation competent. To examine the role of p53 as a transcription factor, we made fusion proteins containing human or mouse p53 sequences fused to the DNA binding domain of a known transcription factor, GAL4. Human and mouse wild-type p53/GAL4 specifically transactivated expression of a chloramphenicol acetyltransferase reporter in HeLa, CHO, and NIH 3T3 cells. Several mutant p53 proteins, including a mouse p53 mutant which is temperature sensitive for suppression, were also analyzed. A p53/GAL4 fusion protein with this mutation was also transcriptionally active only at the permissive temperature. Another mutant p53/GAL4 fusion protein analyzed mimics the mutation inherited in Li-Fraumeni patients. This fusion protein was as active as wild-type p53/GAL4 in our assay. Two human p53 mutants that arose from alterations of the p53 gene in colorectal carcinomas were 30- to 40-fold less effective at activating transcription than wild-type p53/GAL4 fusion proteins. Thus, functional wild-type p53/GAL4 fusion proteins activate transcription, while several transformation competent mutants do so poorly or not at all. Only one mutant p53/GAL4 fusion protein remained transcriptionally active.
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Raycroft, L., J. R. Schmidt, K. Yoas, M. M. Hao, and G. Lozano. "Analysis of p53 mutants for transcriptional activity." Molecular and Cellular Biology 11, no. 12 (December 1991): 6067–74. http://dx.doi.org/10.1128/mcb.11.12.6067-6074.1991.
Full textAbstract:
The wild-type p53 protein functions to suppress transformation, but numerous mutant p53 proteins are transformation competent. To examine the role of p53 as a transcription factor, we made fusion proteins containing human or mouse p53 sequences fused to the DNA binding domain of a known transcription factor, GAL4. Human and mouse wild-type p53/GAL4 specifically transactivated expression of a chloramphenicol acetyltransferase reporter in HeLa, CHO, and NIH 3T3 cells. Several mutant p53 proteins, including a mouse p53 mutant which is temperature sensitive for suppression, were also analyzed. A p53/GAL4 fusion protein with this mutation was also transcriptionally active only at the permissive temperature. Another mutant p53/GAL4 fusion protein analyzed mimics the mutation inherited in Li-Fraumeni patients. This fusion protein was as active as wild-type p53/GAL4 in our assay. Two human p53 mutants that arose from alterations of the p53 gene in colorectal carcinomas were 30- to 40-fold less effective at activating transcription than wild-type p53/GAL4 fusion proteins. Thus, functional wild-type p53/GAL4 fusion proteins activate transcription, while several transformation competent mutants do so poorly or not at all. Only one mutant p53/GAL4 fusion protein remained transcriptionally active.
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Steegenga, W. T., T. van Laar, N. Riteco, A. Mandarino, A. Shvarts, A. J. van der Eb, and A. G. Jochemsen. "Adenovirus E1A proteins inhibit activation of transcription by p53." Molecular and Cellular Biology 16, no. 5 (May 1996): 2101–9. http://dx.doi.org/10.1128/mcb.16.5.2101.
Full textAbstract:
p53 stimulates the transcription of a number of genes, such as MDM2, Waf1, and GADD45. We and others have shown previously that this activity of p53 can be inhibited by adenovirus type 2 or 12 large E1B proteins. Here we show that the adenovirus E1A proteins also can repress the stimulation of transcription by p53, both in transient transfections and in stably transfected cell lines. The inhibition by E1A occurs without a significant effect on the DNA-binding capacity of p53. Furthermore, the activity of a fusion protein containing the N-terminal part of p53 linked to the GAL4 DNA-binding domain can be suppressed by E1A. This indicates that E1A affects the transcription activation domain of p53, although tryptic phosphopeptide mapping revealed that the level of phosphorylation of this domain does not change significantly in E1A-expressing cell lines. Gel filtration studies, however, showed p53 to be present in complexes of increased molecular weight as a result of E1A expression. Apparently, E1A can cause increased homo- or hetero-oligomerization of p53, which might result in the inactivation of the transcription activation domain of p53. Additionally, we found that transfectants stably expressing E1A have lost the ability to arrest in G1 after DNA damage, indicating that E1A can abolish the normal biological function of p53.
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Murray, M. T., G. Krohne, and W. W. Franke. "Different forms of soluble cytoplasmic mRNA binding proteins and particles in Xenopus laevis oocytes and embryos." Journal of Cell Biology 112, no. 1 (January 1, 1991): 1–11. http://dx.doi.org/10.1083/jcb.112.1.1.
Full textAbstract:
To gain insight into the mechanisms involved in the formation of maternally stored mRNPs during Xenopus laevis development, we searched for soluble cytoplasmic proteins of the oocyte that are able to selectively bind mRNAs, using as substrate radiolabeled mRNA. In vitro mRNP assembly in solution was followed by UV-cross-linking and RNase digestion, resulting in covalent tagging of polypeptides by nucleotide transfer. Five polypeptides of approximately 54, 56 60, 70, and 100 kD (p54, p56, p60, p70, and p100) have been found to selectively bind mRNA and assemble into mRNPs. These polypeptides, which correspond to previously described native mRNP components, occur in three different particle classes of approximately 4.5S, approximately 6S, and approximately 15S, as also determined by their reactions with antibodies against p54 and p56. Whereas the approximately 4.5S class contains p42, p60, and p70, probably each in the form of individual molecules or small complexes, the approximately 6S particles appears to consist only of p54 and p56, which occur in a near-stoichiometric ratio suggestive of a heterodimer complex. The approximately 15S particles contain, in addition to p54 and p56, p60 and p100 and this is the single occurring form of RNA-binding p100. We have also observed changes in the in vitro mRNA binding properties of these polypeptides during oogenesis and early embryonic development, in relation to their phosphorylation state and to the activity of an approximately 15S particle-associated protein kinase, suggesting that these proteins are involved in the developmental translational regulation of maternal mRNAs.
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Milner, J., E. A. Medcalf, and A. C. Cook. "Tumor suppressor p53: analysis of wild-type and mutant p53 complexes." Molecular and Cellular Biology 11, no. 1 (January 1991): 12–19. http://dx.doi.org/10.1128/mcb.11.1.12.
Full textAbstract:
It has been suggested that the dominant effect of mutant p53 on tumor progression may reflect the mutant protein binding to wild-type p53, with inactivation of suppressor function. To date, evidence for wild-type/mutant p53 complexes involves p53 from different species. To investigate wild-type/mutant p53 complexes in relation to natural tumor progression, we sought to identify intraspecific complexes, using murine p53. The mutant phenotype p53-246(0) was used because this phenotype is immunologically distinct from wild-type p53-246+ and thus permits immunological analysis for wild-type/mutant p53 complexes. The p53 proteins were derived from genetically defined p53 cDNAs expressed in vitro and also from phenotypic variants of p53 expressed in vivo. We found that the mutant p53 phenotype was able to form a complex with the wild type when the two p53 variants were cotranslated. When mixed in their native states (after translation), the wild-type and mutant p53 proteins did not exhibit any binding affinity for each other in vitro. Under identical conditions, complexes of wild-type human and murine p53 proteins were formed. For murine p53, both the wild-type and mutant p53 proteins formed high-molecular-weight complexes when translated in vitro. This oligomerization appeared to involve the carboxyl terminus, since truncated p53 (amino acids 1 to 343) did not form complexes. We suggest that the ability of the mutant p53 phenotype to complex with wild type during cotranslation may contribute to the transforming function of activated mutants of p53 in vivo.
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Milner, J., E. A. Medcalf, and A. C. Cook. "Tumor suppressor p53: analysis of wild-type and mutant p53 complexes." Molecular and Cellular Biology 11, no. 1 (January 1991): 12–19. http://dx.doi.org/10.1128/mcb.11.1.12-19.1991.
Full textAbstract:
It has been suggested that the dominant effect of mutant p53 on tumor progression may reflect the mutant protein binding to wild-type p53, with inactivation of suppressor function. To date, evidence for wild-type/mutant p53 complexes involves p53 from different species. To investigate wild-type/mutant p53 complexes in relation to natural tumor progression, we sought to identify intraspecific complexes, using murine p53. The mutant phenotype p53-246(0) was used because this phenotype is immunologically distinct from wild-type p53-246+ and thus permits immunological analysis for wild-type/mutant p53 complexes. The p53 proteins were derived from genetically defined p53 cDNAs expressed in vitro and also from phenotypic variants of p53 expressed in vivo. We found that the mutant p53 phenotype was able to form a complex with the wild type when the two p53 variants were cotranslated. When mixed in their native states (after translation), the wild-type and mutant p53 proteins did not exhibit any binding affinity for each other in vitro. Under identical conditions, complexes of wild-type human and murine p53 proteins were formed. For murine p53, both the wild-type and mutant p53 proteins formed high-molecular-weight complexes when translated in vitro. This oligomerization appeared to involve the carboxyl terminus, since truncated p53 (amino acids 1 to 343) did not form complexes. We suggest that the ability of the mutant p53 phenotype to complex with wild type during cotranslation may contribute to the transforming function of activated mutants of p53 in vivo.
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Malaguarnera, R., V. Vella, R. Vigneri, and F. Frasca. "p53 family proteins in thyroid cancer." Endocrine-Related Cancer 14, no. 1 (March 2007): 43–60. http://dx.doi.org/10.1677/erc.1.01223.
Full textAbstract:
At variance with other human malignancies, p53 mutations are not frequent in thyroid cancer and are believed to be responsible mainly for cancer progression to poorly differentiated and aggressive phenotype. p63 and p73, two proteins with a high degree of homology with p53, are overexpressed in thyroid cancer, but their role in cancer initiation or progression is controversial. Regulation of p53 family protein function depends on: (1) the balance between the expression of transcriptionally active (p53, TAp63, and TAp73) and inactive isoforms (ΔNp63 and ΔNp73); (2) their interaction and competition at DNA-responsive elements; (3) their interaction with regulatory proteins, either inhibitory or activating. In thyroid cancer, therefore, although mutations of the p53 oncosuppressor protein family are rare, other mechanisms are present, including aberrant expression of p53 family dominant negative isoforms, up-regulation of inhibitory proteins, and functional inhibition of activating proteins. The overall result is a defective oncosuppressor activity. These inactivating mechanisms may be present in the early stages of thyroid cancer and in different cancer histotypes. A better understanding of this complex network may not only ameliorate our comprehension of cancer biology, but also open the possibility of innovative diagnostic procedures and the development of targeted therapies.
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Chiang, Yen-Ting, Yi-Chung Chien, Yu-Heng Lin, Hui-Hsuan Wu, Dung-Fang Lee, and Yung-Luen Yu. "The Function of the Mutant p53-R175H in Cancer." Cancers 13, no. 16 (August 13, 2021): 4088. http://dx.doi.org/10.3390/cancers13164088.
Full textAbstract:
Wild-type p53 is known as “the guardian of the genome” because of its function of inducing DNA repair, cell-cycle arrest, and apoptosis, preventing the accumulation of gene mutations. TP53 is highly mutated in cancer cells and most TP53 hotspot mutations are missense mutations. Mutant p53 proteins, encoded by these hotspot mutations, lose canonical wild-type p53 functions and gain functions that promote cancer development, including promoting cancer cell proliferation, migration, invasion, initiation, metabolic reprogramming, angiogenesis, and conferring drug resistance to cancer cells. Among these hotspot mutations, p53-R175H has the highest occurrence. Although losing the transactivating function of the wild-type p53 and prone to aggregation, p53-R175H gains oncogenic functions by interacting with many proteins. In this review, we summarize the gain of functions of p53-R175H in different cancer types, the interacting proteins of p53-R175H, and the downstream signaling pathways affected by p53-R175H to depict a comprehensive role of p53-R175H in cancer development. We also summarize treatments that target p53-R175H, including reactivating p53-R175H with small molecules that can bind to p53-R175H and alter it into a wild-type-like structure, promoting the degradation of p53-R175H by targeting heat-shock proteins that maintain the stability of p53-R175H, and developing immunotherapies that target the p53-R175H–HLA complex presented by tumor cells.
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Martinez, Carlos Augusto Real, Denise Gonçalves Priolli, Izilda Aparecida Cardinalli, José Aires Pereira, Adriana Valim Portes, and Nelson Fontana Margarido. "Influência da localização do tumor na expressão tecidual da proteína p53 em doentes com câncer colorretal: estudo de 100 casos." Revista do Colégio Brasileiro de Cirurgiões 35, no. 4 (August 2008): 235–43. http://dx.doi.org/10.1590/s0100-69912008000400006.
Full textAbstract:
OBJETIVO: O objetivo do presente estudo foi verificar, se existem diferenças na expressão tecidual da proteína p53 segundo a localização do tumor em doentes com câncer colorretal. MÉTODO: Foram estudados 100 doentes (54 mulheres), com média de idade de 59,8 anos com adenocarcinoma colorretal. A expressão da proteína p53 foi analisada por imunoistoquímica, com anticorpo monoclonal anti-p53 pela técnica da estreptavidina-biotina-peroxidase. A expressão tecidual da proteína p53 foi relacionada às variáveis: gênero, idade, grau histológico, tipo histológico, tamanho do tumor, estadiamento TNM, profundidade de invasão da parede intestinal, comprometimento linfonodal, invasão angiolinfática, localização do tumor no intestino grosso em relação à flexura esplênica. Na avaliação estatística da relação entre expressão da proteína p53 e as variáveis consideradas empregou-se o teste qui-quadrado, estabelecendo-se nível de significância de 5% (p<0,05). RESULTADOS: A proteína p53 foi positiva em 77% dos casos. Com relação as diferentes variáveis consideradas verificou-se maior tendência de expressão da proteína mutante quando se considerava a idade (p=0,001), grau histológico (p=0,001), tipo histológico (p=0,001), estádios tardios da classificação TNM (p=0,001), maior profundidade de invasão na parede cólica (p=0,001), comprometimento linfonodal (p=0,001), invasão angiolinfática (p=0,02), localização após a flexura esplênica (p=0,001), não se encontrando relação com gênero (p=0,49) e tamanho do tumor (p=0,08). CONCLUSÃO: Os resultados do presente estudo permitem concluir que a expressão da proteína p53 mutante ocorre com maior freqüência nos tumores localizados a partir da flexura esplênica.
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Caamaño, J. H., P. Perez, S. A. Lira, and R. Bravo. "Constitutive expression of Bc1-3 in thymocytes increases the DNA binding of NF-kappaB1 (p50) homodimers in vivo." Molecular and Cellular Biology 16, no. 4 (April 1996): 1342–48. http://dx.doi.org/10.1128/mcb.16.4.1342.
Full textAbstract:
Previous studies have indicated that Bcl-3 interacts through its ankyrin repeats with the transcriptional factors NF-kappaB1 (p50) and NF-kappaB2 (p52), affecting their biological activities. To further investigate the role of Bcl-3 in vivo and its association with the NF-kappaB proteins, we have generated transgenic mice constitutively expressing Bcl-3 in thymocytes. The results indicate that Bcl-3 is associated with endogenous p50 and p52 in nuclear extracts from transgenic animals. Remarkably, constitutive expression of Bcl-3 in these cells augments the DNA binding activity of p52 homodimers. This effect could be reproduced in vitro and is blocked by anti-Bcl-3 antibodies. We have also shown that Bcl-3 is phosphorylated in thymocytes and that its dephosphorylation greatly decreases the effect on p50 homodimers.
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Hwang, Jenn-Kuo, and Chin-Tarng Lin. "Co-localization of Endogenous and Exogenous p53 Proteins in Nasopharyngeal Carcinoma Cells." Journal of Histochemistry & Cytochemistry 45, no. 7 (July 1997): 991–1003. http://dx.doi.org/10.1177/002215549704500709.
Full textAbstract:
Recently, we have established nine nasopharyngeal carcinoma (NPC) cell lines in which only one cell line showed the p53 mutation. For investigation of the p53 mutation in this line, immunostaining using anti-p53 antibody was applied and showed the presence of p53 protein in the cytoplasm but not in the nucleus. Single strand conformation polymorphism analysis of the p53 gene showed one normal and one additional DNA band. Cloning and sequencing of PCR-amplified DNA showed an AGA (arginine) to ACA (threonine) heterozygous point mutation at codon 280. Transfection of the p53 DNA binding sequence and chloramphenicol acetyltransferase assay revealed loss of transcriptional activation function of endogenous p53 protein. Co-localization of the endogenous and the transfected exogenous p53 protein by polyclonal antibodies to anti-p53 protein revealed strong exogenous p53 staining in the transfected nuclei and weak staining of endogenous p53 protein in the cytoplasm. We concluded that (a) a heterozygous point mutation at codon 280 was identified in the NPC-TW 06 cell line; (b) the point mutation may cause the stagnation of mutant p53 protein in the cytoplasm, and loss of its transcriptional activation function; (c) endogenous and exogenous p53 protein can be co-localized at the same time in the transfected cells; and (d) 280 mutant p53 protein in NPC cells does not cause a decrease or increase in sensitivity to chemotherapy.
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Hall, Callum, and Patricia A. J. Muller. "The Diverse Functions of Mutant 53, Its Family Members and Isoforms in Cancer." International Journal of Molecular Sciences 20, no. 24 (December 7, 2019): 6188. http://dx.doi.org/10.3390/ijms20246188.
Full textAbstract:
The p53 family of proteins has grown substantially over the last 40 years. It started with p53, then p63, p73, isoforms and mutants of these proteins. The function of p53 as a tumour suppressor has been thoroughly investigated, but the functions of all isoforms and mutants and the interplay between them are still poorly understood. Mutant p53 proteins lose p53 function, display dominant-negative (DN) activity and display gain-of-function (GOF) to varying degrees. GOF was originally attributed to mutant p53′s inhibitory function over the p53 family members p63 and p73. It has become apparent that this is not the only way in which mutant p53 operates as a large number of transcription factors that are not related to p53 are activated on mutant p53 binding. This raises the question to what extent mutant p53 binding to p63 and p73 plays a role in mutant p53 GOF. In this review, we discuss the literature around the interaction between mutant p53 and family members, including other binding partners, the functional consequences and potential therapeutics.
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Klibanov, S. A., H. M. O'Hagan, and M. Ljungman. "Accumulation of soluble and nucleolar-associated p53 proteins following cellular stress." Journal of Cell Science 114, no. 10 (May 15, 2001): 1867–73. http://dx.doi.org/10.1242/jcs.114.10.1867.
Full textAbstract:
The tumor suppressor p53 is a nucleocytoplasmic shuttling protein that accumulates in the nucleus of cells exposed to various cellular stresses. One important role of nuclear p53 is to mobilize a stress response by transactivating target genes such as the p21(Waf1) gene. In this study, we investigated more closely the localization of p53 in cells following various stresses. Immunocytochemistry of fixed human fibroblasts treated with either UV light, the kinase and transcription inhibitor DRB or the proteasome inhibitor MG132 revealed abundant p53 localized to the nucleus. When cells treated with UV or DRB were permeabilized prior to fixation to allow soluble proteins to diffuse, the nuclear p53 signal was abolished. However, in cells treated with MG132, residual p53 localized to distinct large foci. Furthermore, nucleolin co-localized with p53 to these foci, suggesting that these foci were nucleolar structures. Interestingly, the MDM2 protein was found to co-localize with p53 to nucleolar structures following proteasome inhibition. Our results suggest that the p53 proteins accumulating in the nucleus following UV-irradiation or blockage of transcription are freely soluble and, thus, should be able to roam the nucleus to ensure high occupancy of p53 binding sites. However, inhibition of proteasome activity may be a unique stress in that it leads to the sequestering of p53 proteins to the nucleolus, thereby blunting the p53-mediated transactivation of target genes.
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Thukral, S. K., Y. Lu, G. C. Blain, T. S. Harvey, and V. L. Jacobsen. "Discrimination of DNA binding sites by mutant p53 proteins." Molecular and Cellular Biology 15, no. 9 (September 1995): 5196–202. http://dx.doi.org/10.1128/mcb.15.9.5196.
Full textAbstract:
Critical determinants of DNA recognition by p53 have been identified by a molecular genetic approach. The wild-type human p53 fragment containing amino acids 71 to 330 (p53(71-330)) was used for in vitro DNA binding assays, and full-length human p53 was used for transactivation assays with Saccharomyces cerevisiae. First, we defined the DNA binding specificity of the wild-type p53 fragment by using systematically altered forms of a known consensus DNA site. This refinement indicates that p53 binds with high affinity to two repeats of PuGPuCA.TGPyCPy, a further refinement of an earlier defined consensus half site PuPuPuC(A/T).(T/A) GPyPyPy. These results were further confirmed by transactivation assays of yeast by using full-length human p53 and systematically altered DNA sites. Dimers of the pentamer AGGCA oriented either head-to-head or tail-to-tail bound efficiently, but transactivation was facilitated only through head-to-head dimers. To determine the origins of specificity in DNA binding by p53, we identified mutations that lead to altered specificities of DNA binding. Single-amino-acid substitutions were made at several positions within the DNA binding domain of p53, and this set of p53 point mutants were tested with DNA site variants for DNA binding. DNA binding analyses showed that the mutants Lys-120 to Asn, Cys-277 to Gln or Arg, and Arg-283 to Gln bind to sites with noncanonical base pair changes at positions 2, 3, and 1 in the pentamer (PuGPuCA), respectively. Thus, we implicate these residues in amino acid-base pair contacts. Interestingly, mutant Cys-277 to Gln bound a consensus site as two and four monomers, as opposed to the wild-type p53 fragment, which invariably binds this site as four monomers.
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Steele, Andrew J., Archibald G. Prentice, A. Victor Hoffbrand, Birunthini C. Yogashangary, Stephen M. Hart, Elisabeth P. Nacheva, Julie D. Howard-Reeves, et al. "p53-mediated apoptosis of CLL cells: evidence for a transcription-independent mechanism." Blood 112, no. 9 (November 1, 2008): 3827–34. http://dx.doi.org/10.1182/blood-2008-05-156380.
Full textAbstract:
The p53 protein plays a key role in securing the apoptotic response of chronic lymphocytic leukemia (CLL) cells to genotoxic agents. Transcriptional induction of proapoptotic proteins including Puma are thought to mediate p53-dependent apoptosis. In contrast, recent studies have identified a novel nontranscriptional mechanism, involving direct binding of p53 to antiapoptotic proteins including Bcl-2 at the mitochondrial surface. Here we show that the major fraction of p53 induced in CLL cells by chlorambucil, fludarabine, or nutlin 3a was stably associated with mitochondria, where it binds to Bcl-2. The Puma protein, which was constitutively expressed in a p53-independent manner, was modestly up-regulated following p53 induction. Pifithrin α, an inhibitor of p53-mediated transcription, blocked the up-regulation of Puma and also of p21CIP1. Surprisingly, pifithrin α dramatically augmented apoptosis induction by p53-elevating agents and also accelerated the proapoptotic conformation change of the Bax protein. These data suggest that direct interaction of p53 with mitochondrial antiapoptotic proteins including Bcl-2 is the major route for apoptosis induction in CLL cells and that p53's transcriptional targets include proteins that impede this nontranscriptional pathway. Therefore, strategies that block up-regulation of p53-mediated transcription may be of value in enhancing apoptosis induction of CLL cells by p53-elevating drugs.
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Harris, N., E. Brill, O. Shohat, M. Prokocimer, D. Wolf, N. Arai, and V. Rotter. "Molecular basis for heterogeneity of the human p53 protein." Molecular and Cellular Biology 6, no. 12 (December 1986): 4650–56. http://dx.doi.org/10.1128/mcb.6.12.4650.
Full textAbstract:
The human p53 tumor antigen comprises several physically distinct proteins. Two p53 proteins, separable by polyacrylamide gel electrophoresis, are expressed by the human transformed cell line SV-80. The individual cDNAs which code for these proteins were isolated and constructed into the SP6 transcription vector. The proteins encoded by these clones were identified by in vitro transcription with the SP6 vector and translation in a cell-free system. p53-H-1 and p53-H-19 cDNA clones code for the faster- and slower-migrating p53 protein species, respectively, of SV-80. The in vitro-expressed proteins of p53-H-1 and p53-H-19 had the same antigenic determinants and were structurally indistinguishable from their in vivo counterparts. By expressing defined restricted cDNA fragments in vitro, the region of heterogeneity between the respective cDNAs was located at the 5' end of the cDNAs. Exchanging the 5' fragments of interest and expressing the chimeric clones in vitro confirmed that the DNA heterogeneity was responsible for the difference in the electrophoretic mobility of these proteins. The sequences of the two cDNAs revealed a single base pair difference (G versus C) in the coding region of the clones. This sequence difference resulted in an arginine being coded for in clone p53-H-1 and a proline being coded for at the equivalent position in clone p53-H-19. This variation accounted for the change in the electrophoretic mobility of the individual p53 protein species.
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Harris, N., E. Brill, O. Shohat, M. Prokocimer, D. Wolf, N. Arai, and V. Rotter. "Molecular basis for heterogeneity of the human p53 protein." Molecular and Cellular Biology 6, no. 12 (December 1986): 4650–56. http://dx.doi.org/10.1128/mcb.6.12.4650-4656.1986.
Full textAbstract:
The human p53 tumor antigen comprises several physically distinct proteins. Two p53 proteins, separable by polyacrylamide gel electrophoresis, are expressed by the human transformed cell line SV-80. The individual cDNAs which code for these proteins were isolated and constructed into the SP6 transcription vector. The proteins encoded by these clones were identified by in vitro transcription with the SP6 vector and translation in a cell-free system. p53-H-1 and p53-H-19 cDNA clones code for the faster- and slower-migrating p53 protein species, respectively, of SV-80. The in vitro-expressed proteins of p53-H-1 and p53-H-19 had the same antigenic determinants and were structurally indistinguishable from their in vivo counterparts. By expressing defined restricted cDNA fragments in vitro, the region of heterogeneity between the respective cDNAs was located at the 5' end of the cDNAs. Exchanging the 5' fragments of interest and expressing the chimeric clones in vitro confirmed that the DNA heterogeneity was responsible for the difference in the electrophoretic mobility of these proteins. The sequences of the two cDNAs revealed a single base pair difference (G versus C) in the coding region of the clones. This sequence difference resulted in an arginine being coded for in clone p53-H-1 and a proline being coded for at the equivalent position in clone p53-H-19. This variation accounted for the change in the electrophoretic mobility of the individual p53 protein species.
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Chang, K., I. Ding, F. G. Kern, and M. C. Willingham. "Immunohistochemical analysis of p53 and HER-2/neu proteins in human tumors." Journal of Histochemistry & Cytochemistry 39, no. 9 (September 1991): 1281–87. http://dx.doi.org/10.1177/39.9.1680897.
Full textAbstract:
We examined samples of tumors of human breast, ovary, and colon of various degrees of malignancy for the expression of p53 protein, using a panel of anti-p53 antibodies and peroxidase immunohistochemistry. Of 66 tumor cases (24 cases of ovarian carcinoma, 23 cases of colon adenocarcinoma, and 19 cases of breast carcinoma), 36 (53%) showed high levels of expression of p53 using a human-specific antibody, and 16 (24%) showed high expression of a mutant form of p53. In the mutant p53-positive breast tumor samples, six (86%) were positive for HER-2/neu reactivity, compared with colon (0/4) and ovarian tumors (1/5). The pattern of p53 intracellular localization and tissue distribution, and the relationship between the expression of mutant p53 and cell differentiation, were also examined; poorly differentiated cells showed either overexpression of p53 or higher levels of mutant p53 in comparison with more normal cells.
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Franzoso, Guido, Louise Carlson, Ljiljana Poljak, Elizabeth W. Shores, Suzanne Epstein, Antonio Leonardi, Alex Grinberg, et al. "Mice Deficient in Nuclear Factor (NF)-κB/p52 Present with Defects in Humoral Responses, Germinal Center Reactions, and Splenic Microarchitecture." Journal of Experimental Medicine 187, no. 2 (January 19, 1998): 147–59. http://dx.doi.org/10.1084/jem.187.2.147.
Full textAbstract:
p52 is a subunit of nuclear factor (NF)-κB transcription factors, most closely related to p50. Previously, we have shown that p52, but not p50 homodimers can form transactivating complexes when associated with Bcl-3, an unusual member of the IκB family. To determine nonredundant physiologic roles of p52, we generated mice deficient in p52. Null mutant mice were impaired in their ability to generate antibodies to T-dependent antigens, consistent with an absence of B cell follicles and follicular dendritic cell networks in secondary lymphoid organs, and an inability to form germinal centers. Furthermore, the splenic marginal zone was disrupted. These phenotypes are largely overlapping with those observed in Bcl-3 knockout animals, but distinct from those of p50 knockouts, supporting the notion of a physiologically relevant complex of p52 homodimers and Bcl-3. Adoptive transfer experiments further suggest that such a complex may be critical in accessory cell functions during antigen-specific immune reactions. Possible roles of p52 and Bcl-3 are discussed that may underlie the oncogenic potential of these proteins, as evidenced by recurrent chromosomal translocations of their genes in lymphoid tumors.
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Schmid, Gerald, Jacek Wojciechowski, and Józefa Wesierska-Gadek. "Advantage of a baculovirus expression system for protein-protein interaction studies. Involvement of posttranslational phosphorylation in the interaction between wt p53 protein and poly(ADP-ribose) polymerase-1." Acta Biochimica Polonica 52, no. 3 (August 4, 2005): 713–19. http://dx.doi.org/10.18388/abp.2005_3435.
Full textAbstract:
We recently observed an interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the tumor suppressor p53 protein. However, more extensive studies on both proteins, especially those on characterization of their domains involved in the interaction were difficult due to very low expression levels of p53 in mammalian cells. Therefore, we generated recombinant proteins for such studies. To clarify which domains of human PARP-1 and of human wild-type (wt) p53 were involved in this protein-protein interaction, we generated baculoviral constructs encoding full length or distinct functional domains of both proteins. Full length PARP-1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy-terminal fragments of p53 each were sufficient to confer binding to PARP-1, whereas the amino-terminal part harbouring the transactivation functional domain was dispensable. On the other hand, the amino-terminal and central fragments of PARP-1 were both necessary for complex formation with p53 protein. Since the most important features of p53 protein are regulated by phosphorylation, we addressed the question whether its phosphorylation is essential for the binding between the two proteins. Baculovirally expressed wt p53 was post-translationally modified. At least six distinct p53 isomers were resolved by immunoblotting following two-dimensional separation of baculovirally expressed wt p53 protein. Using specific phospho-serine antibodies, we identified phosphorylation of baculovirally expressed p53 protein at five distinct sites. To define the role of p53 phosphorylation, pull-down assays using untreated and dephosphorylated p53 protein were performed. Dephosphorylated p53 failed to bind PARP-1, indicating that complex formation between the two proteins was regulated by phosphorylation of p53. The marked phosphorylation of p53 at Ser392 observed in unstressed cells suggests that the phosphorylated carboxy-terminal part of p53 undergoes complex formation with PARP-1 resulting in masking of the NES and thereby preventing its export.
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Steegenga, Wilma T., Avi Shvarts, Nicole Riteco, Johannes L. Bos, and Aart G. Jochemsen. "Distinct Regulation of p53 and p73 Activity by Adenovirus E1A, E1B, and E4orf6 Proteins." Molecular and Cellular Biology 19, no. 5 (May 1, 1999): 3885–94. http://dx.doi.org/10.1128/mcb.19.5.3885.
Full textAbstract:
ABSTRACT Multiple adenovirus (Ad) early proteins have been shown to inhibit transcription activation by p53 and thereby to alter its normal biological functioning. Since these Ad proteins affect the activity of p53 via different mechanisms, we examined whether this inhibition is target gene specific. In addition, we analyzed whether the same Ad early proteins have a comparable effect on transcription activation by the recently identified p53 homologue p73. Our results show that the large E1B proteins very efficiently inhibited the activity of p53 on the Bax, p21Waf1, cyclin G, and MDM2 reporter constructs but had no effect on the activation of the same reporter constructs by p73, with the exception of some inhibition of the Bax promoter by Ad12 E1B. The repressive effect of the E1A proteins on p53 activity is less than that seen with the large E1B proteins, but the E1A proteins inhibit the activity of both p53 and p73. We could not detect significant inhibition of p53 functions by E4orf6, but a clear repression of the transcription activation by p73 by this Ad early protein was observed. In addition, we found that stable expression of the Ad5 E1A and that of the E1B protein both caused increased p73 protein expression. The large E1B and the E4orf6 proteins together do not target the p73 protein for rapid degradation after adenoviral infection, as has previously been found for the p53 protein, probably because the large E1B protein does not interact with p73. Our results suggest that the p53 and p73 proteins are both inactivated after Ad infection and transformation but via distinct mechanisms.
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Harms, Kelly Lynn, and Xinbin Chen. "The C Terminus of p53 Family Proteins Is a Cell Fate Determinant." Molecular and Cellular Biology 25, no. 5 (March 1, 2005): 2014–30. http://dx.doi.org/10.1128/mcb.25.5.2014-2030.2005.
Full textAbstract:
ABSTRACT The p53 tumor suppressor is the most commonly mutated gene in human cancers. The ability of p53 to induce cell cycle arrest, apoptosis, DNA repair, and other p53-dependent activities is well known; however, the mechanism by which p53 induces a specific activity over another is unclear. Here, we showed that stringent regulation of and by p53 family isoforms facilitates differential target gene expression and thus determines cell fate. Through the use of engineered deletion mutants, we found that activation domain 2 is required for induction of the proapoptotic target gene insulin-like growth factor binding protein 3 (IGFBP3) by p53 and that the basic domain inhibits induction of this gene by p53. Thus, for the first time we provide evidence that the basic domain of p53 is inhibitory in vivo as has been determined in vitro. We also showed that the in vivo inhibitory activity of the basic domain depends upon activation domain 1, such that combined deletion of activation domain 1 and the basic domain was required to alleviate the inhibition by the basic domain. Importantly, deletion of the inhibitory functional domains, namely N-terminal activation domain 1 and the C-terminal basic domain, is paralleled in nature. We found that the IGFBP3 promoter was activated by p53(ΔNΔBD), which mimics a naturally occurring N- and C-terminally truncated human p53 isoform, and by p53AS, a C-terminally truncated murine p53 isoform generated through alternative splicing, but not by full-length human or murine p53. In addition, we found that the C termini of p63 and p73 inhibit the induction of IGFBP3, such that C-terminally truncated p63 and p73 isoforms induce the expression of IGFBP3, whereas full-length ones cannot. We also demonstrated that IGFBP3 is an important effector of the apoptosis induced by N- and C-terminally truncated p53, such that knockdown of IGFBP3 by using an IGFBP3 neutralizing antibody or IGFBP3 small interfering RNA partially rescues the cell death induced by N- and C-terminally truncated p53. In addition, we identified that histone deacetylase activity, not p53 DNA binding ability, governs the regulation of IGFBP3 by full-length p53 family proteins, as inhibition of histone deacetylases restores the induction of IGFBP3 by exogenous full-length p53, p63, and p73 proteins. Furthermore, we found that activation of p53 or inhibition of histone deacetylases alone was not sufficient to induce IGFBP3; however, combined treatment endowed endogenous p53 with this activity. To better understand the significance of this regulation, we performed a microarray study and identified several target genes differentially regulated by full-length p53 and p53 lacking the N-terminal activation domain 1 and the C-terminal basic domain. Taken together, our data suggest a novel mechanism by which p53 family proteins differentially regulate gene expression and provide an insight for designing a combined therapy for cancer treatment.
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Hu, T., T. Guan, and L. Gerace. "Molecular and functional characterization of the p62 complex, an assembly of nuclear pore complex glycoproteins." Journal of Cell Biology 134, no. 3 (August 1, 1996): 589–601. http://dx.doi.org/10.1083/jcb.134.3.589.
Full textAbstract:
Macromolecular trafficking across the nuclear envelope involves interactions between cytosolic transport factors and nuclear pore complex proteins. The p62 complex, an assembly of 62, 58, 54, and 45-kD O-linked glycoproteins-localized near the central gated channel of the nuclear pore complex, has been directly implicated in nuclear protein import. The cDNA cloning of rat p62 was reported previously. We have now carried out cDNA cloning of rat p58, p54, and p45. We found that p58 contains regions with FG (Phe, Gly) and PA (Pro, Ala) repeats at both its NH2 and COOH termini separated by a predicted alpha-helical coiled-coil region, while p54 has an NH2-terminal FG and PA repeat region and a COOH-terminal predicted coiled-coil region. p45 and p58 appear to be generated by alternative splicing, with p45 containing the NH2-terminal FG repeat region and the coiled-coil region of p58. Using immunogold electron microscopy, we found that p58/p45 and p54 are localized on both sides of the nuclear pore complex, like p62. Previous studies have shown that immobilized recombinant p62 can bind the cytosolic nuclear import factor NTF2 and thereby deplete transport activity from cytosol. We have now found that immobilized recombinant p58 and p54 also can deplete nuclear transport activity from cytosol, and that p62, p58, and p54 bind directly to the cytosolic nuclear import factors p97 and NTF2. At least in the case of p58, this involves FG repeat regions. Moreover, p58 can bind to a complex containing transport ligand, the nuclear localization sequence receptor (Srp1 alpha) and p97. These data support a model in which the p62 complex binds to a multicomponent particle consisting of transport ligand and cytosolic factors to achieve accumulation of ligand near the central gated channel of the nuclear pore complex.
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Shaulsky, G., N. Goldfinger, A. Ben-Ze'ev, and V. Rotter. "Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis." Molecular and Cellular Biology 10, no. 12 (December 1990): 6565–77. http://dx.doi.org/10.1128/mcb.10.12.6565.
Full textAbstract:
The basic carboxy terminus of p53 plays an important role in directing the protein into the nuclear compartment. The C terminus of the p53 molecule contains a cluster of several nuclear localization signals (NLSs) that mediate the migration of the protein into the cell nucleus. NLSI, the most active domain, is highly conserved in genetically diverged species and shares perfect homology with consensus NLS sequences found in other nuclear proteins. The other two NLSs, II and III, appear to be less effective and less conserved. Although nuclear localization is dictated primarily by the NLSs inherent in the primary amino acid sequence, the actual nuclear homing can be modified by interactions with other proteins expressed in the cell. Comparison between wild-type p53 and naturally occurring mutant p53 showed that both protein categories could migrate into the nucleus of rat primary embryonic fibroblasts by essentially similar mechanisms. Nuclear localization of both proteins was totally dependent on the existence of functional NLS domains. In COS cells, however, we found that NLS-deprived wild-type p53 molecules could migrate into the nucleus by complexing with another nuclear protein, simian virus 40 large-T antigen. Wild-type and mutant p53 proteins differentially complexed with viral or cellular proteins, which may significantly affect the ultimate compartmentalization of p53 in the cell; this finding suggests that the actual subcellular compartmentalization of proteins may differ in various cell type milieux and may largely be affected by the ability of these proteins to complex with other proteins expressed in the cell. Experiments designed to test the physiological significance of p53 subcellular localization indicated that nuclear localization of mutant p53 is essential for this protein to enhance the process of malignant transformation of partially transformed cells, suggesting that p53 functions within the cell nucleus.
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Shaulsky, G., N. Goldfinger, A. Ben-Ze'ev, and V. Rotter. "Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis." Molecular and Cellular Biology 10, no. 12 (December 1990): 6565–77. http://dx.doi.org/10.1128/mcb.10.12.6565-6577.1990.
Full textAbstract:
The basic carboxy terminus of p53 plays an important role in directing the protein into the nuclear compartment. The C terminus of the p53 molecule contains a cluster of several nuclear localization signals (NLSs) that mediate the migration of the protein into the cell nucleus. NLSI, the most active domain, is highly conserved in genetically diverged species and shares perfect homology with consensus NLS sequences found in other nuclear proteins. The other two NLSs, II and III, appear to be less effective and less conserved. Although nuclear localization is dictated primarily by the NLSs inherent in the primary amino acid sequence, the actual nuclear homing can be modified by interactions with other proteins expressed in the cell. Comparison between wild-type p53 and naturally occurring mutant p53 showed that both protein categories could migrate into the nucleus of rat primary embryonic fibroblasts by essentially similar mechanisms. Nuclear localization of both proteins was totally dependent on the existence of functional NLS domains. In COS cells, however, we found that NLS-deprived wild-type p53 molecules could migrate into the nucleus by complexing with another nuclear protein, simian virus 40 large-T antigen. Wild-type and mutant p53 proteins differentially complexed with viral or cellular proteins, which may significantly affect the ultimate compartmentalization of p53 in the cell; this finding suggests that the actual subcellular compartmentalization of proteins may differ in various cell type milieux and may largely be affected by the ability of these proteins to complex with other proteins expressed in the cell. Experiments designed to test the physiological significance of p53 subcellular localization indicated that nuclear localization of mutant p53 is essential for this protein to enhance the process of malignant transformation of partially transformed cells, suggesting that p53 functions within the cell nucleus.
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Stein, Yan, Varda Rotter, and Ronit Aloni-Grinstein. "Gain-of-Function Mutant p53: All the Roads Lead to Tumorigenesis." International Journal of Molecular Sciences 20, no. 24 (December 8, 2019): 6197. http://dx.doi.org/10.3390/ijms20246197.
Full textAbstract:
The p53 protein is mutated in about 50% of human cancers. Aside from losing the tumor-suppressive functions of the wild-type form, mutant p53 proteins often acquire inherent, novel oncogenic functions, a phenomenon termed mutant p53 gain-of-function (GOF). A growing body of evidence suggests that these pro-oncogenic functions of mutant p53 proteins are mediated by affecting the transcription of various genes, as well as by protein–protein interactions with transcription factors and other effectors. In the current review, we discuss the various GOF effects of mutant p53, and how it may serve as a central node in a network of genes and proteins, which, altogether, promote the tumorigenic process. Finally, we discuss mechanisms by which “Mother Nature” tries to abrogate the pro-oncogenic functions of mutant p53. Thus, we suggest that targeting mutant p53, via its reactivation to the wild-type form, may serve as a promising therapeutic strategy for many cancers that harbor mutant p53. Not only will this strategy abrogate mutant p53 GOF, but it will also restore WT p53 tumor-suppressive functions.
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Wang, Shang-Kwei, Cheng-Hui Hu, Miao-Chan Lu, Chang-Yih Duh, Pao-Chi Liao, and Yu-Chang Tyan. "Novel virus-associated proteins encoded by UL112–113 of human cytomegalovirus." Journal of General Virology 90, no. 12 (December 1, 2009): 2840–48. http://dx.doi.org/10.1099/vir.0.013037-0.
Full textAbstract:
Evidence suggests that the products of the human cytomegalovirus (HCMV) UL112–113 genes are involved in viral DNA replication during lytic infection. A polyclonal antibody was raised against the UL112 open reading frame (ORF) to characterize its function in detail. Immunoblots utilizing the UL112 antibody identified seven distinct protein bands (p20, p26, p28, p34, p43, p50 and p84) expressed during the HCMV infectious cycle. After screening a cDNA library constructed from cells 72 h after infection with HCMV, only four different cDNA protein-producing constructs were obtained, and their ORFs corresponded to p34, p43, p50 and p84. The proteins p20, p26 and p28 were further shown to be selectively included within mature HCMV particles, virions, non-infectious enveloped particles and dense bodies. Immunoaffinity protein purification was used to prepare the samples for liquid chromatography coupled to tandem mass spectrometry. This analysis revealed that p20, p26 and p28 were derived from the UL112 ORF, most likely through post-translational proteolytic cleavage.
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Hovius, Joppe W. R., K. Emil Hovius, Anneke Oei, Dirk J. Houwers, and Alje P. van Dam. "Antibodies against Specific Proteins of and Immobilizing Activity against Three Strains of Borrelia burgdorferi Sensu Lato Can Be Found in Symptomatic but Not in Infected Asymptomatic Dogs." Journal of Clinical Microbiology 38, no. 7 (2000): 2611–21. http://dx.doi.org/10.1128/jcm.38.7.2611-2621.2000.
Full textAbstract:
In an area where Lyme disease is endemic in The Netherlands all dogs had positive titers by whole-cell enzyme-linked immunosorbent assay and appeared to be naturally infected by Borrelia burgdorferi sensu lato. To compare the antibody responses of symptomatic dogs and asymptomatic controls, we performed Western blots and in vitro immobilization assays to study antibody-dependent bactericidal activity. Strains from three different genospecies were employed as the antigen source: B. burgdorferi strain B31,Borrelia garinii strain A87S, and Borrelia afzelii strain pKo. Antibodies against flagellin (p41) and p39 for three strains were found in sera from both symptomatic and asymptomatic dogs and were therefore considered to be markers of exposure. Antibodies against p56 and p30 of strain B31, against p75, p58, p50, OspC, and p<19 of strain A87S, and against p56, p54, p45, OspB, p31, p26, and p<19 of strain pKo were found significantly more frequently in sera from symptomatic dogs younger than 8 years when the first symptoms were observed than in those from age-matched controls (P < 0.01). These antibodies were not found in preclinical sera and appeared during development of disease. Antibodies against OspA of strains B31 and A87S were only seen in acute-phase and convalescent sera from three dogs that recovered from disease. Incubation with 25% normal canine serum did not result in the immobilization of strains B31 and pKo, but partial immobilization of strain A87S (61% ± 24% [standard deviation] at 5 h) occurred. Seven of 15 sera from symptomatic dogs but none of the sera from 11 asymptomatic dogs had antibody-dependent immobilizing activity against one of the strains. Consecutive sera from one of these dogs immobilized two different strains. Antibody-mediated bactericidal serum was not seen before onset of disease, was strongest in the acute phase of disease, and fluctuated during chronic disease. From seven out of eight symptomatic dogs Borrelia DNA was amplified by PCR; in three of them the bactericidal activity was directed against one of the genospecies amplified from that dog; however, four PCR-positive dogs lacked bactericidal activity. In conclusion, dogs with symptomatic canine borreliosis have more-extensive antibody reactivity against Borrelia, as shown by both Western blotting and immobilization assays.
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Liu, X., C. W. Miller, P. H. Koeffler, and A. J. Berk. "The p53 activation domain binds the TATA box-binding polypeptide in Holo-TFIID, and a neighboring p53 domain inhibits transcription." Molecular and Cellular Biology 13, no. 6 (June 1993): 3291–300. http://dx.doi.org/10.1128/mcb.13.6.3291.
Full textAbstract:
Antioncogene product p53 is a transcriptional transactivator. To investigate how p53 stimulates transcription, we examined the interaction of p53 with general transcription factors in vitro. We found that p53 binds directly to the human TATA box-binding polypeptide (TBP). We also observed a direct interaction between p53 and purified holo-TFIID, a complex composed of TBP and a group of TBP-associated polypeptides known as TAFs. The p53 binding domain on TBP was mapped to the conserved region of TBP, including residues 220 to 271. The TBP binding domain on p53 was mapped to the p53 activation domain between residues 20 and 57. To analyze the significance of the p53-TBP interaction in p53 transactivation, we compared the ability of Gal4-p53 fusion proteins to bind to TBP in vitro and to activate transcription in transient transfection assays. Fusion proteins which bound to TBP activated transcription, and those that did not bind to TBP did not activate transcription to a detectable level, suggesting that a direct interaction between TBP and p53 is required for p53 transactivation. We also found that inclusion of residues 93 to 160 of p53 in a Gal4-p53 fusion repressed transcriptional activation 100-fold. Consequently, this region of p53 inhibits transcriptional activation by the minimal p53 activation domain. Highest levels of activation were observed with sequences 1 to 92 of p53 fused to Gal4, even though this construct bound to TBP in vitro with an affinity similar to that of other Gal4-p53 fusion proteins. We conclude that TBP binding is necessary for p53 transcriptional activation and that p53 sequences outside the TBP binding domain modulate the level of activation.
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Liu, X., C. W. Miller, P. H. Koeffler, and A. J. Berk. "The p53 activation domain binds the TATA box-binding polypeptide in Holo-TFIID, and a neighboring p53 domain inhibits transcription." Molecular and Cellular Biology 13, no. 6 (June 1993): 3291–300. http://dx.doi.org/10.1128/mcb.13.6.3291-3300.1993.
Full textAbstract:
Antioncogene product p53 is a transcriptional transactivator. To investigate how p53 stimulates transcription, we examined the interaction of p53 with general transcription factors in vitro. We found that p53 binds directly to the human TATA box-binding polypeptide (TBP). We also observed a direct interaction between p53 and purified holo-TFIID, a complex composed of TBP and a group of TBP-associated polypeptides known as TAFs. The p53 binding domain on TBP was mapped to the conserved region of TBP, including residues 220 to 271. The TBP binding domain on p53 was mapped to the p53 activation domain between residues 20 and 57. To analyze the significance of the p53-TBP interaction in p53 transactivation, we compared the ability of Gal4-p53 fusion proteins to bind to TBP in vitro and to activate transcription in transient transfection assays. Fusion proteins which bound to TBP activated transcription, and those that did not bind to TBP did not activate transcription to a detectable level, suggesting that a direct interaction between TBP and p53 is required for p53 transactivation. We also found that inclusion of residues 93 to 160 of p53 in a Gal4-p53 fusion repressed transcriptional activation 100-fold. Consequently, this region of p53 inhibits transcriptional activation by the minimal p53 activation domain. Highest levels of activation were observed with sequences 1 to 92 of p53 fused to Gal4, even though this construct bound to TBP in vitro with an affinity similar to that of other Gal4-p53 fusion proteins. We conclude that TBP binding is necessary for p53 transcriptional activation and that p53 sequences outside the TBP binding domain modulate the level of activation.
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Gissi, Davide B., Andrea Gabusi, Dora Servidio, Fabio Cervellati, and Lucio Montebugnoli. "Predictive Role of p53 Protein as a Single Marker or Associated with ki67 Antigen in Oral Leukoplakia: A Retrospective Longitudinal Study." Open Dentistry Journal 9, no. 1 (January 30, 2015): 41–45. http://dx.doi.org/10.2174/1874210601509010041.
Full textAbstract:
Oral leukoplakia (OL) is the most common potentially malignant lesion of the oral cavity. Immunohistochemical analysis of p53 and Ki67 proteins is a simple and inexpensive method widely used in non-dysplastic OLs to reveal lesions predicted to develop oral cancer. The present longitudinal study evaluated the predictive role of p53 and Ki67 proteins alone or in combination in a group of OLs without dysplasia followed for many years. Seventy-seven OL patients referred to our Department between January 2006 and October 2013 underwent histochemical analysis of p53 and Ki67 expression. OLs were considered at high risk in the presence of either high p53 expression (>20%), or low/normal p53 expression associated with high Ki67 expression (Ki67/p53 ratio >3). Seven OLs evolved to OSCC during the follow-up period. Three cases had p53 overexpression, while four had a high Ki67/p53 ratio. Statistical significance was reached when samples with p53 overexpression were combined with samples with high Ki67/p53 ratio (Chi square 5.3; p<0.02). The combined immunohistochemical expression of p53 and Ki67 proteins could be a useful and simple molecular marker for early detection of non-dysplastic OLs at risk of developing oral cancer.