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.

• Klíčová slova
• p53 protein, protein-DNA interaction, transactivation potential,
• MeSH
• G-kvadruplexy * MeSH
• lidé MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• protein - isoformy genetika   metabolismus   MeSH
• proteiny regulující apoptózu genetika   metabolismus   MeSH
• protoonkogenní proteiny genetika   metabolismus   MeSH
• responzivní elementy genetika   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• BBC3 protein, human MeSH Prohlížeč
• nádorový supresorový protein p53 MeSH
• protein - isoformy MeSH
• proteiny regulující apoptózu MeSH
• protoonkogenní proteiny MeSH

BACKGROUND: The Nse1, Nse3 and Nse4 proteins form a tight sub-complex of the large SMC5-6 protein complex. hNSE3/MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and the Nse4 kleisin subunit is related to the EID (E1A-like inhibitor of differentiation) family of proteins. We have recently shown that human MAGE proteins can interact with NSE4/EID proteins through their characteristic conserved hydrophobic pocket. METHODOLOGY/PRINCIPAL FINDINGS: Using mutagenesis and protein-protein interaction analyses, we have identified a new Nse3/MAGE-binding domain (NMBD) of the Nse4/EID proteins. This short domain is located next to the Nse4 N-terminal kleisin motif and is conserved in all NSE4/EID proteins. The central amino acid residues of the human NSE4b/EID3 domain were essential for its binding to hNSE3/MAGEG1 in yeast two-hybrid assays suggesting they form the core of the binding domain. PEPSCAN ELISA measurements of the MAGEC2 binding affinity to EID2 mutant peptides showed that similar core residues contribute to the EID2-MAGEC2 interaction. In addition, the N-terminal extension of the EID2 binding domain took part in the EID2-MAGEC2 interaction. Finally, docking and molecular dynamic simulations enabled us to generate a structure model for EID2-MAGEC2. Combination of our experimental data and the structure modeling showed how the core helical region of the NSE4/EID domain binds into the conserved pocket characteristic of the MAGE protein family. CONCLUSIONS/SIGNIFICANCE: We have identified a new Nse4/EID conserved domain and characterized its binding to Nse3/MAGE proteins. The conservation and binding of the interacting surfaces suggest tight co-evolution of both Nse4/EID and Nse3/MAGE protein families.

• MeSH
• chromozomální proteiny, nehistonové MeSH
• inhibitor diferenciace 2 MeSH
• interakční proteinové domény a motivy MeSH
• intracelulární signální peptidy a proteiny chemie   genetika   metabolismus   MeSH
• jaderné proteiny chemie   MeSH
• konzervovaná sekvence MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• peptidové fragmenty chemie   genetika   metabolismus   MeSH
• počítačová simulace MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• Schizosaccharomyces pombe - proteiny chemie   MeSH
• Schizosaccharomyces MeSH
• sekvence aminokyselin MeSH
• substituce aminokyselin MeSH
• techniky dvojhybridového systému MeSH
• transportní proteiny chemie   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromozomální proteiny, nehistonové MeSH
• EID2 protein, human MeSH Prohlížeč
• EID3 protein, human MeSH Prohlížeč
• inhibitor diferenciace 2 MeSH
• intracelulární signální peptidy a proteiny MeSH
• jaderné proteiny MeSH
• Nse3 protein, S pombe MeSH Prohlížeč
• Nse4 protein, S pombe MeSH Prohlížeč
• NSMCE3 protein, human MeSH Prohlížeč
• peptidové fragmenty MeSH
• proteiny buněčného cyklu MeSH
• rekombinantní proteiny MeSH
• Schizosaccharomyces pombe - proteiny MeSH
• SMC5 protein, human MeSH Prohlížeč
• transportní proteiny MeSH

p53 is one of the most important regulators of cell proliferation and differentiation and of programmed cell death, triggering growth arrest and/or apoptosis in response to different cellular stress signals. The sequence-specific DNA-binding function of p53 protein can be activated by several different stimuli that modulate the C-terminal domain of this protein. The predominant mechanism of activation of p53 sequence-specific DNA binding is phosphorylation at specific sites. For example, phosphorylation of p53 by PKC (protein kinase C) occurs in undamaged cells, resulting in masking of the epitope recognized by monoclonal antibody PAb421, and presumably promotes steady-state levels of p53 activity in cycling cells. In contrast, phosphorylation by cdk2 (cyclin-dependent kinase 2)/cyclin A and by the protein kinase CK2 are both enhanced in DNA-damaged cells. We determined whether one mechanism to account for this mutually exclusive phosphorylation may be that each phosphorylation event prevents modification by the other kinase. We used non-radioactive electrophoretic mobility shift assays to show that C-terminal phosphorylation of p53 protein by cdk2/cyclin A on Ser315 or by PKC on Ser378 can efficiently stimulate p53 binding to DNA in vitro, as well as binding of the monoclonal antibody Bp53-10, which recognizes residues 371-380 in the C-terminus of p53. Phosphorylation of p53 by CK2 on Ser392 induces its DNA-binding activity to a much lower extent than phosphorylation by cdk2/cyclin A or PKC. In addition, phosphorylation by CK2 strongly inhibits PKC-induced activation of p53 DNA binding, while the activation of p53 by cdk2/cyclin A is not affected by CK2. The presence of CK2-mediated phosphorylation promotes PKC binding to its docking site within the p53 oligomerization domain, but decreases phosphorylation by PKC, suggesting that competition between CK2 and PKC does not rely on the inhibition of PKC-p53 complex formation. These results indicate the crucial role of p53 C-terminal phosphorylation in the regulation of its DNA-binding activity, but also suggest that antagonistic relationships exist between different stress signalling pathways.

• MeSH
• buněčné linie MeSH
• cyklin-dependentní kinasa 2 MeSH
• DNA chemie   metabolismus   MeSH
• fosforylace MeSH
• kaseinkinasa II MeSH
• kinasy CDC2-CDC28 metabolismus   MeSH
• lidé MeSH
• monoklonální protilátky farmakologie   MeSH
• nádorový supresorový protein p53 chemie   imunologie   metabolismus   MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteinkinasa C antagonisté a inhibitory   metabolismus   MeSH
• responzivní elementy MeSH
• Spodoptera cytologie   MeSH
• terciární struktura proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CDK2 protein, human MeSH Prohlížeč
• cyklin-dependentní kinasa 2 MeSH
• DNA MeSH
• kaseinkinasa II MeSH
• kinasy CDC2-CDC28 MeSH
• monoklonální protilátky MeSH
• nádorový supresorový protein p53 MeSH
• protein-serin-threoninkinasy MeSH
• proteinkinasa C MeSH

We showed previously that bacterially expressed full-length human wild-type p53b(1-393) binds selectively to supercoiled (sc)DNA in sc/linear DNA competition experiments, a process we termed supercoil-selective (SCS) binding. Using p53 deletion mutants and pBluescript scDNA (lacking the p53 recognition sequence) at native superhelix density we demonstrate here that the p53 C-terminal domain (amino acids 347-382) and a p53 oligomeric state are important for SCS binding. Monomeric p53(361-393) protein (lacking the p53 tetramerization domain, amino acids 325-356) did not exhibit SCS binding while both dimeric mutant p53(319- 393)L344A and fusion protein GCN4-p53(347-393) were effective in SCS binding. Supershifting of p53(320-393)-scDNA complexes with monoclonal antibodies revealed that the amino acid region 375-378, constituting the epitope of the Bp53-10.1 antibody, plays a role in binding of the p53(320-393) protein to scDNA. Using electron microscopy we observed p53-scDNA nucleoprotein filaments produced by all the C-terminal proteins that displayed SCS binding in the gel electrophoresis experiments; no filaments formed with the monomeric p53(361- 393) protein. We propose a model according to which two DNA duplexes are compacted into p53-scDNA filaments and discuss a role for filament formation in recombination.

• MeSH
• biologické modely MeSH
• lidé MeSH
• makromolekulární látky MeSH
• molekulární sekvence - údaje MeSH
• nádorový supresorový protein p53 chemie   metabolismus   ultrastruktura   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční delece MeSH
• superhelikální DNA metabolismus   ultrastruktura   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• makromolekulární látky MeSH
• nádorový supresorový protein p53 MeSH
• rekombinantní fúzní proteiny MeSH
• superhelikální DNA MeSH