TP53 gene abnormalities represent the most important biomarker in chronic lymphocytic leukemia (CLL). Altered protein modifications could also influence p53 function, even in the wild-type protein. We assessed the impact of p53 protein phosphorylations on p53 functions as an alternative inactivation mechanism. We studied p53 phospho-profiles induced by DNA-damaging agents (fludarabine, doxorubicin) in 71 TP53-intact primary CLL samples. Doxorubicin induced two distinct phospho-profiles: profile I (heavily phosphorylated) and profile II (hypophosphorylated). Profile II samples were less capable of activating p53 target genes upon doxorubicin exposure, resembling TP53-mutant samples at the transcriptomic level, whereas standard p53 signaling was triggered in profile I. ATM locus defects were more common in profile II. The samples also differed in the basal activity of the hypoxia pathway: the highest level was detected in TP53-mutant samples, followed by profile II and profile I. Our study suggests that wild-type TP53 CLL cells with less phosphorylated p53 show TP53-mutant-like behavior after DNA damage. p53 hypophosphorylation and the related lower ability to respond to DNA damage are linked to ATM locus defects and the higher basal activity of the hypoxia pathway.

• Klíčová slova
• CLL, p53, phosphorylation,
• MeSH
• ATM protein genetika   metabolismus   MeSH
• chronická lymfatická leukemie * genetika   MeSH
• doxorubicin farmakologie   MeSH
• fosforylace MeSH
• geny p53 MeSH
• hypoxie genetika   MeSH
• lidé MeSH
• nádorový supresorový protein p53 * metabolismus   MeSH
• poškození DNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ATM protein MeSH
• doxorubicin MeSH
• nádorový supresorový protein p53 * MeSH

The p53 family of transcription factors plays key roles in development, genome stability, senescence and tumor development, and p53 is the most important tumor suppressor protein in humans. Although intensively investigated for many years, its initial evolutionary history is not yet fully elucidated. Using bioinformatic and structure prediction methods on current databases containing newly-sequenced genomes and transcriptomes, we present a detailed characterization of p53 family homologs in remote members of the Holozoa group, in the unicellular clades Filasterea, Ichthyosporea and Corallochytrea. Moreover, we show that these newly characterized homologous sequences contain domains that can form structures with high similarity to the human p53 family DNA-binding domain, and some also show similarities to the oligomerization and SAM domains. The presence of these remote homologs demonstrates an ancient origin of the p53 protein family.

• Klíčová slova
• Holozoa, evolution, p53, p63, p73,
• MeSH
• databáze genetické MeSH
• Eukaryota klasifikace   genetika   MeSH
• exony MeSH
• fylogeneze MeSH
• interakční proteinové domény a motivy MeSH
• introny MeSH
• konformace proteinů MeSH
• molekulární evoluce * MeSH
• molekulární modely MeSH
• multigenová rodina * MeSH
• nádorový supresorový protein p53 chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• nádorový supresorový protein p53 MeSH

p53 is a tetrameric protein with a thermodynamically unstable deoxyribonucleic acid (DNA)-binding domain flanked by intrinsically disordered regulatory domains that control its activity. The unstable and disordered segments of p53 allow high flexibility as it interacts with binding partners and permits a rapid on/off switch to control its function. The p53 tetramer can exist in multiple conformational states, any of which can be stabilized by a particular modification. Here, we apply the allostery model to p53 to ask whether evidence can be found that the "activating" C-terminal phosphorylation of p53 stabilizes a specific conformation of the protein in the absence of DNA. We take advantage of monoclonal antibodies for p53 that measure indirectly the following conformations: unfolded, folded, and tetrameric. A double antibody capture enzyme linked-immunosorbent assay was used to observe evidence of conformational changes of human p53 upon phosphorylation by casein kinase 2 in vitro. It was demonstrated that oligomerization and stabilization of p53 wild-type conformation results in differential exposure of conformational epitopes PAb1620, PAb240, and DO12 that indicates a reduction in the "unfolded" conformation and increases in the folded conformation coincide with increases in its oligomerization state. These data highlight that the oligomeric conformation of p53 can be stabilized by an activating enzyme and further highlight the utility of the allostery model when applied to understanding the regulation of unstable and intrinsically disordered proteins.

• Klíčová slova
• CK2, allosteric regulation, conformational change, oligomerization, p53, phosphorylation, protein conformation, protein folding,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• alosterická regulace MeSH
• fosforylace MeSH
• kaseinkinasa II metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• mutace MeSH
• nádorový supresorový protein p53 chemie   genetika   metabolismus   MeSH
• proteinové domény MeSH
• stabilita proteinů MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• kaseinkinasa II MeSH
• nádorový supresorový protein p53 MeSH
• TP53 protein, human MeSH Prohlížeč

Electrochemical enzyme-linked techniques for sequence-specific DNA sensingare presented. These techniques are based on attachment of streptavidin-alkalinephosphatase conjugate to biotin tags tethered to DNA immobilized at the surface ofdisposable screen-printed carbon electrodes (SPCE), followed by production andelectrochemical determination of an electroactive indicator, 1-naphthol. Via hybridizationof SPCE surface-confined target DNAs with end-biotinylated probes, highly specificdiscrimination between complementary and non-complementary nucleotide sequences wasachieved. The enzyme-linked DNA hybridization assay has been successfully applied inanalysis of PCR-amplified real genomic DNA sequences, as well as in monitoring of planttissue-specific gene expression. In addition, we present an alternative approach involvingsequence-specific incorporation of biotin-labeled nucleotides into DNA by primerextension. Introduction of multiple biotin tags per probe primer resulted in considerableenhancement of the signal intensity and improvement of the specificity of detection.

• Klíčová slova
• DNA hybridization, PCR, electrochemical detection, enzyme-linked assay, gene expression, primer extension,
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH