The effect of mutations in the P53 family of transcription factors on their biological functions, including partial or complete loss of transcriptional activity, has been confirmed several times. At present, P53 family proteins showing partial loss of activity appear to be promising potential candidates for the development of novel therapeutic strategies which could restore their transcriptional activity. In this context, it is important to employ tools to precisely monitor their activity; in relation to this, non-canonical DNA secondary structures in promoters including G-quadruplexes (G4s) were shown to influence the activity of transcription factors. Here, we used a defined yeast assay to evaluate the impact of differently modeled G4 forming sequences on a panel of partial function P53 family mutant proteins. Specifically, a 22-mer G4 prone sequence (derived from the KSHV virus) and five derivatives that progressively mutate characteristic guanine stretches were placed upstream of a minimal promoter, adjacent to a P53 response element in otherwise isogenic yeast luciferase reporter strains. The transactivation ability of cancer-associated P53 (TA-P53α: A161T, R213L, N235S, V272L, R282W, R283C, R337C, R337H, and G360V) or Ectodermal Dyplasia syndromes-related P63 mutant proteins (ΔN-P63α: G134D, G134V and inR155) were tested. Our results show that the presence of G4 forming sequences can increase the transactivation ability of partial function P53 family proteins. These observations are pointing to the importance of DNA structural characteristics for accurate classification of P53 family proteins functionality in the context of the wide variety of TP53 and TP63 germline and somatic mutations.

• Klíčová slova
• G-quadruplex, P53 and P63 mutant proteins, Partial functionality, Protein-DNA interactions, Y1H isogenic system,
• MeSH
• aktivace transkripce MeSH
• DNA chemie   MeSH
• G-kvadruplexy * MeSH
• mutantní proteiny genetika   MeSH
• nádorový supresorový protein p53 * metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH
• mutantní proteiny MeSH
• nádorový supresorový protein p53 * MeSH
• transkripční faktory MeSH

Non-canonical secondary structures in DNA are increasingly being revealed as critical players in DNA metabolism, including modulating the accessibility and activity of promoters. These structures comprise the so-called G-quadruplexes (G4s) that are formed from sequences rich in guanine bases. Using a well-defined transcriptional reporter system, we sought to systematically investigate the impact of the presence of G4 structures on transcription in yeast Saccharomyces cerevisiae. To this aim, different G4 prone sequences were modeled to vary the chance of intramolecular G4 formation, analyzed in vitro by Thioflavin T binding test and circular dichroism and then placed at the yeast ADE2 locus on chromosome XV, downstream and adjacent to a P53 response element (RE) and upstream from a minimal CYC1 promoter and Luciferase 1 (LUC1) reporter gene in isogenic strains. While the minimal CYC1 promoter provides basal reporter activity, the P53 RE enables LUC1 transactivation under the control of P53 family proteins expressed under the inducible GAL1 promoter. Thus, the impact of the different G4 prone sequences on both basal and P53 family protein-dependent expression was measured after shifting cells onto galactose containing medium. The results showed that the presence of G4 prone sequences upstream of a yeast minimal promoter increased its basal activity proportionally to their potential to form intramolecular G4 structures; consequently, this feature, when present near the target binding site of P53 family transcription factors, can be exploited to regulate the transcriptional activity of P53, P63 and P73 proteins.

• Klíčová slova
• G-quadruplex, p53, transcriptional activity, yeast,
• MeSH
• DNA metabolismus   MeSH
• G-kvadruplexy * MeSH
• nádorový supresorový protein p53 genetika   MeSH
• promotorové oblasti (genetika) MeSH
• Saccharomyces cerevisiae * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• nádorový supresorový protein p53 MeSH

Impairment of the p53 pathway is a critical event in cancer. Therefore, reestablishing p53 activity has become one of the most appealing anticancer therapeutic strategies. Here, we disclose the p53-activating anticancer drug (3S)-6,7-bis(hydroxymethyl)-5-methyl-3-phenyl-1H,3H-pyrrolo[1,2-c]thiazole (MANIO). MANIO demonstrates a notable selectivity to the p53 pathway, activating wild-type (WT)p53 and restoring WT-like function to mutant (mut)p53 in human cancer cells. MANIO directly binds to the WT/mutp53 DNA-binding domain, enhancing the protein thermal stability, DNA-binding ability, and transcriptional activity. The high efficacy of MANIO as an anticancer agent toward cancers harboring WT/mutp53 is further demonstrated in patient-derived cells and xenograft mouse models of colorectal cancer (CRC), with no signs of undesirable side effects. MANIO synergizes with conventional chemotherapeutic drugs, and in vitro and in vivo studies predict its adequate drug-likeness and pharmacokinetic properties for a clinical candidate. As a single agent or in combination, MANIO will advance anticancer-targeted therapy, particularly benefiting CRC patients harboring distinct p53 status.

• Klíčová slova
• anticancer drug, colorectal cancer, p53 activator, targeted therapy,
• MeSH
• antitumorózní látky chemická syntéza   farmakologie   MeSH
• apoptóza účinky léků   genetika   MeSH
• cisplatina farmakologie   MeSH
• doxorubicin farmakologie   MeSH
• fluorouracil farmakologie   MeSH
• HCT116 buňky MeSH
• kolorektální nádory farmakoterapie   genetika   metabolismus   patologie   MeSH
• kontrolní body buněčného cyklu účinky léků   genetika   MeSH
• lidé MeSH
• myši nahé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 agonisté   genetika   metabolismus   MeSH
• objevování léků MeSH
• proliferace buněk účinky léků   MeSH
• protokoly antitumorózní kombinované chemoterapie farmakologie   MeSH
• pyrroly chemická syntéza   farmakologie   MeSH
• regulace genové exprese u nádorů MeSH
• synergismus léků MeSH
• thiazoly chemická syntéza   farmakologie   MeSH
• vazba proteinů MeSH
• xenogenní modely - testy antitumorózní aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antitumorózní látky MeSH
• cisplatina MeSH
• doxorubicin MeSH
• fluorouracil MeSH
• nádorový supresorový protein p53 MeSH
• pyrroly MeSH
• thiazoly MeSH

P53, P63, and P73 proteins belong to the P53 family of transcription factors, sharing a common gene organization that, from the P1 and P2 promoters, produces two groups of mRNAs encoding proteins with different N-terminal regions; moreover, alternative splicing events at C-terminus further contribute to the generation of multiple isoforms. P53 family proteins can influence a plethora of cellular pathways mainly through the direct binding to specific DNA sequences known as response elements (REs), and the transactivation of the corresponding target genes. However, the transcriptional activation by P53 family members can be regulated at multiple levels, including the DNA topology at responsive promoters. Here, by using a yeast-based functional assay, we evaluated the influence that a G-quadruplex (G4) prone sequence adjacent to the p53 RE derived from the apoptotic PUMA target gene can exert on the transactivation potential of full-length and N-terminal truncated P53 family α isoforms (wild-type and mutant). Our results show that the presence of a G4 prone sequence upstream or downstream of the P53 RE leads to significant changes in the relative activity of P53 family proteins, emphasizing the potential role of structural DNA features as modifiers of P53 family functions at target promoter sites.

• Klíčová slova
• G-quadruplex (G4) prone sequence, P53 family, transactivation potential, wild-type and mutant P53/P63 proteins, yeast,
• MeSH
• apoptóza genetika   MeSH
• DNA genetika   ultrastruktura   MeSH
• G-kvadruplexy * MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• membránové proteiny genetika   ultrastruktura   MeSH
• nádorový supresorový protein p53 genetika   ultrastruktura   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• protein p73 genetika   ultrastruktura   MeSH
• proteiny regulující apoptózu genetika   MeSH
• protoonkogenní proteiny genetika   MeSH
• responzivní elementy genetika   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• BBC3 protein, human MeSH Prohlížeč
• CKAP4 protein, human MeSH Prohlížeč
• DNA MeSH
• membránové proteiny MeSH
• nádorový supresorový protein p53 MeSH
• protein p73 MeSH
• proteiny regulující apoptózu MeSH
• protoonkogenní proteiny MeSH
• TP73 protein, human MeSH Prohlížeč

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

The TP53 gene is the most frequently mutated gene in human cancer and p53 protein plays a crucial role in gene expression and cancer protection. Its role is manifested by interactions with other proteins and DNA. p53 is a transcription factor that binds to DNA response elements (REs). Due to the palindromic nature of the consensus binding site, several p53-REs have the potential to form cruciform structures. However, the influence of cruciform formation on the activity of p53-REs has not been evaluated. Therefore, we prepared sets of p53-REs with identical theoretical binding affinity in their linear state, but different probabilities to form extra helical structures, for in vitro and in vivo analyses. Then we evaluated the presence of cruciform structures when inserted into plasmid DNA and employed a yeast-based assay to measure transactivation potential of these p53-REs cloned at a chromosomal locus in isogenic strains. We show that transactivation in vivo correlated more with relative propensity of an RE to form cruciforms than to its predicted in vitro DNA binding affinity for wild type p53. Structural features of p53-REs could therefore be an important determinant of p53 transactivation function.

• Klíčová slova
• Cruciform structure, Inverted repeat, Protein-DNA interaction, p53 protein,
• MeSH
• aktivace transkripce MeSH
• chromatin genetika   MeSH
• kvasinky genetika   MeSH
• mutace MeSH
• nádorový supresorový protein p53 chemie   genetika   metabolismus   MeSH
• obrácené repetice * MeSH
• počítačová simulace MeSH
• responzivní elementy * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH
• nádorový supresorový protein p53 MeSH