title: Novel simplified yeast-based assays of regulators of p53-MDMX interaction and p53 transcriptional activity.
abstract: Yeast has proven to be an efficient model system for functional and pharmacological studies of the p53 tumour suppressor protein. In this work, the human p53-MDMX regulatory pathway was reconstituted in yeast. Additionally, by using the known inhibitor of p53-MDMX interaction, SJ-172550, the efficacy of a simplified yeast-based screening assay to search for inhibitors of p53-MDMX interaction is demonstrated for the first time. Moreover, further insights on p53 transcriptional activity in yeast are provided. In particular, it is shown that the reported wild-type (wt) p53-induced yeast growth inhibition and cell cycle arrest is associated with actin depolarization and with an increase of actin mRNA and protein expression levels. The increase of actin protein levels was not observed with the p53 R273H mutant (a loss of function p53 mutation hotspot) and was further intensified with the toxic p53 V122A mutant (reported to exhibit higher transcriptional activity than wt p53 for selected p53 target sequences). Moreover, it is shown that the wt p53-induced actin protein levels are modulated by natural (MDM2 and MDMX) and chemical (pifithrin-α, nutlin-3a and SJ-172550) regulators of p53 activity. Furthermore, wt p53 could stimulate transcription from a minimal promoter containing a fragment of the ACT1 upstream sequence. Thus, ACT1 is proposed as a putative endogenous p53 target gene. This finding may open the way for the development of simpler yeast p53 transactivation assays, not based on artificial reporter constructs, for the analysis of the impact of mutants, cofactors and small molecules on p53 transcriptional activity.
authors.affiliation: REQUIMTE, Laboratório de Microbiologia, Departamento de Ciências Biológicas, Universidade do Porto, Portugal.
authors.forename: Mariana
authors.initials: M
authors.lastname: Leão
authors.forename: Sara
authors.initials: S
authors.lastname: Gomes
authors.forename: Joana
authors.initials: J
authors.lastname: Soares
authors.forename: Cláudia
authors.initials: C
authors.lastname: Bessa
authors.forename: Cláudia
authors.initials: C
authors.lastname: Maciel
authors.forename: Yari
authors.initials: Y
authors.lastname: Ciribilli
authors.forename: Clara
authors.initials: C
authors.lastname: Pereira
authors.forename: Alberto
authors.initials: A
authors.lastname: Inga
authors.forename: Lucília
authors.initials: L
authors.lastname: Saraiva
mesh_terms: D000085:Acetates / Q000494:pharmacology; D000199:Actins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D018797:Cell Cycle Proteins; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000187:drug effects*; D057166:High-Throughput Screening Assays; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011720:Pyrazoles / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D000085:Acetates; D000199:Actins; D018797:Cell Cycle Proteins; C482775:MDM4 protein, human; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011720:Pyrazoles; D012333:RNA, Messenger; C549331:SJ 172550; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D008156:Luciferases; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2
keywords: actin; p53; p53-MDMX interaction; screening assays; yeast