TY - JOUR
T1 - Novel simplified yeast-based assays of regulators of p53-MDMX interaction and p53 transcriptional activity
AU - Leão, Mariana
AU - Gomes, Sara
AU - Soares, Joana
AU - Bessa, Cláudia
AU - MacIel, Cláudia
AU - Ciribilli, Yari
AU - Pereira, Clara
AU - Inga, Alberto
AU - Saraiva, Lucília
PY - 2013/12
Y1 - 2013/12
N2 - 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. Yeast-based assays to search for regulators of p53-MDMX interaction and p53 transcriptional activity were developed. Additionally, relevant insights on p53 transcriptional activity in yeast were revealed. Particularly, it was shown that expression of p53 in yeast increased actin mRNA and protein levels and induced actin depolarization. Together, these results showed that ACT1 is an endogenous p53 target gene in yeast.
AB - 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. Yeast-based assays to search for regulators of p53-MDMX interaction and p53 transcriptional activity were developed. Additionally, relevant insights on p53 transcriptional activity in yeast were revealed. Particularly, it was shown that expression of p53 in yeast increased actin mRNA and protein levels and induced actin depolarization. Together, these results showed that ACT1 is an endogenous p53 target gene in yeast.
KW - actin
KW - p53
KW - p53-MDMX interaction
KW - screening assays
KW - yeast
UR - http://www.scopus.com/inward/record.url?scp=84889663663&partnerID=8YFLogxK
U2 - 10.1111/febs.12552
DO - 10.1111/febs.12552
M3 - Article
C2 - 24119020
AN - SCOPUS:84889663663
VL - 280
SP - 6498
EP - 6507
JO - FEBS Journal
JF - FEBS Journal
SN - 1742-464X
IS - 24
ER -