Characterization of transactivational property and coactivator mediation of rat mineralocorticoid receptor activation function-1 (AF-1)

The autonomous activation function-2 (AF-2) in the mineralocorticoid receptor (MR) E/F domain is known to play a major role in the ligand-induced transactivation function of MR; however, it remained unclear about the transactivation function of its A/B domain. We therefore tried to characterize the MR A/B domain as the AF-1 and further studied the actions of known coactivators for AF-2 in the E/F ligand-binding domain in the function of the MR A/B domain. Deletion analyses of rat and human MRs revealed that the A/B domains harbor a transactivation function acting as AF-1. The MR mutant (E959Q) with a point mutation in helix 12, which causes a complete loss of MR AF-2 activity, still retained ligand-induced transactivation function, indicating a significant role for AF-1 in the full activity of the ligand-induced MR function. Among the coactivators tested to potentiate the MR AF-2, TIF2 and p300 potentiated the MR AF-1 through two different core regions [amino acids (a.a.) 1-169, a.a. 451-603] and exhibited functional interactions with the MR A/B domain in the cultured cells. However, such interactions were undetectable in a yeast and in an in vitro glutathione-S-transferase pull-down assay, indicating that the functional interaction of TIF2 and p300 with the MR A/B domain to support the MR AF-1 activity require some unknown nuclear factor(s) or a proper modification of the A/B domain in the cells.     

Modulation of Oxidative Stress by Twist Oncoproteins

Expression of developmental genes Twist1 and Twist2 is reactivated in many human tumors. Among their oncogenic activities, induction of epithelial to mesenchymal transition is believed to increase cell motility and invasiveness and may be related to acquisition of cancer stem cell phenotype. In addition, Twist proteins promote malignant conversion by overriding two oncogene-induced failsafe programs: senescence and apoptosis. Reactive oxygen species (ROS) are also important mediators of apoptosis, senescence and motility and are tightly linked to disease, notably to cancer. We report here that Twist factors and ROS are functionally linked. In wild type cells both Twist1 and Twist2 exhibit antioxidant properties. We show that Twist-driven modulation of oncogene-induced apoptosis is linked to its effects on oxidative stress. Finally, we identify several targets that mediate Twist antioxidant activity. These findings unveil a new function of Twist factors that could be important in explaining their pleiotropic role during carcinogenesis.     

Recombinant human adenoviruses containing hybrid adenovirus type 5 (Ad5)/Ad12 E1A genes: characterization of hybrid E1A proteins and analysis of transforming activity and host range.

Hybrid adenovirus type 12 (Ad12)/Ad5 E1A genes were constructed by homologous recombination in Escherichia coli, a technique which offers several advantages over conventional mutagenesis for genetic analysis of proteins. In particular, functional differences between the proteins can be mapped by correlating the replacement of specific sequences with the acquisition of new properties, and there is no requirement for common unique restriction sites or polymerase chain reaction strategies to construct the hybrids. Recombinant adenoviruses expressing these hybrid E1A proteins were capable of replicating efficiently in HeLa cells, with the exception of one construct which contained a hybrid transactivation domain. The transforming activity of the hybrid E1A constructs was assayed by DNA transfection of primary baby rat kidney cells. Plasmids containing Ad12 E1 were approximately 20-fold less efficient at transformation than those with E1 of Ad5, and it was found that two regions in exon 1 of E1A mediate this difference. No differences were found in the abilities of any hybrid E1A proteins to bind to cellular proteins previously determined to be important for transformation by E1A.