Dissecting the role of TTC5 cofactor in regulation of the Glucocorticoid receptor activities

Abstract

Glucocorticoids are a group of steroid hormones that are secreted by the adrenal cortex in response to stress conditions and regulate vital immunological, developmental, anti-inflammatory, and metabolic cellular functions. These functions of the glucocorticoids are exerted through a nuclear receptor recognized as the glucocorticoid receptor. Glucocorticoid receptor usually resides in the cytoplasm chaperoned with hsp 90 and hsp 70 heat shock proteins in addition to a group of immunophilins. Upon binding the hormone GR is released from the inactivating complex to form a dimer and translocate to the nucleus where it will bind to a specific DNA response sequence GRE. GR regulatory activities are governed by several controlling factors such as ligand, cell type, posttranslational modification and co-regulators that govern the final outcome of GRs gene regulation activities. TTC5 the Tetratricopeptide the stress responsive activator of p300 is a co-regulator of p53, HSF1, and GR. Here we show that TTC5 interacts with GR via four LXXLL and six TPR structural motifs and hence regulates its gene transcription activities in a diverse manner. Using the reporter gene assay on TAT-3 target gene in COS-7 and A549 cells we show that the different LXXLL motifs differentially repress the GR transcription activities in a ligand, cell specific and LXXLL motif position manner and act as probable nuclear receptor CoRNR boxes. Furthermore, measuring GR protein levels and the sub-cellular localizations of both GR and TTC5 show that GR protein intensity and localization are governed by the position of the LXXLL in the TTC5 and presence or absence of hormone. In addition to that, GR was shown to be recruited to GILZ promoter in association with TTC5 within 20 minutes. It was also shown that TTC5 activated by DNA damage possibly precedes GR to the GRE and determines the gene to be bound to and hence transcribed by GR. GR also regulated mRNA levels of SLC19A2 and SDPR influenced by the different LXXLL motifs. We also show by means of the reporter gene assay that the different TPRs comprising the TTC5 structure are required for modulating the GRs gene transcription regulation on. TAT-3 and SLC19A2 promoters in A549 cells and principally work as activators of GR. Immunofluorescent assay revealed that TTC5 is both nuclear and cytoplasmic and can interact with GR in any of the two sub cellular localizations depending on the interaction TPR surface. TTC5 was also show to stabilise GR in presence of hormone with LXXLL motif L247/8 and TPRs 1, and 6. In summary our data suggest that TTC5 modulates the GR gene regulation activities, protein stability, sub-cellular localization, and occupancy on target genes response elements in a motif specific mode. This opens up the possibilities of therapeutic interventions possibly through selective modulation of the activity of individual TTC5 motifs.

Date of Award	1 Aug 2014
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Dean Jackson (Supervisor) & Marija Krstic-Demonacos (Supervisor)