Date of Award
Doctor of Philosophy
Marc B. Cox
The dynamic Hsp70-90 chaperone machinery along with its cochaperone partners are well-characterized for their ability to fold, assemble, and regulate steroid hormone receptors (SHRs). Human small glutamine rich tetratricopeptide repeat (TPR) containing protein alpha (SGTA) is a recently identified protein that has a characteristic Hsp90-binding TPR domain and is a key participant in the androgen, glucocorticoid, and progesterone receptor signaling pathway. In addition, SGTA plays a role in cellular processes such as cell cycle progression and apoptosis. We have demonstrated that SGTA binds directly to both Hsp70 (kd = 6 Î¼M) and Hsp90 (kd = 11 Î¼M). In a cell-free system, SGTA is unable to affect chaperone complex formation and receptor hormone binding. In addition, deletion of the Q-rich region at the C-terminus of SGTA failed to abrogate AR function in yeast reporter assays. To better understand the functional role of SGTA in SHR regulation, we undertook a label-free quantitative proteomics approach to determine unknown interacting proteins with SGTA in LNCaP human prostate cancer cells. Tandem affinity purification followed by Liquid Chromatography Mass Spectrometry (LC-MS/MS) was performed to identify unknown protein interactions. To determine if interactions were transient or strong, a comparison of a 3-hour versus an overnight incubation was used. Our quantitative data suggest that SGTA interacts with proteins involved in a diverse range of pathways. Peroxiredoxin 1 (PRDX1) was present in both conditions and in the three biological replicates. PRDX1 is involved in redox regulation of the cell and has antioxidant properties. In addition, it has been linked to the androgen receptor (AR) pathway where it can bind to AR and enhance its transactivation. Utilizing SGTA and PRDX1 purified, recombinant proteins, we performed an in vitro FLAG pull-down assay using proteins alone or in combination. The in vitro FLAG pull-down assays demonstrated that SGTA directly interacts with PRDX1. In addition, PRDX1 co-immunoprecipitated with SGTA in LNCaP cells. Thus, we sought to determine the functional relevance of the SGTA-PRDX1 interaction. We generated CRISPR/Cas9 SGTAKO HeLa and 22RV1 cell lines and assessed the functional relevance of SGTA and PRDX1. Using luciferase reporter assays we demonstrated that SGTA can antagonize PRDX1 potentiation of AR activity suggesting the mechanism for the negative regulation of AR by SGTA is through competition with PRDX1.
The yeast-based steroid hormone receptor-mediated reporter assay has historically provided much of the evidence regarding the importance of chaperones and cochaperones in steroid hormone receptor signaling pathways and was critical for the characterization of SGTA functional domains in this study. Given our expertise with this system our group modified this assay previously to a short 4-hour assay for use in screening environmental samples for estrogenic activity. Because of the short assay time, sterility is not an issue and samples can be measured directly without extraction or sterilization. As a result, we hypothesized that the assay could be used for the direct measure of estrogenic activity in urine as a novel research tool as well. Thus, we sought to validate its application in human urine samples. The ability and sensitivity of the yeast bioassay to detect estrogenic activity in urine samples from pregnant females in all trimesters was assessed. No toxicity was observed in yeast grown in the presence of human pregnant female urine for 2 hours and the assay was able to accurately detect the increasing estrogenic activity expected with increasing trimester. Upon the addition of Î²-glucoronidase and sulfatase to deconjugate estrogenic metabolites we demonstrated that the assay sensitivity can be increased significantly (i.e. significantly more estrogenic activity was detected). Thus, these assays represent a novel research tool to detect estrogenic activity in animal and human urine samples in a rapid and sensitive manner.
Received from ProQuest
Yenni Alejandra Garcia
Garcia, Yenni Alejandra, "Label-Free Quantitative Proteomics Reveals a Novel SGTA/Peroxiredoxin I Complex That Regulates Androgen Receptor Activity" (2016). Open Access Theses & Dissertations. 849.