Please use this identifier to cite or link to this item: http://hdl.handle.net/2445/99964
Title: Characterization of alternative therapeutic sites on the androgen receptor and novel protein-protein associations
Author: Rodríguez Carbó, Laia
Director: Estébanez Perpiñá, Eva
Keywords: Andrògens
Proteïnes
Càncer de pròstata
Androgens
Proteins
Prostate cancer
Issue Date: 25-Jan-2016
Publisher: Universitat de Barcelona
Abstract: The androgen receptor (AR) is a ligand-activated transcription factor that plays a crucial role in the correct development, differentiation, and function of male reproductive organs. Alterations in the AR protein or in the AR signaling pathway result in pathologies such as prostate cancer (PCa), which is the fifth leading cause of cancer-related death in men in most western industrialized countries. AR represents the major clinical target in the treatment of PCa and, to date, all the FDA-approved drugs against the AR target the ligand-binding pocket of the receptor, competing with the natural hormones. Unfortunately, prolonged treatments with antiandrogens invariably fail, rendering them ineffective and resulting in the development of castration-resistant PCa. Thus, there is pressing need for more potent and selective novel AR antagonists capable of blocking the action of AR in tumors resistant to conventional antiandrogens. Structural and functional analysis highlighted the presence of another site on the ligand-binding domain (LBD) of AR named binding function-3 (BF-3). Importantly, the BF-3 pocket is a hot spot for mutations involved in PCa and androgen insensitivity syndromes, and some FDA-approved drugs have shown to bind at this site, evidencing its pharmacological potential. Our initial hypothesis was that BF-3 may be a protein-protein interaction site, which may play a physiological role in AR action. Therefore, the major goals of this PhD project have been to further determine the biological role of the newly described BF-3 regulatory surface and to identify and better characterize BF-3-interacting proteins. Firstly, we demonstrate that the newly identified BF-3 site in the human AR is highly conserved among the SR subclass, and that naturally occurring mutations associated with pathology and affecting the function of several NRs in vitro colocalize with their putative BF-3 sites. activation function-2 (AF-2) and BF-3 pockets were mutated in order to further elucidate the molecular mechanisms by which point mutations are linked to disease and determine the allosteric responses they induce in the receptor function. We demonstrate that mutations in the BF-3 pocket and residues lying between the AF-2 and BF-3 sites affect AR transactivation and coactivation by GRIP1, as well as AR N/C interaction and binding to NR corepressors NCoR and SMRT. Finally, as the shape and characteristics of the BF-3 pocket in the examined NRs suggest a possible role for protein-protein interactions, we performed several Y2H screens of both an adult human brain and prostate cDNA libraries to identify possible BF-3 novel interactors. In the presence of DHT, four new putative AR binders were identified: ARMC9, MAPK8IP1, Rab11FIP3, and Uba3. Moreover, Rab11FIP3 and Uba3 inhibited the AR LBD transactivation capacity, attenuated its coactivation by GRIP1 and disrupted the N/C interaction, suggesting that these proteins may be AR corepressors not previously described. Furthermore, we show that SRC3, ARMC9 and Uba3 are capable of binding to the AR LBD in the presence of antiandrogens. Our results suggest that targeting the BF-3 surface may open new promising alternatives to current therapeutics. Compounds that may affect allosterically NR function by binding to BF-3 open promising avenues to develop type-specific NR modulators.
URI: http://hdl.handle.net/2445/99964
Appears in Collections:Tesis Doctorals - Departament - Bioquímica i Biologia Molecular (Biologia)

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