Allosteric Conversation in the Androgen Receptor Ligand-Binding Domain Surfaces

Androgen receptor (AR) is a major therapeutic target that plays pivotal roles in prostate cancer (PCa) and androgen insensitivity syndromes. Wepreviously proposed that compounds recruited to ligand-binding domain (LBD) surfaces could regulate AR activity in hormone-refractory PCa and discovered several surface modulators of AR function. Surprisingly, the most effective compounds bound preferentially to a surface of unknown function [binding function 3 (BF-3)] instead of the coactivator-binding site [activation function 2 (AF-2)]. Different BF-3 mutations have been identified in PCa or androgen insensitivity syndrome patients, and they can strongly affect AR activity. Further, comparison of AR x-ray structures with and without bound ligands at BF-3 and AF-2 showed structural coupling between both pockets. Here, we combine experimental evidence and molecular dynamic simulations to investigate whether BF-3 mutations affect AR LBD function and dynamics possibly via allosteric conversation between surface sites. Our data indicate that AF-2 conformation is indeed closely coupled to BF-3 and provide mechanistic proof of their structural interconnection. BF-3 mutations may function as allosteric elicitors, probably shifting the AR LBD conformational ensemble toward conformations that alter AF-2 propensity to reorganize into subpockets that accommodate N-terminal domain and coactivator peptides. The induced conformation may result in either increased or decreased AR activity. Activating BF-3 mutations also favor the formation of another pocket (BF-4) in the vicinity of AF-2 and BF-3, which we also previously identified as a hot spot for a small compound. We discuss the possibility that BF-3 may be a protein-docking site that binds to the N-terminal domain and corepressors. AR surface sites are attractive pharmacological targets to develop allosteric modulators that might be alternative lead compounds for drug design. © 2012 by The Endocrie Society

The Oncoprotein BCL11A Binds to Orphan Nuclear Receptor TLX and Potentiates its Transrepressive Function

Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain

Coregulator Control of Androgen Receptor Action by a Novel Nuclear Receptor-Binding Motif

The androgen receptor (AR) is a ligand-activated transcription factor that is essential for prostate cancer development. It is activated by androgens through its ligand-binding domain (LBD), which consists predominantly of 11 α-helices. Upon ligand binding, the last helix is reorganized to an agonist conformation termed activator function-2 (AF-2) for coactivator binding. Several coactivators bind to the AF-2 pocket through conserved LXXLL or FXXLF sequences to enhance the activity of the receptor. Recently, a small compound-binding surface adjacent to AF-2 has been identified as an allosteric modulator of the AF-2 activity and is termed binding function-3 (BF-3). However, the role of BF-3 in vivo is currently unknown, and little is understood about what proteins can bind to it. Here we demonstrate that a duplicated GARRPR motif at the N terminus of the cochaperone Bag-1L functions through the BF-3 pocket. These findings are supported by the fact that a selective BF-3 inhibitor or mutations within the BF-3 pocket abolish the interaction between the GARRPR motif(s) and the BF-3. Conversely, amino acid exchanges in the two GARRPR motifs of Bag-1L can impair the interaction between Bag-1L and AR without altering the ability of Bag-1L to bind to chromatin. Furthermore, the mutant Bag-1L increases androgen-dependent activation of a subset of AR targets in a genome-wide transcriptome analysis, demonstrating a repressive function of the GARRPR/BF-3 interaction. We have therefore identified GARRPR as a novel BF-3 regulatory sequence important for fine-tuning the activity of the AR

The Oncoprotein BCL11A Binds to Orphan Nuclear Receptor TLX and Potentiates its Transrepressive Function

Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain

Human TLX recruits ATN1.

<p>Y2H screens using TLX-FL and TLX-LBD domain as baits against an adult brain cDNA library identified ATN1 as a TLX-interactor. (A) Schematic diagram of the identified ATN1 clones. All clones share a 378 amino acid overlapping region (residues 813–1190) featuring the ATRO-BOX region. (B) Validation of the interaction between TLX and the identified ATN1 clones by forward one-to-one Y2H assay. TLX constructs FL-TLX (1–385), TLX-LBD (172–385), TLX-H-LBD (94–385) and TLX-DBD (1–95) (baits) were tested for interaction with the ATN1 clones identified (preys). Yeast transformants were plated on a control plate (lacking Trp and Leu) and plated on a selective plate (lacking Trp, Leu, His supplemented with 50 mM 3AT).</p

BCL11A is a TLX corepressor.

<p>(A) Chromatin luciferase [Luc] assay showing corepressor activity of LSD1 with TLX as previosly published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037963#pone.0037963-Yokoyama1" target="_blank">[18]</a>. 293F cells, which have a stably integrated pGL4.31 reporter gene, were transiently transfected with pM or pM TLX LBD vector (200 ng each) and with 400 ng of pcDNA empty vector, and pCMX-FLAG LSD1. (B) Chromatin luciferase [Luc] assay showing corepressor activity of BCL11A with TLX. 293F cells, which have a stably integrated pGL4.31 reporter gene, were transiently transfected with pM or pM TLX LBD vector (200 ng each) and with 400 ng of pcDNA empty vector, pEF1a-BCL11-XL or pEF1a-BCL11-L. The P values were calculated by Student’s t test (n = 3).</p

The oncoprotein BCL11A binds to human TLX.

<p>(A) Schematic diagram of the different isoforms of BCL11A (XL, L, S) and corresponding exons (1, E2, E3, E4, E5L, E5S). All BCL11A identified clones share a 159 amino acid overlapping region (residues 586–744), whose amino acid sequence is shown. Previously BCL11A regions identified to bind to COUP-FT and named ID-1 and ID-2 are highlighted in light grey. Clone 1 features a novel combination of exons, while clones 3, and 4 contain an intronic sequence. All the identified clones contain ID-2, but only some of them also have ID-2 as well. (B) Validation of the interaction between TLX and the identified BCL11A clones by forward one-to-one Y2H assay. TLX constructs FL-TLX (1–385), TLX-LBD (172–385), TLX-H-LBD (94–385) and TLX-DBD (1–95) (baits) were tested for interaction with the BCL11A clones identified (preys). Yeast transformants were plated on a control plate (lacking Trp and Leu) and plated on a selective plate (lacking Trp, Leu, His supplemented with 50 mM 3AT).</p

Coregulator Control of Androgen Receptor Action by a Novel Nuclear Receptor-Binding Motif

The androgen receptor (AR) is a ligand-activated transcription factor that is essential for prostate cancer development. It is activated by androgens through its ligand-binding domain (LBD), which consists predominantly of 11 α-helices. Upon ligand binding, the last helix is reorganized to an agonist conformation termed activator function-2 (AF-2) for coactivator binding. Several coactivators bind to the AF-2 pocket through conserved LXXLL or FXXLF sequences to enhance the activity of the receptor. Recently, a small compound-binding surface adjacent to AF-2 has been identified as an allosteric modulator of the AF-2 activity and is termed binding function-3 (BF-3). However, the role of BF-3 in vivo is currently unknown, and little is understood about what proteins can bind to it. Here we demonstrate that a duplicated GARRPR motif at the N terminus of the cochaperone Bag-1L functions through the BF-3 pocket. These findings are supported by the fact that a selective BF-3 inhibitor or mutations within the BF-3 pocket abolish the interaction between the GARRPR motif(s) and the BF-3. Conversely, amino acid exchanges in the two GARRPR motifs of Bag-1L can impair the interaction between Bag-1L and AR without altering the ability of Bag-1L to bind to chromatin. Furthermore, the mutant Bag-1L increases androgen-dependent activation of a subset of AR targets in a genome-wide transcriptome analysis, demonstrating a repressive function of the GARRPR/BF-3 interaction. We have therefore identified GARRPR as a novel BF-3 regulatory sequence important for fine-tuning the activity of the AR