scholarly journals A Genetically Encoded Bioluminescence Intracellular Nanosensor for Androgen Receptor Activation Monitoring in 3D Cell Models

Sensors ◽  
2021 ◽  
Vol 21 (3) ◽  
pp. 893
Author(s):  
Maria Maddalena Calabretta ◽  
Antonia Lopreside ◽  
Laura Montali ◽  
Luca Cevenini ◽  
Aldo Roda ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Protein Interactions ◽  
Limit Of Detection ◽  
Receptor Activation ◽  
New Drugs ◽  
Hek 293 ◽  
Androgenic Activity ◽  
Half Maximal Effective Concentration ◽  
Compound Screening

In recent years, there has been an increasing demand for predictive and sensitive in vitro tools for drug discovery. Split complementation assays have the potential to enlarge the arsenal of in vitro tools for compound screening, with most of them relying on well-established reporter gene assays. In particular, ligand-induced complementation of split luciferases is emerging as a suitable approach for monitoring protein–protein interactions. We hereby report an intracellular nanosensor for the screening of compounds with androgenic activity based on a split NanoLuc reporter. We also confirm the suitability of using 3D spheroids of Human Embryonic Kidney (HEK-293) cells for upgrading the 2D cell-based assay. A limit of detection of 4 pM and a half maximal effective concentration (EC50) of 1.7 ± 0.3 nM were obtained for testosterone with HEK293 spheroids. This genetically encoded nanosensor also represents a new tool for real time imaging of the activation state of the androgen receptor, thus being suitable for analysing molecules with androgenic activity, including new drugs or endocrine disrupting molecules.

scholarly journals Conazole fungicides inhibit Leydig cell testosterone secretion and androgen receptor activation in vitro

2014 ◽  
Vol 1 ◽  
pp. 271-283 ◽  
Author(s):  
Maarke J.E. Roelofs ◽  
A. Roberto Temming ◽  
Aldert H. Piersma ◽  
Martin van den Berg ◽  
Majorie B.M. van Duursen
Keyword(s):  
Androgen Receptor ◽  
Leydig Cell ◽  
Receptor Activation ◽  
Testosterone Secretion ◽  
Androgen Receptor Activation

scholarly journals Notch ligand activity is modulated by glycosphingolipid membrane composition in Drosophila melanogaster

2010 ◽  
Vol 188 (4) ◽  
pp. 581-594 ◽  
Author(s):  
Sophie Hamel ◽  
Jacques Fantini ◽  
François Schweisguth
Keyword(s):  
Protein Interactions ◽  
Receptor Activation ◽  
Notch Receptor ◽  
Binding Motif ◽  
Membrane Composition ◽  
Notch Receptors ◽  
Notch Ligands ◽  
Ligand Activity ◽  
Signaling Activity

Endocytosis of the transmembrane ligands Delta (Dl) and Serrate (Ser) is required for the proper activation of Notch receptors. The E3 ubiquitin ligases Mindbomb1 (Mib1) and Neuralized (Neur) regulate the ubiquitination of Dl and Ser and thereby promote both ligand endocytosis and Notch receptor activation. In this study, we identify the α1,4-N-acetylgalactosaminyltransferase-1 (α4GT1) gene as a gain of function suppressor of Mib1 inhibition. Expression of α4GT1 suppressed the signaling and endocytosis defects of Dl and Ser resulting from the inhibition of mib1 and/or neur activity. Genetic and biochemical evidence indicate that α4GT1 plays a regulatory but nonessential function in Notch signaling via the synthesis of a specific glycosphingolipid (GSL), N5, produced by α4GT1. Furthermore, we show that the extracellular domain of Ser interacts with GSLs in vitro via a conserved GSL-binding motif, raising the possibility that direct GSL–protein interactions modulate the endocytosis of Notch ligands. Together, our data indicate that specific GSLs modulate the signaling activity of Notch ligands.

In vitro regulation of reporter gene transcription by the androgen receptor AF1 domain

2004 ◽  
Vol 32 (6) ◽  
pp. 1103-1106 ◽  
Author(s):  
M.A. Choudhry ◽  
I.J. McEwan
Keyword(s):  
Gene Expression ◽  
Androgen Receptor ◽  
Reporter Gene ◽  
Protein Interactions ◽  
Transcription Initiation ◽  
Muscular Atrophy ◽  
Activation Function ◽  
Regulation Of Transcription ◽  
Gene Assay

The androgen receptor (AR) is a ligand-activated transcription factor that regulates gene expression in response to the steroids testosterone and dihydrotestosterone. AR-dependent gene expression is likely to play an important role in a number of receptor-associated disorders, such as prostate cancer, spinal bulbar muscular atrophy, male type baldness and hirsutism. The AR contains two transactivation domains, termed AF1 (activation function 1) located in the N-terminus and AF2 (activation function 2) in the C-terminal ligand-binding domain. AF2 exhibits weak transcriptional activity, whereas AF1 is a strong regulator of transcription. Transcriptional regulation by AF1 is thought to be modulated by a number of proteins that interact with this region, and by post-translational modifications. Our focus is on the N-terminal-interacting proteins and their regulation of transcription via interaction with the receptor. To better understand the mechanism of AR-AF1 action, we have reconstituted AR activity in HeLa nuclear extracts using a unique dual reporter gene assay. Multiple LexA-binding sites in the promoter allow transcription to be driven by a recombinant AR-AF1–Lex fusion protein. The findings from initial experiments suggest an increase in transcription initiation and elongation rates by AR-AF1–Lex. The role of protein–protein interactions involving co-activators and basal transcription factors and AR-AF1 activity are discussed.

scholarly journals Several environmental oestrogens are also anti-androgens

1998 ◽  
Vol 158 (3) ◽  
pp. 327-339 ◽  
Author(s):  
P Sohoni ◽  
JP Sumpter
Keyword(s):  
Androgen Receptor ◽  
Endocrine Disruption ◽  
Sex Steroids ◽  
Mechanisms Of Action ◽  
Current Interest ◽  
Androgen Action ◽  
Specific Objective ◽  
Androgenic Activity

There is presently considerable interest in endocrine disruption which is a new area of endocrinology concerned with chemicals that mimic hormones, in particular sex steroids. It has been hypothesised that exposure to such chemicals may be responsible for adverse effects in both humans and wildlife. Until now, chemicals that mimic oestrogens (so-called xenoestrogens) have been the main focus of endocrine disruption research. However, recent evidence suggests that many abnormalities in the male reproductive system may be mediated via the androgen receptor. By blocking androgen action, exposure to an anti-androgen may cause changes similar to those associated with oestrogen exposure. We have used in vitro yeast-based assays to detect oestrogenic, anti-oestrogenic, androgenic and anti-androgenic activities in a variety of chemicals of current interest. We show that many of the so-called 'environmental oestrogens' also possess anti-androgenic activity. The previously reported anti-androgenic activities of vinclozolin and p,p'-1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene (DDE) were confirmed. We also found that o,p'-1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), bisphenol A and butyl benzyl phthalate were anti-androgenic. However, not all xenoestrogens are also anti-androgenic, because nonylphenol was found to be a weak androgen agonist. Our results demonstrate that hormone-mimicking chemicals can have multiple hormonal activities, which may make it difficult to interpret their mechanisms of action in vivo. Although not a specific objective of this study, our results also demonstrate that yeast-based assays are powerful tools with which to investigate both agonist and antagonistic hormonal activities of chemicals.

scholarly journals The stringency and magnitude of androgen-specific gene activation are combinatorial functions of receptor and nonreceptor binding site sequences.

1993 ◽  
Vol 13 (10) ◽  
pp. 6326-6335 ◽  
Author(s):  
A J Adler ◽  
A Scheller ◽  
D M Robins
Keyword(s):  
Androgen Receptor ◽  
Protein Interactions ◽  
Hormonal Regulation ◽  
Steroid Receptors ◽  
Regulation Of Gene Expression ◽  
Gene Activation ◽  
Specific Gene ◽  
Sequence Variations

The mechanism by which specific hormonal regulation of gene expression is attained in vivo is a paradox in that several of the steroid receptors recognize the same DNA element in vitro. We have characterized a complex enhancer of the mouse sex-limited protein (Slp) gene that is activated exclusively by androgens but not by glucocorticoids in transfection. Potent androgen induction requires both the consensus hormone response element (HRE) and auxiliary elements residing within the 120-bp DNA fragment C' delta 9. Multiple nonreceptor factors are involved in androgen specificity, with respect to both the elevation of androgen receptor activity and the inactivity of glucocorticoid receptor (GR), since clustered base changes at any of several sites reduce or abolish androgen induction and do not increase glucocorticoid response. However, moving the HRE as little as 10 bases away from the rest of the enhancer allows GR to function, suggesting that GR is repressed by juxtaposition to particular factors within the androgen-specific complex. Surprisingly, some sequence variations of the HRE itself, within the context of C' delta 9, alter the stringency of specificity, as well as the magnitude, of hormonal response. These HRE sequence effects on expression correspond in a qualitative manner with receptor binding, i.e., GR shows a threefold difference in affinities for HREs amongst which androgen receptor does not discriminate. Altering the HRE orientation within the enhancer also affects hormonal stringency, increasing glucocorticoid but not androgen response. The effect of these subtle variations suggests that they alter receptor position with respect to other factors. Thus, protein-protein interactions that elicit specific gene regulation are established by the array of DNA elements in a complex enhancer and can be modulated by sequence variations within these elements that may influence selection of precise protein contacts.

scholarly journals Corrigendum to “Conazole fungicides inhibit Leydig cell testosterone secretion and androgen receptor activation in vitro” [Toxicol. Rep. 1C (2014) 271–283]

2016 ◽  
Vol 3 ◽  
pp. 784
Author(s):  
Maarke J.E. Roelofs ◽  
A. Roberto Temming ◽  
Aldert H. Piersma ◽  
Martin van den Berg ◽  
Majorie B.M. van Duursen
Keyword(s):  
Androgen Receptor ◽  
Leydig Cell ◽  
Receptor Activation ◽  
Testosterone Secretion ◽  
Androgen Receptor Activation

The stringency and magnitude of androgen-specific gene activation are combinatorial functions of receptor and nonreceptor binding site sequences

1993 ◽  
Vol 13 (10) ◽  
pp. 6326-6335
Author(s):  
A J Adler ◽  
A Scheller ◽  
D M Robins
Keyword(s):  
Androgen Receptor ◽  
Protein Interactions ◽  
Hormonal Regulation ◽  
Steroid Receptors ◽  
Regulation Of Gene Expression ◽  
Gene Activation ◽  
Specific Gene ◽  
Sequence Variations

The mechanism by which specific hormonal regulation of gene expression is attained in vivo is a paradox in that several of the steroid receptors recognize the same DNA element in vitro. We have characterized a complex enhancer of the mouse sex-limited protein (Slp) gene that is activated exclusively by androgens but not by glucocorticoids in transfection. Potent androgen induction requires both the consensus hormone response element (HRE) and auxiliary elements residing within the 120-bp DNA fragment C' delta 9. Multiple nonreceptor factors are involved in androgen specificity, with respect to both the elevation of androgen receptor activity and the inactivity of glucocorticoid receptor (GR), since clustered base changes at any of several sites reduce or abolish androgen induction and do not increase glucocorticoid response. However, moving the HRE as little as 10 bases away from the rest of the enhancer allows GR to function, suggesting that GR is repressed by juxtaposition to particular factors within the androgen-specific complex. Surprisingly, some sequence variations of the HRE itself, within the context of C' delta 9, alter the stringency of specificity, as well as the magnitude, of hormonal response. These HRE sequence effects on expression correspond in a qualitative manner with receptor binding, i.e., GR shows a threefold difference in affinities for HREs amongst which androgen receptor does not discriminate. Altering the HRE orientation within the enhancer also affects hormonal stringency, increasing glucocorticoid but not androgen response. The effect of these subtle variations suggests that they alter receptor position with respect to other factors. Thus, protein-protein interactions that elicit specific gene regulation are established by the array of DNA elements in a complex enhancer and can be modulated by sequence variations within these elements that may influence selection of precise protein contacts.

Effect of organochlorine pesticides on human androgen receptor activation in vitro

2004 ◽  
Vol 196 (2) ◽  
pp. 235-246 ◽  
Author(s):  
Géraldine Lemaire ◽  
Béatrice Terouanne ◽  
Pascale Mauvais ◽  
Serge Michel ◽  
Roger Rahmani
Keyword(s):  
Androgen Receptor ◽  
Organochlorine Pesticides ◽  
Receptor Activation ◽  
Human Androgen Receptor ◽  
Androgen Receptor Activation

scholarly journals CD63 interacts with the carboxy terminus of the colonic H+-K+-ATPase to increase plasma membrane localization and86Rb+uptake

2005 ◽  
Vol 288 (6) ◽  
pp. C1279-C1286 ◽  
Author(s):  
Juan Codina ◽  
Jian Li ◽  
Thomas D. DuBose
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Carboxy Terminus ◽  
Protein Protein Interactions ◽  
Α Subunit ◽  
Hek 293 ◽  
And Function

The carboxy terminus (CT) of the colonic H+-K+-ATPase is required for stable assembly with the β-subunit, translocation to the plasma membrane, and efficient function of the transporter. To identify protein-protein interactions involved in the localization and function of HKα2, we selected 84 amino acids in the CT of the α-subunit of mouse colonic H+-K+-ATPase (CT-HKα2) as the bait in a yeast two-hybrid screen of a mouse kidney cDNA library. The longest identified clone was CD63. To characterize the interaction of CT-HKα2with CD63, recombinant CT-HKα2and CD63 were synthesized in vitro and incubated, and complexes were immunoprecipitated. CT-HKα2protein (but not CT-HKα1) coprecipitated with CD63, confirming stable assembly of HKα2with CD63. In HEK-293 transfected with HKα2plus β1-Na+-K+-ATPase, suppression of CD63 by RNA interference increased cell surface expression of HKα2/NKβ1and86Rb+uptake. These studies demonstrate that CD63 participates in the regulation of the abundance of the HKα2-NKβ1complex in the cell membrane.

scholarly journals Non-competitive androgen receptor inhibition in vitro and in vivo

2009 ◽  
Vol 106 (17) ◽  
pp. 7233-7238 ◽  
Author(s):  
Jeremy O. Jones ◽  
Eric C. Bolton ◽  
Yong Huang ◽  
Clementine Feau ◽  
R. Kiplin Guy ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Prostate Gland ◽  
Benign Prostatic Hypertrophy ◽  
Receptor Activation ◽  
Cellular Mechanisms ◽  
Competitive Antagonism ◽  
Receptor Inhibition ◽  
A Cell

Androgen receptor (AR) inhibitors are used to treat multiple human diseases, including hirsutism, benign prostatic hypertrophy, and prostate cancer, but all available anti-androgens target only ligand binding, either by reduction of available hormone or by competitive antagonism. New strategies are needed, and could have an important impact on therapy. One approach could be to target other cellular mechanisms required for receptor activation. In prior work, we used a cell-based assay of AR conformation change to identify non-ligand inhibitors of AR activity. Here, we characterize 2 compounds identified in this screen: pyrvinium pamoate, a Food and Drug Administration-approved drug, and harmol hydrochloride, a natural product. Each compound functions by a unique, non-competitive mechanism and synergizes with competitive antagonists to disrupt AR activity. Harmol blocks DNA occupancy by AR, whereas pyrvinium does not. Pyrvinium inhibits AR-dependent gene expression in the prostate gland in vivo, and induces prostate atrophy. These results highlight new therapeutic strategies to inhibit AR activity.

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