Isolation and identification of l-dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system

The AR (androgen receptor) is a ligand-regulated transcription factor, which belongs to the steroid receptor family and plays an essential role in growth and development of the prostate. Transcriptional activity of steroid receptors is modulated by interaction with co-regulator proteins and yeast two-hybrid analysis is commonly used to identify these steroid receptor-interacting proteins. However, a limitation of conventional two-hybrid systems for detecting AR protein partners has been that they only allow for analysis of the ligand- and DNA-binding domains of the receptor, as its NTD (N-terminal domain) possesses intrinsic transactivation activity. To identify AR N-terminus-interacting proteins, its NTD was used in the RTA (repressed transactivator) system, which is specifically designed for transactivator bait proteins and was shown to be suitable for two-hybrid analysis with the AR NTD. DDC (l-dopa decarboxylase) was detected multiple times as a novel AR-interacting protein, which was subsequently confirmed in vitro and in vivo. Furthermore, transient transfection of DDC in prostate cancer cells strongly enhanced ligand-dependent AR transcriptional activity, an effect that was antagonized using high concentrations of the anti-androgen bicalutamide. Glucocorticoid receptor activity was also strongly enhanced with DDC co-transfection, while oestrogen receptor activity was only mildly affected. Together, our data demonstrate that DDC interacts with AR to enhance steroid receptor transactivation, which may have important implications in prostate cancer progression.

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BAG-1 diversely affects steroid receptor activity

Biochemical Journal ◽

10.1042/bj20111456 ◽

2011 ◽

Vol 441 (1) ◽

pp. 297-303 ◽

Cited By ~ 9

Author(s):

Regina T. Knapp ◽

Andrea Steiner ◽

Ulrike Schmidt ◽

Kathrin Hafner ◽

Florian Holsboer ◽

...

Keyword(s):

Androgen Receptor ◽

Heat Shock ◽

Heat Shock Protein 70 ◽

Mineralocorticoid Receptor ◽

Transcriptional Activity ◽

Steroid Receptors ◽

Steroid Receptor ◽

Receptor Activity ◽

High Homology ◽

Shed Light

Part of the cellular and physiological functions of BAG-1 (Bcl-2-associated athanogene 1) has been ascribed to the ability of this hsp70 (heat-shock protein 70) co-chaperone to regulate steroid receptor activity. BAG-1 has been reported to inhibit the GR (glucocorticoid receptor) and stimulate the androgen receptor, but to leave the activity of the MR (mineralocorticoid receptor) unchanged. Given the high homology between the MR and GR, this disparity in the actions of BAG-1 is surprising. In the present study, we analysed the effect of BAG-1 on the activity of the closely related PR (progesterone receptor). Similarly to the GR, the transcriptional activity of the PR is inhibited by the long and middle isoforms of BAG-1, BAG-1L and BAG-1M, but not by the short isoform, BAG-1S. We found this inhibition to require the hsp70-binding domain of BAG-1. To shed light on the mechanisms that could explain BAG-1's differential actions on steroid receptors, we tested the binding of BAG-1M to the PR. Mutational analyses of the PR and BAG-1M revealed that the mode of interaction and BAG-1M-mediated inhibition of the PR differs from the reported scenario for the GR. Surprisingly, we also found binding of BAG-1M to the MR. In addition, BAG-1M was able to inhibit the transcriptional activity of the MR. These data entail a reappraisal of the physiological actions of BAG-1M on steroid receptor activity.

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Yeast Two-Hybrid Analysis of PriB-Interacting Proteins in Replication Restart Primosome: A Proposed PriB–SSB Interaction Model

The Protein Journal ◽

10.1007/s10930-013-9509-y ◽

2013 ◽

Vol 32 (6) ◽

pp. 477-483 ◽

Cited By ~ 6

Author(s):

Yen-Hua Huang ◽

Min-Jon Lin ◽

Cheng-Yang Huang

Keyword(s):

Interaction Model ◽

Interacting Proteins ◽

Yeast Two Hybrid ◽

Hybrid Analysis ◽

Replication Restart ◽

Two Hybrid

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Identification of SUMO-Interacting Proteins by Yeast Two-Hybrid Analysis

Methods in Molecular Biology - SUMO Protocols ◽

10.1007/978-1-59745-566-4_7 ◽

2009 ◽

pp. 107-120 ◽

Cited By ~ 26

Author(s):

Mary B. Kroetz ◽

Mark Hochstrasser

Keyword(s):

Interacting Proteins ◽

Yeast Two Hybrid ◽

Hybrid Analysis ◽

Two Hybrid

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The Tomato Metallocarboxypeptidase Inhibitor I, which Interacts with a Heavy Metal-Associated Isoprenylated Protein, Is Implicated in Plant Response to Cadmium

Molecules ◽

10.3390/molecules25030700 ◽

2020 ◽

Vol 25 (3) ◽

pp. 700 ◽

Cited By ~ 2

Author(s):

Anna Manara ◽

Elisa Fasani ◽

Barbara Molesini ◽

Giovanni DalCorso ◽

Federica Pennisi ◽

...

Keyword(s):

Heavy Metal ◽

Active Site ◽

Abiotic Stresses ◽

Lower Amount ◽

Interacting Proteins ◽

Wild Type ◽

Yeast Two Hybrid ◽

Hybrid Analysis ◽

Model Species ◽

Two Hybrid

Metallocarboxypeptidases are metal-dependent enzymes, whose biological activity is regulated by inhibitors directed on the metal-containing active site. Some metallocarboxypeptidase inhibitors are induced under stress conditions and have a role in defense against pests. This paper is aimed at investigating the response of the tomato metallocarboxypeptidase inhibitor (TCMP)-1 to Cd and other abiotic stresses. To this aim, the tomato TCMP-1 was ectopically expressed in the model species Arabidopsis thaliana, and a yeast two-hybrid analysis was performed to identify interacting proteins. We demonstrate that TCMP-1 is responsive to Cd, NaCl, and abscisic acid (ABA) and interacts with the tomato heavy metal-associated isoprenylated plant protein (HIPP)26. A. thaliana plants overexpressing TCMP-1 accumulate lower amount of Cd in shoots, display an increased expression of AtHIPP26 in comparison with wild-type plants, and are characterized by a modulation in the expression of antioxidant enzymes. Overall, these results suggest a possible role for the TCMP-1/HIPP26 complex in Cd response and compartmentalization.

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Novel Interaction between the Co-chaperone Cdc37 and Rho GTPase Exchange Factor Vav3 Promotes Androgen Receptor Activity and Prostate Cancer Growth

Journal of Biological Chemistry ◽

10.1074/jbc.m112.390963 ◽

2012 ◽

Vol 288 (8) ◽

pp. 5463-5474 ◽

Cited By ~ 15

Author(s):

Fayi Wu ◽

Stephanie O. Peacock ◽

Shuyun Rao ◽

Sandra K. Lemmon ◽

Kerry L. Burnstein

Keyword(s):

Prostate Cancer ◽

Androgen Receptor ◽

Rho Gtpase ◽

Receptor Activity ◽

Cancer Growth ◽

Exchange Factor ◽

Androgen Receptor Activity

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A novel DNA methylation signature is associated with androgen receptor activity and patient prognosis in bone metastatic prostate cancer

Clinical Epigenetics ◽

10.1186/s13148-021-01119-0 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Erik Bovinder Ylitalo ◽

Elin Thysell ◽

Mattias Landfors ◽

Maria Brattsand ◽

Emma Jernberg ◽

...

Keyword(s):

Gene Expression ◽

Prostate Cancer ◽

Androgen Receptor ◽

Bone Metastases ◽

Metastatic Prostate Cancer ◽

Tumor Biology ◽

Receptor Activity ◽

Mrna Levels ◽

Androgen Receptor Activity ◽

Patient Prognosis

Abstract Background Patients with metastatic prostate cancer (PC) are treated with androgen deprivation therapy (ADT) that initially reduces metastasis growth, but after some time lethal castration-resistant PC (CRPC) develops. A better understanding of the tumor biology in bone metastases is needed to guide further treatment developments. Subgroups of PC bone metastases based on transcriptome profiling have been previously identified by our research team, and specifically, heterogeneities related to androgen receptor (AR) activity have been described. Epigenetic alterations during PC progression remain elusive and this study aims to explore promoter gene methylation signatures in relation to gene expression and tumor AR activity. Materials and methods Genome-wide promoter-associated CpG methylation signatures of a total of 94 tumor samples, including paired non-malignant and malignant primary tumor areas originating from radical prostatectomy samples (n = 12), and bone metastasis samples of separate patients with hormone-naive (n = 14), short-term castrated (n = 4) or CRPC (n = 52) disease were analyzed using the Infinium Methylation EPIC arrays, along with gene expression analysis by Illumina Bead Chip arrays (n = 90). AR activity was defined from expression levels of genes associated with canonical AR activity. Results Integrated epigenome and transcriptome analysis identified pronounced hypermethylation in malignant compared to non-malignant areas of localized prostate tumors. Metastases showed an overall hypomethylation in relation to primary PC, including CpGs in the AR promoter accompanied with induction of AR mRNA levels. We identified a Methylation Classifier for Androgen receptor activity (MCA) signature, which separated metastases into two clusters (MCA positive/negative) related to tumor characteristics and patient prognosis. The MCA positive metastases showed low methylation levels of genes associated with canonical AR signaling and patients had a more favorable prognosis after ADT. In contrast, MCA negative patients had low AR activity associated with hypermethylation of AR-associated genes, and a worse prognosis after ADT. Conclusions A promoter methylation signature classifies PC bone metastases into two groups and predicts tumor AR activity and patient prognosis after ADT. The explanation for the methylation diversities observed during PC progression and their biological and clinical relevance need further exploration.

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Anti-androgen receptor activity of apoptotic CK2 inhibitor CX4945 in human prostate cancer LNCap cells

Bioorganic & Medicinal Chemistry Letters ◽

10.1016/j.bmcl.2012.07.031 ◽

2012 ◽

Vol 22 (17) ◽

pp. 5470-5474 ◽

Cited By ~ 10

Author(s):

Byung Jun Ryu ◽

Seung-hwa Baek ◽

Jiyeon Kim ◽

Su Jung Bae ◽

Sung-Youn Chang ◽

...

Keyword(s):

Prostate Cancer ◽

Androgen Receptor ◽

Receptor Activity ◽

Human Prostate ◽

Human Prostate Cancer ◽

Lncap Cells ◽

Androgen Receptor Activity ◽

Ck2 Inhibitor

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Reply to Isabel Heidegger, Renate Pichler, and Andreas Pircher's Letter to the Editor re: Erik Bovinder Ylitalo, Elin Thysell, Emma Jernberg, et al. Subgroups of Castration-resistant Prostate Cancer Bone Metastases Defined Through an Inverse Relationship Between Androgen Receptor Activity and Immune Response. Eur Urol 2017;71:776–87

European Urology ◽

10.1016/j.eururo.2017.06.009 ◽

2017 ◽

Vol 72 (4) ◽

pp. e104-e105 ◽

Cited By ~ 1

Author(s):

Elin Thysell ◽

Erik B. Ylitalo ◽

Emma Jernberg ◽

Anders Bergh ◽

Pernilla Wikström

Keyword(s):

Prostate Cancer ◽

Immune Response ◽

Androgen Receptor ◽

Bone Metastases ◽

Inverse Relationship ◽

Receptor Activity ◽

Castration Resistant Prostate Cancer ◽

Castration Resistant ◽

Letter To The Editor ◽

Androgen Receptor Activity

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Specific inhibition of transcriptional activity of the constitutive androstane receptor (CAR) by the splicing factor SF3a3

Biological Chemistry ◽

10.1515/bc.2008.149 ◽

2008 ◽

Vol 389 (10) ◽

Cited By ~ 1

Author(s):

Hye Jin Yun ◽

Jungsun Kwon ◽

Wongi Seol

Keyword(s):

Transcriptional Activity ◽

Constitutive Androstane Receptor ◽

Splicing Factor ◽

Yeast Two Hybrid ◽

Interacting Protein ◽

Functional Studies ◽

Reporter Activity ◽

Inhibitory Function ◽

Two Hybrid ◽

Hybrid Screening

Abstract The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily and plays an important role in the degradation of xenobiotics in the liver. Using yeast two-hybrid screening, we identified SF3a3, a 60-kDa subunit of the splicing factor 3a complex, as a specific CAR-interacting protein. We further confirmed their interaction by both co-immunoprecipitation and GST pull-down assay. Functional studies showed that overexpression of SF3a3 inhibited the reporter activity driven by a promoter containing CAR binding sequences by up to 50%, whereas reduced expression of SF3a3 activated the same reporter activity by approximately three-fold. The inhibitory function of SF3a3 is independent of the presence of TCPOBOP, a CAR ligand. These data suggest that SF3a3 functions as a co-repressor of CAR transcriptional activity, in addition to its canonical function.

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