Under the auspices of the Nuclear Receptor Signaling Atlas (NURSA), we have undertaken to evaluate the feasibility of targeting nuclear receptor-coactivator surfaces for new drug discovery. The underlying objective of this approach is to provide the research community with reagents that can be used to modulate the transcriptional activity of nuclear receptors. Using combinatorial peptide phage display, we have been able to develop peptide antagonists that target specific nuclear receptor (NR)-coactivator binding surfaces. It can be appreciated that reagents of this nature will be of use in the study of orphan nuclear receptors for whom classical ligands have not yet been identified. In addition, because the interaction of coactivators with the receptor is an obligate step for NR transcriptional activity, it is anticipated that peptides that block these interactions will enable the definition of the biological and pharmacological significance of individual NR-coactivator interactions. In this report, we describe the use of this approach to develop antagonists of the androgen receptor by targeting its coactivator-binding pocket and their use to study the coactivator-binding surface of this receptor. Based on our findings, we believe that molecules that function by disrupting the androgen receptor-cofactor interactions will have use in the treatment of prostate cancer.
Exposure of newborn calves to chronic hypoxia causes pulmonary artery (PA) hypertension and remodeling. Previous studies showed that the redox-sensitive transcription factor, early growth response-1 (Egr-1), is upregulated in the PA of chronically hypoxic calves and regulates cell proliferation. Furthermore, we established in mice a correlation between hypoxic induction of Egr-1 and reduced activity of extracellular superoxide dismutase (EC-SOD), an antioxidant that scavenges extracellular superoxide. We now hypothesize that loss of EC-SOD in chronically hypoxic calves leads to extracellular superoxide-mediated upregulation of Egr-1. To validate our hypothesis and identify the signaling pathways involved, we utilized PA tissue from normoxic and chronically hypoxic calves and cultured calf and human PA smooth muscle cells (PASMC). Total SOD activity was low in the PA tissue, and only the extracellular SOD component decreased with hypoxia. PA tissue of hypoxic calves showed increased oxidative stress and increased Egr-1 mRNA. To mimic the in vivo hypoxia-induced extracellular oxidant imbalance, cultured calf PASMC were treated with xanthine oxidase (XO), which generates extracellular superoxide and hydrogen peroxide. We found that 1) XO increased Egr-1 mRNA and protein, 2) XO induced the phosphorylation of ERK1/2 and, 3) pretreatment with an ERK1/2 inhibitor prevented induction of Egr-1 by XO. siRNA knock-down of EC-SOD in human PASMC also upregulated Egr-1 mRNA and protein, activated ERK1/2, and enhanced SMC proliferation and reduced apoptosis. We conclude that an oxidant/antioxidant imbalance arising from loss of EC-SOD in the PA with chronic hypoxia induces Egr-1 via activation of ERK1/2 and contributes to pulmonary vascular remodeling.
The full-length human androgen receptor with an N-terminal biotin acceptor peptide tag was overexpressed in Spodoptera frugiperda cells in the presence of 1 µM dihydrotestosterone. Site-specific biotinylation of BAP was achieved in vivo by co-expression of E. coli biotin holoenzyme synthetase. The androgen receptor was purified by single-step affinity chromatography using Streptavidin Mutein Matrix under native conditions.The resultant protein was active, stable, 95% homogeneous, and we obtained sufficient yield for use in functional and structural studies.
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