A competitive inhibitor that reduces recruitment of androgen receptor to androgen-responsive genes

The androgen receptor (AR) has a critical role in the growth and progression of androgen-dependent and castration-resistant prostate cancers. To identify novel inhibitors of AR transactivation that block growth of prostate cancer cells, a luciferase-based high-throughput screen of ~160,000 small molecules was performed in cells stably expressing AR and a prostate-specific antigen (PSA)-luciferase reporter. CPIC (1-(3-(2-chlorophenoxy) propyl)-1H-indole-3-carbonitrile) was identified as a small molecule that blocks AR transactivation to a greater extent than other steroid receptors. CPIC inhibited AR-mediated proliferation of androgen-sensitive prostate cancer cell lines, with minimal toxicity in AR-negative cell lines. CPIC treatment also reduced the anchorage-independent growth of LAPC-4 prostate cancer cells. CPIC functioned as a pure antagonist by inhibiting the expression of AR-regulated genes in LAPC-4 cells that express wild-type AR and exhibited weak agonist activity in LNCaP cells that express the mutant AR-T877A. CPIC treatment did not reduce AR levels or alter its nuclear localization. We used chromatin immunoprecipitation to identify the site of action of CPIC. CPIC inhibited recruitment of androgen-bound AR to the PSA promoter and enhancer sites to a greater extent than bicalutamide. CPIC is a new therapeutic inhibitor that targets AR-mediated gene activation with potential to arrest the growth of prostate cancer.     

The co-regulators SRC-1 and SMRT are involved in interleukin-6-induced androgen receptor activation

Background

The androgen receptor (AR) can be stimulated by interleukin-6 (IL-6) in the absence of androgens to induce prostate cancer progression. The purpose of this study was to investigate whether the co-activator steroid receptor coactivator-1 (SRC-1) and co-repressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) are involved in IL-6-induced AR activation.

Methods

The effects of IL-6 on LNCaP cell proliferation were monitored using real-time cell analysis (RTCA) iCELLigence system. The impacts of IL-6 on the association of the AR with SRC-1 and SMRT were investigated using the mammalian two-hybrid assay.

Results

IL-6 increased the proliferation of LNCaP cells with maximal induction at 50 ng/mL. The AR-SRC-1interaction was enhanced by IL-6, with maximal induction at the concentration of 50 ng/mL (P<0.05). IL-6 decreased theAR-SMRT interaction and a marked reduction was detected at 50 ng/mL (P<0.05).

Conclusions

IL-6 enhances LNCaP cells proliferation, which suggests that IL-6 might cause AR-positive prostate cancer growth through activation of the AR. The mechanism of IL-6-inducedARactivation is mediated through enhancing AR-SRC-1 interaction and inhibiting AR-SMRT interaction. We have shown a significant role for SRC-1 and SMRT in modulating IL-6-induced AR transactivation.

     

Identification of a protein that interacts with the vanilloid receptor

The vanilloid receptor (VR1 or TRPV1) is a capsaicin (CAP)-sensitive non-selective cation channel. Although its channel activity is reportedly modulated through protein-protein interactions, to date very few VR1 interacting proteins have been identified. To address this issue, a yeast two-hybrid screening technique using the C-terminus of rVR1 as bait was employed. Upon interrogation of a mouse brain library, one gene product that interacts with VR1 and is highly homologous to human eferin was found. Its interaction with VR1 was confirmed by GST-pull-down and co-immunoprecipitation. When cotransfected into HEK cells, VR1 and eferin largely colocalize. Furthermore, in rat dorsal root ganglion cells, the rat eferin homologue also colocalizes with rVR1. However, this protein had no significant effect on VR1 channel activity in response to CAP. This was determined by two-electrode recording of oocytes and whole cell recording of HEK cells that were cotransfected with VR1 and human eferin.     

Membrane glycoprotein M6a interacts with the micro-opioid receptor and facilitates receptor endocytosis and recycling

Using a yeast two-hybrid screen, the neuronal membrane glycoprotein M6a, a member of the proteolipid protein family, was identified to be associated with the mu-opioid receptor (MOPr). Bioluminescence resonance energy transfer and co-immunoprecipitation experiments confirmed that M6a interacts agonist-independently with MOPr in human embryonic kidney 293 cells co-expressing MOPr and M6a. Co-expression of MOPr with M6a, but not with M6b or DM20, exists in many brain regions, further supporting a specific interaction between MOPr and M6a. After opioid treatment M6a co-internalizes and then co-recycles with MOPr to cell surface in transfected human embryonic kidney 293 cells. Moreover, the interaction of M6a and MOPr augments constitutive and agonist-dependent internalization as well as the recycling rate of mu-opioid receptors. On the other hand, overexpression of a M6a-negative mutant prevents mu-opioid receptor endocytosis, demonstrating an essential role of M6a in receptor internalization. In addition, we demonstrated the interaction of M6a with a number of other G protein-coupled receptors (GPCRs) such as the delta-opioid receptor, cannabinoid receptor CB1, and somatostatin receptor sst2A, suggesting that M6a might play a general role in the regulation of certain GPCRs. Taken together, these data provide evidence that M6a may act as a scaffolding molecule in the regulation of GPCR endocytosis and intracellular trafficking.     

The vitamin D receptor interacts preferentially with DRIP205-like LxxLL motifs

The vitamin D receptor (VDR) mediates the biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) through its capacity to recruit coregulatory proteins. This interaction is mediated via a coregulatory LxxLL motif. We screened a combinatorial (x)7LxxLL(x)7 phage library with purified VDR to identify peptides that displayed high affinity and selectivity for VDR. These peptides contained the consensus sequence Lx E/H x H/F P L/M/I LxxLL and exhibited significant sequence similarity to the active LxxLL box found in DRIP205. Nearly all LxxLL peptides interacted in a ligand-dependent manner directly with human VDR. However, a pattern of selectivity of the peptides for other members of the nuclear receptor family was also observed. Interestingly, the interaction between the VDR and many of the peptides was differentially sensitive to a broad assortment of VDR ligands. Finally, several of these peptides were shown to inhibit activation of a 1,25(OH)2D3-sensitive reporter gene. These studies suggest that the LxxLL motif can interact directly with the VDR and that this interaction is regulated by chemically diverse vitamin D ligands.     

TTIP is a novel protein that interacts with the truncated T1 TrkB neurotrophin receptor

Alternative splicing of the TrkB gene produces a full length tyrosine kinase receptor as well as two truncated isoforms that contain extracellular and transmembrane domains but lack the kinase domain and have unique C terminal tails. The function of the truncated TrkB isoforms is unclear and to gain insights into their function, we have isolated a protein from 15N neuroblastoma cells that specifically binds the TrkB.T1 isoform. Pulldown experiments using a GST fusion protein containing the TrkB.T1 intracellular domain identified a 61 kDa protein from radiolabeled 15N lysates. Coimmunoprecipitation experiments showed that the 61 kDa protein interacted with epitope-tagged TrkB.T1 overexpressed in 15N cells as well as with TrkB.T1 which was endogenously expressed. Peptide competition experiments revealed that the protein, designated TTIP (for Truncated TrkB Interacting Protein), showed specific binding to the TrkB.T1 tail. MALDI MS and MS/MS analysis has revealed that TTIP is a novel protein not yet listed in the current databases.     

The DNA-dependent protein kinase interacts with DNA to form a protein-DNA complex that is disrupted by phosphorylation

DNA double-strand breaks are a serious threat to genome stability and cell viability. One of the major pathways for the repair of DNA double-strand breaks in human cells is nonhomologous end-joining. Biochemical and genetic studies have shown that the DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, and Artemis are essential components of the nonhomologous end-joining pathway. DNA-PK is composed of a large catalytic subunit, DNA-PKcs, and a heterodimer of Ku70 and Ku80 subunits. Current models predict that the Ku heterodimer binds to ends of double-stranded DNA, then recruits DNA-PKcs to form the active protein kinase complex. XRCC4 and DNA ligase IV are subsequently required for ligation of the DNA ends. Magnesium-ATP and the protein kinase activity of DNA-PKcs are essential for DNA double-strand break repair. However, little is known about the physiological targets of DNA-PK. We have previously shown that DNA-PKcs and Ku undergo autophosphorylation, and that this correlates with loss of protein kinase activity. Here we show, using electron spectroscopic imaging, that DNA-PKcs and Ku interact with multiple DNA molecules to form large protein-DNA complexes that converge at the base of multiple DNA loops. The number of large protein complexes and the amount of DNA associated with them were dramatically reduced under conditions that promote phosphorylation of DNA-PK. Moreover, treatment of autophosphorylated DNA-PK with the protein phosphatase 1 catalytic subunit restored complex formation. We propose that autophosphorylation of DNA-PK plays an important regulatory role in DNA double-strand break repair by regulating the assembly and disassembly of the DNA-PK-DNA complex.     

Identification of a sequence in the matricellular protein SPARC that interacts with the scavenger receptor stabilin-1

SPARC (osteonectin/BM-40), a secreted matricellular protein that promotes cellular deadhesion and motility in wound healing, carcinogenesis, and inflammation, binds to the scavenger receptor stabilin-1 in alternatively activated macrophages and undergoes endocytosis and clearance from the extracellular space. Both SPARC and stabilin-1 are expressed by endothelial cells during inflammation, but their interaction in this context is unknown. We have identified a binding site on SPARC for stabilin-1 by a solid-state peptide array coupled with a modified enzyme-linked immunosorbent assay. A monoclonal antibody that recognizes the identified binding site was also characterized that could be an inhibitor for the SPARC-stabilin-1 interaction in macrophages or endothelial cells.