The Cochaperone Bag-1L Enhances Androgen Receptor Action via Interaction with the NH(2)-Terminal Region of the Receptor

Members of the Bag-1 family of cochaperones regulate diverse cellular processes including the action of steroid hormone receptors. The largest member of this family, Bag-1L, enhances the transactivation function of the androgen receptor. This occurs primarily through interaction with the NH(2) and COOH termini of the receptor. At the NH(2) terminus of the receptor, Bag-1L interacts with a region termed τ5. Bag-1M, a naturally occurring variant of Bag-1L that binds to τ5 but is defective in the COOH-terminal interaction, is less efficient in enhancing the transactivation function of the receptor. Surface plasmon resonance and transfection studies showed that the molecular chaperone Hsp70 contributes to the binding of Bag-1L to τ5 and to the regulation of the transactivation function of the androgen receptor. Chromatin immunoprecipitation studies demonstrated that the androgen receptor, Hsp70, and Bag-1L are all targeted to the androgen response elements of the gene that encodes prostate-specific antigen. These studies demonstrate the regulation of transcriptional activity of androgen receptor by a molecular chaperone-cochaperone complex

A Peptidic Unconjugated GRP78/BiP Ligand Modulates the Unfolded Protein Response and Induces Prostate Cancer Cell Death

<div><p>The molecular chaperone GRP78/BiP is a key regulator of protein folding in the endoplasmic reticulum, and it plays a pivotal role in cancer cell survival and chemoresistance. Inhibition of its function has therefore been an important strategy for inhibiting tumor cell growth in cancer therapy. Previous efforts to achieve this goal have used peptides that bind to GRP78/BiP conjugated to pro-drugs or cell-death-inducing sequences. Here, we describe a peptide that induces prostate tumor cell death without the need of any conjugating sequences. This peptide is a sequence derived from the cochaperone Bag-1. We have shown that this sequence interacts with and inhibits the refolding activity of GRP78/BiP. Furthermore, we have demonstrated that it modulates the unfolded protein response in ER stress resulting in PARP and caspase-4 cleavage. Prostate cancer cells stably expressing this peptide showed reduced growth and increased apoptosis in <em>in vivo</em> xenograft tumor models. Amino acid substitutions that destroyed binding of the Bag-1 peptide to GRP78/BiP or downregulation of the expression of GRP78 compromised the inhibitory effect of this peptide. This sequence therefore represents a candidate lead peptide for anti-tumor therapy.</p> </div

Bag-1 proteins interact with the molecular chaperone GRP78/BiP.

<p>A. The Bag-1 family of proteins binds GRP78/BiP. GST pull-down assay was performed incubating 5 µg each of GST-fused Bag-1 proteins with 400 µg of 22Rv.1 cell lysate. Western blotting with a specific antibodiy against GRP78/BiP was used to detect the binding and an anti-GST antibody was used to determine the amount of bacterially purified protein employed in the assay. B. Bag-1 and GRP78 interact <i>in vivo</i>. GRP78 was immunoprecipitated with an anti-GRP78/BiP antibody or IgG as control in 22Rv.1 cells. Western blotting with an anti-Bag-1 and anti-GRP78 antibody was performed to determine GRP78-Bag-1 interaction. C. Bag-1 and GRP78/BiP show a perinuclear colocalization. Confocal microscopic analyses were carried out in 22Rv.1 cells that have been paraformaldehyde-fixed and stained with a Bag-1 antibody to detect Bag-1 (green channel) and a GRP78/BiP specific antibody to detect GRP78/BiP (red channel). The orange staining in the merge of the two channels indicates the degree of co-localization of the two proteins. All images (40X) were acquired with a Leica TCS SPE confocal microscope (Leica Microsystems; Scale bars indicate 25 µm). D. Diagrammatic representation of the domains of GRP78. E. Bag-1 binds to multiple sites on GRP78. GST-pull down assay performed incubating 500 µg lysate of HEK-293 cells transfected with an HA-tagged Bag-1 and 10 µg GST-fused GRP78 full length and its ATPase and substrate binding domain (SBD). Western blot analysis was carried out using an HA antibody to detect the Bag-1-tagged protein. Shown is a Coomassie blue staining of the bacterially purified GST proteins to demonstrate equal loading of the GST proteins.</p

An N-terminal unfolded fragment of the Bag-1 peptide is sufficient to reduce tumor cell growth.

<p>A. Schematic diagram of the Bag-1 peptide and its deletion mutants. The ubiquitin-like domain is represented in blue and the BAG domain in red. The numbers refer to the amino acid positions of the different domains. The amino acid residues are numbered following the numbering for Bag-1L. B. Prediction of the secondary structure of the Bag-1 peptide, showing an N-terminal β-hairpin from the ubiquitin-like domain (blue) and a C-terminal α-helix from the BAG domain (red). C. Normalized circular dichroism spectra of the Bag-1 peptides. 30 µM of the Bag-1 peptide were measured in 20 mM KHPO₄ buffer, pH 6.8 (black line). Its α-helical content was estimated to be approximately 25% by deconvolution of the spectra (red line). 12 µM of the N-term peptide (green line) and 11 µM of C-term (blue line) were measured under the same conditions. D. ¹H¹⁵N-HSQC NMR spectrum of ¹⁵N-labeled Bag-1 peptide (202–269) in 20 mm KHPO₄ buffer, pH 6.8, at 23°C. The narrow spectral dispersion indicates that the peptide does not exhibit a folded globular structure. The H_δ and H_ε side chain signals of asparagine and glutamine are connected by thin lines. E. The N-terminal region of the Bag-1 peptide is important for GRP78 binding. 400 µg of 22Rv.1 cell lysate were incubated with glutathione-agarose beads carrying 15 µg GST-N-term peptide (lane 2), GST-C-term peptide (lane 3), GST-ΔUbi peptide (lane 4), GST-Bag-1(202–269) peptide (lane 5) and GST (lane 6). The beads were washed and the bound proteins were separated by 10% SDS-PAGE and subjected to Western blotting using antibodies directed against GRP78 or GST. The input lane shows 1/10 aliquot of cell lysate used for the study. F. Clonogenic assay of the Bag-1 peptides expressed in 22Rv.1 cells. Cells transfected with the indicated constructs were selected in medium containing neomycin and the colonies formed were quantified. Shown are the mean value ±SEM of at least three independent experiments using three different plasmid preparations (*p<0.05). G-I. The N-terminal peptide reduces tumor growth <i>in vivo</i>. Six-week old athymic nude mice were injected subcutaneously on both flanks with 5×10⁶ cells of each stable clone. Tumor size was measured once per week using a caliper and expressed as tumor volume in mm³. Shown are the tumor volumes of clones transfected with the N-terminal peptide (G), the C-terminal peptide (H) and the ΔUbi peptide (I). Each point represents the mean volume and standard deviation of at least 5 to 10 tumors.</p

Selectivity of action of the Bag-1 peptide.

<p>A Amino acid sequence of the 19mer peptide and its deletion mutants. B GST pull-down assay performed incubating 400 µg of 22Rv.1 cell lysate and 25 µg of GST-fused deletion mutants of the 19mer peptide. After the pull-down experiment, Western blots were carried out with anti-GRP78/BiP and GST antibodies. C. Fluorescence polarization (FP) assays with a 10 nM FITC-labeled 7-mer Bag-1 peptide and dilutions of the GST-fused substrate binding domain of GRP78 in 60 µl of Tris-GSH buffer (50 mM Tris, 10 mM reduced glutathione and 0.1% BSA, pH 8.0). Triplicate samples were measured after 5 h of incubation at room temperature with an Infinite F-200 reader (Tecan) with excitation at 490 nm and emission at 535 nm. Binding constants were determined from three independent experiments using non-linear regression in Sigma Plot 10.0. D. Competition assays with a complex of 10 nM of FITC-labeled Bag-1 7-mer peptide and 0.75 mg/ml GST-fused GRP78 substrate binding domain was measured against a dilution series from 100 µM to 0.1 nM of wild-type Bag-1 7-mer peptide (filled line) or triple mutant peptide (dotted line) in 40 µl of Tris-GSH buffer. FITC-peptide and the FITC-peptide-protein complex were used as controls. Fluorescence polarization was determined as described above and IC₅₀ values were determined in Sigma Plot. E. Clonogenic assay in 22Rv.1 cells with pcDNA3 based vectors coding for the N-terminal Bag-1 peptide (positive control), the 19mer and the 19mer mutant fragments as well as the empty expression vector (negative control). Cells were transfected with the indicated constructs and selected in medium containing neomycin. The colonies were stained and quantified. Shown as bar chats are the mean ± SEM of at least three independent experiments with three different plasmid preparations. (*p<0.05). F. <i>In vivo</i> luciferase refolding assay was performed in HEK-293 cells transfected at 70% confluency with 2 µg β-actin-fire fly luciferase construct, 3 µg pcDNA3 empty vector as control or pCMV6-GRP78, 6 µg pcDNA3.1-HA-19-mer or 19-mer mutant peptide and 0.5 µg Renilla luciferase construct to determine the transfection efficiency. Shown as bar charts is the mean of at least three different experiments ± SEM (*p<0.05). G. Clonogenic assay with the indicated malignant (LNCaP, DU145, PC3) and benign (BPH1, PNT2, RWPE1) prostate cell lines transfected with the vector control (black bars) and a vector expressing the 19-mer peptide (white bars). Shown are the mean ± SD of at least three different experiments performed with three different plasmid preparations (*p<0.05). H. Western blot analysis of cell lysates derived from the prostate cancer cells used for the colony forming assay with specific antibodies against GRP78 and β-actin.</p

Effect of the Bag-1 peptide on tumor weights in 22Rv.1 and LNCaP xenograft tumor mouse models.

<p>Tumor weights and time of euthanasia of mice are recorded. The values represent the mean weights and standard deviation calculated from 4–10 tumors and the time of euthanasia.</p

Identification of Bag-1 peptide interfering with the action of GRP78/BiP.

<p>A. Diagrammatic representation of Bag-1 and its deletion mutants used for the identification of sequences required for binding the SBD of GRP78/BiP. Depicted in blue and red are the ubiquitin-like domain and the BAG domain respectively. B. Identification of the Bag-1 peptide binding to GRP78/BiP. GST-pull down assay performed incubating 500 µg lysates from HEK-293 cells transfected with the indicated Bag-1 constructs and 10 µg of GST-GRP78(SBD) or GST as control. Western blot analysis with an HA antibody to detected the Bag-1 mutants is shown. Coomassie blue staining was performed to demonstrate equal loading of the GST fusion proteins. C. The Bag-1 68 amino acid peptide binds <i>in vivo</i> to GRP78. HEK 293 cells were co-transfected with pcDNA-HA-tagged Bag-1 peptide. The transfected cells were treated with 2 mM Dithiobis(succinimidylpropionate) to cross-link the proteins and the cell lysates were immunoprecipitated with an anti-HA antibody or IgG as control, followed by Western blotting with an anti-GRP78/BiP antibody. D. The Bag-1 68 amino acid peptide inhibits GRP78/BiP refolding activity. <i>In vivo</i> luciferase refolding assay was performed in HEK-293 cells transfected at 70% confluency with 2 µg β-actin-fire fly luciferase construct, 3 µg pcDNA3 empty vector as control or pCMV6-GRP78/Bip, 6 µg of the indicated pcDNA3.1-Bag-1 or mutant constructs and 0.5 µg Renilla luciferase construct to determine the transfection efficiency. The transfected cells were divided into two. One half was left untreated and the other half was heat shocked at 45°C for 30 min. Thereafter luciferase activity was measured. Results are expressed as percentage of refolding activity relative to non-heat-shocked cells and presented as bar charts of the average of three independent experiments ± SEM. *p<0.05.</p

The Bag-1 peptide modulates the UPR and sensitizes 22Rv.1 cells to ER-stress.

<p>A. UPR modulation upon Bag-1 peptide overexpression. Pooled clones of 22Rv.1 cells transfected with pcDNA3.1-HA-Bag-1 68 amino acid peptide or an empty expression vector were treated with 300 nM thapsigargin (TG) for the indicated time points. Cells were lysed and subjected to Western blot analysis using the indicated antibodies or phospho-specific antibodies. B. The Bag-1 peptide sensitizes 22Rv.1 cells to ER-stress induced apoptosis. Pooled clones of 22Rv.1 transfected with the Bag-1 peptide or the empty expression vector were treated with thapsigargin (TG) or glucose-starved (GS) for 24 h. The cells were lysed and subjected to Western blot analysis using anti-PARP and caspase 4 specific antibodies. Anti-HA antibody was used to detect the HA-Bag-1 peptide. Anti-β-actin antibody was used to demonstrate equal loading of the protein samples. C. GRP78 downregulation increases PARP cleavage. Pooled clones of 22Rv.1 expressing HA-tagged Bag-1 peptide or an empty expression vector were transfected with GRP78/BiP siRNA or control GFP siRNA. The cells were lysed and Western blot was carried out with anti-PARP, anti-GRP78 and anti-HA antibodies. β-actin antibody was used to determine the level of protein loaded on the gel.</p