The cancer/testis antigen prostate-associated gene 4 (PAGE4) is a highly intrinsically disordered protein

Abstract

The cancer/testis antigens (CTAs) are an important group of heterogeneous proteins that are predominantly expressed in the testis in the normal human adult but are aberrantly expressed in several types of cancers. Prostate-associated gene 4 (PAGE4) is a member of the CT-X family of CTAs that in addition to testis, is highly expressed in the fetal prostate, and may also play an important role both in benign and malignant prostate diseases. However, the function of this gene remains poorly understood. Here, we show that PAGE4 is a highly (100%) intrinsically disordered protein (IDP). The primary protein sequence conforms to the features of a typical IDP sequence and the secondary structure prediction algorithm metaPrDOS strongly supported this prediction. Furthermore, SDS-gel electrophoresis and analytical size exclusion chromatography of the recombinant protein revealed an anomalous behavior characteristic of IDPs. UV circular dichroism (CD) and NMR spectroscopy confirmed that PAGE4 is indeed a highly disordered protein. In further bioinformatic analysis, the PredictNLS algorithm uncovered a potential nuclear localization signal, whereas the algorithm DBS-Pred returned a 99.1% probability that PAGE4 is a DNA-binding protein. Consistent with this prediction, biochemical experiments showed that PAGE4 preferentially binds a GC-rich sequence. Silencing PAGE4 expression induced cell death via apoptosis and in mice carrying PCa xenografts, siRNA-mediated knockdown of the PAGE4 mRNA attenuated tumor growth in vivo. Furthermore, overexpressing PAGE4 protected cells from stress-induced death. To our knowledge, PAGE4 is the first example of a CTA that is an IDP with an anti-apoptotic function.

FIGURE 1.

Bioinformatic predictions of disorder in the PAGE4 protein structure. A, amino acid sequence of PAGE4. Polar residues are indicated in green and non-polar residues are in red. The putative NLS and DNA-binding region are indicated by asterisks and underlines, respectively. Disorder in the PAGE4 protein was predicted by FoldIndex (B), RONN (C), and metaPrDOS (D) as described under “Experimental Procedures.”

FIGURE 2.

Anomalous behavior of PAGE4 protein in gel electrophoresis and analytical gel filtration chromatography. A, SDS-PAGE of recombinant PAGE4 synthesized in bacteria. B, immunoblot showing anomalous migration of the endogenous PAGE4 protein obtained from LNCaP cells (lane 1, 50 μg) and 5 μg of recombinant PAGE4 (lane 2) using a anti-PAGE4-specific antibody. C and D, analytical size exclusion chromatography. Absorptions at 280 and 220 nm were monitored as a function of retention volume and plotted with units shown on the left and right axis, respectively.

FIGURE 3.

Biophysical characterization of PAGE4 disorder. A, circular dichroism spectra of PAGE4 as a function of temperature. CD spectra were recorded at the following temperatures: 5 (cyan), 10 (orange), 15 (green), 20 (red), and 25 °C (blue). A protein concentration of 0.33 mg/ml was used in 10 mm sodium phosphate buffer containing 150 mm sodium chloride, pH 7.4. CD measurements were made using a 0.1-cm path length. B, one-dimensional ¹H NMR spectra of PAGE4 as a function of temperature. The amide proton region is shown. Spectra were recorded on a Bruker DMX-600 using a protein concentration of 1 mg/ml and the same buffer conditions as above. A water flip-back pulse sequence was used for solvent suppression.

FIGURE 4.

PAGE4 binds to a GC-rich DNA sequence. The DNA-binding assay using purified FLAG-tagged PAGE4 protein was done as described. A, dsDNA obtained by PCR. Lane 1, vector control; lane 2, PAGE4; and lane 3, p53 positive control. B, the GC-rich consensus sequences using the WebLogo software obtained from sequencing 33 independent clones.

FIGURE 5.

Silencing PAGE4 expression inhibits cell survival and enhances chemo-cytotoxicity in prostate cancer cells. A, LNCaP, LNCaP-96, and CWR22rv1 cells were transfected with 50 nm PAGE4 SMARTpool siRNA. Cell viability was evaluated by WST-1 assay 3–7 days after transfection. B, crystal violet staining of CWR22rv1 cells 5 days after siRNA transfection. C, LNCaP cells was stained with propidium iodide 72 h after siRNA transfection and subjected to cell cycle analysis. D, apoptosis of LNCaP cells was evaluated by TUNEL assay 72 h after siRNA transfection. E, immunofluorescence staining of γ-H2A.X in LNCaP cells 72 h after siRNA transfection. Magnification is ×200 in all panels. F, the whole lysate of LNCaP cells were subjected to immunoblotting for p21, phospho-p53 (Ser¹⁵) (pS-p53), and γ-H2A.X 72 h after siRNA transfection. Actin was used as a control for protein loading. G, LNCaP, LNCaP-96, and CWR22rv1 cells were transfected with 50 nm PAGE4 SMARTpool siRNA. Forty-eight hours after transfection, cells were treated with ADM at the indicated dose for 4 h, and then moved back to normal medium for another 16 h. Cell viability was evaluated by WST-1 assay. All experiments were biological replicates repeated a minimum of three times. Data are represented as mean ± S.D. *, p < 0.05 in comparison to scrambled-siRNA; #, p < 0.05 in comparison to normal controls without any treatment.

FIGURE 6.

PAGE4 siRNA administration inhibits cell growth of LNCaP xenografts in SCID mice. A, SCID mice (seven in each group) were subcutaneously injected with 2 × 10⁶ LNCaP cells suspended in PBS and Matrigel, and treated with PAGE4 siRNA by local injection every 5 days for 3 times. Mean relative tumor volume of each group was plotted over time. B, two mice in each group were sacrificed 2 days after the last administration of siRNA. Tumor tissues were removed and subjected to RT-PCR to evaluate PAGE4 mRNA expression. Data are represented as mean ± S.D. *, p < 0.05 in comparison to scrambled siRNA treatment; #, p < 0.05 in comparison to PBS treatment.

FIGURE 7.

Overexpression of PAGE4 protected cells from stress-induced death. HEK293T (A) and CWR22rv1 (B) cells were seeded in 6-well plates and transfected with 4 μg of pCMV6-PAGE4-GFP vector (■) or empty vector pCMV6-GFP (□). The number of living cells was counted using a hemocytometer after excluding dead cells by trypan blue staining 1–5 days after transfection. C, Western blot for PARP-1, pS-p53, and PAGE4 protein in the cell lysate of the two cell lines 72 h after transfection. Actin was blotted as loading control. D, CWR22rv1 cells were seeded in 12-well plates and transfected with pCMV6-GFP, pCMV6-PAGE4-GFP, or pCMV6-PAGE4-GFP followed by 50 nm PAGE4 siRNA. Forty-eight hours after transfection, cells were treated with 1 μg/ml of ADM for 4 h, and then moved back to normal medium for another 19 h. Cell viability was evaluated by WST-1 assay. ■, ADM treatment; □, normal medium control. *, p < 0.05 as compared with GFP-transfected cells; #, p < 0.05 as compared with PAGE4-GFP plus PAGE4 siRNA-transfected cells. E, cells were lysed 24 h after ADM treatment and subjected to Western blot for PARP-1. Data are represented as mean ± S.D. All experiments were biological replicates repeated a minimum of three times.