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 DNAbinding protein. Consistent with this prediction, biochemical experiments showed that PAGE4 preferentially binds a GCrich 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.

Phosphorylation-induced Conformational Ensemble Switching in an Intrinsically Disordered Cancer/Testis Antigen

“…However, the PAGE4 protein is up-regulated in both symptomatic benign prostatic hyperplasia, which is related to prostate cancer (13), and in prostate cancer itself (12,15,16), indicating a potential role for PAGE4 in disease pathology. Consistent with this hypothesis, knocking down PAGE4 expression results in cell death, whereas its overexpression leads to a growth advantage of prostate cancer cells (17).…”

Resilience of death: intrinsic disorder in proteins involved in the programmed cell death

“…48 NMR analysis revealed that an important regulator of proliferation and apoptosis, cAMPresponsive (CRE)-binding (CREB) protein (CBP), contains an intrinsically disordered ACTR-binding domain (residues 2059-2117) that completely folds upon binding. 49 Combined experimental and computational analysis revealed that N-and C-terminal regions of the human Nogo proteins 50 and C-terminal domain of a mitochondrial pro-apoptotic protein ARTS 51 are typical IDPRs, and that the prostate apoptosis response factor-4 (PAR-4), 52 prostate-associated gene 4 (PAGE4) protein, 53 and important oncoprotein c-Myc 54 are mostly disordered apoptosis-related proteins.…”

Phosphorylation-induced conformational dynamics in an intrinsically disordered protein and potential role in phenotypic heterogeneity

“…PAGE4 is a cancer/testis antigen that is intrinsically disordered (>95%) (29) and is remarkably prostate-specific in the human male (30). It has the hallmarks of a protooncogene, with important roles in development and differentiation.…”

“…33). However, PAGE4 protein expression is elevated in prostate cancer (PCa) (31,(34)(35)(36), where it is upregulated in response to a variety of stress factors (29,31). PAGE4 is phosphorylated at S9 and T51 by HomeodomainInteracting Protein Kinase 1 (HIPK1), a component of the cellular stress-response pathway, and functions as a strong potentiator of the oncoprotein c-Jun (32,37).…”