Abstract 2456: The transmembrane domain of prostate specific membrane antigen: Deciphering the role of the Small-XXX-Small motif in oligomerization

Abstract

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related deaths among males in the United States. The prevalence of this disease and the benefits associated with early detection and treatment call for closer investigation into potential biomarkers and therapeutics targets. The high expression of the integral membrane protein called prostate specific membrane antigen (PSMA, EC 3.4.17.21) is implicated in PCa invasiveness as well as neovasculature metastasis of nonprostatic solid tumors. Little is known about the exact role of PSMA in PCa progression albeit it is one of the most validated biomarker for the diagnosis and detection of PCa. We have demonstrated that the isolated PSMA transmembrane domain (TMD) is capable of oligomerization under reducing gel electrophoresis conditions, interacting independently of extracellular domain dimerization. Following this discovery, we proceeded to determine the factor(s) responsible for its oligomerization. Since it is known that certain motifs are critical for TMD oligomerization of many transmembrane proteins, we hypothesized that the Small-XXX-Small motif in PSMA TMD may play a role in the oligomerization and subsequent activation of PSMA. Thus, to gain insight into the assembly of PSMA and the factors responsible for this assembly, we investigated if mutations at this TM motif will disrupt TM oligomerization. Our initial studies revealed that PSMA TMD has two repeat units of the Small-XXX-Small motif. This motif is known to be crucial in inducing a strong peptide self-assembly, analogous to the bitopic protein Glycophorin A. We synthesized the PSMA TMD mutant peptides using Biotage Alstra microwave peptide synthesizer, purified by high performance liquid chromatography, and characterized by MALDI-TOF mass spectrometry to establish homogeneity. SDS-PAGE gel shift assays revealed a direct correlation of oligomerization propensity and the presence of the motif, while motif knockdown led to observed loss of this property. ToxR reporter assay that qualitatively assesses relative TMD interactions revealed that oligomerization of PSMA TMD is ∼150% stronger than Glycophorin A. These findings will serve as a road map in the design of tight-binding exogenous anti-TMD peptides that target this oligomerization hot spot for possible diagnostic and therapeutic applications. Citation Format: Brianna S. Berg, Brandan M. Cook, Jack R. Hyder, James I. Godfroy, Hubert Yin, Jonel P. Saludes. The transmembrane domain of prostate specific membrane antigen: Deciphering the role of the Small-XXX-Small motif in oligomerization. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2456. doi:10.1158/1538-7445.AM2015-2456