Abstract 694: Structure-function analysis of RPL18A, a putative binding target of rigosertib

Rigosertib (ON 01910.Na; RGS) is a clinical stage anticancer agent that causes spindle abnormalities and mitotic arrest in neoplastic cells. The drug inhibits PI3K and PLK1 signaling pathways, down regulates cyclin D1 expression and induces apoptosis. Previously, we reported identification of RPL18A (L18A), a protein from the large ribosomal subunit, as a putative binding target of RGS [Proc. AACR 2014, #4595]. Knock-down of L18A with siRNA caused apoptosis in cancer cell lines. Role of L18A in the function of the ribosome is not known. Goal of this study was to conduct structure-function analysis of L18A and create deficiency mutant(s) for future experiments. Human L18A is predicted to adopt the following linear order of secondary structural elements - alpha helices (a) and beta strands (b) - when assembled into the ribosome - b1b2a1b3b4b5b6a2a3b7a4 (from www.rcsb.org/pdb/protein/Q02543). The predicted 3D structure of L18A suggests overall bilobal architecture with a flanking C-terminal tail. Packed in tandem, each lobe contains one alpha helix surrounded by a beta sheet formed by 4 (N-lobe) or 3 (C-lobe) beta strands. To map the potential binding site for RGS, we engineered multiple mutants of L18A including truncations at its N and C termini. Our approach involved successive removal of the structural elements and a few substitutions with alanine. Mutant proteins were expressed in E.coli as GST fusions, and derivative cell lysates were tested in pull-down assay with biotin-...
Source: Cancer Research - Category: Cancer & Oncology Authors: Tags: Experimental and Molecular Therapeutics Source Type: research