title: Structural studies of UBXN2A and mortalin interaction and the putative role of silenced UBXN2A in preventing response to chemotherapy.
abstract: Overexpression of the oncoprotein mortalin in cancer cells and its protein partners enables mortalin to promote multiple oncogenic signaling pathways and effectively antagonize chemotherapy-induced cell death. A UBX-domain-containing protein, UBXN2A, acts as a potential mortalin inhibitor. This current study determines whether UBXN2A effectively binds to and occupies mortalin's binding pocket, resulting in a direct improvement in the tumor's sensitivity to chemotherapy. Molecular modeling of human mortalin's binding pocket and its binding to the SEP domain of UBXN2A followed by yeast two-hybrid and His-tag pull-down assays revealed that three amino acids (PRO442, ILE558, and LYS555) within the substrate-binding domain of mortalin are crucial for UBXN2A binding to mortalin. As revealed by chase experiments in the presence of cycloheximide, overexpression of UBXN2A seems to interfere with the mortalin-CHIP E3 ubiquitin ligase and consequently suppresses the C-terminus of the HSC70-interacting protein (CHIP)-mediated destabilization of p53, resulting in its stabilization in the cytoplasm and upregulation in the nucleus. Overexpression of UBXN2A causes a significant inhibition of cell proliferation and the migration of colon cancer cells. We silenced UBXN2A in the human osteosarcoma U2OS cell line, an enriched mortalin cancer cell, followed by a clinical dosage of the chemotherapeutic agent 5-fluorouracil (5-FU). The UBXN2A knockout U2OS cells revealed that UBXNA is essential for the cytotoxic effect achieved by 5-FU. UBXN2A overexpression markedly increased the apoptotic response of U2OS cells to the 5-FU. In addition, silencing of UBXN2A protein suppresses apoptosis enhanced by UBXN2A overexpression in U2OS. The knowledge gained from this study provides insights into the mechanistic role of UBXN2A as a potent mortalin inhibitor and as a potential chemotherapy sensitizer for clinical application.
authors.affiliation: Division of Basic Biomedical Sciences, Sanford School of Medicine, The University of South Dakota, 414 E. Clark Street, Lee Medical Building, Vermillion, SD, 57069, USA.
authors.forename: Sanam
authors.initials: S
authors.lastname: Sane
authors.affiliation: Division of Basic Biomedical Sciences, Sanford School of Medicine, The University of South Dakota, 414 E. Clark Street, Lee Medical Building, Vermillion, SD, 57069, USA.
authors.forename: Ammara
authors.initials: A
authors.lastname: Abdullah
authors.affiliation: Division of Basic Biomedical Sciences, Sanford School of Medicine, The University of South Dakota, 414 E. Clark Street, Lee Medical Building, Vermillion, SD, 57069, USA.
authors.forename: Morgan E
authors.initials: ME
authors.lastname: Nelson
authors.affiliation: Division of Basic Biomedical Sciences, Sanford School of Medicine, The University of South Dakota, 414 E. Clark Street, Lee Medical Building, Vermillion, SD, 57069, USA.
authors.forename: Hongmin
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Pharmaceutical Sciences and Center for Cancer Research, University of Tennessee Health Science Center, Memphis, TN, USA.
authors.forename: Subhash C
authors.initials: SC
authors.lastname: Chauhan
authors.affiliation: Division of Basic Biomedical Sciences, Sanford School of Medicine, The University of South Dakota, 414 E. Clark Street, Lee Medical Building, Vermillion, SD, 57069, USA.
authors.forename: Samuel S
authors.initials: SS
authors.lastname: Newton
authors.affiliation: Division of Basic Biomedical Sciences, Sanford School of Medicine, The University of South Dakota, 414 E. Clark Street, Lee Medical Building, Vermillion, SD, 57069, USA. khosrow.rezvani@usd.edu.
authors.forename: Khosrow
authors.initials: K
authors.lastname: Rezvani
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001665:Binding Sites; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D005472:Fluorouracil / Q000494:pharmacology*; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D062105:Molecular Docking Simulation; D009369:Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D012516:Osteosarcoma / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D011485:Protein Binding; D000072417:Protein Domains; D034622:RNA Interference; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D014452:Ubiquitins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000964:Antimetabolites, Antineoplastic; D018840:HSP70 Heat-Shock Proteins; D016159:Tumor Suppressor Protein p53; C000609706:UBXN2A protein, human; D014452:Ubiquitins; C080352:mortalin; D005472:Fluorouracil
keywords: Chemotherapy; Colon cancer; Mortalin; Osteosarcoma; UBXN2A; p53