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-domaincontaining 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 pulldown 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.

UBXN2A enhances CHIP‐mediated proteasomal degradation of oncoprotein mortalin‐2 in cancer cells

“…Based on previous report (Lu et al ., ), we also examined the reduction in mot‐2 protein in UBXN2A‐induced cells in the presence of a chemotherapeutic agent, 5‐Fluorouracil (5‐FU). While the combination of UBXN2A expression induced by DOX and 5‐FU synergistically increased apoptosis events (Sane et al ., ), there was no further reduction in mot‐2 protein levels in combined treatment with DOX and 5‐FU (Fig. D).…”

“…In another previous study, we found that UBXN2A binds CHIP and facilitates its function as an E3 ubiquitin ligase (Teng et al ., ). Based on the above evidence, we hypothesized that UBXN2A as a ubiquitin‐like protein not only binds and interrupts the mot‐2 protein interaction network (Sane et al ., ) but also increases ubiquitination and proteasomal degradation of mot‐2 via the CHIP E3.…”

UBXD Proteins: A Family of Proteins with Diverse Functions in Cancer

“…UBXN2A binds to several E3 ubiquitin ligases, including CHIP (carboxyl terminus of Hsc70 interacting protein) ubiquitin E3 ligase [28]. Binding to different E3 ubiquitin ligases enables UBXN2A to regulate the stability of several diverse substrates in a cell- and tissue-dependent manner [28,41]. …”

“…The SEP domain of UBXN2A attaches to the protein binding pocket located within the SBD domain of mot-2 [41]. By binding to mot-2, UBXN2A unsequesters p53 tumor suppressor proteins, which leads to activation of the apoptotic pathway downstream of p53 [55].…”

“…Induction of UBXN2A induces cell apoptosis and reverses tumor progression in both in vitro and xenograft mouse models [56]. In addition, overexpression or silencing of UBXN2A increases response to conventional chemotherapy in cancer cells with enriched mot-2 [41]. Induction of UBXN2A shows minimal effect on normal cells because the large portion of mot-2 is located in mitochondria [55,56].…”