A N-Terminal Domain in the Guanine Nucleotide Exchange Factor LEDP/132 Regulates ADP-Ribosylation Factor 1-Mediated Protections of Lens Epithelial Cells Against ER Stress

Abstract

Purpose: : ADP-ribosylation factors (ARFs) and their activating guanine nucleotide exchange factors (GEFs) play key roles in signaling. Previously, we have reported that Lens Epithelium-Derived Protein (LEDP/132) maintains Golgi and ER homeostasis. Herein, we demonstrated that N-terminal GEF domain of LEDP/132 is required for binding and activation of ARF1, and overexpression of LEDP/132 positively correlated with ARF1 activation and cell survival during oxidative/ER stress, and this activity in LECs was abrogated by LEDP/132 silencing.

Methods: : A full length of cDNA was cloned into prokaryotic (pGEX-5x-3) and eukaryotic (pGFP) expression vectors. LEDP/132-specific siRNA was constructed using pSilencer 4.1-CMV hygro vector. Deletion mutants linked to GFP/GST were engineered to determine functional motifs, and localization was ascertained using cell organelle specific markers and immunocytochemistry with LEDP/132 and organelle specific antibodies. Expression analysis of LEDP/132 in aging lenses or LECs was done by real-time PCR and Western blot using LEDP/132 specific probes. Cotransfection coupled with pull-down/coimmunoprecipitation assays determined interaction of N- or C-LEDP/132 and ARF1. ARF1 activation assay was done by commercial kit. Oxidative/ER stress was induced by UVB exposure, H₂O₂, tunicamycin and Brefeldin A (BFA) in cells over- or under-expressing LEDP/132. Brdu/MTS and TUNEL assays were used to define cell growth/survival and apoptosis, respectively.

Results: : LEDP/132 contained three coil-coil with ER (amino acid [aa],⁵²⁵KDEL⁵²⁸) and Golgi (aa,¹³⁶RRXXKKXXRR⁴³⁰) retention motifs and localized in Golgi, ER and transporting vesicles. Domain based deletion mutants of LEDP/132 revealed that N-terminal (aa, 1/610) localized in ER and Golgi and GEF domain (aa, 244/377) is responsible for binding and activation of ARF1 and promotion of GDP to GTP exchange. Cells overexpressing LEDP/132 or N-terminal-LEDP/132 had enhanced growth and resistance to oxidative/ER stressors by optimizing expression of Bip, a marker of ER stress. Overexpression of N-terminal or Full LEDP/132 was insensitive to BFA, suggesting that LEDP/132 is BFA resistant. Underexpression of LEDP/132 with siRNA significantly reduced cell survival and increased apoptosis following tunicamycin treatment. Expression analysis of LEDP/132 revealed lower expression in aging lenses/LECs.

Conclusions: : We identify a novel functional GEF domain, a BFA-resistant ARF1-GEF for LEDP/132, and show that expression level is essential for ARF1 activation-mediated survival signaling and protection against oxidative/ER stresses.

Keywords: protein purification and characterization • protein structure/function • protective mechanisms