N-terminus of pro-EMAP II regulates its binding with C-terminus, Arginyl-tRNA Synthetase, and Neurofilament light protein

Pro-EMAP II, one component of the MultiAminoacyl tRNA Synthetase (MSC) Complex, plays multiple roles in physiological and pathological processes of protein translation, signal transduction, immunity, lung development and tumor growth. Recent studies determined that pro-EMAP II has an essential role in maintaining axon integrity in central and peripheral neural systems where deletion of pro-EMAP II’s Cterminus was reported in a consanguineous Israeli Bedouin kindred suffering from PelizaeusMerzbacher-like disease. We hypothesized that pro-EMAP II’s N-terminus had an important role in the regulation of protein-protein interactions. Using a GFP reporter system, we defined a putative leucine-zipper in the N-terminus of human pro-EMAP II protein (amino acid residues 1-70), which can form specific strip-like punctate structures. Through GFP punctate analysis, we uncovered that pro-EMAP II’s C-terminus (147312 amino acid residues) can repress the GFP punctate formation. Pull-down assays confirmed the binding between pro-EMAP II N-terminus and its C-terminus is mediated by a putative leucine-zipper. Furthermore, the pro-EMAP II 170 aa region was identified as the binding partner of the arginyl-tRNA synthetase (RARS), a polypeptide of MSC complex. We also determined that the punctate GFP pro-EMAP II 1-70aa aggregate co-localizes and binds to the neurofilament light (NFL) subunit protein that is associated with pathologic neurofilament network disorganization and degeneration of motor neurons. These findings indicate the structure and binding interaction of Pro-EMAP II protein and suggest a role of this protein in the pathological neurodegenerative diseases. ___________________________________________ Endothelial Monocyte-Activating Peptide II (EMAP II) was initially isolated and identified as a 22kD proinflammatory cytokine secreted from a murine methylcholanthrene A-induced fibrosarcoma (1-4). Later studies confirmed that EMAP II was the Cterminus (147-312aa) of pro-EMAP II cleaved under apoptosis and by protease inhibitors (5-7). ProEMAP II, also known as p43, AIMP1 and Scye I, encodes one component of Multi-Aminoacyl tRNA Synthetase Complex (MSC) and is involved in protein translation. Mechanistically, the C-terminus of EMAP II has been determined to induce apoptosis in endothelial cells, inhibit angiogenesis and tumor growth through inhibition of vascular endothelial growth factor (VEGF) mediated signaling and http://www.jbc. org/cgi/doi/10.1074/jbc.M114.630533 The latest version is at JBC Papers in Press. Published on February 27, 2015 as Manuscript M114.630533 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. at Iniana U niersity Shool of M eicine on July 1, 2015 hp://w w w . jb.org/ D ow nladed from Putative Leucine-Zipper of EMAP II 2 alpha5beta1 integrin mediated deposition of the extracellular matrix protein fibronectin (8-10). Expression and secretion of EMAP II is relevant to prognosis of some cancer patients and found to be involved in diverse biological process such as lung development, immune responses and glucose metabolism in pancreas (11-15). These imply the importance of EMAP II in the diagnosis and treatment of cancer and other diseases (16-20). Intracellularly, pro-EMAP II directly interacts with other components of MSC such as p38, arginyl-tRNA synthetase (RARS) and glutaminyl-tRNA synthetase (QARS) (21-24). Such interaction plays an important role in the assembly and function of MSC. In addition, through C-terminus binding with E3 ubiquitin ligase Smurf2, pro-EMAP II stabilizes Smurf2 and down regulates the TGF-beta signaling pathway (25). Additional studies unveiled functional mechanisms and domains of pro-EMAP II protein (26,27). Park SG et al identified pro-EMAP II ’s Nterminus 4-46aa region to be important in promoting fibroblast cell proliferation and wound repair (28). Pro-EMAP II was also determined to bind with heat shock protein HSP90B1/gp96, which plays critical role in innate and adaptive immunity (16,29). Based on sequence analysis, Guo M et al (2010) (30) introduced a novel concept that during evolution, the N-terminus of pro-EMAP II was preserved due to evolutionary pressure and the C-terminus gained cytokine functions through the accumulation of mutations and this may be the reason for the diverse functions of EMAP II. Recently studies have determined some biological roles for pro-EMAP II. Zhu X et al (2009) (31) found that pro-EMAP II directly interacts with neurofilament light (NFL) chain and down-regulates neurofilament phosphorylation. Depletion of proEMAP II resulted in neurofilament network disorganization and degeneration of motor neurons. In 2010, pro-EMAP II truncation at the C-terminus was identified in consanguineous Israeli Bedouin kindred who suffered from Pelizaeus-Merzbacherlike disease (PMLD) (32,33) (34), indicating the requirement of further evaluation of the structure and functionality of pro-EMAP II in different biological processes. The role of pro-EMAP II in MSC, regulation of gene expression and modification, the interaction with other proteins and the functionality in different cell types requires further and detailed exploration. Here we established an approach to study the structure and function of pro-EMAP II using GFP punctate analysis and pull-down assay. We found that the N-terminus of pro-EMAP II can form specific strip like punctate structure. We characterized this structure as a putative leucinezipper of pro-EMAP II. We also determined that the C-terminus of pro-EMAP II could bind with its Nterminus to repress punctate structure formation, and co-localizes to the neurofilament light subunit protein. Furthermore, our studies indicate that the 170aa region in the N-terminus of pro-EMAP II is responsible for its binding to the MSC subunits. EXPERIMENTAL PROCEDURES Cells, reagents and antibodies: Human lung adenocarcinoma A549, Human Embryonic Kidney 293 and human neuroblastoma SH-SY5Y cells utilized in studies for neuronal function and differentiation. Cells were maintained in DMEM containing 10% fetal bovine serum and 5 mg/ml L-glutamine at 37C in 10% CO2. Human fetal kidney HEK293 cells were maintained in DMEM containing 5% fetal bovine serum at 37C in 5% CO2. GeneExpresso 8000 In Vitro DNA Transfection Reagent was purchased from InnoVita Incoporation, Gaithersburg, MD; Lipofectamine 2000 and Hoechst 33342 from Invitrogen, Carlsbad, CA; TransIT LT 1 from Mirus Bio, LLC, Madison, WI, PS-341 from Millenium Pharmaceuticals, Cambridge, MA; Bredfeldin A, B-1080 Bafilomycin A1 (Baf A1) and Chloroquine were from SigmaAldrich St. Louis, MO and Bio-Rad Protein Assay Dye Reagent Concentrate was from Bio-Rad Laboratories, Inc., Hercules, CA. Disuccinyl suberate (DSS) was purchased from Thermo Fisher Scientific Rockford, IL and BL21 DE3 strain was purchased from Stratagene (Cat.230255). Antibodies against GFP were purchased from Invitrogen (cat#A11122) and Santa Cruz Biotecnologies (Santa Cruz, CA) (cat#sc-9996). Antibody against EMAP II was raised from rabbit in our lab (ref). Antibodies against GM130 (sc-55590) and GST (sc-33613) were purchased from Santa Cruz. PCR cloning and detection: For cloning, PCR reactions were carried out using high fidelity DNA amplification kit (Sigma-Aldrich) containing 10 pmol of each primer and 10 ng of DNA template. For RT-PCR and real-time PCR, total RNA was isolated from cells using Trizol reagent. Reverse Transcription was carried out using Superscipt III RT-PCR System according to manufacture’s instruction (Invitrogen Corp). Oligonucleotides were at Iniana U niersity Shool of M eicine on July 1, 2015 hp://w w w . jb.org/ D ow nladed from Putative Leucine-Zipper of EMAP II 3 synthesized by Invitrogen and Sigma-Aldrich. Nterminus 1-102aa of RARS gene was amplified using oligos 5’AACGAATTCAACCATGGACGTACTGGTGTCT GAGT-3’ and 5’CTTGTCGACCACTAGCAGAGGAGGATTTTCC A-3’. N-terminus 1-108aa of human QARS gene was amplified using oligos 5’AACGAATTCAACCATGGCGGCTCTAGACTCC CTGT-3’ and