Neurofibromatosis type 1 (NF1) is an autosomal dominant disease that predisposes individuals to developing benign neurofibromas and malignant peripheral nerve sheath tumors (MPNST). The mechanism of NF1-tumoigenesis or the curatives have not been established. Using iPEACH (Kobayashi et al, MCP 2013), we previously found that translationally controlled tumor protein (TCTP) is a novel biological target for NF1-associated tumors (Kobayashi et al. JBC 2014), and the complex formation of TCTP and protein elongation factors (EF1A2 and GEF proteins for EF1A2) was identified as a translational regulator specific in NF1 associated tumors (Kobayashi et al, MCP 2019). In this study, cross-linking mass spectrometry combined with affinity purification (AP) using FLAG-antibody was utilized to analyze the structural insight in the complex of TCTP and elongation factors.
For AP analysis, FLAG-tagged TCTP and EF1A2 were overexpressed into Hela cells, and the Flag-TCTP- and Flag-EF1A2-binding proteins were co-immunoprecipitated using magnetic beads coupled with FLAG-antibody. These proteins were treated with cross-linker disuccinimidyl sulfoxide (DSSO) and digested with trypsin on beads. The tryptic cross-linked peptides were analyzed by XlinkX workflow (Lui et al. Nat Methods 2015) using nanoLC-Orbitrap Fusion ETD system.
Cross-linking mass spectrometry analyses specifically identified the 76 and 140 DSSO-crosslinked peptides in TCTP- and EF1A2-FLAG-IP fractions (FDR < 1% and XlinkX score > 20). Totally, eleven peptides crosslinked in the complex of TCTP-EF1A2-elongation factors were identified with high confidence. Among them, the crosslinking between K19 in TCTP and K41/K44 in EF1A2 was observed. These data suggest that TCTP inhibits the EF1A2 homodimer formation and mediates the GDP/GTP exchange reaction on EF1A2 via the direct binding to EF1A2 near the GTP-binding site.
These results could provide a novel treatment strategy for the NF1-associated tumors targeting the translational machinery driven by the specific TCTP-EF1A2 interaction.