Tryptophanyl-tRNA synthetase (WRS) is a cytosolic amino-acyl-tRNA synthetase essential for protein synthesis. WRS is also one of a growing number of intracellular proteins that are attributed distinct noncanonical “moonlighting” functions in the extracellular milieu. Moonlighting aminoacyl-tRNA synthetases regulate processes such as inflammation, but how these multifunctional enzymes are themselves regulated remains unclear. Here, we demonstrate that WRS is secreted from human macrophages, fibroblasts, and endothelial cells in response to the proinflammatory cytokine interferon (IFN). WRS signaled primarily through Toll-like receptor 2 (TLR2) in macrophages, leading to phosphorylation of the p65 subunit of NF-B with associated loss of NF-B inhibitor (IB-) protein. This signaling initiated secretion of tumor necrosis factor (TNF) and CXCL8 (IL8) from macrophages. We also demonstrated that WRS is a potent monocyte chemoattractant. Of note, WRS increased matrix metalloproteinase (MMP) activity in the conditioned medium of macrophages in a TNF-depen-dent manner. Using purified recombinant proteins and LC-MS/MS to identify proteolytic cleavage sites, we demonstrated that multiple MMPs, but primarily macrophage MMP7 and neutrophil MMP8, cleave secreted WRS at several sites. Loss of the WHEP domain following cleavage at Met48 generated a WRS proteoform that also results from alternative splicing, designated 1- 47 WRS. The MMP-cleaved WRS lacked TLR signaling and proinflammatory activities. Thus, our results suggest that moonlighting WRS promotes IFN proinflammatory activities, and these responses can be dampened by MMPs.
Bibliographical noteFunding Information:
1Supported by a Vancouver Coastal Health Research Institute (VCHRI)-CIHR-University of British Columbia (UBC) M.D./Ph.D. studentship, CIHR Vanier Canada graduate scholarship, UBC 4-year doctoral fellowship, and a Centre for Blood Research graduate award.
This work was supported by Canadian Institutes of Health Research (CIHR) Grant MOP-111055 (to C. M. O. and G. S. B.), CIHR Foundation Grant FDN-148408 (to C. M. O.), Michael Smith Foundation for Health Research (MSFHR) Grant IN-NPG-00105 to establish the British Columbia Proteomics Network (to C. M. O.), and Canada Foundation for Innovation Grant 31059 (to C. M. O.). The authors declare that they have no conflicts of interest with the contents of this article.
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All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology