Tryptophanyl-tRNA synthetase (WRS) and tyrosyl-tRNA synthetase (YRS) ligate their cargo amino acids (Trp and Tyr, respectively) to their cognate tRNAs in the cytoplasm and, surprisingly, are secreted through a non-canonical pathway. WRS and YRS are also two of a growing number of intracellular proteins that are attributed distinct non-canonical “moonlighting” functions in the extracellular milieu. Specifically, aminoacyl-tRNA synthetases regulate several processes such as inflammation, but how these multifunctional enzymes are themselves regulated remains unclear.
Using secretion assays, purified recombinant proteins, LC–MS/MS to identify proteolytic cleavage sites, and TLR-reporter cells, we show that WRS and YRS are secreted from human macrophages, fibroblasts, and endothelial cells in response to proinflammatory cytokine interferon γ initiated loss of NF-kB inhibitor α(IkBα) protein. This initiated secretion of tumor necrosis factor α (TNFα),MIP-1a/b, CXCL8 (IL8), and CXCL1 (KC) from THP1 monocyte and PBMC-derived macrophages.
Of note, both WRS and YRS increased matrix metalloproteinase (MMP) activity in the conditioned medium of macrophages in a TNFa-dependent manner. Since MMPs process a variety of intracellular proteins that also exhibit extracellular moonlighting functions, we profiled 10 recombinant MMPs we expressed forcleavage of WRS and YRS.We identified 55 MMP cleavage sites in YRS by ATOMS positional proteomics and Edman degradation. Stable proteoforms resulted from cleavages near the start of the YRS C-terminal EMAPII domain: All of the MMPs tested cleaved at ADS386↓387LYV and VSG405↓406LVQ, generating 43-kDa and 45-kDa fragments, butmacrophage MMP7 and neutrophil MMP8showed the greatest activity. Notably, MMP-cleaved YRS enhancedTLR2 signaling, increased TNFasecretion from macrophages, and amplified monocyte/macrophage chemotaxis compared to unprocessed YRS.
In contrast, MMP cleavage of WRS occurred at several sites and with the loss of the WHEP domain following cleavage at 48Met, generated a WRS proteoform that also results from alternative splicing. Designated δ1-47 WRS, the MMP-cleaved WRS lackedTLR signalling and proinflammatory activities.
Overall, the proinflammatory activity of YRS is enhanced by MMP cleavage forming a feed-forward mechanism that enhances inflammation whereas inflammatory MMP cleavage of WRS then dampens inflammation. Thus, simply identifying proteins in cells or the extracellular environment without knowledge of cleavage status and N-termini leads to lack of critical functional information.