Abstract
Tyrosyl-tRNA synthetase ligates tyrosine to its cognate tRNA in the cytoplasm, but it can also be secreted through a noncanonical pathway. We found that extracellular tyrosyl-tRNA synthetase (YRS) exhibited proinflammatory activities. In addition to acting as a monocyte/macrophage chemoattractant, YRS initiated signaling through Toll-like receptor 2 (TLR2) resulting in NF-κB activation and release of tumor necrosis factor α (TNFα) and multiple chemokines, including MIP-1α/β, CXCL8 (IL8), and CXCL1 (KC) from THP1 monocyte and peripheral blood mononuclear cell–derived macrophages. Furthermore, YRS up-regulated matrix metalloproteinase (MMP) activity in a TNFα-dependent manner in M0 macrophages. Because MMPs process a variety of intracellular proteins that also exhibit extracellular moonlighting functions, we profiled 10 MMPs for YRS cleavage and identified 55 cleavage sites by amino-terminal oriented mass spectrometry of substrates (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- and 45-kDa fragments. The highest catalytic efficiency for YRS was demonstrated by MMP7, which is highly expressed by monocytes and macrophages, and by neutrophil-specific MMP8. MMP-cleaved YRS enhanced TLR2 signaling, increased TNFα secretion from macrophages, and amplified monocyte/macrophage chemotaxis compared with unprocessed YRS. The cleavage of YRS by MMP8, but not MMP7, was inhibited by tyrosine, a substrate of the YRS aminoacylation reaction. Overall, the proinflammatory activity of YRS is enhanced by MMP cleavage, which we suggest forms a feed-forward mechanism to promote inflammation.
Highlights
In the cytoplasm, but it can be secreted through a noncanonical pathway
We localized the major MMP7 and MMP8 cleavage sites on the 3D structure of YRS that we modeled from two partial YRS structures, the N-terminal Rossmann fold catalytic and anticodon recognition domains, which occur as a dimer [29, 67], and the isolated endothelial monocyte-activating polypeptide II-like (EMAPII)-like C-terminal domain (Fig. 10C) [66]
matrix metalloproteinase (MMP) cleavage of YRS was identified in murine skin inflammation from our earlier terminal amine isotopic labeling of substrates (TAILS) proteomic analyses of inflamed versus normal skin in WT versus Mmp2Ϫ/Ϫ mice [45], murine Mmp2Ϫ/Ϫ fibroblasts [40], and murine Mmp12Ϫ/Ϫ macrophage secretomes [6] with added MMP2 or MMP12
Summary
The recruitment of monocytes is a key component of the inflammatory response [54]. We expressed and purified human YRS in Escherichia coli ClearColi BL21 (DE3) [55, 56]. Treatment of THP1 M0 macrophages with recombinant YRS for 3 h induced the release of TNF␣ into the medium (N ϭ 2; Fig. 1C). This effect was maximal at 50 nM YRS. A TLR2-neutralizing antibody and TLR2 pathway inhibitors blocked NF-B activation in the HEK293 TLR2 reporter cells (N ϭ 2; Fig. 2, E and F) We used these antibodies and inhibitors to demonstrate that YRS initiates NF-B signaling through TLR2 in macrophages. Blocking either the extracellular domain of TLR2 with an ␣TLR2 antibody or interfering with intracellular components of the NF-B–signaling pathway using chemical inhibitors disrupted YRS-induced TNF␣ secretion, demonstrating that TLR2/NF-B mediates this proinflammatory response to YRS in human macrophages. Incubation led to a strong reduction in the intensity of the 62-kDa
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