Department of Biochemistry and Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada; Centre for Blood Research, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada.
Centre for Blood Research, University of British Columbia, 2350 Health Sciences Mall, Vancouver, British Columbia V6T 1Z3, Canada; Department of Oral Biological and Medical Sciences, University of British Columbia, 2199 Wesbrook Mall, Faculty of Dentistry, Vancouver, British Columbia V6T 1Z3, Canada.
J Biol Chem. 2019 Aug 30;294(35):12866-12879. doi: 10.1074/jbc.RA119.009584. Epub 2019 Jul 19.
Tryptophanyl-tRNA synthetase (WRS) is a cytosolic aminoacyl-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 γ (IFNγ). WRS signaled primarily through Toll-like receptor 2 (TLR2) in macrophages, leading to phosphorylation of the p65 subunit of NF-κB with associated loss of NF-κB inhibitor α (IκB-α) protein. This signaling initiated secretion of tumor necrosis factor α (TNFα) 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 TNFα-dependent 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 Met generated a WRS proteoform that also results from alternative splicing, designated Δ1-47 WRS. The MMP-cleaved WRS lacked TLR signaling and proinflammatory activities. Thus, our results suggest that moonlighting WRS promotes IFNγ proinflammatory activities, and these responses can be dampened by MMPs.
色氨酰-tRNA 合成酶（WRS）是细胞质中必需的氨酰-tRNA 合成酶，参与蛋白质合成。WRS 也是越来越多的具有独特非典型“月光”功能的细胞内蛋白质之一，在细胞外环境中发挥作用。月光氨基酸酰-tRNA 合成酶调节炎症等过程，但这些多功能酶本身是如何被调节的尚不清楚。在这里，我们证明 WRS 可被人巨噬细胞、成纤维细胞和内皮细胞在促炎细胞因子干扰素 γ（IFNγ）的刺激下分泌。WRS 主要通过巨噬细胞中的 Toll 样受体 2（TLR2）信号转导，导致 NF-κB 的 p65 亚基磷酸化，同时伴有 NF-κB 抑制剂 α（IκB-α）蛋白的丢失。这种信号转导启动了肿瘤坏死因子 α（TNFα）和 CXCL8（IL8）从巨噬细胞中的分泌。我们还证明 WRS 是一种有效的单核细胞趋化剂。值得注意的是，WRS 以 TNFα 依赖的方式增加了巨噬细胞条件培养基中的基质金属蛋白酶（MMP）活性。通过使用纯化的重组蛋白和 LC-MS/MS 鉴定蛋白水解切割位点，我们证明了多种 MMP，但主要是巨噬细胞 MMP7 和中性粒细胞 MMP8，在几个位点切割分泌的 WRS。在 Met 切割后失去 WHEP 结构域会产生一种 WRS 蛋白水解产物，该产物也来自选择性剪接，命名为 Δ1-47 WRS。MMP 切割的 WRS 缺乏 TLR 信号和促炎活性。因此，我们的研究结果表明，具有双重功能的 WRS 促进了 IFNγ 的促炎活性，而这些反应可以被 MMP 减弱。
