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Moonlighting tRNA synthetases as extracellular targets of matrix metalloproteinases Jobin, Parker


Protease activity is of particular interest because of its irreversible nature and hence commitment by living systems to post-translationally truncate or remove by degradation its substrates. Degradomics, a combination of approaches used to study proteases, their inhibitors, and their substrates, allows powerful analyses of proteolytic networks. Degradomics has allowed the identification of a vast number of novel protease substrates, leading to speculation of molecular partnerships previously unknown to biology. For the matrix metalloproteinase (MMP) family of extracellular proteases, degradomic screens have led us to realize that many proteins with intracellular roles are secreted by non-canonical means to perform novel extracellular functions that may be modulated by MMPs. Where it was once thought MMPs only degraded extracellular matrix (ECM), they are now known to process diverse signaling substrates. Interesting multifunctional targets of MMP processing are “moonlighting” proteins that have more than one unique activity and can shuttle between intracellular and extracellular compartments to exhibit different functions in each. Recently, intracellular tRNA synthetases have been identified as extracellular moonlighting proteins. Despite the lack of signal peptides, six tRNA synthetases have been found to be secreted and perform different functions in the extracellular environment, notably activation of the immune response. I hypothesized that MMP processing of tryptophanyl-tRNA synthetase (WRS), a cytokine, and tyrosyl-tRNA synthetase (YRS), fragments of which are proinflammatory, would modify the inflammatory activities of these tRNA synthetases. First, WRS and YRS secretion from human cells was confirmed. I then expressed and purified full-length WRS and YRS to evaluate MMP processing of these proteins. MMP cleavage sites within WRS and YRS were determined, revealing that MMPs cut the N-terminus from WRS but cleave within the YRS C-terminus, generating stable proteoforms. Cell culture assays revealed that both WRS and YRS have proinflammatory functions, each activating Toll-like receptors (TLRs). While removal of the N-terminus of WRS by MMP processing attenuated these activities, conversely, MMP cleavage of YRS increased proinflammatory functions, suggesting that MMPs play differing roles depending on the substrate being processed. This research exposes the exciting biology that awaits in tapping a previously unknown well of moonlighting MMP substrates with diverse bioactive roles.

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