Christopher Mark Overall

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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Primarily controlled by gene expression and fine-tuned by translation and degradation rates, protein activity is governed by a plethora of post-translation modifications such as phosphorylation and glycosylation, which generate a diversity of protein species and thereby control complex biological phenotypes. Protease processing by proteases is a particular modification leading to the irreversible generation of stable protein truncations. Well understood in examples such as signal- or propeptide removal, recent analyses consistently identify >50% of N-terminal peptides mapping inside the protein sequence as predicted by genomics, indicating an important regulatory role of proteases. All proteins undergo protease cleavage as part of processing or degradation, a second biological process controlled by proteases. Proteases are involved in numerous pathologies and commonly considered as drug targets. However, protease research and drug development is complicated, in part due to widespread crosstalk between proteases. Proteases regulate other proteases through direct cleavage or cleavage of protease inhibitors in a complex network of protease interactions, the protease web. Yet, a comprehensive analysis of the protease web has not been performed, hampering assignment of proteases to clear biological roles, their direct substrates, and protease inhibitor drug targeting. A second problem in the identification of protein processing is the potential confound between protein termini generated by protease processing, alternative splicing, and alternative translation. In this thesis, I computationally analyzed large and diverse datasets of protease interactions and protein truncations to gain insight into complex proteolytic processes and to guide biochemical follow- up experiments. Analyzing protease cleavage, alternative splicing and alternative translation data incorporated into our database TopFIND, I found that protease cleavage and alternative translation likely generate most protein truncations. Combining protease cleavage and inhibition data in a graph model of the protease web, I demonstrated extensive protease crosstalk and then predicted and validated a proteolytic pathway. Finally, investigating strategies for the prediction of protease inhibition, I predicted hundreds of protease-inhibitor interactions, and validated inhibition of kallikrein-5 by serpin B12. This work thus generated predictions for biochemical follow-up as well as important insights into the regulation of biological systems through proteases.

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Recruitment of leukocytes is a hallmark feature of inflammation, as is the dissipation of the infiltrate for healing. Continual recruitment and leukocyte activation results in host tissue damage that is pathognomonic of chronic inflammatory disease. Matrix metalloproteases (MMPs) are an important family of endopeptidases that are elevated and associated with inflammatory diseases. Historically considered to promote cellular invasion by degrading components of the extracellular matrix, it is now recognized that MMPs specifically process bioactive molecules to alter the function of these proteins. A novel substrate discovery method identified that MMP truncation of the first 4 amino acid residues of the monocyte chemoattractant cytokine (chemokine) CCL7 produced a receptor antagonist that is capable of reversing the inflammatory response in vivo. Since this first discovery, nearly half of all chemokines have been identified as MMP substrates, resulting in products that promote and inhibit neutrophil recruitment, and inhibit monocyte recruitment and so implicating MMPs as major regulators of innate immunity.I hypothesized that MMP processing of select chemokines would promote monocyte recruitment, and that neutrophil-specific membrane-type (MT)6-MMP processes chemokines and other inflammatory mediators during neutrophil migration through the endothelium and stroma. To identify chemokines activated by MMP-processing, I systematically evaluated all monocyte attracting CC chemokines, finding that all are cleaved by at least one MMP. Moreover, in vitro functional assays showed that MMP processing of CCL16 increases glycosaminoglycan binding of the chemokine, whereas CCL15 and CCL23 products have enhanced agonist activity. To identify substrates of MT6-MMP, chemokine cleavage was evaluated in vitro, and the proteomics method terminal amino isotopic labeling of substrates (TAILS) was applied to soluble and membrane-associated human lung fibroblast and human microendothelial cell proteomes. 14 chemokines, as well as vimentin, insulin-like growth factor binding protein-7, cystatin C, and galectin-1 were confirmed to be substrates of MT6-MMP. I propose that MT6-MMP has pleiotropic roles in inflammation by potentiating and then inhibiting neutrophil recruitment, contributing to monocyte recruitment, and promoting wound healing. Contributing to our understanding of the roles of MMPs in inflammation, my work also suggests new modalities whereby perturbing MMP regulation promotes inflammatory disease.

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Inflammation is an essential process in wound healing and for the elimination of invadingpathogens. However, unregulated inflammation can lead to numerous pathologies includingautoimmunities, tumorigenesis, and atherosclerosis. Matrix metalloproteinases (MMPs),once thought to be only extracellular matrix degrading enzymes, are now known to be keyregulators of inflammatory and immune responses through proteolysis of bioactivemolecules. MMP-8, a neutrophil-specific MMP, is protective in skin cancer models whereMMP-8 knockout mice have an initial delay in neutrophil infiltration followed by a massiveaccumulation at the site of treatment. We investigated this delay in a murine air pouchmodel of acute inflammation, where MMP-8 deficiency caused decreased neutrophilmigration in response to LPS. This was attributed to MMP-8 processing and activation ofLPS-inducible CXC chemokine (LIX), a murine neutrophil chemoattractant. Indeed, MMP-8knockout mice had normal neutrophil infiltration in response to synthetic analogs of cleavedLIX. Furthermore, homologous pathways with human chemokines CXCL5 and CXCL8 weredescribed. In vivo, an indirect interaction between MMP-8 and LIX also occurs, wherebyMMP-8 processes and inactivates cLl-proteinase inhibitor causing increased neutrophilelastase activity, which then efficiently cleaves and activates LIX. MMP-8 was protective in amodel of rheumatoid arthritis where synovial tissues from MMP-8 deficient mice had anabundance of neutrophils. This prolonged neutrophil accumulation correlated with a loss ofcaspase-1 1 expression, consequent decreased caspase-3 activity and reduced apoptosis.MMP-8 shedding of TNF-a was also decreased in MMP-8 deficient leukocytes, potentiallydampening a key apoptotic pathway in neutrophils. The role of MMPs in processing the Thicell CXCR3-binding chemokines CXCL9, CXCL1O, and CXCL11 was investigated. Theleukocytic MMPs -7, -8, -9, and -12 cleaved CXCL11 at both the amino and carboxyterminus. N-terminal cleavage resulted in the conversion of a receptor agonist to antagonistwhereas C-terminal cleavage by MMP-8 caused a significant loss in glycosaminoglycanbinding, demonstrating for the first time that direct chemokine proteolysis can regulate theformation of haptotactic gradients. Therefore, MMP-8 is a pivotal regulator in the onset andtermination of inflammation, and has multifaceted roles in innate and acquired immunity aswell as the autoimmune disorder rheumatoid arthritis.

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