Many snake venom toxins are serine proteases but their specific in vivo targets are mostly unknown. Various act on components of the coagulation cascade, and fibrinolytic and kallikrein-kinin systems to trigger various pathological effects observed in the envenomation. Despite showing high similarity in terms of primary structure snake venom serine proteinases (SVSPs) show exquisite specificity towards macromolecular substrates. Therefore, the characterization of their peptide bond specificity is important for understanding the active site preference associated with effective proteolysis as well as for the design of peptide substrates and inhibitors. Bothrops jararaca contains various SVSPs among which Bothrops protease A is a specific fibrinogenolytic agent and PA-BJ is a platelet-activating enzyme. In this study we used proteome derived peptide libraries in the Proteomic Identification of protease Cleavage Sites (PICS) approach to explore the peptide bond specificity of Bothrops protease A and PA-BJ in order to determine their individual peptide cleavage sequences. A total of 371 cleavage sites (208 for Bothrops protease A and 163 for PA-BJ) were detected and both proteinases displayed a clear preference for arginine at the P1 position. Moreover, the analysis of the specificity profiles of Bothrops protease A and PA-BJ revealed subtle differences in the preferences along P6-P6', despite a common yet unusual preference for Pro at P2. Taken together, these results map the subsite specificity of both SVSPs and shed light in the functional differences between these proteinases.

The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)-the constitutive α-secretase-is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin β, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin(-/-) mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.

Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6' in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1' (with a ~32-93% preference for leucine depending on the MMP), and basic and small residues in P2' and P3', respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1' leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and non-prime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. These caveats make it critical to check for these biologically highly important effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1'-Leu only 5 shared both features, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced non-prime side flexibility constraining accommodation of the rigidifying P3 proline with leucine locked in S1'. Similar negative cooperativity between P3 proline and the novel preference for asparagine in P1 cements our conclusion that non-prime side flexibility greatly impacts MMP binding affinity and cleavage efficiency. Thus, unexpected sequence cooperativity consequences were revealed by PICS that uniquely encompasses both the non-prime and prime sides flanking the proteomic-pinpointed scissile bond.

Controlled macrophage differentiation and activation in the initiation and resolution of inflammation is crucial for averting progression to chronic inflammatory and autoimmune diseases. Here we show a negative feedback mechanism for proinflammatory IFN-γ activation of macrophages driven by macrophage-associated matrix metalloproteinase 12 (MMP12). Through C-terminal truncation of IFN-γ at 135Glu↓Leu136 the IFN-γ receptor-binding site was efficiently removed thereby reducing JAK-STAT1 signaling and IFN-γ activation of proinflammatory macrophages. In acute peritonitis this signature was absent in Mmp12 -/- mice and recapitulated in Mmp12 +/+ mice treated with a MMP12-specific inhibitor. Similarly, loss-of-MMP12 increases IFN-γ-dependent proinflammatory markers and iNOS+/MHC class II+ macrophage accumulation with worse lymphadenopathy, arthritic synovitis and lupus glomerulonephritis. In active human systemic lupus erythematosus, MMP12 levels were lower and IFN-γ higher compared to treated patients or healthy individuals. Hence, macrophage proteolytic truncation of IFN-γ attenuates classical activation of macrophages as a prelude for resolving inflammation.

Agrin is the key inducer of postsynaptic differentiations at the neuromuscular junction. The multidomain heparan sulfate proteoglycan is mediating via its N-terminal segment the interaction with laminin, whereas the C-terminal portion is responsible for Dystroglycan binding and clustering of the Acetylcholine receptor. Matrix metalloproteinases (MMP) are known to play essential roles in matrix remodeling, degradation and regulation of extracellular signaling networks.

Tumor growth and metastasis are determined by the complex interplay of factors, including those intrinsic to tumor cells and extrinsic factors associated with the tumor microenvironment. Our previous work demonstrated key roles for CD34 in the maintenance of vascular integrity and eosinophil and mast cell homing. Since both of these functions affect tumor development, we characterized the effect of CD34 ablation on tumor growth using the B16F1 melanoma model. Intriguingly, we found that CD34 plays a biphasic role in tumor progression. In early growth, both subcutaneous-injected tumors and intravenous-injected lung metastases grew more slowly in Cd34(-/-) mice. This correlated with abnormal vessel morphology and increased vascular permeability in these mice. Bone marrow transplantation experiments confirmed that this reflects a non-hematopoietic function of CD34. At later stages, subcutaneous tumor growth was accelerated in Cd34(-/-) mice and surpassed growth in wildtype mice. Bone marrow chimera experiments demonstrated this difference was due to a hematopoietic function for CD34 and, correspondingly we found reduced intra-tumor mast cell numbers in Cd34(-/-) mice. In aggregate, our analysis reveals a novel role for CD34 in both early and late tumor growth and provides novel insights into the role of the tumor microenvironment in tumor progression.

The neutrophil-specific protease membrane-type 6 matrix metalloproteinase (MT6-MMP)/MMP-25/leukolysin is implicated in multiple sclerosis and cancer yet remains poorly characterized. To characterize the biological roles of MT6-MMP, it is critical to identify its substrates for which only seven are currently known. Here, we biochemically characterized MT6-MMP, profiled its tissue inhibitor of metalloproteinase inhibitory spectrum, performed degradomics analyses, and screened 26 chemokines for cleavage using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. MT6-MMP processes seven each of the CXC and CC chemokine subfamilies. Notably, cleavage of the neutrophil chemoattractant CXCL5 activates the chemokine, thereby increasing its agonist activity, indicating a feed-forward mechanism for neutrophil recruitment. Likewise, cleavage also activated CCL15 and CCL23 to increase monocyte recruitment. Utilizing the proteomics approach proteomic identification of cleavage site specificity (PICS), we identified 286 peptidic cleavage sites spanning from P6 to P6' from which an unusual glutamate preference in P1 was identified. The degradomics screen terminal amine isotopic labeling of substrates (TAILS), which enriches for neo-N-terminal peptides of cleaved substrates, was used to identify 58 new native substrates in fibroblast secretomes after incubation with MT6-MMP. Vimentin, cystatin C, galectin-1, IGFBP-7, and secreted protein, acidic and rich in cysteine (SPARC) were among those substrates we biochemically confirmed. An extracellular "moonlighting" form of vimentin is a chemoattractant for THP-1 cells, but MT6-MMP cleavage abolished monocyte recruitment. Unexpectedly, the MT6-MMP-cleaved vimentin potently stimulated phagocytosis, which was not a property of the full-length protein. Hence, MT6-MMP regulates neutrophil and monocyte chemotaxis and by generating "eat-me" signals upon vimentin cleavage potentially increases phagocytic removal of neutrophils to resolve inflammation.

Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses using iTRAQ-TAILS links gelatinases with new mechanisms of action in angiogenesis and reveals unpredicted restrictions in substrate repertoires for these two very similar proteases.

We developed a bioinformatics-led substrate discovery workflow to expand the known substrate repertoire of MALT1. Our approach, termed GO-2-Substrates, integrates protein function information, including GO terms from known substrates, with protein sequences to rank substrate candidates by similarity. We applied GO-2-Substrates to MALT1, a paracaspase and master regulator of NF-κB signalling in adaptive immune responses. With only 12 known substrates, the evolutionarily conserved paracaspase functions and phenotypes of Malt1 -/- mice strongly implicate the existence of undiscovered substrates. We tested the ranked predictions from GO-2-Substrates of new MALT1 human substrates by co-expression of candidates transfected with the oncogenic constitutively active cIAP2-MALT1 fusion protein or CARD11/BCL10/MALT1 active signalosome. We identified seven new MALT1 substrates by the co-transfection screen: TANK, TAB3, CASP10, ZC3H12D, ZC3H12B, CILK1 and ILDR2. Using catalytically inactive cIAP2-MALT1 (Cys464Ala), a MALT1 inhibitor, MLT-748, and noncleavable P1-Arg to Ala mutant versions of each substrate in dual transfections, we validated the seven new substrates in vitro. We confirmed the cleavage of endogenous TANK and the RNase ZC3H12D in B cells by Western blotting and mining TAILS N-terminomics datasets, where we also uncovered evidence for these and 12 other candidate substrates by endogenous MALT1. Thus, protein function information improves substrate predictions. The new substrates and other high-ranked MALT1 candidate substrates should open new biological frontiers for further validation and exploration of the function of MALT1 within and beyond NF-κB regulation.

Viruses have evolved diverse strategies to evade the host innate immune response and promote infection. The retinoic acid-inducible gene I (RIG-I)-like receptors RIG-I and MDA5 are antiviral factors that sense viral RNA and trigger downstream signal via mitochondrial antiviral-signaling protein (MAVS) to activate type I interferon expression. 14-3-3ε is a key component of the RIG-I translocon complex that interacts with MAVS at the mitochondrial membrane; however, the exact role of 14-3-3ε in this pathway is not well understood. In this study, we demonstrate that 14-3-3ε is a direct substrate of both the poliovirus and coxsackievirus B3 (CVB3) 3C proteases (3Cpro) and that it is cleaved at Q236↓G237, resulting in the generation of N- and C-terminal fragments of 27.0 and 2.1 kDa, respectively. While the exogenous expression of wild-type 14-3-3ε enhances IFNB mRNA production during poly(I:C) stimulation, expression of the truncated N-terminal fragment does not. The N-terminal 14-3-3ε fragment does not interact with RIG-I in co-immunoprecipitation assays, nor can it facilitate RIG-I translocation to the mitochondria. Probing the intrinsically disordered C-terminal region identifies key residues responsible for the interaction between 14-3-3ε and RIG-I. Finally, overexpression of the N-terminal fragment promotes CVB3 infection in mammalian cells. The strategic enterovirus 3Cpro-mediated cleavage of 14-3-3ε antagonizes RIG-I signaling by disrupting critical interactions within the RIG-I translocon complex, thus contributing to evasion of the host antiviral response. IMPORTANCE Host antiviral factors work to sense virus infection through various mechanisms, including a complex signaling pathway known as the retinoic acid-inducible gene I (RIG-I)-like receptor pathway. This pathway drives the production of antiviral molecules known as interferons, which are necessary to establish an antiviral state in the cellular environment. Key to this antiviral signaling pathway is the small chaperone protein 14-3-3ε, which facilitates the delivery of a viral sensor protein, RIG-I, to the mitochondria. In this study, we show that the enteroviral 3C protease cleaves 14-3-3ε during infection, rendering it incapable of facilitating this antiviral response. We also find that the resulting N-terminal cleavage fragment dampens RIG-I signaling and promotes virus infection. Our findings reveal a novel viral strategy that restricts the antiviral host response and provides insights into the mechanisms underlying 14-3-3ε function in RIG-I antiviral signaling.

Left bundle branch area pacing (LBBAP) is a novel approach for cardiac resynchronization therapy (CRT), but the impact of myocardial substrate on its effect is poorly understood. This study aims to assess the association of cardiac magnetic resonance (CMR)-derived scar burden and the response of CRT via LBBAP.

The data described provide a comprehensive resource for the family-wide active site specificity portrayal of the human matrix metalloproteinase family. We used the high-throughput proteomic technique PICS (Proteomic Identification of protease Cleavage Sites) to comprehensively assay 9 different MMPs. We identified more than 4300 peptide cleavage sites, spanning both the prime and non-prime sides of the scissile peptide bond allowing detailed subsite cooperativity analysis. The proteomic cleavage data were expanded by kinetic analysis using a set of 6 quenched-fluorescent peptide substrates designed using these results. These datasets represent one of the largest specificity profiling efforts with subsequent structural follow up for any protease family and put the spotlight on the specificity similarities and differences of the MMP family. A detailed analysis of this data may be found in Eckhard et al. (2015) [1]. The raw mass spectrometry data and the corresponding metadata have been deposited in PRIDE/ProteomeXchange with the accession number PXD002265.

Intestinal tumorigenesis is a result of mutations in signaling pathways that control cellular proliferation, differentiation, and survival. Mutations in the Wnt/β-catenin pathway are associated with the majority of intestinal cancers, while dysregulation of the Hippo/Yes-Associated Protein (YAP) pathway is an emerging regulator of intestinal tumorigenesis. In addition, these closely related pathways play a central role during intestinal regeneration. We have previously shown that methylation of the Hippo transducer YAP by the lysine methyltransferase SETD7 controls its subcellular localization and function. We now show that SETD7 is required for Wnt-driven intestinal tumorigenesis and regeneration. Mechanistically, SETD7 is part of a complex containing YAP, AXIN1, and β-catenin, and SETD7-dependent methylation of YAP facilitates Wnt-induced nuclear accumulation of β-catenin. Collectively, these results define a methyltransferase-dependent regulatory mechanism that links the Wnt/β-catenin and Hippo/YAP pathways during intestinal regeneration and tumorigenesis.

Antigen receptor signalling activates the canonical NF-κB pathway via the CARD11/BCL10/MALT1 (CBM) signalosome involving key, yet ill-defined roles for linear ubiquitination. The paracaspase MALT1 cleaves and removes negative checkpoint proteins, amplifying lymphocyte responses in NF-κB activation and in B-cell lymphoma subtypes. To identify new human MALT1 substrates, we compare B cells from the only known living MALT1(mut/mut) patient with healthy MALT1(+/mut) family members using 10-plex Tandem Mass Tag TAILS N-terminal peptide proteomics. We identify HOIL1 of the linear ubiquitin chain assembly complex as a novel MALT1 substrate. We show linear ubiquitination at B-cell receptor microclusters and signalosomes. Late in the NF-κB activation cycle HOIL1 cleavage transiently reduces linear ubiquitination, including of NEMO and RIP1, dampening NF-κB activation and preventing reactivation. By regulating linear ubiquitination, MALT1 is both a positive and negative pleiotropic regulator of the human canonical NF-κB pathway-first promoting activation via the CBM--then triggering HOIL1-dependent negative-feedback termination, preventing reactivation.

Clostridial collagenases are among the most efficient degraders of collagen. Most clostridia are saprophytes and secrete proteases to utilize proteins in their environment as carbon sources; during anaerobic infections, collagenases play a crucial role in host colonization. Several medical and biotechnological applications have emerged utilizing their high collagenolytic efficiency. However, the contribution of the functionally most important peptidase domain to substrate specificity remains unresolved. We investigated the active site sequence specificity of the peptidase domains of collagenase G and H from Clostridium histolyticum and collagenase T from Clostridium tetani. Both prime and non-prime cleavage site specificity were simultaneously profiled using Proteomic Identification of protease Cleavage Sites (PICS), a mass spectrometry-based method utilizing database searchable proteome-derived peptide libraries. For each enzyme we identified >100 unique-cleaved peptides, resulting in robust cleavage logos revealing collagen-like specificity patterns: a strong preference for glycine in P3 and P1', proline at P2 and P2', and a slightly looser specificity at P1, which in collagen is typically occupied by hydroxyproline. This specificity for the classic collagen motifs Gly-Pro-X and Gly-X-Hyp represents a remarkable adaptation considering the complex requirements for substrate unfolding and presentation that need to be fulfilled before a single collagen strand becomes accessible for cleavage.

Members of the species Rickettsia are obligate intracellular, gram-negative, arthropod-borne pathogens of humans and other mammals. The life-threatening character of diseases caused by many Rickettsia species and the lack of reliable protective vaccine against rickettsioses strengthens the importance of identifying new protein factors for the potential development of innovative therapeutic tools. Herein, we report the identification and characterization of a novel membrane-embedded retropepsin-like homologue, highly conserved in 55 Rickettsia genomes. Using R. conorii gene homologue RC1339 as our working model, we demonstrate that, despite the low overall sequence similarity to retropepsins, the gene product of rc1339 APRc (for Aspartic Protease from Rickettsia conorii) is an active enzyme with features highly reminiscent of this family of aspartic proteases, such as autolytic activity impaired by mutation of the catalytic aspartate, accumulation in the dimeric form, optimal activity at pH 6, and inhibition by specific HIV-1 protease inhibitors. Moreover, specificity preferences determined by a high-throughput profiling approach confirmed common preferences between this novel rickettsial enzyme and other aspartic proteases, both retropepsins and pepsin-like. This is the first report on a retropepsin-like protease in gram-negative intracellular bacteria such as Rickettsia, contributing to the analysis of the evolutionary relationships between the two types of aspartic proteases. Additionally, we have also shown that APRc is transcribed and translated in R. conorii and R. rickettsii and is integrated into the outer membrane of both species. Finally, we demonstrated that APRc is sufficient to catalyze the in vitro processing of two conserved high molecular weight autotransporter adhesin/invasion proteins, Sca5/OmpB and Sca0/OmpA, thereby suggesting the participation of this enzyme in a relevant proteolytic pathway in rickettsial life-cycle. As a novel bona fide member of the retropepsin family of aspartic proteases, APRc emerges as an intriguing target for therapeutic intervention against fatal rickettsioses.

A goal of the Chromosome-centric Human Proteome Project is to identify all human protein species. With 3844 proteins annotated as "missing", this is challenging. Moreover, proteolytic processing generates new protein species with characteristic neo-N termini that are frequently accompanied by altered half-lives, function, interactions, and location. Enucleated and largely void of internal membranes and organelles, erythrocytes are simple yet proteomically challenging cells due to the high hemoglobin content and wide dynamic range of protein concentrations that impedes protein identification. Using the N-terminomics procedure TAILS, we identified 1369 human erythrocyte natural and neo-N-termini and 1234 proteins. Multiple semitryptic N-terminal peptides exhibited improved mass spectrometric identification properties versus the intact tryptic peptide enabling identification of 281 novel erythrocyte proteins and six missing proteins identified for the first time in the human proteome. With an improved bioinformatics workflow, we developed a new classification system and the Terminus Cluster Score. Thereby we described a new stabilizing N-end rule for processed protein termini, which discriminates novel protein species from degradation remnants, and identified protein domain hot spots susceptible to cleavage. Strikingly, 68% of the N-termini were within genome-encoded protein sequences, revealing alternative translation initiation sites, pervasive endoproteolytic processing, and stabilization of protein fragments in vivo. The mass spectrometry proteomics data have been deposited to ProteomeXchange with the data set identifier .

The knowledgebase TopFIND is an analysis platform focussed on protein termini, their origin, modification and hence their role on protein structure and function. Here, we present a major update to TopFIND, version 3, which includes a 70% increase in the underlying data to now cover a 90,696 proteins, 165,044 N-termini, 130,182 C-termini, 14,382 cleavage sites and 33,209 substrate cleavages in H. sapiens, M. musculus, A. thaliana, S. cerevisiae and E. coli. New features include the mapping of protein termini and cleavage entries across protein isoforms and significantly, the mapping of protein termini originating from alternative transcription and alternative translation start sites. Furthermore, two analysis tools for complex data analysis based on the TopFIND resource are now available online: TopFINDer, the TopFIND ExploRer, characterizes and annotates proteomics-derived N- or C-termini sets for their origin, sequence context and implications for protein structure and function. Neo-termini are also linked to associated proteases. PathFINDer identifies indirect connections between a protease and list of substrates or termini thus supporting the evaluation of complex proteolytic processes in vivo. To demonstrate the utility of the tools, a recent N-terminomics data set of inflamed murine skin has been re-analyzed. In re-capitulating the major findings originally performed manually, this validates the utility of these new resources. The point of entry for the resource is http://clipserve.clip.ubc.ca/topfind from where the graphical interface, all application programming interfaces (API) and the analysis tools are freely accessible.

Cardiac resynchronization therapy results in improved ejection fraction in patients with heart failure. We sought to determine whether these effects were mediated by changes in contractility, afterload, or volumes.

We identify matrix metalloproteinase (MMP)-8, the polymorphonuclear (PMN) leukocyte collagenase, as a critical mediator initiating lipopolysaccharide (LPS)-responsiveness in vivo. PMN infiltration towards LPS is abrogated in Mmp8-null mice. MMP-8 cleaves LPS-induced CXC chemokine (LIX) at Ser(4)-Val(5) and Lys(79)-Arg(80). LIX bioactivity is increased upon N-terminal cleavage, enhancing intracellular calcium mobilization and chemotaxis upon binding its cognate receptor, CXCR2. As there is no difference in PMN chemotaxis in Mmp8-null mice compared with wild-type mice towards synthetic analogues of MMP-8-cleaved LIX, MMP-8 is not essential for extravasation or cell migration in collagenous matrices in vivo. However, with biochemical redundancy between MMPs 1, 2, 9, and 13, which also cleave LIX at position 4 approximately 5, it was surprising to observe such a markedly reduced PMN infiltration towards LPS and LIX in Mmp8-/- mice. This lack of physiological redundancy in vivo identifies MMP-8 as a key mediator in the regulation of innate immunity. Comparable results were found with CXCL8/IL-8 and CXCL5/ENA-78, the human orthologues of LIX. MMP-8 cleaves CXCL8 at Arg(5)-Ser(6) and at Val(7)-Leu(8) in CXCL5 to activate respective chemokines. Hence, rather than collagen, these PMN chemoattractants are important MMP-8 substrates in vivo; PMN-derived MMP-8 cleaves and activates LIX to execute an in cis PMN-controlled feed-forward mechanism to orchestrate the initial inflammatory response and promote LPS responsiveness in tissue.