Amanda E. Starr

Isotopic labeling of terminal amines in complex samples identifies protein N-termini and protease cleavage products

Effective proteome-wide strategies that distinguish the N-termini of proteins from the N-termini of their protease cleavage products would accelerate identification of the substrates of proteases with broad or unknown specificity. Our approach, named terminal amine isotopic labeling of substrates (TAILS), addresses this challenge by using dendritic polyglycerol aldehyde polymers that remove tryptic and C-terminal peptides. We analyze unbound naturally acetylated, cyclized or labeled N-termini from proteins and their protease cleavage products by tandem mass spectrometry, and use peptide isotope quantification to discriminate between the substrates of the protease of interest and the products of background proteolysis. We identify 731 acetylated and 132 cyclized N-termini, and 288 matrix metalloproteinase (MMP)-2 cleavage sites in mouse fibroblast secretomes. We further demonstrate the potential of our strategy to link proteases with defined biological pathways in complex samples by analyzing mouse inflammatory bronchoalveolar fluid and showing that expression of the poorly defined breast cancer protease MMP-11 in MCF-7 human breast cancer cells cleaves both endoplasmin and the immunomodulator and apoptosis inducer galectin-1.

LPS responsiveness and neutrophil chemotaxis in vivo require PMN MMP-8 activity.

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 Ser4∼Val5 and Lys79∼Arg80. 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∼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 Arg5-Ser6 and at Val7-Leu8 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.

Macrophage-specific metalloelastase (MMP-12) truncates and inactivates ELR CXC chemokines and generates CCL2, -7, -8, and -13 antagonists: potential role of the macrophage in terminating polymorphonuclear leukocyte influx

Through the activity of macrophage-specific matrix metalloproteinase-12 (MMP-12), we found that macrophages dampen the lipopolysaccharide (LPS)-induced influx of polymorphonuclear leukocytes (PMNs)-thus providing a new mechanism for the termination of PMN recruitment in acute inflammation. MMP-12 specifically cleaves human ELR(+) CXC chemokines (CXCL1, -2, -3, -5, and -8) at E-LR, the critical receptor-binding motif or, for CXCL6, carboxyl-terminal to it. Murine (m) MMP-12 also cleaves mCXCL1, -2, and -3 at E-LR. MMP-12-cleaved mCXCL2 (macrophage-inflammatory protein-2 [MIP-2]) and mCXCL3 (dendritic cell inflammatory protein-1 [DCIP-1]) lost chemotactic activity. Furthermore, MMP-12 processed and inactivated monocyte chemotactic proteins CCL2, -7, -8, and -13 at position 4-5 generating CCR antagonists. Indeed, PMNs and macrophages in bronchoalveolar lavage fluid were significantly increased 72 hours after intranasal instillation of LPS in Mmp12(-/-) mice compared with wild type. Specificity occurred at 2 levels. Macrophage MMP-1 and MMP-9 did not cleave in the ELR motif. Second, unlike human ELR(+)CXC chemokines, mCXCL5 (LPS-induced CXC chemokine [LIX]) was not inactivated. Rather, mMMP-12 cleavage at Ser4-Val5 activated the chemokine, promoting enhanced PMN early infiltration in wild-type mice compared with Mmp12(-/-) mice 8 hours after LPS challenge in air pouches. We propose that the macrophage, specifically through MMP-12, assists in orchestrating the regulation of acute inflammatory responses by precise proteolysis of ELR(+)CXC and CC chemokines.

Network Analyses Reveal Pervasive Functional Regulation Between Proteases in the Human Protease Web

Network modeling of interactions between proteases and their inhibitors reveals a network of new protein connections and cascades in the protease web.

Stromal cell-derived factors 1α and 1β, inflammatory protein-10 and interferon-inducible T cell chemo-attractant are novel substrates of dipeptidyl peptidase 8

N‐terminal truncation of chemokines by proteases including dipeptidyl peptidase (DP) IV significantly alters their biological activity; generally ablating cognate G‐protein coupled receptor engagement and often generating potent receptor antagonists. DP8 is a recently recognised member of the prolyl oligopeptidase gene family that includes DPIV. Since DPIV is known to process chemokines we surveyed 27 chemokines for cleavage by DP8. We report DP8 cleavage of the N‐terminal two residues of IP10 (CXCL10), ITAC (CXCL11) and SDF‐1 (CXCL12). This has implications for DP8 substrate specificity. Chemokine cleavage and inactivation may occur in vivo upon cell lysis and release of DP8 or in the inactivation of internalized chemokine/receptor complexes.

Biochemical Characterization and N-terminomics Analysis of Leukolysin, the Membrane-type 6 Matrix Metalloprotease (MMP25) CHEMOKINE AND VIMENTIN CLEAVAGES ENHANCE CELL MIGRATION AND MACROPHAGE PHAGOCYTIC ACTIVITIES

Background: Neutrophil-specific membrane-type 6 matrix metalloproteinase (MT6-MMP)/leukolysin has seven known substrates. Results: We identified 72 new MT6-MMP substrates by proteomics and family-wide chemokine screens. Cell membrane-bound vimentin chemoattracts macrophages, whereas MT6-MMP-cleaved vimentin is an “eat-me” signal greatly increasing phagocytosis. Conclusion: MT6-MMP substrates indicate a role for clearance of apoptotic neutrophils. Significance: MT6-MMP cleaves many bioactive proteins important in innate immunity. 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.

Matrix metalloproteinase 8 deficiency in mice exacerbates inflammatory arthritis through delayed neutrophil apoptosis and reduced caspase 11 expression

OBJECTIVE Neutrophil accumulation is balanced by both cell infiltration and cell clearance, the controls of which are pivotal in the pathogenesis of rheumatoid arthritis (RA) and other chronic inflammatory diseases. Of the neutrophil-specific proteases, matrix metalloproteinase 8 (MMP-8; also known as neutrophil collagenase or collagenase 2) is traditionally viewed as being crucial for collagen degradation and hence cell migration and infiltration. This study was undertaken to examine the role of MMP-8 in a murine model of spontaneous RA. METHODS MMP-8(-/-) mice were backcrossed onto the Fas-defective MRL/lpr background, a mouse strain characterized by systemic autoimmunity including spontaneous autoimmune arthritis. Arthritis was induced with Freunds complete adjuvant and clinical disease and histologic parameters were assessed. RESULTS MMP-8(-/-) mice had earlier and more severe joint inflammation than their MMP-8(+/+) counterparts, coupled with a massive accumulation of neutrophils in synovial tissue, an unexpected result considering the commonly held view that MMP-8 has important extracellular matrix-degradative functions. Protease and protease inhibitor analysis of MMP-8(-/-) mouse neutrophils by CLIP-CHIP microarray revealed very little additional change in protease levels except for low expression of the apoptosis initiator caspase 11. This was confirmed at the protein level in unstimulated, lipopolysaccharide-treated, and interferon-γ-treated MMP-8(-/-) mouse neutrophils. Downstream of caspase 11, the activity of the apoptosis executioner caspase 3 was consequently reduced in MMP-8(-/-) mouse neutrophils, translating to reduced neutrophil apoptosis and cell accumulation compared with wild-type mouse cells. CONCLUSION Our findings indicate that MMP-8 is not essential for neutrophil migration in arthritis and likely other autoimmune diseases. Rather, MMP-8 is important for normal rates of neutrophil apoptosis and hence regulates cell clearance. Because MMP-8 deficiency leads to an exaggerated accumulation of neutrophil infiltrates due to delayed apoptosis and concurrent pathologic changes associated with dramatically increased neutrophil infiltration, MMP-8 is antiinflammatory and therefore a drug antitarget in the treatment of arthritis.

Biochemical analysis of matrix metalloproteinase activation of chemokines CCL15 and CCL23 and increased glycosaminoglycan binding of CCL16

Background: Proteases responsible for a CCL15-(25–92) product have not been elucidated. Results: All 14 CC monocyte chemoattractants, including CCL15, are processed by multiple MMPs. Conclusion: MMP-processing of CCL15, CCL23, and CCL16 functional activity is altered by MMP processing. Significance: This is the first study showing MMPs can activate CC chemokines and hence monoycte chemoattraction with potential to propagate inflammation. Leukocyte migration and activation is orchestrated by chemokines, the cleavage of which modulates their activity and glycosaminoglycan binding and thus their roles in inflammation and immunity. Early research identified proteolysis as a means of both activating or inactivating CXC chemokines and inactivating CC chemokines. Recent evidence has shown activating cleavages of the monocyte chemoattractants CCL15 and CCL23 by incubation with synovial fluid, although the responsible proteases could not be identified. Herein we show that CCL15 is processed in human synovial fluid by matrix metalloproteinases (MMPs) and serine proteases. Furthermore, a family-wide investigation of MMP processing of all 14 monocyte-directed CC chemokines revealed that each is precisely cleaved by one or more MMPs. By MALDI-TOF-MS, 149 cleavage sites were sequenced including the first reported instance of CCL1, CCL16, and CCL17 proteolysis. Full-length CCL15-(1–92) and CCL23-(1–99) were cleaved within their unique 31 and 32-amino acid residue extended amino termini, respectively. Unlike other CCL chemokines that lose activity and become receptor antagonists upon MMP cleavage, the prominent MMP-processed products CCL15-(25–92, 28–92) and CCL23-(26–99) are stronger agonists in calcium flux and Transwell CC receptor transfectant and monocytic THP-1 migration assays. MMP processing of CCL16-(1–97) in its extended carboxyl terminus yields two products, CCL16-(8–77) and CCL16-(8–85), with both showing unexpected enhanced glycosaminoglycan binding. Hence, our study reveals for the first time that MMPs activate the long amino-terminal chemokines CCL15 and CCL23 to potent forms that have potential to increase monocyte recruitment during inflammation.

Cysteine Cathepsins Activate ELR Chemokines and Inactivate Non-ELR Chemokines

Background: Chemokine function is regulated by proteolytic processing. Results: Cysteine cathepsins activate signaling by ELR CXC chemokines and terminate signaling by non-ELR chemokines. Conclusion: Cysteine cathepsins process CXC chemokines and promote inflammation by recruitment of CXCR2-expressing cells. Significance: This is the first comprehensive study on the processing of CXC chemokines by cysteine cathepsins. Cysteine cathepsins are primarily lysosomal proteases involved in general protein turnover, but they also have specific proteolytic functions in antigen presentation and bone remodeling. Cathepsins are most stable at acidic pH, although growing evidence indicates that they have physiologically relevant activity also at neutral pH. Post-translational proteolytic processing of mature chemokines is a key, yet underappreciated, level of chemokine regulation. Although the role of selected serine proteases and matrix metalloproteases in chemokine processing has long been known, little has been reported about the role of cysteine cathepsins. Here we evaluated cleavage of CXC ELR (CXCL1, -2, -3, -5, and -8) and non-ELR (CXCL9–12) chemokines by cysteine cathepsins B, K, L, and S at neutral pH by high resolution Tris-Tricine SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Whereas cathepsin B cleaved chemokines especially in the C-terminal region, cathepsins K, L, and S cleaved chemokines at the N terminus with glycosaminoglycans modulating cathepsin processing of chemokines. The functional consequences of the cleavages were determined by Ca2+ mobilization and chemotaxis assays. We show that cysteine cathepsins inactivate and in some cases degrade non-ELR CXC chemokines CXCL9–12. In contrast, cathepsins specifically process ELR CXC chemokines CXCL1, -2, -3, -5, and -8 N-terminally to the ELR motif, thereby generating agonist forms. This study suggests that cysteine cathepsins regulate chemokine activity and thereby leukocyte recruitment during protective or pathological inflammation.

Chapter 13 Characterizing Proteolytic Processing of Chemokines by Mass Spectrometry, Biochemistry, Neo‐Epitope Antibodies and Functional Assays

The nature, sequence, and length of the carboxy and amino termini of chemokines are important determinants of chemokine function, being essential for both efficient haptotactic gradient formation and cognate receptor activation events of these chemotactic cytokines. Chemokines are susceptible to proteolytic cleavage in both of these regions, which usually results in dramatic changes to the chemokine bioactivity. Herein we provide techniques to assess, detect, and characterize protease activity on chemokines and the biologic outcomes.