42 results found
Minns AF, Qi Y, Yamamoto K, et al., 2023, The C-terminal domains of ADAMTS1 contain exosites involved in its proteoglycanase activity, Journal of Biological Chemistry, Vol: 299, ISSN: 0021-9258
A disintegrin-like and metalloproteinase with thrombospondin type 1 motifs (ADAMTS1) is a protease involved in fertilization, cancer, cardiovascular development, and thoracic aneurysms. Proteoglycans such as versican and aggrecan have been identified as ADAMTS1 substrates, and Adamts1 ablation in mice typically results in versican accumulation; however, previous qualitative studies have suggested that ADAMTS1 proteoglycanase activity is weaker than that of other family members such as ADAMTS4 and ADAMTS5. Here, we investigated the functional determinants of ADAMTS1 proteoglycanase activity. We found that ADAMTS1 versicanase activity is approximately 1000-fold lower than ADAMTS5 and 50-fold lower than ADAMTS4 with a kinetic constant (kcat/Km) of 3.6 × 103 M-1 s-1 against full-length versican. Studies on domain-deletion variants identified the spacer and cysteine-rich domains as major determinants of ADAMTS1 versicanase activity. Additionally, we confirmed that these C-terminal domains are involved in the proteolysis of aggrecan as well as biglycan, a small leucine-rich proteoglycan. Glutamine scanning mutagenesis of exposed positively charged residues on the spacer domain loops and loop substitution with ADAMTS4 identified clusters of substrate-binding residues (exosites) in β3-β4 (R756Q/R759Q/R762Q), β9-β10 (residues 828-835), and β6-β7 (K795Q) loops. This study provides a mechanistic foundation for understanding the interactions between ADAMTS1 and its proteoglycan substrates and paves the way for development of selective exosite modulators of ADAMTS1 proteoglycanase activity.
Kwok HF, Yamamoto K, de Groot R, et al., 2023, Editorial: ADAM, ADAMTS and astacin proteases: Challenges and breakthroughs in the -Omics era-Volume II, Frontiers in Molecular Biosciences, Vol: 10
Cuffaro D, Ciccone L, Rossello A, et al., 2022, Targeting Aggrecanases for Osteoarthritis Therapy: From Zinc Chelation to Exosite Inhibition, JOURNAL OF MEDICINAL CHEMISTRY, Vol: 65, Pages: 13505-13532, ISSN: 0022-2623
Yamamoto K, Scavenius C, Meschis MM, et al., 2022, A top-down approach to uncover the hidden ligandome of low-density lipoprotein receptor-related protein 1 in cartilage, Matrix Biology, Vol: 112, Pages: 190-218, ISSN: 0174-173X
The low-density lipoprotein receptor-related protein 1 (LRP1) is a cell-surface receptor ubiquitously expressed in various tissues. It plays tissue-specific roles by mediating endocytosis of a diverse range of extracellular molecules. Dysregulation of LRP1 is involved in multiple conditions including osteoarthritis (OA) but little information is available about the specific profile of direct binding partners of LRP1 (ligandome) for each tissue, which would lead to a better understanding of its role in disease states. Here, we investigated adult articular cartilage where impaired LRP1-mediated endocytosis leads to tissue destruction. We used a top-down approach involving proteomic analysis of the LRP1 interactome in human chondrocytes, direct binding assays using purified LRP1 and ligand candidates, and validation in LRP1-deficient fibroblasts and human chondrocytes, as well as a novel Lrp1 conditional knockout (KO) mouse model. We found that inhibition of LRP1 and ligand interaction results in cell death, alteration of the entire secretome and transcriptional modulations in human chondrocytes. We identified a chondrocyte-specific LRP1 ligandome consisting of more than 50 novel ligand candidates. Surprisingly, 23 previously reported LRP1 ligands were not regulated by LRP1-mediated endocytosis in human chondrocytes. We confirmed direct LRP1 binding of HGFAC, HMGB1, HMGB2, CEMIP, SLIT2, ADAMTS1, TSG6, IGFBP7, SPARC and LIF, correlation between their affinity for LRP1 and the rate of endocytosis, and some of their intracellular localization. Moreover, a conditional LRP1 KO mouse model demonstrated a critical role of LRP1 in regulating the high-affinity ligands in cartilage in vivo. This systematic approach revealed the specificity and the extent of the chondrocyte LRP1 ligandome and identified potential novel therapeutic targets for OA.
Pirillo C, Birch F, Tissot FS, et al., 2022, Metalloproteinase inhibition reduces AML growth, prevents stem cell loss, and improves chemotherapy effectiveness, BLOOD ADVANCES, Vol: 6, Pages: 3126-3141, ISSN: 2473-9529
Teraz-Orosz A, Gierula M, Petri A, et al., 2022, Laminin G1 residues of protein S mediate its TFPI cofactor function and are competitively regulated by C4BP., Blood Advances, Vol: 6, Pages: 704-715, ISSN: 2473-9529
Protein S is a cofactor in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. It enhances TFPIα-mediated inhibition of factor (F)Xa activity and generation. The enhancement is dependent on a TFPIα-protein S interaction, involving TFPIα Kunitz 3 and protein S laminin G-type (LG)-1. C4b binding protein (C4BP), which binds to protein S LG1, almost completely abolishes its TFPI cofactor function. However, neither the amino acids involved in TFPIα enhancement, nor the mechanisms underlying the reduced TFPI cofactor function of C4BP-bound protein S, are known. To screen for functionally important regions within protein S LG1 we generated seven variants with inserted N-linked glycosylation attachment sites. Protein S D253T and Q427N/K429T, displayed severely reduced TFPI cofactor function while showing normal activated protein C (APC) cofactor function and C4BP binding. Based on these results, we designed four protein S variants in which 4-6 surface exposed charged residues were substituted for alanine. One variant, protein S K255A/E257A/D287A/R410A/K423A/E424A, exhibited either abolished or severely reduced TFPI cofactor function in plasma and FXa inhibition assays, both in the presence or absence of FV-short, but retained normal APC cofactor function and high affinity C4BP-binding. The C4BP β-chain was expressed to determine the mechanisms behind the reduced TFPI cofactor function of C4BP-bound protein S. Like C4BP-bound protein S, C4BP β-chain-bound protein S had severely reduced TFPI cofactor function. These results show that protein S Lys255, Glu257, Asp287, Arg410, Lys423 and Glu424 are critical for protein S-mediated enhancement of TFPIα and that binding of the C4BP β-chain blocks this function.
Santamaria S, Martin DR, Dong X, et al., 2021, Post-translational regulation and proteolytic activity of the metalloproteinase ADAMTS8, Journal of Biological Chemistry, Vol: 297, Pages: 1-17, ISSN: 0021-9258
A disintegrin-like and metalloprotease domain with thrombospondin type 1 motifs (ADAMTS)8 is a secreted protease, which was recently implicated in pathogenesis of pulmonary arterial hypertension (PAH). However, the substrate repertoire of ADAMTS8 and regulation of its activity are incompletely understood. Although considered a proteoglycanase because of high sequence similarity and close phylogenetic relationship to the proteoglycan-degrading proteases ADAMTS1, 4, 5, and 15, as well as tight genetic linkage with ADAMTS15 on human chromosome 11, its aggrecanase activity was reportedly weak. Several post-translational factors are known to regulate ADAMTS proteases such as autolysis, inhibition by endogenous inhibitors, and receptor-mediated endocytosis, but their impacts on ADAMTS8 are unknown. Here, we show that ADAMTS8 undergoes autolysis at six different sites within its spacer domain. We also found that in contrast to ADAMTS4 and 5, ADAMTS8 levels were not regulated through low-density lipoprotein receptor-related protein 1 (LRP1)-mediated endocytosis. Additionally, ADAMTS8 lacked significant activity against the proteoglycans aggrecan, versican, and biglycan. Instead, we found that ADAMTS8 cleaved osteopontin, a phosphoprotein whose expression is upregulated in PAH. Multiple ADAMTS8 cleavage sites were identified using liquid chromatography–tandem mass spectrometry. Osteopontin cleavage by ADAMTS8 was efficiently inhibited by TIMP-3, an endogenous inhibitor of ADAMTS1, 4, and 5, as well as by TIMP-2, which has no previously reported inhibitory activity against other ADAMTS proteases. These differences in post-translational regulation and substrate repertoire differentiate ADAMTS8 from other family members and may help to elucidate its role in PAH.
Martin DR, Santamaria S, Koch CD, et al., 2021, Identification of novel ADAMTS1, ADAMTS4 and ADAMTS5 cleavage sites in versican using a label-free quantitative proteomics approach., Journal of Proteomics, Vol: 249, Pages: 1-8, ISSN: 0165-022X
The chondroitin sulfate proteoglycan versican is important for embryonic development and several human disorders. The versican V1 splice isoform is widely expressed and cleaved by ADAMTS proteases at a well-characterized site, Glu441-Ala442. Since ADAMTS proteases cleave the homologous proteoglycan aggrecan at multiple sites, we hypothesized that additional cleavage sites existed within versican. We report a quantitative label-free approach that ranks abundance of liquid chromatography-tandem mass spectrometry (LC-MS/MS)-identified semi-tryptic peptides after versican digestion by ADAMTS1, ADAMTS4 and ADAMTS5 to identify site-specific cleavages. Recombinant purified versican V1 constructs were digested with the recombinant full-length proteases, using catalytically inactive mutant proteases in control digests. Semi-tryptic peptide abundance ratios determined by LC-MS/MS in ADAMTS:control digests were compared to the mean of all identified peptides to obtain a z-score by which outlier peptides were ranked, using semi-tryptic peptides identifying Glu441 -Ala442 cleavage as the benchmark. Tryptic peptides with higher abundance in control:ADAMTS digests supported cleavage site identification. We identified several novel cleavage sites supporting the ADAMTS1/4/5 cleavage site preference for a P1-Glu residue in proteoglycan substrates. Digestion of proteins in vitro and application of this z-score approach is potentially widely applicable for mapping protease cleavage sites using label-free proteomics. SIGNIFICANCE: Versican abundance and turnover are relevant to the pathogenesis of several human disorders. Versican is cleaved by A Disintegrin-like And Metalloprotease with Thrombospondin type 1 motifs (ADAMTS) family members at Glu441-Ala442, generating a bioactive proteoform called versikine, but additional cleavage sites and the site-specificity of individual ADAMTS proteases is unexplored. Here, we used label-free proteomics approach to identify versican cleavage sites
Yamamoto K, de Groot R, Scilabra SD, et al., 2021, Editorial: ADAM, ADAMTS and Astacin Proteases: Challenges and Breakthroughs in the -Omics Era, FRONTIERS IN MOLECULAR BIOSCIENCES, Vol: 8
- Author Web Link
- Citations: 1
Santamaria S, Buemi F, Nuti E, et al., 2021, Development of a fluorogenic ADAMTS-7 substrate, Journal of Enzyme Inhibition and Medicinal Chemistry, Vol: 36, ISSN: 1475-6366
The extracellular protease ADAMTS-7 has been identified as a potential therapeutic target in atherosclerosis and associated diseases such as coronary artery disease (CAD). However, ADAMTS-7 inhibitors have not been reported so far. Screening of inhibitors has been hindered by the lack of a suitable peptide substrate and, consequently, a convenient activity assay. Here we describe the first fluorescence resonance energy transfer (FRET) substrate for ADAMTS-7, ATS7FP7. ATS7FP7 was used to measure inhibition constants for the endogenous ADAMTS-7 inhibitor, TIMP-4, as well as two hydroxamate-based zinc chelating inhibitors. These inhibition constants match well with IC50 values obtained with our SDS-PAGE assay that uses the N-terminal fragment of latent TGF-β–binding protein 4 (LTBP4S-A) as a substrate. Our novel fluorogenic substrate ATS7FP7 is suitable for high throughput screening of ADAMTS-7 inhibitors, thus accelerating translational studies aiming at inhibition of ADAMTS-7 as a novel treatment for cardiovascular diseases such as atherosclerosis and CAD.
Santamaria S, Cuffaro D, Nuti E, et al., 2021, Exosite inhibition of ADAMTS-5 by a glycoconjugated arylsulfonamide, Scientific Reports, Vol: 11, ISSN: 2045-2322
ADAMTS-5 is a major protease involved in the turnover of proteoglycans such as aggrecan and versican. Dysregulated aggrecanase activity of ADAMTS-5 has been directly linked to the etiology of osteoarthritis (OA). For this reason, ADAMTS-5 is a pharmaceutical target for the treatment of OA. ADAMTS-5 shares high structural and functional similarities with ADAMTS-4, which makes the design of selective inhibitors particularly challenging. Here we exploited the ADAMTS-5 binding capacity of β-N-acetyl-d-glucosamine to design a new class of sugar-based arylsulfonamides. Our most promising compound, 4b, is a non-zinc binding ADAMTS-5 inhibitor which showed high selectivity over ADAMTS-4. Docking calculations combined with molecular dynamics simulations demonstrated that 4b is a cross-domain inhibitor that targets the interface of the metalloproteinase and disintegrin-like domains. Furthermore, the interaction between 4b and the ADAMTS-5 Dis domain is mediated by hydrogen bonds between the sugar moiety and two lysine residues (K532 and K533). Targeted mutagenesis of these two residues confirmed their importance both for versicanase activity and inhibitor binding. This positively-charged cluster of ADAMTS-5 represents a previously unknown substrate-binding site (exosite) which is critical for substrate recognition and can therefore be targeted for the development of selective ADAMTS-5 inhibitors.
Santamaria S, 2021, Targeting the PI3K/AKT pathway: a potential new weapon in the global fight against SARS-CoV-2?, INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES, Vol: 17, Pages: 2770-2771, ISSN: 1449-2288
- Author Web Link
- Citations: 3
Santamaria S, de Groot R, 2020, ADAMTS proteases in cardiovascular physiology and disease, Open Biology, Vol: 10, Pages: 1-18, ISSN: 2046-2441
The a disintegrin-like and metalloproteinase with thrombospondin motif (ADAMTS) family comprises 19 proteases that regulate the structure and function of extracellular proteins in the extracellular matrix and blood. The best characterized cardiovascular role is that of ADAMTS-13 in blood. Moderately low ADAMTS-13 levels increase the risk of ischeamic stroke and very low levels (less than 10%) can cause thrombotic thrombocytopenic purpura (TTP). Recombinant ADAMTS-13 is currently in clinical trials for treatment of TTP. Recently, new cardiovascular roles for ADAMTS proteases have been discovered. Several ADAMTS family members are important in the development of blood vessels and the heart, especially the valves. A number of studies have also investigated the potential role of ADAMTS-1, -4 and -5 in cardiovascular disease. They cleave proteoglycans such as versican, which represent major structural components of the arteries. ADAMTS-7 and -8 are attracting considerable interest owing to their implication in atherosclerosis and pulmonary arterial hypertension, respectively. Mutations in the ADAMTS19 gene cause progressive heart valve disease and missense variants in ADAMTS6 are associated with cardiac conduction. In this review, we discuss in detail the evidence for these and other cardiovascular roles of ADAMTS family members, their proteolytic substrates and the potential molecular mechanisms involved.
Santamaria S, De Groot R, 2020, ADAMTS proteases in cardiovascular physiology and disease: ADAMTS, cardiovascular roles, Open Biology, Vol: 10
The a disintegrin-like and metalloproteinase with thrombospondin motif (ADAMTS) family comprises 19 proteases that regulate the structure and function of extracellular proteins in the extracellular matrix and blood. The best characterized cardiovascular role is that of ADAMTS-13 in blood. Moderately low ADAMTS-13 levels increase the risk of ischeamic stroke and very low levels (less than 10%) can cause thrombotic thrombocytopenic purpura (TTP). Recombinant ADAMTS-13 is currently in clinical trials for treatment of TTP. Recently, new cardiovascular roles for ADAMTS proteases have been discovered. Several ADAMTS family members are important in the development of blood vessels and the heart, especially the valves. A number of studies have also investigated the potential role of ADAMTS-1,-4 and-5 in cardiovascular disease. They cleave proteoglycans such as versican, which represent major structural components of the arteries. ADAMTS-7 and-8 are attracting considerable interest owing to their implication in atherosclerosis and pulmonary arterial hypertension, respectively. Mutations in the ADAMTS19 gene cause progressive heart valve disease and missense variants in ADAMTS6 are associated with cardiac conduction. In this review, we discuss in detail the evidence for these and other cardiovascular roles of ADAMTS family members, their proteolytic substrates and the potential molecular mechanisms involved.
Salvatore S, Cuffaro D, Nuti E, et al., 2020, Exosite inhibition of A Disintegrin And Metalloproteinase with Thrombospondin motif (ADAMTS)-5 by a glycoconjugated arylsulfonamide, Publisher: Cold Spring Harbor Laboratory
ADAMTS-5 is a major protease involved in the turnover of proteoglycans such as aggrecan and versican. Its aggrecanase activity has been directly linked to the etiology of osteoarthritis (OA), identifying ADAMTS-5 as a pharmaceutical target for OA treatment. However, most existing ADAMTS-5 inhibitors target its active site and therefore suffer from poor selectivity. Here, using a novel approach, we have designed a new class of sugar-based arylsulfonamide inhibitors, which are selective for ADAMTS-5 through binding to a previously unknown substrate-binding site (exosite). Docking calculations combined with molecular dynamics simulations demonstrated that our lead compound is a cross-domain inhibitor that targets the interface of the metalloproteinase and disintegrin-like domains. Targeted mutagenesis identified disintegrin-like domain residues K532 and K533 as an exosite which is critical for substrate recognition. Furthermore, we show that this exosite acts as major determinant for inhibitor binding and, therefore, can be targeted for development of selective ADAMTS-5 inhibitors.
Santamaria S, 2020, ADAMTS-5: A difficult teenager turning 20, International Journal of Experimental Pathology: mechanisms and models of disease, Vol: 101, Pages: 4-20, ISSN: 0959-9673
A Disintegrin And Metalloproteinase with ThromboSpondin motif (ADAMTS)‐5 was identified in 1999 as one of the enzymes responsible for cleaving aggrecan, the major proteoglycan in articular cartilage. Studies in vitro, ex vivo and in vivo have validated ADAMTS‐5 as a target in osteoarthritis (OA), a disease characterized by extensive degradation of aggrecan. For this reason, it attracted the interest of many research groups aiming to develop a therapeutic treatment for OA patients. However, ADAMTS‐5 proteoglycanase activity is not only involved in the dysregulated aggrecan proteolysis, which occurs in OA, but also in the physiological turnover of other related proteoglycans. In particular, versican, a major ADAMTS‐5 substrate, plays an important structural role in heart and blood vessels and its proteolytic processing by ADAMTS‐5 must be tightly regulated. On the occasion of the 20th anniversary of the discovery of ADAMTS‐5, this review looks at the evidence for its detrimental role in OA, as well as its physiological turnover of cardiovascular proteoglycans. Moreover, the other potential functions of this enzyme are highlighted. Finally, challenges and emerging trends in ADAMTS‐5 research are discussed.
Santamaria S, 2020, Chemical Modification of Proteoglycanases with Biotin, ADAMTS PROTEASES: METHODS AND PROTOCOLS, Editors: Apte, Publisher: HUMANA PRESS INC, Pages: 113-123, ISBN: 978-1-4939-9697-1
- Citations: 1
Santamaria S, Yamamoto K, 2020, Analysis of Aggrecanase Activity Using Neoepitope Antibodies, ADAMTS PROTEASES: METHODS AND PROTOCOLS, Editors: Apte, Publisher: HUMANA PRESS INC, Pages: 125-136, ISBN: 978-1-4939-9697-1
- Citations: 5
Gierula M, SallesCrawley II, Santamaria S, et al., 2019, The roles of factor Va and protein S in formation of the activated protein C/protein S/factor Va inactivation complex, Journal of Thrombosis and Haemostasis, ISSN: 1538-7933
Background: Activated protein C (APC)-mediated inactivation of factor (F)Va is greatlyenhanced by protein S. For inactivation to occur, a trimolecular complex between FVa,APC and protein S must form on the phospholipid membrane. However, directdemonstration of complex formation has proven elusive.Objectives:To elucidate the nature of the phospholipid-dependent interactions betweenAPC, protein S and FVa.Methods:We evaluated binding of active site blocked APC to phospholipid-coatedmagnetic beads in the presence and absence of protein S and/or FVa. The importanceof protein S and FV residues were evaluated functionally.Results: APC alone bound weakly to phospholipids. Protein S mildly enhanced APCbinding to phospholipid surfaces, whereas FVa did not. However, FVa together withproteinS enhanced APC binding(>14-fold), demonstrating formation of an APC/proteinS/FVa complex. C4b binding protein-bound protein S failed to enhance APC binding,agreeing with its reduced APC cofactor function. Protein S variants (E36A and D95A)with reduced APC cofactor function exhibited essentially normal augmentation of APCbinding to phospholipids, but diminished APC/protein S/FVa complex formation,suggesting involvement in interactions dependent upon FVa. Similarly, FVaNara(W1920R), an APC resistant FV variant, also did not efficiently incorporate into thetrimolecular complex as efficiently as wild-type FVa. FVa inactivation assays suggestedthat the mutation impairs its affinity for phospholipid membranes and with protein Swithin the complex. Conclusions: FVa plays a central role in the formation of its inactivation complex.Furthermore, membrane proximal interactions between FVa, APC and protein S areessential for its cofactor function.
Santamaria S, Yamamoto Y, Teraz-Orosz A, et al., 2019, Exosites in hypervariable loops of ADAMTS dpacer domains control substrate recognition and proteolysis, Scientific Reports, Vol: 9, ISSN: 2045-2322
ADAMTS (A Disintegrin-like and Metalloproteinase domain with Thrombospondin type 1 Motif)-1, -4 and -5 share the abilities to cleave large aggregating proteoglycans including versican and aggrecan. These activities are highly relevant to cardiovascular disease and osteoarthritis and during development. Here, using purified recombinant ADAMTS-1, -4 and -5, we quantify, compare, and define the molecular basis of their versicanase activity. A novel sandwich-ELISA detecting the major versican cleavage fragment was used to determine, for the first time, kinetic constants for versican proteolysis. ADAMTS-5 (kcat/Km 35 × 105 M−1 s−1) is a more potent (~18-fold) versicanase than ADAMTS-4 (kcat/Km 1.86 × 105 M−1 sec−1), whereas ADAMTS-1 versicanase activity is comparatively low. Deletion of the spacer domain reduced versicanase activity of ADAMTS-5 19-fold and that of ADAMTS-4 167-fold. Co-deletion of the ADAMTS-5 cysteine-rich domain further reduced versicanase activity to a total 153-fold reduction. Substitution of two hypervariable loops in the spacer domain of ADAMTS-5 (residues 739–744 and 837–844) and ADAMTS-4 (residues 717–724 and 788–795) with those of ADAMTS-13, which does not cleave proteoglycans, caused spacer-dependent reductions in versicanase activities. Our results demonstrate that these loops contain exosites critical for interaction with and processing of versican. The hypervariable loops of ADAMTS-5 are shown to be important also for its aggrecanase activity. Together with previous work on ADAMTS-13 our results suggest that the spacer domain hypervariable loops may exercise significant control of ADAMTS proteolytic activity as a general principle. Identification of specific exosites also provides targets for selective inhibitors.
Colige A, Monseur C, Crawley J, et al., 2019, Proteomic discovery of substrates of the cardiovascular protease ADAMTS7, Journal of Biological Chemistry, Vol: 294, Pages: 8037-8045, ISSN: 0021-9258
The protease ADAMTS7 functions in the extracellular matrix (ECM) of the cardiovascular system. However, its physiological substrate specificity and mechanism of regulation remain to be explored. To address this, we conducted an unbiased substrate analysis using terminal amine isotopic labeling of substrates (TAILS). The analysis identified candidate substrates of ADAMTS7 in the human fibroblast secretome, including proteins with a wide range of functions, such as collagenous and noncollagenous extracellular matrix proteins, growth factors, proteases, and cell-surface receptors. It also suggested that autolysis occurs at Glu-729–Val-730 and Glu-732–Ala-733 in the ADAMTS7 Spacer domain, which was corroborated by N-terminal sequencing and Western blotting. Importantly, TAILS also identified proteolysis of the latent TGF-β–binding proteins 3 and 4 (LTBP3/4) at a Glu-Val and Glu-Ala site, respectively. Using purified enzyme and substrate, we confirmed ADAMTS7-catalyzed proteolysis of recombinant LTBP4. Moreover, we identified multiple additional scissile bonds in an N-terminal linker region of LTBP4 that connects fibulin-5/tropoelastin and fibrillin-1–binding regions, which have an important role in elastogenesis. ADAMTS7-mediated cleavage of LTBP4 was efficiently inhibited by the metalloprotease inhibitor TIMP-4, but not by TIMP-1 and less efficiently by TIMP-2 and TIMP-3. As TIMP-4 expression is prevalent in cardiovascular tissues, we propose that TIMP-4 represents the primary endogenous ADAMTS7 inhibitor. In summary, our findings reveal LTBP4 as an ADAMTS7 substrate, whose cleavage may potentially impact elastogenesis in the cardiovascular system. We also identify TIMP-4 as a likely physiological ADAMTS7 inhibitor.
Santamaria S, de Groot R, 2019, Monoclonal antibodies against metzincin targets, British Journal of Pharmacology, Vol: 176, Pages: 52-66, ISSN: 1476-5381
The metzincin clan of metalloproteinases includes the MMP, disintegrin and metalloproteinase (ADAM) and ADAM with thrombospondin motifs families, which cleave extracellular targets in a wide range of (patho)physiological processes. Antibodies constitute a powerful tool to modulate the activity of these enzymes for both therapeutic and research purposes. In this review, we give an overview of monoclonal antibodies (mAbs) that have been tested in preclinical disease models, human trials and important studies of metzincin structure and function. Initial attempts to develop therapeutic small molecule inhibitors against MMPs were hampered by structural similarities between metzincin active sites and, consequently, off‐target effects. Therefore, more recently, mAbs have been developed that do not bind to the active site but bind to surface‐exposed loops that are poorly conserved in closely related family members. Inhibition of protease activity by these mAbs occurs through a variety of mechanisms, including (i) barring access to the active site, (ii) disruption of exosite binding, and (iii) prevention of protease activation. These different modes of inhibition are discussed in the context of the antibodies' potency, selectivity and, importantly, the effects in models of disease and clinical trials. In addition, various innovative strategies that were used to generate anti‐metzincin mAbs are discussed.
Santamaria S, Nagase H, 2018, Measurement of Protease Activities Using Fluorogenic Substrates, PROTEASES AND CANCER: METHODS AND PROTOCOLS, Editors: Cal, Obaya, Publisher: HUMANA PRESS INC, Pages: 107-122, ISBN: 978-1-4939-7594-5
- Citations: 4
Yamamoto K, Santamaria S, Botkjaer KA, et al., 2017, Inhibition of Shedding of Low-Density Lipoprotein Receptor-Related Protein 1 Reverses Cartilage Matrix Degradation in Osteoarthritis, ARTHRITIS & RHEUMATOLOGY, Vol: 69, Pages: 1246-1256, ISSN: 2326-5191
- Author Web Link
- Citations: 33
Santamaria S, Reglińska-Matveyev N, Gierula M, et al., 2017, Factor V anticoagulant cofactor activity that targets the early phase of coagulation, Journal of Biological Chemistry, Vol: 292, Pages: 9335-9344, ISSN: 0021-9258
Tissue factor pathway inhibitor (TFPI), the main inhibitor of initiation of coagulation, exerts an important anticoagulant role through the factor Xa (FXa)-dependent inhibition of tissue factor/factor VIIa (FVIIa). Protein S is a TFPI cofactor, enhancing the efficiency of FXa inhibition. TFPI can also inhibit prothrombinase assembly by directly interacting with coagulation factor V (FV) which has been activated by FXa. Since full-length TFPI associates with FV in plasma, we hypothesized that FV may influence TFPI inhibitory function. Using pure component FXa inhibition assays, we found that while FV alone did not influence TFPI-mediated FXa inhibition, it further enhanced TFPI in the presence of protein S, resulting in an ~8-fold reduction in Ki compared with TFPI alone. A FV variant (R709Q/R1018Q/R1545Q, FVΔIIa) that cannot be cleaved/activated by thrombin or FXa, also enhanced TFPI-mediated inhibition of FXa ~12-fold in the presence of protein S. In contrast, neither activated FV (FVa) nor recombinant B-domain-deleted FV could enhance TFPI-mediated inhibition of FXa in the presence of protein S, suggesting a functional contribution of the B domain. Using TFPI and protein S variants we show further that the enhancement of TFPI-mediated FXa inhibition by protein S and FV depends on a direct protein S/TFPI interaction and that the TFPI C-terminal tail is not essential for this enhancement. In FXa-catalyzed prothrombin activation assays, both FV and FVΔIIa (but not FVa) enhanced TFPI function in the presence of protein S. These results demonstrate a new anticoagulant (cofactor) function of FV that targets the early phase of coagulation before prothrombinase assembly.
Santamaria S, Fedorov O, McCafferty J, et al., 2017, Development of a monoclonal anti-ADAMTS-5 antibody that specifically blocks the interaction with LRP1, MABS, Vol: 9, Pages: 595-602, ISSN: 1942-0862
- Author Web Link
- Citations: 7
Yamamoto K, Okano H, Miyagawa W, et al., 2016, MMP-13 is constitutively produced in human chondrocytes and co-endocytosed with ADAMTS-5 and TIMP-3 by the endocytic receptor LRP1, MATRIX BIOLOGY, Vol: 56, Pages: 57-73, ISSN: 0945-053X
- Author Web Link
- Citations: 95
Nuti E, Cuffaro D, D'Andrea F, et al., 2016, Sugar-Based Arylsulfonamide Carboxylates as Selective and Water-Soluble Matrix Metalloproteinase-12 Inhibitors, CHEMMEDCHEM, Vol: 11, Pages: 1626-1637, ISSN: 1860-7179
- Author Web Link
- Citations: 29
Botkjaer KA, Kwok HF, Terp MG, et al., 2016, Development of a specific affinity-matured exosite inhibitor to MT1-MMP that efficiently inhibits tumor cell invasion in vitro and metastasis in vivo, ONCOTARGET, Vol: 7, Pages: 16773-16792
- Author Web Link
- Citations: 33
Santamaria S, Yamamoto K, Botkjaer K, et al., 2015, Antibody-based exosite inhibitors of ADAMTS-5 (aggrecanase-2), BIOCHEMICAL JOURNAL, Vol: 471, Pages: 391-401, ISSN: 0264-6021
- Author Web Link
- Citations: 30
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