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The ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin motifs) enzymes are secreted, multi-domain matrix-associated zinc metalloendopeptidases that have diverse roles in tissue morphogenesis and patho-physiological remodeling, in inflammation and in vascular biology. The human family includes 19 members that can be sub-grouped on the basis of their known substrates, namely the aggrecanases or proteoglycanases (ADAMTS1, 4, 5, 8, 9, 15 and 20), the procollagen N-propeptidases (ADAMTS2, 3 and 14), the cartilage oligomeric matrix protein-cleaving enzymes (ADAMTS7 and 12), the von-Willebrand Factor proteinase (ADAMTS13) and a group of orphan enzymes (ADAMTS6, 10, 16, 17, 18 and 19). Control of the structure and function of the extracellular matrix (ECM) is a central theme of the biology of the ADAMTS, as exemplified by the actions of the procollagen-N-propeptidases in collagen fibril assembly and of the aggrecanases in the cleavage or modification of ECM proteoglycans. Defects in certain family members give rise to inherited genetic disorders, while the aberrant expression or function of others is associated with arthritis, cancer and cardiovascular disease. In particular, ADAMTS4 and 5 have emerged as therapeutic targets in arthritis. Multiple ADAMTSs from different sub-groupings exert either positive or negative effects on tumorigenesis and metastasis, with both metalloproteinase-dependent and -independent actions known to occur. The basic ADAMTS structure comprises a metalloproteinase catalytic domain and a carboxy-terminal ancillary domain, the latter determining substrate specificity and the localization of the protease and its interaction partners; ancillary domains probably also have independent biological functions. Focusing primarily on the aggrecanases and proteoglycanases, this review provides a perspective on the evolution of the ADAMTS family, their links with developmental and disease mechanisms, and key questions for the future.

Integrin-mediated binding is important for the metastatic dissemination of different types of cancer cells. Snake venom disintegrins obtustatin and echistatin are potent, irreversible, and selective inhibitors of α1β1 and αvβ3 integrins, respectively. Obtustatin is one of the shortest disintegrins yet described, containing 41 amino acids. It has a similar pattern of cysteines to the other disintegrin echistatin but with a KTS motif rather than a classic RGD in its active site. A surface acoustic wave biosensor was applied to prove the molecular recognition of disintegrins by their substrates. The human erythrocyte ghost cells were immobilized at the sensors to allow for the detection of kinetic binding constants of disintegrins compared to the surface of giant unilamellar vesicles (GUVs). Obtustatin binds to the erythrocyte ghost membrane with affinity in the mid-nanomolar range (2.32 × 10–7 M), and echistatin in the low micromolar range, which indicates specific molecular recognition for both disintegrins, but the higher response for obtustatin. The data directly confirm that disintegrins bind to the erythrocyte ghost membrane, thereby supporting the previously overlooked presence of integrins in red blood cell membranes.

Snake venom contains mixture of bioactive proteins and polypeptides. Most of these proteins and polypeptides exist as monomers, but some of them form complexes in the venom. These complexes exhibit much higher levels of pharmacological activity compared to individual components and play an important role in pathophysiological effects during envenomation. They are formed through covalent and/or non-covalent interactions. The subunits of the complexes are either identical (homodimers) or dissimilar (heterodimers; in some cases subunits belong to different families of proteins). The formation of complexes, at times, eliminates the non-specific binding and enhances the binding to the target molecule. On several occasions, it also leads to recognition of new targets as protein-protein interaction in complexes exposes the critical amino acid residues buried in the monomers. Here, we describe the structure and function of various protein complexes of snake venoms and their role in snake venom toxicity.

Introduction Sclerostin is a Wnt inhibitor produced by osteocytes that regulates bone formation. Because bone tissue contributes to the development of osteoarthritis (OA), we investigated the role of sclerostin in bone and cartilage in a joint instability model in mice. Methods Ten-week-old SOST-knockout (SOST-KO) and wild-type (WT) mice underwent destabilization of the medial meniscus (DMM). We measured bone volume at the medial femoral condyle and osteophyte volume and determined the OA score and expression of matrix proteins. Primary murine chondrocytes were cultured with Wnt3a and sclerostin to assess the expression of matrix proteins, proteoglycan release and glycosaminoglycan accumulation. Results Sclerostin was expressed in calcified cartilage of WT mice with OA. In SOST-KO mice, cartilage was preserved despite high bone volume. However, SOST-KO mice with DMM had a high OA score, with increased expression of aggrecanases and type X collagen. Moreover, SOST-KO mice with OA showed disrupted anabolic–catabolic balance and cartilage damage. In primary chondrocytes, sclerostin addition abolished Wnt3a-increased expression of a disintegrin and metalloproteinase with thrombospondin motifs, matrix metalloproteinases and type X collagen by inhibiting the canonical Wnt pathway. Moreover, sclerostin inhibited Wnt-phosphorylated c-Jun N-terminal kinase (JNK) and rescued the expression of anabolic genes. Furthermore, sclerostin treatment inhibited both Wnt canonical and non-canonical JNK pathways in chondrocytes, thus preserving metabolism. Conclusion Sclerostin may play an important role in maintaining cartilage integrity in OA.

Remodeling of the extracellular matrix supports tissue and organ development, by regulating cellular morphology and tissue integrity. However, proper extracellular matrix remodeling requires spatiotemporal regulation of extracellular metalloproteinase activity. Members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family, including MIG-17 and GON-1, are evolutionarily conserved, secreted, zinc-requiring metalloproteinases. Although these proteases are required for extracellular matrix remodeling during gonadogenesis in Caenorhabditis elegans, their in vivo regulatory mechanisms remain to be delineated. Therefore, we focused on the C. elegans tissue inhibitors of metalloproteinases (TIMPs), TIMP-1 and CRI-2. Analysis of the transcription and translation products for GFP/Venus fusions, with TIMP-1 or CRI-2, indicated that these inhibitors were secreted and localized to the basement membrane of gonads and the plasma membrane of germ cells. A timp-1 deletion mutant exhibited gonadal growth defects and sterility, and the phenotypes of this mutant were fully rescued by a TIMP-1::Venus construct, but not by a TIMP-1(C21S)::Venus mutant construct, in which the inhibitor coding sequence had been mutated. Moreover, genetic data suggested that TIMP-1 negatively regulates proteolysis of the α1 chain of type IV collagen. We also found that the loss-of-function observed for the mutants timp-1 and cri-2 involves a partial suppression of gonadal defects found for the mutants mig-17/ADAMTS and gon-1/ADAMTS, and that this suppression was canceled upon overexpression of gon-1 or mig-17, respectively. Based on these results, we propose that both TIMP-1 and CRI-2 act as inhibitors of MIG-17 and GON-1 ADAMTSs to regulate gonad development in a noncell-autonomous manner.

During development of the Caenorhabditis elegans gonad, the gonadal leader cells, called distal tip cells (DTCs), migrate in a U-shaped pattern to form the U-shaped gonad arms. The ADAMTS ( a d isintegrin a nd m etalloprotease with t hrombo s pondin motifs) family metalloproteases MIG-17 and GON-1 are required for correct DTC migration. Mutations in mig-17 result in misshapen gonads due to the misdirected DTC migration, and mutations in gon-1 result in shortened and swollen gonads due to the premature termination of DTC migration. Although the phenotypes shown by mig-17 and gon-1 mutants are very different from one another, mutations that result in amino acid substitutions in the same basement membrane protein genes, emb-9/collagen IV a1 , let-2 / collagen IV a2 and fbl-1/fibulin-1 , were identified as genetic suppressors of mig-17 and gon-1 mutants. To understand the roles shared by these two proteases, we examined the effects of the mig-17 suppressors on gon-1 and the effects of the gon-1 suppressors and enhancers on mig-17 gonadal defects. Some of the emb-9 , let-2 and fbl-1 mutations suppressed both mig-17 and gon-1 , whereas others acted only on mig-17 or gon-1 . These results suggest that mig-17 and gon-1 have their specific functions as well as functions commonly shared between them for gonad formation. The levels of collagen IV accumulation in the DTC basement membrane were significantly higher in the gon-1 mutants as compared with wild type and were reduced to the wild-type levels when combined with suppressor mutations, but not with enhancer mutations, suggesting that the ability to reduce collagen IV levels is important for gon-1 suppression.

Chondroitin, a class of glycosaminoglycan polysaccharides, is found as proteoglycans in the extracellular matrix, plays a crucial role in tissue morphogenesis during development and axonal regeneration. Ingestion of chondroitin prolongs the lifespan of C. elegans . However, the roles of endogenous chondroitin in regulating lifespan and healthspan mostly remain to be investigated. Here, we demonstrate that a gain-of-function mutation in MIG-22, the chondroitin polymerizing factor (ChPF), results in elevated chondroitin levels and a significant extension of both the lifespan and healthspan in C. elegans . Importantly, the remarkable longevity observed in mig-22(gf) mutants is dependent on SQV-5/chondroitin synthase (ChSy), highlighting the pivotal role of chondroitin in controlling both lifespan and healthspan. Additionally, the mig-22(gf) mutation effectively suppresses the reduced healthspan associated with the loss of MIG-17/ADAMTS metalloprotease, a crucial for factor in basement membrane (BM) remodeling. Our findings suggest that chondroitin functions in the control of healthspan downstream of MIG-17, while regulating lifespan through a pathway independent of MIG-17.

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.

The epidermal growth factor (EGF) family and the EGF receptor (EGFR, ErbB) tyrosine kinase family have been spearheading the studies of signal transduction events that determine cell fate and behavior in vitro and in vivo. The EGFR family and their signaling pathways are giving us tremendous advantages in developing fascinating molecular target strategies for cancer therapy. Currently, two important types of EGFR inhibitors are in clinical use: neutralizing antibodies of EGFR or ErbB2, and synthetic small compounds of tyrosine kinase inhibitors designed for receptors. On the other hand, basic research of the EGF family ligands presents new challenges as membrane‐anchored growth factors. All members of the EGF family have important roles in development and diseases and are shed from the plasma membrane by metalloproteases. The ectodomain shedding of the ligands has emerged as a critical component in the functional transactivation of EGFRs in interreceptor cross‐talk in response to various shedding stimulants such as G‐protein coupled receptor agonists, growth factors, cytokines, and various physicochemical stresses. Among the EGFR‐ligands, heparin‐binding EGF‐like growth factor (HB‐EGF) is a prominent ligand in our understanding of the pathophysiological roles of ectodomain shedding in cancer, wound healing, cardiac diseases, etc. Here we focus on ectodomain shedding of the EGF family ligands, especially HB‐EGF by disintegrin and metalloproteases, which are not only key events of receptor cross talk, but also novel intercellular signaling by their carboxy‐terminal fragments to regulate gene expression directly. (Cancer Sci 2008; 99: 214–220)

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.