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Dieter Hartmann – One of the best experts on this subject based on the ideXlab platform.

Andreas Ludwig – One of the best experts on this subject based on the ideXlab platform.

  • discovery of an enzyme and substrate selective inhibitor of adam10 using an exosite binding glycosylated substrate
    Scientific Reports, 2016
    Co-Authors: Franck Madoux, Andreas Ludwig, Christoph Beckerpauly, Daniela Dreymuller, Jeanphillipe Pettiloud, Radleigh Santos, Gregg B Fields, Thomas D Bannister, Timothy P Spicer, Mare Cudic
    Abstract:

    ADAM10 and ADAM17 have been shown to contribute to the acquired drug resistance of HER2-positive breast cancer in response to trastuzumab. The majority of ADAM10 and ADAM17 inhibitor development has been focused on the discovery of compounds that bind the active site zinc, however, in recent years, there has been a shift from active site to secondary substrate binding site (exosite) inhibitor discovery in order to identify non-zinc-binding molecules. In the present work a glycosylated, exosite-binding substrate of ADAM10 and ADAM17 was utilized to screen 370,276 compounds from the MLPCN collection. As a result of this uHTS effort, a selective, time-dependent, non-zinc-binding inhibitor of ADAM10 with Ki = 883 nM was discovered. This compound exhibited low cell toxicity and was able to selectively inhibit shedding of known ADAM10 substrates in several cell-based models. We hypothesize that differential glycosylation of these cognate substrates is the source of selectivity of our novel inhibitor. The data indicate that this novel inhibitor can be used as an in vitro and, potentially, in vivo, probe of ADAM10 activity. Additionally, results of the present and prior studies strongly suggest that glycosylated substrate are applicable as screening agents for discovery of selective ADAM probes and therapeutics.

  • adam10 is the major sheddase responsible for the release of membrane associated meprin a
    Journal of Biological Chemistry, 2014
    Co-Authors: C Herzog, Andreas Ludwig, Randy S Haun, Sudhir V Shah, Gur P Kaushal
    Abstract:

    Meprin A, composed of α and β subunits, is a membrane-bound metalloproteinase in renal proximal tubules. Meprin A plays an important role in tubular epithelial cell injury during acute kidney injury (AKI). The present study demonstrated that during ischemia-reperfusion-induced AKI, meprin A was shed from proximal tubule membranes, as evident from its redistribution toward the basolateral side, proteolytic processing in the membranes, and excretion in the urine. To identify the proteolytic enzyme responsible for shedding of meprin A, we generated stable HEK cell lines expressing meprin β alone and both meprin α and meprin β for the expression of meprin A. Phorbol 12-myristate 13-acetate and ionomycin stimulated ectodomain shedding of meprin β and meprin A. Among the inhibitors of various proteases, the broad spectrum inhibitor of the ADAM family of proteases, tumor necrnecrosis factor-α protease inhibitor (TAPI-1), was most effective in preventing constitutive, phorbol 12-myristate 13-acetate-, and ionomycin-stimulated shedding of meprin β and meprin A in the medium of both transfectants. The use of differential inhibitors for ADAM10 and ADAM17 indicated that ADAM10 inhibition is sufficient to block shedding. In agreement with these results, small interfering RNA to ADAM10 but not to ADAM9 or ADAM17 inhibited meprin β and meprin A shedding. Furthermore, overexpression of ADAM10 resulted in enhanced shedding of meprin β from both transfectants. Our studies demonstrate that ADAM10 is the major ADAM metalloproteinase responsible for the constitutive and stimulated shedding of meprin β and meprin A. These studies further suggest that inhibiting ADAM 10 activity could be of therapeutic benefit in AKI.

  • The role of ADAM-mediated shedding in vascular biology.
    European journal of cell biology, 2011
    Co-Authors: Daniela Dreymueller, Jessica Pruessmeyer, Ester Groth, Andreas Ludwig
    Abstract:

    Within the vasculature the disintegrins and metalloproteinases (ADAMs) 8, 9, 10, 12, 15, 17, 19, 28 and 33 are expressed on endothelial cells, smooth muscle cells and on leukocytes. As surface-expressed proteases they mediate cleavage of vascular surface molecules at an extracellular site close to the membrane. This process is termed shedding and leads to the release of a soluble substrate ectodomain thereby critically modulating the biological function of the substrate. In the vasculature several surface molecules undergo ADAM-mediated shedding including tumour necrosis factor (TNF) α, interleukin (IL) 6 receptor α, L-seleselectin, vascular endothelial (VE)-cadherin, the transmembrane CX3C-chemokine ligand (CX3CL) 1, Notch, transforming growgrowth factor (TGF) and heparin-binding epidermal growth factor (HB-EGF). These substrates play distinct roles in vascular biology by promoting inflammation, permeability changes, leukocyte recruitment, resolution of inflammation, regeneration and/or neovascularisation. Especially ADAM17 and ADAM10 are capable of cleaving many substrates with diverse function within the vasculature, whereas other ADAMs have a more restricted substrate range. Therefore, targeting ADAM17 or ADAM10 by pharmacologic inhibition or gene knockout not only attenuates the inflammatory response in animal models but also affects tissue regeneration and neovascularisation. Recent discoveries indicate that other ADAMs (e.g. ADAM8 and 9) also play important roles in vascular biology but appear to have more selective effects on vascular responses (e.g. on neovascularisation only). Although, targeting of ADAM17 and ADAM10 in inflammatory diseases is still a promising approach, temporal and spatial as well as substrate-specific inhibition approaches are required to minimise undesired side effects on vascular cells.

Paul Saftig – One of the best experts on this subject based on the ideXlab platform.

  • 230: Unraveling the role of Adam17 in IL-6 trans-signaling
    Cytokine, 2013
    Co-Authors: Jeanette Schwarz, Paul Saftig, Isabell Yan, Olga Will, Stefan-rose-john, Hans-willi Mittrücker, Athena Chalaris
    Abstract:

    IL-6 is a key regulator of immune responses after bacterial infection. IL-6 signaling is mediated via the receptor subunits IL-6R and gp130. The signal transducing subunit gp130 is ubiquitously expressed whereas IL-6R expression is restricted to hepatocytes and some leukocytes. The IL-6R can be cleaved from the cell surface by ADAM proteases. The soluble form of the IL-6R (sIL-6R) has the same IL-6 binding affinity as the membrane-bound receptor. The resulting IL-6/sIL-6R complex activates cells, which only express gp130 on their cell surface, a process called trans-signaling. Thus, IL-6-transsignaling following IL-6R proteolysis essentially renders all cells of the body responsive to IL-6. The proteolytic web controlling IL-6R shedding in vivo under pathophysiological conditions is poorly characterized. Recently, it was demonstrated that human IL-6R is a substrate for the protease ADAM17 in contrast to the murine ADAM17, which is mainly cleaved by ADAM10. However, these data were raised in cell culture systems. We analyzed IL-6R shedding in vivo on leukocytes after infection with Listeria monocytogenes or LPS-challenge of ADAM17 and ADAM10-deficient mice. ADAM17 and ADAM10 knockout mice are not viable. For this reason we generated a hypomorphic ADAM17 mouse model which expresses only 5% of the normal ADAM17 levels. Additionally we use conditional ADAM10 mice with ADAM10 deleted either in monocytes or Tcells. Listeria infection led to massive shedding of the IL-6R on Tcells and inflammatory monocytes. Further we could demonstrate that Listeria -induced IL-6R cleavage is abrogated in hypomorphic ADAM17 ex/ex mice. Additionally we can show that serum levels of sIL-6R are elevated after LPS challenge of wildtype but not ADAM17 ex/ex mice. These results strongly suggest that ADAM17 is the main IL-6R sheddase under pathophysiological conditions. In future experiments we will evaluate the contribution of the protease ADAM10 in mediating IL-6R shedding to uncover the proteolytic steps involved in IL-6 trans-signaling.

  • the a disintegrin and metalloproteases adam10 and adam17 novel drug targets with therapeutic potential
    European Journal of Cell Biology, 2011
    Co-Authors: Paul Saftig, Karina Reiss
    Abstract:

    Proteolytic ectodomain release, a process known as “shedding”, has been recognised as a key mechanism for regulating the function of a diversity of cell surface proteins. A Disintegrin And Metalloproteinases (ADAMs) have emerged as the major proteinase family that mediates ectodomain shedding. Dysregulation of ectodomain shedding is associated with autoimmune and cardiovascular diseases, neurodegeneration, infection, inflammation and cancer. Therefore, ADAMs are increasingly regarded as attractive targets for novel therapies. ADAM10 and its close relative ADAM17 (TNF-alpha converting enzyme (TACE)) have been studied in particular in the context of ectodomain shedding and have been demonstrated as key molecules in most of the shedding events characterised to date. Whereas the level of expression of ADAM10 may be of importance in cancer and neurodegenerative disorders, ADAM17 mainly coordinates pro- and anti-inflammatory activities during immune response. Despite the high therapeutical potential of ADAM inhibition, all clinical trials using broad-spectrum metalloprotease inhibitors have failed so far. This review will cover the emerging roles of both ADAM10 and ADAM17 in the regulation of major physiological and developmental pathways and will discuss the suitability of specifically modulating the activities of both proteases as a feasible way to inhibit inflammatory states, cancer and neurodegeneration.

  • metalloproteases regulate t cell proliferation and effector function via lag 3
    The EMBO Journal, 2007
    Co-Authors: Nianyu Li, Dieter Hartmann, Paul Saftig, Yao Wang, Karen Forbes, Kate M Vignali, Bret S E Heale, Roy A. Black, John J. Rossi
    Abstract:

    Tight control of T‐cell proliferation and effector function is essential to ensure an effective but appropriate immune response. Here, we reveal that this is controlled by the metalloprotease‐mediated cleavage of LAG‐3, a negative regulatory protein expressed by all activated T cells. We show that LAG‐3 cleavage is mediated by two transmembrane metalloproteases, ADAM10 and ADAM17, with the activity of both modulated by two distinct T‐cell receptor (TCR) signaling‐dependent mechanisms. ADAM10 mediates constitutive LAG‐3 cleavage but increases ∼12‐fold following T‐cell activation, whereas LAG‐3 shedding by ADAM17 is induced by TCR signaling in a PKCθ‐dependent manner. LAG‐3 must be cleaved from the cell surface to allow for normal T‐cell activation as noncleavable LAG‐3 mutants prevented proliferation and cytokine production. Lastly, ADAM10 knockdown reduced wild‐type but not LAG‐3 −/− T‐cell proliferation. These data demonstrate that LAG‐3 must be cleaved to allow efficient T‐cell proliferation and cytokine production and establish a novel paradigm in which T‐cell expansion and function are regulated by metalloprotease cleavage with LAG‐3 as its sole molecular target.

P Saftig – One of the best experts on this subject based on the ideXlab platform.

  • ADAMs 10 and 17 Represent Differentially Regulated Components of a General Shedding Machinery for Membrane Proteins Such as Transforming Growth Factor α, L-Selectin, and Tumor Necrosis Factor α
    Molecular biology of the cell, 2009
    Co-Authors: Sylvain Le Gall, Karina Reiss, Pierre Bobe, Xiao Da Niu, Daniel Lundell, David R. Gibb, Daniel Conrad, P Saftig
    Abstract:

    Protein ectodomain shedding is a critical regulator of many membrane proteins, including epidermal growth factor receptor-ligands and tumor necrnecrosis factor (TNF)-alpha, providing a strong incentive to define the responsible sheddases. Previous studies identified ADAM17 as principal sheddase for transforming growgrowth factor (TGF)-alpha and heparin-binding epidermal growth factor, but Ca++ influx activated an additional sheddase for these epidermal growth factor receptor ligands in Adam17-/- cells. Here, we show that Ca++ influx and stimulation of the P2X7R signaling pathway activate ADAM10 as sheddase of many ADAM17 substrates in Adam17-/- fibroblasts and primary B cells. Importantly, although ADAM10 can shed all substrates of ADAM17 tested here in Adam17-/- cells, acute treatment of wild-type cells with a highly selective ADAM17 inhibitor (SP26) showed that ADAM17 is nevertheless the principal sheddase when both ADAMs 10 and 17 are present. However, chronic treatment of wild-type cells with SP26 promoted processing of ADAM17 substrates by ADAM10, thus generating conditions such as in Adam17-/- cells. These results have general implications for understanding the substrate selectivity of two major cellular sheddases, ADAMs 10 and 17.

  • evaluation of the contribution of different adams to tumor necrosis factor α tnfα shedding and of the function of the tnfα ectodomain in ensuring selective stimulated shedding by the tnfα convertase tace adam17
    Journal of Biological Chemistry, 2004
    Co-Authors: Yufang Zheng, P Saftig, Dieter Hartmann
    Abstract:

    Abstract Tumor necrosis factor-α (TNFα), a potent pro-inflammatory cytokine, is released from cells by proteolytic cleavage of a membrane-anchored precursor. The TNF-α converting enzyme (TACE; a disintegrin and metalloprotease17; ADAM17) is known to have a key role in the ectodomain shedding of TNFα in several cell types. However, because purified ADAMs 9, 10, and 19 can also cleave a peptide corresponding to the TNFα cleavage site in vitro, these enzymes are considered to be candidate TNFα sheddases as well. In this study we used cells lacking ADAMs 9, 10, 17 (TACE), or 19 to address the relative contribution of these ADAMs to TNFα shedding in cell-based assays. Our results corroborate that ADAM17, but not ADAM9, -10, or -19, is critical for phorbol ester- and pervanadate-stimulated release of TNFα in mouse embryonic fibroblasts. However, overexpression of ADAM19 increased the constitutive release of TNFα, whereas overexpression of ADAM9 or ADAM10 did not. This suggests that ADAM19 may contribute to TNFα shedding, especially in cells or tissues where it is highly expressed. Furthermore, we used mutagenesis of TNFα to explore which domains are important for its stimulated processing by ADAM17. We found that the cleavage site of TNFα is necessary and sufficient for cleavage by ADAM17. In addition, the ectodomain of TNFα makes an unexpected contribution to the selective cleavage of TNFα by ADAM17: it prevents one or more other enzymes from cleaving TNFα following PMA stimulation. Thus, selective stimulated processing of TNFα by ADAM17 in cells depends on the presence of an appropriate cleavage site as well as the inhibitory role of the TNF ectodomain toward other enzymes that can process this site.

Jurgen Scheller – One of the best experts on this subject based on the ideXlab platform.

  • human and murine interleukin 23 receptors are novel substrates for a disintegrin and metalloproteases adam10 and adam17
    Journal of Biological Chemistry, 2016
    Co-Authors: Manuel Franke, Christoph Garbers, Doreen M Floss, Christoph Beckerpauly, Bjorn Rabe, Jutta Schroder, Niloufar Monhasery, Theresa Ackfeld, Thorben M Hummel, Jurgen Scheller
    Abstract:

    IL-23 (interleukin 23) regulates immune responses against pathogens and plays a major role in the differentiation and maintenance of TH17 cells and the development of autoimmune diseases and cancer. The IL-23 receptor (IL-23R) complex consists of the unique IL-23R and the common IL-12 receptor β1 (IL-12Rβ1). Differential splicing generates antagonistic soluble IL-23R (sIL-23R) variants, which might limit IL-23-mediated immune responses. Here, ectodomain shedding of human and murine IL-23R was identified as an alternative pathway for the generation of sIL-23R. Importantly, proteolytically released sIL-23R has IL-23 binding activity. Shedding of IL-23R was induced by stimulation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), but not by ionomycin. PMA-induced shedding was abrogated by an ADAM (A disintegrin and metalloprotease) 10 and 17 selective inhibitor, but not by an ADAM10 selective inhibitor. ADAM17-deficient but not ADAM10-deficient HEK293 cells failed to shed IL-23R after PMA stimulation, demonstrating that ADAM17 but not ADAM10 cleaves the IL-23R. Constitutive shedding was, however, inhibited by an ADAM10 selective inhibitor. Using deletions and specific amino acid residue exchanges, we identified critical determinants of ectodomain shedding within the stalk region of the IL-23R. Finally, interaction studies identified domains 1 and 3 of the IL-23R as the main ADAM17 binding sites. In summary, we describe human and murine IL-23R as novel targets for protein ectodomain shedding by ADAM10 and ADAM17.

  • t cell immunoglobulin and mucin domain 2 tim 2 is a target of adam10 mediated ectodomain shedding
    FEBS Journal, 2014
    Co-Authors: Christin Dewitz, Jurgen Scheller, Athena Chalaris, Karina Reiss, Katja Mollerhackbarth, Olga Schweigert, Stefan Rosejohn
    Abstract:

    T-cell immunoglobulin and mucin domain (TIM)-2 is expressed on activated B cells. Here, we provide evidence that murine TIM-2 is a target of ADAM10-mediated ectodomain shedding, resulting in the generation of a soluble form of TIM-2. We identified ADAM10 but not ADAM17 as the major sheddase of TIM-2, as shown by pharmacological ADAM10 inhibition and with ADAM10-deficient and ADAM17-deficient murine embryonic fibroblasts. Ionomycin-induced or 2′(3′)-O-(4-benzoylbenzoyl) ATP triethylammonium salt-induced shedding of TIM-2 was abrogated by deletion of 10 juxtamembrane amino acids from the stalk region but not by deletion of two further N-terminally located blocks of 10 amino acids, indicating a membrane-proximal cleavage site. TIM-2 lacking the intracellular domain was cleaved after ionomycin or 2′ (3′)-O-(4-benzoylbenzoyl) ATP triethylammonium salt treatment, indicating that this domain was not involved in the regulation of ectodomain shedding. Moreover, TIM-2 shedding was negatively controlled by calmodulin. Shed and soluble TIM-2 interacted with H-ferritin. In summary, we describe TIM-2 as a novel target for ADAM10-mediated ectodomain shedding, and reveal the involvement of ADAM proteases in cellular iron homeostasis.

  • a disintegrin and metalloprotease adam 10 and adam17 are major sheddases of t cell immunoglobulin and mucin domain 3 tim 3
    Journal of Biological Chemistry, 2013
    Co-Authors: Katja Mollerhackbarth, Christoph Garbers, Stefan Rosejohn, Christin Dewitz, Olga Schweigert, Ahmad Trad, Jurgen Scheller
    Abstract:

    T cell immunoglobulin and mucin domain 3 (Tim-3) dampens the response of CD4+ and CD8+ effector T cells via induction of cell death and/or T cell exhaustion and enhances the ability of macrophages to clear pathogens via binding to galectin 9. Here we provide evidence that human Tim-3 is a target of A disintegrin and metalloprotease (ADAM)-mediated ectodomain shedding resulting in a soluble form of Tim-3. We identified ADAM10 and ADAM17 as major sheddases of Tim-3 as shown by ADAM-specific inhibitors and the ADAM10 pro-domain in HEK293 cells and ADAM10/ADAM17-deficient murine embryonic fibroblasts. PMA-induced shedding of Tim-3 was abrogated by deletion of amino acids Glu181–Asp190 of the stalk region and Tim-3 lacking the intracellular domain was not efficiently cleaved after PMA stimulation. Surprisingly, a single lysine residue within the intracellular domain rescues shedding of Tim-3. Shedding of endogenous Tim-3 was found in primary human CD14+ monocytes after PMA and ionomycin stimulation. Importantly, the recently described down-regulation of Tim-3 from Toll-like receptor-activated CD14+ monocytes was caused by ADAM10- and ADAM17-mediated shedding. Inhibition of Tim-3 shedding from lipopolysaccharide-induced monocytes did not influence lipopolysaccharide-induced TNFα and IL-6 but increases IL-12 expression. In summary, we describe Tim-3 as novel target for ADAM-mediated ectodomain shedding and suggest a role of Tim-3 shedding in TLR-mediated immune responses of CD14+ monocytes.