Die membranproximale Domäne von ADAM17 - Ein Schlüssel zur Regulation der Proteaseaktivität

Ektodomänen-Shedding stellt eine irreversible, posttranslationale Modifikation dar, die unter anderem zur Freisetzung von biologisch aktiven Ektodomänen führt. A Disintegrin And Metalloprotease 17 (ADAM17) ist mit über 80 Substraten eine, für physiologische Prozesse wie Entzündungsreaktionen oder Regenerationsprozesse, wichtige Sheddase. Trotz der bedeutenden Rolle von ADAM17 ist bis heute nur wenig über die Regulation ihrer Shedding-Aktivität bekannt. In früheren Studien wurde gezeigt, dass extrazelluäre Proteindisulfidisomerasen (PDIs) mit ADAM17 interagieren und eine strukturelle Änderung innerhalb der Ektodomäne von ADAM17 herbeiführen. Dies hat eine Inaktivierung von ADAM17 zur Folge. In dieser Arbeit wurde gezeigt, dass die PDIs mit der membranproximalen Domäne von ADAM17 (MPD17) interagieren und diese durch die Isomerisierung zweier Disulfidbrücken von einer offenen in eine geschlossene Form überführen. Dabei liegt im aktiven ADAM17 die offene, flexible MPD17 vor, während im inaktiven Zustand die MPD17 die geschlossene Form einnimmt. Der Wechsel von der offenen zu der geschlossenen Form reguliert CANDIS (Conserved ADAM-SeventeeN Dynamic Interaction Sequence), ein hochkonservierter Bereich innerhalb der Stalk-Region von ADAM17, der in der Lage ist die beiden Substrate IL-6R und IL-1RII zu binden. Nur wenn die MPD17 in der offenen Form vorliegt, werden diese Substrate von CANDIS gebunden. Die geschlossene MPD17 verhindert hingegen den Zugang der Substrate zu CANDIS. Neue Daten zeigen, dass das ADAM17-vermittelte Shedding-Ereignis durch eine transiente Phosphatidylserin-Translokation ausgelöst wird. In dieser Arbeit wurde in diesem Zusammenhang geklärt, dass nur die offene MPD17, und nicht die geschlossene Form, spezifisch Ortho-Phospho-L-Serin (OPLS) bindet. Daraus lässt sich schließen, dass das Shedding-Ereignis in Zusammenhang mit einer Bindung der offenen MPD17 an die Phosphatidylserinpräsentierende Zellmembran steht. Diese Bindung ist in der geschlossenen, inaktiven Form nicht möglich. Die Ergebnisse dieser Arbeit zeigen die fundamentale Bedeutung der MPD17 und CANDIS für die Shedding-Aktivität von ADAM17 und ermöglichen so ein tieferes Verständnis der ADAM17-Regulation.Ectodomain shedding is an irreversible posttranslational modification which leads to the release of biologically active ectodomains of transmembrane proteins. A disintegrin and metalloprotease 17 (ADAM17) is an important sheddase which can cleave more than 80 different substrates and is therefore involved in various physiological and pathophysiological processes, including inflammation and regeneration. Despite the importance of ADAM17, the regulation of the shedding activity is scarcely understood. It is known from previous studies that extracellular protein disulphide isomerases (PDIs) interact with ADAM17 which results in a structural change in the ectodomain of the protease. This interaction leads to the inactivation of ADAM17. In this study, it was shown that the PDIs interact with the membrane-proximal domain of ADAM17 (MPD17) and that this domain can be converted from an open conformation into a closed one through isomerisation of disulphide-bonds. Thereby, the open and more flexible form relates to the active ADAM17 while the MPD17 shows the closed and rather rigid conformation in the inactive protease. The shift from the open to the closed conformation regulates CANDIS (Conserved ADAM Seventeen Dynamic Interaction Sequence), a highly conserved sequence within the stalk region of ADAM17 which is able to bind the two substrates IL-6R and IL-1RII. This interaction can only occur when the MPD17 is in the open conformation while the closed form prevents the interaction between CANDIS and the substrates. Recent results show that ADAM17 mediated shedding can be triggered by transient exposure of phosphatidylserine (PS). Along that line, it was shown in this study that solely the active form of MPD17, but not the closed one, can bind to OPLS (ortho-phospho-l-serine). This leads to the conclusion that an ADAM17 mediated shedding event includes the binding of the open conformation of the MPD17 to the PS-presenting plasma membrane. This binding does not occur when MPD17 is in the closed conformation. In conclusion, this study shows the fundamental relevance of the MPD17 and CANDIS for the shedding activity and allows a deeper understanding of the regulation of ADAM17

Interleukin-11 (IL-11) receptor cleavage by the rhomboid protease RHBDL2 induces IL-11 trans-signaling

Interleukin-11 (IL-11) is a pleiotropic cytokine with both pro- and anti-inflammatory properties. It activates its target cells via binding to the membrane-bound IL-11 receptor (IL-11R), which then recruits a homodimer of the ubiquitously expressed, signal-transducing receptor gp130. Besides this classic signaling pathway, IL-11 can also bind to soluble forms of the IL-11R (sIL-11R), and IL-11/sIL-11R complexes activate cells via the induction of gp130 homodimerization (trans-signaling). We have previously reported that the metalloprotease ADAM10 cleaves the membrane-bound IL-11R and thereby generates sIL-11R. In this study, we identify the rhomboid intramembrane protease RHBDL2 as a so far unrecognized alternative sheddase that can efficiently trigger IL-11R secretion. We determine the cleavage site used by RHBDL2, which is located in the extracellular part of the receptor in close proximity to the plasma membrane, between Ala-370 and Ser-371. Furthermore, we identify critical amino acid residues within the transmembrane helix that are required for IL-11R proteolysis. We also show that ectopically expressed RHBDL2 is able to cleave the IL-11R within the early secretory pathway and not only at the plasma membrane, indicating that its subcellular localization plays a central role in controlling its activity. Moreover, RHBDL2-derived sIL-11R is biologically active and able to perform IL-11 trans-signaling. Finally, we show that the human mutation IL-11R-A370V does not impede IL-11 classic signaling, but prevents RHBDL2-mediated IL-11R cleavage

The iRhom2/ADAM17 Axis Attenuates Bacterial Uptake by Phagocytes in a Cell Autonomous Manner

Uptake of bacteria by phagocytes is a crucial step in innate immune defence. Members of the disintegrin and metalloproteinase (ADAM) family critically control the immune response by limited proteolysis of surface expressed mediator molecules. Here, we investigated the significance of ADAM17 and its regulatory adapter molecule iRhom2 for bacterial uptake by phagocytes. Inhibition of metalloproteinase activity led to increased phagocytosis of pHrodo labelled Gram-negative and -positive bacteria (E. coli and S. aureus, respectively) by human and murine monocytic cell lines or primary phagocytes. Bone marrow-derived macrophages showed enhanced uptake of heat-inactivated and living E. coli when they lacked either ADAM17 or iRhom2 but not upon ADAM10-deficiency. In monocytic THP-1 cells, corresponding short hairpin RNA (shRNA)-mediated knockdown confirmed that ADAM17, but not ADAM10, promoted phagocytosis of E. coli. The augmented bacterial uptake occurred in a cell autonomous manner and was accompanied by increased release of the chemokine CXCL8, less TNFα release and only minimal changes in the surface expression of the receptors TNFR1, TLR6 and CD36. Inhibition experiments indicated that the enhanced bacterial phagocytosis after ADAM17 knockdown was partially dependent on TNFα-activity but not on CXCL8. This novel role of ADAM17 in bacterial uptake needs to be considered in the development of ADAM17 inhibitors as therapeutics

The metalloproteinase ADAM10 requires its activity to sustain surface expression

The metalloproteinase ADAM10 critically contributes to development, inflammation, and cancer and can be controlled by endogenous or synthetic inhibitors. Here, we demonstrate for the first time that loss of proteolytic activity of ADAM10 by either inhibition or loss of function mutations induces removal of the protease from the cell surface and the whole cell. This process is temperature dependent, restricted to mature ADAM10, and associated with an increased internalization, lysosomal degradation, and release of mature ADAM10 in extracellular vesicles. Recovery from this depletion requires de novo synthesis. Functionally, this is reflected by loss and recovery of ADAM10 substrate shedding. Finally, ADAM10 inhibition in mice reduces systemic ADAM10 levels in different tissues. Thus, ADAM10 activity is critically required for its surface expression in vitro and in vivo. These findings are crucial for development of therapeutic ADAM10 inhibition strategies and may showcase a novel, physiologically relevant mechanism of protease removal due to activity loss

Secreted Frizzled-related protein 3 (sFRP3)-mediated suppression of Interleukin-6 receptor release by A disintegrin and Metalloprotease 17 (ADAM17) is abrogated in the osteoarthritis-associated rare double variant of sFRP3

To avoid malformation and disease, tissue development and homeostasis are coordinated precisely in time and space. Secreted Frizzled-related protein 3 (sFRP3), encoded by the Frizzled-related protein gene (FRZB), acts as an antagonist of Wnt signalling in bone development by delaying the maturation of proliferative chondrocytes into hypertrophic chondrocytes. A disintegrin and metalloprotease 17 (ADAM17) is a transmembrane protease that is essential for developmental processes and promotes cartilage maturation into bone. sFRP3 is chondroprotective and is expressed in chondrocytes of healthy articular cartilage. Upon damage to cartilage, sFRP3 is down-regulated. Rare variants of sFRP3 are associated with osteoarthritis. This study demonstrates a novel function of sFRP3 in suppression of the enzymatic activity of ADAM17 which results in the inhibition of ADAM17-meditated interleukin-6 receptor (IL-6R) shedding. By contrast, the rare double variant of sFRP3 failed to suppress ADAM17. The shed soluble IL-6R is linked to inflammation, cartilage degeneration, and osteolysis. Accordingly, enhanced activity of ADAM17 in cartilage, caused by the expression of the rare double sFRP3 variant, provides an explanation for the genetic effect of sFRP3 variants in joint disease. The finding that sFRP3 interacts with the ADAM17 substrate IL-6R also suggests a new regulatory mechanism by which the substrate is protected against shedding.status: publishe