Prof. Dr. Joachim Grötzinger

Joachim Grötzinger
Joachim Grötzinger
Structure and Function of Proteins
Why control ADAM17 activity?

To avoid malformation and disease, tissue development and homeostasis have to be precisely coordinated in time and space. This requires networks of tightly regulated signaling and interaction events, which are formed by exact interactions of proteins. To ensure proper coordination, proteins, functioning as messengers, receptors, agonists, antagonists, or adhesion molecules, have to be expressed and released at specific time-points. The transmembrane metalloprotease A Disintegrin and Metalloprotease 17 (ADAM17) ensures the timely release of cell surface molecules by a rapid, irreversible proteolytic switch, called shedding. This process generates two of the most important initiators of immune responses, namely, TNFα [1,2] and soluble (s)IL-6R [3], from their membrane-bound counterparts. Notably, prior to shedding, proTNF-α and IL-6R conduct regenerative processes [4-6], highlighting the drastic changes that ADAM17 activity elicits.

Figure 1
Figure 1: ADAM17 activity has to be tightly controlled to allow physiological processes and to prevent pathological situations. ADAM17 sheds more than 70 different substrates [5]; therefore, the enzyme acts as a major sheddase in various physiological and protective processes, such as immune defence, coagulation, and regeneration. By contrast, the uncontrolled release of such potent substrates is associated with numerous pathological conditions, such as uncontrolled inflammation (autoimmune diseases and chronic inflammation) and cancer progression. Hence, ADAM17 is initially kept in an inactive state, e.g., by interaction with inhibitory proteins such as β1-integrin [7-10]. Only upon activation by various stimuli, such as LPS, ATP, and thrombin, does ADAM17 shed its substrates from the cell surface, thereby changing their activities. Later, the enzyme is switched off. This switching off is catalysed by protein disulfide isomerases (PDIs) and is accompanied by a structural change within the extracellular part of ADAM17, which prevents substrate recognition and shedding.

Another prominent class of ADAM17 substrates are growth factors of the EGFR ligand family [11]. These substrates are expressed as membrane-bound preforms; therefore, they are not active prior to shedding. The significance of this process in development is displayed by the perinatality of ADAM17-deficient mice, which show comparable phenotypes to EGFR ligand-deficient mice, including malformations of the skin, hair, eye, heart, and lung [11]. In adult organisms, generation of soluble EGFR ligands is essential for proper regeneration processes [12].
These few examples (and ADAM17 processes more than 70 substrates) illustrate the necessity of this enzyme for appropriate physiological processes such as immune defence and regeneration. However, they also indicate that uncontrolled ADAM17 activity supports misguided inflammation, such as occurs in autoimmune diseases (e.g., rheumatoid arthritis), chronic inflammation (e.g., Crohn's disease), and cancer progression, due to mistimed and enhanced release of pro-inflammatory mediators and growth factors. Hence, ADAM17 has to be tightly controlled to avoid pathophysiological situations and, accordingly, it is of extraordinarily high therapeutic interest. A prerequisite to modulate ADAM17 activity is an in-depth knowledge of the molecular basis of its functioning, including its on and off switches, because ADAM17 has to be activated to become proteolytically active [13, 14] and inactivated to prevent damage. The cytoplasmic tail of ADAM17 is dispensable for its activation by numerous stimuli [15-17]; therefore, we focus on its extracellular part.
Figur 2 Figure 2: Schematic draw­ing of atypical and typical ADAMs.
(A) ADAM17 and its closest rel­ative ADAM10 are atypi­cal mem­bers of the ADAM family because their extra­cel­lular regions comprise a pro-domain, a catalytic do­main, a disintegrin domain, and a membrane-proximal domain (MPD), as well as a small stalk region [18-20].
(B) By contrast, typical members of the ADAM family comprise a cysteine-rich domain and an EGF-like domain in­stead of the MPD [20]. According to the type-I transmembrane protein topology of all ADAM proteases, the extracel­lular part ends in a single transmembrane region and a cytoplasmic tail [20].
The extracellular part of ADAM17
In its mature form, ADAM17 lacks its pro-domain, which retains the zymogen in an inactive state and is removed during maturation in the Golgi apparatus [21,22]. As the names suggest, the catalytic domain cleaves the substrates and the disintegrin domain interacts with β1-integrin. This interaction (Figure 3) supports cell-cell contacts between fibroblasts and tumour cells [10] and keeps the enzyme inactive, most likely due to steric hindrance of substrate recognition [7-10]. Activation of β1-integrin leads to the release of ADAM17 accompanied by the catalytically active enzyme [8,9].
Figur 3 Figure 3: (A) Inactive ADAM17 in­teracts with β1-integrin.
(B) This interaction supports cell-cell ad­hesion between lung carcinoma cells (NCI-H292) and murine embry­onic fibroblasts (MEFs), since the pres­ence of soluble disintegrin do­main reduces adhesion between these cells [10].
(C) Upon stimulation, the ADAM17-β1-integrin complex dissociates and both molecules become active.
(D) If this precise interaction is in­deed mediated by the disintegrin do­main, addition of soluble re­com­bi­nant disintegrin domain should in­crease the shedding activity of ADAM­17 due to competitive binding of soluble disintegrin domain to β1-integrin.
(E) Soluble disintegrin domain in­creases constitutive background shedding of amphiregulin from the cell surface of a breast cancer cell line (MDA-MB-231). These results con­firm that the inhibitory interaction between β1-integrin and ADAM17 is mediated by this specific domain, but that dissociation of the ADAM17-β1-integrin complex is not the only re­quirement for ADAM17 activation [10].
The MPD is supposed to play a key role in the regulation of this enzyme [18, 19, 23-26, 17, 27-30]. In line with this, the MPD controls and coordinates multimerisation [25] and substrate recognition [24] (Figure 4). Notably, ADAM17 recognises its substrates in distinct ways, depending on their membrane topology. An ADAM17 deletion mutant, which solely comprises the MPD and lacks the catalytic and disintegrin domains and the transmembrane and cytoplasmic regions, is sufficient to bind the IL-6R and IL-1RII substrates, both of which have a type-I protein topology, in contrast to the type-II transmembrane protein TNFα (Figure 4B–D). In accordance, replacement of the MPD of ADAM17 with the EGF-like domain of ADAM22 generates a shedding-incompetent chimera (Figure 4I).

Figur 4

Figure 4: Substrate binding and multimerisation of ADAM17 is driven by its MPD/stalk region.
(A) Full-length HA-tagged ADAM17 can recognise, bind, and precipitate its substrate, myc-tagged IL-1RII [24].
(B) A GPI-anchored construct containing the MPD and stalk region of ADAM17, termed MPD17-GPI, tagged with PC can precipitate the type-I proteins IL-1RII (C) and IL-6R (D), but not the type-II protein TNFα. These data indicate that there are marked differences in the recognition mode of at least type-I and type-II class substrates [24].
(E) MPD17-GPI interacts with ADAM17 substrates; therefore, it functions as a competitive inhibitor of the full-length molecule. To examine this, HEK293 cells, which endogenously express ADAM17, were transfected with control or MPD17-GPI-encoding vectors. To obtain proper shedding, cells were stimulated with PMA, DMSO as a control, or PMA plus a metalloprotease inhibitor (either GM6001 or marimastat). As indicated by the red bar, MPD17-GPI significantly decreases the shedding of all tested substrates. The shedding activity of cells transfected with control DNA was set at 100%.
(F) An interaction between MPD17-GPI and TNFα could not be detected by coimmunoprecipitation experiments; therefore, this inhibitory effect might be due to dimerisation of ADAM17, thereby generating inactive ADAM17-MPD17-GPI complexes. Hence, we tested whether ADAM17 forms multimers by cotransfecting and coimmuno­pre­cipitating PC- and HA-tagged full-length molecules, and it was found that ADAM17 indeed forms multimers or at least dimers.
(G) Next, we tested in the same experimental setting whether the MPD with the stalk region (MPD17-GPI) is able to do so, and in fact the MPD17-GPI alone is able to dimerise.
(H) To prove that the MPD is needed for shedding, a chimera was generated in which the MPD of ADAM17 was replaced by the EGF-like domain of ADAM22 (ADAM17EGF22).
(I) The enzymatic activity of this chimera was tested in comparison to that of wild-type ADAM17. The constructs were transfected together with IL-1RII, as the ADAM17 sub­strate, into ADAM17ex/ex MEFs. In these MEFs, ADAM17 expression is reduced by 95%. After 1 day, shedding activity was tested as in HEK293 cells (E). The resulting data indicate that ADAM17 in which the MPD is replaced completely lacks shedding competency because it shows comparable results to eGFP-transfected control cells.

The specific thiol switch within ADAM17
Due to the impact of the MPD in ADAM17 regulation and activity, we were interested in what it actually looks like. Hence, we produced recombinant MPD using bacterial expression systems for structural determination by multidimensional heteronuclear NMR spectroscopy. Soluble expressed MPD is a disulfide isomer that is only partially structured and is a flexible elongated domain (Figure 5C). The MPD comprises a classical thioredoxin motif (C600KVC), which is essential for ADAM17 shedding activity [23], and exogenous PDI supresses ADAM17 activity [18,30]. Therefore, we hypothesise that PDI targets this specific motif. Indeed, PDI catalyses disulfide isomerisation within the flexible open MPD [18]. Two disulfide bonds thereby undergo specific isomerisation. This process changes the affected disulfide pattern from a sequential arrangement (C600-C630 and C635-C641), which is present in the elongated MPD structure, to an overlapping pattern (C600-635 and C630-641), which exists in a compact, rigid-structured MPD. Whereas the latter isoform is associated with the inactive enzyme, the flexible form is associated with active ADAM17 [18]. Hence, the MPD acts as the off-switch of ADAM17, which is operated by exogenous PDI [18].

Figur 5

Figure 5: PDI operates the structural off-switch of ADAM17, its MPD.
(A) The MPD of ADAM17 exists in two conformers, which differ in terms of their disulfide bond pattern and can be se­pa­rated by reverse phase HPLC. Intriguingly, the open MPD can be converted into the closed MPD by reduced PDI [18].
(B) The HSQC spectrum of the open conformer shows fewer signals than the closed conformer. These signals re­pre­sent amide groups of the protein in a defined environment; therefore, these data indicate that the open MPD is less struc­tured than the closed MPD. Intriguingly, signals present in the HSQC of the flexible open MPD appear at the same position as those of the closed isomer, indicating that these parts have identical structures.
(C/D) The N-terminal regions of both isomers show identical structures [18].
(C) By contrast, the C-terminal part is flex­ible in the open MPD, but (D) well-structured in the closed MPD.
(E) The differences in the MPD structures are due to different disulfide bond connections. The open flexible MPD con­tains a sequential arrangement of the two disulphide bond connections (C600-C630 and C635-C640). PDIs catalyse the isomerisation of these specific disulphide bonds into an overlaying arrangement (C600-C635 and C630-C640) [18].
(F) The closed MPD is linked to inactivated ADAM17. Overexpression of PDIA6 reduces ADAM17 activity after PMA stimulation compared to that in mock-transfected control cells.
(G) PDI abrogates the substrate recognition of ADAM17. Treatment of an inactive ADAM17 variant (HE-ADAM17) with PDI abolishes binding of ADAM17, as shown by Western blotting.

Model of ADAM17 inactivation by PDI
Our current knowledge allows us to propose the following model of ADAM17 activity regarding type-I substrates, which can be possibly not fulfil by type-II substrates due to the contrariwise orientation of the protein backbone (Figure 6).


Figure 6: Current model of ADAM17 substrate recognition and inactivation.
  1. Black R. A., Rauch C. T., Kozlosky C. J., Peschon J. J., Slack J. L., Wolfson M. F., Castner B. J., Stocking K. L., Reddy P., Srinivasan S., Nelson N., Boiani N., Schooley K. A., Gerhart M., Davis R., Fitzner J. N., Johnson R. S., Paxton R. J., March C. J. and Cerretti D. P. (1997) A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature. 385: 729-33.
  2. Moss M. L., Jin S. L., Milla M. E., Bickett D. M., Burkhart W., Carter H. L., Chen W. J., Clay W. C., Didsbury J. R., Hassler D., Hoffman C. R., Kost T. A., Lambert M. H., Leesnitzer M. A., McCauley P., McGeehan G., Mitchell J., Moyer M., Pahel G., Rocque W., Overton L. K., Schoenen F., Seaton T., Su J. L., Becherer J. D. and al. e. (1997) Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha. Nature. 385: 733-736.
  3. Müllberg J., Dittrich E., Graeve L., Gerhartz C., Yasukawa K., Taga T., Kishimoto T., Heinrich P. C. and Rose-John S. (1993) Differential shedding of the two subunits of the interleukin-6 receptor. FEBS Lett. 332: 174-8.
  4. Jones S. A., Scheller J. and Rose-John S. (2011) Therapeutic strategies for the clinical blockade of IL-6/gp130 signaling. J Clin Invest. 121: 3375-83.
  5. Scheller J., Chalaris A., Garbers C. and Rose-John S. (2011) ADAM17: a molecular switch to control inflammation and tissue regeneration. Trends Immunol. 32 380-387.
  6. Van Hauwermeiren F., Vandenbroucke R. E. and Libert C. (2011) Treatment of TNF mediated diseases by selective inhibition of soluble TNF or TNFR1. Cytokine Growth Factor Rev. 22: 311-9.
  7. Bax D. V., Messent A. J., Tart J., van Hoang M., Kott J., Maciewicz R. A. and Humphries M. J. (2004) Integrin alpha5beta1 and ADAM-17 interact in vitro and co-localize in migrating HeLa cells. J Biol Chem. 279: 22377-22386.
  8. Göoz P., Dang Y., Higashiyama S., Twal W. O., Haycraft C. J. and Gooz M. (2012) A disintegrin and metalloenzyme (ADAM) 17 activation is regulated by alpha5beta1 integrin in kidney mesangial cells. PLoS One. 7: e33350.
  9. Saha A., Backert S., Hammond C. E., Gooz M. and Smolka A. J. (2010) Helicobacter pylori CagL activates ADAM17 to induce repression of the gastric H, K-ATPase alpha subunit. Gastroenterology. 139: 239-48.
  10. Trad A., Riese M., Shomali M., Hedeman N., Effenberger T., Grötzinger J. and Lorenzen I. (2013) The disintegrin domain of ADAM17 antagonises fibroblastcarcinoma cell interactions. Int J Oncol. 42: 1793-800.
  11. Peschon J. J., Slack J. L., Reddy P., Stocking K. L., Sunnarborg S. W., Lee D. C., Russell W. E., Castner B. J., Johnson R. S., Fitzner J. N., Boyce R. W., Nelson N., Kozlosky C. J., Wolfson M. F., Rauch C. T., Cerretti D. P., Paxton R. J., March C. J. and Black R. A. (1998) An Essential Role for Ectodomain Shedding in Mammalian Development. Science. 282: 1281-1284.
  12. Chalaris A., Adam N., Sina C., Rosenstiel P., Lehmann-Koch J., Schirmacher P., Hartmann D., Cichy J., Gavrilova O., Schreiber S., Jostock T., Matthews V., Hasler R., Becker C., Neurath M. F., Reiss K., Saftig P., Scheller J. and Rose-John S. (2010) Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice. J Exp Med. 207: 1617-24.
  13. Le Gall S. M., Bobé P., Reiss K., Horiuchi K., Niu X. D., Lundell D., Gibb D. R., Conrad D., Saftig P. and Blobel C. P. (2009) ADAMs 10 and 17 Represent Differentially Regulated Components of a General Shedding Machinery for Membrane Proteins such as TGF{alpha}, L-Selectin and TNF{alpha}. Mol Biol Cell. 20: 1785-1794.
  14. Le Gall S. M., Maretzky T., Issuree P. D., Niu X. D., Reiss K., Saftig P., Khokha R., Lundell D. and Blobel C. P. (2010) ADAM17 is regulated by a rapid and reversible mechanism that controls access to its catalytic site. J Cell Sci. 123: 3913-3922.
  15. Hall K. C. and Blobel C. P. (2012) Interleukin-1 Stimulates ADAM17 through a Mechanism Independent of its Cytoplasmic Domain or Phosphorylation at Threonine 735. PLoS One. 7: e31600.
  16. Maretzky T., Zhou W., Huang X. Y. and Blobel C. P. (2011) A transforming Src mutant increases the bioavailability of EGFR ligands via stimulation of the cell-surface metalloproteinase ADAM17. Oncogene. 30: 611-8.
  17. Reddy P., Slack J. L., Davis R., Cerretti D. P., Kozlosky C. J., Blanton R. A., Shows D., Peschon J. J. and Black R. A. (2000) Functional analysis of the domain structure of tumor necrosis factor-alpha converting enzyme. J Biol Chem. 275: 14608-14.
  18. Düsterhöft S., Jung S., Hung C. W., Tholey A., Sönnichsen F. D., Grötzinger J. and Lorenzen I. (2013) Membrane-proximal domain of a disintegrin and metalloprotease-17 represents the putative molecular switch of its shedding activity operated by protein-disulfide isomerase. J Am Chem Soc. 135: 5776-81.
  19. Janes P. W., Saha N., Barton W. A., Kolev M. V., Wimmer-Kleikamp S. H., Nievergall E., Blobel C. P., Himanen J. P., Lackmann M. and Nikolov D. B. (2005) Adam meets Eph: an ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans. Cell. 123: 291-304.
  20. Takeda S. (2009) Three-dimensional domain architecture of the ADAM family proteinases. Semin Cell Dev Biol. 20: 146-52.
  21. Endres K., Anders A., Kojro E., Gilbert S., Fahrenholz F. and Postina R. (2003) Tumor necrosis factor-alpha converting enzyme is processed by proprotein-convertases to its mature form which is degraded upon phorbol ester stimulation. Eur J Biochem. 270: 2386-93.
  22. Srour N., Lebel A., McMahon S., Fournier I., Fugere M., Day R. and Dubois C. M. (2003) TACE/ADAM-17 maturation and activation of sheddase activity require proprotein convertase activity. FEBS Lett. 554: 275-83.
  23. Li X. and Fan H. (2004) Loss of ectodomain shedding due to mutations in the metalloprotease and cysteine-rich/disintegrin domains of the tumor necrosis factor-alpha converting enzyme (TACE). J Biol Chem. 279: 27365-75.
  24. Lorenzen I., Lokau J., Düsterhoft S., Trad A., Garbers C., Scheller J., Rose-John S. and Grötzinger J. (2012) The membrane-proximal domain of A Disintegrin and Metalloprotease 17 (ADAM17) is responsible for recognition of the interleukin-6 receptor and interleukin-1 receptor II. FEBS Lett. 586: 1093-1100.
  25. Lorenzen I., Trad A. and Grötzinger J. (2011) Multimerisation of A disintegrin and metalloprotease protein-17 (ADAM17) is mediated by its EGF-like domain. Biochem Biophys Res Commun. 415: 330-6.
  26. Milla M. E., Leesnitzer M. A., Moss M. L., Clay W. C., Carter H. L., Miller A. B., Su J. L., Lambert M. H., Willard D. H., Sheeley D. M., Kost T. A., Burkhart W., Moyer M., Blackburn R. K., Pahel G. L., Mitchell J. L., Hoffman C. R. and Becherer J. D. (1999) Specific sequence elements are required for the expression of functional tumor necrosis factor-alpha-converting enzyme (TACE). J Biol Chem. 274: 30563-30570.
  27. Takeda S., Igarashi T., Mori H. and Araki S. (2006) Crystal structures of VAP1 reveal ADAMs' MDC domain architecture and its unique C-shaped scaffold. EMBO J. 25: 2388-96.
  28. Tape C. J., Willems S. H., Dombernowsky S. L., Stanley P. L., Fogarasi M., Ouwehand W., McCafferty J. and Murphy G. (2011) Cross-domain inhibition of TACE ectodomain. Proc Natl Acad Sci U S A. 108: 5578-83.
  29. Wang Y., Herrera A. H., Li Y., Belani K. K. and Walcheck B. (2009) Regulation of mature ADAM17 by redox agents for L-selectin shedding. J Immunol. 182: 2449-2457.
  30. Willems S. H., Tape C. J., Stanley P. L., Taylor N. A., Mills I. G., Neal D. E., McCafferty J. and Murphy G. (2010) Thiol isomerases negatively regulate the cellular shedding activity of ADAM17. Biochem J. 428: 439-50.
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