Structural basis for membrane-proximal proteolysis of substrates by ADAM10.

The endopeptidase ADAM10 is a critical catalyst for the regulated proteolysis of key drivers of mammalian development, physiology, and non-amyloidogenic cleavage of APP as the primary α-secretase. ADAM10 function requires the formation of a complex with a C8-tetraspanin protein, but how tetraspanin binding enables positioning of the enzyme active site for membrane-proximal cleavage remains unknown. We present here a cryo-EM structure of a vFab-ADAM10-Tspan15 complex, which shows that Tspan15 binding relieves ADAM10 autoinhibition and acts as a molecular measuring stick to position the enzyme active site about 20 Å from the plasma membrane for membrane-proximal substrate cleavage. Cell-based assays of N-cadherin shedding establish that the positioning of the active site by the interface between the ADAM10 catalytic domain and the bound tetraspanin influences selection of the preferred cleavage site. Together, these studies reveal the molecular mechanism underlying ADAM10 proteolysis at membrane-proximal sites and offer a roadmap for its modulation in disease.

ADAM10 mediates shedding of carbonic anhydrase IX ectodomain non­redundantly to ADAM17.

Carbonic anhydrase IX (CA IX) is a transmembrane enzyme participating in adaptive responses of tumors to hypoxia and acidosis. CA IX regulates pH, facilitates metabolic reprogramming, and supports migration, invasion and metastasis of cancer cells. Extracellular domain (ECD) of CA IX can be shed to medium and body fluids by a disintegrin and metalloproteinase (ADAM) 17. Here we show for the first time that CA IX ECD shedding can be also executed by ADAM10, a close relative of ADAM17, via an overlapping cleavage site in the stalk region of CA IX connecting its exofacial catalytic site with the transmembrane region. This finding is supported by biochemical evidence using recombinant human ADAM10 protein, colocalization of ADAM10 with CA IX, ectopic expression of a dominant­negative mutant of ADAM10 and RNA interference­mediated suppression of ADAM10. Induction of the CA IX ECD cleavage with ADAM17 and/or ADAM10 activators revealed their additive effect. Similarly, additive effect was observed with an ADAM17­inhibiting antibody and an ADAM10­preferential inhibitor GI254023X. These data indicated that ADAM10 is a CA IX sheddase acting on CA IX non­redundantly to ADAM17.

Protein secondary structure determines the temporal relationship between folding and disulfide formation.

How and when disulfide bonds form in proteins relative to the stage of their folding is a fundamental question in cell biology. Two models describe this relationship: the folded precursor model, in which a nascent structure forms before disulfides do, and the quasi-stochastic model, where disulfides form prior to folding. Here we investigated oxidative folding of three structurally diverse substrates, ß2-microglobulin, prolactin, and the disintegrin domain of ADAM metallopeptidase domain 10 (ADAM10), to understand how these mechanisms apply in a cellular context. We used a eukaryotic cell-free translation system in which we could identify disulfide isomers in stalled translation intermediates to characterize the timing of disulfide formation relative to translocation into the endoplasmic reticulum and the presence of non-native disulfides. Our results indicate that in a domain lacking secondary structure, disulfides form before conformational folding through a process prone to nonnative disulfide formation, whereas in proteins with defined secondary structure, native disulfide formation occurs after partial folding. These findings reveal that the nascent protein structure promotes correct disulfide formation during cotranslational folding.

ADAM proteases: Emerging role and targeting of the non-catalytic domains.

ADAM proteases are multi domain transmembrane metalloproteases that cleave a range of cell surface proteins and activate signaling pathways implicated in tumor progression, including those mediated by Notch, EFGR, and the Eph receptors. Consequently, they have emerged as key therapeutic targets in the efforts to inhibit tumor initiation and progression. To that end, two main approaches have been taken to develop ADAM antagonists: (i) small molecule inhibitors, and (ii) monoclonal antibodies. In this mini-review we describe the distinct features of ADAM proteases, particularly of ADAM10 and ADAM17, their domain organization, conformational rearrangements, regulation, as well as their emerging importance as therapeutic targets in cancer. Further, we highlight an anti-ADAM10 monoclonal antibody that we have recently developed, which has shown significant promise in inhibiting Notch signaling and deterring growth of solid tumors in pre-clinical settings.

Meprin ß induces activities of A disintegrin and metalloproteinases 9, 10, and 17 by specific prodomain cleavage.

Meprin ß is a membrane-bound metalloprotease involved in extracellular matrix assembly and inflammatory processes in health and disease. A disintegrin and metalloproteinase (ADAM)10 and ADAM17 are physiologic relevant sheddases of inactive promeprin ß, which influences its substrate repertoire and subsequent biologic functions. Proteomic analysis also revealed several ADAMs as putative meprin ß substrates. Here, we demonstrate specific N-terminal processing of ADAM9, 10, and 17 by meprin ß and identify cleavage sites within their prodomains. Because ADAM prodomains can act as specific inhibitors, we postulate a role for meprin ß in the regulation of ADAM activities. Indeed, prodomain cleavage by meprin ß caused increased ADAM protease activities, as observed by peptide-based cleavage assays and demonstrated by increased ectodomain shedding activity. Direct interaction of meprin ß and ADAM proteases could be shown by immunofluorescence microscopy and immunoprecipitation experiments. As demonstrated by a bacterial activator of meprin ß and additional measurement of TNF-α shedding on bone marrow-derived macrophages, meprin ß/ADAM protease interactions likely influence inflammatory conditions. Thus, we identified a novel proteolytic pathway of meprin ß with ADAM proteases to control protease activities at the cell surface as part of the protease web.-Wichert, R., Scharfenberg, F., Colmorgen, C., Koudelka, T., Schwarz, J., Wetzel, S., Potempa, B., Potempa, J., Bartsch, J. W., Sagi, I., Tholey, A., Saftig, P., Rose-John, S., Becker-Pauly, C. Meprin ß induces activities of A disintegrin and metalloproteinases 9, 10, and 17 by specific prodomain cleavage.

Regulation of the alternative ß-secretase meprin ß by ADAM-mediated shedding.

Alzheimer's Disease (AD) is the sixth-leading cause of death in industrialized countries. Neurotoxic amyloid-ß (Aß) plaques are one of the pathological hallmarks in AD patient brains. Aß accumulates in the brain upon sequential, proteolytic processing of the amyloid precursor protein (APP) by ß- and γ-secretases. However, so far disease-modifying drugs targeting ß- and γ-secretase pathways seeking a decrease in the production of toxic Aß peptides have failed in clinics. It has been demonstrated that the metalloproteinase meprin ß acts as an alternative ß-secretase, capable of generating truncated Aß2-x peptides that have been described to be increased in AD patients. This indicates an important ß-site cleaving enzyme 1 (BACE-1)-independent contribution of the metalloprotease meprin ß within the amyloidogenic pathway and may lead to novel drug targeting avenues. However, meprin ß itself is embedded in a complex regulatory network. Remarkably, the anti-amyloidogenic α-secretase a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is a direct competitor for APP at the cell surface, but also a sheddase of inactive pro-meprin ß. Overall, we highlight the current cellular, molecular and structural understanding of meprin ß as alternative ß-secretase within the complex protease web, regulating APP processing in health and disease.

ADAM10 sheddase activation is controlled by cell membrane asymmetry.

Dysregulation of the disintegrin-metalloproteinase ADAM10 may contribute to the development of diseases including tumorigenesis and Alzheimer's disease. The mechanisms underlying ADAM10 sheddase activation are incompletely understood. Here, we show that transient exposure of the negatively charged phospholipid phosphatidylserine (PS) is necessarily required. The soluble PS headgroup was found to act as competitive inhibitor of substrate cleavage. Overexpression of the Ca2+-dependent phospholipid scramblase Anoctamin-6 (ANO6) led to increased PS externalization and substrate release. Transfection with a constitutively active form of ANO6 resulted in maximum sheddase activity in the absence of any stimulus. Calcium-dependent ADAM10 activation could not be induced in lymphocytes of patients with Scott syndrome harbouring a missense mutation in ANO6. A putative PS-binding motif was identified in the conserved stalk region. Replacement of this motif resulted in strong reduction of sheddase activity. In conjunction with the recently described 3D structure of the ADAM10 extracellular domain, a model is advanced to explain how surface-exposed PS triggers ADAM10 sheddase function.

Levels of ADAM10 are reduced in Alzheimer's disease CSF.

BACKGROUND: The disintegrin metalloproteinase 10 (ADAM10) is the main α-secretase acting in the non-amyloidogenic processing of the amyloid precursor protein. This study assesses whether ADAM10 is present in cerebrospinal fluid (CSF), and whether it has potential as a biomarker for Alzheimer's disease (AD). METHODS: ADAM10 was characterized in human CSF samples by immunoprecipitation and western blotting using antibodies specific for different domains of the protein and by ultracentrifugation in sucrose density gradients. Samples from AD patients (n = 20) and age-matched non-AD controls (n = 20) were characterized for classical CSF biomarkers, Aß42, T-tau, or P-tau by ELISA, and assayed for soluble ADAM10 levels by western blotting. RESULTS: We found that ADAM10 is present in human CSF as several distinct species: an immature form retaining the prodomain (proADAM10; ~ 80 kDa), a mature unprocessed full-length form (ADAM10f; ~ 55 kDa), and a truncated large soluble form released from the membrane (sADAM10; ~ 50 kDa). Fractionation by ultracentrifugation on sucrose density gradients showed that the ADAM10f and sADAM10 species form large complexes. Immunoblotting revealed a significant decrease in ADAM10f and sADAM10 in AD CSF compared to control CSF, while proADAM10 levels remained unaltered. CONCLUSIONS: Several forms of ADAM10 are present in CSF, mainly assembled as high-molecular weight complexes. The determination of the levels of mature forms of CSF-ADAM10 may be useful as a biomarker for AD.

Structural Basis for Regulated Proteolysis by the α-Secretase ADAM10.

Cleavage of membrane-anchored proteins by ADAM (a disintegrin and metalloproteinase) endopeptidases plays a key role in a wide variety of biological signal transduction and protein turnover processes. Among ADAM family members, ADAM10 stands out as particularly important because it is both responsible for regulated proteolysis of Notch receptors and catalyzes the non-amyloidogenic α-secretase cleavage of the Alzheimer's precursor protein (APP). We present here the X-ray crystal structure of the ADAM10 ectodomain, which, together with biochemical and cellular studies, reveals how access to the enzyme active site is regulated. The enzyme adopts an unanticipated architecture in which the C-terminal cysteine-rich domain partially occludes the enzyme active site, preventing unfettered substrate access. Binding of a modulatory antibody to the cysteine-rich domain liberates the catalytic domain from autoinhibition, enhancing enzymatic activity toward a peptide substrate. Together, these studies reveal a mechanism for regulation of ADAM activity and offer a roadmap for its modulation.

Regulation of the trafficking and the function of the metalloprotease ADAM10 by tetraspanins.

By interacting directly with partner proteins and with one another, tetraspanins organize a network of interactions referred to as the tetraspanin web. ADAM10 (A Disintegrin And Metalloprotease 10), an essential membrane-anchored metalloprotease that cleaves off the ectodomain of a large variety of cell surface proteins including cytokines, adhesion molecules, the precursor of the ß-amyloid peptide APP or Notch, has emerged as a major component of the tetraspanin web. Recent studies have shown that ADAM10 associates directly with all members (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17 and Tspan33) of a subgroup of tetraspanins having eight cysteines in the large extracellular domain and referred to as TspanC8. All TspanC8 regulate ADAM10 exit from the endoplasmic reticulum, but differentially regulate its subsequent trafficking and its function, and have notably a different impact on Notch signaling. TspanC8 orthologs in invertebrates also regulate ADAM10 trafficking and Notch signaling. It may be possible to target TspanC8 tetraspanins to modulate in a tissue- or substrate-restricted manner ADAM10 function in pathologies such as cardiovascular diseases, cancer or Alzheimer's disease.

Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients.

A humoral immune response against aberrant tumor proteins can be elicited in cancer patients, resulting in the production of auto-antibodies (Abs). By serological proteome analysis we identified the surface membrane protein ADAM10, a metalloproteinase that has a role in epithelial-tumor progression and invasion, as a target of the immune response in colorectal cancer (Crc). A screening carried out on the purified protein using testing cohorts of sera (Crc patients n = 57; control subjects n = 39) and validation cohorts of sera (Crc patients n = 49; control subjects n = 52) indicated that anti-ADAM10 auto-Abs were significantly induced in a large group (74%) of colon cancer patients, in particular in patients at stage II and III of the disease. Interestingly, in Crc patients classified as stage III disease, the presence of anti-ADAM10 auto-Abs in the sera was associated with a favourable follow-up with a significant shifting of the recurrence-free survival median time from 23 to 55 months. Even though the ADAM10 protein was expressed in Crc regardless the presence of auto-Abs, the immature/non-functional isoform of ADAM10 was highly expressed in the tumor of anti-ADAM10-positive patients and was the isoform targeted by the auto-Abs. In conclusion, the presence of anti-ADAM10 auto-Abs seems to reflect the increased tumor expression of the immunogenic immature-ADAM10 in a group of Crc patients, and is associated with a favourable prognosis in patients at stage III of the disease.

An activated form of ADAM10 is tumor selective and regulates cancer stem-like cells and tumor growth.

The transmembrane metalloprotease ADAM10 sheds a range of cell surface proteins, including ligands and receptors of the Notch, Eph, and erbB families, thereby activating signaling pathways critical for tumor initiation and maintenance. ADAM10 is thus a promising therapeutic target. Although widely expressed, its activity is normally tightly regulated. We now report prevalence of an active form of ADAM10 in tumors compared with normal tissues, in mouse models and humans, identified by our conformation-specific antibody mAb 8C7. Structure/function experiments indicate mAb 8C7 binds an active conformation dependent on disulfide isomerization and oxidative conditions, common in tumors. Moreover, this active ADAM10 form marks cancer stem-like cells with active Notch signaling, known to mediate chemoresistance. Importantly, specific targeting of active ADAM10 with 8C7 inhibits Notch activity and tumor growth in mouse models, particularly regrowth after chemotherapy. Our results indicate targeted inhibition of active ADAM10 as a potential therapy for ADAM10-dependent tumor development and drug resistance.

Dissecting the role of ADAM10 as a mediator of Staphylococcus aureus α-toxin action.

Staphylococcus aureus is a leading cause of bacterial infections in humans, including life-threatening diseases such as pneumonia and sepsis. Its small membrane-pore-forming α-toxin is considered an important virulence factor. By destroying cell-cell contacts through cleavage of cadherins, the metalloproteinase ADAM10 (a disintegrin and metalloproteinase 10) critically contributes to α-toxin-dependent pathology of experimental S. aureus infections in mice. Moreover, ADAM10 was proposed to be a receptor for α-toxin. However, it is unclear whether the catalytic activity or specific domains of ADAM10 are involved in mediating binding and/or subsequent cytotoxicity of α-toxin. Also, it is not known how α-toxin triggers ADAM10's enzymatic activity, and whether ADAM10 is invariably required for all α-toxin action on cells. In the present study, we show that efficient cleavage of the ADAM10 substrate epithelial cadherin (E-cadherin) requires supra-cytotoxic concentrations of α-toxin, leading to significant increases in intracellular [Ca(2+)]; the fall in cellular ATP levels, typically following membrane perforation, became observable at far lower concentrations. Surprisingly, ADAM10 was dispensable for α-toxin-dependent xenophagic targeting of S. aureus, whereas a role for α-toxin attack on the plasma membrane was confirmed. The catalytic site of ADAM10, furin cleavage site, cysteine switch and intracellular domain of ADAM10 were not required for α-toxin binding and subsequent cytotoxicity. In contrast, an essential role for the disintegrin domain and the prodomain emerged. Thus, co-expression of the prodomain with prodomain-deficient ADAM10 reconstituted binding of α-toxin and susceptibility of ADAM10-deficient cells. The results of the present study may help to inform structural analyses of α-toxin-ADAM10 interactions and to design novel strategies to counteract S. aureus α-toxin action.

Discovery of an enzyme and substrate selective inhibitor of ADAM10 using an exosite-binding glycosylated substrate.

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.