Development of flow cytometry assays for measuring cell-membrane enzyme activity on individual cells.

Cell-membrane expressing enzymes such as ADAM (a disintegrin and metalloproteinase) superfamily members are thought to be key catalysts of vital cellular functions. To directly measure these enzymes and determine their association with particular cells and functions, individual-cell membrane-bound enzyme activity assays are required, but unavailable. We developed two such assays, using a fluorescence resonance energy transfer (FRET) peptide substrate (FPS) and flow cytometry.

ADAM10 Sheddase Activity is a Potential Lung-Cancer Biomarker.

: Increases in expression of ADAM10 and ADAM17 genes and proteins are inconsistently found in cancer lesions, and are not validated as clinically useful biomarkers. The enzyme-specific proteolytic activities, which are solely mediated by the active mature enzymes, directly reflect enzyme cellular functions and might be superior biomarkers than the enzyme gene or protein expressions, which comprise the inactive proenzymes and active and inactivated mature enzymes. Using a recent modification of the proteolytic activity matrix analysis (PrAMA) measuring specific enzyme activities in cell and tissue lysates, we examined the specific sheddase activities of ADAM10 (ADAM10sa) and ADAM17 (ADAM17sa) in human non-small cell lung-carcinoma (NSCLC) cell lines, patient primary tumors and blood exosomes, and the noncancerous counterparts.

Recent Advances in ADAM17 Research: A Promising Target for Cancer and Inflammation.

Since its discovery, ADAM17, also known as TNF converting enzyme or TACE, is now known to process over 80 different substrates. Many of these substrates are mediators of cancer and inflammation. The field of ADAM metalloproteinases is at a crossroad with many of the new potential therapeutic agents for ADAM17 advancing into the clinic.

Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates.

Increases in expression of ADAM10 and ADAM17 genes and proteins have been evaluated, but not validated as cancer biomarkers. Specific enzyme activities better reflect enzyme cellular functions, and might be better biomarkers than enzyme genes or proteins. However, no high throughput assay is available to test this possibility.

Fluorescent substrates for ADAM15 useful for assaying and high throughput screening.

A disintegrin and metalloproteinase 15 (ADAM15), also known as metargidin, plays important roles in regulating inflammation, wound healing, neovascularization, and is an attractive drug target. Fluorescence resonance energy transfer (FRET)-based peptide substrates were tested to identify candidate reagents for high throughput screening and detection of ADAM15 in biological samples. ADAM15 exhibits a unique and diverse activity profile compared to other metalloproteinases.

An improved fluorescent substrate for assaying soluble and membrane-associated ADAM family member activities.

A fluorescent resonance energy transfer substrate with improved sensitivity for ADAM17, -10, and -9 (where ADAM represents a disintegrin and metalloproteinase) has been designed. The new substrate, Dabcyl-Pro-Arg-Ala-Ala-Ala-Homophe-Thr-Ser-Pro-Lys(FAM)-NH2, has specificity constants of 6.3 (±0.

CD30 on extracellular vesicles from malignant Hodgkin cells supports damaging of CD30 ligand-expressing bystander cells with Brentuximab-Vedotin, in vitro.

The goal of targeted immunotherapy in cancer is to damage both malignant and tumor-supporting cells of the microenvironment but spare unaffected tissue. The malignant cells in classical Hodgkin lymphoma (cHL) selectively express CD30. They release this receptor on extracellular vesicles (EVs) for the tumor-supporting communication with CD30 ligand (CD30L)-positive bystander cells.

Targeting autocrine HB-EGF signaling with specific ADAM12 inhibition using recombinant ADAM12 prodomain.

Dysregulation of ErbB-family signaling underlies numerous pathologies and has been therapeutically targeted through inhibiting ErbB-receptors themselves or their cognate ligands. For the latter, "decoy" antibodies have been developed to sequester ligands including heparin-binding epidermal growth factor (HB-EGF); however, demonstrating sufficient efficacy has been difficult. Here, we hypothesized that this strategy depends on properties such as ligand-receptor binding affinity, which varies widely across the known ErbB-family ligands.

A colorimetric-based amplification system for proteinases including MMP2 and ADAM8.

We have developed a new amplification system for proteinases that is sensitive, simple, and inexpensive to run, exemplified by a horseradish peroxidase (HRP)-conjugated, dual MMP2 (matrix metalloproteinase 2) and ADAM8 (a disintegrin and metalloproteinase 8) peptide substrate assay presented herein. The HRP-conjugated substrate is attached to beads through a 6× histidine tag and then incubated with the target enzyme, cleaving the HRP reporter. This product is subsequently removed from the unreacted bound portions of the substrate by magnetic deposition of the beads.

The function of the milk-clotting enzymes bovine and camel chymosin studied by a fluorescence resonance energy transfer assay.

Enzymatic coagulation of bovine milk can be divided in 2 steps: an enzymatic step, in which the Phe105-Met106 bond of the milk protein bovine κ-casein is cleaved, and an aggregation step. The aspartic peptidases bovine and camel chymosin (EC 3.4.

ADAM8 as a drug target in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a grim prognosis with <5% survivors after 5 years. High expression levels of ADAM8, a metalloprotease disintegrin, are correlated with poor clinical outcome. We show that ADAM8 expression is associated with increased migration and invasiveness of PDAC cells caused by activation of ERK1/2 and higher MMP activities.

EphrinA/EphA-induced ectodomain shedding of neural cell adhesion molecule regulates growth cone repulsion through ADAM10 metalloprotease.

EphrinA/EphA-dependent axon repulsion is crucial for synaptic targeting in developing neurons but downstream molecular mechanisms remain obscure. Here, it is shown that ephrinA5/EphA3 triggers proteolysis of the neural cell adhesion molecule (NCAM) by the metalloprotease a disintegrin and metalloprotease (ADAM)10 to promote growth cone collapse in neurons from mouse neocortex. EphrinA5 induced ADAM10 activity to promote ectodomain shedding of polysialic acid-NCAM in cortical neuron cultures, releasing a ~ 250 kDa soluble fragment consisting of most of its extracellular region.

Potential of fluorescent metalloproteinase substrates for cancer detection.

Objectives: MMP-2, MMP-9, their complexes and ADAM12 are detected in the urine of breast cancer patients and predict disease status. We assessed the use of FRET-based substrates in an assay to distinguish breast cancer patients from controls.

Design And Methods: Substrates with varying specificities for MMP-9 and MMP-2 and several ADAMs were screened.

ADAM9 inhibition increases membrane activity of ADAM10 and controls α-secretase processing of amyloid precursor protein.

Prodomains of A disintegrin and metalloproteinase (ADAM) metallopeptidases can act as highly specific intra- and intermolecular inhibitors of ADAM catalytic activity. The mouse ADAM9 prodomain (proA9; amino acids 24-204), expressed and characterized from Escherichia coli, is a competitive inhibitor of human ADAM9 catalytic/disintegrin domain with an overall inhibition constant of 280 ± 34 nM and high specificity toward ADAM9. In SY5Y neuroblastoma cells overexpressing amyloid precursor protein, proA9 treatment reduces the amount of endogenous ADAM10 enzyme in the medium while increasing membrane-bound ADAM10, as shown both by Western and activity assays with selective fluorescent peptide substrates using proteolytic activity matrix analysis.

Species specificity of ADAM10 and ADAM17 proteins in interleukin-6 (IL-6) trans-signaling and novel role of ADAM10 in inducible IL-6 receptor shedding.

Hypomorphic ADAM17(ex/ex) mice showed defects in mucosal regeneration due to inefficient enhanced GFR shedding. ADAM17 is the main sheddase of interleukin-6 receptor (IL-6R) to induce IL-6 trans-signaling. However, serum levels of soluble murine IL-6R were not reduced in ADAM17(ex/ex) mice, and murine ADAM17 was not the major sheddase of murine IL-6R.

Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous determination of multiple protease activities.

Matrix metalloproteinases (MMPs) and A Disintegrin and Metalloproteinases (ADAMs) are two related protease families that play key roles in matrix remodeling and growth factor ligand shedding. Directly ascertaining the proteolytic activities of particular MMPs and ADAMs in physiological environments in a non-invasive, real-time, multiplex manner remains a challenge. This work describes Proteolytic Activity Matrix Analysis (PrAMA), an integrated experimental measurement and mathematical analysis framework for simultaneously determining the activities of particular enzymes in complex mixtures of MMPs and ADAMs.

Tumor necrosis factor-alpha (TNF-alpha) regulates shedding of TNF-alpha receptor 1 by the metalloprotease-disintegrin ADAM8: evidence for a protease-regulated feedback loop in neuroprotection.

Tumor necrosis factor alpha (TNF-alpha) is a potent cytokine in neurodegenerative disorders, but its precise role in particular brain disorders is ambiguous. In motor neuron (MN) disease of the mouse, exemplified by the model wobbler (WR), TNF-alpha causes upregulation of the metalloprotease-disintegrin ADAM8 (A8) in affected brain regions, spinal cord, and brainstem. The functional role of A8 during MN degeneration in the wobbler CNS was investigated by crossing WR with A8-deficient mice: a severely aggravated neuropathology was observed for A8-deficient WR compared with WR A8(+/-) mice, judged by drastically reduced survival [7 vs 81% survival at postnatal day 50 (P50)], accelerated force loss in the forelimbs, and terminal akinesis.

A disintegrin and metalloproteinase-10 (ADAM-10) mediates DN30 antibody-induced shedding of the met surface receptor.

Met, the tyrosine kinase receptor for the hepatocyte growth factor is a prominent regulator of cancer cell invasiveness and has emerged as a promising therapeutic target. Binding of the anti-Met monoclonal antibody DN30 to its epitope induces the proteolytic cleavage of Met, thereby impairing the invasive growth of tumors. The molecular mechanism controlling this therapeutic shedding process has so far been unknown.

Fluorescent substrates useful as high-throughput screening tools for ADAM9.

Fluorescence resonance energy transfer substrates were designed and tested as substrates for ADAM9. The donor/quencher pair used were 5-carboxy fluorescein (Fam) and 4-(4-dimethyl-aminophenylazo)benzoyl (Dabcyl) since they have been well studied sensitive fluorescent probes. The peptides based on precursor TNF-alpha, Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(Fam)- NH2 and Dabcyl-Leu-Ala-Gln-Ala-HomoPhe-Arg-Ser-Lys(Fam)- NH2, and C-terminal TGF-alpha, Dabcyl-Glu-His-Ala-Asp-Leu-Leu-Ala-Val-Val-Ala-Ala-Lys(Fam)- NH2 cleavage sites were effectively processed by ADAM9 with turnover numbers of 100 +/- 20 x 10(-2) min(-1), 20 +/- 10 x 10(-2) min(-1), and 10 +/- 3 x 10(-2) min(-1).

ADAM10 as a therapeutic target for cancer and inflammation.

Both cancer and chronic inflammatory diseases are often marked by homeostatic signal transduction pathways run amok. Cleavage of membrane-bound substrates by extracellular metalloproteinases is frequently the rate limiting step in activating many of these pathways, resulting either in liberation of active ligands (shedding) or initiating further processing into bioactive cytoplasmic domains (regulated intramembrane proteolysis or RIP). ADAM10 is a member of the ADAM (A Disintegrin And Metalloproteinase) family of transmembrane metalloproteinases implicated in the RIPing and shedding of dozens of substrates that drive cancer progression and inflammatory disease, including Notch, E-cadherin, EGF, ErbB2 and inflammatory cytokines.

Metastasis-associated C4.4A, a GPI-anchored protein cleaved by ADAM10 and ADAM17.

Metalloproteases play a complex role in tumor progression. While the activity of some ADAM, ADAMTS and matrix metalloproteases (MMPs) seems to be protumorigenic, the activity of others seems to prevent tumor progression. The identification of the array of substrates of a given metalloprotease (degradome) seems an adequate approach to predict the effect of the inhibition of a metalloprotease in tumors.

Drug insight: tumor necrosis factor-converting enzyme as a pharmaceutical target for rheumatoid arthritis.

The success of agents that inhibit tumor necrosis factor (TNF), such as infliximab, adalimumab and etanercept, has led to a desire for orally available small molecules that have a better safety profile and are less costly to produce than current agents. One target for anti-TNF therapy that is currently under investigation is TNF-converting enzyme, which promotes the release of soluble TNF from its membrane-bound precursor. Inhibitors of this enzyme with drug-like properties have been made and tested in the clinic.

ADAM10 as a target for anti-cancer therapy.

There is a great unmet medical need in the area of cancer treatment. A potential therapeutic target for intervention in cancer is ADAM10. ADAM10 is a disintegrin-metalloproteinase that processes membrane bound proteins from the cell surface to yield soluble forms.