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

Abstract

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. The PrAMA method interprets dynamic signals from panels of moderately specific FRET-based polypeptide protease substrates to deduce a profile of specific MMP and ADAM proteolytic activities. Deconvolution of signals from complex mixtures of proteases is accomplished using prior data on individual MMP/ADAM cleavage signatures for the substrate panel measured with purified enzymes. We first validate PrAMA inference using a compendium of roughly 4000 measurements involving known mixtures of purified enzymes and substrates, and then demonstrate application to the live-cell response of wildtype, ADAM10−/−, and ADAM17−/− fibroblasts to phorbol ester stimulation. Results indicate PrAMA can distinguish closely related enzymes from each other with high accuracy, even in the presence of unknown background proteolytic activity. PrAMA offers a valuable tool for applications ranging from live-cell in vitro assays to high-throughput inhibitor screening with complex enzyme mixtures. Moreover, our approach may extend to other families of proteases, such as caspases and cathepsins, that also can lack highly-specific substrates.

Regulated ADAM17-dependent EGF family ligand release by substrate-selecting signaling pathways

“…We measured basal and TPA-induced protease activity by using proteolytic activity matrix analysis (PrAMA). PrAMA allows measurement of particular protease activities independently of measuring specific physiological substrate cleavage (30). This technique uses a panel of FRET-based peptide substrates that emit fluorescence upon cleavage.…”

Studying Cellular Signal Transduction with OMIC Technologies

“…Methods that have sufficient throughput and coverage to be considered “OMIC” approaches are rare, but some exist and certainly others are in development. For example, assays have been developed to facilitate high-throughput quantification of phospho-protein specific phosphatase activity [66], as well as matrix metalloprotease activity [67]. …”

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

“…While the results described in this paper are a proof of concept to demonstrate the utility of amplification systems based on attaching a reporter to a substrate followed by physical separation of product from unreacted substrate for quantification, we also tested whether the 14 HRP would be affected by the presence of urine in the assay.…”

“…This substrate, Flsub13 or PEPDAB013, was cleaved at a higher rate in urine samples from patients with invasive or metastatic breast cancer when compared to normal controls [13]. Flsub13 has also been used in complex biological mixtures in combination with other FRET substrates, to quantify individual levels of ADAM and MMP family members using a technique termed proteolytic activity matrix analysis (PrAMA) [14].…”