Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous multiple protease activities
Name
Lauffenburger-2011-Proteolytic Activity Matrix Analysis.pdf
Size
2.11 MB
Format
Adobe PDF
Checksum (MD5)
826760730ab9e968aea58886706bf6d8
Author(s)
Miller, Aaron
Barkal, Layla J.
Jeng, Karen
Herrlich, Andreas
Moss, Marcia L.
Griffith, Linda G.
Lauffenburger, Douglas A.
Date Issued
December 2010
Journal
Integrative Biology
Publisher
Royal Society of Chemistry
Citation
Miller, Miles A. et al. “Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous determination of multiple protease activities.” Integrative Biology 3 (2011): 422. Web. 16 Nov. 2011. © 2011 Royal Society of Chemistry
Version
Author's final manuscript
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 and ionomycin 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.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Whitehead Institute for Biomedical Research
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Creative Commons Attribution-Noncommercial-Share Alike 3.0
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DOI of Published Version
https://doi.org/10.1039/c0ib00083c
