Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous 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...
| Main Authors: | , , , , , , |
|---|---|
| Other Authors: | |
| Format: | Article |
| Language: | en_US |
| Published: |
Royal Society of Chemistry
2011
|
| Online Access: | http://hdl.handle.net/1721.1/67035 https://orcid.org/0000-0002-1801-5548 |
| _version_ | 1826192340582137856 |
|---|---|
| author | Miller, Aaron Barkal, Layla J. Jeng, Karen Herrlich, Andreas Moss, Marcia L. Griffith, Linda G. Lauffenburger, Douglas A. |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Miller, Aaron Barkal, Layla J. Jeng, Karen Herrlich, Andreas Moss, Marcia L. Griffith, Linda G. Lauffenburger, Douglas A. |
| author_sort | Miller, Aaron |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T09:10:17Z |
| format | Article |
| id | mit-1721.1/67035 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T09:10:17Z |
| publishDate | 2011 |
| publisher | Royal Society of Chemistry |
| record_format | dspace |
| spelling | mit-1721.1/670352022-09-26T10:55:20Z Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous multiple protease activities Miller, Aaron Barkal, Layla J. Jeng, Karen Herrlich, Andreas Moss, Marcia L. Griffith, Linda G. Lauffenburger, Douglas A. Massachusetts Institute of Technology. Department of Biological Engineering Whitehead Institute for Biomedical Research Lauffenburger, Douglas A. Aaron, Miles Barkal, Layla J. Jeng, Karen Herrlich, Andreas Griffith, Linda G. Lauffenburger, Douglas A. 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. Andrew and Edna Viterbi Fellowship in Computational Biology National Science Foundation (U.S.). Graduate Research Fellowship Program National Science Foundation (U.S.) (grant 1R01EB010246-01) National Science Foundation (U.S.) (grant 5R01GM081336-02) 2011-11-16T19:10:16Z 2011-11-16T19:10:16Z 2010-12 2010-08 Article http://purl.org/eprint/type/JournalArticle 1757-9708 1757-9694 http://hdl.handle.net/1721.1/67035 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 https://orcid.org/0000-0002-1801-5548 en_US http://dx.doi.org/10.1039/c0ib00083c Integrative Biology Creative Commons Attribution-Noncommercial-Share Alike 3.0 http://creativecommons.org/licenses/by-nc-sa/3.0/ application/pdf Royal Society of Chemistry PubMed Central |
| spellingShingle | Miller, Aaron Barkal, Layla J. Jeng, Karen Herrlich, Andreas Moss, Marcia L. Griffith, Linda G. Lauffenburger, Douglas A. Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous multiple protease activities |
| title | Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous multiple protease activities |
| title_full | Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous multiple protease activities |
| title_fullStr | Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous multiple protease activities |
| title_full_unstemmed | Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous multiple protease activities |
| title_short | Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous multiple protease activities |
| title_sort | proteolytic activity matrix analysis prama for simultaneous multiple protease activities |
| url | http://hdl.handle.net/1721.1/67035 https://orcid.org/0000-0002-1801-5548 |
| work_keys_str_mv | AT milleraaron proteolyticactivitymatrixanalysispramaforsimultaneousmultipleproteaseactivities AT barkallaylaj proteolyticactivitymatrixanalysispramaforsimultaneousmultipleproteaseactivities AT jengkaren proteolyticactivitymatrixanalysispramaforsimultaneousmultipleproteaseactivities AT herrlichandreas proteolyticactivitymatrixanalysispramaforsimultaneousmultipleproteaseactivities AT mossmarcial proteolyticactivitymatrixanalysispramaforsimultaneousmultipleproteaseactivities AT griffithlindag proteolyticactivitymatrixanalysispramaforsimultaneousmultipleproteaseactivities AT lauffenburgerdouglasa proteolyticactivitymatrixanalysispramaforsimultaneousmultipleproteaseactivities |