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 availab...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
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Ivyspring International Publisher
2018
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| Online Access: | http://hdl.handle.net/1721.1/117613 https://orcid.org/0000-0002-1801-5548 https://orcid.org/0000-0002-0050-989X |
| _version_ | 1826198811398111232 |
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| author | Yoneyama, Toshie Gorry, Michael Gaither-Davis, Autumn Lin, Yan Moss, Marcia L. Stabile, Laura P. Herman, James G. Vujanovic, Nikola L. Miller, Miles Aaron Griffith, Linda G Lauffenburger, Douglas A |
| author2 | Massachusetts Institute of Technology. Biotechnology Process Engineering Center |
| author_facet | Massachusetts Institute of Technology. Biotechnology Process Engineering Center Yoneyama, Toshie Gorry, Michael Gaither-Davis, Autumn Lin, Yan Moss, Marcia L. Stabile, Laura P. Herman, James G. Vujanovic, Nikola L. Miller, Miles Aaron Griffith, Linda G Lauffenburger, Douglas A |
| author_sort | Yoneyama, Toshie |
| collection | MIT |
| description | 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. Recent studies have developed the high throughput real-time proteolytic activity matrix analysis (PrAMA) that integrates the enzymatic processing of multiple enzyme substrates with mathematical-modeling computation. The original PrAMA measures with significant accuracy the activities of individual metalloproteinases expressed on live cells. To make the biomarker assay usable in clinical practice, we modified PrAMA by testing enzymatic activities in cell and tissue lysates supplemented with broad-spectrum non-MP enzyme inhibitors, and by maximizing the assay specificity using systematic mathematical-modeling analyses. The modified PrAMA accurately measured the absence and decreases of ADAM10 sheddase activity (ADAM10sa) and ADAM17sa in ADAM10-/-and ADAM17-/-mouse embryonic fibroblasts (MEFs), and ADAM10- and ADAM17-siRNA transfected human cancer cells, respectively. It also measured the restoration and inhibition of ADAM10sa in ADAM10-cDNA-transfected ADAM10-/-MEFs and GI254023X-treated human cancer cell and tissue lysates, respectively. Additionally, the modified PrAMA simultaneously quantified with significant accuracy ADAM10sa and ADAM17sa in multiple human tumor specimens, and showed the essential characteristics of a robust high throughput multiplex assay that could be broadly used in biomarker studies. Selectively measuring specific enzyme activities, this new clinically applicable assay is potentially superior to the standard protein- and gene-expression assays that do not distinguish active and inactive enzyme forms. |
| first_indexed | 2024-09-23T11:10:23Z |
| format | Article |
| id | mit-1721.1/117613 |
| institution | Massachusetts Institute of Technology |
| last_indexed | 2024-09-23T11:10:23Z |
| publishDate | 2018 |
| publisher | Ivyspring International Publisher |
| record_format | dspace |
| spelling | mit-1721.1/1176132022-09-27T17:37:22Z Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates Yoneyama, Toshie Gorry, Michael Gaither-Davis, Autumn Lin, Yan Moss, Marcia L. Stabile, Laura P. Herman, James G. Vujanovic, Nikola L. Miller, Miles Aaron Griffith, Linda G Lauffenburger, Douglas A Massachusetts Institute of Technology. Biotechnology Process Engineering Center Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemical Engineering Massachusetts Institute of Technology. Department of Mechanical Engineering Miller, Miles Aaron Griffith, Linda G Lauffenburger, Douglas A 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. Recent studies have developed the high throughput real-time proteolytic activity matrix analysis (PrAMA) that integrates the enzymatic processing of multiple enzyme substrates with mathematical-modeling computation. The original PrAMA measures with significant accuracy the activities of individual metalloproteinases expressed on live cells. To make the biomarker assay usable in clinical practice, we modified PrAMA by testing enzymatic activities in cell and tissue lysates supplemented with broad-spectrum non-MP enzyme inhibitors, and by maximizing the assay specificity using systematic mathematical-modeling analyses. The modified PrAMA accurately measured the absence and decreases of ADAM10 sheddase activity (ADAM10sa) and ADAM17sa in ADAM10-/-and ADAM17-/-mouse embryonic fibroblasts (MEFs), and ADAM10- and ADAM17-siRNA transfected human cancer cells, respectively. It also measured the restoration and inhibition of ADAM10sa in ADAM10-cDNA-transfected ADAM10-/-MEFs and GI254023X-treated human cancer cell and tissue lysates, respectively. Additionally, the modified PrAMA simultaneously quantified with significant accuracy ADAM10sa and ADAM17sa in multiple human tumor specimens, and showed the essential characteristics of a robust high throughput multiplex assay that could be broadly used in biomarker studies. Selectively measuring specific enzyme activities, this new clinically applicable assay is potentially superior to the standard protein- and gene-expression assays that do not distinguish active and inactive enzyme forms. 2018-09-04T15:45:07Z 2018-09-04T15:45:07Z 2017-10 2017-04 2018-08-30T17:23:41Z Article http://purl.org/eprint/type/JournalArticle 1837-9664 http://hdl.handle.net/1721.1/117613 Yoneyama, Toshie, Michael Gorry, Miles A Miller, Autumn Gaither-Davis, Yan Lin, Marcia L. Moss, Linda G. Griffith, et al. “Modification of Proteolytic Activity Matrix Analysis (PrAMA) to Measure ADAM10 and ADAM17 Sheddase Activities in Cell and Tissue Lysates.” Journal of Cancer 8, no. 19 (2017): 3916–3932. https://orcid.org/0000-0002-1801-5548 https://orcid.org/0000-0002-0050-989X http://dx.doi.org/10.7150/JCA.20779 Journal of Cancer Creative Commons Attribution-NonCommercial 4.0 International http://creativecommons.org/licenses/by-nc/4.0/ application/pdf Ivyspring International Publisher Journal of Cancer |
| spellingShingle | Yoneyama, Toshie Gorry, Michael Gaither-Davis, Autumn Lin, Yan Moss, Marcia L. Stabile, Laura P. Herman, James G. Vujanovic, Nikola L. Miller, Miles Aaron Griffith, Linda G Lauffenburger, Douglas A Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates |
| title | Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates |
| title_full | Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates |
| title_fullStr | Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates |
| title_full_unstemmed | Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates |
| title_short | Modification of proteolytic activity matrix analysis (PrAMA) to measure ADAM10 and ADAM17 sheddase activities in cell and tissue lysates |
| title_sort | modification of proteolytic activity matrix analysis prama to measure adam10 and adam17 sheddase activities in cell and tissue lysates |
| url | http://hdl.handle.net/1721.1/117613 https://orcid.org/0000-0002-1801-5548 https://orcid.org/0000-0002-0050-989X |
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