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Published on: May 25, 2018
Proteolytic Activity Matrix Analysis (PrAMA) for simultaneous determination of multiple protease activities
Miles A Miller1, Layla Barkal, Karen Jeng
1Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Integrative Biology : Quantitative Biosciences From Nano to Macro
|December 25, 2010
Summary
Proteolytic Activity Matrix Analysis (PrAMA) enables simultaneous, real-time measurement of matrix metalloproteinase (MMP) and A Disintegrin and Metalloproteinase (ADAM) activities. This novel framework accurately distinguishes closely related enzymes in complex biological mixtures.
Area of Science:
- Biochemistry and Molecular Biology
- Enzymology
- Protease Activity Profiling
Background:
- Matrix metalloproteinases (MMPs) and A Disintegrin and Metalloproteinases (ADAMs) are crucial protease families involved in matrix remodeling and growth factor shedding.
- Accurately measuring specific MMP and ADAM activities in real-time, non-invasively, and in multiplex within physiological environments presents a significant challenge.
Purpose of the Study:
- To develop and validate an integrated framework, Proteolytic Activity Matrix Analysis (PrAMA), for simultaneously determining specific MMP and ADAM activities in complex mixtures.
- To enable real-time, non-invasive, and multiplexed assessment of protease activity in physiological settings.
Main Methods:
- PrAMA utilizes a panel of moderately specific Förster Resonance Energy Transfer (FRET)-based polypeptide protease substrates.
- Dynamic FRET signals are interpreted using mathematical analysis and prior cleavage signature data from purified enzymes.
- The method was validated using approximately 4000 measurements of known enzyme-substrate mixtures and applied to live-cell assays.
Main Results:
- PrAMA accurately deconvolutes and quantifies proteolytic activities of specific MMPs and ADAMs, even in the presence of unknown background activity.
- The framework demonstrated high accuracy in distinguishing closely related enzymes.
- Successful application to live-cell responses in wildtype and knockout fibroblast models (ADAM10-/-, ADAM17-/-) stimulated with phorbol ester and ionomycin.
Conclusions:
- PrAMA provides a robust and valuable tool for simultaneously assessing specific protease activities in complex biological samples.
- The method is applicable to various settings, including live-cell assays and high-throughput inhibitor screening.
- The PrAMA approach holds potential for extension to other protease families like caspases and cathepsins.
