Abstract:
Fluorescence imaging methods integrated with substrate-based reporter assays (both
genetic and synthetic substrate) are routinely used to study the function of “active
protease” in the (patho) physiological processes. However, most of the substrate-based
reporters lack target specificity in the in vivo conditions. Recently, the activity-based
fluorescent probe (ABFP) method is used for monitoring protease function in vivo. This
method provides an opportunity to back-track the signal produced by the target enzyme and
other proteases. However, this is achieved through post-processing of cell or tissue lysate
followed by in-gel fluorescence studies. ABFP method is labor-intensive and cannot be
translated to high-throughput imaging studies. To address the drawbacks of existing
techniques, herein, we disclose the design and development of a new technology called
“Activity-based Reporter Gene Technology” (AbRGT). It uses a reporter protein-tagged
protease of interest (PoI) and an activity-based fluorescent probe (ABFP). The specific
activation of PoI is determined by measuring the fluorescence resonance energy transfer
(FRET) signal that occurs only upon labeling of ABFP to the reporter protein-tagged
PoI. In this manner, the method allows the imaging of an active protease with an exquisite
specificity in the presence of highly homogenous proteins within a cell. As a proof-of concept, we have applied this method to study the function of individual caspase protease in
both intrinsic and extrinsic apoptosis signaling pathways. We demonstrate that the same
method can be used for profiling of compounds that can inhibit caspases activity. We have
also shown the design and potential use of the BRET approach of AbRGT in the high throughput screening of protease inhibitors. Altogether, this method holds huge potential for
applications in the area of diagnostics, screening of drugs, and other discovery efforts.
