Imaging Proteolytic Activity in Live Cells and Animal Models

Abstract

In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors); their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulated activation of apoptosis by the caspase family of proteases has been linked to diseases such as cancer, autoimmunity and inflammation. In an effort to better understand the role of proteases in health and disease, a luciferase biosensor is described which can quantitatively report proteolytic activity in live cells and mouse models. The biosensor, hereafter referred to as GloSensor Caspase 3/7 has a robust signal to noise (50–100 fold) and dynamic range such that it can be used to screen for pharmacologically active compounds in high throughput campaigns as well as to study cell signaling in rare cell populations such as isolated cancer stem cells. The biosensor can also be used in the context of genetically engineered mouse models of human disease wherein conditional expression using the Cre/loxP technology can be implemented to investigate the role of a specific protease in living subjects. While the regulation of apoptosis by caspase's was used as an example in these studies, biosensors to study additional proteases involved in the regulation of normal and pathological cellular processes can be designed using the concepts presented herein.

Live cell evaluation of granzyme delivery and death receptor signaling in tumor cells targeted by human natural killer cells

“…Recent developments in murine systems now permit examination of the kinetics of Grz and caspase activation during CTL-mediated apoptosis, 23 and visualization of tumoricidal caspase 3/7 activity in animals. 29,54 Here, we have used luciferase biosensors to measure GrzB, GrzA, GrzK, and pro-apoptotic caspase activation in real time after human NK cell cytotoxicity induced by CD16 ligation and NK receptor-mediated recognition.…”

A cyclic HSV1-TK reporter for real-time PET imaging of apoptosis

“…So far, there are several caspase-3 responsive reporters available for in vivo imaging of apoptosis, most of which use a luciferase protein (23)(24)(25)(26) or a fusion protein (27,28) separated by the DEVD sequence. In these linear designs, the deactivation or "silence" of the probes is usually achieved by fusing other proteins or protein domains on each side of the reporter proteins.…”

“…Consequently, fluorescence and bioluminescence reporter systems have been developed based on a small peptide aspartylglutamylvalanyl aspartic acid (DEVD), which can be recognized and cleaved by caspase-3 (17,(20)(21)(22). Although optical imaging with these reporter probes has been demonstrated to be useful for in vivo monitoring of apoptosis especially in cell culture or s.c. animal models (23)(24)(25)(26)(27)(28), further applications of these approaches are thwarted by tissue attenuation of photons and lack of fully quantitative and tomographic capabilities. Therefore, there is an urgent need to develop inherently more sensitive, quantitative, and translatable reporter systems for noninvasive apoptosis imaging.…”

Glycoengineering of Therapeutic Antibodies Enhances Monocyte/Macrophage-Mediated Phagocytosis and Cytotoxicity

“…In addition to LDH release, ADCC was determined by luminescence in an assay based on the assessment of active caspase-3/7 pathway in tumor target cells using 1833-POPP233 cells stably transfected with plasmid coding for split luciferase (C-Luc and N-Luc domain fused with intervening caspase-3/7 cleavage motif [DEVD]). The cleavage of the reporter molecule by activated caspase-3/7 enables interaction of C-Luc and N-Luc and reconstitutes luciferase activity (32,33). 1833-POPP233 target and effector cells (human M1 or M2c macrophages of CD16 + monocytes) were incubated for 8 or 24 h in the presence or absence of WT or GE GA201 (1 mg/ml), and luminescence was quantified by the addition of D-Luciferin (Biosynth).…”