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.
Highlights
Development of new agents that can be used to treat a wide variety of human diseases including cancer, stroke, cardiovascular and neurodegenerative diseases such as Alzheimer’s and Parkinson’s continues unabated
The variety and complexity of signaling events associated with cell death programs which can lead to mitotic catastrophe, apoptosis, necrosis, necroptosis, pyrosis and autophagy exemplify the importance of developing generalizable biomarker readouts of the cell death process
We have developed a second generation reporter which detects the activation of Caspase 3 and 7 during apoptosis, Caspase 3/7 GloSensor
Summary
Development of new agents that can be used to treat a wide variety of human diseases including cancer, stroke, cardiovascular and neurodegenerative diseases such as Alzheimer’s and Parkinson’s continues unabated. While for example, anticancer agents are designed as inducers of cell death for tumor cell eradication, anti-neurodegenerative agents are sought to ameliorate neuronal dropout through inhibition of cell death processes. The variety and complexity of signaling events associated with cell death programs which can lead to mitotic catastrophe, apoptosis, necrosis, necroptosis, pyrosis and autophagy exemplify the importance of developing generalizable biomarker readouts of the cell death process. In an effort to assess and quantify efficacy of drug interventions, assays distinguishing induction and inhibition of signaling events have been developed predominantly for cell culture screens [1]. Caspase 3/7 activation is considered a key surrogate marker for assessment of apoptosis and the ability to image this process in intact cells and live animals has gained increasing interest with the onslaught of molecularly targeted agents. Caspases mediate the early stages of apoptosis by proteolytically processing their substrates such as PARP their proteolytic function can be used in the design of radioactive, fluorescent and luminescent assays for assessing apoptosis [2]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
