AI Article Synopsis
- Caspases are important proteases involved in cell death and inflammation, and new biosensors called NanoLocks, based on NanoLuc luciferase, were developed to detect their activity.
- The NanoLock sensors operate on a dark-to-bright model and show significant activation responses, achieving a 1233-fold increase in signal with tobacco etch virus protease and over 500-fold with caspase 3.
- These sensors outperform commercial kits in terms of signal-to-noise ratio and convenience, with successful testing in human cells and the establishment of a cell line for screening caspase 6 modulators.
Article Abstract
Caspases are a family of evolutionary conserved cysteine proteases that play key roles in programmed cell death and inflammation. Among the methods for the detection of caspase activity, biosensors based on luciferases have advantages in genetical encoding and convenience in assay. In this study, we constructed a new set of caspase biosensors based on NanoLuc luciferase. This kind of sensors, named NanoLock, work in dark-to-bright model, with the help of a NanoLuc quencher peptide (HiBiT-R/D) mutated from HiBiT. Optimized NanoLock responded to proteases with high signal to noise ratio (S/N), 1233-fold activation by tobacco etch virus protease in HEK293 cells and > 500-fold induction to caspase 3 in vitro. We constructed NanoLocks for the detection of caspase 1, 3, 6, 7, 8, 9, and 10, and assays in HEK293 cells demonstrated that these sensors performed better than commercial kits in the aspect of S/N and convenience. We further established a cell line stably expressing NanoLock-casp 6 and provided a proof-of-concept for the usage of this cell line in the high throughput screening of caspase 6 modulator.
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| http://dx.doi.org/10.1016/j.jbiotec.2022.08.005 | DOI Listing |
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