A Novel Bioluminescent Biosensor Quantifying Intramolecular Interaction and Levels of Pyroptosis Effector GSDMD.

Gasdermin D (GSDMD) is a key executor of pyroptosis, a form of inflammation-induced programmed cell death. Recently, GSDMD has been shown to play important roles in the development of various inflammatory-related human diseases including heart failure and cancer, suggesting that it is a promising therapeutic target for these diseases. While extensive studies on GSDMD's role in pyroptosis have been reported, it is challenging to study its function due to the lack of enzymatic activity of GSDMD. In this study, we used the NanoBiT technology to develop a novel GSDMD bioluminescent biosensor (GSDMD-BS) that detects the amount of non-cleaved GSDMD. This sensor allows us to quantify GSDMD's intramolecular interactions, the amounts of uncleaved GSDMD after caspase-1 cleavage, and expression levels in living cells. In vitro experiments using purified GSDMD-BS also confirmed the sensor's accuracy in reporting GSDMD levels and cleavage. Moreover, the potential for in vivo application was demonstrated in a xenograft mouse model. In conclusion, we have developed a GSDMD biosensor that is a valuable tool for real-time monitoring of GSDMD dynamics and pyroptosis. This biosensor will significantly expedite pyroptosis research and can be used for high-throughput screening for drugs targeting GSDMD for the therapy of many inflammation-related diseases.

Development of Novel Bioluminescent Biosensors Monitoring the Conformation and Activity of the Merlin Tumour Suppressor.

Solid tumours can universally evade contact inhibition of proliferation (CIP), a mechanism halting cell proliferation when cell-cell contact occurs. Merlin, an ERM-like protein, crucially regulates CIP and is frequently deactivated in various cancers, indicating its significance as a tumour suppressor in cancer biology. Despite extensive investigations into Merlin's role in cancer, its lack of intrinsic catalytic activity and frequent conformation changes have made it notoriously challenging to study. To address this challenge, we harnessed innovative luciferase technologies to create and validate a NanoBiT split-luciferase biosensor system in which Merlin is cloned between two split components (LgBiT and SmBiT) of NanoLuc luciferase. This system enables precise quantification of Merlin's conformation and activity both in vitro and within living cells. This biosensor significantly enhances the study of Merlin's molecular functions, serving as a potent tool for exploring its contributions to CIP and tumorigenesis.