iGLuc: a luciferase-based inflammasome and protease activity reporter

Abstract

Measurement of protease activity in living cells or organisms remains a challenging task. We here present a transgene-encoded biosensor that reports the proteolytic activity of caspase-1 in the course of inflammasome activation and that of other proteases in a highly sensitive and specific manner. This protease reporter is based on the biological activity of a pro-interleukin (IL)-1β-Gaussia luciferase (iGLuc) fusion construct, in which pro-IL-1β-dependent formation of protein aggregates renders GLuc enzyme inactive. Cleavage leads to monomerization of this biosensor protein, resulting in a strong gain in luciferase activity. Exchange of the canonical caspase-1 cleavage site in this reporter construct allows the generation of protease biosensors with additional specificities. The high sensitivity, signal-to-background ratio and specificity of the iGLuc system renders it a useful tool to study proteolytic events in mouse and human cells at high throughput and to monitor protease activity in mice in vivo.Inflammasomes are large multiprotein signaling platforms that form in the cytoplasm upon sensing microbe or damage-associated molecular patterns 1 . Several NLR (NOD-like receptor) proteins (such as NLRP3) and the PYHIN (pyrin and HIN domain-containing) protein family member AIM2 (absent in melanoma 2) have been shown to function as initiators of inflammasome activation 2 . Via their shared adaptor protein-ASC, or apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD)-they recruit the cysteine protease caspase-1, which is in turn activated by proximity-induced dimerization leading to its autocatalytic cleavage into two heterodimerforming subunits. One of the main targets of active caspase-1 is IL-1β, a highly proCorrespondence should be addressed to V.H. (veit.hornung@uni-bonn.de).

Stepwise processing analyses of the single-turnover PCSK9 protease reveal its substrate sequence specificity and link clinical genotype to lipid phenotype

“…Similar approaches using a release of luciferase from pro-IL-1- protein aggregates have been used to probe protease activity in the inflammasome. (43) We applied our system to evaluate clinical SNPs found in PCSK9, a key modulator of serum LDL-C and an attractive cardiovascular target. Our findings suggest that alteration in PCSK9 proteolytic function is a frequent effect of SNPs which present in a clinical setting.…”

Suppression of NLRP3 inflammasome by γ-tocotrienol ameliorates type 2 diabetes

“…from Dr. Hornung (hereafter referred to as iJ774 macrophages) ( 19 ). iJ774 macrophages were cultured in DMEM supplemented with L -glutamine, sodium pyruvate, and 10% (v/v) FBS (Gibco).…”

Development of a Gaussia Luciferase-Based Human Norovirus Protease Reporter System: Cell Type-Specific Profile of Norwalk Virus Protease Precursors and Evaluation of Inhibitors

“…Recently, Gaussia luciferase (GLuc) fusion proteins have been demonstrated to be simple and useful tools in cell-based, protease-dependent biological assays (25)(26)(27). Isolated from the marine copepod Gaussia princeps, GLuc is the smallest, yet brightest, luciferase cloned to date.…”

“…Upon induction of apoptosis or autophagy, the fusion protein is cleaved by the activated caspases (apoptosis) or Atg4B (autophagy), leading to the release and secretion of GLuc (26,27). In another report, iGLuc, which is a fusion protein between prointerleukin-1␤ (pro-IL-1␤) and GLuc, was created as a biosensor that is cleaved by the protease activity of caspase-1 during the course of inflammasome activation (25).…”