Regulation of inflammasome activity by NLRP3 ubiquitination – UbInfla

We characterize the regulation of NLRP3 ubiquitination. In particular, we identify among the activation signals and the inhibitors of the inflammasome, the stimuli that control NLRP3 ubiquitination. We identify the enzymes and their cofactors responsible for NLRP3 ubiquitination and deubiquitination through combined candidate-based and global approaches. We test the impact of NLRP3 ubiquitination in the regulation of inflammation in vivo using mouse models and patient studies. Another major objective is to decipher the molecular mechanisms responsible for the inactivation of ubiquitinated NLRP3. We identify the ubiquitination sites and the type of ubiquitin chains associated with NLRP3. We also study the impact of ubiquitination in the subcellular localization and the interactome of NLRP3.

This project is still ongoing. Results are so far confidential.

Our results will improve our understanding of the regulation of inflammation. Identification of novel signaling pathways, enzymes and cofactors that regulate inflammasome will lead to the discovery of novel susceptibility genes and improve the diagnosis of inflammatory diseases. In addition, the understanding of the molecular mechanisms of inflammation will lead to the identification of new therapeutical targets for anti-inflammatory drug discovery.

Caspase-11 controls IL-1b release through degradation of TRPC1. Py BF et al, Cell Reports, 2014

The innate immunity system constitutes a highly efficient barrier to diverse insults by rapidly detecting pathogens as well as tissue damage through pattern recognition receptors (PRRs). NLRP3 is an important PRR for anti-viral, -bacterial, and -fungal innate immunity. On the other hand, NLRP3 inappropriate activation contributes to deleterious inflammatory syndromes as tragically exemplified in inherited Cryopyrin-Associated Periodic Syndromes (CAPS) and in numerous conditions fueled by inappropriate stimuli-mediated NLRP3 activation including septic shock, gout disease, atherosclerosis, diabetes, Alzheimer’s disease and ischemia-reperfusion injuries. In macrophages, NLRP3 activation requires priming signal, usually provided by TLR ligands, and activation signals, provided by structurally diverse molecular patterns. These activating signals may trigger common intracellular change(s) sensed by NLRP3, but NLRP3 activation mechanisms remain poorly characterized. Upon activation, NLRP3 assembles a typical multimeric inflammasome complex serving as activation platform for caspase-1. Caspase-1 is an important pro-inflammatory caspase directly involved in processing and release of cytosolic pro-cytokines, including pro-IL-1ß, and cell death. We recently evidenced that inflammasome assembly requires NLRP3 deubiquitination by the deubiquiting (DUB) enzyme BRCC3. The aim of this proposal is to decipher this new and uncharacterized ubiquitin-dependent regulatory pathway critical for NLRP3 activation.
We will first aim at characterizing the upstream elements controlling NLRP3 ubiquitination level. We will identify the upstream stimuli that modulate NLRP3 activity through BRCC3-dependent NLRP3 deubiquitination by testing regularly used priming and activating signals, as well as inhibitors of the inflammasome. We will take advantage of the detailed elucidation of their signaling pathways to determine the key signaling elements regulating BRCC3 activity. In order to understand the molecular mechanisms regulating NLRP3 deubiquitination, we will first characterize the BRCC3-dependent NLRP3 deubiquitinating complex, and then follow the expression level, the subcellular localization, and the assembly of its constituents during inflammatory conditions. In parallel, we will attempt to identify the ubiquitin ligase(s) mediating NLRP3 ubiquitination by a combination of candidates screening and global approaches. We will also test the in vivo relevance of NLRP3 ubiquitination in inflammasome regulation using both mouse model and data analysis from patients suffering from CAPS-like syndromes.
Another objective of this proposal is to decipher the molecular mechanisms of the loss of activity of NLRP3 following its ubiquitination. We will characterize NLRP3 ubiquitination by identifying the ubiquitination sites and the type of ubiquitin chains conjugated to NLRP3. We will then investigate how ubiquitination affects NLRP3 subcellular localization and its ability to interact with its partners.
From this project, we expect to get a better understanding of inflammasome regulation and identify new therapeutic targets for future anti-inflammatory drug discovery.