Periodic Report Summary 2 - UBINFLAM (Regulation of inflammasome activity through NLRP3 ubiquitination level)
The innate immunity system constitutes a highly efficient barrier to diverse insults by rapidly detecting pathogens as well as tissue damages. Activation of pattern recognition receptors by pathogen- and damage-associated molecular patterns leads to the early local detection of the insult and production of pro-inflammatory cytokines. NLRP3 is an important PRR for anti-viral, -bacterial, and -fungal innate immunity. On the other hand, NLRP3 inappropriate activation can also contribute to deleterious inflammatory syndromes as tragically exemplified in Cryopyrin-Associated Periodic Syndromes (CAPS) associated with mutations in the NLRP3 gene leading to the expression of constitutively activated NLRP3. Moreover inappropriate stimuli-mediated NLRP3 activation is involved in diverse other conditions including septic shock, gout disease, atherosclerosis, diabetes, Alzheimer’s disease and ischemia-reperfusion injuries. Consistent with inflammation being a double edge sword for the organism, NLRP3 activity is tightly regulated by two signals referred to as priming and activation signals. The priming signal is generally provided by Toll-like receptor (TLR) ligands such as LPS. Activation signals consist in structurally diverse molecular patterns including bacterial toxin nigericin, extracellular ATP, crystals, hyperglycemia, nucleic acid and alum that may trigger common intracellular change(s) sensed by NLRP3. NLRP3 activation mechanisms remain poorly characterized but lead to a change in NLRP3 oligomer conformation followed by the assembly a typical multimeric inflammasome complex comprising the adaptor ASC and the effector pro-caspase-1 to mediate the trans-activation of caspase-1. Caspase-1 is an important pro-inflammatory caspase directly involved in processing and release of cytosolic pro-cytokines, including pro-IL-1β and pro-IL-18 and other inflammatory factors through an unconventional secretion pathway. In some conditions, caspase-1 triggers pyroptosis, a highly inflammatory form of cell death.
The activation mechanism of NLRP3 remains poorly understood. Our previous work evidenced that the inflammasome assembly required NLRP3 deubiquitination by the deubiquitinase BRCC3. The aim of our project is to decipher this new ubiquitin-dependent regulatory pathway critical for NLRP3 activation. During this past mid-term period, we focused on understanding the molecular consequences of NLRP3 ubiquitination responsible for its inactivation. Using diverse approaches, we identified more than a dozen of post-translationally modified sites in NLRP3. We developed cell-based reconstitution assays to analyze the activity of mutant NLRP3 bearing substitutions of these modified sites, and evidenced several sites critical for NLRP3 activity. Further work on that part will aim at (1) characterizing the impact of these particular sites in inflammatory response in in vivo model and at (2) understanding at the molecular level the consequences of these modifications on NLRP3 structure, sub-localization, interaction partners. In addition, we investigated the enzymes regulating NLRP3 post-translational modifications. First, we generated mouse lines deficient for the deubiquitinase BRCC3. This transgenic animal model is currently instrumental in deciphering the impact of BRCC3-mediated NLRP3 regulation in spontaneous auto-inflammation, and inflammation. In parallel we performed genetic-based screen to identify other enzymes involved in this regulation and several hits are currently under characterization. Finally, technical resource developed during this project have been adapted for translational research and are now instrumental for (1) the set up of functional assay to help diagnosis of auto-inflammatory patients and (2) the screen of small compounds inhibiting NLRP3 activation.
The activation mechanism of NLRP3 remains poorly understood. Our previous work evidenced that the inflammasome assembly required NLRP3 deubiquitination by the deubiquitinase BRCC3. The aim of our project is to decipher this new ubiquitin-dependent regulatory pathway critical for NLRP3 activation. During this past mid-term period, we focused on understanding the molecular consequences of NLRP3 ubiquitination responsible for its inactivation. Using diverse approaches, we identified more than a dozen of post-translationally modified sites in NLRP3. We developed cell-based reconstitution assays to analyze the activity of mutant NLRP3 bearing substitutions of these modified sites, and evidenced several sites critical for NLRP3 activity. Further work on that part will aim at (1) characterizing the impact of these particular sites in inflammatory response in in vivo model and at (2) understanding at the molecular level the consequences of these modifications on NLRP3 structure, sub-localization, interaction partners. In addition, we investigated the enzymes regulating NLRP3 post-translational modifications. First, we generated mouse lines deficient for the deubiquitinase BRCC3. This transgenic animal model is currently instrumental in deciphering the impact of BRCC3-mediated NLRP3 regulation in spontaneous auto-inflammation, and inflammation. In parallel we performed genetic-based screen to identify other enzymes involved in this regulation and several hits are currently under characterization. Finally, technical resource developed during this project have been adapted for translational research and are now instrumental for (1) the set up of functional assay to help diagnosis of auto-inflammatory patients and (2) the screen of small compounds inhibiting NLRP3 activation.