Innate immunity is the first line of defence against pathogens. However, activation of certain receptors of innate immunity can also occur in the absence of microbes or their components. NLRP3 is a cytosolic innate immunity sensor that allows the detection of a wide range of pathogenic and endogenous molecular patterns. Upon activation by a variety of activators, NLRP3 oligomerises and recruits the adaptor protein ASC and the effector protein pro-caspase-1, leading to the autoproteolytic activation of pro-caspase-1, which in turn cleaves the cytokines pro-IL-1β and pro-IL-18 into their mature form. The substrate of caspase-1 is also the protein gasdermin D, the N-terminal part of which forms pores in the membrane, leading to cell death called pyroptosis. Dysregulated NLRP3 inflammasome activity is associated with many chronic inflammatory, metabolic and neurodegenerative diseases. The mechanism of NLRP3 inflammasome activation is still unclear. In recent years, a number of studies have suggested the importance of NLRP3 cellular location in its activation. It has been shown that NLRP3 inflammasome assembly can also occur at the disassembled trans-Golgi apparatus, to which NLRP3 is thought to localise via a positively charged segment of NLRP3(127-130). This segment is located in a region of NLRP3 crucial for the detection of potassium ion efflux, a common cellular event of many canonical NLRP3 activators.
Since it is unclear what the mechanism of NLRP3 inflammasome activation is and whether the cellular location of the protein also contributes to this, we wanted to define the role of the NLRP3(127-130) segment in the activation of differentially localised NLRP3. To clarify whether the role of the NLRP3(127-130) segment is relevant only for NLRP3 localisation or also for the appropriate response to activators, we mutated the positively charged NLRP3(127-130) segment of NLRP3(127 130) in previously prepared NLRP3 constructs with engineered localisation to different organelles, into a sequence of four alanines and stably integrated these NLRP3 variants into macrophages. We found that the artificially localised NLRP3 variants can be activated despite the presence of the 4×A mutation, while introducing the same change in a non-localised NLRP3 prevents its activation. The mutation had no effect on the activity of the constitutively active NLRP3 variants. In the master thesis, we showed that the NLRP3(127-130) segment does not affect the ability to activate the localised NLRP3 upon stimulation with different activators of the canonical pathway. We can conclude that NLRP3(127-130) segment enables binding of NLRP3 to organelles and thus facilitates an increase in local concentration of NLRP3 molecules and further activation steps.
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