Proteolysis is an evolutionarily conserved mechanism of activating NLRP1 inflammasomes.

Author(s): Chavarria-Smith, Joseph Edward | Advisor(s): Vance, Russell E. | Abstract: Inflammasomes are cytosolic protein complexes that serve as platforms for the recruitment and activation of the pro-inflammatory CASPASE-1 protease (CASP1). CASP1 activation leads to processing and maturation of the cytokines interleukin-1b and -18, and a lytic form of cell death termed pyroptosis. Inflammasome assembly is initiated by cytosolic proteins in response to microbial infections, and many of these sensor proteins belong to the Nucleotide-binding domain, Leucine-rich Repeat containing protein (NLR) family. NLRP1 (NLR family, Pyrin domain containing 1) was the first NLR described to form an inflammasome, but until recently, its mechanism of activation and physiological functions in host defense have remained unclear.In Chapter 1 we extensively review the literature on the proposed mechanisms of NLRP1 activation in humans and rodents. We discuss the activation of NLRP1 by various stimuli, including Bacillus anthracis Lethal Toxin, Toxoplasma gondii, muramyl dipeptide (MDP), and host intracellular ATP depletion. The role NLRP1 plays in pathogen recognition and resistance during infection is also discussed, as is the regulation of NLRP1 by host and viral proteins. We also discuss the unexpected differences in the mechanism of NLRP1 inflammasome activation as compared to the activation of other inflammasomes, such as the NAIP/NLRC4 inflammasomes.In Chapter 2 we cover our discovery regarding the mechanism of mouse NLRP1B activation by Lethal Toxin (LeTx), which is composed of Lethal Factor (LF) and Protective Antigen (PA). We made the critical observation that LF cleaves NLRP1B directly near the N- terminus. We then demonstrated that LF cleavage of NLRP1B is required for NLRP1B activation and inflammasome formation. Most importantly, we were able to show that proteolysis is sufficient to activate NLRP1B when we replaced the activity of LF with a Tobacco Etch Virus (TEV) protease. We conclude by proposing that NLRP1B has evolved to respond to other proteases derived from other pathogens.In Chapter 3 we test the hypothesis that other variants of NLRP1 expressed in mice and human are also activated by proteolysis. We first demonstrate that another allele of NLRP1B, which has no known agonist, is able to respond to cleavage and induce inflammasome formation. We then demonstrate that this activity can also be seen with the NLRP1A paralog. Lastly, we extend this work by analyzing the human ortholog of NLRP1. Surprisingly, proteolysis is also a conserved mechanism of activating human NLRP1. Collectively, these results suggest that NLRP1 might be broadly conserved as a protease sensor in mammals, and provide an important host defense mechanism.In Chapter 4 we report on the development of a new method of analyzing CASP1 activation. We generated a CASP1 dimerization reporter that generates the fluorescent Venus protein when an inflammasome is activated and does not induce cell death. We generated a cell line that stably expresses the reporter, NLRP1B, and ASC. This line responds to LeTx, and can be analysed by flow cytometry and live cell microscopy. This reporter also works in macrophage-like cells that endogenously express inflammasome components. As a proof of principle we demonstrate that cells with an activated inflammasome can be enriched by FACS. We believe this reporter is an ideal tool for the discovery of novel positive and negative regulators of inflammasomes. The largest advantage of the reporter is that it does not induce cell death. This feature allows for the recovery of cells from a complex mixture that would be amenable for a high thorough put screen.