Doctor of Philosophy
Date of Defense
Dr. Michael R. Nichols
Dr. Cynthia M. Dupureur
Dr. Chung F. Wong
Dr. Wendy M. Olivas
The innate immune system is the first line of defense in response to invasion by pathogens. One of the major pathways in the innate immune system involves a three-protein complex known as the NLRP3 inflammasome. This complex comprises of NLRP3, ASC, and procaspase-1. In response to stimuli, the inflammasome assembles to activate caspase-1 which subsequently facilitates production of interleukin-1β (IL-1β), an inflammatory cytokine. The NLRP3 inflammasome has been implicated in a variety of inflammatory disorders including Alzheimer’s disease (AD). Amyloid beta (Aβ) is the protein that causes AD and Aβ deposits in the brain activate microglia resulting in chronic inflammation.
However, the actual events by which Aβ activates the inflammasome are still unknown. In addition, the actual structure of the inflammatory complex and how that relates to caspase-1 activation is yet to be determined. Hence, my research focused on determining the interactions between Aβ and the NLRP3 inflammasome, and how they lead to caspase-1 activation. To conduct these studies, I expressed full-length, mutants, and truncated versions of the inflammasome proteins using a mammalian ExpiCHO-S system. This was the first time anyone had ever attempted to use this cell line to express these proteins. Recombinant proteins were successfully expressed, purified by affinity and size-exclusion chromatography then characterized. Biochemical and biophysical techniques were used to study the sizes, structures, and stoichiometry of the proteins. The proteins also exhibited aggregation and efforts to break the aggregates involved stringent conditions such as thermal and chemical denaturation, detergents, and reducing agents.
Caspase-1 activity assays were conducted to determine which exact domains of the protein are required for enzyme activity. Using a fluorescence assay, I observed transient enzymatic activity with procaspase-1 but not with the reconstituted p20p10 and CARDp20 domains This discovery was unexpected since the proenzyme had been previously dubbed inactive. Cell stimulation assays and solution interactions with recombinant proteins confirmed direct interactions between Aβ and NLRP3 protein in agreement with previous findings. Thus, these studies provide insightful findings regarding how Aβ activates the NLRP3 inflammasome and also offer new perspectives relating to the actual structure of active caspase-1.
Makoni, Nyasha, "Interactions of the NLRP3 Inflammasome Complex" (2020). Dissertations. 1002.
Additional FilesNyashaMakoni 3MT.pptx (509 kB)
3 Minute Thesis slide