Date of Award
Spring 4-18-2024
Document Type
Thesis (Ph.D.)
Department or Program
Microbiology and Immunology
First Advisor
James Bliska
Abstract
The mammalian innate immune defense employs sophisticated mechanisms, such as effector triggered immunity (ETI), to discern between pathogenic and non-pathogenic bacteria and initiate a protective response. ETI is involved in immune detection of virulent activities of bacterial effector proteins in the host cytosol. Inflammasome sensors, such as pyrin, detect cellular infection or stress as part of ETI, leading to the assembly of multiprotein complexes, caspase-1 activation and proinflammatory cytokine release. Pyrin uniquely initiates inflammatory responses against RhoA-inactivating bacterial toxins and effectors such as Yersinia's YopE and YopT, and C. difficile’s toxins TcdA/B. Understanding pyrin regulation is crucial due to its association with dysregulated autoinflammatory responses, including Familial Mediterranean Fever (FMF), linked to pyrin gene mutations. Pyrin regulation mirrors the guard hypothesis observed in plants, whereby negative regulation is achieved through phosphorylation by RhoA-PKN signaling and binding of 14-3-3 proteins, maintaining an inactive state. Upon RhoA inactivation, pyrin is dephosphorylated and interacts with the inflammasome adapter protein ASC to assemble a caspase-1 inflammasome. Research into positive regulation mechanisms of pyrin carried out in this dissertation underscores the importance of 1) phosphoserine phosphatase (PPP) activity; 2) oligomerization; and 3) microtubules (MTs) in facilitating inflammasome assembly in phagocytic cells. Murine pyrin is phosphorylated at S205 before inflammasome assembly, and PP2A catalytic subunits dephosphorylate this site in macrophages. Results with Blue-Native (BN)-PAGE show that both human and murine pyrin form dimers and higher-order oligomers which are phosphorylated when inactive. Interestingly, gain of function codon changes associated with autoinflammatory diseases like FMF do not alter steady state oligomerization of human pyrin. Results confirm that intact MTs positively regulate pyrin post-dephosphorylation but upstream of inflammasome assembly. However, MTs do not appear to regulate pyrin oligomerization. Additionally, experiments designed to confirm previous results did not establish a role for the MT-associated protein HDAC6, or localization to MT organizing centers, for murine pyrin inflammasome assembly in macrophages, as shown by live cell imaging. In summary, these findings offer insights into mechanisms of positive regulation of pyrin and a roadmap to further investigate the regulation of oligomeric pyrin and the balance of kinase and phosphatase activity along with MT dynamics in pyrin-associated infectious and autoinflammatory diseases.
Original Citation
Malik HS, Magnotti F, Loeven NA, Delgado JM, Kettenbach AN, Henry T, Bliska JB. 2023. Phosphoprotein phosphatase activity positively regulates oligomeric pyrin to trigger inflammasome assembly in phagocytes. mBio doi:10.1128/mbio.02066-23:e0206623
Recommended Citation
Malik, Haleema Sadia, "Mechanistic Insights on Positive Regulation of the Pyrin Inflammasome in Macrophages" (2024). Dartmouth College Ph.D Dissertations. 260.
https://digitalcommons.dartmouth.edu/dissertations/260