Structural investigation of macromolecular assembly by adaptive and innate immune regulators using cryo-EM

• Main Author: Huynh, Phuong Thao
• Other Authors: Luo Dahai
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2022
• Subjects: Science::Medicine
• Online Access: https://hdl.handle.net/10356/159552
• Institution: Nanyang Technological University

The immune system works to protect individuals from infections through an array of mechanisms, which are classified into two main groups, namely, innate immunity and adaptive immunity. Innate immunity is constituted by a set of germline-encoded responses that handle molecules commonly found in many microbes or toxins but not in the host, while the adaptive system refers to responses that are encoded by gene elements that somatically rearrange to assemble antigen-binding complexes with high specificity to target invasive foreign molecules. Although their fundamental modes of action are distinctive yet complementary at the same time, members of both innate and adaptive immunity rely on the formation of well-organized macromolecular complexes to perform their functions properly. Disruption in the complex assembly causes the malfunction of proteins, subsequently leading to life-threatening disorders, with autoimmune diseases being one of the most common yet complicated diseases to address. Some severe autoimmune diseases were reported to be the consequences of specific protein malfunction, such as the NLR family pyrin domain containing 6 (NLRP6), an innate immune receptor that was reported to be linked with Crohn’s disease; or Autoimmune Regulator (AIRE), a transcriptional regulator that was found to be the cause of APS-1; and members of speckled protein (SP), a family of chromatin readers which were associated with primary biliary cholangitis and multiple sclerosis. However, how the underlying molecular basis in the complex assembly of these proteins facilitates their proper functions, protecting the human body from autoimmune diseases, remains unclear. This work shows several reconstructed structures of the functional domains in NLRP6, AIRE and SPs using cryo-electron microscopy (cryo-EM) at resolutions that are useful to provide insightful information, explaining how these proteins maintain their functions. The cryo-EM reconstructed 3D model of NLRP6 reveals how it undergoes conformational changes to facilitate inflammasome recruitment and propagation, suggesting its molecular basis in the protection of the gut. Other proteins, including AIRE and SPs, have caspase activation and recruitment domain (CARD) structures resolved at a resolution of ~4 Å, revealing a distinctive mode of assembly of nuclear CARDs that facilitates the phase transition of nuclear bodies. Mutations that disrupt filament formation by these nuclear speckled proteins can lead to a complete loss of function, as demonstrated in the case of the R12E mutant of the AIRE protein. In general, the findings presented in this work contribute to the existing knowledge of the modulation of self- and nonself-discrimination by innate and adaptive immune systems. This work can be used as a starting point for more in vivo assays that aim to establish a comprehensive connection between NLRP6, AIRE, and SPs and their related autoimmune diseases. The reconstructed structures are also insightful in guiding the identification of potential protein-based drug targets to dampen the chronic effects and the long-term consequences brought to patients who suffer from life-threatening autoimmune disorders.