The research program of the Structural Immunobiology Unit (SIBU) is focused on two integrated themes: the study of large macromolecular signaling platforms known as the inflammasomes and the mechanisms of nucleic acid recognition by innate immune receptors. We use X-ray crystallography as the primary method to dissect the molecular details of innate receptors and signaling adapters, integrated with chemical tools that target these signaling pathways for potential therapeutic applications. Several families of the pattern-recognition receptors (PRRs) play important roles in the innate immune system, such as the Toll-like receptors (TLRs), the RIG-I-like receptors (RLRs), the NOD-like receptors (NLRs), the PYHIN family of receptors and the C-type lectin receptors (CLRs). A subset of the NLR and PYHIN family of receptors form large macromolecular signaling scaffolds known as the inflammasomes. Inflammasomes are large signaling platforms composed of the receptor (such as NLRP1, NLRP3, NLRP6, NLRP7, NLRC4/NAIP, AIM2 and IFI16), the adapter protein ASC, and the effector enzyme procaspase-1. The assembly of the inflammasomes employs homotypic interactions by the death domain superfamily that includes the pyrin domain (PYD) and caspase recruitment domain (CARD). Activation of the inflammasomes results in maturation of proinflammatory cytokines IL-1βand IL-18, and a unique type of cell death called pyroptosis. A major unresolved issue in the field has been the lack of definitive evidence for direct receptor:ligand association for most inflammasomes: seemingly unrelated stimuli are all capable of activating inflammasomes. Therefore, the true identities of the respective ligands and a unifying mechanism of inflammasome activation remain elusive. Nucleic acids are among the most potent activators of cells in the innate immune system that subsequently induce robust adaptive immunity. However, immune responses to nucleic acids, the universal genetic material, pose a unique challenge. By definition, PRRs from the host (self) typically recognize structural and chemical signatures that are specific for microorganisms (non-self). In contrast, innate DNA recognition is largely independent of sequences or modifications, and DNA from host, microbial, and synthetic sources are all known to induce inflammatory responses. It is therefore not surprising that innate nucleic acid receptors play fundamental roles in anti-viral and anti-bacterial defense, as well as being an important contributor to autoimmune diseases such as lupus and psoriasis. During 2012-2013 we have focused on the following area of research. First, we studied the signaling domains PYD and CARD that are responsible for the assembly of the AIM2 inflammasome. The innate immune system responds to the presence of cytosolic DNA molecules through the secretion of interferons and proinflammatory cytokines as a host defense mechanism. The PYHIN family members AIM2 contains a C-terminal DNA-binding HIN domain and an N-terminal PYD domain that belongs to the death domain superfamily.
AIM2 is predominantly a cytosolic protein that responds to dsDNA from both host and pathogens.
The AIM2 inflammasome controls the activation of caspase-1 and subsequent maturation and secretion of IL-1βand IL-18.
AIM2 plays crucial roles in host defense against intracellular bacteria such as Francisella tularensis and Listeria monocytogenes and DNA viruses such as vaccinia virus and HSV, as well as autoimmune diseases such as systemic lupus erythematosus (SLE) in which DNA is a major autoimmune target. Following our previous work on the structures of the AIM2 and IFI16 HIN domains in complex with DNA, we have determined the crystal structure of the AIM2 PYD and identified its interface with the AIM2 HIN domain and the PYD of the downstream adapter ASC. Our structural, biochemical and computational analyses have revealed that the AIM2 HIN domain and ASC PYD both bind the AIM2 PYD, and these interface overlap with each other to preclude simultaneous binding. Such steric hindrance would allow for downstream signaling from AIM2 to ASC only upon release of the AIM2 autoinhibition state by the DNA ligand, consistent with our previous reports of the resting state of the AIM2 inflammasome. In addition to our work on the PYD structure, we have deciphered the structures of the CARD that is responsible for association of ASC with procaspase-1 and another inflammasome receptor NLRP1 with procaspase-1. The structures illustrate a bipolar distribution of electrostatic charges on the surface of the CARD, and suggest that salt bridges may play a major role in the assembly of the inflammasomes through CARD-CARD homotypic interactions. In addition to our work on DNA sensors in the cytosol, we investigated the recognition of nucleic acids by the membrane bound protein RAGE during lung inflammation. Endosomal DNA receptor such as TLR9 has limited access to extracellular nucleic acids, which is largely delivered by cell surface receptor RAGE and its co-receptor HMGB1 (high-mobility group protein B1). In collaboration with the Latz group at Germany, we found that RAGE enhanced immune response to DNA by promoting DNA uptake to the endosomal compartment, and RAGE-deficient mice can not mount typical lung inflammatory response to DNA. Fluorescence polarization, EMSA and confocal microscopy studies confirmed the direct binding of RAGE extracellular V-C1 domains to DNA both in solution and on cell surface, as well as DNA-mediated oligomerization and clustering of RAGE. Interestingly, the ability of DNA to oligomerize RAGE appeared to be directly related to the kinetics of its trafficking through the endosomal network. In agreement, our crystal structure of the RAGE extracellular domain in complex with DNA demonstrated that DNA interacted with RAGE dimers through electrostatic attraction, similar to the DNA interaction with the HIN domains. In addition, the dimerization of the RAGE extracellular domains is mediated by a hydrophobic surface. Because the charge-based binding modality may be applicable to other RAGE ligands involved in inflammation, diminishing RAGE receptor association and clustering by targeting this hydrophobic surface may be a viable method to modulate a number of other inflammatory conditions involving RAGE signaling. Lastly, our recent work has shed light on innate immune signaling mechanisms downstream of the inflammasome activation pathway, as well as the subversion of such signaling by pathogens. MyD88 is a central adaptor molecule for the Toll-like receptors and the IL-1 receptor. A number of bacterial TIR domain-containing proteins have been shown to modulate the MyD88-mediated signaling pathways as a mechanism to subvert immune responses. We have determined the crystal structure of the MyD88 TIR domain with distinct loop conformations that underscore the functional specialization of the adapter, receptor, and microbial TIR domains. We demonstrate that TcpC directly associates with MyD88 and TLR4 through its predicted DD and BB loops to impair the TLR-induced cytokine induction. Furthermore, NMR titration experiments identify the unique CD, DE and EE loops from MyD88 at the TcpC-interacting surface, suggesting that TcpC specifically engages these MyD88 structural elements for immune suppression. These findings thus provide a molecular basis for the subversion of TLR and IL-1R signaling by the uropathogenic E coli virulence factor TcpC, and furnish a framework for the design of novel therapeutic agents that modulate immune activation.
|Hong, Jingjun; Feng, Hanqiao; Wang, Feng et al. (2014) The catalytic subunit of the SWR1 remodeler is a histone chaperone for the H2A.Z-H2B dimer. Mol Cell 53:498-505|
|Abdi, Kaveh; Singh, Nevil J; Spooner, Eric et al. (2014) Free IL-12p40 monomer is a polyfunctional adaptor for generating novel IL-12-like heterodimers extracellularly. J Immunol 192:6028-36|
|Jin, Tengchuan; Huang, Mo; Smith, Patrick et al. (2013) The structure of the CARD8 caspase-recruitment domain suggests its association with the FIIND domain and procaspases through adjacent surfaces. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:482-7|
|Jin, Tengchuan; Huang, Mo; Smith, Patrick et al. (2013) Structure of the caspase-recruitment domain from a zebrafish guanylate-binding protein. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:855-60|
|Jin, Tengchuan; Curry, James; Smith, Patrick et al. (2013) Structure of the NLRP1 caspase recruitment domain suggests potential mechanisms for its association with procaspase-1. Proteins 81:1266-70|
|Jin, Tengchuan; Perry, Andrew; Smith, Patrick et al. (2013) Structure of the absent in melanoma 2 (AIM2) pyrin domain provides insights into the mechanisms of AIM2 autoinhibition and inflammasome assembly. J Biol Chem 288:13225-35|
|Snyder, Greg A; Cirl, Christine; Jiang, Jiansheng et al. (2013) Molecular mechanisms for the subversion of MyD88 signaling by TcpC from virulent uropathogenic Escherichia coli. Proc Natl Acad Sci U S A 110:6985-90|
|Latz, Eicke; Xiao, T Sam; Stutz, Andrea (2013) Activation and regulation of the inflammasomes. Nat Rev Immunol 13:397-411|
|Xiao, T Sam; Fitzgerald, Katherine A (2013) The cGAS-STING pathway for DNA sensing. Mol Cell 51:135-9|
|Xiao, Tsan (2009) Innate immune recognition of nucleic acids. Immunol Res 43:98-108|
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