Our research program is focused on the detailed molecular mechanisms underlying the initiation of the innate immunity and the ensuing inflammatory responses. The innate immune system is the first line of defense against infection that also orchestrates the proper function of the adaptive immune response. Its central components are the sensors or receptors residing in the extracellular or intracellular compartments that respond to microbial invasion or tissue damage. The ligands or stimuli for such receptors are generally conserved chemical signatures from microbes such lipopolysacchride, but can also include universal molecules of life such as DNA and RNA. Stringent regulation of the location/expression of the receptors and/or ligands is thus essential for the proper function of the innate immune system, and unregulated innate immune activation can result in autoimmune or autoinflammatory disorders involving nucleic acids. During 2010-2011, we focused our efforts on the following specific area of research. First, we studied the cytosolic DNA receptors AIM2 that form inflammasome and IFI16 that induces IFN production. 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. Recently, a family of DNA-recognizing innate receptors was identified from the HIN-200 proteins (hematopoietic interferon-inducible nuclear proteins with a 200-amino-acid repeat, also named p200 proteins encoded by the IFI200 genes), such as AIM2 and IFI16. Both contain C-terminal DNA-binding HIN domain(s) and an N-terminal pyrin (PYD) domain that belongs to the death domain superfamily of signaling domains.
AIM2 is predominantly a cytosolic protein that responds to dsDNA from both host and pathogens to form large signaling platforms known as the inflammasomes, which also contain adapter proteins ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) and effector enzymes caspase-1. These macromolecular complexes control the activation of caspase-1 and subsequent maturation and secretion of IL-1βand IL-18. Five innate receptors, including NLRP1, NLRP3, NLRC4, NAIP and AIM2, are known to form inflammasomes. Many dysfunctional mutations of these receptors are located within their oligomerization domains, except for AIM2 that lacks such a domain. IFI16 was originally identified as an anti-proliferative and pro-apoptotic nuclear protein. It was recently shown to be also present in the cytosol and was linked to interferon production in response to dsDNA. The precise signaling mechanisms downstream of IFI16 are less clear, although its ability to engage the ER resident protein STING (stimulator of interferon genes) appeared to be a prerequisite. Double-stranded DNA from various sources was capable of stimulating both AIM2 and IFI16 irrespective of their nucleotide sequences and GC contents, facilitating innate immune responses to both pathogenic invasion and cellular stress. As such, these innate receptors play 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. We are studying the formation of receptor-DNA complexes using dsDNA of various lengths and sequences. Experiments are underway to study the affinities of the HIN domains to dsDNA using fluorescence polarization assay and electrophoretic mobility shift assay (EMSA), as well as the activation of IFN reporter genes and AIM2 inflammasomes by dsDNA. Complementary to our work on cytosolic DNA receptors, we are investigating the roles of membrane bound protein RAGE in the induction of inflammation by nucleic acid. 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 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. Experiments are underway to study the interaction of RAGE with DNA of various sequences, and the oligomerization states of RAGE induced by DNA. We have continued our study of the signaling mechanisms by the adapter protein MyD88. MyD88 is a central adapter molecule in the TLR and IL-1R signaling pathways. It contains a C-terminal TIR domain that binds another TIR domain-containing protein TIRAP from host. Uropathogenic E. coli and Brucella encode two TIR domain-containing proteins TcpC and TcpB that directly target MyD88 and TIRAP, respectively. In collaboration with Thomas Miethke from Germany and Nico Tjandra from NHBLI, we found that TcpC inhibits innate immune signaling through MyD88, and the two TIR domains bind each other directly in solution. We are characterizing the specific TIR-TIR domain interface using peptides representing several long loops of the TIR domains, and experiments are underway to produce stable TIR-TIR domain complexes for biophysical studies. Our general interest in inflammatory responses leads to a collaboration with the Daniel Kastner lab at NHGRI on the role of Pyrin in autoinflammatory disorder FMF. Most patients with FMF, an inherited autoinflammatory disorder characterized by recurrent fever and inflammation, carry missense mutations in the Pyrin protein. Recent work from the Kastner lab demonstrated that Pyrin interacts with adapter protein ASC and regulate the maturation of pro-caspase-1 and pro-IL-1βprocessing, perhaps through the formation of large signaling platforms similar to the inflammasomes. The coiled-coil (CC) domain of Pyrin has been reported to mediate its oligomerization and may be involved in the regulation of pro-caspase-1 maturation. Experiments are underway to study the oligomerization states of the CC domain and formation of stable, homogeneous oligomers for biophysical studies. We have established a collaboration with Fadila Bouamr from the Laboratory of Molecular Microbiology, NIAID, to study the mechanisms of HIV budding process. HIV binds host Bro1 domain-containing proteins such as Alix, HD-PTP and Brox to gain access to the host ESCRT (endosomal sorting complexes required for transport) machinery, thus facilitating the release of progenitor viruses from infected cells. We have succeeded in obtaining the crystal structures of the Bro1 domains of HD-PTP and Brox and found unique loop structures for both Brox and Alix. Functional studies identified the Phe105 loop of Alix as a crucial structural element for Alix-mediated HIV budding. Interestingly, Brox also contains a unique long loop on the same surface of its Bro1 domain structure. We are currently working to identify the binding partners for both loops from Alix and Brox, respectively. All the three Bro1 domains bind CHMP (charged multivesicular body protein) proteins from the ESCRT-III complex, which is essential for HIV budding. We are in the process of characterizing the similarities and differences in CHMP binding by the three Bro1 domain-containing proteins to gain deeper understanding of their common and unique roles in HIV budding.
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