Supramolecular organizing centers (SMOCs) as signaling machines in innate immune activation.

Innate immunity offers the first line of defense against infections and other types of danger such as tumorigenesis. Its discovery provides tremendous therapeutic opportunities for numerous human diseases. Delving into the structural basis of signal transduction by innate immune receptors, our lab has recently helped to establish the new paradigm in which innate immune receptors transduce ligand-binding signals through formation of higher-order assemblies containing intracellular adapters, signaling enzymes and their substrates. These large signalosome assemblies may be visible under light microscopy as punctate structures in the µm scale, connecting to the underlying molecular structures in the nm scale. They drive proximity-induced enzyme activation, and provide a mechanism for signaling amplification by nucleated polymerization. These supramolecular signaling complexes also open new questions on their cellular organization and mode of regulation, pose challenges to our methodology, and afford valuable implications in drug discovery against these medically important pathways.

The role of spartin and its novel ubiquitin binding region in DALIS occurrence.

Troyer syndrome is an autosomal recessive hereditary spastic paraplegia (HSP) caused by frameshift mutations in the SPG20 gene that results in a lack of expression of the truncated protein. Spartin is a multifunctional protein, yet only two conserved domains--a microtubule-interacting and trafficking domain and a plant-related senescence domain involved in cytokinesis and mitochondrial physiology, respectively--have been defined. We have shown that overexpressed spartin binds to the Ile44 hydrophobic pocket of ubiquitin, suggesting spartin might contain a ubiquitin-binding domain. In the present study, we demonstrate that spartin contributes to the formation of dendritic aggresome-like induced structures (DALIS) through a unique ubiquitin-binding region (UBR). Using short hairpin RNA, we knocked down spartin in RAW264.7 cells and found that DALIS frequency decreased; conversely, overexpression of spartin increased the percentage of cells containing DALIS. Using nuclear magnetic resonance spectroscopy, we characterized spartin's UBR and defined the UBR's amino acids that are key for ubiquitin binding. We also found that spartin, via the UBR, binds Lys-63-linked ubiquitin chains but does not bind Lys-48-linked ubiquitin chains. Finally, we demonstrate that spartin's role in DALIS formation depends on key residues within its UBR.

Molecular cell biology and immunobiology of mammalian rod/ring structures.

Nucleotide biosynthesis is a highly regulated process necessary for cell growth and replication. Cytoplasmic structures in mammalian cells, provisionally described as rods and rings (RR), were identified by human autoantibodies and recently shown to include two key enzymes of the CTP/GTP biosynthetic pathways, cytidine triphosphate synthetase (CTPS) and inosine monophosphate dehydrogenase (IMPDH). Several studies have described CTPS filaments in mammalian cells, Drosophila, yeast, and bacteria. Other studies have identified IMPDH filaments in mammalian cells. Similarities among these studies point to a common evolutionarily conserved cytoplasmic structure composed of a subset of nucleotide biosynthetic enzymes. These structures appear to be a conserved metabolic response to decreased intracellular GTP and/or CTP pools. Antibodies to RR were found to develop in some hepatitis C patients treated with interferon-α and ribavirin. Additionally, the presence of anti-RR antibodies was correlated with poor treatment outcome.

Expression of hypoallergenic Der f 2 derivatives with altered intramolecular disulphide bonds induces the formation of novel ER-derived protein bodies in transgenic rice seeds.

House dust mites (HDM) are the most common source of indoor allergens and are associated with allergic diseases worldwide. To benefit allergic patients, safer and non-invasive mucosal routes of oral administration are considered to be the best alternative to conventional allergen-specific immunotherapy. In this study, transgenic rice was developed expressing derivatives of the major HDM allergen Der f 2 with reduced Der f 2-specific IgE reactivity by disrupting intramolecular disulphide bonds in Der f 2. These derivatives were produced specifically as secretory proteins in the endosperm tissue of seeds under the control of the endosperm-specific glutelin GluB-1 promoter. Notably, modified Der f 2 derivatives aggregated in the endoplasmic reticulum (ER) lumen and were deposited in a unique protein body (PB)-like structure tentatively called the Der f 2 body. Der f 2 bodies were characterized by their intracellular localization and physico-chemical properties, and were distinct from ER-derived PBs (PB-Is) and protein storage vacuoles (PB-IIs). Unlike ER-derived organelles such as PB-Is, Der f 2 bodies were rapidly digested in simulated gastric fluid in a manner similar to that of PB-IIs. Oral administration in mice of transgenic rice seeds containing Der f 2 derivatives encapsulated in Der f 2 bodies suppressed Der f 2-specific IgE and IgG production compared with that in mice fed non-transgenic rice seeds, and the effect was dependent on the type of Der f 2 derivative expressed. These results suggest that engineered hypoallergenic Der f 2 derivatives expressed in the rice seed endosperm could serve as a basis for the development of viable strategies for the oral delivery of vaccines against HDM allergy.

The cell biology of TRIM5α.

The tripartite motif (TRIM)-containing proteins are involved in many cellular functions such as cell signaling, apoptosis, cell differentiation, and immune modulation. TRIM5 proteins, including TRIM5α and TRIM-Cyp, are known to possess antiretroviral activity against many different retroviruses. Besides being retroviral restriction factors, TRIM5 proteins participate in other cellular functions that have recently emerged in the study of TRIM5α. In this review, we discuss properties of TRIM5α such as cytoplasmic body formation, protein turnover, and trafficking. Also, we discuss recent insights into innate immune modulation mediated by TRIM5α, highlighting the various functions TRIM5α has in cellular processes.

Autoantibodies to GW bodies and other autoantigens in primary biliary cirrhosis.

Autoantibodies to intracellular targets in mitochondria and nuclei are serological hallmarks of primary biliary cirrhosis (PBC). One of the most recently identified cellular targets of PBC autoantibodies is a novel cytoplasmic structure referred to as GW bodies [GWB, G (glycine) W (tryptophan)-containing bodies (GWB)]. GWB are indentified as discrete cytoplasmic domains that are involved in mRNA processing via the RNA interference (RNAi) pathway. Key components of GWB include the proteins GW182, Ago2, RNA-associated protein 55 (RAP55) and Ge-1/Hedls. The primary objective was to study the frequency and clinical association of antibodies directed to GWB components, in 109 PBC patients. Autoantibodies to mitochondrial antigen-pyruvate dehydrogenase complex (M2), branched-chain 2-oxo-acid dehydrogenase complex and 2-oxo glutarate dehydrogenase complex (3E-BPO), gp210, sp100, promyelocytic leukaemia cell antigen (PML) and liver kidney microsomal-1 antigen (LKM-1) were detected by a line immunoassay and antibodies to GWB (GW182, RAP55, Ge-1, GW2, GW3) and glutamate receptor interacting protein (GRIP)-associated protein-1 (GRASP-1), by an addressable laser bead immunoassay (ALBIA). The most common GWB autoantigen targets were: RAP55-28%, GW182-12%, GW2-2% and antibodies to GRASP-1-17%. By comparison, the frequency of reactivity to established PBC autoantigens was: gp210, 27%; sp100, 27% and PML, 17%. None of the autoantibodies were associated with differences in Mayo risk score or liver decompensation. This study is the first study to show that antibodies to RAP55, GW182 and GRASP-1 are the most common GWB targets in PBC.

Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella.

Intracellular pathogens and endogenous danger signals in the cytosol engage NOD-like receptors (NLRs), which assemble inflammasome complexes to activate caspase-1 and promote the release of proinflammatory cytokines IL-1beta and IL-18. However, the NLRs that respond to microbial pathogens in vivo are poorly defined. We show that the NLRs NLRP3 and NLRC4 both activate caspase-1 in response to Salmonella typhimurium. Responding to distinct bacterial triggers, NLRP3 and NLRC4 recruited ASC and caspase-1 into a single cytoplasmic focus, which served as the site of pro-IL-1beta processing. Consistent with an important role for both NLRP3 and NLRC4 in innate immune defense against S. typhimurium, mice lacking both NLRs were markedly more susceptible to infection. These results reveal unexpected redundancy among NLRs in host defense against intracellular pathogens in vivo.

Formation of GW/P bodies as marker for microRNA-mediated regulation of innate immune signaling in THP-1 cells.

GW bodies (GWB or P bodies) are cytoplasmic foci thought to result from microRNA (miRNA) regulation of messenger RNA (mRNA) targets and subsequent mRNA degradation. The purpose of this study is to examine the effects of lipopolysaccharide (LPS) stimulation of human monocytes on GWB formation, miRNA induction, miRNA target regulation and downstream cytokine and chemokine expression. In response to LPS stimulation, the number of GWB consistently increased by twofold at 8 h after stimulation and this increase was abolished when the miRNA-effector proteins Rck/p54 or argonaute 2 were depleted. As the level of miR-146a increased from 19-fold up to 100-fold during LPS stimulation, the transfection of a miR-146a mimic into THP-1 cells was examined to determine whether miR-146a alone can induce similar changes in GWB. The results showed transfected miR-146a could produce a comparable increase in the number of GWB and this was accompanied by a reduction in major cytokines/chemokines induced by LPS. These data show that the increase in size and number of GWB may serve as a biomarker for miRNA-mediated gene regulation, and miR-146a has a significant role in the regulation of LPS-induced cytokine production in THP-1 cells.

Dendritic cell aggresome-like-induced structure formation and delayed antigen presentation coincide in influenza virus-infected dendritic cells.

Influenza virus infection induces maturation of murine dendritic cells (DCs), which is most important for the initiation of an immune response. However, in contrast to EL-4 and MC57 cells, DCs present viral CTL epitopes with a delay of up to 10 h. This delay in Ag presentation coincides with the up-regulation of MHC class I molecules as well as costimulatory molecules on the cell surface and the accumulation of newly synthesized ubiquitinated proteins in large cytosolic structures, called DC aggresome-like-induced structures (DALIS). These structures were observed previously after LPS-induced maturation of DCs, and it was speculated that they play a role in the regulation of MHC class I Ag presentation. Our findings provide the first evidence for a connection between DC maturation, MHC class I-restricted Ag presentation, and DALIS formation, which is further supported by the observation that DALIS contain ubiquitinated influenza nucleoprotein.