UNC93B1 is essential for the plasma membrane localization and signaling of Toll-like receptor 5

The proper trafficking and localization of Toll-like receptors (TLRs) are important for specific ligand recognition and efficient signal transduction. The TLRs sensing bacterial membrane components are expressed on the cell surface and recruit signaling adaptors to the plasma membrane upon stimulation. On the contrary, the nucleotide-sensing TLRs are mostly found inside cells and signal from the endolysosomes in an acidic pH-dependent manner. Trafficking of the nucleotide-sensing TLRs from the endoplasmic reticulum to the endolysosomes strictly depends on UNC93B1, and their signaling is completely abolished in the 3d mutant mice bearing the H412R mutation of UNC93B1. In contrast, UNC93B1 was considered to have no role for the cell surface-localized TLRs and signaling via TLR1, TLR2, TLR4, and TLR6 is normal in the 3d mice. Unexpectedly, we discovered that TLR5, a cell surface receptor for bacterial protein flagellin, also requires UNC93B1 for plasma membrane localization and signaling. TLR5 physically interacts with UNC93B1, and the cells from the 3d or UNC93B1-deficient mice not only lack TLR5 at the plasma membrane but also fail to secret cytokines and to up-regulate costimulatory molecules upon flagellin stimulation, demonstrating the essential role of UNC93B1 in TLR5 signaling. Our study reveals that the role of UNC93B1 is not limited to the TLRs signaling from the endolysosomes and compels the further probing of the mechanisms underlying the UNC93B1-assisted differential targeting of TLRs.Toll-like receptors (TLRs) sense unique microbial structures or host-derived molecules released from stressed or dying cells to initiate the innate immune responses (1). TLRs are composed of three domains: the leucine-rich repeat (LRR) domain responsible for ligand binding, a single transmembrane domain, and the cytoplasmic Toll/IL-1 receptor homology domain by which TLRs recruit adaptor molecules for downstream signal transduction. Activated TLRs stimulate the NF-κB, MAPK, and IFN regulatory factor pathways, leading to the expression of diverse inflammatory cytokines, chemokines, and type I interferons. TLRs also activate antigen presenting cells to induce costimulatory molecules and coordinate various aspects of adaptive immune responses (2).The members of the TLR family can be classified into two groups based on their subcellular localization patterns (3–5). TLR1, TLR2, TLR4, and TLR6, which mainly recognize the components of bacterial cell membrane, are located on the cell surface and initiate signaling thereat. In contrast, the nucleotide-sensing TLRs such as TLR3, TLR7, TLR8, TLR9, and TLR13 are largely found in endolysosomes and require an acidic environment for their efficient signaling. Additionally, TLR11 and TLR12, the sensors for Toxoplasma protein profilin, are also expressed inside cells and transmit signals in an acidic pH-dependent manner (6–8). All the intracellular TLRs commonly bind to a multispanning membrane protein UNC93B1, which is required for their proper localization and signaling (6–13). One missense mutation (H412R) of UNC93B1, found in a chemically mutagenized mouse strain called 3d, hinders binding of UNC93B1 with TLRs and prevents their exit from the endoplasmic reticulum (ER) (9–11). Consequently, signaling by all endosomal TLRs is abolished in the cells from 3d mice. In contrast, trafficking and signaling of the cell surface-localized TLRs such as TLR2 and TLR4 are not affected by the UNC93B1 mutation (9, 11).The proper localization of TLRs is critical not only for efficient signaling but also for preventing undesirable receptor hyperactivation (14, 15). Especially, sequestration of the nucleotide-sensing TLRs in endolysosomes significantly contributes to attenuating the immune stimulation by host-derived nucleotides abundant in the extracellular spaces (14). Structural discrimination of microbial vs. mammalian nucleotides is not straightforward, and a mutant TLR9 protein, engineered to artificially localize at the plasma membrane, responds to mammalian DNA as well as the CpG oligonucleotides mimicking bacterial DNA. As a result, mice expressing such mutant TLR9 succumb to systemic autoinflammation and die prematurely (15). Therefore, regulatory mechanisms for localization and trafficking of TLRs need to be tightly controlled.TLR5 recognizes flagellin, the major protein subunit of bacterial flagellum, and functions as a critical innate sensor for flagellated bacteria in all mucous organs (16–18). TLR5 plays an important role in intestinal homeostasis mediating the immune adaptation to symbiotic microflora as well as defense against pathogenic bacterial infection (19–21). In addition, systemic injection of flagellin confers protection against ionizing radiation in a TLR5-dependent manner, implying that TLR5 agonism might be clinically used for radioprotection (22). TLR5 overexpressed in the intestinal epithelial cells was exclusively found on the basolateral surface, accounting for the selective induction of proinflammatory cytokine by basolateral but not by apical flagellin (17). Also, we recently demonstrated that endogenous TLR5 is expressed at the cell surface of mouse neutrophils, monocytes, and dendritic cells (DCs) in a TLR-specific chaperone PRAT4A-dependnet manner (23). However, other regulatory mechanisms for the localization of TLR5 at the plasma membrane are unknown. Here, we show that UNC93B1 binds to TLR5, travels to the plasma membrane with the receptor, and is required for flagellin-induced signaling at the cell surface.     

Evidence for erythrocyte-binding antigen 175 as a component of a ligand-blocking blood-stage malaria vaccine

The ligands that pathogens use to invade their target cells have often proven to be good targets for vaccine development. However, Plasmodium falciparum has redundant ligands that mediate invasion of erythrocytes. The first requirement for the development of a successful ligand-blocking malaria vaccine is the demonstration that antibodies induced to each ligand can block the erythrocyte invasion of parasites with polymorphic sequences. Because of P. falciparum's redundancy in erythrocyte invasion, each ligand needs to be studied under artificial conditions in which parasite invasion is restricted in its use of alternative pathways. Here we investigate the role of erythrocyte-binding antigen 175 (EBA-175), a parasite ligand that binds to sialic acid on glycophorin A, in the invasion of erythrocytes by 10 P. falciparum clones under conditions in which invasion is partially limited to the EBA-175-glycophorin A pathway, using chymotrypsin-treated erythrocytes. We show that the ability to invade erythrocytes for both sialic acid-independent and sialic acid-dependent pathways requires the EBA-175-glycophorin A pathway for erythrocyte invasion. Importantly, antibodies against region II of EBA-175 from the 3D7 clone blocked invasion of chymotrypsin-treated erythrocytes by >50% by all parasite clones studied, including those with multiple different mutations described in the literature. The one exception was FCR3, which had a similar sequence to 3D7 but only 30% inhibition of invasion of chymotrypsin-treated erythrocytes, indicating alternative pathways for invasion of chymotrypsin-treated erythrocytes. Our findings suggest that antibodies to region II of EBA-175, as one component of a ligand-blocking malaria vaccine, are largely unaffected by polymorphism in EBA-175.     

Functional interaction between native G protein-coupled purinergic receptors in Xenopus follicles

Purinergic receptors are expressed in the membrane of the follicular cell layer that communicates with the Xenopus oocyte. Adenosine (Ado) generates a cAMP-dependent K⁺ current (I_K,cAMP), whereas ATP activates a Cl⁻ current (F_Cl) and has a dual effect on I_K,cAMP, provoking both its activation and inhibition. Here, purinergic responses were studied electrophysiologically, first in the whole follicle (w.f.), and then in the same follicle after removal of its epithelium/theca layers (e.t.r. follicle). Responses were analyzed as the ratio of the current amplitudes (i_etr/i_wf) in the two preparations. For ATP activation of I_K,cAMP and F_Cl, the ratios i_etr/i_wf were 0.053 and 22, respectively, whereas that for Ado was 0.75. Thus, epithelium/theca removal drastically altered the ATP response, suggesting a change in the signaling pathway that correlated with changes in the pharmacological characteristics: the half-maximal effective concentration for activation of the main current in w.f. (I_K,cAMP) was 14 ± 3.8 μM [Hill coefficient (nH) = 2.7 ± 0.61], and that in e.t.r. follicles (F_Cl) was 1.8 ± 0.68 μM (nH = 0.76 ± 0.09), whereas Ado-response parameters did not change. Responses to UTP and β,γ-methylene-ATP, specific agonists for I_K,cAMP inhibition and activation, respectively, indicated that in e.t.r. follicles inhibition increased and activation decreased drastically. Thus, purinergic responses were not independent; instead, they were functionally linked. We hypothesize that this property was due to direct interactions between receptors for Ado (A2 subtype) and ATP (P2Y subtype) in the Xenopus follicle.     

Modulation of ERα transcriptional activity by the orphan nuclear receptor ERRβ and evidence for differential effects of long- and short-form splice variants

Oestrogen receptor related proteins (ERRs) affect target gene expression without binding oestradiol. We investigated the functional activity of two splice variant isoforms of ERRβ (ERRβS [short], ERRβL [long]) expressed in human endometrium, where they are coexpressed with the oestrogen receptor alpha (ERα). Over-expression of ERRβL enhanced ERα-dependent ligand-induced activation of an ERE-luciferase reporter construct, altered the induction of c-myc mRNA and increased proliferation of Ishikawa cells whereas ERRβS was found to reduce these endpoints.     

Structural basis for alcohol modulation of a pentameric ligand-gated ion channel

Despite its long history of use and abuse in human culture, the molecular basis for alcohol action in the brain is poorly understood. The recent determination of the atomic-scale structure of GLIC, a prokaryotic member of the pentameric ligand-gated ion channel (pLGIC) family, provides a unique opportunity to characterize the structural basis for modulation of these channels, many of which are alcohol targets in brain. We observed that GLIC recapitulates bimodal modulation by n-alcohols, similar to some eukaryotic pLGICs: methanol and ethanol weakly potentiated proton-activated currents in GLIC, whereas n-alcohols larger than ethanol inhibited them. Mapping of residues important to alcohol modulation of ionotropic receptors for glycine, γ-aminobutyric acid, and acetylcholine onto GLIC revealed their proximity to transmembrane cavities that may accommodate one or more alcohol molecules. Site-directed mutations in the pore-lining M2 helix allowed the identification of four residues that influence alcohol potentiation, with the direction of their effects reflecting α-helical structure. At one of the potentiation-enhancing residues, decreased side chain volume converted GLIC into a highly ethanol-sensitive channel, comparable to its eukaryotic relatives. Covalent labeling of M2 positions with an alcohol analog, a methanethiosulfonate reagent, further implicated residues at the extracellular end of the helix in alcohol binding. Molecular dynamics simulations elucidated the structural consequences of a potentiation-enhancing mutation and suggested a structural mechanism for alcohol potentiation via interaction with a transmembrane cavity previously termed the "linking tunnel." These results provide a unique structural model for independent potentiating and inhibitory interactions of n-alcohols with a pLGIC family member.     

TOLL-LIKE RECEPTORS (TLR) 2 AND 4 EXPRESSION OF KERATINOCYTES FROM PATIENTS WITH LOCALIZED AND DISSEMINATED DERMATOPHYTOSIS

There are few studies on the role of innate immune response in dermatophytosis. An investigation was conducted to define the involvement of Toll-Like Receptors (TLRs) 2 and 4 in localized (LD) and disseminated (DD) dermatophytosis due to T. rubrum. Fifteen newly diagnosed patients, eight patients with LD and seven with DD, defined by involvement of at least three body segments were used in this study. Controls comprised twenty skin samples from healthy individuals undergoing plastic surgery. TLR2 and TLR4 were quantified in skin lesions by immunohistochemistry. A reduced expression of TLR4 in the lower and upper epidermis of both LD and DD patients was found compared to controls; TLR2 expression was preserved in the upper and lower epidermis of all three groups. As TLR4 signaling induces the production of inflammatory cytokines and neutrophils recruitment, its reduced expression likely contributed to the lack of resolution of the infection and the consequent chronic nature of the dermatophytosis. As TLR2 expression acts to limit the inflammatory process and preserves the epidermal structure, its preserved expression may also contribute to the persistent infection and limited inflammation that are characteristic of dermatophytic infections.     

Highly sensitive and selective odorant sensor using living cells expressing insect olfactory receptors

This paper describes a highly sensitive and selective chemical sensor using living cells (Xenopus laevis oocytes) within a portable fluidic device. We constructed an odorant sensor whose sensitivity is a few parts per billion in solution and can simultaneously distinguish different types of chemicals that have only a slight difference in double bond isomerism or functional group such as ─OH, ─CHO and ─C(═O)─. We developed a semiautomatic method to install cells to the fluidic device and achieved stable and reproducible odorant sensing. In addition, we found that the sensor worked for multiple-target chemicals and can be integrated with a robotic system without any noise reduction systems. Our developed sensor is compact and easy to replace in the system. We believe that the sensor can potentially be incorporated into a portable system for monitoring environmental and physical conditions.     

TRPA channels distinguish gravity sensing from hearing in Johnston's organ

Although many animal species sense gravity for spatial orientation, the molecular bases remain uncertain. Therefore, we studied Drosophila melanogaster, which possess an inherent upward movement against gravity-negative geotaxis. Negative geotaxis requires Johnston's organ, a mechanosensory structure located in the antenna that also detects near-field sound. Because channels of the transient receptor potential (TRP) superfamily can contribute to mechanosensory signaling, we asked whether they are important for negative geotaxis. We identified distinct expression patterns for 5 TRP genes; the TRPV genes nanchung and inactive were present in most Johnston's organ neurons, the TRPN gene nompC and the TRPA gene painless were localized to 2 subpopulations of neurons, and the TRPA gene pyrexia was expressed in cap cells that may interact with the neurons. Likewise, mutating specific TRP genes produced distinct phenotypes, disrupting negative geotaxis (painless and pyrexia), hearing (nompC), or both (nanchung and inactive). Our genetic, physiological and behavioral data indicate that the sensory component of negative geotaxis involves multiple TRP genes. The results also distinguish between different mechanosensory modalities and set the stage for understanding how TRP channels contribute to mechanosensation.     

Molecular cloning, characterization and expression profiles of three estrogen receptors in protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides)

Estrogen plays key roles in vertebrate reproductive system via estrogen receptors (ERs) as mediating pathways. In the present study, three full-length ERs cDNA sequences were isolated from a protogynous teleost, the orange-spotted grouper (Epinephelus coioides), and were 2235 bp for gERα, 1967 bp for gERβ1 and 2158 bp for gERβ2, respectively. Phylogenetic and amino acid alignment analyses showed that each gER was clustered in the corresponding taxonomic groups of the perciformes and exhibited high evolutional conservation in functional domains. RT-PCR revealed that gERs expressed at different levels in all the obtained tissues. gERα highly expressed in mature ovaries, gERβ1 mainly expressed in immature ovaries and gERβ2 varied greatly during ovarian development. During female to male sex reversal induced by 17α-methyltestosterone (MT) implantation, gERα decreased gradually, gERβ1 increased gradually, and gERβ2 decreased firstly and recovered subsequently in male stage. The present study speculated the potential roles of gERs during female maturation and female to male sex reversal induced by MT in the protogynous grouper E. coioides.     

Mutations in insulin signaling pathway alter juvenile hormone synthesis in Drosophila melanogaster

Juvenile hormone (JH) is a key endocrine regulator of insect metamorphosis, reproduction, and aging. The synthesis of JH is regulated by neuropeptides and biogenic amines, but the molecular and cellular basis of this control remains largely unknown. Genetic analysis of JH synthesis in Drosophila melanogaster mutant for insulin signaling may provide new and powerful insights. Mutants of the insulin receptor (InR) are slow to develop, small, infertile, and long-lived. We previously reported that mutants of InR had reduced JH synthesis as young adults, and that normal longevity and vitellogenesis were restored by topical application of a JH analog [Science 292 (2001) 107]. Here, we describe the 10-day adult age course of JH synthesis from isolated corpus allatum (CA) of InR and of chico, the insulin receptor substrate homolog. JH synthesis increased in wildtype flies to a maximum of 30fmol/gland/h at day 10. In contrast, homozygous InR mutants produced no more than 3 fmol/gland/h JH within the first 5 days, and only 7 fmol/gland/h at day 10. InR mutation disproportionately reduced the synthesis of JH III-bisepoxide, the major JH subtype of the fly. Mutation of chico also reduces body size and extends longevity [Science 292 (2001) 104; Aging Cell 1 (2002a) 75]. Both homozygous and heterozygous chico genotypes reduced JH synthesis, but only to 47 and 67%, respectively, of wildtype and without influencing the ratio of JH subtypes. Because JH synthetic rate does not correlate with the size of CA, it is not likely that insulin signaling mediates JH by impeding endocrine tissue development. Alternatively, we find allatotropin-positive axons to be abundant in the adult brain and in the corpora cardiaca-corpus allatum complex but these neurons are less immunoreactive in the InR mutant genotype, suggesting that insulin signaling may affect JH synthesis through control of JH regulatory neuropeptides.     

Identification and characterization of mesotocin and V1a-like vasotocin receptors in a urodele amphibian, Taricha granulosa

The cDNA sequences encoding the mesotocin receptor (MTR) and vasotocin 1a receptor (VTR-1a) were identified in a urodele amphibian, the rough-skinned newt, Taricha granulosa. Saturation binding of [³H]oxytocin (OT) to the Taricha MTR (tMTR) was best fit by a two-state model; a high affinity-low abundance site and a lower affinity-high abundance site. Competition-binding studies found the following rank-order affinities for the tMTR: mesotocin (MT) > OT ≈ vasotocin (VT) > vasopressin (VP) > isotocin (IT). Inositol phosphate (IP) accumulation studies demonstrated functional activity of both the tMTR and Taricha VTR-1a (tVTR-1a) in a heterologous cell culture system. The rank-order potencies for the tMTR were MT > OT > VT ≈ VP > IT. The combined binding and IP results indicate that VT may act as a partial agonist of the tMTR. Rank-order potencies for the tVTR-1a were VT > VP > MT ≈ OT > IT. For both receptors, stimulation of IP accumulation was blocked by d(CH₂)₅[Tyr(Me)²]AVP (Manning compound) and d(CH₂)₅[Tyr(Me)²,Thr⁴,Tyr-NH₂]OVT (OTA). OTA was a more potent antagonist for the transiently expressed tMTR while Manning compound was relatively more potent at inhibiting IP accumulation in tVTR-1a expressing cells. In contradiction to earlier assumptions, the absolute IC₅₀ of Manning compound was lower for the tMTR (27 nM ± 13) than the tVTR-1a (586 nM ± 166) indicating its potential higher affinity for the tMTR, a finding with special relevance to interpretation of comparative studies investigating the behavioral and physiological actions of neurohypophysial peptides in non-mammalian species.     

Functional expression of frog and rainbow trout melanocortin 2 receptors using heterologous MRAP1s

Analysis of the functional expression of the melanocortin 2 receptor (MC2R) from a rather broad spectrum of vertebrates indicates that MC2R is exclusively selective for the ligand, ACTH, and the melanocortin receptor accessory protein 1 (MRAP1) is required for high affinity ACTH binding and activation of MC2R. A phylogenetic analysis of MRAP1 suggested that tetrapod sequences and bony fish sequences may represent two distinct trends in the evolution of the mrap1 gene. To test this hypothesis, a frog (Xenopus tropicalis) MC2R was expressed in CHO cells either in the presence of a tetrapod (mouse) MRAP1 or a bony fish (zebrafish) MRAP1. The response of frog MC2R to different concentrations of human ACTH(1-24) was more robust in the presence of mouse MRAP1 than in the presence of zebrafish MRAP1. Conversely, the cAMP response mediated by the rainbow trout (Oncorhynchusmykiss) MC2R was almost twofold higher and occurred at 1000-fold lower ACTH concentration in the presence of zebrafish MRAP1 than in the presence of mouse MRAP1. Collectively, these experiments raise the possibility that at least two distinct trends have emerged in the co-evolution of MC2R/MRAP1 interactions during the radiation of the vertebrates.     

Genetic variability in ESAG6 genes among Trypanosoma evansi isolates and in comparison to other Trypanozoon members

Bloodstream trypanosomes take up iron needed for their propagation through the transferrin receptor that, in Trypanosoma brucei, is encoded by expression-site-associated genes (ESAGs), ESAG6 and 7 genes located in variant surface glycoprotein expression sites. ESAG6 and 7 genes in different expression sites have been shown to encode transferrin receptors with varying affinities for polymorphic transferrins. T. brucei could cope with the different host transferrins by switching between expression sites. ESAG6- and 7-encoded transferrin receptor appear to be present in Trypanosoma evansi but the genes have not yet been characterized. In this study, we cloned and sequenced different members of ESAG6 genes in seven isolates of T. evansi from geographically distinct localities in Thailand. We assessed the intra- and inter-species genetic variability in the transferrin receptor gene regions involved in transferrin binding and established that T. evansi, like T. brucei, has widely diverse ESAG6 genes. In addition, T. evansi possess a clade of ESAG6 variants not observed in T. brucei and different T. evansi strains share at least two conserved variants. We further noted that T. evansi possesses all the reported T. equiperdum ESAG6 variants as a subset. Our findings depict a correlation between the genetic diversity in the transferrin-binding regions of ESAG6 genes with the broad host range of T. evansi and T. brucei compared to the narrow host range of Trypanosoma equiperdum.     

Molecular cloning and characterization of a gonadotropin-releasing hormone receptor in the guinea pig, Cavia porcellus

Guinea pig gonadotropin-releasing hormone (gpGnRH) is predicted to have a unique structure among all known forms of GnRH molecule [Endocrinology 138 (1997) 4123] and it is of great interest to determine whether the unique structure of gpGnRH is manifested in the characteristics of the guinea pig GnRH receptor. In the present study, we isolated a full-length cDNA for a GnRH receptor from the pituitary gland of the guinea pig. The putative guinea pig GnRH receptor protein has an amino acid identity of 79-87% with mammalian type I GnRH receptors. The amino acid residues which have been demonstrated to be important for ligand binding and signal transduction were conserved in the guinea pig GnRH receptor. However, there are several specific amino acid substitutions among mammalian type I GnRH receptors. Moreover, though the guinea pig has generally been classified as a rodent, the putative GnRH receptor protein did not have some rodent-specific characteristics. Total IP assays demonstrated that the cloned guinea pig GnRH receptor is a functional GnRH receptor and that it shows different preference of ligand sensitivities from the rat GnRH receptor.