Molecular evolution and expression pattern of Toll-like receptor 3 from the lamprey Lampetra japonica

Toll-like receptors(TLRs)are type I transmembrane proteins that are important components of innate immunity and play essential roles in inducing acquired immune responses[1].These proteins consist of three parts:the cytoplasmic domain,transmembrane domain,and extracellular domain.The extracellular domain is composed of 18–33 leucine-rich repeat(LRR)sequences that enable the host to specifically recognize pathogen-associated molecular patterns and are the core of TLR recognition ligands[2].The cytoplasmic domain is homologous with the interleukin 1 receptor(IL-1R)family and known as the Toll-IL-1 receptor homology domain,which is highly conserved and plays a key role in signal transduction[1].TLR3 recognizes viral dsRNA during immune protection from viruses[3].After the host is infected by a virus,the LRR region of TLR3 recognizes the viral dsRNA,and the Toll/IL-1 receptor(TIR)domain recruits the adaptor protein TIR domain-containing molecule 1(also known as TRIF)for signal cascade transmission[4].The activation of TLR3 finally leads to cytokine secretion,especially the production of type I interferon[4].Lamprey,the most primitive marine jawless vertebrate,is an ideal model for studying vertebrate embryo development,organ differentiation,and immune system evolution[5].Although jawless vertebrates contain three variable lymphocyte receptors,they have not been found to possess the recombinational antigen receptors shared by all jawed vertebrates[6].Compared with that in higher vertebrates,the mechanism of acquired immunity in lampreys is still not complete,and lampreys rely mainly on innate immunity to fight against pathogenic microorganisms.Although TLR3 has been extensively studied in jawed vertebrates,little is known about the molecular evolutionary history and expression patterns of TLR3 in jawless vertebrates.     

Expressional and Biochemical Characterization of Rice Disease Resistance Gene Xa3/Xa26 Family

The rice （Oryza sativa L.） Xa3/Xa26 gene, conferring race-specific resistance to bacterial blight disease and encoding a leucine-rich repeat （LRR） receptor kinase-like protein, belongs to a multigene family consisting of tandem clustered homologous genes, colocalizing with several uncharacterized genes for resistance to bacterial blight or fungal blast. To provide more information on the expressional and biochemical characteristics of the Xa3/Xa26 family, we analyzed the family members. Four Xa3/Xa26 family members in the indica rice variety Teqing, which carries a bacterial blight resistance gene with a chromosomal location tightly linked to Xa3/Xa26, and five Xa3/Xa26 family members in the japonica rice variety Nipponbare, which carries at least one uncharacterized blast resistance gene, were constitutively expressed in leaf tissue. The result suggests that some of the family members may be candidates of these uncharacterized resistance genes. At least five putative N-glycosylation sites in the LRR domain of XA3/XA26 protein are not glycosylated. The XA3/XA26 and its family members MRKa and MRKc all possess the consensus sequences of paired cysteines, which putatively function in dimerization of the receptor proteins for signal transduction, immediately before the first LRR and immediately after the last LRR. However, no homo-dimer between the XA3/XA26 molecules or hetero-dimer between XA3/XA26 and MRKa or MRKc were formed, indicating that XA3/XA26 protein might function either as a monomer or a hetero-dimer formed with other protein outside of the XA3/XA26 family. These results provide valuable information for further extensive investigation into this multiple protein family.     

Reversible acetylation regulates vascular endothelial growth factor receptor-2 activity

The tyrosine kinase receptor vascular endothelial growth factor receptor 2 （VEG FR2） is a key regulator of angiogenesis. Here we show that VEGFR2 is acetylated in endothelial cells both at four lysine residues forming a dense cluster in the kinase insert domain and at a single lysine located in the receptor activation loop. These modifications are under dynamic control of the acetyltransferase p300 and two deacetyiases HDAC5 and HDAC6. We demonstrate that VEGFR2 acetylation essentially regulates receptor phosphorylation. In par- ticular, VEGFR2 acetylation significantly alters the kinetics of receptor phosphorylation after ligand binding, allowing receptor phos- phoryiation and intraceUular signaling upon proLonged stimulation with VEGF. Molecular dynamics simulations indicate that acetylation of the lysine in the activation loop contributes to the transition to an open active state, in which tyrosine phosphorylation is favored by better exposure of the kinase target residues. These findings indicate that post-translational modification by acetyiation is a critical mechanism that directLy affects VEGFR2 function.     

Receptor tyrosine kinases positively regulate BACE activity and Amyloid-β production through enhancing BACE internalization

Amyloid-β （Aβ） peptide, the primary constituent of senile plaques in Alzheimer＇s disease （AD）, is generated by β-secretase- and y-secretase-mediated sequential proteolysis of the amyloid precursor protein （APP）. The aspartic protease, β -site APP cleavage enzyme （BACE）, has been identified as the main β-secretase in brain but the regulation of its activity is largely unclear. Here, we demonstrate that both BACE activity and subsequent Aβ production are enhanced after stimulation of receptor tyrosine kinases （RTKs）, such as the receptors for epidermal growth factor （EGF） and nerve growth factor （NGF）, in cultured cells as well as in mouse hippocampus. Furthermore, stimulation of RTKs also induces BACE internalization into endosomes and Golgi apparatus. This enhancement of BACE activity and A β production upon RTK activation could be specifically inhibited by Src family kinase inhibitors and by depletion of endogenous c-Src with RNAi, and could be mimicked by over-expressed c-Src. Moreover, blockage of BACE internalization by a dominant negative form of Rab5 also abolished the enhancement of BACE activity and Aβ production, indicating the requirement of BACE internalization for the enhanced activity. Taken together, our study presents evidence that BACE activity and Aβ production are under the regulation of RTKs and this is achieved via RTK-stimulated BACE internalization, and suggests that an aberration of such regulation might contribute to pathogenic Aβ production.     

Receptor-Like Kinases in Plant Innate Immunity

Plants employ a highly effective surveillance system to detect potential pathogens, which is critical for the success of land plants in an environment surrounded by numerous microbes. Recent efforts have led to the identification of a number of immune receptors and components of immune receptor complexes. It is now clear that receptor-like kinases （RLKs） and receptor-like proteins （RLPs） are key pattern-recognition receptors （PRRs） for microbe- and plant-derived molecular patterns that are associated with pathogen invasion. RLKs and RLPs involved in immune signaling belong to large gene families in plants and have undergone lineage specific expansion. Molecular evolution and population studies on phytopathogenic molecular signatures and their receptors have provided crucial insight into the co-evolution between plants and pathogens.     

OsHK3 is a crucial regulator of abscisic acid signaling involved in antioxidant defense in rice

In this study, the role of the rice(Oryza sativa L.)histidine kinase Os HK3 in abscisic acid(ABA)-induced antioxidant defense was investigated. Treatments with ABA, H2O2,and polyethylene glycol(PEG) induced the expression of Os HK3 in rice leaves, and H2O2 is required for ABA-induced increase in the expression of Os HK3 under water stress. Subcellular localization analysis showed that Os HK3 is located in the cytoplasm and the plasma membrane. The transient expression analysis and the transient RNA interference test in rice protoplasts showed that Os HK3 is required for ABA-induced upregulation in the expression of antioxidant enzymes genes and the activities of antioxidant enzymes. Further analysis showed that Os HK3 functions upstream of the calcium/calmodulin-dependent protein kinase Os DMI3 and the mitogen-activated protein kinase Os MPK1 to regulate the activities of antioxidant enzymes in ABA signaling. Moreover, Os HK3was also shown to regulate the expression of nicotinamide adenine dinucleotide phosphate oxidase genes, Osrboh B and Osrboh E, and the production of H2O2 in ABA signaling. Our data indicate that Os HK3 play an important role in the regulation of ABA-induced antioxidant defense and in the feedback regulation of H2O2 production in ABA signaling.     

RasGRP Ras guanine nucleotide exchange factors in cancer

RasGRP proteins are activators of Ras and other related small GTPases by the virtue of functioning as guanine nucleotide exchange factors （GEFs）. In vertebrates, four RasGRP family members have been described. RasGRP-1 through -4 share many structural domains but there are also subtle differences between each of the different family members. Whereas SOS RasGEFs are ubiquitously expressed, RasGRP proteins are expressed in distinct patterns, such as in different cells of the hematopoietic system and in the brain. Most studies have concentrated on the role of RasGRP proteins in the development and function of immune cell types because of the predominant RasGRP expression profiles in these cells and the immune phenotypes of mice deficient for Rasgrp genes. However, more recent studies demonstrate that RasGRPs also play an important role in tumorigenesis. Examples are skin- and hematological- cancers but also solid malignancies such as melanoma or prostate cancer. These novel studies bring up many new and unanswered questions related to the molecular mechanism of RasGRP-driven oncogenesis, such as new receptor systems that RasGRP appears to respond to as well as regulatory mechanisms for RasGRP expression that appear to be perturbed in these cancers. Here we will review some of the known aspects of RasGRP biology in lymphocytes and will discuss the exciting new notion that RasGRP Ras exchange factors play a role in oncogenesis downstream of various growth factor receptors.     

Roles of IκB kinase ε in the innate immune defense and beyond

IκB kinase ε(IKKε) is a non-canonical IκB kinase that is extensively studied in the context of innate immune response. Recently, significant progress has been made in understanding the role of IKKεin interferon(IFN) signaling. In addition to its roles in innate immunity, recent studies also demonstrate that IKKε is a key regulator of the adaptive immune response. Specifically, IKKεfunctions as a negative feedback kinase to curtail CD8 T cell response, implying that it can be a potential therapeutic target to boost antiviral and antitumor T cell immunity. In this review, we highlight the roles of IKKε in regulating IFN signaling and T cell immunity, and discuss a few imminent questions that remain to be answered.     

Downregulation of CD4＋CD25＋ regulatory T cells may underlie enhanced Th1 immunity caused by immunization with activated autologous T cells

Regulatory T cells （Treg） play important roles in immune system homeostasis, and may also be involved in tumor immunotolerance by suppressing Th1 immune response which is involved in anti-tumor immunity. We have previously reported that immunization with attenuated activated autologous T cells leads to enhanced anti-tumor immunity and upregulated Thl responses in vivo. However, the underlying molecular mechanisms are not well understood. Here we show that Treg function was significantly downregulated in mice that received immunization of attenuated activated autologous T cells. We found that Foxp3 expression decreased in CD4＋CD25＋ T cells from the immunized mice. Moreover, CD4＋CD25＋Foxp3＋ Treg obtained from immunized mice exhibited diminished immunosuppression ability compared to those from naive mice. Further analysis showed that the serum of immunized mice contains a high level ofanti-CD25 antibody （about 30 ng/ml, p〈0.01 vs controls）. Consistent with a role ofanti-CD25 response in the downregulation of Treg, adoptive transfer of serum from immunized mice to naive mice led to a significant decrease in Treg population and function in recipient mice. The triggering of anti-CD25 response in immunized mice can be explained by the fact that CD25 was induced to a high level in the ConA activated autologous T cells used for immunization. Our results demonstrate for the first time that immunization with attenuated activated autologous T cells evokes anti-CD25 antibody production, which leads to impeded CD4＋CD25＋Foxp3＋ Treg expansion and function in vivo. We suggest that dampened Treg function likely contributes to enhanced Thl response in immunized mice and is at least part of the mechanism underlying the boosted anti-tumor immunity.     

Salmonella enterica Flagellin Is Recognized via FLS2 and Activates PAMP-Triggered Immunity in Arabidopsis thaliana

Infections with Salmonella enterica belong to the most prominent causes of food poisoning and infected fruits and vegetables represent important vectors for salmonellosis. Recent evidence indicates that plants recognize S. enterica and raise defense responses. Nonetheless, the molecular mechanisms controlling the interaction of S. enterica with plants are still largely unclear. Here, we show that flagellin from S. enterica represents a prominent pathogenassociated molecular pattern （PAMP） in Arabidopsis thaliana, which induces PAMP-triggered immunity （PTI） via the recognition of the fig22 domain by the receptor kinase FLS2. The Arabidopsis fls2 mutant shows reduced though not abolished PTI activation, indicating that plants rely also on recognition of other S. enterica PAMPs. Interestingly, the S. enterica type III secretion system （T3SS） mutant prgH- induced stronger defense gene expression than wild-type bacteria in Arabidopsis, suggesting that T3SS effectors are involved in defense suppression. Furthermore, we observe that S. enterica strains show variation in the fig22 epitope, which results in proteins with reduced PTI-inducing activity. Altogether, these results show that S. enterica activates PTI in Arabidopsis and suggest that, in order to accomplish plant colonization, S. enterica evolved strategies to avoid or suppress PTI.     

Immune regulation by protein ubiquitination: roles of the E3 ligases VHL and Itch

Protein ubiquitination is an important means of posttranslational modification which plays an essential role in the regulation of various aspects of leukocyte development and function. The specificity of ubiquitin tagging to a protein substrate is determined by E3 ubiquitin ligases via defined E3-substrate interactions. In this review, we will focus on two E3 ligases, VHL and Itch, to discuss the latest progress in understanding their roles in the differentiation and function of CD4+ T helper cell subsets, the stability of regulatory T cells, effector function of CD8+ T cells, as well as the development and maturation of innate lymphoid cells. The biological implications of these E3 ubiquitin ligases will be highlighted in the context of normal and dysregulated immune responses including the control of homeostasis, inflammation, auto-immune responses and anti-tumor immunity. Further elucidation of the ubiquitin system in immune cells will help in the design of new therapeutic interventions for human immunological diseases and cancer.     

Identification of a sugar gustatory receptor and its effect on fecundity of the brown planthopper Nilaparvata lugens

In insects, the gustatory system plays a crucial role in multiple physiological behaviors, including feeding, toxin avoidance, courtship, mating and oviposition. Gustatory stimuli from the environment are recognized by gustatory receptors. To date, little is known about the function of gustatory receptors in agricultural pest insects. In this study, we cloned a sugar gustatory receptor gene, N/Gr11, from the brown planthopper (BPH), Nilaparvata lugens (Stal), a serious pest of rice in Asia;we then identified its ligands, namely, fructose, galactose and arabinose, by calcium imaging assay. After injection of N/Gr11 doublestranded RNA, we found that the number of eggs laid by BPH decreased. Moreover, we found that N/Gr11 inhibited the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and promoted the phosphorylation of protein kinase B (AKT). These findings demonstrated that N/Gr11 could accelerate the fecundity of BPH through AMPK- and AKT-mediated signaling pathways. This is the first report to indicate that a gustatory receptor modulates the fecundity of insects and that the receptor could be a potential target for pest control.     

CaMKIIα and caveolin-1 cooperate to drive ATP-induced membrane delivery of the P2X3 receptor

The P2X3 receptor plays a vital role in sensory processing and transmission. The assembly and trafficking of the P2X3 receptor are important for its function in primary sensory neurons. As an important inflammation mediator, ATP is released from different cell types around primary sensory neurons, especially under pathological pain conditions. Here, we showthat α, β-MeATP dramatically promoted membrane delivery of the P2X3 receptor both in HEK293T celts expressing recombinant P2X3 receptor and in rat primary sensory neurons. α, β-MeATP induced P2X3 receptor-mediated Ca^2＋ influx, which further activated Ca^2＋/calmodulin-dependent protein kinase Ilec （CaMKIIα）. The N terminus of the P2X3 receptor was responsible for CaMKIleα binding, whereas Thr38s in the C terminus was phosphorylated by CaMKIIα. Thr^388 phosphorylation increased P2X3 receptor binding to caveoUn-1. CaveoUn-1 knockdown abrogated the α, β-MeATP-induced membrane insertion of the P2X3 receptor. Moreover,α, β-MeATP drove the CaMKIlec-mediated membrane coinsertion of the P2X2 receptor with the P2X3 receptor. The increased P2X3 receptors on the cell membrane that are due to Thr388 phosphorytation facilitated P2X3 receptor-mediated signal transduction. Together, our data indicate that CaMKIIoL and caveoUn-1 cooperate to drive Ugand-induced membrane delivery of the P2X3 receptor and may provide a mechanism of P2X3 receptor sensitization in pain development.     

Engineering plants to secrete affinity‐tagged pathogen elicitors for deciphering immune receptor complex or inducing enhanced immunity

Plant cells mount plenty of pattern-recognition receptors(PRRs) to detect the microbe-associated molecular patterns(MAMPs) from potential microbial pathogens.MAMPs are overrepresented by proteinaneous patterns,such as the flg22 peptide from bacterial flagellin. Identification of PRR receptor complex components by forward or reverse genetics can be time/labor-consuming, and be confounded by functional redundancies. Here, we present a strategy for identifying PRR complex components by engineering plants to inducibly secrete affinity-tagged proteinaneous MAMPs to the apoplast. The PRR protein complexes bound to self-secreted MAMPs are enriched through affinity purification and dissected by mass spectrometry. As a proof of principle, we could capture the flg22 receptor FLS2 and co-receptor BAK1 using Arabidopsis plants secreting FLAG-tagged flg22 under estradiolinduction. Moreover, we identified receptor-like kinases LIK1 and PEPR1/PEPR2 as potential components in the FLS2 receptor complex, which were further validated by protein–protein interaction assays and the reverse genetics approach. Our study showcases a simple way to biochemically identify endogenous PRR complex components without overexpressing the PRR or using chemical crosslinkers, and suggests a possible crosstalk between different immune receptors in plants. A modest dose of estradiol can also be applied to inducing enhanced immunity in engineered plants to both bacterial and fungal pathogens.     

Rig-I^-/- mice develop colitis associated with downregulation of Gαi2

RIG-I （retinoid acid-inducible gene-I）, a putative RNA helicase with a cytoplasmic caspase-recrultment domain （CARD）, was identified as a pattem-recognition receptor （PRR） that mediates antiviral immunity by inducing type I interferon production. To further study the biological function of RIG-I, we generated Rig-I^-/- mice through homologous recombination, taking a different strategy to the previously reported strategy. Our Rig-I^-/- mice are viable and fertile. Histological analysis shows that Rig-I^-/ mice develop a colitis-like phenotype and increased susceptibility to dextran sulfate sodium-induced colitis. Accordingly, the size and number of Peyer＇s patches dramatically decreased in mutant mice. The peripheral T-cell subsets in mutant mice are characterized by an increase in effector T cells and a decrease in naive T cells, indicating an important role for Rig-I in the regulation ofT-cell activation. It was further found that Rig-I deficiency leads to the downregulation of G protein αi2 subunit （Gαi2） in various tissues, including T and B lymphocytes. By contrast, upregulation of Rig-I in NB4 cells that are treated with ATRA is accompanied by elevated Gαi2 expression. Moreover, Gαi2 promoter activity is increased in co-transfected NIH3T3 cells in a Rig-I dose-dependent manner. All these findings suggest that Rig-I has crucial roles in the regulation of Gαi2 expression and T-cell activation. The development of colitis may be, at least in part, associated with downregulation of Gαi2 and disturbed T-cell homeostasis.     

An OsPRMT5-OsAGO2/miR1875-OsHXK1 module regulates rice immunity to blast disease

Rice ARGONAUTE2(OsAGO2) is a core component of the rice RNA-induced silencing complex(RISC), which is repressed by Magnaporthe oryzae(M. oryzae) infection. Whether and how OsAGO2-mediated gene silencing plays a role in rice blast resistance and which sRNAs participate in this process are unknown. Our results indicate that OsAGO2 is a key immune player that manipulates rice defense responses against blast disease.OsAGO2 associates with the 24-nt miR1875 and binds to the promoter region of HEXOKIN...     

Arabidopsis RING Peroxins are E3 Ubiquitin Ligases that Interact with Two Homologous Ubiquitin Receptor Proteins

Peroxisomes are essential eukaryotic organelles that mediate various metabolic processes. Peroxisome import depends on a group of peroxisome biogenesis factors called peroxins, many of which are evolutionarily conserved. PEX2, PEX10, and PEX12 are three RING-finger-domain-containing integral membrane peroxins crucial for protein import. In yeast (Saccharomyces cerevisae), RING peroxins act as E3 ligases, facilitating the recycling of the peroxisome import receptor protein PEX5 through ubiquitination. In plants, RING peroxins are essential to plant vitality. To elucidate the mode of action of the plant RING peroxins, we employed in vitro assays to show that the Arabidopsis RING peroxins also have E3 ligase activities. We also identified a PEX2-interacting protein, DSK2b, which is a member of the ubiquitin receptor family known to function as shuttle factors ferrying polyubiquitinated substrates to the proteasome for degradation. DSK2b and its tandem duplicate DSK2a are localized in the cytosol and the nucleus, and both interact with the RING domain of PEX2 and PEX12. DSK2 artificial microRNA lines did not display obvious defects in plant growth or peroxisomal processes, indicating functional redundancies among Arabidopsis ubiquitin receptor proteins. Our results suggest that Arabidopsis RING peroxins can function as E3 ligases and act together with the ubiquitin receptor protein DSK2 in the peroxisomal membrane-associated protein degradation system.     

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.     

PINK1 kinase dysfunction triggers neurodegeneration in the primate brain without impacting mitochondrial homeostasis

In vitro studies have established the prevalent theory that the mitochondrial kinase PINK1 protects neurodegeneration by removing damaged mitochondria in Parkinson's disease(PD).However,difficulty in detecting endogenous PINK1 protein in rodent brains and cell lines has prevented the rigorous investigation of the in vivo role of PINK1.Here we report that PINK1 kinase form is selectively expressed in the human and monkey brains.CRISPR/Cas9-mediated deficiency of PINK1 causes similar neurodegeneration in the brains of fetal and adult monkeys as well as cultured monkey neurons without affecting mitochondrial protein expression and morphology.Importantly,PINK1 mutations in the primate brain and human cells reduce protein phosphorylation that is important for neuronal function and survival.Our findings suggest that PINK1 kinase activity rather than its mitochondrial function is essential for the neuronal survival in the primate brains and that its kinase dysfunction could be involved in the pathogenesis of PD.