Ping Jin

The evolution and origin of animal Toll-like receptor signaling pathway revealed by network-level molecular evolutionary analyses.

Genes carry out their biological functions through pathways in complex networks consisting of many interacting molecules. Studies on the effect of network architecture on the evolution of individual proteins will provide valuable information for understanding the origin and evolution as well as functional conservation of signaling pathways. However, the relationship between the network architecture and the individual protein sequence evolution is yet little known. In current study, we carried out network-level molecular evolution analysis on TLR (Toll-like receptor ) signaling pathway, which plays an important role in innate immunity in insects and mammals, and we found that: 1) The selection constraint of genes was negatively correlated with its position along TLR signaling pathway; 2) all genes in TLR signaling pathway were highly conserved and underwent strong purifying selection; 3) the distribution of selective pressure along the pathway was driven by differential nonsynonymous substitution levels; 4) The TLR signaling pathway might present in a common ancestor of sponges and eumetazoa, and evolve via the TLR, IKK, IκB and NF-κB genes underwent duplication events as well as adaptor molecular enlargement, and gene structure and conservation motif of NF-κB genes shifted in their evolutionary history. Our results will improve our understanding on the evolutionary history of animal TLR signaling pathway as well as the relationship between the network architecture and the sequences evolution of individual protein.

Identification and characterization of a putative lipopolysaccharide-induced TNF-α factor (LITAF) gene from Amphioxus (Branchiostoma belcheri): an insight into the innate immunity of Amphioxus and the evolution of LITAF.

Innate immunity defenses against infectious agent in all multicultural organisms. TNF-α is an important cytokine that can be stimulated by Lipopolysaccharide (LPS) to regulate the innate immunity. The lipopolysaccharide-induced TNF-α factor (LITAF) functions as a transcription factor for regulating the expression of TNF-α as well as various inflammatory cytokines in response to LPS stimulation. The physiological significance of LITAF gene in the innate immunity of various animals has recently been reported. However, no LITAF gene has yet been identified in amphioxus, which is the best available stand-in for the proximate invertebrate ancestor of the vertebrates. In this study, we identified and characterized an amphioxus LITAF gene (designated as AmphiLITAF). First, we identified the AmphiLITAF from the amphioxus and found that AmphiLITAF gene with ~1.6 kb in length has a 827bp cDNA transcription product which encodes a putative protein with 127 amino acids containing conserved LITAF-domain, and the deduced amino acid of AmphiLITAF shared 37-60% similarity with the LITAFs from other species; second, we uncovered the spatial distribution of the LITAF in different tissues, the expression level of AmphiLITAF mRNA was the highest in hepatic cecum and intestine, moderate in muscles, gills and gonad, and the lowest in notochord. Our findings provide an insight into the innate immune response in the amphioxus and the evolution of the LITAF family.

Evolutionary rate patterns of genes involved in the Drosophila Toll and Imd signaling pathway

BackgroundTo survive in a hostile environment, insects have evolved an innate immune system to defend against infection. Studies have shown that natural selection may drive the evolution of immune system-related proteins. Yet, how network architecture influences protein sequence evolution remains unclear. Here, we analyzed the molecular evolutionary patterns of genes in the Toll and Imd innate immune signaling pathways across six Drosophila genomes within the context of a functional network.ResultsBased on published literature, we identified 50 genes that are directly involved in the Drosophila Toll and Imd signaling pathways. Of those genes, only two (Sphinx1 and Dnr1) exhibited signals of positive selection. There existed a negative correlation between the strength of purifying selection and gene position within the pathway; the downstream genes were more conserved, indicating that they were subjected to stronger evolutionary constraints. Interestingly, there was also a significantly negative correlation between the rate of protein evolution and the number of regulatory microRNAs, implying that genes regulated by more miRNAs experience stronger functional constraints and therefore evolve more slowly.ConclusionTaken together, our results suggested that both network architecture and miRNA regulation affect protein sequence evolution. These findings improve our understanding of the evolutionary patterns of genes involved in Drosophila innate immune pathways.

Genome-wide miRNA screening reveals miR-310 family members negatively regulate the immune response in Drosophila melanogaster via co-targeting Drosomycin

ABSTRACT Although innate immunity mediated by Toll signaling has been extensively studied in Drosophila melanogaster, the role of miRNAs in regulating the Toll‐mediated immune response remains largely unknown. In this study, following Gram‐positive bacterial challenge, we identified 93 differentially expressed miRNAs via genome‐wide miRNA screening. These miRNAs were regarded as immune response related (IRR). Eight miRNAs were confirmed to be involved in the Toll‐mediated immune response upon Gram‐positive bacterial infection through genetic screening of 41 UAS‐miRNA lines covering 60 miRNAs of the 93 IRR miRNAs. Interestingly, four out of these eight miRNAs, miR‐310, miR‐311, miR‐312 and miR‐313, are clustered miRNAs and belong to the miR‐310 family. These miR‐310 family members were shown to target and regulate the expression of Drosomycin, an antimicrobial peptide produced by Toll signaling. Taken together, our study implies important regulatory roles of miRNAs in the Toll‐mediated innate immune response of Drosophila upon Gram‐positive bacterial infection. HIGHLIGHTSWe investigated immune response in Drosophila infected with Gram‐positive bacteria.Ninety‐three immune‐response‐related miRNAs were identified.Eight of the miRNAs were involved in Toll‐mediated immune response.miR‐310 family members regulate the immune response by co‐targeting Drosomycin.

Involvement of AmphiREL, a Rel-like gene identified in Brachiastoma belcheri, in LPS-induced response: implication for evolution of Rel subfamily genes.

Rel/NF-κB family genes are important transcriptional factors regulating vital activities of immunity response, but no Rel/NF-κB gene has been identified in amphioxus. In this study, we have not only identified and characterized a Rel-like gene from Brachiastoma belcheri, but also extensively studied the evolution of Rel gene subfamily. We found that: 1) the amphioxus genome contains an AmphiREL gene encoding a Rel/NF-κB homolog, and AmphiREL gene was involved in the innate immune response of LPS stimulation in amphioxus. 2) Gene synteny comparison and structure comparison suggested that AmphiREL is an orthologous gene of human RELB, and is a paralogous gene of human RELA and REL. 3) Structural changes of Rel subfamily proteins are diverse during the evolution process, and imply their functional diversity. 4) The Rel subfamily genes have undergone very strong purifying selection. Together, our results provide important clues for understanding the evolution and function of Rel subfamily genes.

Particularity and universality of a putative Gram-negative bacteria-binding protein (GNBP) gene from amphioxus (Branchiostoma belcheri): Insights into the function and evolution of GNBP

Gram-negative bacteria-binding proteins (GNBPs) are important pattern recognition proteins (PRPs), which can initiate host defense in response to pathogen surface molecules. The roles of GNBP in innate immunity of arthropods and molluscs have recently been reported. However, the GNBP gene has not been characterized in the species of higher evolutionary status yet. In this study, we identified and characterized an amphioxus GNBP gene (designated as AmphiGNBP). First, we identified and cloned the AmphiGNBP and found that the AmphiGNBP encodes a putative protein with 558 amino acids, which contains a conserved β-1, 3-glucan recognizing and binding domain. Second, we found that the AmphiGNBP encodes two extra WSC (cell Wall integrity and Stress response Component) domains, which are unique in AmphiGNBP protein. The two WSC domains of AmphiGNBP protein coupled with the expansion of amphioxus immunity repertoire might undergo intensive domain shuffling during the age of the Cambrian explosion. Finally, we found that the AmphiGNBP was mainly expressed in immune tissues, such as hepatic cecum and intestine, and the expression of AmphiGNBP was affected after LPS stimulation. In conclusion, our findings disclose the particularity and universality of AmphiGNBP and provide profound insights into the function and evolution of GNBP.

Identification and characterization of a p38-like gene from amphioxus (Branchiostoma belcheri): An insight into amphioxus innate immunity and evolution

p38 MAP kinases, members of mitogen-activated protein kinases (MAPKs) activated by environmental stresses and cytokines, play important roles in transcription regulation and inflammatory responses. However, the p38 MAP kinase gene has not been identified in amphioxus to date. Here, we identified and characterized a p38 MAP kinase gene from Branchiostoma belcheri (designed as Amphip38). First, we cloned the full length of Amphip38 gene and found that the deduced amino acid sequence of Amphip38 has 80.5-84% similarity and 67.2-72.5% identity to those from other species. Second, we found that Amphip38 contained the conserved TGY motif, ATP binding site (GXGXXG), substrate binding site (ATRW) and ED site in known p38 MAP kinases. The predicted 3D structure of Amphip38 was found to be similar to human p38 MAP kinases. These results indicate that Amphip38 belongs to p38 MAP kinase gene family. Third, we found that the Amphip38 was ubiquitously and differentially expressed in five investigated tissues (intestine, gills, notochord, muscles, and hepatic cecum). Finally, we found that LPS stimulation induced the expression of Amphip38 gene, and lead to increase of phosphorylation-p38 MAP kinase. These results indicate that Amphip38 is involved in innate immunity response in amphioxus. In addition, we found that Amphip38 gene might be an ancestor of vertebrate p38 MAP kinase gene via evolutionary analysis. In conclusion, our results provided an insight into the innate immunity response and the evolution of the vertebrate p38 MAP kinase gene family.

Identification and evolution of an NFAT gene involving Branchiostoma belcheri innate immunity.

The Nuclear Factor of Activated T cells (NFAT) plays an important role in innate and adaptive immunity, but no NFAT genes have yet been identified in amphioxus species. Here we identified and characterized an NFAT-like gene from Branchiostoma belcheri, and also studied extensively the evolutionary history of NFAT family genes. We found that the amphioxus genome contains an AmphiNFAT gene encoding an NFAT homolog. The AmphiNFAT gene was found to be involved in the innate immune response to LPS stimulation in B. belcheri and was ubiquitously and differentially expressed in all investigated tissues. The NFAT family genes were present in a common ancestor with cnidaria, and NFAT1-4 paralogs were lost early in Branchiostoma and Strongylocentrotus genomes. We discovered that NFAT family genes underwent strong purifying selection. Taken together, our findings provide an insight into the innate immune response of amphioxus and the evolution of the NFAT gene family.

Transcriptome-wide analysis of microRNAs in Branchiostoma belcheri upon Vibrio parahemolyticus infection

&NA; MicroRNAs (miRNAs) are endogenous small non‐coding RNAs that participate in diverse biological processes via regulating expressions of target genes at post‐transcriptional level. Amphioxus, as modern survivor of an ancient chordate lineage, is a model organism for comparative genomics study. However, miRNAs involved in regulating immune responses in Branchiostoma belcheri are largely unclear. Here, we systematically investigated the microRNAs (miRNAs) involved in regulating immune responses in the cephalochordate amphioxus (Branchiostoma belcheri) through next‐generation deep sequencing of amphioxus samples infected with Vibrio parahemolyticus. We identified 198 novel amphioxus miRNAs, consisting of 12 conserved miRNAs, 33 candidate star miRNAs and 153 potential amphioxus‐specific‐miRNAs. Using microarray profiling, 14 miRNAs were differentially expressed post infection, suggesting they are immune‐related miRNAs. Eight miRNAs (bbe‐miR‐92a‐3p, bbe‐miR‐92c‐3p, bbe‐miR‐210‐5p, bbe‐miR‐22‐3p, bbe‐miR‐1˜bbe‐miR‐133 and bbe‐miR‐217˜bbe‐miR‐216 clusters) were significantly increased at 12 h post‐infection, while bbe‐miR‐2072‐5p was downregulated at 6 h and 12 h. Three miRNAs, bbe‐miR‐1‐3p, bbe‐miR‐22‐3p and bbe‐miR‐92a‐3p, were confirmed to be involved in immune responses to infection by qRT‐PCR. Our findings further clarify important regulatory roles of miRNAs in the innate immune response to bacterial infection in amphioxus. HighlightsAmphioxus miRNAs were identified via systematic analysis using deep RNA sequencing.Ten increased & 4 decreased immune‐related miRNAs were identified using microarray.Three immune related amphioxus miRNAs were further validated by qRT‐PCR.Immune‐related amphioxus miRNAs were functional annotated.

miR-958 inhibits Toll signaling and Drosomycin expression via direct targeting of Toll and Dif in Drosophila melanogaster

Drosophila melanogaster is widely used as a model system to study innate immunity and signaling pathways related to innate immunity, including the Toll signaling pathway. Although this pathway is well studied, the precise mechanisms of posttranscriptional regulation of key components of the Toll signaling pathway by microRNAs (miRNAs) remain obscure. In this study, we used an in silico strategy in combination with the Gal80ts-Gal4 driver system to identify microRNA-958 (miR-958) as a candidate Toll pathway regulating miRNA in Drosophila We report that overexpression of miR-958 significantly reduces the expression of Drosomycin, a key antimicrobial peptide involved in Toll signaling and the innate immune response. We further demonstrate in vitro and in vivo that miR-958 targets the Toll and Dif genes, key components of the Toll signaling pathway, to negatively regulate Drosomycin expression. In addition, a miR-958 sponge rescued the expression of Toll and Dif, resulting in increased expression of Drosomycin. These results, not only revealed a novel function and modulation pattern of miR-958, but also provided a new insight into the underlying molecular mechanisms of Toll signaling in regulation of innate immunity.