Qian Cong

Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation

Innate immune receptor signaling, united Innate immune receptors such as RIG-I, cGAS, and Toll-like receptors bind microbial fragments and alert the immune system to an infection. Each receptor type signals through a different adapter protein. These signals activate the protein kinase TBK1 and the transcription factor IRF3, which tells cells to secrete interferon proteins (IFNs) important for host defense. Liu et al. now report a common signaling mechanism used by all three types of innate immune receptor-adaptor protein pairs to activate IRF3 and generate IFNs. This is important because cells must regulate their IFN production carefully to avoid inflammation and autoimmunity. Science, this issue 10.1126/science.aaa2630 Diverse innate immune receptors use a common signaling mechanism to activate type I interferons. INTRODUCTION Sensing of pathogenic microbes and tissue damage by the innate immune system triggers immune cells to secrete cytokines that promote host defense. Viral RNA, cytosolic DNA, and the bacterial cell wall component lipopolysaccharide activate signaling cascades through a number of pattern recognition receptor (PRR)–adaptor protein pairs, including RIG-I–MAVS, cGAS-STING, and TLR3/4-TRIF (TLR3/4, Toll-like receptors 3 and 4). Activation of these signaling modules results in the production of type I interferons (IFNs), a family of cytokines that are essential for host protection. The adaptor proteins MAVS, STING, and TRIF each activate the downstream protein kinase TBK1, which then phosphorylates the transcription factor interferon regulatory factor 3 (IRF3), which drives type I IFN production. Although much progress has been made in our understanding of PRR and adaptor protein activation, the mechanism by which the adaptor proteins activate TBK1 and IRF3 remains unclear. RATIONALE Other signaling pathways besides the RIG-I–MAVS, cGAS-STING, and TLR3/4-TRIF pathways activate TBK1. However, IRF3 phosphorylation by TBK1 is observed only in the IFN-producing pathways that use MAVS, STING, or TRIF as the adaptor protein. The discrepant activation of TBK1 and IRF3 implies the existence of a kinase-substrate specification mechanism exclusive to the IFN-producing pathways. Specification of TBK1-mediated IRF3 activation is essential for the tight regulation of IFN production, which would otherwise lead to autoimmune diseases. RESULTS Using biochemical and mouse cell– and human cell–based assays, we found that both MAVS and STING interacted with IRF3 in a phosphorylation-dependent manner. We show that both MAVS and STING are phosphorylated in response to stimulation at their respective C-terminal consensus motif, pLxIS (p, hydrophilic residue; x, any residue; S, phosphorylation site). This phosphorylation event then recruits IRF3 to the active adaptor protein and is essential for IRF3 activation. Point mutations that impair the phosphorylation of MAVS or STING at their consensus motif abrogated IRF3 binding and subsequent IFN induction. We found that MAVS is phosphorylated by the kinases TBK1 and IKK, whereas STING is phosphorylated by TBK1. Phosphorylated MAVS and STING subsequently bind to conserved, positively charged surfaces of IRF3, thereby recruiting IRF3 for its phosphorylation and activation by TBK1. Point mutations at IRF3’s positively charged surfaces abrogated IRF3 binding to MAVS and STING and subsequent IRF3 phosphorylation and activation. We further show that TRIF-mediated activation of IRF3 depends on TRIF phosphorylation at the pLxIS motif commonly found in MAVS, STING, and IRF3. These results reveal that phosphorylation of innate immune adaptor proteins is an essential and conserved mechanism that selectively recruits IRF3 to activate type I IFN production. CONCLUSION We uncovered a general mechanism of IRF3 activation by the innate immune adaptor proteins MAVS, STING, and TRIF, which functions in three distinct pattern recognition pathways. Following its activation, each adaptor protein recruits and activates downstream kinase TBK1, which phosphorylates the cognate upstream adaptor protein at a consensus motif. Phosphorylated MAVS, STING, or TRIF in turn recruits IRF3 through its conserved, positively charged phospho-binding domain, allowing IRF3 phosphorylation by TBK1. Phosphorylated IRF3 subsequently dissociates from the adaptor protein and dimerizes though the same phospho-binding domain before translocating into the nucleus to induce IFN. These results elucidate how IRF3 activation and IFN production are tightly controlled and explain why TBK1 is necessary but not sufficient to phosphorylate IRF3: Phosphorylation of IRF3 by TBK1 occurs only with the assistance of an adaptor protein such as MAVS, STING, or TRIF, which also must be phosphorylated. Phosphorylation of innate immune adaptor proteins licenses IRF3 activation. MAVS, STING, and TRIF—which are activated by viral RNA, cytosolic DNA, and bacterial lipopolysaccharide (LPS), respectively—activate the kinases IKK and TBK1. These kinases then phosphorylate the adaptor proteins, which in turn recruit IRF3, thereby licensing IRF3 for phosphorylation (P) by TBK1. Phosphorylated IRF3 dissociates from the adaptor proteins, dimerizes, and then enters the nucleus to induce IFNs. During virus infection, the adaptor proteins MAVS and STING transduce signals from the cytosolic nucleic acid sensors RIG-I and cGAS, respectively, to induce type I interferons (IFNs) and other antiviral molecules. Here we show that MAVS and STING harbor two conserved serine and threonine clusters that are phosphorylated by the kinases IKK and/or TBK1 in response to stimulation. Phosphorylated MAVS and STING then bind to a positively charged surface of interferon regulatory factor 3 (IRF3) and thereby recruit IRF3 for its phosphorylation and activation by TBK1. We further show that TRIF, an adaptor protein in Toll-like receptor signaling, activates IRF3 through a similar phosphorylation-dependent mechanism. These results reveal that phosphorylation of innate adaptor proteins is an essential and conserved mechanism that selectively recruits IRF3 to activate the type I IFN pathway.

CASP9 assessment of free modeling target predictions

We present an overview of the ninth round of Critical Assessment of Protein Structure Prediction (CASP9) “Template free modeling” category (FM). Prediction models were evaluated using a combination of established structural and sequence comparison measures and a novel automated method designed to mimic manual inspection by capturing both global and local structural features. These scores were compared to those assigned manually over a diverse subset of target domains. Scores were combined to compare overall performance of participating groups and to estimate rank significance. Moreover, we discuss a few examples of free modeling targets to highlight the progress and bottlenecks of current prediction methods. Notably, a server prediction model for a single target (T0581) improved significantly over the closest structure template (44% GDT increase). This accomplishment represents the “winner” of the CASP9 FM category. A number of human expert groups submitted slight variations of this model, highlighting a trend for human experts to act as “meta predictors” by correctly selecting among models produced by the top‐performing automated servers. The details of evaluation are available at http://prodata.swmed.edu/CASP9/.

CASP9 target classification.

The Critical assessment of protein structure prediction round 9 (CASP9) aimed to evaluate predictions for 129 experimentally determined protein structures. To assess tertiary structure predictions, these target structures were divided into domain‐based evaluation units that were then classified into two assessment categories: template based modeling (TBM) and template free modeling (FM). CASP9 targets were split into domains of structurally compact evolutionary modules. For the targets with more than one defined domain, the decision to split structures into domains for evaluation was based on server performance. Target domains were categorized based on their evolutionary relatedness to existing templates as well as their difficulty levels indicated by server performance. Those target domains with sequence‐related templates and high server prediction performance were classified as TMB, whereas those targets without identifiable templates and low server performance were classified as FM. However, using these generalizations for classification resulted in a blurred boundary between CASP9 assessment categories. Thus, the FM category included those domains without sequence detectable templates (25 target domains) as well as some domains with difficult to detect templates whose predictions were as poor as those without templates (five target domains). Several interesting examples are discussed, including targets with sequence related templates that exhibit unusual structural differences, targets with homologous or analogous structure templates that are not detectable by sequence, and targets with new folds. Proteins 2011;

Tiger Swallowtail Genome Reveals Mechanisms for Speciation and Caterpillar Chemical Defense

Predicting phenotype from genotype represents the epitome of biological questions. Comparative genomics of appropriate model organisms holds the promise of making it possible. However, the high heterozygosity of many Eukaryotes currently prohibits assembling their genomes. Here, we report the 376 Mb genome sequence of Papilio glaucus (Pgl), the first sequenced genome from the Papilionidae family. We obtained the genome from a wild-caught specimen using a cost-effective strategy that overcomes the high (2%) heterozygosity problem. Comparative analyses suggest the molecular bases of various phenotypic traits, including terpene production in the Papilionidae-specific organ, osmeterium. Comparison of Pgl and Papilio canadensis transcriptomes reveals mutation hotspots (4% genes) associated with their divergence: four key circadian clock proteins are enriched in inter-species mutations and likely responsible for the difference in pupal diapause. Finally, the Pgl genome confirms Papilio appalachiensis as a hybrid of Pgl and Pca, but suggests it inherited 3/4 of its genes from Pca.

MESSA: MEta-Server for protein Sequence Analysis

BackgroundComputational sequence analysis, that is, prediction of local sequence properties, homologs, spatial structure and function from the sequence of a protein, offers an efficient way to obtain needed information about proteins under study. Since reliable prediction is usually based on the consensus of many computer programs, meta-severs have been developed to fit such needs. Most meta-servers focus on one aspect of sequence analysis, while others incorporate more information, such as PredictProtein for local sequence feature predictions, SMART for domain architecture and sequence motif annotation, and GeneSilico for secondary and spatial structure prediction. However, as predictions of local sequence properties, three-dimensional structure and function are usually intertwined, it is beneficial to address them together.ResultsWe developed a MEta-Server for protein Sequence Analysis (MESSA) to facilitate comprehensive protein sequence analysis and gather structural and functional predictions for a protein of interest. For an input sequence, the server exploits a number of select tools to predict local sequence properties, such as secondary structure, structurally disordered regions, coiled coils, signal peptides and transmembrane helices; detect homologous proteins and assign the query to a protein family; identify three-dimensional structure templates and generate structure models; and provide predictive statements about the proteins function, including functional annotations, Gene Ontology terms, enzyme classification and possible functionally associated proteins. We tested MESSA on the proteome of Candidatus Liberibacter asiaticus. Manual curation shows that three-dimensional structure models generated by MESSA covered around 75% of all the residues in this proteome and the function of 80% of all proteins could be predicted.AvailabilityMESSA is free for non-commercial use at http://prodata.swmed.edu/MESSA/

Complete genomes of Hairstreak butterflies, their speciation, and nucleo-mitochondrial incongruence

Comparison of complete genomes of closely related species enables research on speciation and how phenotype is determined by genotype. Lepidoptera, an insect order of 150,000 species with diverse phenotypes, is well-suited for such comparative genomics studies if new genomes, which cover additional Lepidoptera families are acquired. We report a 729 Mbp genome assembly of the Calycopis cecrops, the first genome from the family Lycaenidae and the largest available Lepidoptera genome. As detritivore, Calycopis shows expansion in detoxification and digestion enzymes. We further obtained complete genomes of 8 Calycopis specimens: 3 C. cecrops and 5 C. isobeon, including a dry specimen stored in the museum for 30 years. The two species differ subtly in phenotype and cannot be differentiated by mitochondrial DNA. However, nuclear genomes revealed a deep split between them. Genes that can clearly separate the two species (speciation hotspots) mostly pertain to circadian clock, mating behavior, transcription regulation, development and cytoskeleton. The speciation hotspots and their function significantly overlap with those we previously found in Pterourus, suggesting common speciation mechanisms in these butterflies.

Speciation in Cloudless Sulphurs Gleaned from Complete Genomes

For 200 years, zoologists have relied on phenotypes to learn about the evolution of animals. A glance at the genotype, even through several gene markers, revolutionized our understanding of animal phylogeny. Recent advances in sequencing techniques allow researchers to study speciation mechanisms and the link between genotype and phenotype using complete genomes. We sequenced and assembled a complete genome of the Cloudless Sulphur (Phoebis sennae) from a single wild-caught specimen. This genome was used as reference to compare genomes of six specimens, three from the eastern populations (Oklahoma and north Texas), referred to as a subspecies Phoebis sennae eubule, and three from the southwestern populations (south Texas) known as a subspecies Phoebis sennae marcellina. While the two subspecies differ only subtly in phenotype and mitochondrial DNA, comparison of their complete genomes revealed consistent and significant differences, which are more prominent than those between tiger swallowtails Pterourus canadensis and Pterourus glaucus. The two sulphur taxa differed in histone methylation regulators, chromatin-associated proteins, circadian clock, and early development proteins. Despite being well separated on the whole-genome level, the two taxa show introgression, with gene flow mainly from P. s. marcellina to P. s. eubule. Functional analysis of introgressed genes reveals enrichment in transmembrane transporters. Many transporters are responsible for nutrient uptake, and their introgression may be of selective advantage for caterpillars to feed on more diverse food resources. Phylogenetically, complete genomes place family Pieridae away from Papilionidae, which is consistent with previous analyses based on several gene markers.

The ABC transporters in Candidatus Liberibacter asiaticus

Candidatus Liberibacter asiaticus (Ca. L. asiaticus) is a Gram‐negative bacterium and the pathogen of Citrus Greening disease (Huanglongbing, HLB). As a parasitic bacterium, Ca. L. asiaticus harbors ABC transporters that play important roles in exchanging chemical compounds between Ca. L. asiaticus and its host. Here, we analyzed all the ABC transporter‐related proteins in Ca. L. asiaticus. We identified 14 ABC transporter systems and predicted their structures and substrate specificities. In‐depth sequence and structure analysis including multiple sequence alignment, phylogenetic tree reconstruction, and structure comparison further support their function predictions. Our study shows that this bacterium could use these ABC transporters to import metabolites (amino acids and phosphates) and enzyme cofactors (choline, thiamine, iron, manganese, and zinc), resist to organic solvent, heavy metal, and lipid‐like drugs, maintain the composition of the outer membrane (OM), and secrete virulence factors. Although the features of most ABC systems could be deduced from the abundant experimental data on their orthologs, we reported several novel observations within ABC system proteins. Moreover, we identified seven nontransport ABC systems that are likely involved in virulence gene expression regulation, transposon excision regulation, and DNA repair. Our analysis reveals several candidates for further studies to understand and control the disease, including the type I virulence factor secretion system and its substrate that are likely related to Ca. L. asiaticus pathogenicity and the ABC transporter systems responsible for bacterial OM biosynthesis that are good drug targets. Proteins 2012.

An automatic method for CASP9 free modeling structure prediction assessment

MOTIVATION Manual inspection has been applied to and is well accepted for assessing critical assessment of protein structure prediction (CASP) free modeling (FM) category predictions over the years. Such manual assessment requires expertise and significant time investment, yet has the problems of being subjective and unable to differentiate models of similar quality. It is beneficial to incorporate the ideas behind manual inspection to an automatic score system, which could provide objective and reproducible assessment of structure models. RESULTS Inspired by our experience in CASP9 FM category assessment, we developed an automatic superimposition independent method named Quality Control Score (QCS) for structure prediction assessment. QCS captures both global and local structural features, with emphasis on global topology. We applied this method to all FM targets from CASP9, and overall the results showed the best agreement with Manual Inspection Scores among automatic prediction assessment methods previously applied in CASPs, such as Global Distance Test Total Score (GDT_TS) and Contact Score (CS). As one of the important components to guide our assessment of CASP9 FM category predictions, this method correlates well with other scoring methods and yet is able to reveal good-quality models that are missed by GDT_TS. AVAILABILITY The script for QCS calculation is available at http://prodata.swmed.edu/QCS/. CONTACT grishin@chop.swmed.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Nuclear genomes distinguish cryptic species suggested by their DNA barcodes and ecology

Significance Thirteen years of mitochondrial DNA barcoding of 15,000+ species of Lepidoptera and their parasitoids living in Area de Conservación Guanacaste, northwestern Costa Rica, indicate several thousand cases where barcodes combined with ecology suggest unrecognized cryptic species, substantially increasing species counts. Here, we show that the slightly different barcodes of three extremely similar parapatric–sympatric species of butterflies covary not only with ecology and subtle morphological traits but also with nuclear genomes—a finding that we predict will be commonplace and a method that we predict will be widely used. The barcodes of the century-old type specimens of Udranomia kikkawai from Venezuela reveal that this name applies to one of the three Costa Rican cryptic species; the others we describe as new. DNA sequencing brings another dimension to exploration of biodiversity, and large-scale mitochondrial DNA cytochrome oxidase I barcoding has exposed many potential new cryptic species. Here, we add complete nuclear genome sequencing to DNA barcoding, ecological distribution, natural history, and subtleties of adult color pattern and size to show that a widespread neotropical skipper butterfly known as Udranomia kikkawai (Weeks) comprises three different species in Costa Rica. Full-length barcodes obtained from all three century-old Venezuelan syntypes of U. kikkawai show that it is a rainforest species occurring from Costa Rica to Brazil. The two new species are Udranomia sallydaleyae Burns, a dry forest denizen occurring from Costa Rica to Mexico, and Udranomia tomdaleyi Burns, which occupies the junction between the rainforest and dry forest and currently is known only from Costa Rica. Whereas the three species are cryptic, differing but slightly in appearance, their complete nuclear genomes totaling 15 million aligned positions reveal significant differences consistent with their 0.00065-Mbp (million base pair) mitochondrial barcodes and their ecological diversification. DNA barcoding of tropical insects reared by a massive inventory suggests that the presence of cryptic species is a widespread phenomenon and that further studies will substantially increase current estimates of insect species richness.