Understanding the Myddosome Complex: An Exploration of How Homotypic Death Domain Interfaces Contribute to Overall Structure.

The Myddosome is a large multimeric complex assembled through homotypic interactions between the Death Domains (DDs) of adjacent proteins. It acts as a scaffold bringing downstream signaling elements into proximity with each other. This allows for the subsequent activation of downstream transcription factors, such as IRFs (Interferon Regulatory Factors) and NFκB. The crystal structure of the Myddosome was published by Lin et al [1], by combining the Death Domains of MyD88, IRAK4 and IRAK2. The complex was shown to form a left-handed helical tower as it assembles in a staggered hexagonal pattern, due to the three characteristic types of homotypic Death Domain (DD:DD) interactions seen in complexes of the Death Domain Superfamily. The work discussed in this thesis aimed to expand upon our understanding of the Myddosome complex. An initial bioinformatic analysis was carried out to further investigate the different homotypic Death Domain interfaces responsible for Myddosome assembly. The physical and geometric properties of the complex were examined in detail to identify how they contribute towards the complex’s final structure. Additional computational work provides evolutionary context for the development of the Myddosome, while also drawing links to known non- communicable diseases. A new type of homotypic Death Domain interface was potentially identified, while examining the interactions formed during Myddosome assembly in silico. It appears that this interface is exclusive to members of the IRAK family. Subsequent in vitro work was initiated to verify these observations and future research possibilities were discussed. Points of further clarification were identified when examining the 3-dimensional Myddosome structure as published by Lin et al. Unit cell contacts caused by the presence of un-cleaved GST purification tags were found to be responsible for structural distortion seen in the crystallographic structure. Additionally, the MyD88 Intermediate Domain was not included in the structure of the Myddosome, despite studies characterising the MyD88 Intermediate Domain as vital for functional Myddosome assembly. Further in vitro biophysical analysis was proposed to both rectify the distortion and to further characterise the interactions involved in Myddosome assembly in the presence of the MyD88 Intermediate Domain

Pan-Vertebrate Toll-Like Receptors During Evolution

Human toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) to raise innate immune responses. The human TLR family was discovered because of its sequence similarity to fruit fly (Drosophila) Toll, which is involved in an anti-fungal response. In this review, we focus on the origin of the vertebrate TLR family highlighted through functional and phylogenetic analyses of TLRs in non-mammalian vertebrates. Recent extensive genome projects revealed that teleosts contain almost all subsets of TLRs that correspond to human TLRs (TLR1, 2, 3, 4, 5, 7, 8, and 9), whereas the urochordate Ciona intestinalis contains only a few TLR genes. Therefore, mammals likely obtained almost all TLR family members at the beginning of vertebrate evolution. This premise is further supported by several functional analyses of non-mammalian TLRs. We have summarized several teleost TLRs with unique properties distinct from mammalian TLRs to outline their specific roles. According to Takifugu rubripes genome project, the puffer fish possesses fish-specific TLR21 and 22. Surprisingly, phylogenetic analyses indicate that TLR21 and 22 emerged during an early period of vertebrate evolution in parallel with other TLRs and that the mammalian ancestor lost TLR21 and 22 during evolution. Our laboratory recently revealed that TLR22 recognizes double-strand RNA and induces interferon production through the TICAM-1 adaptor, as in TLR3, but unlike TLR3, TLR22 localizes to the cell surface. Therefore, differential expression of TLR3 and TLR22, rather than simple redundancy of RNA sensors, may explain the effective protection of fish from RNA virus infection in the water. In this review, we summarize the similarities and differences of the TLR family in various vertebrates and introduce these unique TLRs for a possible application to the field of clinical practices for cancer or virus infection

Comparative Genomics Reveals the Origins and Diversity of Arthropod Immune Systems.

Insects are an important model for the study of innate immune systems, but remarkably little is known about the immune system of other arthropod groups despite their importance as disease vectors, pests, and components of biological diversity. Using comparative genomics, we have characterized the immune system of all the major groups of arthropods beyond insects for the first time--studying five chelicerates, a myriapod, and a crustacean. We found clear traces of an ancient origin of innate immunity, with some arthropods having Toll-like receptors and C3-complement factors that are more closely related in sequence or structure to vertebrates than other arthropods. Across the arthropods some components of the immune system, such as the Toll signaling pathway, are highly conserved. However, there is also remarkable diversity. The chelicerates apparently lack the Imd signaling pathway and beta-1,3 glucan binding proteins--a key class of pathogen recognition receptors. Many genes have large copy number variation across species, and this may sometimes be accompanied by changes in function. For example, we find that peptidoglycan recognition proteins have frequently lost their catalytic activity and switch between secreted and intracellular forms. We also find that there has been widespread and extensive duplication of the cellular immune receptor Dscam (Down syndrome cell adhesion molecule), which may be an alternative way to generate the high diversity produced by alternative splicing in insects. In the antiviral short interfering RNAi pathway Argonaute 2 evolves rapidly and is frequently duplicated, with a highly variable copy number. Our results provide a detailed analysis of the immune systems of several important groups of animals for the first time and lay the foundations for functional work on these groups.This project was funded by a Royal Society University Research Fellowship and a European Research Council grant DrosophilaInfection (281668) to F.M.J., and a Medical Research Council studentship to W.J.P.This is the final published version. It first appeared at http://mbe.oxfordjournals.org/content/early/2015/05/12/molbev.msv093.long

The innate immune repertoire in Cnidaria - ancestral complexity and stochastic gene loss

Analysis of genomic resources available for cnidarians revealed that several key components of the vertebrate innate immune repertoire are present in representatives of the basal cnidarian class Anthozoa, but are missing in Hydra, a member of the class Hydrozoa, indicating ancient origins for many components of the innate immune system

IL-17 signaling components in bivalves: Comparative sequence analysis and involvement in the immune responses

The recent discovery of soluble immune-regulatory molecules in invertebrates takes advantage of the rapid growth of next generation sequencing datasets. Following protein domain searches in the transcriptomes of 31 bivalve spp. and in few available mollusk genomes, we retrieved 59 domains uniquely identifying interleukin 17 (IL-17) and 96 SEFIR domains typical of IL-17 receptors and CIKS/ACT1 proteins acting downstream in the IL-17 signaling pathway. Compared to the Chordata IL-17 family members, we confirm a separate clustering of the bivalve domain sequences and a consistent conservation pattern of amino acid residues. Analysis performed at transcript and genome level allowed us to propose an updated view of the components outlining the IL-17 signaling pathway in Mytilus galloprovincialis and Crassostrea gigas (in both species, homology modeling reduced the variety of IL-17 domains to only two 3D structures). Digital expression analysis indicated more heterogeneous expression levels for the mussel and oyster IL-17 ligands than for IL-17 receptors and CIKS/CIKSL proteins. Besides, new qPCR analyses confirmed such gene expression trends in hemocytes and gills of mussels challenged with heat-killed bacteria. These results uphold the involvement of an ancient IL-17 signaling pathway in the bivalve immune responses and, likewise in humans, suggest the possibility of distinctive modulatory roles of individual IL-17s/IL-17 receptors. Overall, the common evidence of pro-inflammatory cytokines and inter-related intracellular signaling pathways in bivalves definitely adds complexity to the invertebrate immunity

Massively Parallel RNA Sequencing Identifies a Complex Immune Gene Repertoire in the lophotrochozoan Mytilus edulis

The marine mussel Mytilus edulis and its closely related sister species are distributed world-wide and play an important role in coastal ecology and economy. The diversification in different species and their hybrids, broad ecological distribution, as well as the filter feeding mode of life has made this genus an attractive model to investigate physiological and molecular adaptations and responses to various biotic and abiotic environmental factors. In the present study we investigated the immune system of Mytilus, which may contribute to the ecological plasticity of this species. We generated a large Mytilus transcriptome database from different tissues of immune challenged and stress treated individuals from the Baltic Sea using 454 pyrosequencing. Phylogenetic comparison of orthologous groups of 23 species demonstrated the basal position of lophotrochozoans within protostomes. The investigation of immune related transcripts revealed a complex repertoire of innate recognition receptors and downstream pathway members including transcripts for 27 toll-like receptors and 524 C1q domain containing transcripts. NOD-like receptors on the other hand were absent. We also found evidence for sophisticated TNF, autophagy and apoptosis systems as well as for cytokines. Gill tissue and hemocytes showed highest expression of putative immune related contigs and are promising tissues for further functional studies. Our results partly contrast with findings of a less complex immune repertoire in ecdysozoan and other lophotrochozoan protostomes. We show that bivalves are interesting candidates to investigate the evolution of the immune system from basal metazoans to deuterostomes and protostomes and provide a basis for future molecular work directed to immune system functioning in Mytilus

Innate immune genes in the zebrafish, Danio rerio

The zebrafish embryo offers the in vertebrates so far unparalled opportunity to study innate immune responses without the influence of an acquired response, which starts to operate much later in development. In the recent past the zebrafish has proven to be a versatile tool to study host-pathogen interactions and an excellent model for the study of human diseases. However, it has been unclear whether the defense mechanisms employed in the embryo are comparable to that known from mammals. In order to determine the extent of conservation between mammalian and fish innate immune pathways, a phylogenetic analysis was conducted to search the zebrafish genome and those of two pufferfish for putative orthologs of known mammalian innate immune genes. This revealed that those components of the intracellular signaling cascades, that transduce the signal downstream of the receptors, are conserved between fish and mammals. However, for the class II cytokines and their receptors the assembled data showed that orthologous relationships cannot easily be established. Within the class II cytokines it could be shown that fish have their own set of interferons (named interferon-φ), which are more closely related to each other than to the mammalian class II cytokine genes or to those shared by mammals and fish, suggesting that interferon genes expanded independently in zebrafish and mammals. The class II cytokine receptor genes are highly divergent. Mammals and fish have approximately the same number of receptor genes. Since syntenic relationships were found only between pufferfish and mammals and appeared to have been lost in the zebrafish, no reliable orthologies could be established. IFNγ is a key regulator of immune responses in mammals, but a possible role of the two zebrafish ifnγ genes during innate immunity is not well established. Infection studies in zebrafish embryos showed that both genes mediate responses to protect the embryo against pathogenic bacterial infection, but seem to function in a redundant manner. Analysis of putative interferon receptor genes revealed that zebrafish Ifnγ1 and Ifnγ2 use distinct receptor chains to transduce the signal to the conserved intracellular signaling pathway. By conducting knockdown experiments in combination with infection assays it was shown that ifnγ2 depends on crfb13 to protect the embryo against infection with Yersinia ruckeri. Among the NLR protein family, a large fish-specific expansion was found. The NLRs are intracellular pathogen sensors that have only recently been described in mammals. They react to various pathogens and are implicated in several human autoimmune disorders, such as Morbus Crohn. More than 200 novel zebrafish genes encoding NLRs were identified in this study. They were found to be closely related to each other but lack the identifiable effector domains that mediate the downstream events in mammals. A new conserved domain was identified, which is present in all fish NLRs and which was termed Fisna. Its functional role is however unknown. Taken together the results from the phylogentic analyses suggest that the signaling mediators are conserved in fish, whereas those proteins that possibly respond to or interact with pathogens appear to have diverged. In addition to the fish-specific NLRs, Nwd1/NACHT-P1 was identified as a novel NLR gene in fish and mammals. Analysis of the genomic organization and transcript structure of zebrafish nwd1 revealed that it encodes a predicted protein of 1620 amino acids that does not contain any known N-terminal effector domain. In situ hybridization in zebrafish showed that it is specifically expressed in neuromasts, which are mechanosensory organs found only in fish and amphibians. In RT-PCR studies, mouse Nwd1 was found expressed in tissues such as brain, thymus, or testis. These data suggest a functional role for this gene, which awaits further analysis

Intracellular interferons in fish : a unique means to combat viral infection

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