The STIR-domain superfamily in signal transduction,development and immunity

We have identified a conserved sequence segment in transmembrane receptors (including SEFs, IL17Rs) and soluble factors (including CIKS/ACT1) in eukaryotes and bacteria - the SEFIR domain. This sequence domain is part of the new STIR domain superfamily comprising also the TIR domain known to mediate TIR-TIR homotypic interactions. In TOLL/IL1R-like pathways, the cytoplasmically localized TIR domain of a receptor and the TIR domain of a soluble adaptor interact physically and activate signalling. The similarity between the SEFIR and TIR domains involves the conserved boxes 1 and 2 of the TIR domain that are implicated in homotypic dimerization, but there is no sequence similarity between SEFIR domains and the TIR sequence box 3. By analogy, we suggest that SEFIR-domain proteins function as signalling components of Toll/IL-1R-similar pathways and that their SEFIR domain mediates physical protein-protein interactions between pathway components.     

Crystal Structure of TIR Domain of TLR6 Reveals Novel Dimeric Interface of TIR–TIR Interaction for Toll-Like Receptor Signaling Pathway

Toll-like receptors (TLRs) are responsible for recognition of particular pathogens during the innate immune response and cytoplasmic Toll/interleukin-1 receptor (TIR) domain responsible for downstream signaling. TLR6 working with TLR2 can detect bacterial lipoprotein leading signal for nuclear factor-kappaB activation for immune response. To better understand TLR-mediated signaling event in the innate immune system, in this study, we report the first crystal structure of the TIR domain of TLR6 at 2.2 Å resolution. Our structure reveals novel homo-dimerization interfaces, which might be a critical for the interaction with TIR-containing adaptor proteins and itself. We also report structural similarities and differences of TLR6 with those of other TIR domains, which may be functionally relevant.     

Evolution of Vertebrate Immunity: Sequence and Functional Analysis of the SEFIR Domain Family Member Act1

SEF/IL-17R/CIKS/ACT1 homology (SEFIR) domain containing proteins, which include the IL-17 receptors and an adaptor protein Act1, have essential roles in vertebrate immunity. However, the molecular mechanisms of Act1 function remain largely unexplored. In this article, we employed an evolutionary analysis to discover novel structural and functional properties of Act1. Firstly, we have found that the previously identified helix-loop-helix and Ufd2-box domains in human Act1 have relatively recent evolutionary origins in higher vertebrates. Zebrafish Act1, which lacks these domains, is unable to induce JNK phosphorylation and activate cytokine expression when expressed in human cells. Secondly, we have established that Act1-like proteins contain DEATH-domains in basal animals, such as Hydra and primitive chordates, but lack this domain in vertebrates. Finally, we have shown that Act1-TRAF6 interactions are conserved throughout vertebrate evolution: Act1 derived from zebrafish can bind to TRAF6 and activate NF-κB in human cells. Moreover, we have identified a novel highly conserved motif at the amino-terminus of Act1, which is critical for binding to TRAF6 and activating NF-κB-dependent gene expression. We propose a model of evolutionary changes in Act1-mediated signalling, which contributes to a better understanding of evolution of the vertebrate immunity.     

与精神发育迟滞有关的IL1RAPL1基因研究进展

IL1RAPL1基因缺失、倒置以及突变会导致非特异性精神发育迟滞,因而与人类认知能力密切相关。研究该基因的生物学功能与认知功能将为临床诊断和防治精神发育迟滞提供参考。本文综述了IL1RAPL1基因产物、基因的生理功能与认知功能的研究现状,并对今后的进一步研究工作进行了展望。     

细菌外毒素序列中特有模体的识别及其基因本体注释分析

【目的】识别细菌外毒素序列中特有模体,进一步理解外毒素的致病机制。【方法】构建非致病性细菌蛋白质数据库,利用InterProScan对数据库中非致病菌蛋白质序列以及收集的经实验确认的89条细菌外毒素蛋白质序列进行模体搜索。【结果】在89条细菌外毒素序列中,分析得到了39个细菌外毒素特有模体。【结论】得到的外毒素特有模体与外毒素功能密切相关,为在致病性细菌基因组内搜索外毒素序列奠定了基础;同时通过对外毒素特有模体的基因本体(Gene ontology,GO)注释分析,进一步阐明了细菌外毒素的致病机制。     

SPRY Domain-Containing SOCS Box Protein 2: Crystal Structure and Residues Critical for Protein Binding

The four mammalian SPRY (a sequence repeat in dual-specificity kinase splA and ryanodine receptors) domain-containing suppressor of cytokine signalling (SOCS) box proteins (SSB-1 to -4) are characterised by a C-terminal SOCS box and a central SPRY domain. The latter is a protein interaction module found in over 1600 proteins, with more than 70 encoded in the human genome. Here we report the crystal structure of the SPRY domain of murine SSB-2 and compare it with the SSB-2 solution structure and crystal structures of other B30.2/SPRY domain-containing family proteins. The structure is a bent β-sandwich, consisting of two seven-stranded β-sheets wrapped around a long loop that extends from the centre strands of the inner or concave β-sheet; it closely matches those of GUSTAVUS and SSB-4. The structure is also similar to those of two recently determined Neuralized homology repeat (NHR) domains (also known as NEUZ domains), with detailed comparisons, suggesting that the NEUZ/NHR domains form a subclass of SPRY domains. The binding site on SSB-2 for the prostate apoptosis response-4 (Par-4) protein has been mapped in finer detail using mutational analyses. Moreover, SSB-1 was shown to have a Par-4 binding surface similar to that identified for SSB-2. Structural perturbations of SSB-2 induced by mutations affecting its interaction with Par-4 and/or c-Met have been characterised by NMR. These comparisons, in conjunction with previously published dynamics data from NMR relaxation studies and coarse-grained dynamics simulation using normal mode analysis, further refine our understanding of the structural basis for protein recognition of SPRY domain-containing proteins.     

Evolution of double MutT/Nudix domain-containing proteins: similar domain architectures from independent gene duplication-fusion events

The MutT/Nudix superfamily proteins repair DNA damage and play a role in human health and disease. In this study, we examined two different cases of double MutT/Nudix domain-containing proteins from eukaryotes and prokaryotes. Firstly, these double domain proteins were discovered in Drosophila, but only single Nudix domain proteins were found in other animals. The phylogenetic tree was constructed based on the protein sequence of Nudix_N and Nudix_C from Drosophila, and Nudix from other animals. The phylogenetic analysis suggested that the double Nudix domain proteins might have undergone a gene duplication-speciation-fusion process. Secondly, two genes of the MutT family, DR0004 and DR0329, were fused by two mutT gene segments and formed double MutT domain protein genes in Deinococcus radiodurans. The evolutionary tree of bacterial MutT proteins suggested that the double MutT domain proteins in D. radiodurans probably resulted from a gene duplication-fusion event after speciation. Gene duplication-fusion is a basic and important gene innovation mechanism for the evolution of double MutT/Nudix domain proteins. Independent gene duplication-fusion events resuited in similar domain architectures of different double MutT/Nudix domain proteins.     

The MYND domain-containing protein BRAM1 inhibits lymphotoxin beta receptor-mediated signaling through affecting receptor oligomerization

MYND (myeloid-Nervy-DEAF-1) domains exist in a large number of proteins that are functionally important in development or associated with cancers. We have previously demonstrated that a MYND domain-containing protein, the bone morphogenesis protein receptor-associated molecule 1 (BRAM1), is able to interact with Epstein-Barr virus-encoded latent membrane protein 1 (LMP1), which acts as a constitutively activated tumor necrosis factor receptor (TNFR). Herein we further demonstrated that BRAM1 additionally associates with the TNFR-superfamily member, the lymphotoxin beta receptor (LTβR), and hence inhibits LTβR-mediated function. Using the yeast two-hybrid assay, we demonstrated that BRAM1 interacts with LTβR mainly through the self-association domain of LTβR (aa 336-398). The co-immunoprecipitation experiment further revealed that BRAM1 as well as MYND domain-containing proteins, MTG8 and DEAF-1, interacts with LTβR via their MYND domains. The BRAM1-LTβR interaction impedes the self-association of LTβR and the recruitment of TNFR-associated factors 2 and 3 (TRAF2 and TRAF3), leading to abolishment of LTβR-induced NF-κB signaling, JNK activation, and caspase-dependent cell death. In sum, our data demonstrate that the MYND-containing protein BRAM1 abrogates LTβR function through a protein-protein interaction. These findings may provide a direction for the treatment of dysregulation of LTβR-mediated signaling.     

IL‐1 signaling is critical for expansion but not generation of autoreactive GM‐CSF+ Th17 cells

Interleukin‐1 (IL‐1) is implicated in numerous pathologies, including multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). However, the exact mechanism by which IL‐1 is involved in the generation of pathogenic T cells and in disease development remains largely unknown. We found that following EAE induction, pertussis toxin administration leads to IL‐1 receptor type 1 (IL‐1R1)‐dependent IL‐1β expression by myeloid cells in the draining lymph nodes. This myeloid‐derived IL‐1β did not vitally contribute to the generation and plasticity of Th17 cells, but rather promoted the expansion of a GM‐CSF⁺ Th17 cell subset, thereby enhancing its encephalitogenic potential. Lack of expansion of GM‐CSF‐producing Th17 cells led to ameliorated disease in mice deficient for IL‐1R1 specifically in T cells. Importantly, pathogenicity of IL‐1R1‐deficient T cells was fully restored by IL‐23 polarization and expansion in vitro. Therefore, our data demonstrate that IL‐1 functions as a mitogenic mediator of encephalitogenic Th17 cells rather than qualitative inducer of their generation.     

IL-17 mediates estrogen-deficient osteoporosis in an Act1-dependent manner

Estrogen-deficient osteoporosis may be an inflammatory disorder and we therefore asked if IL-17 participates in its pathogenesis. Deletion of the principal IL-17 receptor (IL-17RA) protects mice from ovariectomy (OVX)-induced bone loss. Further supporting a central role of IL-17 in its pathogenesis, OVX-induced osteoporosis is prevented by a blocking antibody targeting the cytokine. IL-17 promotes osteoclastogenesis by stimulating RANK ligand (RANKL) expression by osteoblastic cells, mediated by the IL-17RA SEFIR/TILL domain. Estrogen deprivation, however does not enhance IL-17RA mRNA expression by osteoblasts or in bone, but augments that of Act1, an IL-17RA-interacting protein and signaling mediator. Similar to IL-17RA(-/-) mice, those lacking Act1 are protected from OVX-induced bone loss. Also mirroring IL-17RA-deficiency, absence of Act1 in osteoblasts, but not osteoclasts, impairs osteoclastogenesis via dampened RANKL expression. Transduction of WT Act1 into Act1(-/-) osteoblasts substantially rescues their osteoclastogenic capacity. The same construct, however, lacking its E3 ligase U-box or its SEFIR domain, which interacts with its counterpart in IL-17RA, fails to do so. Estrogen deprivation, therefore, promotes RANKL expression and bone resorption in association with upregulation of the IL-17 effector, Act1, supporting the concept that post-menopausal osteoporosis is a disorder of innate immunity.     

SEF/IL-17R (SEFIR) Is Not Enough: AN EXTENDED SEFIR DOMAIN IS REQUIRED FOR IL-17RA-MEDIATED SIGNAL TRANSDUCTION*

IL-17, the hallmark cytokine of the Th17 population, mediates immunity to extracellular pathogens and promotes autoimmune immunopathology. The signaling mechanisms triggered by the IL-17 receptor (IL-17RA) and related receptors are strikingly different from other cytokine subclasses. Namely, IL-17Rs contain a conserved SEF/IL-17R (SEFIR) subdomain that engages Act1, leading to activation of TRAF6, NF-κB, and other events. Although the SEFIR is critical for signaling, the molecular details of the functional subdomains within IL-17RA remain poorly characterized. Here, we provide a detailed structure-function analysis delineating the C-terminal boundary of the SEFIR-containing region of IL-17RA. We show that functionality of this domain requires a large extension to the previously identified SEFIR motif. In contrast to the SEFIR, this extension is not conserved among IL-17R family members. Surprisingly, Act1 recruitment is not sufficient for downstream signaling activation, whereas ubiquitination of TRAF6 correlates tightly with functional receptors. We further demonstrate that IL-17RA exhibits signaling properties that are nonredundant with other IL-17R family members. Finally, we report that IL-17 signals synergistically with lymphotoxin-α3, using the same signaling motifs within IL-17RA. These studies provide new insight into the structure-function relationships of IL-17RA and reveal distinct signaling differences among IL-17R family members.     

Structural and functional differences of SWIRM domain subtypes

SWIRM is a conserved domain found in several chromatin-associated proteins. Based on their sequences, the SWIRM family members can be classified into three subfamilies, which are represented by Swi3, LSD1, and Ada2. Here we report the SWIRM structure of human MYb-like, Swirm and Mpn domain-containing protein-1 (MYSM1). The MYSM1 SWIRM structure forms a compact HTH-related fold comprising five alpha-helices, which best resembles the Swi3 SWIRM structure, among the known SWIRM structures. The MYSM1 and Swi3 SWIRM structures are more similar to the LSD1 structure than the Ada2alpha structure. The SWIRM domains of MYSM1 and LSD1 lacked DNA binding activity, while those of Ada2alpha and the human Swi3 counterpart, SMARCC2, bound DNA. The dissimilarity in the DNA-binding ability of the MYSM1 and SMARCC2 SWIRM domains might be due to a couple of amino acid differences in the last helix. These results indicate that the SWIRM family has indeed diverged into three structural subfamilies (Swi3/MYSM1, LSD1, and Ada2 types), and that the Swi3/MYSM1-type subfamily has further diverged into two functionally distinct groups. We also solved the structure of the SANT domain of MYSM1, and demonstrated that it bound DNA with a similar mode to that of the c-Myb DNA-binding domain.     

Scaffolding role of TcpB in disrupting TLR4-Mal interactions: Three to tango

Toll/interleukin-1 like receptors (TLRs) are membrane-spanning proteins crucially involved in innate immunity. On activation, the cytoplasmic toll/interleukin-1 receptor (TIR) domains of these receptors undergo homo- or heterodimerization. Brucella sp. are bacterial pathogens that affect the immune system by suppressing the TLR signaling pathway. They enact this by encoding a TIR domain–containing protein, TcpB, which suppresses NF-κB activation and proinflammatory cytokine secretion mediated by TLR4 receptors. TcpB has been shown to target the Mal-mediated pathway to suppress TLR signaling. The recent identification of its mechanism of interference with TLR4 signaling involving Mal prompted us to further study the structural aspects of TcpB binding with TLR4 and Mal. Our triprotein model displays the overall scaffolding role of TcpB in anchoring TLR4 and Mal thereby inhibiting their interaction leading to the attenuation of the TLR4 pathway.     

Th17 can regulate silica‐induced lung inflammation through an IL‐1β‐dependent mechanism

Silicosis is an occupational lung disease caused by the inhalation of silica dust and characterized by lung inflammation and fibrosis. Interleukin (IL)‐1β is induced by silica and functions as the key pro‐inflammatory cytokine in this process. The Th17 response, which is induced by IL‐1β, has been reported very important in chronic human lung inflammatory diseases. To elucidate the underlying mechanisms of IL‐1β and IL‐17 in silicosis, we used anakinra and an anti‐IL‐17 monoclonal antibody (mAb) to block the receptor of IL‐1β (IL‐RI) and IL‐17, respectively, in a mouse model of silicosis. We observed increased IL‐1β expression and an enhanced Th17 response after silica instillation. Treatment with an IL‐1 type I receptor (IL‐1RI) antagonist anakinra substantially decreased silica‐induced lung inflammation and the Th17 response. Lung inflammation and the accumulation of inflammatory cells were attenuated in the IL‐17‐neutralized silicosis group. IL‐17 may promote lung inflammation by modulating the differentiation of Th1 and regulatory T cells (Tregs) and by regulating the production of IL‐22 and IL‐1β during the lung inflammation of silicosis. Silica may induce IL‐1β production from alveolar macrophages and promote inflammation by initiating a Th17 response via an IL‐1β/IL‐1RI‐dependent mechanism. The Th17 response could induce lung inflammation during the pathogenesis of silicosis by regulating the homoeostasis of the Th immune responses and affecting the production of IL‐22 and IL‐1β. This study describes a potentially important inflammatory mechanism of silicosis that may bring about novel therapies for this inflammatory and fibrotic disease.     

Effects of myeloid differentiation primary response gene 88 (MyD88) activation on Helicobacter infection in vivo and induction of a Th17 response

Background:  Helicobacter pylori is a spiral‐shaped Gram‐negative microaerophilic bacterium associated with a number of gastrointestinal disorders, including gastritis, peptic ulcers, and gastric cancer. Several studies have implicated a Th17 response as a key to protective immunity against Helicobacter. Materials and Methods:  Wild type (WT) and MyD88‐deficient (MyD88^?/?) mice in the C57BL/6 background were infected with H. felis for 6 and 25 weeks and colonization density and host response evaluated. Real‐time PCR was used to determine the expression of cytokines and antimicrobial peptides in the gastric tissue of mice. Results:  mRNA expression levels of the Th17 cytokines interleukin‐17A (IL‐17A) and IL‐22 were markedly up‐regulated in WT compared with MyD88^?/? mice both at 6 and at 25 weeks in response to infection with H. felis, indicating that induction of Th17 responses depends on MyD88 signaling. Furthermore, reduction in the expression of Th17‐dependent intestinal antimicrobial peptide lipocalin‐2 was linked with increased bacterial burden in the absence of MyD88 signaling. Conclusion:  We provide evidence showing that MyD88‐dependent signaling is required for the host to induce a Th17 response for the control of Helicobacter infection.     

IL‐17A is not a treatment target in progressive vitiligo

Multiple reports confirm elevated circulating IL‐17 levels and increased numbers of Th17 lymphocytes in patients with non‐segmental vitiligo. Additionally, melanocyte damaging characteristics have been ascribed to IL‐17. A single‐arm pilot study using secukinumab in active non‐segmental vitiligo was conducted. The large majority of patients developed additional skin depigmentations limiting further enrollment. Overall, laboratory analysis revealed no change in secreted chemokines or Th subsets. Th17 lymphocytes correlated with Th2, Th9, and Th22 cells while an inverse link with Th1 cells and serum sCD25 levels was observed. In contrast, Th17.1 cells correlated positively with Th1 lymphocytes. Confirmatory results were found in an independent group of patients with vitiligo showing a significant increase in Th17.1 and Th1 lymphocytes in progressive vitiligo patients compared to healthy controls, which was not found for Th17 cells. These results do not support a direct pathogenic role of IL‐17 or Th17 cells in vitiligo. Nonetheless, a delicate Th17/Th17.1/Th1 balance seems evident which changes markedly according to disease activity. This may offer new treatment options by interfering with cytokines that drive differentiation of Th17 cells toward Th1.     

Crystal Structures of the Toll/Interleukin-1 Receptor (TIR) Domains from the Brucella Protein TcpB and Host Adaptor TIRAP Reveal Mechanisms of Molecular Mimicry

The Toll/IL-1 receptor (TIR) domains are crucial innate immune signaling modules. Microbial TIR domain-containing proteins inhibit Toll-like receptor (TLR) signaling through molecular mimicry. The TIR domain-containing protein TcpB from Brucella inhibits TLR signaling through interaction with host adaptor proteins TIRAP/Mal and MyD88. To characterize the microbial mimicry of host proteins, we have determined the X-ray crystal structures of the TIR domains from the Brucella protein TcpB and the host adaptor protein TIRAP. We have further characterized homotypic interactions of TcpB using hydrogen/deuterium exchange mass spectrometry and heterotypic TcpB and TIRAP interaction by co-immunoprecipitation and NF-κB reporter assays. The crystal structure of the TcpB TIR domain reveals the microtubule-binding site encompassing the BB loop as well as a symmetrical dimer mediated by the DD and EE loops. This dimerization interface is validated by peptide mapping through hydrogen/deuterium exchange mass spectrometry. The human TIRAP TIR domain crystal structure reveals a unique N-terminal TIR domain fold containing a disulfide bond formed by Cys⁸⁹ and Cys¹³⁴. A comparison between the TcpB and TIRAP crystal structures reveals substantial conformational differences in the region that encompasses the BB loop. These findings underscore the similarities and differences in the molecular features found in the microbial and host TIR domains, which suggests mechanisms of bacterial mimicry of host signaling adaptor proteins, such as TIRAP.     

Identification of interleukin‐16 (IL‐16) and interleukin‐17D (IL‐17D) genes from Dabry's sturgeon (Acipenser dabryanus)

Dabry's sturgeon (Acipenser dabryanus) represents an ancient Actinopterygian lineage that are termed “living fossils”. Many diseases have been found in Dabry's sturgeon. In the present study, genes encoding interleukin (IL)‐16 and IL‐17D in Dabry's sturgeon were identified by RNA‐sequencing. Phylogenetic tree analysis suggested that they clustered together with the corresponding pro‐IL‐16 proteins and IL‐17D proteins from other fish. Sequence analysis revealed that IL‐17D protein was more conserved than pro‐IL‐16. Dabry's sturgeon pro‐IL‐16 contains four putative PDZ domains and do not include signal peptides, while IL‐17D only possesses signal peptides (1–25 aa). The expression patterns of IL‐16 and IL‐17D genes were investigated in Dabry's sturgeon to reveal their functions in disease. The expression level of IL‐16 showed no significant changes in embryos; however, the high expression level of IL‐17D at 0–14 hpf (hours post fertilization) implied the existence of maternal expression in the oocyte and an association with embryonic development. Tissue distribution analysis revealed that IL‐16 and IL‐17D proteins have potential functions in immune and non‐immune tissue compartments. IL‐16 and IL‐17D had different fold changes in primary spleen leukocytes after polyinosinic:polycytidylic acid (poly I:C) and lipopolysaccharide (LPS) administration, which suggested that IL‐16 has a stronger antiviral capability compared with its antibacterial response, and IL‐17D has a stronger antibacterial capability compared with its antiviral response. IL‐16 showed an earlier response to virus compared with IL‐17D, and IL‐17D showed earlier and shorter response to bacteria compared with IL‐16. Our findings suggested the roles of IL‐16 and IL‐17D in Dabry's sturgeon, and provided the theoretical basis for the prevention and control of diseases of Dabry's sturgeon.     

Characterization of gene structure and expression of two toll-like receptors from Japanese flounder, <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

We cloned the cDNAs and genes of two different types of toll-like receptors from Japanese flounder. The results of homology searches suggested that these genes (designated JF-TLR2 and JF-TLR22) are homologues of human TLR2 and fugu TLR22, respectively. The cDNAs of JF-TLR2 and JF-TLR22 encoded 818 and 961 amino acid residues, respectively. JF-TLR2 and JF-TLR22 contained two distinct structural/functional motifs of the TLR family, such as a leucine-rich repeat (LRR) domain in the extracellular portion and a toll/interleukin-1 receptor (TIR) domain in the intracellular portion. The genes of JF-TLR2 and JF-TLR22 consisted of 12 exons (4.9 kb in total length) and four exons (4.3 kb in total length), respectively. The first exon of each gene is a non-coding exon. Southern blot hybridization indicated that both JF-TLR2 and JF-TLR22 exist as single copies in the genome. These genes were mainly expressed in peripheral blood leukocytes (PBLs) and weakly expressed in PBL-rich organs such as kidney, spleen and gill. Expression of these genes was induced by both peptidoglycan and polyI:C, although the number of JF-TLR-expressing cells were not changed after induction. All of these results suggest that they are involved in the innate immune system.