Universal Adaptor Protein Research Articles

Identification and functional analysis of myeloid differentiation factor 88 (MyD88) in early development of Haliotis diversicolor

Myeloid differentiation factor 88 (MyD88) is a universal adaptor protein and plays an important role in the signal transduction of Toll like receptors (TLR) family. In this study, the MyD88 gene from the Haliotis diversicolor (hdMyD88) was identified. The full-length cDNA of hdMyD88 has a 1927 base pairs (bp), with an open reading frame of 1314 bp encoding 437 amino acids including a death domain (DD) at the N-terminus and TIR domain at the C-terminus which are typical features of MyD88 family proteins. Three conserved boxes are also found in the hdMyD88, which are similar to MyD88 in vertebrates. The expression levels of hdMyD88 mRNA at different early embryonic developmental stages of abalone were measured by qPCR revealed that their constitutive expression at all developmental stages analyzed with the considerably highest values at 8 cell stage and the lowest level at the trochosphere stage. Additionally, the mRNA expression of hdMyD88 decreased significantly (P < 0.05) after MyD88-dsRNA soak in the stage of trochosphere and veliger than EGFP-dsRNA group and blank control group. Whole embryo in situ hybridization showed that the positive signals of hdMyD88 were in visceral mass of trochophore larvae and veliger larvae. These results indicate hdMyD88 may could respond to pathogenic infection and may play an important role in early innate immunity in the process of abalone larval development.

Role of Small Heat Shock Proteins in the Remodeling of Actin Microfilaments.

Small heat shock proteins (sHsps) play an important role in the maintenance of proteome stability and, particularly, in stabilization of the cytoskeleton and cell contractile apparatus. Cell exposure to different types of stress is accompanied by the translocation of sHsps onto actin filaments; therefore, it is commonly believed that the sHsps are true actin-binding proteins. Investigations of last years have shown that this assumption is incorrect. Stress-induced translocation of sHsp to actin filaments is not the result of direct interaction of these proteins with intact actin, but results from the chaperone-like activity of sHsps and their interaction with various actin-binding proteins. HspB1 and HspB5 interact with giant elastic proteins titin and filamin thus providing an integrity of the contractile apparatus and its proper localization in the cell. HspB6 binds to the universal adapter protein 14-3-3 and only indirectly affects the structure of actin filament. HspB7 interacts with filamin C and controls actin filament assembly. HspB8 forms tight complex with the universal regulatory and adapter protein Bag3 and participates in the chaperone-assisted selective autophagy (CASA) of actin-binding proteins (e.g., filamin), as well as in the actin-depending processes taking place in mitoses. Hence, the mechanisms of sHsp participation in the maintenance of the contractile apparatus and cytoskeleton are much more complicated and diverse than it has been postulated earlier and are not limited to direct interactions of sHsps with actin. The old hypothesis on the direct binding of sHsps to intact actin should be revised and further detailed investigation on the sHsp interaction with minor proteins participating in the formation and remodeling of actin filaments is required.

Mechanism of MyD88S mediated signal termination

BackgroundA universal adaptor protein, MyD88, orchestrates the innate immune response by propagating signals from toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R). Receptor activation seeds MyD88 dependent formation of a signal amplifying supramolecular organizing center (SMOC)—the myddosome. Alternatively spliced variant MyD88S, lacking the intermediate domain (ID), exhibits a dominant negative effect silencing the immune response, but the mechanistic understanding is limited.MethodsLuciferase reporter assay was used to evaluate functionality of MyD88 variants and mutants. The dimerization potential of MyD88 variants and myddosome nucleation process were monitored by co-immunoprecipitation and confocal microscopy. The ID secondary structure was characterized in silico employing I-TASSER server and in vitro using nuclear magnetic resonance (NMR) and circular dichroism (CD).ResultsWe show that MyD88S is recruited to the nucleating SMOC and inhibits its maturation by interfering with incorporation of additional components. Biophysical analysis suggests that important functional role of ID is not supported by a well-defined secondary structure. Mutagenesis identifies Tyr116 as the only essential residue within ID required for myddosome nucleation and signal propagation (NF-κB activation).ConclusionsOur results argue that the largely unstructured ID of MyD88 is not only a linker separating toll-interleukin-1 receptor (TIR) homology domain and death domain (DD), but contributes intermolecular interactions pivotal in MyD88-dependent signaling. The dominant negative effect of MyD88S relies on quenching the myddosome nucleation and associated signal transduction.Dn-xKRix_iGoWWk4uZnJcGVideo abstract

A bacterial kinase phosphorylates OSK1 to suppress stomatal immunity in rice

The Xanthomonas outer protein C2 (XopC2) family of bacterial effectors is widely found in plant pathogens and Legionella species. However, the biochemical activity and host targets of these effectors remain enigmatic. Here we show that ectopic expression of XopC2 promotes jasmonate signaling and stomatal opening in transgenic rice plants, which are more susceptible to Xanthomonas oryzae pv. oryzicola infection. Guided by these phenotypes, we discover that XopC2 represents a family of atypical kinases that specifically phosphorylate OSK1, a universal adaptor protein of the Skp1-Cullin-F-box ubiquitin ligase complexes. Intriguingly, OSK1 phosphorylation at Ser53 by XopC2 exclusively increases the binding affinity of OSK1 to the jasmonate receptor OsCOI1b, and specifically enhances the ubiquitination and degradation of JAZ transcription repressors and plant disease susceptibility through inhibiting stomatal immunity. These results define XopC2 as a prototypic member of a family of pathogenic effector kinases and highlight a smart molecular mechanism to activate jasmonate signaling.

Ctenopharyngodon idella Tollip regulates MyD88-induced NF-κB activation

Toll-interacting protein (Tollip) and MyD88 are key components of the TLR/IL-1R signaling pathway in mammals. MyD88 is known as a universal adaptor protein involving in TLR/IL-1R-induced NF-κB activation. Tollip is a crucial negative regulator of TLR-mediated innate immune responses. Previous studies have demonstrated that teleost Tollip served as a negative regulator of MyD88-dependent TLR signaling pathway. However, the mechanism is still unclear. In particular, the effect of TBD, C2, and CUE domains of Tollip on MyD88-NF-κB signaling pathway remains to be elucidated. In this study, we found that the response of grass carp Tollip (CiTollip) to LPS stimulation was faster and stronger than that of poly I:C treatment, and CiTollip diminished the expression of tnf-α induced by LPS. Further assays indicated that except for the truncated mutant of △CUE2 (1–173 aa), wild type CiTollip and other truncated mutants (△N-(52–276 aa), △C2-(173–276 aa) and △CUE1-(1–231 aa)) could associate with MyD88 and negatively regulate MyD88-induced NF-κB activation. It suggested that the C-terminal (173–276 aa), in particular the connection section between C2 and CUE domains (173–231 aa), played a pivotal role in suppressing MyD88-induced activation of NF-κB.

Conformational Changes Induced by S34Y and R98C Variants in the Death Domain of Myd88.

Myeloid differentiating factor 88 (Myd88) is a universal adaptor protein that plays a critical role in innate immunity by mediating TLR downstream signaling. Myd88 death domain (DD) forms an oligomeric complex by association with other DD-containing proteins such as IRAK4. Despite its universal role, polymorphisms in Myd88 can result in several diseases. Previous studies have suggested that, out of several non-synonymous single-nucleotide polymorphisms (nsSNPs), the variants S34Y and R98C in the DD of Myd88 disrupt the formation of the Myddosome complex. Therefore, we performed molecular dynamics (MD) simulations on wild-type (Myd88WT) and mutant (Myd88S34Y, Myd88R98C) DDs to evaluate the subtle conformational changes induced by these mutations. Our results suggest that the S34Y variant induces large structural transitions compared to the R98C variant as evidenced by residual flexibility at the variable loop regions, particularly in the H1–H2 loop, and variations in the collective modes of motion observed for wild-type and mutant Myd88 DDs. The residue interaction network strongly suggests a distortion in the interaction pattern at the location of the mutated residue between the wild type and mutants. Moreover, betweenness centrality values indicate that variations in the distribution of functionally important residues may be reflected by distinct residue signal transductions in both wild-type and mutant Myd88 DDs, which may influence the interaction with other DDs in TLR downstream signaling.

Different Vinculin Binding Sites Use the Same Mechanism to Regulate Directional Force Transduction

Vinculin is a universal adaptor protein that transiently reinforces the mechanical stability of adhesion complexes. It stabilizes mechanical connections that cells establish between the actomyosin cytoskeleton and the extracellular matrix via integrins or to neighboring cells via cadherins, yet little is known regarding its mechanical design. Vinculin binding sites (VBSs) from different nonhomologous actin-binding proteins use conserved helical motifs to associate with the vinculin head domain. We studied the mechanical stability of such complexes by pulling VBS peptides derived from talin, α-actinin, and Shigella IpaA out of the vinculin head domain. Experimental data from atomic force microscopy single-molecule force spectroscopy and steered molecular dynamics (SMD) simulations both revealed greater mechanical stability of the complex for shear-like than for zipper-like pulling configurations. This suggests that reinforcement occurs along preferential force directions, thus stabilizing those cytoskeletal filament architectures that result in shear-like pulling geometries. Large force-induced conformational changes in the vinculin head domain, as well as protein-specific fine-tuning of the VBS sequence, including sequence inversion, allow for an even more nuanced force response.

Functional relationship between CgMyD88-1 and CgMyD88-2 in the Pacific oyster

MyD88 is a universal adapter protein for the Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) signaling pathway. Since invertebrates are believed to lack MyD88-independent pathways, MyD88 appears more critical in oyster TLR signaling pathway. In the Pacific oyster (Crassostrea gigas), two complete paralogues, named as CgMyD88-1 and CgMyD88-2, have been identified. In the current study, we indicated that CgMyD88-1 and CgMyD88-2 might act synergistically to increase the efficiency of immune signaling by activating NF-κB transcription factor. However, we found that upon stimulation with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid [poly (I:C)], CgMyD88-1 and CgMyD88-2 show differences in their response: CgMyD88-1 accumulated as large spots in the cytoplasm, while CgMyD88-2 assembled in the cytoplasm and in the membrane. Our results support the theory that expansion of these immune genes is associated with functional diversity.

Characterization, subcellular localization and function analysis of myeloid differentiation factor 88 (Pt-MyD88) in swimming crab, Portunus trituberculatus

Myeloid differentiation factor 88 (MyD88) is a universal and essential adaptor protein required for the Toll-like receptors (TLRs) pathway activation in invertebrates as well as in vertebrates. Herein, we characterized a MyD88 (Pt-MyD88) cDNA sequence in the swimming crab (Portunus trituberculatus). The Pt-MyD88 ORF is predicted to encode 469 peptides with an N-terminal death domain and a typical C-terminal TIR domain. Real-Time quantitative PCR analysis showed that the Pt-MyD88 transcriptions were constitutively expressed in hemocytes, gill, intestine, heart and muscle in normal crab. The expressions of Pt-MyD88 would be down-regulated by V. alginolyticus or LPS challenge, and be up-regulated by WSSV infection in hemocytes. Intracellular localization showed Pt-MyD88 was distributed mainly in the cytoplasm when it was over-expressed in human cell HEK293T or in Drosophila Schneider 2 (S2). Functionally, over-expression of Pt-MyD88 could either activate the NF-κB in HEK293T cells or activate the promoters of Drosophila antimicrobial peptide genes (AMPs) in S2 cell. In primary cultured hemocytes of swimming crab, after Pt-MyD88 was knocked-down by specific long double strand RNA, the expression of anti-lipopolysaccharide factor1 (ALF1), hyastatin3, crustin1 and crustin3 have been significantly inhibited, while the expression of other AMPs is normal compared to non-specific dsRNA treated cells.

Receptor tyrosine kinase MerTK suppresses an allogenic type I IFN response to promote transplant tolerance.

Recipient infusion of donor apoptotic cells is an emerging strategy for inducing robust transplantation tolerance. Daily clearance of billions of self-apoptotic cells relies on homeostatic engagement of phagocytic receptors, in particular, receptors of the tyrosine kinase family TAM (Tyro3, Axl, and MerTK), to maintain self-tolerance. However, an outstanding question is if allogeneic apoptotic cells trigger the same receptor system for inducing allogeneic tolerance. Here, we employed allogeneic apoptotic splenocytes and discovered that the efferocytic receptor MerTK on recipient phagocytes is a critical mediator for transplantation tolerance induced by this strategy. Our findings indicate that the tolerogenic properties of allogeneic apoptotic splenocytes require MerTK transmission of intracellular signaling to suppress the production of inflammatory cytokine interferon α (IFN-α). We further demonstrate that MerTK is crucial for subsequent expansion of myeloid-derived suppressor cells and for promoting their immunomodulatory function, including maintaining graft-infiltrating CD4+ CD25+ Foxp3+ regulatory T cells. Consequently, recipient MerTK deficiency resulted in failure of tolerance by donor apoptotic cells, and this failure could be effectively rescued by IFN-α receptor blockade. These findings underscore the importance of the efferocytic receptor MerTK in mediating transplantation tolerance by donor apoptotic cells and implicate MerTK agonism as a promising target for promoting transplantation tolerance.

Mechanism for the Regulated Control of Bacterial Transcription Termination by a Universal Adaptor Protein

NusG/Spt5 proteins are the only transcription factors utilized by all cellular organisms. In enterobacteria, NusG antagonizes the transcription termination activity of Rho, a hexameric helicase, during the synthesis of ribosomal and actively translated mRNAs. Paradoxically, NusG helps Rho act on untranslated transcripts, including non-canonical antisense RNAs and those arising from translational stress; how NusG fulfills these disparate functions is unknown. Here, we demonstrate that NusG activates Rho by assisting helicase isomerization from an open-ring, RNA-loading state to a closed-ring, catalytically active translocase. A crystal structure of closed-ring Rho in complex with NusG reveals the physical basis for this activation and further explains how Rho is excluded from translationally competent RNAs. This study demonstrates how a universally conserved transcription factor acts to modulate the activity of a ring-shaped ATPase motor and establishes how the innate sequence bias of a termination factor can be modulated to silence pervasive, aberrant transcription.

Mechanism for the Regulated Control of Transcription Termination by a Universal Adapter Protein

NusG/Spt5 proteins are the only transcription factors utilized by all cellular organisms. In enterobacteria, NusG antagonizes the transcription termination activity of Rho, a hexameric helicase and functional analog of eukaryotic Sen1, during transcription of ribosomal and actively translated mRNAs. Paradoxically, NusG helps Rho act on untranslated transcripts, including non-canonical antisense RNAs or those arising from translational stress; how NusG fulfills these disparate functions is unknown. Here, we demonstrate that NusG activates Rho by assisting helicase isomerization from an open-ring, RNA-loading state to a closed-ring, catalytically-active translocase. A crystal structure of closed-ring Rho in complex with NusG reveals the physical basis for this activation, and further explains how Rho is excluded from translationally-competent RNAs. This study demonstrates how a universally conserved transcription factor acts to modulate the activity of a ring-shaped ATPase motor, and establishes how the innate sequence-bias of a termination factor can be modulated to silence pervasive, aberrant transcription.

38P - MYD88 mutations are associated with a high tumor mutational burden

Aim/Background: MYD88 is a universal adaptor protein used by almost all toll-like receptors (TLRs) and is critical for IL1 receptor innate immunity signaling. Activating mutations in MYD88 have been described in the majority of patients with lymphoplasmocytic lymphoma. MYD88 mutations have not been characterized in solid tumors. Methods: Whole-genome somatic tumor mutation data were downloaded from the resource project TCGA (Broad GDAC website (https://gdac.broadinstitute.org/)). 8,495 samples corresponding to unique patients (32 tumor types) were curated. Lists of significant variants were generated using the MutSig2CV algorithm (http://www.broadinstitute.org/cancer/cga/mutsig). All non-synonymous missense, nonsense, del/insertions, frameshift or splicing site mutations within MYD88 were kept. Association between MYD88 non-silent mutations and the overall tumor mutational burden (TMB) was assessed by univariate analysis using the Mann-Whitney’s test. Results: Of the 8,475 samples curated, 22 were mutated for MYD88. Tumor histologies included 7 diffuse large B-cell lymphoma (DLBCL) (32%), as well as 15 solid tumors (68%): stomach (18% of 22), colorectal (14%), endometrial (9%) and others (30%). Tumors with mutated versus wild-type MYD88 demonstrated a median of 811 vs 66 mutations (p = <0.0001). Solid tumors, excluding hematologic malignancies, mutated for MYD88 (n = 15) harbored a median of 1,528 mutations (range, 50-12,312) vs 68 (range, 1-23,950) for wild-type MYD88 (p = <0.0001) (Table). Table Open table in a new tab Conclusions: MYD88 non-silent alterations are rare in solid tumors. However, solid tumors with MYD88 non-silent alterations are associated with very high overall TMB compared to tumors with no MYD88 mutation. Signaling through MYD88 can induce NFKB expression, which may result in high TMB via aberrant expression of activation-induced cytidine deaminase. The relationship between MYD88 alterations and high TMB merits further exploration. Legal entity responsible for the study: Aaron Goodman Funding: No funding Disclosure: A.M. Goodman: Fellowship Funding from Pfizer. R. Kurzrock: Genentech, Merck, Serono, Pfizer, Sequenom, Foundation Medicine, and Guardant, as well as consultant fees from Sequenom, X Biotech, and Actuate Therapeutics and an ownership interest in Novena, Inc. and Curematch Inc. All other authors have declared no conflicts of interest.

PREVALENCE OF MYD88 L265P MUTATION IN HISTOLOGICALLY PROVEN, DIFFUSE LARGE B-CELL VITREORETINAL LYMPHOMA

Myeloid differentiation primary response gene 88 (MYD88) is a universal adaptor protein in the innate immune system. When associated with a proline for leucine substitution mutation at position 265 (L265P), the protein becomes constitutively activated, amplifying the intracellular pro-inflammatory signal. Recently, we reported two cases of vitreoretinal lymphoma (VRL) that were positive for the mutation. The purpose of this study was to determine prevalence of the MYD88 L265P mutation in a larger series of VRL. Retrospective chart review of 25 patients with histologically confirmed VRL evaluated at Mayo Clinic, Rochester, between January 2000 and March 2015. Paraffin-embedded blocks from the vitreous were submitted for polymerase chain reaction testing of the L265P mutation. The mutation was positive in 82.4% of all VRL cases and 86.7% of primary VRL cases. The minimum necessary DNA concentration needed for the polymerase chain reaction assay was 4.93 ng/mL. MYD88 gene analysis is a helpful ancillary tool for diagnosing VRL. It often requires fewer cells than flow cytometry or cytology and may be especially useful in early cases where a sufficient number of cells may not be available.

Antimony-Resistant Leishmania donovani Exploits miR-466i To Deactivate Host MyD88 for Regulating IL-10/IL-12 Levels during Early Hours of Infection.

Infection with antimony-resistant Leishmania donovani (Sb(R)LD) induces aggressive pathology in the mammalian hosts as compared with ones with antimony-sensitive L. donovani (Sb(S)LD) infection. Sb(R)LD, but not Sb(S)LD, interacts with TLR2/TLR6 to induce IL-10 by exploiting p50/c-Rel subunits of NF-κB in infected macrophages (Mϕs). Most of the TLRs exploit the universal adaptor protein MyD88 to activate NF-κB. We now show that infection of Mϕs from MyD88(-/-) mice with Sb(R)LD gave rise to significantly higher intracellular parasite number coupled with elevated IL-10/IL-12 ratio in the culture supernatant as compared with infection in wild type (WT) Mϕs. Τhese attributes were not seen with Sb(S)LD in similar experiments. Further, Sb(R)LD infection upregulated miR-466i, which binds with 3'-untranslated region, leading to the downregulation of MyD88. Infection of MyD88(-/-) Mϕ or IL-12(-/-) Mϕ with Sb(R)LD induced IL-10 surge at 4 h, whereas the same in WT Mϕ started from 12 h. Thus, absence of IL-12 in MyD88(-/-) mice favored early binding of NF-κB subunits to the IL-10 promoter, resulting in IL-10 surge. Infection of MyD88(-/-) mice with Sb(R)LD showed significantly higher organ parasites coupled with ill-defined and immature hepatic granulomas, whereas in WT mice there were less organ parasites and the granulomas were well defined. From the survival kinetics it was observed that Sb(R)LD-infected MyD88(-/-) mice died by 60 d postinfection, whereas the WT mice continued to survive. Our results demonstrate that Sb(R)LD has evolved a unique strategy to evade host antileishmanial immune repertoire by manipulating host MyD88 to its advantage.

Hidden disorder propensity of the N-terminal segment of universal adapter protein 14-3-3 is manifested in its monomeric form: Novel insights into protein dimerization and multifunctionality

The multiplicity of functions of 14-3-3 proteins, integrated into many cellular interactions and signaling networks, is primarily based upon their dimeric α-helical structure that is capable of binding phosphorylated protein partners as well as displaying a “moonlighting” chaperone-like activity. The structure and functions of 14-3-3 proteins are regulated in different ways, including Ser58 phosphorylation in the interface, which shifts equilibrium towards the formation of protein monomers whose role is poorly understood. While modification of Ser58 induced only partial dissociation, the engineered triple mutation of human 14-3-3ζ located in the first α-helix deeply monomerized the protein, allowing for a structural analysis of the monomeric form. Dimer-incapable 14-3-3 proteins retained binding capacity and specificity towards some phosphopartners, and also demonstrated increased chaperone-like activity on various substrates. Here, we found a substantial propensity of the N-terminal segment (~40 residues) of 14-3-3 proteins to intrinsic disorder, showing remarkable conservation across different isoforms and organisms. We hypothesized that this intrinsic disorder propensity, hidden in the α-helical 14-3-3 dimer, can be manifested upon its dissociation and interrogated novel monomeric 14-3-3ζ carrying both monomerizing and S58E mutations (14-3-3ζmS58E). CD spectroscopy showed that, at physiological temperatures, this protein has ~10–15% reduced helicity relative to the wild type protein, corresponding to roughly 40 residues. Along with the known flexibility of C-terminus, SAXS-based modeling of the 14-3-3ζmS58E structure strongly suggested pliability of its N-terminus. The unraveled disorder propensity of the N-terminal tails of 14-3-3 proteins provides new clues for better understanding of the molecular mechanisms of dimerization and multifunctionality of these universal adapter proteins.

MyD88 Polymorphisms and Association with Susceptibility to Salmonella Pullorum.

Myeloid differentiation primary response gene 88 (MYD88), a universal adapter protein, plays an important role in activating the nuclear factor-κB (NF-κB) and regulating the expression of proinflammatory genes like tumor necrosis factor (TNF) and interleukin-1 (IL-1), which were highly involved in Salmonella Pullorum infection. To detect the relationship between polymorphisms of the MyD88 gene and Salmonella Pullorum disease, we screened the coding region (CDS) of the MYD88 gene by DNA pool construction and sequencing based on case-control study. Eight single nucleotide polymorphisms (SNPs) in the sequenced fragment (5 exons), 7 known loci and one novel mutation named G4810372T (SNP8), were found in the fifth exon. In addition, we found 7 nonsynonymous substitutions. The allele frequency of only one SNP, g.4810191C > T (SNP1), was significantly different (P < 0.05) between case and control groups. The genotype frequencies of SNP1 (g.4810191C > T) and SNP3 (g.4810257G > T) were of significant difference between the case and the control groups (P < 0.05). Collectively, SNPs of the MyD88 gene were significantly associated with susceptibility to Salmonella Pullorum infection, which can be used as a disease-resistant marker in chicken. These results provided a theoretical basis for future research on chicken breeding by marker-assisted selection.

Novel myeloid differentiation factor 88, EsMyD88, exhibits EsTube-binding activity in Chinese mitten crab Eriocheir sinensis

Myeloid differentiation factor 88 (MyD88) is a universal and essential adapter protein that participates in the activation of the Toll-like receptor/interleukin-1 receptor-mediated signaling pathway. In the present study, a new MyD88 gene (named EsMyD88) was identified in the Chinese mitten crab Eriocheir sinensis. The cDNA of EsMyD88 was 2210 bp long with a 1416 bp open reading frame that encoded a protein with 472 amino acids. Predicted EsMyD88 protein had a death domain at the N-terminal and a TIR domain at the C-terminal. BLASTP and phylogenetic analysis results showed that EsMyD88 was clustered in one group together with other crustaceans MyD88 (SpMyD88, FcMyD88, LvMyD88, and LvMyD88-1). EsMyD88 was detected in all the examined tissues of healthy crabs, and was mainly expressed in the hemocytes and nerves. When normal crabs were challenged with lipopolysaccharide, peptidoglycan, Staphylococcus aureus, Vibrio parahaemolyticus, or Aeromonas hydrophila, the expression levels of EsMyD88 significantly increased either in the hepatopancreas or hemocytes. Results of the pull-down assay showed that EsMyD88 could bind to downstream cytosolic adaptor EsTube. Overexpression of EsMyD88 protein in Drosophila Schneider 2 cells led to the activation of antimicrobial peptide genes. RNA interference assay showed that EsMyD88 is involved in regulating the transcription of ALF1 and ALF2, Cru1 and Cru2, and Lys in crab challenged with V. parahaemolyticus. All the results mentioned earlier indicated that EsMyD88 gene has a key function in antibacterial innate immune defense.

Molecular Cloning, Characterization, and Expression of Two Myeloid Differentiation Factor 88 (Myd88) in Pacific Oyster, Crassostrea gigas

AbstractMyeloid differentiation factor 88 (MyD88) is a universal adaptor protein and plays an important role in the signal transduction of Toll/interleukin‐1 receptor (TIR/IL‐1R) family. In this study, two novel molluskan MyD88 family members (named as CgMyD88‐1 and CgMyD88‐2) were identified from the Pacific oyster, Crassostrea gigas. The full‐length cDNA of CgMyD88‐1 was of 2453 base pairs (bp), with an open reading frame (ORF) of 1077 bp encoding 358 amino acids. The cDNA of CgMyD88‐2 consists of 1374 bp ORF encoding 457 amino acids. Both CgMyD88s contain death domain (DD) and TIR domain which are typical features of MyD88 family proteins. Three conserved boxes were also found in the two CgMyD88s which were similar to MyD88s in vertebrates. The expression level of both CgMyD88 mRNAs in selected tissues was measured by quantitative real‐time polymerase chain reaction (qRT‐PCR). Both CgMyD88 transcripts were constitutively expressed in all tested tissues. Gill and hemocytes were shown with highest expression levels while adductor muscle and gonad were with lower levels. The CgMyD88 transcripts in hemocytes after ostreid herpesvirus 1 (OsHV‐1) challenge were also determined by qRT‐PCR. Both CgMyD88s were significantly upregulated in hemocytes after OsHV‐1 challenge and had significant differences (P &lt; 0.05) at some time points compared with the control. Our results as previously reported suggest that MyD88‐dependent pathway exists in C. gigas, and its critical adaptor CgMyD88 plays important roles in oysters' innate immune defense against OsHV‐1 infection.

Identification and function of two myeloid differentiation factor 88 variants in triangle-shell pearl mussel (Hyriopsis cumingii)

Myeloid differentiation factor 88 (MyD88) is a universal and essential adapter protein that participates in the activation of the Toll-like receptor (TLR)/interleukin-1 receptor-mediated signaling pathway. In this study, two MyD88 genes (HcMyD88-1 and HcMyD88-2) were identified from triangle-shell pearl mussel (Hyriopsis cumingii). Both HcMyD88-1 and HcMyD88-2 proteins were determined to have a death domain at the N-terminal and a TIR domain at the C-terminal. Both HcMyD88-1 and HcMyD88-2 genes were mainly expressed in the hepatopancreas of healthy mussels. HcMyD88-1 and HcMyD88-2 slightly responded to Gram-negative bacterial challenge. Upon bacterial challenge with Gram-positive Staphyloccocus aureus, HcMyD88-1 and HcMyD88-2 transcription levels remarkably increased at 2 and 6h, respectively. Overexpression of HcMyD88-1 and HcMyD88-2 proteins in Drosophila Schneider 2 cells led to the activation of antimicrobial peptide genes. These results indicated that HcMyD88-2 had higher activity than HcMyD88-1 during the activation of attacin A, drosomycin, and metchnikowin genes, suggesting that HcMyD88 genes may play a role in antibacterial innate immune defense.