Biomedical subjects

Hiroyuki Oshiumi

Publications and source records attributed to Hiroyuki Oshiumi.

13 recordsLinked to original sources

PubMed2005

Regulator of complement activation (RCA) locus in chicken: identification of chicken RCA gene cluster and functional RCA proteins.

A 150-kb DNA fragment, which contains the gene of the chicken complement regulatory protein CREM (formerly named Cremp), was isolated from a microchromosome by screening bacterial artificial chromosome library. Within 100 kb of the cloned region, three complete genes encoding short consensus repeats (SCRs, motifs with tandemly arranged 60 aa) were identified by exon-trap method and 3'- or 5'-RACE. A chicken orthologue of the human gene 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2, which exists in close proximity to the regulator of complement activation genes in humans and mice, was located near this chicken SCR gene cluster. Moreover, additional genes encoding SCR proteins appeared to be present in this region. Three distinct transcripts were detected in RNA samples from a variety of chicken organs and cell lines. Two novel genes named complement regulatory secretory protein of chicken (CRES) and complement regulatory GPI-anchored protein of chicken (CREG) besides CREM were identified by cloning corresponding cDNA. Based on the predicted primary structures and properties of the expressed molecules, CRES is a secretory protein, whereas CREG is a GPI-anchored membrane protein. CREG and CREM were protected host cells from chicken complement-mediated cytolysis. Likewise, a membrane-bound form of CRES, which was artificially generated, also protected host cells from chicken complement. Taken together, the chicken possesses an regulator of complement activation locus similar to those of the mammals, and the gene products function as complement regulators.

Amino Acid Sequence↗

PubMed2005

Cutting Edge: NF-kappaB-activating kinase-associated protein 1 participates in TLR3/Toll-IL-1 homology domain-containing adapter molecule-1-mediated IFN regulatory factor 3 activation.

TLRs signal the presence of microbial patterns and activate transcription factors. In TLR3 and TLR4, the adapter Toll-IL-1R homology domain-containing adapter molecule (TICAM-1) (also called Toll/IL-1R domain-containing adapter inducing IFN-beta (TRIF)) mediates IFN regulatory factor 3 (IRF3) phosphorylation followed by IFN-beta production. The regulatory subunit TNFR-associated factor family member-associated NF-kappaB activator (TANK) couples with the kinase complex IkappaB kinase-related kinase epsilon/NF-kappaB-activating kinase (NAK) (TANK-binding kinase 1 (TBK1)) that involveTICAM-1-dependent IFN-beta induction. There are several TANK-homologous proteins. We tested whether TICAM-1 binds and coprecipitates with TANK or its family proteins. The results are: 1) the TANK family protein NAK-associated protein 1 (NAP1), but not TANK, coprecipitates withTICAM-1; 2) NAP1 overexpression markedly enhances TBK1-mediated IFN-beta promoter activation; 3) a dominant-negative form, NAP (158-270), suppresses IRF3 activation in response to poly(I:C) or LPS; 4) RNA interference targeting of the NAP1 message results in a failure of poly(I:C)-mediated IRF3 polymerization and IFN-beta production. Thus, NAP1 is the kinase subunit responsible for TLR3/4-mediated IFN-beta induction in the TICAM-1 pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2005

TICAM-1 and TICAM-2: toll-like receptor adapters that participate in induction of type 1 interferons.

Toll-like receptor (TLR) 3 and 4 mediate the expression of many genes, including NF-kappaB- and interferon-regulatory factor (IRF)-3/interferon (IFN)-inducible genes, in macrophages and dendritic cells (DCs) in response to their ligand stimuli, polyI:C and lipopolysaccharide (LPS). Toll-IL-1 receptor homology domain (TIR)-containing adapter molecule 1 (TICAM-1) facilitates expression of IFN-inducible genes via TLR3. Although MyD88 and Mal/TIRAP adapters function downstream of TLR4, they barely induce IFN-beta. In addition, DC maturation as well as IFN-beta induction are largely independent of MyD88 and Mal/TIRAP. TICAM-1 is the functional adapter for both TLR3 and TLR4 that induces type 1 IFN and MyD88-independent DC maturation. In LPS-mediated TLR4 activation, a complex of TICAM-1 and an additional TLR4-binding adapter serves as the adapter. We named this TLR4-TICAM-1-bridging adapter TICAM-2. Our results reveal the details of MyD88-independent pathways which separately recruit the distinct adapters downstream of TLR3 and TLR4 and variations of the TLR output are in part regulated by the two additional adapters in DCs.

Adaptor Proteins, Signal Transducing↗

PubMed2004

A protein complex containing Mei5 and Sae3 promotes the assembly of the meiosis-specific RecA homolog Dmc1.

Meiotic recombination requires the meiosis-specific RecA homolog Dmc1 as well as the mitotic RecA homolog Rad51. Here, we show that the two meiosis-specific proteins Mei5 and Sae3 are necessary for the assembly of Dmc1, but not for Rad51, on chromosomes including the association of Dmc1 with a recombination hot spot. Mei5, Sae3, and Dmc1 form a ternary and evolutionary conserved complex that requires Rad51 for recruitment to chromosomes. Mei5, Sae3, and Dmc1 are mutually dependent for their chromosome association, and their absence prevents the disassembly of Rad51 filaments. Our results suggest that Mei5 and Sae3 are loading factors for the Dmc1 recombinase and that the Dmc1-Mei5-Sae3 complex is integrated onto Rad51 ensembles and, together with Rad51, plays both catalytic and structural roles in interhomolog recombination during meiosis.

Amino Acid Sequence↗

PubMed2004

Sensing bacterial flagellin by membrane and soluble orthologs of Toll-like receptor 5 in rainbow trout (Onchorhynchus mikiss).

Rainbow trout (Onchorhynchus mikiss) possess two genes encoding putative leucine-rich repeat (LRR)-containing proteins similar to human TLR5. Molecular cloning of these two LRR proteins suggested the presence of a TLR5-like membrane form (rtTLR5M) and a soluble form (rtTLR5S). Here we elucidated the primary structures and the unique combinational functions of these fish versions of TLR5. The LRR regions of rtTLR5S and rtTLR5M exhibited 81% homology and relatively high (35.6 and 33.7%) homology to the extracellular domains of human TLR5 (huTLR5). Thus, two distinct genes encode the TLR5 orthologs in fish, one of which has a consensus intracellular domain (TIR). In order to test their functions, we constructed fusion proteins with the LRR region of rtTLR5S (S-chimera) or that of rtTLR5M and the TIR of huTLR5 (M-chimera). The S- and M-chimeras expressed in HeLa or CHO cells signaled the presence of Vibrio anguillarum flagellin, resulting in NF-kappaB activation. rtTLR5M was ubiquitously expressed, whereas rtTLR5S was predominantly expressed in the liver. In the hepatoma cell lines of the rainbow trout RTH-149, stimulation of rtTLR5M with V. anguillarum or its flagellin allowed the up-regulation of rtTLR5S. Flagellin-mediated NF-kappaB activation was more significant in the presence of or simultaneous expression of rtTLR5S. Therefore, a two-step flagellin response occurred for host defense against bacterial infection in fish: (a) flagellin first induced basal activation of NF-kappaB via membrane TLR5, facilitating the production of soluble TLR5 and minimal acute phase proteins, and (b) the inducible soluble TLR5 amplifies membrane TLR5-mediated cellular responses in a positive feedback fashion.

Amino Acid Sequence↗

PubMed2004

A short consensus repeat-containing complement regulatory protein of lamprey that participates in cleavage of lamprey complement 3.

The prototype of the short consensus repeat (SCR)-containing C regulatory protein is of interest in view of its evolutionary significance with regard to the origin of the C regulatory system. Lamprey is an agnathan fish that belongs to the lowest class of vertebrates. Because it does not possess lymphocytes, it lacks Ig and consequently the classical C pathway. We identified an SCR-containing C regulatory protein from the lamprey. The primary structure predicted from the cDNA sequence showed that this is a secretary protein consisting of eight SCRs. This framework is similar to the alpha-chain of C4b-binding protein (C4bp). SCR2 and -3 of human C4bp are essential for C4b inactivation, and this region is fairly well conserved in the lamprey protein. However, the other SCRs of this protein are similar to those of other human C regulatory proteins. The lamprey protein binds to the previously reported lamprey C3b/C3bi deposited on yeast and cleaves lamprey C3b-like C3 together with a putative serum protease. The scheme resembles the C regulatory system of mammals, where factor I and its cofactor inactivate C3b. Unlike human cofactors, the lamprey protein requires divalent cations for C3b-like C3 cleavage. Its artificial membrane-anchored form protects host cells from lamprey C attack via the lectin pathway. Thus, the target of this protein appears to be C3b and/or its family. We named this protein Lacrep, the lamprey C regulatory protein. Lacrep is a member of SCR-containing C regulators, the first of its kind identified in the lowest vertebrates.

Amino Acid Sequence↗

PubMed2004

The cytoplasmic 'linker region' in Toll-like receptor 3 controls receptor localization and signaling.

Toll-like receptor 3 (TLR3) recognizes double-stranded RNA and transmits signals to activate NF-kappaB and the interferon (IFN)-beta promoter via the newly identified adaptor, TICAM-1. The extracellular LRR domain of TLR3 is engaged in the ligand recognition, while the intracellular TIR domain is crucial for the adaptor binding and signal transduction upon ligand stimulation. Here, we analyzed TLR3 localization in human monocyte-derived immature dendritic cells (iDCs) and stable transfectants expressing human TLR3 by immunofluorescence staining and confocal microscopy. TLR3 was predominantly localized in specific but as yet unidentified intracellular vesicles where TLR3 signaling was initiated. Expression analysis of TLR3-tail-truncated mutants revealed that the cytoplasmic 'linker' region (residues 730-755) determines the intracellular localization of TLR3. Site-directed mutagenesis of the linker region allowed us to identify the relevant determinants as Arg(740) and Val(741) residues for intracellular expression of TLR3. Furthermore, alanine scanning of the linker region demonstrated that the Phe(732), Leu(742) and Gly(743) in the TLR3 cytoplasmic linker region are essential for ligand-induced NF-kappaB and IFN-beta promoter activation. Thus, the cytoplasmic linker region of TLR3 regulates receptor retention inside the organelle and signaling, which may be closely linked to TLR3 function in DCs.

Adaptor Proteins, Vesicular Transport↗

PubMed2004

Toll-like receptor 3: a link between toll-like receptor, interferon and viruses.

Production of type I interferon (IFN-alpha/beta) by virus-infected cells is the central event in their antiviral immune responses. In mammalian cells, IFN-alpha/beta gene transcription is induced through distinct signaling pathways by viral infection or by treatment with double-stranded (ds) RNA, which is an intermediate of virus replication. Toll-like receptor 3 (TLR3) was found to recognize dsRNA and transmit signals to activate NF-kappaB and the IFN-beta promoter. Recent identification of the TLR3-adaptor protein and its downstream signaling molecules, which are involved in IFN-alpha/beta production, revealed a novel IFN-inducing pathway for an anti-viral immune response. Here, we summarize the current knowledge of TLR3-mediated immune responses.

Immunity, Cellular↗

PubMed2003

TIR-containing adapter molecule (TICAM)-2, a bridging adapter recruiting to toll-like receptor 4 TICAM-1 that induces interferon-beta.

Lipopolysaccharide (LPS) is an agonist for Toll-like receptor (TLR) 4 and expresses many genes including NF-kappaB- and interferon regulatory factor (IRF)-3/IFN-inducible genes in macrophages and dendritic cells (DCs). TICAM-1/TRIF was identified as an adapter that facilitates activation of IRF-3 followed by expression of interferon (IFN)-beta genes in TLR3 signaling, but TICAM-1 does not directly bind TLR4. Although MyD88 and Mal/TIRAP adapters functions downstream of TLR4, DC maturation and IFN-beta induction are independent of MyD88 and Mal/TIRAP. In this investigation, we report the identification of a novel adapter, TICAM-2, that physically bridges TLR4 and TICAM-1 and functionally transmits LPS-TLR4 signaling to TICAM-1, which in turn activates IRF-3. In its structural features, TICAM-2 resembled Mal/TIRAP, an adapter that links TLR2/4 and MyD88. However, TICAM-2 per se exhibited minimal ability to activate NF-kappaB and the IFN-beta promoter. Hence, in LPS signaling TLR4 recruits two types of adapters, TIRAP and TICAM-2, to its cytoplasmic domain that are indirectly connected to two effective adapters, MyD88 and TICAM-1, respectively. We conclude that for LPS-TLR4-mediated activation of IFN-beta, the adapter complex of TICAM-2 and TICAM-1 plays a crucial role. This results in the construction of MyD88-dependent and -independent pathways separately downstream of the two distinct adapters.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Subcellular localization of Toll-like receptor 3 in human dendritic cells.

Toll-like receptor (TLR)3 recognizes dsRNA and transduces signals to activate NF-kappaB and IFN-beta promoter. Type I IFNs (IFN-alpha/beta) function as key cytokines in anti-viral host defense. Human fibroblasts express TLR3 on the cell surface, and anti-TLR3 mAb inhibits dsRNA-induced IFN-beta secretion by fibroblasts, suggesting that TLR3 acts on the cell surface to sense viral infection. In this study, we examined the expression and localization of human TLR3 in various DC subsets using anti-TLR3 mAb. In monocyte-derived immature dendritic cells (iDCs), TLR3 predominantly resided inside the cells but not on the cell surface. iDCs produced IL-12p70 and IFN-alpha and -beta in response to poly(I:C). Similar response was observed in iDCs treated with rotavirus-derived dsRNA. These responses could not be blocked by pretreatment of the cells with anti-TLR3 mAb. In CD11c(+) blood DCs, cytoplasmic retention of TLR3 was also observed as in monocyte-derived iDCs, again endorsing a different TLR3 distribution profile from fibroblasts. In precursor DC2, however, TLR3 could not be detected inside or outside the cells. Of note, there was a putative centrosomal protein that shared an epitope with TLR3 in myeloid DCs and precursor DC2, but not peripheral blood monocytes. Immunoelectron microscopic analysis revealed that TLR3, when stably expressed in the murine B cell line Ba/F3, was specifically accumulated in multivesicular bodies, a subcellular compartment situated in endocytic trafficking pathways. Thus, regulation and localization of TLR3 are different in each cell type, which may reflect participation of cell type-specific multiple pathways in antiviral IFN induction via TLR3.

Animals↗

PubMed2003

TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-beta induction.

Human Toll-like receptor (TLR) 3 recognizes double-stranded (ds) RNA and induces production of interferon (IFN)-beta independent of the adaptor molecules MyD88 and TIRAP. Thus, another adaptor must exist that preferentially mediates TLR3-dependent production of IFN-beta. We have identified an alternative adaptor, designated Toll-interleukin 1 receptor domain (TIR)-containing adaptor molecule (TICAM)-1, that can physically bind the TIR domain of TLR3 and activate the IFN-beta promoter in response to poly(I):poly(C). Thus, dsRNA-TLR3-dependent production of IFN-beta is mediated mainly by TICAM-1. This TICAM-1-dependent pathway may have a role in other TLR-IFN-beta pathways, which form part of the MyD88-independent cellular immune response.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Prediction of the prototype of the human Toll-like receptor gene family from the pufferfish, Fugu rubripes, genome.

The insect Toll family of proteins and their mammalian counterparts seemingly shared one common ancestor and evolved independently. Here we demonstrated that the prototype of the mammalian-type (M-type) Toll family is shared by the fish and humans. According to the draft of the pufferfish Fugu genome project, the signature Toll-IL-1 receptor homology domain (TIR domain) has been conserved during evolution. FuguTLR2, 3, 5, 7, 8 and 9 members correspond structurally to respective mammalian TLRs. One Fugu TLR showed equally high amino acid identity to human TLR1, 6 and 10, and we named it FuguTLR1. Fugu rubripes has genes for TLR21 and 22, which are unique to fish. One possible interpretation of these findings is that TLR1, 2, 3, 4, 5, 7, 8, 9, 21 and 22 existed in the ancestral genome common to fish and mammals, and that TLR4 was lost in the fish lineage, while TLR21 and 22 were lost in the mammalian lineage. Strikingly, a solitary ascidian, Halocynthia roretzi, has only a few Toll-like proteins, which, like Caenorhabditis elegans Toll, represent primitive ones before the expansion of the Toll family. Therefore, the expansion of TLR genes should have occurred earlier than fish, but not C. intestinalis, separated evolutionarily from mammals. These results infer that the appearance of the M-type innate system was completed before or concomitant with the appearance of acquired immunity. We interpret the present data to mean that the differences of TLRs identified in this study between fishes and humans may be rather peripheral, partially due to selection pressure exerted by pathogens in distinct environments.

Amino Acid Sequence↗

PubMed2002

[RNA interference for mammalian cells].

Knock-out of mammalian genes is technically troublesome and time-consuming compared to those of lower animals. RNA interference (RNAi) is a strategy of sequence-specific post-translational gene silencing, and it has been successfully applied for disruption of gene transcripts in C. elegans. Gene silencing by RNAi had not been accomplished in mammalian cells until recently, because bystander activation of DNA-dependent protein kinase (PKR) coincides to disturb gene silencing by long double-stranded RNA (dsRNA), resulting in non-specific repression of translation of many proteins. A breakthrough has come recently with a report suggesting that 20- or 21-bp duplex RNA with 2 bp 3' overhang are made by DICER protein that cleaves long dsRNA during RNAi reactions in vivo and the resultant short dsRNAs mediate RNAi. Those short dsRNAs, namely, small interference RNA (siRNA), barely activate PKR. Using synthesized 21 bp siRNA, T. Tuschl's group has challenged to establish an artificial RNAi method suitable for mammalian cells. Their report was favorable in that siRNA specifically suppressed targeted gene translation in mammalian cells during culture without activation of PKR. Recently K. Taira's group developed the vector-based siRNA expression system by which RNAi is feasible in mammalian cells. Almost all genes can be targeted by RNAi. RNAi methods require minimal time and labor; therefore, mammalian gene knockdown by RNAi will become popular in the near future.

Gene Silencing↗