Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Protein ubiquitination, which is catalyzed by ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, and ubiquitin ligases, is a crucial post-translational modification to regulate numerous cellular functions in a spatio-temporal-specific manner. The human genome encodes ~100 deubiquitinating enzymes (DUBs), which antagonistically regulate the ubiquitin system. OTUD1, an ovarian tumor protease (OTU) family DUB, has an N-terminal-disordered alanine-, proline-, glycine-rich region (APGR), a catalytic OTU domain, and a ubiquitin-interacting motif (UIM). OTUD1 preferentially hydrolyzes lysine-63-linked ubiquitin chains in vitro; however, recent studies indicate that OTUD1 cleaves various ubiquitin linkages, and is involved in the regulation of multiple cellular functions. Thus, OTUD1 predominantly functions as a tumor suppressor by targeting p53, SMAD7, PTEN, AKT, IREB2, YAP, MCL1, and AIF. Furthermore, OTUD1 regulates antiviral signaling, innate and acquired immune responses, and cell death pathways. Similar to Nrf2, OTUD1 contains a KEAP1-binding ETGE motif in its APGR and regulates the reactive oxygen species (ROS)-mediated oxidative stress response and cell death. Importantly, in addition to its association with various cancers, including multiple myeloma, OTUD1 is involved in acute graft-versus-host disease and autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and ulcerative colitis. Thus, OTUD1 is an important DUB as a therapeutic target for a variety of diseases.

DOI： 10.3390/antiox12020350

PubMed

Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

In neurodegenerative diseases such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), the progressive accumulation of ubiquitin-positive cytoplasmic inclusions leads to proteinopathy and neurodegeneration. Along with the seven types of Lys-linked ubiquitin chains, the linear ubiquitin chain assembly complex (LUBAC)-mediated Met1-linked linear ubiquitin chain, which activates the canonical NF-κB pathway, is also involved in cytoplasmic inclusions of tau in AD and TAR DNA-binding protein 43 in ALS. Post-translational modifications, including heterologous ubiquitination, affect proteasomal and autophagic degradation, inflammatory responses, and neurodegeneration. Single nucleotide polymorphisms (SNPs) in SHARPIN and RBCK1 (which encodes HOIL-1L), components of LUBAC, were recently identified as genetic risk factors of AD. A structural biological simulation suggested that most of the SHARPIN SNPs that cause an amino acid replacement affect the structure and function of SHARPIN. Thus, the aberrant LUBAC activity is related to AD. Protein ubiquitination and ubiquitin-binding proteins, such as ubiquilin 2 and NEMO, facilitate liquid-liquid phase separation (LLPS), and linear ubiquitination seems to promote efficient LLPS. Therefore, the development of therapeutic approaches that target ubiquitination, such as proteolysis-targeting chimeras (PROTACs) and inhibitors of ubiquitin ligases, including LUBAC, is expected to be an additional effective strategy to treat neurodegenerative diseases.

DOI： 10.3389/fmolb.2023.1089213

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1186/s40478-022-01486-6

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.3892/ol.2022.13514

PubMed

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

健康人の唾液蛋白質(SP)がアンジオテンシン変換酵素2(ACE2)に結合する能力を評価し、新型コロナウイルス(SARS-CoV-2)スパイク蛋白質S1(S1)受容体結合ドメインとACE2の間の結合に及ぼすSPの影響を測定した。SPはACE2に結合し、S1のACE2への結合を妨害し、S1-ACE2相互作用を阻害するカチオン性ヒストンH2Aと好中球エラスターゼを含む4種類のACE2結合性SPを同定した。ウシ胸腺ヒストン(CTH)もH2Aと同じく効果的に結合を阻害した。CTHはACE2発現宿主細胞へのSARS-CoV-2偽ウイルス性侵入を抑制した。ε-ポリ-L-リジンなどの合成ポリペプチドもS1-ACE2結合を妨害し、ACE2結合におけるSPの重要性が示された。以上より、正荷電のSPはACE2の負荷電表面をクローキングしてSARS-CoV-2の侵入に対する障壁になり、カチオン性ポリペプチドは新型コロナウイルス感染症に対する予防的・治療的手法になり得ると考えられた。

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Saliva contributes to the innate immune system, which suggests that it can prevent SARS-CoV-2 entry. We studied the ability of healthy salivary proteins to bind to angiotensin-converting enzyme 2 (ACE2) using biolayer interferometry and pull-down assays. Their effects on binding between the receptor-binding domain of the SARS-CoV-2 spike protein S1 (S1) and ACE2 were determined using an enzyme-linked immunosorbent assay. Saliva bound to ACE2 and disrupted the binding of S1 to ACE2 and four ACE2-binding salivary proteins were identified, including cationic histone H2A and neutrophil elastase, which inhibited the S1-ACE2 interaction. Calf thymus histone (ct-histone) also inhibited binding as effectively as histone H2A. The results of a cell-based infection assay indicated that ct-histone suppressed SARS-CoV-2 pseudoviral invasion into ACE2-expressing host cells. Manufactured polypeptides, such as ε-poly-L-lysine, also disrupted S1-ACE2 binding, indicating the importance of the cationic properties of salivary proteins in ACE2 binding. Overall, we demonstrated that positively-charged salivary proteins are a barrier against SARS-CoV-2 entry by cloaking the negatively-charged surface of ACE2, and provided a view that the cationic polypeptides represent a preventative and therapeutic treatment against COVID-19.

DOI： 10.1093/jb/mvac054

PubMed

Publishing type：Research paper (scientific journal)  

Abstract

Deubiquitinating enzymes (DUBs) regulate numerous cellular functions by removing ubiquitin modifications. We examined the effects of 88 human DUBs on linear ubiquitin chain assembly complex (LUBAC)-induced NF-κB activation, and identified OTUD1 as a potent suppressor. OTUD1 regulates the canonical NF-κB pathway by hydrolyzing K63-linked ubiquitin chains from NF-κB signaling factors, including LUBAC. OTUD1 negatively regulates the canonical NF-κB activation, apoptosis, and necroptosis, whereas OTUD1 upregulates the interferon (IFN) antiviral pathway. Mass spectrometric analysis showed that OTUD1 binds KEAP1, and the N-terminal intrinsically disordered region of OTUD1, which contains an ETGE motif, is indispensable for the KEAP1-binding. Indeed, OTUD1 is involved in the KEAP1-mediated antioxidant response and reactive oxygen species (ROS)-induced cell death, oxeiptosis. In Otud1^−/−-mice, inflammation, oxidative damage, and cell death were enhanced in inflammatory bowel disease, acute hepatitis, and sepsis models. Thus, OTUD1 is a crucial regulator for the inflammatory, innate immune, and oxidative stress responses and ROS-associated cell death pathways.

DOI： 10.1038/s41419-022-05145-5

PubMed

Other URL： https://www.nature.com/articles/s41419-022-05145-5

Publishing type：Research paper (scientific journal)  

DOI： 10.3390/cells11152398

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Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

DOI： 10.1073/pnas.2202125119.

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.redox.2022.102286

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Pancreatic cancer is a highly challenging malignancy with extremely poor prognosis. Cytoglobin (CYGB), a hemeprotein involved in liver fibrosis and cancer development, is expressed in pericytes of all organs. Here, we examined the role of CYGB in the development of pancreatic cancer. CYGB expression appeared predominately in the area surrounding adenocarcinoma and negatively correlated with tumor size in patients with pancreatic cancer. Directly injecting 7, 12-dimethylbenz[a]anthracene into the pancreatic tail in wild-type mice resulted in time-dependent induction of severe pancreatitis, fibrosis, and oxidative damage, which was rescued by Cygb overexpression in transgenic mice. Pancreatic cancer incidence was 93% in wild-type mice but only 55% in transgenic mice. Enhanced CYGB expression in human pancreatic stellate cells in vitro reduced cellular collagen synthesis, inhibited cell activation, increased expression of antioxidant-related genes, and increased CYGB secretion into the medium. Cygb-overexpressing or recombinant human CYGB (rhCYGB) -treated MIA PaCa-2 cancer cells exhibited dose-dependent cell cycle arrest at the G1 phase, diminished cell migration, and reduction in colony formation. RNA sequencing in rhCYGB-treated MIA PaCa-2 cells revealed downregulation of cell cycle and oxidative phosphorylation pathways. An increase in MIA PaCa-2 cell proliferation and reactive oxygen species production by H2O2 challenge was blocked by rhCYGB treatment or Cygb overexpression. PANC-1, OCUP-A2, and BxPC-3 cancer cells showed similar responses to rhCYGB. Known antioxidants N-acetyl cysteine and glutathione also inhibited cancer cell growth. These results demonstrate that CYGB suppresses pancreatic stellate cell activation, pancreatic fibrosis, and tumor growth, suggesting its potential therapeutic application against pancreatic cancer.

DOI： 10.1038/s41389-022-00389-4

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Ubiquitin regulates numerous aspects of biology via a complex ubiquitin code. The linear ubiquitin chain is an atypical code that forms a unique structure, with the C-terminal tail of the distal ubiquitin linked to the N-terminal Met1 of the proximal ubiquitin. Thus far, LUBAC is the only known ubiquitin ligase complex that specifically generates linear ubiquitin chains. LUBAC-induced linear ubiquitin chains regulate inflammatory responses, cell death and immunity. Genetically modified mouse models and cellular assays have revealed that LUBAC is also involved in embryonic development in mice. LUBAC dysfunction is associated with autoimmune diseases, myopathy, and neurodegenerative diseases in humans, but the underlying mechanisms are poorly understood. In this review, we focus on the roles of linear ubiquitin chains and LUBAC in immune and neurodegenerative diseases. We further discuss LUBAC inhibitors and their potential as therapeutics for these diseases.

DOI： 10.1042/BST20211078

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Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41598-021-02522-6

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

The anti-apoptotic myeloid cell leukemia 1 (MCL1) protein belongs to the pro-survival BCL2 family and is frequently amplified or elevated in human cancers. MCL1 is highly unstable, with its stability being regulated by phosphorylation and ubiquitination. Here, we identify acetylation as another critical post-translational modification regulating MCL1 protein stability. We demonstrate that the lysine acetyltransferase p300 targets MCL1 at K40 for acetylation, which is counteracted by the deacetylase sirtuin 3 (SIRT3). Mechanistically, acetylation enhances MCL1 interaction with USP9X, resulting in deubiquitination and subsequent MCL1 stabilization. Therefore, ectopic expression of acetylation-mimetic MCL1 promotes apoptosis evasion of cancer cells, enhances colony formation potential, and facilitates xenografted tumor progression. We further demonstrate that elevated MCL1 acetylation sensitizes multiple cancer cells to pharmacological inhibition of USP9X. These findings reveal that acetylation of MCL1 is a critical post-translational modification enhancing its oncogenic function and provide a rationale for developing innovative therapeutic strategies for MCL1-dependent tumors.

DOI： 10.1016/j.celrep.2021.109988

PubMed

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

BACKGROUND & AIMS: Anti-fibrotic therapy remains an unmet medical need in human chronic liver disease. We report the anti-fibrotic properties of cytoglobin (CYGB), a respiratory protein expressed in hepatic stellate cells (HSCs), the main cell type involved in liver fibrosis. APPROACH & RESULTS: Cygb-deficient mice which had bile duct ligation (BDL)-induced liver cholestasis or choline-deficient L-amino acid-defined (CDAA) diet-induced steatohepatitis significantly exacerbated liver damage, fibrosis and reactive oxygen species (ROS) formation. All these manifestations were attenuated in Cygb-overexpressing mice. We produced 6His-tagged recombinant human CYGB (His-CYGB), traced its bio-distribution and assessed its function in HSCs or in mice with advanced liver cirrhosis using thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). In cultured HSCs, extracellular His-CYGB was endocytosed and accumulated in endosomes via clathrin-mediated pathway. His-CYGB significantly impeded ROS formation spontaneously or in the presence of ROS inducers in HSCs, thus leading to the attenuation of collagen type I alpha 1 production and alpha-smooth muscle actin expression. Replacement the iron centre of the heme group with cobalt nullified the effect of His-CYGB. In addition, His-CYGB induced interferon-β secretion by HSCs which partly contributed to its anti-fibrotic function. Momelotinib incompletely reversed the effect of His-CYGB. Intravenously injected His-CYGB markedly suppressed liver inflammation, fibrosis and oxidative cell damage in TAA- or DDC-administered mice without adverse effects. RNA-seq analysis revealed the downregulation of inflammation and fibrosis-related genes and the upregulation of antioxidant genes in both cell culture and liver tissues. The injected His-CYGB predominantly localised to HSCs but not to macrophages, suggesting specific targeting effects. His-CYGB exhibited no toxicity in humanised liver chimeric PXB mice. CONCLUSIONS: His-CYGB could have anti-fibrotic clinical applications for human chronic liver diseases.

DOI： 10.1002/hep.31752

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

OBJECTIVE: Histamine derived from mast cells and basophils plays important roles in inducing allergic symptoms. Although T cells also produce histamine, the involvement of the histamine produced from T cells has remained enigmatic. We sought to reveal the roles of T helper 2 (Th2) cell-derived histamine in nasal allergic disorders. METHODS: The histamine production from Th2 cells was measured by EIA. The mRNA expression of histidine decarboxylase (HDC) was measured by real-time PCR. To investigate the roles of Th2 cell-derived histamine in vivo, we analyzed an antigen-specific Th2 cell transfer mouse model. RESULTS: Th2 cells produced histamine by T cell receptor stimulation, and these properties were specific for Th2 cells, but not Th1 cells and naïve CD4 T cells. The histamine produced from Th2 cells was involved in the infiltrations of Th2 cells in response to antigen exposure. CONCLUSION: These results suggest that Th2 cell-derived histamine play important roles in nasal allergic disorders.

DOI： 10.1007/s00011-021-01458-x

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s00011-021-01458-x

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

DOI： 10.1016/j.jaad.2020.05.051

PubMed

Publishing type：Research paper (scientific journal)  

BACKGROUND & AIMS: Anti-fibrotic therapy remains an unmet medical need in human chronic liver disease. We report the anti-fibrotic properties of cytoglobin (CYGB), a respiratory protein expressed in hepatic stellate cells (HSCs), the main cell type involved in liver fibrosis. APPROACH & RESULTS: Cygb-deficient mice which had bile duct ligation (BDL)-induced liver cholestasis or choline-deficient L-amino acid-defined (CDAA) diet-induced steatohepatitis significantly exacerbated liver damage, fibrosis and reactive oxygen species (ROS) formation. All these manifestations were attenuated in Cygb-overexpressing mice. We produced 6His-tagged recombinant human CYGB (His-CYGB), traced its bio-distribution and assessed its function in HSCs or in mice with advanced liver cirrhosis using thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). In cultured HSCs, extracellular His-CYGB was endocytosed and accumulated in endosomes via clathrin-mediated pathway. His-CYGB significantly impeded ROS formation spontaneously or in the presence of ROS inducers in HSCs, thus leading to the attenuation of collagen type I alpha 1 production and alpha-smooth muscle actin expression. Replacement the iron centre of the heme group with cobalt nullified the effect of His-CYGB. In addition, His-CYGB induced interferon-β secretion by HSCs which partly contributed to its anti-fibrotic function. Momelotinib incompletely reversed the effect of His-CYGB. Intravenously injected His-CYGB markedly suppressed liver inflammation, fibrosis and oxidative cell damage in TAA- or DDC-administered mice without adverse effects. RNA-seq analysis revealed the downregulation of inflammation and fibrosis-related genes and the upregulation of antioxidant genes in both cell culture and liver tissues. The injected His-CYGB predominantly localised to HSCs but not to macrophages, suggesting specific targeting effects. His-CYGB exhibited no toxicity in humanised liver chimeric PXB mice. CONCLUSIONS: His-CYGB could have anti-fibrotic clinical applications for human chronic liver diseases.

DOI： 10.1002/hep.31752

PubMed

Publishing type：Research paper (scientific journal)  

Mind bomb 2 (MIB2) is an E3 ligase involved in Notch signalling and attenuates TNF-induced apoptosis through ubiquitylation of receptor-interacting protein kinase 1 (RIPK1) and cylindromatosis. Here we show that MIB2 bound and conjugated K48- and K63-linked polyubiquitin chains to a long-form of cellular FLICE-inhibitory protein (cFLIPL), a catalytically inactive homologue of caspase 8. Deletion of MIB2 did not impair the TNF-induced complex I formation that mediates NF-κB activation but significantly enhanced formation of cytosolic death-inducing signalling complex II. TNF-induced RIPK1 Ser166 phosphorylation, a hallmark of RIPK1 death-inducing activity, was enhanced in MIB2 knockout cells, as was RIPK1 kinase activity-dependent and -independent apoptosis. Moreover, RIPK1 kinase activity-independent apoptosis was induced in cells expressing cFLIPL mutants lacking MIB2-dependent ubiquitylation. Together, these results suggest that MIB2 suppresses both RIPK1 kinase activity-dependent and -independent apoptosis, through suppression of RIPK1 kinase activity and ubiquitylation of cFLIPL, respectively.

DOI： 10.1038/s42003-020-01603-y

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Mind bomb 2 (MIB2) is an E3 ligase involved in Notch signalling and attenuates TNF-induced apoptosis through ubiquitylation of receptor-interacting protein kinase 1 (RIPK1) and cylindromatosis. Here we show that MIB2 bound and conjugated K48- and K63-linked polyubiquitin chains to a long-form of cellular FLICE-inhibitory protein (cFLIPL), a catalytically inactive homologue of caspase 8. Deletion of MIB2 did not impair the TNF-induced complex I formation that mediates NF-κB activation but significantly enhanced formation of cytosolic death-inducing signalling complex II. TNF-induced RIPK1 Ser166 phosphorylation, a hallmark of RIPK1 death-inducing activity, was enhanced in MIB2 knockout cells, as was RIPK1 kinase activity-dependent and -independent apoptosis. Moreover, RIPK1 kinase activity-independent apoptosis was induced in cells expressing cFLIPL mutants lacking MIB2-dependent ubiquitylation. Together, these results suggest that MIB2 suppresses both RIPK1 kinase activity-dependent and -independent apoptosis, through suppression of RIPK1 kinase activity and ubiquitylation of cFLIPL, respectively.

DOI： 10.1038/s42003-020-01603-y

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2020.12.045

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2020.12.045

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.3389/fimmu.2021.635475

PubMed

Publishing type：Research paper (scientific journal)  

Histamine, which is mainly produced by mast cells and basophils, participates in various allergic symptoms, and some studies have reported that macrophages also produce histamine. Moreover, recent studies have revealed that macrophages, especially alternatively activated macrophages (M2) induced by T helper 2 (Th2) cytokines, such as interleukin (IL)-4 and IL-13, participate in the pathogenesis of allergic diseases. The major source of Th2 cytokines is antigen-specific Th2 cells. To elucidate the relationship between histamine, macrophages, and Th2 cells in allergic inflammation, we established a macrophage-Th2 cell co-culture model in vitro and an antigen-specific Th2 cell transfer mouse model of rhinitis. In vitro analyses indicated that macrophages produce histamine by interacting with antigen-specific Th2 cells through the antigen. Furthermore, Th2 cells and macrophages cooperatively elicited rhinitis in the mouse model. We determined that histamine induces Th2- and macrophage-elicited sneezing responses through H1 receptor signaling, whereas it induces nasal eosinophil infiltrations through H4 receptor signaling. Collectively, these results indicate a novel histamine production mechanism by macrophages, in which Th2 cells and macrophages cooperatively induce nasal allergic inflammation through histamine signaling.

DOI： 10.1371/journal.pone.0248158

PubMed

Publishing type：Part of collection (book)  

DOI： 10.1007/978-981-16-4866-3_14

Publishing type：Research paper (scientific journal)  

DOI： 10.15036/arerugi.70.1239

CiNii Article

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Nuclear dot protein 52 kDa (NDP52, also known as CALCOCO2) functions as a selective autophagy receptor. The linear ubiquitin chain assembly complex (LUBAC) specifically generates the N-terminal Met1-linked linear ubiquitin chain, and regulates innate immune responses, such as nuclear factor-κB (NF-κB), interferon (IFN) antiviral, and apoptotic pathways. Although NDP52 and LUBAC cooperatively regulate bacterial invasion-induced xenophagy, their functional crosstalk remains enigmatic. Here we show that NDP52 suppresses canonical NF-κB signaling through the broad specificity of ubiquitin-binding at the C-terminal UBZ domain. Upon TNF-α-stimulation, NDP52 associates with LUBAC through the HOIP subunit, but does not disturb its ubiquitin ligase activity, and has a modest suppressive effect on NF-κB activation by functioning as a component of TNF-α receptor signaling complex I. NDP52 also regulates the TNF-α-induced apoptotic pathway, but not doxorubicin-induced intrinsic apoptosis. A chemical inhibitor of LUBAC (HOIPIN-8) cancelled the increased activation of the NF-κB and IFN antiviral pathways, and enhanced apoptosis in NDP52-knockout and -knockdown HeLa cells. Upon Salmonella-infection, colocalization of Salmonella, LC3, and linear ubiquitin was detected in parental HeLa cells to induce xenophagy. Treatment with HOIPIN-8 disturbed the colocalization and facilitated Salmonella expansion. In contrast, HOIPIN-8 showed little effect on the colocalization of LC3 and Salmonella in NDP52-knockout cells, suggesting that NDP52 is a weak regulator in LUBAC-mediated xenophagy. These results indicate that the crosstalk between NDP52 and LUBAC regulates innate immune responses, apoptosis, and xenophagy.

DOI： 10.3389/fimmu.2021.635475

PubMed

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Histamine, which is mainly produced by mast cells and basophils, participates in various allergic symptoms, and some studies have reported that macrophages also produce histamine. Moreover, recent studies have revealed that macrophages, especially alternatively activated macrophages (M2) induced by T helper 2 (Th2) cytokines, such as interleukin (IL)-4 and IL-13, participate in the pathogenesis of allergic diseases. The major source of Th2 cytokines is antigen-specific Th2 cells. To elucidate the relationship between histamine, macrophages, and Th2 cells in allergic inflammation, we established a macrophage-Th2 cell co-culture model in vitro and an antigen-specific Th2 cell transfer mouse model of rhinitis. In vitro analyses indicated that macrophages produce histamine by interacting with antigen-specific Th2 cells through the antigen. Furthermore, Th2 cells and macrophages cooperatively elicited rhinitis in the mouse model. We determined that histamine induces Th2- and macrophage-elicited sneezing responses through H1 receptor signaling, whereas it induces nasal eosinophil infiltrations through H4 receptor signaling. Collectively, these results indicate a novel histamine production mechanism by macrophages, in which Th2 cells and macrophages cooperatively induce nasal allergic inflammation through histamine signaling.

DOI： 10.1371/journal.pone.0248158

PubMed

Publishing type：Research paper (scientific journal)  

The LUBAC ubiquitin ligase complex, composed of the HOIP, HOIL-1L, and SHARPIN subunits, stimulates the canonical nuclear factor-κB (NF-κB) activation pathways through its Met1-linked linear ubiquitination activity. Here we performed cellular and mathematical modeling analyses of the LUBAC involvement in the T cell receptor (TCR)-mediated NF-κB activation pathway, using the Jurkat human T cell line. LUBAC is indispensable for TCR-induced NF-κB and T cell activation, and transiently associates with and linearly ubiquitinates the CARMA1-BCL10-MALT1 (CBM) complex, through the catalytic HOIP subunit. In contrast, the linear ubiquitination of NEMO, a substrate of the TNF-α-induced canonical NF-κB activation pathway, was limited during the TCR pathway. Among deubiquitinases, OTULIN, but not CYLD, plays a major role in downregulating LUBAC-mediated TCR signaling. Mathematical modeling indicated that linear ubiquitination of the CBM complex accelerates the activation of IκB kinase (IKK), as compared with the activity induced by linear ubiquitination of NEMO alone. Moreover, simulations of the sequential linear ubiquitination of the CBM complex suggested that the allosteric regulation of linear (de)ubiquitination of CBM subunits is controlled by the ubiquitin-linkage lengths. These results indicated that, unlike the TNF-α-induced NF-κB activation pathway, the TCR-mediated NF-κB activation in T lymphocytes has a characteristic mechanism to induce LUBAC-mediated NF-κB activation.

DOI： 10.3389/fimmu.2020.601926

PubMed

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Protein ubiquitinations play pivotal roles in many cellular processes, including homeostasis, responses to various stimulations, and progression of diseases. Deubiquitinating enzymes (DUBs) remove ubiquitin molecules from ubiquitinated proteins and cleave the polyubiquitin chain, thus negatively regulating numerous ubiquitin-dependent processes. Dysfunctions of many DUBs reportedly cause various diseases; therefore, DUBs are considered as important drug targets, although the biochemical characteristics and cellular functions of many DUBs are still unclear. Here, we established a human DUB protein array to detect the activity and linkage specificity of almost all human DUBs. Using a wheat cell-free protein synthesis system, 88 full-length recombinant human DUB proteins were prepared and termed the DUB array. In vitro DUB assays were performed with all of these recombinant DUBs, using eight linkage types of diubiquitins as substrates. As a result, 80 DUBs in the array showed DUB activities, and their linkage specificities were determined. These 80 DUBs included many biochemically uncharacterized DUBs in the past. In addition, taking advantage of these active DUB proteins, we applied the DUB array to evaluate the selectivities of DUB inhibitors. We successfully developed a high-throughput and semi-quantitative DUB assay based on AlphaScreen technology, and a model study using two commercially available DUB inhibitors revealed individual selectivities to 29 DUBs, as previously reported. In conclusion, the DUB array established here is a powerful tool for biochemical analyses and drug discovery for human DUBs.

DOI： 10.3390/biomedicines8060152

PubMed

Publishing type：Research paper (scientific journal)  

Protein ubiquitinations play pivotal roles in many cellular processes, including homeostasis, responses to various stimulations, and progression of diseases. Deubiquitinating enzymes (DUBs) remove ubiquitin molecules from ubiquitinated proteins and cleave the polyubiquitin chain, thus negatively regulating numerous ubiquitin-dependent processes. Dysfunctions of many DUBs reportedly cause various diseases; therefore, DUBs are considered as important drug targets, although the biochemical characteristics and cellular functions of many DUBs are still unclear. Here, we established a human DUB protein array to detect the activity and linkage specificity of almost all human DUBs. Using a wheat cell-free protein synthesis system, 88 full-length recombinant human DUB proteins were prepared and termed the DUB array. In vitro DUB assays were performed with all of these recombinant DUBs, using eight linkage types of diubiquitins as substrates. As a result, 80 DUBs in the array showed DUB activities, and their linkage specificities were determined. These 80 DUBs included many biochemically uncharacterized DUBs in the past. In addition, taking advantage of these active DUB proteins, we applied the DUB array to evaluate the selectivities of DUB inhibitors. We successfully developed a high-throughput and semi-quantitative DUB assay based on AlphaScreen technology, and a model study using two commercially available DUB inhibitors revealed individual selectivities to 29 DUBs, as previously reported. In conclusion, the DUB array established here is a powerful tool for biochemical analyses and drug discovery for human DUBs.

DOI： 10.3390/biomedicines8060152

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jaad.2020.05.051

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

The linear ubiquitin chain assembly complex (LUBAC) is a ubiquitin ligase composed of the Heme-oxidized IRP2 ubiquitin ligase-1L (HOIL-1L), HOIL-1L-interacting protein (HOIP), and Shank-associated RH domain interactor (SHARPIN) subunits. LUBAC specifically generates the N-terminal Met1-linked linear ubiquitin chain and regulates acquired and innate immune responses, such as the canonical nuclear factor-κB (NF-κB) and interferon antiviral pathways. Deubiquitinating enzymes, OTULIN and CYLD, physiologically bind to HOIP and control its function by hydrolyzing the linear ubiquitin chain. Moreover, proteins containing linear ubiquitin-specific binding domains, such as NF-κB-essential modulator (NEMO), optineurin, A20-binding inhibitors of NF-κB (ABINs), and A20, modulate the functions of LUBAC, and the dysregulation of the LUBAC-mediated linear ubiquitination pathway induces cancer and inflammatory, autoimmune, and neurodegenerative diseases. Therefore, inhibitors of LUBAC would be valuable to facilitate investigations of the molecular and cellular bases for LUBAC-mediated linear ubiquitination and signal transduction, and for potential therapeutic purposes. We identified and characterized α,β-unsaturated carbonyl-containing chemicals, named HOIPINs (HOIP inhibitors), as LUBAC inhibitors. We summarize recent advances in elucidations of the pathophysiological functions of LUBAC-mediated linear ubiquitination and identifications of its regulators, toward the development of LUBAC inhibitors.

DOI： 10.3390/ijms21093381

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Publishing type：Research paper (scientific journal)  

DOI： 10.3390/ijms21093381

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

The NF-κB and interferon antiviral signaling pathways play pivotal roles in inflammatory and innate immune responses. The LUBAC ubiquitin ligase complex, composed of the HOIP, HOIL-1L, and SHARPIN subunits, activates the canonical NF-κB pathway through Met1-linked linear ubiquitination. We identified small-molecule chemical inhibitors of LUBAC, HOIPIN-1 and HOIPIN-8. Here we show that HOIPINs down-regulate not only the proinflammatory cytokine-induced canonical NF-κB pathway, but also various pathogen-associated molecular pattern-induced antiviral pathways. Structural analyses indicated that HOIPINs inhibit the RING-HECT-hybrid reaction in HOIP by modifying the active Cys885, and residues in the C-terminal LDD domain, such as Arg935 and Asp936, facilitate the binding of HOIPINs to LUBAC. HOIPINs effectively induce cell death in activated B cell-like diffuse large B cell lymphoma cells, and alleviate imiquimod-induced psoriasis in model mice. These results reveal the molecular and cellular bases of LUBAC inhibition by HOIPINs, and demonstrate their potential therapeutic uses.

DOI： 10.1038/s42003-020-0882-8

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DOI： 10.1016/j.bbrc.2019.12.049

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Publishing type：Research paper (scientific journal)  

DOI： 10.1093/jnen/nlz135

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Neuronal cytoplasmic inclusions (NCIs) containing TAR DNA-binding protein of 43 kDa (TDP-43) are pathological hallmarks of amyotrophic lateral sclerosis (ALS) and are known to be ubiquitinated. Eight linkage types of polyubiquitin chains have been reported, each type of chain exerting different intracellular actions. The linkage type of polyubiquitin chain involved in the formation of NCIs in sporadic ALS (sALS), however, has not yet been elucidated. We performed immunohistochemical study of the spinal cords of 12 patients with sALS and on those of 6 control subjects. Virtually all ubiquitinated NCIs were immunolabeled with lysine 48-linked polyubiquitin chain (K48-Ub). Although the majority of NCIs were triple-immunoreactive for K48-Ub, linear polyubiquitin chain (L-Ub), and lysine 63-linked polyubiquitin chain (K63-Ub), thin parts of K48-Ub-immunopositive NCIs were not labeled for K63-Ub or L-Ub. We also detected HOIP and SHARPIN, components of linear ubiquitin chain assembly complex, colocalizing with L-Ub on NCIs. Moreover, the immunosignal of optineurin, an autophagy receptor working with L-Ub, and that of activated NF-κB p65, were observed to be colocalizing with L-Ub on certain parts of NCIs. The L-Ub modification of TDP-43-positive NCIs may function as an inducer of autophagic clearance of NCIs, neuroinflammation, and neurodegeneration in sALS.

DOI： 10.1093/jnen/nlz135

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.14952/seikagaku.2020.920064

CiNii Article

Publishing type：Research paper (scientific journal)  

DOI： 10.14952/seikagaku.2020.920028

CiNii Article

Publishing type：Research paper (scientific journal)   Kind of work：Single Work  

ユビキチンは,タンパク質に結合して選択的にプロテアソーム分解へ導く目印として発見されたが,ユビキチン分子間で多様な連結を形成することで分解以外にも数多くの生理機能制御を担うことが明らかになってきた.本稿では,我々が見出した直鎖状ユビキチン鎖を生成する酵素（LUBAC，linear ubiquitin chain assembly complex)の生理機能と疾患との連関,および創薬を目指した阻害剤開発の現状を紹介したい.

DOI： 10.14894/faruawpsj.56.1_26

CiNii Article

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

The LUBAC ubiquitin ligase complex, composed of the HOIP, HOIL-1L, and SHARPIN subunits, stimulates the canonical nuclear factor-κB (NF-κB) activation pathways through its Met1-linked linear ubiquitination activity. Here we performed cellular and mathematical modeling analyses of the LUBAC involvement in the T cell receptor (TCR)-mediated NF-κB activation pathway, using the Jurkat human T cell line. LUBAC is indispensable for TCR-induced NF-κB and T cell activation, and transiently associates with and linearly ubiquitinates the CARMA1-BCL10-MALT1 (CBM) complex, through the catalytic HOIP subunit. In contrast, the linear ubiquitination of NEMO, a substrate of the TNF-α-induced canonical NF-κB activation pathway, was limited during the TCR pathway. Among deubiquitinases, OTULIN, but not CYLD, plays a major role in downregulating LUBAC-mediated TCR signaling. Mathematical modeling indicated that linear ubiquitination of the CBM complex accelerates the activation of IκB kinase (IKK), as compared with the activity induced by linear ubiquitination of NEMO alone. Moreover, simulations of the sequential linear ubiquitination of the CBM complex suggested that the allosteric regulation of linear (de)ubiquitination of CBM subunits is controlled by the ubiquitin-linkage lengths. These results indicated that, unlike the TNF-α-induced NF-κB activation pathway, the TCR-mediated NF-κB activation in T lymphocytes has a characteristic mechanism to induce LUBAC-mediated NF-κB activation.

DOI： 10.3389/fimmu.2020.601926

PubMed

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

ユビキチンは,タンパク質に結合して選択的にプロテアソーム分解へ導く目印として発見されたが,ユビキチン分子間で多様な連結を形成することで分解以外にも数多くの生理機能制御を担うことが明らかになってきた.本稿では,我々が見出した直鎖状ユビキチン鎖を生成する酵素（LUBAC，linear ubiquitin chain assembly complex)の生理機能と疾患との連関,および創薬を目指した阻害剤開発の現状を紹介したい.

DOI： 10.14894/faruawpsj.56.1_26

CiNii Article

Publishing type：Research paper (scientific journal)  

脱ユビキチン化酵素(DUB)は,タンパク質分解やシグナル伝達の活性化などのユビキチン化修飾を介した細胞応答を負に制御する重要な因子として注目されている.また,DUBの高発現や異常な活性化が,がんや神経疾患などを誘導することが明らかとなっており,創薬ターゲットとしても注目されている.我々はコムギ無細胞系を用いてヒトのDUBの約8割を網羅する83種類の組換えタンパク質を合成し,DUBタンパク質アレイを作製した.このDUBタンパク質アレイについて,8種類の重合様式の二量体ユビキチンを基質として,76種類のDUBについて活性を検出し,そのユビキチン鎖特異性を明らかにした.さらに,このDUBタンパク質アレイを応用することで,DUBを阻害する化合物のDUB特異性評価パネルを作製した.(著者抄録)

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2019.12.049

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.RA119.010119

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

The tumor suppressor CYLD is a deubiquitinating enzyme that suppresses polyubiquitin-dependent signaling pathways, including the proinflammatory and cell growth-promoting NF-κB pathway. Missense mutations in the CYLD gene are present in individuals with syndromes such as multiple familial trichoepithelioma (MFT), but the pathogenic roles of these mutations remain unclear. Recent studies have shown that CYLD interacts with a RING finger domain protein, mind bomb homologue 2 (MIB2), in the regulation of NOTCH signaling. However, whether MIB2 is an E3 ubiquitin ligase that acts on CYLD is unknown. Here, using the cell-free-based AlphaScreen and pulldown assays to detect protein-protein interactions, along with immunofluorescence assays and murine Mib2 knockout cells and animals, we demonstrate that MIB2 promotes proteasomal degradation of CYLD and enhances NF-κB signaling. Of note, arthritic inflammation was suppressed in Mib2-deficient mice. We further observed that the ankyrin repeat in MIB2 interacts with the third CAP domain in CYLD and that MIB2 catalyzes Lys-48-linked polyubiquitination of CYLD at Lys-338 and Lys-530. MIB2-dependent CYLD degradation activated NF-κB signaling via tumor necrosis factor alpha (TNFα) stimulation and the linear ubiquitination assembly complex (LUBAC). Mib2-knockout mice had reduced serum interleukin-6 (IL-6) and exhibited suppressed inflammatory responses in the K/BxN serum-transfer arthritis model. Interestingly, MIB2 significantly enhanced the degradation of a CYLDP904L variant identified in an individual with MFT, although the molecular pathogenesis of the disease was not clarified here. Together, these results suggest that MIB2 enhances NF-κB signaling in inflammation by promoting the ubiquitin-dependent degradation of CYLD.

DOI： 10.1074/jbc.RA119.010119

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Characteristic neuropathological structures observed in Alzheimer's disease, such as neurofibrillary tangles and dystrophic neurites of senile plaques, are universally ubiquitinated. Eight linkage types of polyubiquitin chains are known, and each type of chain exerts different intracellular action. Among them, linear polyubiquitin chain was recently reported to be associated with the pathomechanism of neuronal cell death. We therefore generated a novel antibody that specifically recognizes linear polyubiquitin chain. We immunohistochemically examined the brains of patients with Alzheimer's disease. A subset of neurofibrillary tangles, dystrophic neurites of senile plaques, and neuropil threads were evident to be immunopositive for linear polyubiquitin chains, but their number was fewer than those recognized by the antibody against K48-linked polyubiquitin chains. Double immunofluorescence investigation showed that in certain neurofibrillary tangles, a part of K48-linked polyubiquitin-immunopositive structures were immunolabeled for linear polyubiquitin chains. Our results imply that linear polyubiquitination follows K48-linked polyubiquitination in abnormally accumulated tau proteins in Alzheimer's disease, and imply involvement in its neurodegeneration.

DOI： 10.1016/j.neulet.2019.03.017

PubMed

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

DOI： 10.1016/j.neulet.2019.03.017

PubMed

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

DOI： 10.1016/j.bbrc.2018.12.164

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2018.12.164

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1177/2472555218793066

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1177/2472555218793066

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1038/s41467-018-06922-7

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

ENPP1 (Ecto-nucleotide pyrophosphatase phosphodiesterase 1), a type II transmembrane glycoprotein, hydrolyzes ATP to produce AMP and diphosphate, thereby inhibiting bone mineralization. A recent study showed that ENPP1 also preferentially hydrolyzes 2'3'-cGAMP (cyclic GMP-AMP) but not its linkage isomer 3'3'-cGAMP, and negatively regulates the cGAS-STING pathway in the innate immune system. Here, we present the high-resolution crystal structures of ENPP1 in complex with 3'3'-cGAMP and the reaction intermediate pA(3',5')pG. The structures revealed that the adenine and guanine bases of the dinucleotides are recognized by nucleotide- and guanine-pockets, respectively. Furthermore, the structures indicate that 2'3'-cGAMP, but not 3'3'-cGAMP, binds to the active site in a conformation suitable for catalysis, thereby explaining the specific degradation of 2'3'-cGAMP by ENPP1. Our findings provide insights into how ENPP1 hydrolyzes both ATP and cGAMP to participate in the two distinct biological processes.

DOI： 10.1038/s41467-018-06922-7

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1089/mab.2018.0023

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1089/mab.2018.0023

PubMed

Publishing type：Research paper (scientific journal)  

KIT遺伝子はまだら症の原因遺伝子であり、これまでに60以上の変異が同定されているが、変異KIT蛋白質の機能と細胞内動態については十分に解明されていないことから、遺伝子解析とin vivo研究を行った。まだら症の発端者である3歳の日本人女児とその家族(両親、母方の祖母・祖父)の末梢血細胞を用いてKIT遺伝子の遺伝子解析を行った。さらに、野生型または変異KIT遺伝子でトランスフェクトしたHEK293T細胞における変異KIT蛋白質の細胞内局在を解析した。遺伝子解析により、KITの細胞外ドメインにおいて、Val216およびSer217のインフレーム欠失をもたらす新規ヘテロ接合変異c.645_650delTGTGTCが明らかになった。免疫沈降分析により、幹細胞因子(SCF)による処理後に野生型および変異KITがヘテロ二量体を形成することが確認されたが、下流のシグナル伝達因子のリン酸化は減少した。免疫蛍光研究では、変異KITは主に小胞体に蓄積し、細胞表面上にまばらに発現していることが示された。グリコシダーゼ消化研究により、変異KITは主にERに小胞体に局在することが明らかになった。

Publishing type：Research paper (scientific journal)  

Background: Piebaldism is a pigmentary disorder characterized by a white forelock and depigmented patches. Although the loss-of-function mutations in the KIT gene underlie the disease, the intracellular dynamics of the mutant KIT are largely unknown. We herein report a Japanese family with piebaldism in which the affected members showed a mild phenotype. Objective: The objective of this study is to investigate the functions and intracellular dynamics of the mutant KIT protein. Methods: We performed genetic analyses of the KIT gene using peripheral blood cells. We analyzed the intracellular localization of the mutant KIT protein in HEK293T cells transfected with wild-type (Wt) and/or mutant KIT genes. Immunoprecipitation analyses, immunoblotting and immunofluorescence studies were performed using antibodies against KIT and downstream signaling proteins. Glycosidase digestion analysis was performed to clarify the intracellular localization of KIT protein. Results: A genetic analysis revealed a novel heterozygous mutation c.645_650delTGTGTC which results in the in-frame deletion of Val216 and Ser217 in the extracellular domain of KIT. Immunoprecipitation analyses confirmed that the wild and mutant KIT formed a heterodimer after treatment with stem cell factor (SCF)
however, the phosphorylation of the downstream signaling factors was decreased. In an immunofluorescence study, the mutant KIT accumulated predominantly in the endoplasmic reticulum (ER) and was sparsely expressed on the cell surface. A glycosidase digestion study revealed that the mutant KIT is predominantly localized in the ER. Conclusion: These data reveal an aberrant function and intracellular localization of mutant KIT protein in piebaldism.

DOI： 10.1016/j.jdermsci.2018.03.012

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1089/mab.2018.0005

PubMed

Publishing type：Research paper (scientific journal)  

DAZ interacting zinc finger 3 (DZIP3), an RNA-binding RING-type ubiquitin ligase, has been reported to be involved in multiple physiological functions, including the regulation of chemokine- or estradiol-induced gene expression, self-renewal, and maintaining pluripotency in mouse embryonic stem cells. However, the precise cellular functions of DZIP3 remain elusive. In this study, we report the establishment of DZIP3-specific monoclonal antibodies (MAbs), using the rat medial iliac lymph node method. In immunoblotting analyses, our antibodies detected endogenous human and mouse DZIP3. In addition, immunoprecipitation analyses revealed the availability of these antibodies for human or mouse DZIP3. Thus, these MAbs will be available to elucidate cellular functions of DZIP3.

DOI： 10.1089/mab.2018.0005

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1111/1346-8138.14149

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1111/1346-8138.14149

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jdermsci.2018.03.012

PubMed

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

DOI： 10.1111/bjd.15342

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

DOI： 10.1111/bjd.15342

PubMed

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/J.bbrc.2017.02.040

PubMed

Publishing type：Research paper (scientific journal)  

NF-kappa B is crucial to regulate immune and inflammatory responses and cell survival. LUBAC generates a linear ubiquitin chain and activates NF-kappa B through ubiquitin ligase (E3) activity in the HOIP subunit. Here, we show that HOIP is predominantly cleaved by caspase at Asp390 upon apoptosis, and that is subjected to proteasomal degradation. We identified that FADD, as well as NEMO, is a substrate for LUBAC. Although the C-terminal fragment of HOIP retains NF-kappa B activity, linear ubiquitination of NEMO and FADD decreases upon apoptosis. Moreover, the N-terminal fragment of HOIP binds with deubiquitinases, such as OTULIN and CYLD-SPATA2. These results indicate that caspase-mediated cleavage of HOIP divides critical functional regions of HOIP, and that this regulates linear (de)ubiquitination of substrates upon apoptosis. (C) 2017 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2017.02.040

PubMed

Publishing type：Research paper (scientific journal)  

Linear ubiquitin chain assembly complex (LUBAC), composed of SHARPIN (SHANK-associated RH domain-interacting protein), HOIL-1L (longer isoform of heme-oxidized iron-regulatory protein 2 ubiquitin ligase-1), and HOIP (HOIL-1L interacting protein), forms linear ubiquitin on nuclear factor-B (NF-B) essential modulator (NEMO) and induces NF-B pathway activation. SHARPIN expression and LUBAC formation were significantly reduced in the livers of mice 24 h after the injection of either carbon tetrachloride (CCl4) or acetaminophen (APAP), both of which produced the fulminant hepatitis phenotype. To elucidate its pathological significance, hepatic SHARPIN expression was suppressed in mice by injecting shRNA adenovirus via the tail vein. Seven days after this transduction, without additional inflammatory stimuli, substantial inflammation and fibrosis with enhanced hepatocyte apoptosis occurred in the livers. A similar but more severe phenotype was observed with suppression of HOIP, which is responsible for the E3 ligase activity of LUBAC. Furthermore, in good agreement with these in vivo results, transduction of Hepa1-6 hepatoma cells with SHARPIN, HOIL-1L, or HOIP shRNA adenovirus induced apoptosis of these cells in response to tumor necrosis factor- (TNF) stimulation. Thus, LUBAC is essential for the survival of hepatocytes, and it is likely that reduction of LUBAC is a factor promoting hepatocyte death in addition to the direct effect of drug toxicity.

DOI： 10.3390/ijms18020326

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.3390/ijms18020326

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.ppat.1006162

PubMed

Publishing type：Research paper (scientific journal)  

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is crucial for the development of adult T-cell leukemia (ATL), a highly malignant CD4(+) T cell neoplasm. Among the multiple aberrant Tax-induced effects on cellular processes, persistent activation of transcription factor NF-kappa B,which is activated only transiently upon physiological stimulation, is essential for leukemogenesis. We and others have shown that Tax induces activation of the I kappa B kinase (IKK) complex, which is a critical step in NF-kappa B activation, by generating Lys63-linked polyubiquitin chains. However, the molecular mechanism underlying Tax-induced IKK activation is controversial and not fully understood. Here, we demonstrate that Tax recruits linear (Met1-linked) ubiquitin chain assembly complex (LUBAC) to the IKK complex and that Tax fails to induce IKK activation in cells that lack LUBAC activity. Mass spectrometric analyses revealed that both Lys63-linked and Met1-linked polyubiquitin chains are associated with the IKK complex. Furthermore, treatment of the IKK-associated polyubiquitin chains with Met1-linked-chain-specific deubiquitinase (OTULIN) resulted in the reduction of high molecular weight polyubiquitin chains and the generation of short Lys63-linked ubiquitin chains, indicating that Tax can induce the generation of Lys63-and Met1-linked hybrid polyubiquitin chains. We also demonstrate that Tax induces formation of the active macromolecular IKK complex and that the blocking of Tax-induced polyubiquitin chain synthesis inhibited formation of the macromolecular complex. Taken together, these results lead us to propose a novel model in which the hybrid-chain-dependent oligomerization of the IKK complex triggered by Tax leads to trans-autophosphorylation- mediated IKK activation.

DOI： 10.1371/journal.ppat.1006162

PubMed

Publishing type：Research paper (scientific journal)   Kind of work：Single Work  

LRBA(LPS-Responsive and Beige-like Anchor protein)は炎症・免疫制御に関わるタンパク質であり、その欠損がB細胞に機能不全をきたし、抗体産生低下を主徴とする分類不能型免疫不全(CVID)を発症するのではないかと考え検討を行った。CRISPR/Cas9法を用いてLrba遺伝子をノックアウトしたマウス胎児性線維芽細胞(MEF)を構築し、種々の刺激に対するシグナル応答性を評価した。各種自然・獲得免疫刺激によって惹起するシグナル伝達経路活性化への影響を野生型MEFとLrba-/-MEF間で比較した。その結果、polyI:C刺激で誘導されるIFN産生経路がLrba-/-MEFでは亢進していることが明らかとなり、Lrbaは生理的にはIFN産生経路を抑制することが示された。次いで、CRISPR/Cas9法を用いてLrba-KOマウスを作製し、Lrba mRNAの発現を解析したところ、Lrbaは各臓器で普遍的に発現しているが、免疫応答に関わる組織では特に発現レベルが高い可能性が示唆された。

Publishing type：Research paper (scientific journal)  

LRBA(LPS-Responsive and Beige-like Anchor protein)は炎症・免疫制御に関わるタンパク質であり、その欠損がB細胞に機能不全をきたし、抗体産生低下を主徴とする分類不能型免疫不全(CVID)を発症するのではないかと考え検討を行った。CRISPR/Cas9法を用いてLrba遺伝子をノックアウトしたマウス胎児性線維芽細胞(MEF)を構築し、種々の刺激に対するシグナル応答性を評価した。各種自然・獲得免疫刺激によって惹起するシグナル伝達経路活性化への影響を野生型MEFとLrba-/-MEF間で比較した。その結果、polyI:C刺激で誘導されるIFN産生経路がLrba-/-MEFでは亢進していることが明らかとなり、Lrbaは生理的にはIFN産生経路を抑制することが示された。次いで、CRISPR/Cas9法を用いてLrba-KOマウスを作製し、Lrba mRNAの発現を解析したところ、Lrbaは各臓器で普遍的に発現しているが、免疫応答に関わる組織では特に発現レベルが高い可能性が示唆された。

CiNii Article

Publishing type：Research paper (scientific journal)  

LRBA(LPS-Responsive and Beige-like Anchor protein)は炎症・免疫制御に関わるタンパク質であり、その欠損がB細胞に機能不全をきたし、抗体産生低下を主徴とする分類不能型免疫不全(CVID)を発症するのではないかと考え検討を行った。CRISPR/Cas9法を用いてLrba遺伝子をノックアウトしたマウス胎児性線維芽細胞(MEF)を構築し、種々の刺激に対するシグナル応答性を評価した。各種自然・獲得免疫刺激によって惹起するシグナル伝達経路活性化への影響を野生型MEFとLrba-/-MEF間で比較した。その結果、polyI:C刺激で誘導されるIFN産生経路がLrba-/-MEFでは亢進していることが明らかとなり、Lrbaは生理的にはIFN産生経路を抑制することが示された。次いで、CRISPR/Cas9法を用いてLrba-KOマウスを作製し、Lrba mRNAの発現を解析したところ、Lrbaは各臓器で普遍的に発現しているが、免疫応答に関わる組織では特に発現レベルが高い可能性が示唆された。

Publishing type：Research paper (scientific journal)  

DOI： 10.1038/srep34756

PubMed

Publishing type：Research paper (scientific journal)  

In the innate immune system, pattern recognition receptors (PRRs) specifically recognize ligands derived from bacteria or viruses, to trigger the responsible downstream pathways. DEAD box protein 41 (DDX41) is an intracellular PRR that triggers the downstream pathway involving the adapter STING, the kinase TBK1, and the transcription factor IRF3, to activate the type I interferon response. DDX41 is unique in that it recognizes two different ligands; i.e., double-stranded DNA (dsDNA) and cyclic dinucleotides (CDN), via its DEAD domain. However, the structural basis for the ligand recognition by the DDX41 DEAD domain has remained elusive. Here, we report two crystal structures of the DDX41 DEAD domain in apo forms, at 1.5 and 2.2 angstrom resolutions. A comparison of the two crystal structures revealed the flexibility in the ATP binding site, suggesting its formation upon ATP binding. Structure-guided functional analyses in vitro and in vivo demonstrated the overlapped binding surface for dsDNA and CDN, which is distinct from the ATP-binding site. We propose that the structural rearrangement of the ATP binding site is crucial for the release of ADP, enabling the fast turnover of DDX41 for the dsDNA/CDN-induced STING activation pathway.

DOI： 10.1038/srep34756

PubMed

Publishing type：Research paper (scientific journal)  

Optineurin (OPTN) mutations cause neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and glaucoma. Although the ALS-associated E478G mutation in the UBAN domain of OPTN reportedly abolishes its NF-kappa B suppressive activity, the precise molecular basis in ALS pathogenesis still remains unclear. Here we report that the OPTN-UBAN domain is crucial for NF-kappa B suppression. Our crystal structure analysis reveals that OPTN-UBAN binds linear ubiquitin with homology to NEMO. TNF-alpha-mediated NF-kappa B activation is enhanced in OPTN-knockout cells, through increased ubiquitination and association of TNF receptor (TNFR) complex I components. Furthermore, OPTN binds caspase 8, and OPTN deficiency accelerates TNF-alpha-induced apoptosis by enhancing complex II formation. Immunohistochemical analyses of motor neurons from OPTN-associated ALS patients reveal that linear ubiquitin and activated NF-kappa B are partially co-localized with cytoplasmic inclusions, and that activation of caspases is elevated. Taken together, OPTN regulates both NF-kappa B activation and apoptosis via linear ubiquitin binding, and the loss of this ability may lead to ALS.

DOI： 10.1038/ncomms12547

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1038/ncomms12547

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1038/leu.2015.267

Publishing type：Research paper (scientific journal)  

The Skp1-Cul1-F-box protein (SCF) complex catalyzes protein ubiquitination in diverse cellular processes and is one of the best-characterized ubiquitin ligases. F-box proteins determine the substrate specificities of SCF ubiquitin ligases. Among these, Fbs1/FBG1/FBXO2, Fbs2/FBG2/FBXO6, and Fbs3/FBG5/FBXO27 recognize the N-glycans of glycoproteins, whereas FBG3/FBXO44 has no sugar-binding activity, despite the high sequence homology and conservation of the residues necessary for oligosaccharide binding between Fbs1-3 and FBG3. Here we determined the crystal structure of the Skp1-FBG3 complex at a resolution of 2.6 angstrom. The substrate-binding domain of FBG3 is composed of a 10-stranded antiparallel beta-sandwich with three helices. Although the overall structure of FBG3 is similar to that of Fbs1, the residues that form the Fbs1 carbohydrate-binding pocket failed to be superposed with the corresponding residues of FBG3. Structure-based mutational analysis shows that distinct hydrogen bond networks of four FBG3 loops, i.e., beta 2-beta 3, beta 5-beta 6, beta 7-beta 8, and beta 9-beta 10, prevent the formation of the carbohydrate-binding pocket shown in Fbs1.

DOI： 10.1371/journal.pone.0140366

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1038/jid.2015.12

PubMed

Publishing type：Research paper (scientific journal)  

Monilethrix is a hair shaft anomaly characterized by beaded hair with periodic changes in hair thickness. Mutations in the desmoglein 4 (DSG4) gene reportedly underlie the autosomal recessive form of the disease. However, the pathogenesis and cellular basis for the DSG4 mutation induced monilethrix remained largely unknown. We report a Japanese female patient with monilethrix. Observation of her hair shaft by means of transmission electron microscopy showed fewer desmosomes and abnormal keratinization. Genetic analysis revealed a homozygous mutation, c.2119delG (p.Asp707Ilefs*109), in the DSG4 gene, which was predicted to cause a frameshift and premature termination in the intracellular region of the DSG4 protein. The mutation has not been reported previously. In the patient's hair shaft, we detected reduced but partial expression of the mutant DSG4 protein. Cellular analyses demonstrated that the mutant DSG4 lost its affinity to plakoglobin and accumulated in the endoplasmic reticulum (ER). The amounts of mutant DSG4 were increased by proteasome inhibitor treatment, and the expression of an ER chaperone, GRP78/BiP, was elevated in the patient's skin. Collectively, these results suggest that the dysfunctional mutated DSG4, tethered in the ER, undergoes ER-associated degradation, leading to unfolded protein response induction, and thus ER stress may have a role in the pathogenesis of monilethrix.

DOI： 10.1038/jid.2015.12

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1038/nsmb.2970

PubMed

Publishing type：Research paper (scientific journal)  

The tumor suppressor CYLD belongs to a ubiquitin (Ub)-specific protease (USP) family and specifically cleaves Met1- and Lys63-linked polyubiquitin chains to suppress inflammatory signaling pathways. Here, we report crystal structures representing the catalytic states of zebrafish CYLD for Met1- and Lys63-linked Ub chains and two distinct precatalytic states for Met1-linked chains. In both catalytic states, the distal Ub is bound to CYLD in a similar manner, and the scissile bond is located close to the catalytic residue, whereas the proximal Ub is bound in a manner specific to Met1- or Lys63-linked chains. Further structure-based mutagenesis experiments support the mechanism by which CYLD specifically cleaves both Met1- and Lys63-linked chains and provide insight into tumor-associated mutations of CYLD. This study provides new structural insight into the mechanisms by which USP family deubiquitinating enzymes recognize and cleave Ub chains with specific linkage types.

DOI： 10.1038/nsmb.2970

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1155/2015/125380

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pone.0140366

PubMed

Publishing type：Research paper (scientific journal)  

Nonalcoholic steatohepatitis (NASH) is a disorder characterized by hepatic lipid accumulation followed by the inflammation-induced death of hepatocytes and fibrosis. In this process, oxidative stress contributes to the induction of several inflammatory cytokines including TNF-alpha and IL-1 beta in macrophages, while, in hepatocytes, NF-kappa B reportedly induces the expressions of cell survival genes for protection from apoptosis. Recently, it was reported that the new ubiquitin ligase complex termed linear ubiquitin chain assembly complex (LUBAC), composed of SHARPIN (SHANK-associated RH domain-interacting protein), HOIL-1L (longer isoform of heme-oxidized iron-regulatory protein 2 ubiquitin ligase-1), and HOIP (HOIL-1L interacting protein), forms linear ubiquitin on NF-kappa B essential modulator (NEMO) and thereby induces NF-kappa B pathway activation. In this study, we demonstrated the formation of LUBAC to be impaired in the livers of NASH rodent models produced by methionine and choline deficient (MCD) diet feeding, first by either gel filtration or Blue Native-PAGE, with subsequent confirmation by western blotting. The reduction of LUBAC is likely to be attributable to markedly reduced expression of SHARPIN, one of its components. Thus, impaired LUBAC formation, which would result in insufficient NF-kappa B activation, may be one of the molecular mechanisms underlying the enhanced apoptotic response of hepatocytes in MCD diet-induced NASH livers.

DOI： 10.1155/2015/125380

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Publishing type：Research paper (scientific journal)  

DOI： 10.1007/978-4-431-55561-2_12

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)   Kind of work：Single Work  

B細胞リンパ腫におけるユビキチン系の寄与について検討した。NF-κB経路の制御に関わることが報告されていたOTU型のA20(TNFAIP3)とCezanne、USP型のCYLDに着目して抑制能を解析した。A20とCYLDは発現量を増加させるとユビキチンリガーゼ(LUBAC)発現によるNF-κB活性化を強く阻害するが、Cezanneは抑制効果が低かった。CYLDは脱ユビキチン化酵素活性依存的にLUBACによるNF-κB活性化を抑制した。A20がZF7を介して直鎖状ユビキチンに特異的に結合することでNF-κB活性を制御することが示唆された。A20はZF7の直鎖状ユビキチン結合能を介して受容体近傍に集積していること、その不全はNF-κB活性の持続的亢進となりB細胞リンパ腫を発症させることが示唆された。

Publishing type：Research paper (scientific journal)  

B細胞リンパ腫におけるユビキチン系の寄与について検討した。NF-κB経路の制御に関わることが報告されていたOTU型のA20(TNFAIP3)とCezanne、USP型のCYLDに着目して抑制能を解析した。A20とCYLDは発現量を増加させるとユビキチンリガーゼ(LUBAC)発現によるNF-κB活性化を強く阻害するが、Cezanneは抑制効果が低かった。CYLDは脱ユビキチン化酵素活性依存的にLUBACによるNF-κB活性化を抑制した。A20がZF7を介して直鎖状ユビキチンに特異的に結合することでNF-κB活性を制御することが示唆された。A20はZF7の直鎖状ユビキチン結合能を介して受容体近傍に集積していること、その不全はNF-κB活性の持続的亢進となりB細胞リンパ腫を発症させることが示唆された。

CiNii Article

Publishing type：Research paper (scientific journal)  

B細胞リンパ腫におけるユビキチン系の寄与について検討した。NF-κB経路の制御に関わることが報告されていたOTU型のA20(TNFAIP3)とCezanne、USP型のCYLDに着目して抑制能を解析した。A20とCYLDは発現量を増加させるとユビキチンリガーゼ(LUBAC)発現によるNF-κB活性化を強く阻害するが、Cezanneは抑制効果が低かった。CYLDは脱ユビキチン化酵素活性依存的にLUBACによるNF-κB活性化を抑制した。A20がZF7を介して直鎖状ユビキチンに特異的に結合することでNF-κB活性を制御することが示唆された。A20はZF7の直鎖状ユビキチン結合能を介して受容体近傍に集積していること、その不全はNF-κB活性の持続的亢進となりB細胞リンパ腫を発症させることが示唆された。

Publishing type：Research paper (scientific journal)  

DOI： 10.1083/jcb.201304188

PubMed

Publishing type：Research paper (scientific journal)   Kind of work：Single Work  

DOI： 10.1093/jb/mvt079

PubMed

Publishing type：Research paper (scientific journal)  

Ubiquitination is a post-translational modification involved in the regulation of a broad variety of cellular functions, such as protein degradation and signal transduction, including nuclear factor-kappa B (NF-kappa B) signalling. NF-kappa B is crucial for inflammatory and immune responses, and aberrant NF-kappa B signalling is implicated in multiple disorders. We found that linear ubiquitin chain assembly complex (LUBAC), composed of HOIL-1L, HOIP and SHARPIN, generates a novel type of Met1 (M1)-linked linear polyubiquitin chain and specifically regulates the canonical NF-kappa B pathway. Moreover, specific deubiquitinases, such as CYLD, A20 (TNFAIP3) and OTULIN/gumby, inhibit LUBAC-induced NF-kappa B activation by different molecular mechanisms, and several M1-linked ubiquitin-specific binding domains have been structurally defined. LUBAC and these linear ubiquitination-regulating factors contribute to immune and inflammatory processes and apoptosis. Functional impairments of these factors are correlated with multiple disorders, including autoinflammation, immunodeficiencies, dermatitis, B-cell lymphomas and Parkinson's disease. This review summarizes the molecular basis and the pathophysiological implications of the linear ubiquitination-mediated NF-kappa B activation pathway regulation by LUBAC.

DOI： 10.1093/jb/mvt079

PubMed

Publishing type：Research paper (scientific journal)  

The detection of cytosolic DNA, derived from pathogens or host cells, by cytosolic receptors is essential for appropriate host immune responses. Cyclic GMP-AMP synthase (cGAS) is a newly identified cytosolic DNA receptor that produces cyclic GMP-AMP, which activates stimulator of interferon genes (STING), resulting in TBK1-IRF3 pathway activation followed by the production of type I interferons. Here we report the crystal structure of human cGAS. The structure revealed that a cluster of lysine and arginine residues forms the positively charged DNA binding surface of human cGAS, which is important for the STING-dependent immune activation. A structural comparison with other previously determined cGASs and our functional analyses suggested that a conserved zinc finger motif and a leucine residue on the DNA binding surface are crucial for the DNA-specific immune response of human cGAS, consistent with previous work. These structural features properly orient the DNA binding to cGAS, which is critical for DNA-induced cGAS activation and STING-dependent immune activation. Furthermore, we showed that the cGAS-induced activation of STING also involves the activation of the NF-kappa B and IRF3 pathways. Our results indicated that cGAS is a DNA sensor that efficiently activates the host immune system by inducing two distinct pathways.

DOI： 10.1371/journal.pone.0076983

PubMed

Publishing type：Research paper (scientific journal)  

Although ubiquitin is thought to be important for the autophagic sequestration of invading bacteria (also called xenophagy), its precise role remains largely enigmatic. Here we determined how ubiquitin is involved in this process. After invasion, ubiquitin is conjugated to host cellular proteins in endosomes that contain Salmonella or transfection reagent-coated latex (polystyrene) beads, which mimic invading bacteria. Ubiquitin is recognized by the autophagic machinery independently of the LC3-ubiquitin interaction through adaptor proteins, including a direct interaction between ubiquitin and Atg16L1. To ensure that invading pathogens are captured and degraded, Atg16L1 targeting is secured by two backup systems that anchor Atg16L1 to ubiquitin-decorated endosomes. Thus, we reveal that ubiquitin is a pivotal molecule that connects bacteria-containing endosomes with the autophagic machinery upstream of LC3.

DOI： 10.1083/jcb.201304188

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Publishing type：Research paper (scientific journal)  

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.pone.0076983

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DOI： 10.1038/emboj.2012.241

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s12104-011-9350-1

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

HOIL-1L and its binding partner, HOIL-1L interacting protein (HOIP), are essential components of linear ubiquitin (Ub) chain assembly complex (LUBAC), a 600-kDa enzyme complex catalyzing elongation of a tandemly connected Ub chain, which serve as a regulator of NF-κB activation. Specific interaction between the N-terminal Ub-like domain (UBL) of HOIL-1L and the Ub-associated domain (UBA) located at the central region of HOIP is shown to be involved in the formation of LUBAC. For better understanding of the mechanisms underlying the generation of the linear Ub chains by LUBAC, it is necessary to characterize the UBL-UBA interaction on the basis of structural data, which, however, is not available to date. Here we report backbone and side chain NMR assignments of the UBL of human HOIL-1L. By inspection of chemical shift index, it was predicted that HOIL-1L-UBL assumes a Ub fold followed by an α-helical segment, offering the basis for determination its 3D structure and interaction with HOIP-UBA in solution.

DOI： 10.1007/s12104-011-9350-1

PubMed

Publishing type：Research paper (scientific journal)  

LUBAC (linear ubiquitin chain assembly complex) activates the canonical NF-kappa B pathway through linear polyubiquitination of NEMO (NF-kappa B essential modulator, also known as IKK gamma) and RIP1. However, the regulatory mechanism of LUBAC-mediated NF-kappa B activation remains elusive. Here, we show that A20 suppresses LUBAC-mediated NF-kappa B activation by binding linear polyubiquitin via the C-terminal seventh zinc finger (ZF7), whereas CYLD suppresses it through deubiquitinase (DUB) activity. We determined the crystal structures of A20 ZF7 in complex with linear diubiquitin at 1.70-1.98 angstrom resolutions. The crystal structures revealed that A20 ZF7 simultaneously recognizes the Met1-linked proximal and distal ubiquitins, and that genetic mutations associated with B cell lymphomas map to the ubiquitin-binding sites. Our functional analysis indicated that the binding of A20 ZF7 to linear polyubiquitin contributes to the recruitment of A20 into a TNF receptor (TNFR) signalling complex containing LUBAC and I kappa B kinase (IKK), which results in NF-kappa B suppression. These findings provide new insight into the regulation of immune and inflammatory responses. The EMBO Journal (2012) 31, 3856-3870. doi:10.1038/emboj.2012.241; Published online 28 August 2012

DOI： 10.1038/emboj.2012.241

PubMed

Publishing type：Research paper (scientific journal)  

The NF-kappa B pathway is a central signaling pathway for inflammatory and immune responses, and aberrant NF-kappa B signaling is implicated multiple disorders, such as cancer and autoimmune, chronic inflammatory and metabolic diseases. NF-kappa B is regulated by various post-translational modifications, including phosphorylation and multiple ubiquitinations. We determined that LUBAC (linear ubiquitin chain assembly complex), composed of SHARPIN, HOIL-IL and HOIP, generates a novel type of Met1-linked linear polyubiquitin chain and specifically regulates the canonical NF-kappa B pathway via the linear ubiquitination of NEMO and R1P1. In the absence of LUBAC components, NF-kappa B signaling was attenuated and induced apoptosis and inflammation. Many studies on the pathophysiological functions of LUBAC, such as in B cell development, innate immune response, carcinogenesis, and osteogenesis, have been performed recently. This review summarizes these new findings on LUBAC- and linear ubiquitination-mediated NF-kappa B regulation and their implications in disorders.

DOI： 10.1507/endocrj.EJ12-0148

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DOI： 10.1074/jbc.M112.347195

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Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.micinf.2012.01.011

PubMed

Publishing type：Research paper (scientific journal)  

Nuclear factor-kappa B (NF-kappa B) essential modulator (NEMO), a component of the inhibitor of kappa B kinase (IKK) complex, controls NF-kappa B signaling by binding to ubiquitin chains. Structural studies of NEMO provided a rationale for the specific binding between the UBAN (ubiquitin binding in ABIN and NEMO) domain of NEMO and linear (Met-1-linked) di-ubiquitin chains. Full-length NEMO can also interact with Lys-11-, Lys-48-, and Lys-63-linked ubiquitin chains of varying length in cells. Here, we show that purified full-length NEMO binds preferentially to linear ubiquitin chains in competition with lysine-linked ubiquitin chains of defined length, including long Lys-63-linked deca-ubiquitins. Linear di-ubiquitins were sufficient to activate both the IKK complex in vitro and to trigger maximal NF-kappa B activation in cells. In TNF alpha-stimulated cells, NEMO chimeras engineered to bind exclusively to Lys-63-linked ubiquitin chains mediated partial NF-kappa B activation compared with cells expressing NEMO that binds to linear ubiquitin chains. We propose that NEMO functions as a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. This phenomenon could explain quantitatively distinct NF-kappa B activation patterns in response to numerous cell stimuli.

DOI： 10.1074/jbc.M112.347195

PubMed

Publishing type：Research paper (scientific journal)  

LUBAC (linear ubiquitin chain assembly complex) is a ubiquitin ligase complex composed of SHARPIN, HOIL-IL and HOIP that generates linear polyubiquitin chains and regulates the NF-kappa B pathway, which is pivotal in inflammatory and immune responses. Recent findings on the regulation of NF-kappa B by LUBAC and the diseases associated with this process are the focus of this review. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

DOI： 10.1016/j.micinf.2012.01.011

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Publishing type：Research paper (scientific journal)  

DOI： 10.1038/embor.2012.24

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

HOIL-1L and its binding partner HOIP are essential components of the E3-ligase complex that generates linear ubiquitin (Ub) chains, which are critical regulators of NF-κB activation. Using crystallographic and mutational approaches, we characterize the unexpected structural basis for the specific interaction between the Ub-like domain (UBL) of HOIL-1L and the Ub-associated domain (UBA) of HOIP. Our data indicate the functional significance of this non-canonical mode of UBA-UBL interaction in E3 complex formation and subsequent NF-κB activation. This study highlights the versatility and specificity of protein-protein interactions involving Ub/UBLs and their cognate proteins.

DOI： 10.1038/embor.2012.24

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Publishing type：Research paper (scientific journal)  

DOI： 10.3892/ijo.2011.1209

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Publishing type：Research paper (scientific journal)  

NF-kappa B is involved in the metastasis of malignant cells. We have shown that NF-kappa B activation is involved in the pulmonary metastasis of LM8 cells, a highly metastatic subclone of Dunn murine osteosarcoma cells. Recently, it was determined that a newly identified type of polyubiquitin chain, a linear polyubiquitin chain, which is specifically generated by the linear ubiquitin chain assembly complex (LUBAC), plays a critical role in NF-kappa B activation. Here, we have evaluated the roles of LUBAC-mediated NF-kappa B activation in the development of lung metastasis of osteosarcoma cells. All three components of LUBAC (HOIL-1L, HOIP and SHARPIN) were highly expressed in LM8 cells compared to Dunn cells. Attenuation of LUBAC expression by stable knockdown of HOIL-1L in LM8 cells significantly suppressed NF-kappa B activity, invasiveness in vitro and lung metastasis. Induction of intracellular adhesion molecule-1 (ICAM-1) expression by LUBAC is involved in cell retention in the lungs after an intravenous inoculation of tumor cells. Moreover, we found that knockdown of LUBAC decreased not only the number but also the size of the metastatic nodules of LM8 cells in the lungs. These results indicate that LUBAC-mediated NF-kappa B activation plays crucial roles in several steps involved in metastasis, including extravasation and growth of osteosarcoma cells in the lung, and that suppression of LUBAC-mediated linear polyubiquitination activity may be a new approach to treat this life-threatening disease of young adolescents.

DOI： 10.3892/ijo.2011.1209

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DOI： 10.1126/science.1211667

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PubMed

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DOI： 10.1038/nature09814

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DOI： 10.1038/nature09815

PubMed

Publishing type：Research paper (scientific journal)  

SHARPIN is a ubiquitin-binding and ubiquitin-like-domain-containing protein which, when mutated in mice, results in immune system disorders and multi-organ inflammation(1,2). Here we report that SHARPIN functions as a novel component of the linear ubiquitin chain assembly complex (LUBAC) and that the absence of SHARPIN causes dysregulation of NF-kappa B and apoptotic signalling pathways, explaining the severe phenotypes displayed by chronic proliferative dermatitis (cpdm) in SHARPIN-deficient mice. Upon binding to the LUBAC subunit HOIP (also known as RNF31), SHARPIN stimulates the formation of linear ubiquitin chains in vitro and in vivo. Coexpression of SHARPIN and HOIP promotes linear ubiquitination of NEMO (also known as IKBKG), an adaptor of the I kappa B kinases (IKKs) and subsequent activation of NF-kappa B signalling, whereas SHARPIN deficiency in mice causes an impaired activation of the IKK complex and NF-kappa B in B cells, macrophages and mouse embryonic fibroblasts (MEFs). This effect is further enhanced upon concurrent downregulation of HOIL-1L (also known as RBCK1), another HOIP-binding component of LUBAC. In addition, SHARPIN deficiency leads to rapid cell death upon tumour-necrosis factor alpha (TNF-alpha) stimulation via FADD- and caspase-8-dependent pathways. SHARPIN thus activates NF kappa B and inhibits apoptosis via distinct pathways in vivo.

DOI： 10.1038/nature09814

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Publishing type：Research paper (scientific journal)  

Cpdm (chronic proliferative dermatitis) mice develop chronic dermatitis and an immunodeficiency with increased serum IgM(1-3), symptoms that resemble those of patients with X-linked hyper-IgM syndrome and hypohydrotic ectodermal dysplasia (XHM-ED), which is caused by mutations in NEMO (NF-kappa B essential modulator; also known as IKBKG)(4-6). Spontaneous null mutations in the Sharpin (SHANK-associated RH domain interacting protein in postsynaptic density)(7) gene are responsible for the cpdm phenotype in mice(8). SHARPIN shows significant similarity to HOIL-1L (also known as RBCK1)(8,9), a component of linear ubiquitin chain assembly complex (LUBAC), which induces NF-kappa B activation through conjugation of linear polyubiquitin chains to NEMO(10-13). Here, we identify SHARPIN as an additional component of LUBAC. SHARPIN-containing complexes can linearly ubiquitinate NEMO and activated NF-kappa B. Thus, we re-define LUBAC as a complex containing SHARPIN, HOIL-1L, and HOIP (also known as RNF31). Deletion of SHARPIN drastically reduced the amount of LUBAC, which resulted in attenuated TNF-alpha- and CD40-mediated activation of NF-kappa B in mouse embryonic fibroblasts (MEFs) or B cells from cpdm mice. Considering the pleomorphic phenotype of cpdm mice, these results confirm the predicted role of LUBAC-mediated linear polyubiquitination in NF-kappa B activation induced by various stimuli, and strongly suggest the involvement of LUBAC-induced NF-kappa B activation in various disorders.

DOI： 10.1038/nature09815

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Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.molcel.2010.12.029

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Publishing type：Research paper (scientific journal)  

Upon detection of viral RNA, retinoic acid-inducible gene I (RIG-I) undergoes TRIM25-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that the linear ubiquitin assembly complex (LUBAC), comprised of two RING-IBR-RING (RBR)-containing E3 ligases, HOIL-1L and HOIP, independently targets TRIM25 and RIG-I to effectively suppress virus-induced IFN production. RBR E3 ligase domains of HOIL-1L and HOIP bind and induce proteasomal degradation of TRIM25, whereas the NZF domain of HOIL-1L competes with TRIM25 for RIG-I binding. Consequently, both actions by the HOIL-1L/HOIP LUBAC potently inhibit RIG-I ubiquitination and antiviral activity, but in a mechanistically separate manner. Conversely, the genetic deletion or depletion of HOIL-1L and HOIP robustly enhances virus-induced type I IFN production. Taken together, the HOIL-1L/HOIP LUBAC specifically suppresses RIG-I ubiquitination and activation by inducing TRIM25 degradation and inhibiting TRIM25 interaction with RIG-I, resulting in the comprehensive suppression of the IFN-mediated antiviral signaling pathway.

DOI： 10.1016/j.molcel.2010.12.029

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DOI： 10.1107/S1744309109050581

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Publishing type：Research paper (scientific journal)  

F-box proteins are the substrate-recognition components of Skp1-Cullin1-F-box protein-Rbx1 (SCF) ubiquitin ligase complexes. Fbs1, an F-box protein, binds specifically to proteins modified with high-mannose oligosaccharides. Fbg3, another F-box protein, has 51% sequence identity to Fbs1. Although the residues that are necessary for binding to oligosaccharides are conserved between Fbs1 and Fbg3, Fbg3 does not bind glycoproteins. Skp1 and Fbg3 were co-expressed in Escherichia coli and their complex was purified to homogeneity and crystallized. Microseeding combined with the sandwiched hanging-drop technique improved the quality of the resulting crystals. The plate-shaped crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 34.1, b = 76.6, c = 193.9 angstrom and one molecule per asymmetric unit.

DOI： 10.1107/S1744309109050581

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DOI： 10.1038/embor.2009.144

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Publishing type：Research paper (scientific journal)  

The ubiquitin-conjugation system regulates a vast range of biological phenomena by affecting protein function mostly through polyubiquitin conjugation. The type of polyubiquitin chain that is generated seems to determine how conjugated proteins are regulated, as they are recognized specifically by proteins that contain chain-specific ubiquitin-binding motifs. An enzyme complex that catalyses the formation of newly described linear polyubiquitin chains-known as linear ubiquitin chain-assembly complex (LUBAC)-has recently been characterized, as has a particular ubiquitin-binding domain that specifically recognizes linear chains. Both have been shown to have crucial roles in the canonical nuclear factor-kappa B (NF-kappa B)-activation pathway. The ubiquitin system is intimately involved in regulating the NF-kappa B pathway, and the regulatory roles of K63-linked chains have been studied extensively. However, the role of linear chains in this process is only now emerging. This article discusses the possible mechanisms under lying linear polyubiquitin-mediated activation of NF-kappa B, and the different roles that K63-linked and linear chains have in NF-kappa B activation. Future directions for linear polyubiquitin research are also discussed.

DOI： 10.1038/embor.2009.144

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PubMed

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DOI： 10.1038/ncb1821

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Publishing type：Research paper (scientific journal)  

Nuclear factor-kappa B (NF-kappa B) is a key transcription factor in inflammatory, anti-apoptotic and immune processes. The ubiquitin pathway is crucial in regulating the NF-kappa B pathway. We have found that the LUBAC ligase complex, composed of the two RING finger proteins HOIL-1L and HOIP, conjugates a head-to-tail- linked linear polyubiquitin chain to substrates. Here, we demonstrate that LUBAC activates the canonical NF-kappa B pathway by binding to NEMO (NF-kappa B essential modulator, also called IKK gamma) and conjugates linear polyubiquitin chains onto specific Lys residues in the CC2-LZ domain of NEMO in a Ubc13-independent manner. Moreover, in HOIL-1 knockout mice and cells derived from these mice, NF-kappa B signalling induced by pro-inflammatory cytokines such as TNF-alpha and IL-1 beta was suppressed, resulting in enhanced TNF-alpha-induced apoptosis in hepatocytes of HOIL-1 knockout mice. These results indicate that LUBAC is involved in the physiological regulation of the canonical NF-kappa B activation pathway through linear polyubiquitylation of NEMO.

DOI： 10.1038/ncb1821

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DOI： 10.1073/pnas.0806215105

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DOI： 10.1074/jbc.M710599200

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Publishing type：Research paper (scientific journal)  

Oxygen-dependent ubiquitination of the alpha-subunit of hypoxia-inducible factor (HIF-alpha) by the (von Hippel-Lindau protein)-Elongin B/C-Cullin2-Rbx1 (VBC-Cul2) ubiquitin ligase, a member of the cullin-RING ubiquitin ligases (CRLs), plays a central role in controlling oxygen metabolism. Nedd8 conjugation of cullins enhances the ligase activity of CRLs, and the COP9/ signalosome (CSN) enhances the degradation of several CRL substrates, although it removes Nedd8 from cullins. Here we demonstrate that CSN increased the efficiency of the VBC-Cul2 complex for recognizing and ubiquitinating substrates by facilitating the dissociation of ubiquitinated substrates from the pVHL subunit of the complex. Moreover CSN enhanced HIF-1 alpha degradation by promoting the dissociation of HIF-1 alpha from pVHL in cells. The length of the polyubiquitin chain conjugated to the substrate appeared to be involved in CSN-mediated dissociation of the substrate from pVHL. In contrast to other mechanisms underlying CSN-mediated activation of CRLs, the dissociation of ubiquitinated substrates from pVHL did not require the deneddylation activity of CSN, implying that CSN enhances degradation of CRL substrates by multiple mechanisms.

DOI： 10.1074/jbc.M710599200

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Publishing type：Research paper (scientific journal)  

DOI： 10.1091/mbc.E07-06-0601

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Publishing type：Research paper (scientific journal)  

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin-proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTR Delta F508, by specifically promoting ubiquitylation of CFTR Delta F508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTR Delta F508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTR Delta F508 and catalyzes further polyubiquitylation of CFTR Delta F508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTR Delta F508.

DOI： 10.1091/mbc.E07-06-0601

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1128/MCB.0241-06

Publishing type：Research paper (scientific journal)  

The transcription repressor Bach1 is a sensor and effector of heme that regulates the expression of heme oxygenase I and globin genes. Heme binds to Bach1, inhibiting its DNA binding activity and inducing its nuclear export. We found that hemin further induced the degradation of endogenous Bach1 in NIH 3T3 cells, murine embryonic fibroblasts, and murine erythroleukemia cells. In contrast, succinylacetone, an inhibitor of heme synthesis, caused accumulation of Bach1 in murine embryonic fibroblasts, indicating that physiological levels of heme regulated the Bach1 turnover. Polyubiquitination and rapid degradation of overexpressed Bach1 were induced by hemin treatment. HOIL-1, an ubiquitin-protein ligase which recognizes heme-bound, oxidized iron regulatory protein 2, was found to bind with Bach1 when both were overexpressed in NIH 3T3 cells. HOIL-1 stimulated the polyubiquitination of Bach1 in a purified in vitro ubiquitination system depending on the intact heme binding motifs of Bach1. Expression of dominant-negative HOIL-1 in murine erythroleukemia cells resulted in higher stability of endogenous Bach1, raising the possibility that the heme-regulated degradation involved HOIL-1 in murine erythroleukemia cells. These results suggest that heme within a cell regulates the polyubiquitination and degradation of Bach1.

DOI： 10.1128/MCB.0241-06

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Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2006.09.163

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Publishing type：Research paper (scientific journal)  

Several isoforms of protein kinase C (PKC) are degraded by the ubiquitin-proteasome pathway after phorbol ester-mediated activation. However, little is known about the ubiquitin ligase (E3) that targets activated PKCs. We recently showed that an E3 complex composed of HOIL-1L and HOW (LUBAC) generates linear polyubiquitin chains and induces the proteasomal degradation of a model substrate. HOIL-1L has also been characterized as a PKC-binding protein. Here we show that LUBAC preferentially binds activated conventional PKCs and their constitutively active mutants. LUBAC efficiently ubiquitinated activated PKC in vitro, and degradation of activated PKC alpha was delayed in HOIL-1L-deficient cells. Conversely, PKC activation induced cleavage of HOIL-1L and led to down-regulation of the ligase activity of LUBAC. These results indicate that LUBAC is an E3 for activated conventional PKC, and that PKC and LUBAC regulate each other for proper PKC signaling. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2006.09.163

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Publishing type：Research paper (scientific journal)  

DOI： 10.1038/sj.emboj.7601360

PubMed

Publishing type：Research paper (scientific journal)  

The ubiquitin system plays important roles in the regulation of numerous cellular processes by conjugating ubiquitin to target proteins. In most cases, conjugation of polyubiquitin to target proteins regulates their function. In the polyubiquitin chains reported to date, ubiquitin monomers are linked via isopeptide bonds between an internal Lys and a C-terminal Gly. Here, we report that a protein complex consisting of two RING finger proteins, HOIL-1L and HOIP, exhibits ubiquitin polymerization activity by recognizing ubiquitin moieties of proteins. The polyubiquitin chain generated by the complex is not formed by Lys linkages, but by linkages between the C- and N-termini of ubiquitin, indicating that the ligase complex possesses a unique feature to assemble a novel head-to-tail linear polyubiquitin chain. Moreover, the complex regulates the stability of Ub-GFP (a GFP fusion protein with an N-terminal ubiquitin). The linear polyubiquitin chain generated post-translationally may function as a new modulator of proteins.

DOI： 10.1038/sj.emboj.7601360

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Publishing type：Research paper (scientific journal)   Kind of work：Single Work  

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DOI： 10.1016/j.thromres.2005.02.017

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Publishing type：Research paper (scientific journal)  

Introduction: Misfolded and unassembled glycoproteins are eliminated from the endoplasmic reticulum (ER) lumen by the ER-associated degradation (ERAD). We previously identified a Tyr595Cys (Y595C) mutation of protein S (PS) in a family of a quantitative PS deficiency. The mutation causes intracettular degradation and decreased secretion of the Y595C mutant PS. The aim of the present study was to further characterize the molecular basis of the intracellular degradation of the mutant.
Materials and methods: We stably Expressed the mutant in mammalian cells, and analyzed the intracellular localization of the protein. The intracellular degradation pathway was determined by pulse-chase analyses in the presence of various inhibitors of ERAD.
Results and conclusions: Endoglycosidase H digestion and immunofluorescence staining reveated the mutant being retained in the ER. Epoxomicin, a potent and specific proteasome inhibitor, and Ala-Ala-Phe-CH(2)Cl (AAF), an inhibitor of tripeptidyl peptidase II (TPPII), suppressed the intracellular degradation of the mutant by about 65% and 50%, respectively. When epoxomicin was combined with AAF, the inhibitory effect was substantially enhanced. Although castanospermine, an inhibitor of glucosidases I and II, did not affect the degradation, kifunensine, an inhibitor of ER mannosidase 1, suppressed it. Thus, it appears that the Y595C mutant is degraded through more than one pathway of ERAD, including the proteasome-dependent pathway and an alternate proteasome-independent pathway where proteases such as TPPII may be involved. Production of the critical B isoform of Man(8)GlcNAC(2) targets the mutant for ERAD, however, the interaction with calnexin/ catreticulin through monoglucosytated oLigosaccharides may not be required for the degradation of the mutant. (c) 2005 Etsevier Ltd. All rights reserved.

DOI： 10.1016/j.thromres.2005.02.017

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Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.molcel.2005.05.027

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Publishing type：Research paper (scientific journal)  

yIron regulatory protein 2 (IRP2), a regulator of iron metabolism, is modulated by ubiquitination and degradation. We have shown that IRP2 degradation is triggered by heme-mediated oxidation. We report here that not only Cys201, an invariant residue in the heme regulatory motif (HRM), but also His204 is critical for IRP2 degradation. Spectroscopic studies revealed that Cys201 binds ferric heme, whereas His204 is a ferrous heme binding site, indicating the involvement of these residues in sensing the redox state of the heme iron and in generating the oxidative modification. Moreover, the HRM in IRP2 has been suggested to play a critical role in its recognition by the HOIL-1 ubiquitin ligase. Although HRMs are known to sense heme concentration by simply binding to heme, the HRM in IRP2 specifically contributes to its oxidative modification, its recognition by the ligase, and its sensing of iron concentration after iron is integrated into heme.

DOI： 10.1016/j.molcel.2005.05.027

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Publishing type：Research paper (scientific journal)  

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Publishing type：Research paper (scientific journal)   Kind of work：Single Work  

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DOI： 10.1074/jbc.M304157200

PubMed

Publishing type：Research paper (scientific journal)  

F-box proteins are substrate recognition components of Skp1-Cullin1-F-box protein-Roc1 (SCF) E3 ubiquitin-protein ligases. We reported previously that Fbs1 (F-box protein that recognizes sugar chains; equivalent to Fbx2 or NFB42) binds specifically to proteins attached with high mannose oligosaccharides and subsequently contributes to elimination of N-glycoproteins in cytosol (Yoshida, Y., Chiba, T., Tokunaga, F., Kawasaki, H., Iwai, K., Suzuki, T., Ito, Y., Matsuoka, K., Yoshida, M., Tanaka, K., and Tai, T. (2002) Nature 418, 438 - 442). Here we report the identification of another F-box protein that recognizes N-glycan, Fbs2 (called Fbx6b or FBG2 previously). Although the expression of Fbs1 was restricted to the adult brain and testis, the Fbs2 transcript was widely expressed. The Fbs2 protein forms an SCFFbs2 ubiquitin-ligase complex that targets sugar chains in N-glycoproteins for ubiquitylation. Only glycoproteins bound to concanavalin A lectin and not to wheat germ agglutinin or Ricinus communis agglutinin interacted with Fbs2 in various tissues and cell lines. Pull-down analysis using various oligosaccharides revealed that Man(3-9)GlcNAc(2) glycans were required for efficient Fbs2 binding, whereas modifications of mannose residues by other sugars or deletion of inner GlcNAc reduced Fbs2 binding. Fbs2 interacted with N-glycans of T-cell receptor alpha-subunit (TCRalpha), a typical substrate of the endoplasmic reticulum-associated degradation (ERAD) pathway, and the forced expression of mutant Fbs2DeltaF, which lacks the F-box domain essential for forming the SCF complex, and decrease of endogenous Fbs2 by small interfering RNA led to inhibition of TCRalpha degradation in cells. Thus, Fbs2 is a novel member of F-box protein family that recognizes N-glycans and plays a role in ERAD.

DOI： 10.1074/jbc.M304157200

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Publishing type：Research paper (scientific journal)  

DOI： 10.1038/ncb952

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Publishing type：Research paper (scientific journal)  

The ubiquitin system is involved in several basic cellular functions(1-3). Ubiquitination is carried out by a cascade of three reactions catalysed by the El, E2 and E3 enzymes. Among these, the E3 ubiquitin-protein ligases have a pivotal role in determining the specificity of the system by recognizing the target substrates through defined targeting motifs(1-3). Although RING finger proteins constitute an important family of E3 ligases(4), only a few post-transcriptional modifications, including phosphorylation(1), proline hydroxylation(5,6) and glycosylation(7), are known to function as recognition signals for E3. Iron regulatory protein 2 (IRP2), a modulator of iron metabolism, is regulated by iron-induced ubiquitination and degradation(8). Here we show that the RING finger protein HOIL-1 functions as an E3 ligase for oxidized IRP2, suggesting that oxidation is a specific recognition signal for ubiquitination. The oxidation of IRP2 is generated by haem, which binds to IRP2 in iron-rich cells, and by oxygen, indicating that the iron sensing of IRP2 depends on the synthesis and availability of haem.

DOI： 10.1038/ncb952

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Publishing type：Research paper (scientific journal)  

DOI： 10.1016/s0003-9861(02)00717-8

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Publishing type：Research paper (scientific journal)  

Previously we showed that two antithrombin mutants were degraded through an endoplasmic reticulum (ER)-associated degradation (ERAD) pathway [F. Tokunaga et al., FEBS Lett. 412 (1997) 65]. Here, we examined the combined effects of inhibitors of glycosidases, protein synthesis, proteasome, and tyrosine phosphatase on ERAD of a Glu313-deleted (DeltaGlu) mutant of antithrombin. We found that kifunensine, an ER marmosidase I inhibitor, suppressed ERAD, indicating that specific mannose trimming plays a critical role. Cycloheximide and puromycin, inhibitors of protein synthesis, also suppressed ERAD, the effects being cancelled by pretreatment with castanospermine. In contrast, kifunensine suppressed ERAD even in castanospermine-treated cells, suggesting that suppression of ERAD does not always require the binding of lectin-like ER chaperones-like calnexin and/or calreticulin. These results indicate that, besides proteasome inhibitors, inhibitors of ER mannosidase I and protein synthesis suppress ERAD of the antithrombin DeltaGlu mutant at different stages, and processing of N-linked oligosaccharides highly correlated with the efficiency of ERAD. (C) 2003 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0003-9861(02)00717-8

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Publishing type：Research paper (scientific journal)  

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Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M206773200

PubMed

Publishing type：Research paper (scientific journal)  

Monoclonal antibodies were raised against hemocytes of the horseshoe crab Tachypleus tridentatus. All of the antibodies obtained reacted with the same protein bands on SDS-PAGE of hemocyte lysate. Flow cytometry and biotinylation of surface substances on the hemocytes indicated that the antigens are major peripheral proteins of hemocytes. The antigens were purified from hemocyte lysate and were good substrates for the horseshoe crab hemocyte transglutaminase (HcTGase). Transglutaminases play an important role during the final stage of blood coagulation in mammals and crustaceans. Although HcTGase did not intermolecularly cross-link a clottable protein coagulogen or its proteolytic product coagulin, HcTGase promoted the cross-linking of coagulin with the surface antigens, resulting in the formation of a stable polymer. We determined the nucleotide sequences for two isoproteins of the antigens. The two proteins containing 271 and 284 residues (66% identity) were composed of tandem repeats of proline-rich segments. We named them proxins-1 and -2 after proline-rich proteins for protein cross-linking. Proxins may form a stable physical barrier against invading pathogens in cooperation with hemolymph coagulation at injured sites.

DOI： 10.1074/M206773200

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Publishing type：Research paper (scientific journal)  

DOI： 10.1038/nature00890

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Publishing type：Research paper (scientific journal)  

N-glycosylation of proteins in the endoplasmic reticulum (ER) has a central role in protein quality control(1-3). Here we report that N-glycan serves as a signal for degradation by the Skp1-Cullin1-Fbx2-Roc1 (SCF(Fbx2)) ubiquitin ligase complex. The F-box protein Fbx2 (ref. 4) binds specifically to proteins attached to N-linked high-mannose oligosaccharides and subsequently contributes to ubiquitination of N-glycosylated proteins. Pre-integrin beta1 is a target of Fbx2; these two proteins interact in the cytosol after inhibition of the proteasome. In addition, expression of the mutant Fbx2DeltaF, which lacks the F-box domain that is essential for forming the SCF complex, appreciably blocks degradation of typical substrates of the ER-associated degradation pathway(5,6). Our results indicate that SCF(Fbx2) ubiquitinates N-glycosylated proteins that are translocated from the ER to the cytosol by the quality control mechanism.

DOI： 10.1038/nature00890

PubMed

Publishing type：Research paper (scientific journal)  

To examine the role of early carbohydrate recognition/trimming reactions in targeting endoplasmic reticulum (ER)-retained, misfolded glycoproteins for ER-associated degradation (ERAD), we have stably expressed the cog thyroglobulin (Tg) mutant cDNA in Chinese hamster ovary cells. We found that inhibitors of ER mannosidase I (but not other glycosidases) acutely suppressed Cog Tg degradation and also perturbed the ERAD process for Tg reduced with dithiothreitol as well as for gamma -carboxylation-deficient protein C expressed in warfarin-treated baby hamster kidney cells. Kifunensine inhibition of ER mannosidase I also suppressed ERAD in castanospermine-treated cells; thus, suppression of ERAD does not require lectin-like binding of ER chaperones calnexin and calreticulin to monoglucosylated oligosaccharides. Notably, the undegraded protein fraction remained completely microsome-associated. In pulse-chase studies, kifunensine-sensitive degradation was still inhibitable even 1 h after Tg synthesis. Intriguingly, chronic treatment with kifunensine caused a 3-fold accumulation of Cog Tg in Chinese hamster ovary cells and did not lead to significant induction of the ER unfolded protein response. We hypothesize that, in a manner not requiring lectin-like activity of calnexin/ calreticulin, the recognition or processing of a specific branched N-linked mannose structure enhances the efficiency of glycoprotein retrotranslocation from the ER lumen.

Publishing type：Research paper (scientific journal)  

To examine the role of early carbohydrate recognition/trimming reactions in targeting endoplasmic reticulum (ER)-retained, misfolded glycoproteins for ER-associated degradation (ERAD), we have stably expressed the cog thyroglobulin (Tg) mutant cDNA in Chinese hamster ovary cells. We found that inhibitors of ER mannosidase I (but not other glycosidases) acutely suppressed Cog Tg degradation and also perturbed the ERAD process for Tg reduced with dithiothreitol as well as for gamma -carboxylation-deficient protein C expressed in warfarin-treated baby hamster kidney cells. Kifunensine inhibition of ER mannosidase I also suppressed ERAD in castanospermine-treated cells; thus, suppression of ERAD does not require lectin-like binding of ER chaperones calnexin and calreticulin to monoglucosylated oligosaccharides. Notably, the undegraded protein fraction remained completely microsome-associated. In pulse-chase studies, kifunensine-sensitive degradation was still inhibitable even 1 h after Tg synthesis. Intriguingly, chronic treatment with kifunensine caused a 3-fold accumulation of Cog Tg in Chinese hamster ovary cells and did not lead to significant induction of the ER unfolded protein response. We hypothesize that, in a manner not requiring lectin-like activity of calnexin/ calreticulin, the recognition or processing of a specific branched N-linked mannose structure enhances the efficiency of glycoprotein retrotranslocation from the ER lumen.

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

A glycoprotein (M(r)=43000) from horseshoe crab hemocytes with antimicrobial activity against Gram-negative bacteria was purified, The internal peptide sequences coincided exactly with the deduced amino acid sequence of a cDNA clone, designated limulus factor D, which was isolated by screening a hemocyte cDNA library with an anti-human plasminogen antibody, The open reading frame codes for a precursor of factor D of 394 amino acid residues, including an NH2-terminal signal sequence. The COOH-terminal domain of factor D has significant sequence homology with the catalytic domain of mammalian serine proteases, in particular with human tissue plasminogen activator (32% identity), except for the substitution of Ser of the active site tried to Gly. Factor D has a unique NH2-terminal domain with weak sequence homology with part of the mammalian interleukin-6 receptor alpha-chain. Factor D is likely to have an important role in host defense mechanisms.

DOI： 10.1016/S0014-5793(96)01224-0

PubMed

Publishing type：Research paper (scientific journal)  

A glycoprotein (M(r)=43000) from horseshoe crab hemocytes with antimicrobial activity against Gram-negative bacteria was purified, The internal peptide sequences coincided exactly with the deduced amino acid sequence of a cDNA clone, designated limulus factor D, which was isolated by screening a hemocyte cDNA library with an anti-human plasminogen antibody, The open reading frame codes for a precursor of factor D of 394 amino acid residues, including an NH2-terminal signal sequence. The COOH-terminal domain of factor D has significant sequence homology with the catalytic domain of mammalian serine proteases, in particular with human tissue plasminogen activator (32% identity), except for the substitution of Ser of the active site tried to Gly. Factor D has a unique NH2-terminal domain with weak sequence homology with part of the mammalian interleukin-6 receptor alpha-chain. Factor D is likely to have an important role in host defense mechanisms.

DOI： 10.1016/S0014-5793(96)01224-0

PubMed

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Heparin cofactor II (HCII) is a serine proteinase inhibitor in human plasma that rapidly inhibits thrombin in the presence of dermatan sulfate or heparin. To understand the molecular mechanism for HCII deficiency in a patient with reduced circulating HCII antigen, we studied a Japanese patient with type I HCII deficiency who suffered from angina pectoris and coronary artery disease, Polymerase chain reaction (PCR)-based sequence analysis showed that the propositus' gene for HCII (HCII Awaji gene) had a thymine insertion after codon (GAT) for Asp88 in exon II, resulting in a frameshift mutation. Consequently, the abnormal HCII Awaji protein was suggested to have an altered amino acid sequence from position 89 and terminate at 107, thus being composed of the NH2-terminal one fifth of normal HCII and dysfunctional for thrombin inhibition, The molecular weight and pI value of HCII Awaji were calculated to be 12,040 and 3.6, respectively, without posttranslational modification. Mutagenic PCR followed by the Tsp5091 digestion showed that a half of the PCR products derived from the propositus and his sister was cleaved, suggesting that his sister also has the same mutant allele, Crossed-immunoelectrophoresis and Western blot analyses of plasma and urine from the propositus and of plasma from his sister did not provide evidence for the existence of the abnormal HCII, suggesting that little truncated HCII was circulating in the patient's blood. However, stable expression assay using human kidney 293 cells transfected with the expression vector containing cDNA encoding wild-type or Awaji-type HCII showed that mutant as well as wildtype HCII was secreted into culture medium normally, These results suggest that the abnormal HCII Awaji protein is secreted normally, but rapidly degraded in the circulating blood. (C) 1996 by The American Society of Hematology.

Publishing type：Research paper (scientific journal)  

Heparin cofactor II (HCII) is a serine proteinase inhibitor in human plasma that rapidly inhibits thrombin in the presence of dermatan sulfate or heparin. To understand the molecular mechanism for HCII deficiency in a patient with reduced circulating HCII antigen, we studied a Japanese patient with type I HCII deficiency who suffered from angina pectoris and coronary artery disease, Polymerase chain reaction (PCR)-based sequence analysis showed that the propositus' gene for HCII (HCII Awaji gene) had a thymine insertion after codon (GAT) for Asp88 in exon II, resulting in a frameshift mutation. Consequently, the abnormal HCII Awaji protein was suggested to have an altered amino acid sequence from position 89 and terminate at 107, thus being composed of the NH2-terminal one fifth of normal HCII and dysfunctional for thrombin inhibition, The molecular weight and pI value of HCII Awaji were calculated to be 12,040 and 3.6, respectively, without posttranslational modification. Mutagenic PCR followed by the Tsp5091 digestion showed that a half of the PCR products derived from the propositus and his sister was cleaved, suggesting that his sister also has the same mutant allele, Crossed-immunoelectrophoresis and Western blot analyses of plasma and urine from the propositus and of plasma from his sister did not provide evidence for the existence of the abnormal HCII, suggesting that little truncated HCII was circulating in the patient's blood. However, stable expression assay using human kidney 293 cells transfected with the expression vector containing cDNA encoding wild-type or Awaji-type HCII showed that mutant as well as wildtype HCII was secreted into culture medium normally, These results suggest that the abnormal HCII Awaji protein is secreted normally, but rapidly degraded in the circulating blood. (C) 1996 by The American Society of Hematology.

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Warfarin is known to disrupt the microsomal vitamin K cycle, which results in a decrease of the plasma level of protein C, an anticoagulant factor, as well as some other vitamin K-dependent coagulation factors. We examined the effect of warfarin on secretion of recombinant protein C expressed in human kidney 293 or BHK cells. In transient expression, warfarin caused a two- to four-fold decrease in the quantity of protein C secreted, compared to findings with vitamin K-treated cells. Pulse-chase experiments using stable cells showed that, although recombinant protein C was secreted in the presence of vitamin K, the decrease in total amount of the radioactivity in the warfarin-treated cells suggested intracellular degradation. This degradation depended on the concentration of warfarin and was not inhibited by an endoplasmic reticulum (ER)-Golgi transport inhibitor or by lysosomotropic inhibitors. Thus, protein C synthesized in the presence of warfarin is selectively degraded and the degradation occurs in a pre-Golgi, nonlysosomal compartment. Among the protease inhibitors tested, N-acetyl-Leu-Leu-methioninal and N-acetyl-Leu-Leu-norleucinal blocked the degradation of protein C synthesized in the presence of warfarin and the protein C accumulated intracellularly, in a dose-dependent manner. Both inhibitors, however, did not disturb the secretion of protein C in the vitamin K-treated cells. Thus, a cysteine protease(s) appeared to be responsible for the degradation. These results suggest that protein C synthesized in the presence of warfarin was selectively degraded by a cysteine protease(s) in the ER through a 'quality control' mechanism.

PubMed

Publishing type：Research paper (international conference proceedings)  

Publishing type：Research paper (international conference proceedings)  

Warfarin is known to disrupt the microsomal vitamin K cycle, which results in a decrease of the plasma level of protein C, an anticoagulant factor, as well as some other vitamin K-dependent coagulation factors. We examined the effect of warfarin on secretion of recombinant protein C expressed in human kidney 293 or BHK cells. In transient expression, warfarin caused a two- to four-fold decrease in the quantity of protein C secreted, compared to findings with vitamin K-treated cells. Pulse-chase experiments using stable cells showed that, although recombinant protein C was secreted in the presence of vitamin K, the decrease in total amount of the radioactivity in the warfarin-treated cells suggested intracellular degradation. This degradation depended on the concentration of warfarin and was not inhibited by an endoplasmic reticulum (ER)-Golgi transport inhibitor or by lysosomotropic inhibitors. Thus, protein C synthesized in the presence of warfarin is selectively degraded and the degradation occurs in a pre-Golgi, nonlysosomal compartment. Among the protease inhibitors tested, N-acetyl-Leu-Leu-methioninal and N-acetyl-Leu-Leu-norleucinal blocked the degradation of protein C synthesized in the presence of warfarin and the protein C accumulated intracellularly, in a dose-dependent manner. Both inhibitors, however, did not disturb the secretion of protein C in the vitamin K-treated cells. Thus, a cysteine protease(s) appeared to be responsible for the degradation. These results suggest that protein C synthesized in the presence of warfarin was selectively degraded by a cysteine protease(s) in the ER through a 'quality control' mechanism.

PubMed

Publishing type：Research paper (scientific journal)  

A new subunit, named RC6-I, of the rat 20 S proteasome was purified and the partial amino acid sequences of several peptide fragments obtained by digestion with lysyl-endopeptidase were determined by Edman degradation. Amplification of cDNAs encoding RC6-I by the polymerase chain reaction (PCR) technique revealed two types of cDNA, tentatively designated as RC6-IL and RC6-IS in order of size. The nucleotide sequences of the two cDNAs are identical except that RC6-IL contains an insertion of 18 nucleotides in the coding region compared with RC6-IS. The polypeptide predicted from the open reading frame of RC6-IS cDNA consists of 248 amino acid residues with a calculated molecular weight of 27783. These values are consistent with those obtained by protein chemical analyses. Computer-assisted homology analysis showed that RC6-I belongs to the α-type subfamily of the proteasome gene family, which shows similarity to the α-subunit of the archaebacterium Thermoplasma acidophilum proteasome, and that the 18 nucleotide insert, encoding six amino acid residues, VVASVS, appears to be unique to RC6-IL, because this motif has not been conserved in any other α-type subunit. By reverse transcription (RT)-PCR analysis, the mRNAs for both RC6-IL and RC6-IS were found in all the rat tissues examined. These results suggest that proteasomes are present as a heterogeneous population, possibly for acquisition of diversity of functions. © 1995.

DOI： 10.1016/0167-4781(95)00113-U

PubMed

Publishing type：Research paper (scientific journal)  

A new subunit, named RC6-I, of the rat 20 S proteasome was purified and the partial amino acid sequences of several peptide fragments obtained by digestion with lysyl-endopeptidase were determined by Edman degradation. Amplification of cDNAs encoding RC6-I by the polymerase chain reaction (PCR) technique revealed two types of cDNA, tentatively designated as RC6-IL and RC6-IS in order of size. The nucleotide sequences of the two cDNAs are identical except that RC6-IL contains an insertion of 18 nucleotides in the coding region compared with RC6-IS. The polypeptide predicted from the open reading frame of RC6-IS cDNA consists of 248 amino acid residues with a calculated molecular weight of 27783. These values are consistent with those obtained by protein chemical analyses. Computer-assisted homology analysis showed that RC6-I belongs to the α-type subfamily of the proteasome gene family, which shows similarity to the α-subunit of the archaebacterium Thermoplasma acidophilum proteasome, and that the 18 nucleotide insert, encoding six amino acid residues, VVASVS, appears to be unique to RC6-IL, because this motif has not been conserved in any other α-type subunit. By reverse transcription (RT)-PCR analysis, the mRNAs for both RC6-IL and RC6-IS were found in all the rat tissues examined. These results suggest that proteasomes are present as a heterogeneous population, possibly for acquisition of diversity of functions. © 1995.

DOI： 10.1016/0167-4781(95)00113-U

PubMed

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

The nucleotide sequence of a cDNA that encodes anew subunit, named RC10-II, of the 20S proteasome of rat embryonic brain has been determined. The polypeptide predicted from the open reading frame consists of 205 amino acid residues with a calculated molecular weight of 22,965 and isoelectric point of 6.15. Computer analysis showed that RC10-II belongs to the β-type subgroup of proteasomes, differing clearly from α-type subunits of the proteasome gene family. The primary structure of RC10-II was found to contain the partial amino acid sequences of several fragments of subunit 13, which has a cysteinyl residue critical for the trypsin-like catalytic activity, as reported by L.R. Dick et al. [Biochemistry 31, 7347-7355, 1992], suggesting that RC10-II is a proteasomal subunit necessary for the expression of tryptic activity. © 1993.

DOI： 10.1016/0014-5793(93)80856-P

PubMed

Publishing type：Research paper (scientific journal)  

The nucleotide sequence of a cDNA that encodes anew subunit, named RC10-II, of the 20S proteasome of rat embryonic brain has been determined. The polypeptide predicted from the open reading frame consists of 205 amino acid residues with a calculated molecular weight of 22,965 and isoelectric point of 6.15. Computer analysis showed that RC10-II belongs to the β-type subgroup of proteasomes, differing clearly from α-type subunits of the proteasome gene family. The primary structure of RC10-II was found to contain the partial amino acid sequences of several fragments of subunit 13, which has a cysteinyl residue critical for the trypsin-like catalytic activity, as reported by L.R. Dick et al. [Biochemistry 31, 7347-7355, 1992], suggesting that RC10-II is a proteasomal subunit necessary for the expression of tryptic activity. © 1993.

DOI： 10.1016/0014-5793(93)80856-P

PubMed

Publishing type：Research paper (scientific journal)  

The nucleotide sequence of a cDNA that encodes a new subunit, named RC7-I, of the 20 S proteasome of rat hepatoma cells has been determined. The polypeptide predicted from the open reading frame consists of 201 amino acid residues with a calculated molecular weight of 22,912 and isoelectric point of 7.25. Approximately 80% of the partial amino acid sequences of several fragments of RC7-I, determined by protein chemical analyses, were found to be in excellent accordance with those deduced from the cDNA sequence. Computer analysis showed that RC7-I belongs to the beta-type subgroup of proteasomes with similarity to the beta-subunit of the archaebacterial proteasome, differing clearly from alpha-type subunits of the proteasome gene family. The overall structure of RC7-I was found to be homologous to that of yeast PRE1, which is necessary for chymotryptic activity.

Publishing type：Research paper (scientific journal)  

The nucleotide sequence of a cDNA that encodes a new subunit, named RC7-I, of the 20 S proteasome of rat hepatoma cells has been determined. The polypeptide predicted from the open reading frame consists of 201 amino acid residues with a calculated molecular weight of 22,912 and isoelectric point of 7.25. Approximately 80% of the partial amino acid sequences of several fragments of RC7-I, determined by protein chemical analyses, were found to be in excellent accordance with those deduced from the cDNA sequence. Computer analysis showed that RC7-I belongs to the beta-type subgroup of proteasomes with similarity to the beta-subunit of the archaebacterial proteasome, differing clearly from alpha-type subunits of the proteasome gene family. The overall structure of RC7-I was found to be homologous to that of yeast PRE1, which is necessary for chymotryptic activity.

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

The molecular properties of an ATP/ubiquitin-dependent ‗26S‘ proteasome complex purified from rat liver were examined by physicochemical, biochemical, and morphological analyses. On ultracentrifugation, the proteasome complex sedimented as almost a single component with a sedimentation coefficient of 30.3S. Dynamic light-scattering measurements indicated that it has a diffusion coefficient of 1.38 x 10-7 cm2/sec and a Stokes radius of 15.5 nm. From these two coefficients, the protein complex was estimated to have the high molecular weight of 2.02 x 106. Static light-scattering analysis indicated a molecular weight of 1.91 x 106 and a radius of gyration of 16.8 nm. The proteasome complex was found to be composed of multiple subunits of the 20S proteasome with molecular weights of 2.1-3.1 x 104 and 15-20 protein species with molecular weights of 3.5-11.0 x 104, which were directly associated with the 20S proteasome. The electron micrographic finding that the 26S proteasome complex had a caterpillar shape, direct electron-microscopic observations on the subunit arrangement of the 20S proteasome, and classification of the subunits of the latter into two groups with respect to sequence homology suggested that the 26S complex is a symmetrical assembly of two domains, each containing a large terminal subset and half the central 20S subset of components. For clarification of the molecular structure of the 26S proteasome complex in solution, its physicochemical parameters were calculated theoretically using a model based on this caterpillar-shaped complex. The values obtained for the Stokes radius and radius of gyration of 12.2 and 14.9 nm were consistent with the experimental values. These results provide evidence that the 26S proteasome complex is a cylindrical caterpillar-like structure of ‗30S‘ in solution, consisting of a 20S proteasome component with proteolytic function and multiple other components, which possibly have regulatory roles. © 1994 Academic Press. All rights reserved.

DOI： 10.1006/jsbi.1993.1050

Publishing type：Research paper (international conference proceedings)  

Publishing type：Research paper (scientific journal)  

The molecular properties of an ATP/ubiquitin-dependent ‗26S‘ proteasome complex purified from rat liver were examined by physicochemical, biochemical, and morphological analyses. On ultracentrifugation, the proteasome complex sedimented as almost a single component with a sedimentation coefficient of 30.3S. Dynamic light-scattering measurements indicated that it has a diffusion coefficient of 1.38 x 10-7 cm2/sec and a Stokes radius of 15.5 nm. From these two coefficients, the protein complex was estimated to have the high molecular weight of 2.02 x 106. Static light-scattering analysis indicated a molecular weight of 1.91 x 106 and a radius of gyration of 16.8 nm. The proteasome complex was found to be composed of multiple subunits of the 20S proteasome with molecular weights of 2.1-3.1 x 104 and 15-20 protein species with molecular weights of 3.5-11.0 x 104, which were directly associated with the 20S proteasome. The electron micrographic finding that the 26S proteasome complex had a caterpillar shape, direct electron-microscopic observations on the subunit arrangement of the 20S proteasome, and classification of the subunits of the latter into two groups with respect to sequence homology suggested that the 26S complex is a symmetrical assembly of two domains, each containing a large terminal subset and half the central 20S subset of components. For clarification of the molecular structure of the 26S proteasome complex in solution, its physicochemical parameters were calculated theoretically using a model based on this caterpillar-shaped complex. The values obtained for the Stokes radius and radius of gyration of 12.2 and 14.9 nm were consistent with the experimental values. These results provide evidence that the 26S proteasome complex is a cylindrical caterpillar-like structure of ‗30S‘ in solution, consisting of a 20S proteasome component with proteolytic function and multiple other components, which possibly have regulatory roles. © 1994 Academic Press. All rights reserved.

DOI： 10.1006/jsbi.1993.1050

Publishing type：Research paper (scientific journal)  

The nucleotide sequence of a cDNA that encodes a new subunit, named RC1, of rat proteasomes (multicatalytic proteinase complexes) has been determined. The polypeptide predicted from the open reading frame consisted of 208 amino acid residues with a calculated molecular mass of 23,130, which is consistent with the size obtained by electrophoretic analysis of purified RC1. The partial amino acid sequences of several fragments of RC1, obtained by protein chemical analyses, were found to be in excellent accordance with those deduced from the cDNA sequence. Surprisingly, the overall structure of RC1 was found to be almost identical to that of recently isolated RING10, whose gene is located in the class II region of the human MHC gene cluster. This finding suggests that RC1 is a homologue of human RING10, supporting the proposal that proteasomes are involved in the antigen processing pathway. © 1992.

DOI： 10.1016/0014-5793(92)80211-X

PubMed

Publishing type：Research paper (scientific journal)  

The nucleotide sequence of a cDNA that encodes a new subunit, named RC1, of rat proteasomes (multicatalytic proteinase complexes) has been determined. The polypeptide predicted from the open reading frame consisted of 208 amino acid residues with a calculated molecular mass of 23,130, which is consistent with the size obtained by electrophoretic analysis of purified RC1. The partial amino acid sequences of several fragments of RC1, obtained by protein chemical analyses, were found to be in excellent accordance with those deduced from the cDNA sequence. Surprisingly, the overall structure of RC1 was found to be almost identical to that of recently isolated RING10, whose gene is located in the class II region of the human MHC gene cluster. This finding suggests that RC1 is a homologue of human RING10, supporting the proposal that proteasomes are involved in the antigen processing pathway. © 1992.

DOI： 10.1016/0014-5793(92)80211-X

PubMed

Publishing type：Research paper (scientific journal)  

Seventeen murine monoclonal antibodies (mAbs) against horseshoe crab clotting factor, factor C, were prepared and characterized. When the binding sites of these mAbs were analyzed by immunoblotting, ten mAbs recognized nonreduced factor C, five mAbs were directed against the heavy chain, and two mAbs were directed against the B chain. Three mAbs, 1H4, 2C12, and 2A7, one selected from each group, were used for further study. The mAb 1H4, which recognized only nonreduced factor C molecule, inhibited the factor C activity in a dose-dependent manner. It also inhibited lipopolysaccharide (LPS)- and α-chymotrypsln-mediated activations of the zymogen factor C, suggesting that 1H4 binds close to the active site and/or the substrate-binding site located in the serine protease domain (B chain) of factor C. On the other hand, 2C12 and 2A7 recognized, respectively, an epitope located in the heavy and the B chains, and inhibited LPS-mediated activation of factor C, but not α-chymotrypsin-mediated activation of factor C or factor C¯ activity. Both F(ab')2 and Fab' fragments derived from 2C12 inhibited LPS-mediated activation in the same manner. These three mAbs did not bind with LPS, although a factor C-mAb complex was able to bind LPS, suggesting that the LPS-mediated activation of the zymogen factor C was induced through Intermolecular interaction between the LPS-bound factor C mole cules. The dissociation constants (Kd) for 1H4, 2C12, and 2A7 binding to factor C were determined as 1.9×10-90.6×10-10and 1.8×10 respectively. By using 2C12 and polyclonal antibody against factor C¯, an enzyme-linked immunosorbent assay for quantitative determination of factor C was established. © 1992 BY THE JOURNAL OF BIOCHEMISTRY.

DOI： 10.1093/oxfordjournals.jbchem.a123924

PubMed

Publishing type：Research paper (scientific journal)  

Seventeen murine monoclonal antibodies (mAbs) against horseshoe crab clotting factor, factor C, were prepared and characterized. When the binding sites of these mAbs were analyzed by immunoblotting, ten mAbs recognized nonreduced factor C, five mAbs were directed against the heavy chain, and two mAbs were directed against the B chain. Three mAbs, 1H4, 2C12, and 2A7, one selected from each group, were used for further study. The mAb 1H4, which recognized only nonreduced factor C molecule, inhibited the factor C activity in a dose-dependent manner. It also inhibited lipopolysaccharide (LPS)- and α-chymotrypsln-mediated activations of the zymogen factor C, suggesting that 1H4 binds close to the active site and/or the substrate-binding site located in the serine protease domain (B chain) of factor C. On the other hand, 2C12 and 2A7 recognized, respectively, an epitope located in the heavy and the B chains, and inhibited LPS-mediated activation of factor C, but not α-chymotrypsin-mediated activation of factor C or factor C¯ activity. Both F(ab')2 and Fab' fragments derived from 2C12 inhibited LPS-mediated activation in the same manner. These three mAbs did not bind with LPS, although a factor C-mAb complex was able to bind LPS, suggesting that the LPS-mediated activation of the zymogen factor C was induced through Intermolecular interaction between the LPS-bound factor C mole cules. The dissociation constants (Kd) for 1H4, 2C12, and 2A7 binding to factor C were determined as 1.9×10-90.6×10-10and 1.8×10 respectively. By using 2C12 and polyclonal antibody against factor C¯, an enzyme-linked immunosorbent assay for quantitative determination of factor C was established. © 1992 BY THE JOURNAL OF BIOCHEMISTRY.

DOI： 10.1093/oxfordjournals.jbchem.a123924

PubMed

CiNii Article

Publishing type：Research paper (scientific journal)  

The structure of hemocytes in the normal state and during blood coagulation, and the intracellular localization of three clotting factors and two antimicrobial factors were examined in the Japanese horseshoe crab Tachypleus tridentatus. Two types of hemocytes were found in the circulating blood: non-granular and granular hemocytes. The latter contained numerous dense granules classed into two major types: L- and D-granules. The L-granules were larger (up to 1.5-mu-m in diameter) and less electron-dense than the D-granules (less than 0.6-mu-m in diameter). The L-granules contained three clotting factors and one antimicrobial factor, whereas the D-granules exclusively contained the other antimicrobial factor. After treatment with endotoxin, the L-granules were released more rapidly than the D-granules, although almost all granules were finally exocytosed. The granular hemocyte possessed a single Golgi complex; possible precursor granules of L-granules and D-granules contained tubular and condensed dense material, respectively. These data are discussed in relation to the self-defense mechanisms of the horseshoe crab.

Publishing type：Research paper (scientific journal)  

The structure of hemocytes in the normal state and during blood coagulation, and the intracellular localization of three clotting factors and two antimicrobial factors were examined in the Japanese horseshoe crab Tachypleus tridentatus. Two types of hemocytes were found in the circulating blood: non-granular and granular hemocytes. The latter contained numerous dense granules classed into two major types: L- and D-granules. The L-granules were larger (up to 1.5-mu-m in diameter) and less electron-dense than the D-granules (less than 0.6-mu-m in diameter). The L-granules contained three clotting factors and one antimicrobial factor, whereas the D-granules exclusively contained the other antimicrobial factor. After treatment with endotoxin, the L-granules were released more rapidly than the D-granules, although almost all granules were finally exocytosed. The granular hemocyte possessed a single Golgi complex; possible precursor granules of L-granules and D-granules contained tubular and condensed dense material, respectively. These data are discussed in relation to the self-defense mechanisms of the horseshoe crab.

Publishing type：Research paper (scientific journal)  

Direct virus inactivation of tachyplesin I and related isopeptides, which are antimicrobial peptides isolated from the hemocytes of the horseshoe crab (Tachypleus tridentatus and Limulus polyphemus), was examined against several viruses. Vesicular stomatitis virus (VSV) was inactivated by incubation with tachyplesin I and its isopeptides. Influenza A (H1N1) virus was slightly inactivated by tachyplesin I, whereas herpes simplex virus 1 and 2, adenovirus 1, reovirus 2 and poliovirus 1 were resistant to inactivation. The inactivation of VSV by tachyplesin I depended on the concentration, the time and the temperature of incubation. Pretreatment of tachyplesin I with trypsin or lipopolysaccharide of gram-negative bacteria entirely abolished the antiviral activity. Electron microscopy of VSV treated with tachyplesin I showed naked and damaged virions. These data suggest that tachyplesin I directly inactivates the VSV by destroying its envelope subunits.

Publishing type：Research paper (scientific journal)  

A novel thermolabile β-2 macroglycoprotein ('thermolabile substance' (TLS) or 'Hakata antigen' (HA)), which was detected by the precipitating (auto) antibodies of patients with systemic lupus erythematosus, was isolated and characterized. The purification procedure entailed the following steps: isoelectric precipitation in the range between pH 5.2-6.1, hydroxyapatite absorption chromatography, 35% saturated ammonium sulfate precipitation, Sephadex G-200 gel filtration, Pevikon block electrophoresis, lentil lectin affinity chromatography and immobilized rabbit anti-human whole serum IgG column chromatography. Utilizing these procedures, 0.1 mg of HA was purified from 3 I of pooled human serum. The molecular mass of HA was determined as 650 kDa by Sepharose 4B gel filtration. On SDS-PAGE analysis, HA showed a single band at 35 kDa under reduced conditions and numerous ladder bands between 35 kDa to more than 300 kDa under nonreduced conditions. On analytical ultracentrifugation, Ha gave a molecular mass of 520 kDa with a single meniscus and a sedimentation constant of 12.0. The amino acid and carbohydrate analysis of reduced and S-pyridylethylated HA revealed that it contained five residues of hydroxyproline and an N-linked type sugar chain. © 1991.

DOI： 10.1016/0167-4838(91)90158-V

PubMed

Publishing type：Research paper (scientific journal)  

A novel thermolabile β-2 macroglycoprotein ('thermolabile substance' (TLS) or 'Hakata antigen' (HA)), which was detected by the precipitating (auto) antibodies of patients with systemic lupus erythematosus, was isolated and characterized. The purification procedure entailed the following steps: isoelectric precipitation in the range between pH 5.2-6.1, hydroxyapatite absorption chromatography, 35% saturated ammonium sulfate precipitation, Sephadex G-200 gel filtration, Pevikon block electrophoresis, lentil lectin affinity chromatography and immobilized rabbit anti-human whole serum IgG column chromatography. Utilizing these procedures, 0.1 mg of HA was purified from 3 I of pooled human serum. The molecular mass of HA was determined as 650 kDa by Sepharose 4B gel filtration. On SDS-PAGE analysis, HA showed a single band at 35 kDa under reduced conditions and numerous ladder bands between 35 kDa to more than 300 kDa under nonreduced conditions. On analytical ultracentrifugation, Ha gave a molecular mass of 520 kDa with a single meniscus and a sedimentation constant of 12.0. The amino acid and carbohydrate analysis of reduced and S-pyridylethylated HA revealed that it contained five residues of hydroxyproline and an N-linked type sugar chain. © 1991.

DOI： 10.1016/0167-4838(91)90158-V

PubMed

Publishing type：Research paper (scientific journal)  

Factor C is an endotoxin-sensitive, intracellular serine protease zymogen which initiates the coagulation cascade system in the limulus hemolymph. We have determined the entire amino acid sequence of factor C using recombinant DNA technique. The zymogen consisted of 994 amino acid residues with a calculated molecular mass of 109,648 Da. Most interestingly, factor C has five repeating units ("Sushi" domain or short consensus repeat) of about 60 amino acid residues each, which have been found in many proteins participating in the mammalian complement system. In addition to a typical serine protease domain in the carboxyl-terminal portion, characteristic segments with an epidermal growth factor-like, a lectin-like, a cysteine-rich, and a proline-rich domain were also found, revealing a unique mosaic protein structure. The serine protease domain was most analogous to human thrombin. Factor C was identified to localize in large granules in the cell, indicating that it is released from the cell by lipopolysaccharide stimulation. Furthermore, we identified a transcript possibly derived by alternative splicing of factor C mRNA, which encodes a protein sharing the amino-terminal portion of factor C. We suggest that factor C, a newly discovered type of serine protease zymogen, is a "coagulation-complement factor" which may play important roles in both hemostasis and host defense mechanisms.

Publishing type：Research paper (scientific journal)  

Factor C is an endotoxin-sensitive, intracellular serine protease zymogen which initiates the coagulation cascade system in the limulus hemolymph. We have determined the entire amino acid sequence of factor C using recombinant DNA technique. The zymogen consisted of 994 amino acid residues with a calculated molecular mass of 109,648 Da. Most interestingly, factor C has five repeating units ("Sushi" domain or short consensus repeat) of about 60 amino acid residues each, which have been found in many proteins participating in the mammalian complement system. In addition to a typical serine protease domain in the carboxyl-terminal portion, characteristic segments with an epidermal growth factor-like, a lectin-like, a cysteine-rich, and a proline-rich domain were also found, revealing a unique mosaic protein structure. The serine protease domain was most analogous to human thrombin. Factor C was identified to localize in large granules in the cell, indicating that it is released from the cell by lipopolysaccharide stimulation. Furthermore, we identified a transcript possibly derived by alternative splicing of factor C mRNA, which encodes a protein sharing the amino-terminal portion of factor C. We suggest that factor C, a newly discovered type of serine protease zymogen, is a "coagulation-complement factor" which may play important roles in both hemostasis and host defense mechanisms.

Publishing type：Research paper (scientific journal)  

米国産カブトガニ(Limulus polyphemus)よりC因子を精製し,日本産カブトガニのC因子と比較した.米国産C因子は分子量およびアミノ酸組成,サブユニット構成のほか,免疫化学的にも日本産のそれと相同であった.日本産C因子同様,米国産C因子もリポ多糖や合成リピドAにより自己触媒的に活性化され,興味深いことに両C因子はトリプシンではなくα-キモトリプシンによって活性化され,活性型C因子はα-トロンビン様の基質特異性を示した.一方,体液凝固系に関与するすべての因子を含む血球抽出液もキモトリプシンにより活性化され,ゲルを形成した.哺乳類の凝固・線溶・補体系がすべてトリプシン様酵素によって活性化されるのに対して,カブドガニ凝固系はキモトリプシンによって開始される新しいタイプのカスケード系である

Publishing type：Research paper (scientific journal)  

米国産カブトガニ(Limulus polyphemus)よりC因子を精製し,日本産カブトガニのC因子と比較した.米国産C因子は分子量およびアミノ酸組成,サブユニット構成のほか,免疫化学的にも日本産のそれと相同であった.日本産C因子同様,米国産C因子もリポ多糖や合成リピドAにより自己触媒的に活性化され,興味深いことに両C因子はトリプシンではなくα-キモトリプシンによって活性化され,活性型C因子はα-トロンビン様の基質特異性を示した.一方,体液凝固系に関与するすべての因子を含む血球抽出液もキモトリプシンにより活性化され,ゲルを形成した.哺乳類の凝固・線溶・補体系がすべてトリプシン様酵素によって活性化されるのに対して,カブドガニ凝固系はキモトリプシンによって開始される新しいタイプのカスケード系である

Publishing type：Research paper (scientific journal)  

米国産カブトガニ(Limulus polyphemus)よりC因子を精製し,日本産カブトガニのC因子と比較した.米国産C因子は分子量およびアミノ酸組成,サブユニット構成のほか,免疫化学的にも日本産のそれと相同であった.日本産C因子同様,米国産C因子もリポ多糖や合成リピドAにより自己触媒的に活性化され,興味深いことに両C因子はトリプシンではなくα-キモトリプシンによって活性化され,活性型C因子はα-トロンビン様の基質特異性を示した.一方,体液凝固系に関与するすべての因子を含む血球抽出液もキモトリプシンにより活性化され,ゲルを形成した.哺乳類の凝固・線溶・補体系がすべてトリプシン様酵素によって活性化されるのに対して,カブドガニ凝固系はキモトリプシンによって開始される新しいタイプのカスケード系である

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (international conference proceedings)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/0006-291X(90)91199-3

Other URL： http://orcid.org/0000-0003-2409-6556

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/0006-291X(90)91199-3

DOI： 10.1016/0006-291x(90)91199-3

Publishing type：Research paper (scientific journal)  

オルニチントランスカルバミラーゼ(OTC)とOTC前駆体の性質を比較する目的で,ラットOTCを大腸菌で発現させ,沈澱画分より尿素またはグアニジン塩酸で抽出し,ほぼ均一に精製した.塩酸グアニジンで可溶化した成熟型OTC希釈後0°Cでインキュベートすることにより,比活性170 μmol/min/mg protein (native酵素の18%)まで活性を回復した.同様の条件下で,OTC前駆体は125 μmol/min/mgまで活性化された.nativeおよび再活性化OTCの沈降係数は6.2Sであるのに対し,活性化OTC前駆体は5.2Sと少し異なるが,ともに三量体を形成していることが示唆された.したがって,前駆体に附着している延長ペプチドは必ずしも前駆体のfoldingを阻害しないことが示された.精製前駆体を8M尿素で可溶化後希釈すると直ちに不溶化して沈澱するが,網状赤血球ライセート存在下では沈降係数6〜11Sの可溶性複合体を形成した.成熟OTCではこのような複合体の形成は見られない.この複合体形成は精製前駆体および化学合成したOTCの延長ペプチドによって阻害されたが,成熟型OTCでは阻害されなかった.これらの結果より,網状赤血球ライセートのなかには,OTCの延長ペプチド部分に結合して可溶性の複合体を形成するサイトソル因子が存在することが示唆された

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

オルニチントランスカルバミラーゼ(OTC)とOTC前駆体の性質を比較する目的で,ラットOTCを大腸菌で発現させ,沈澱画分より尿素またはグアニジン塩酸で抽出し,ほぼ均一に精製した.塩酸グアニジンで可溶化した成熟型OTC希釈後0°Cでインキュベートすることにより,比活性170 μmol/min/mg protein (native酵素の18%)まで活性を回復した.同様の条件下で,OTC前駆体は125 μmol/min/mgまで活性化された.nativeおよび再活性化OTCの沈降係数は6.2Sであるのに対し,活性化OTC前駆体は5.2Sと少し異なるが,ともに三量体を形成していることが示唆された.したがって,前駆体に附着している延長ペプチドは必ずしも前駆体のfoldingを阻害しないことが示された.精製前駆体を8M尿素で可溶化後希釈すると直ちに不溶化して沈澱するが,網状赤血球ライセート存在下では沈降係数6〜11Sの可溶性複合体を形成した.成熟OTCではこのような複合体の形成は見られない.この複合体形成は精製前駆体および化学合成したOTCの延長ペプチドによって阻害されたが,成熟型OTCでは阻害されなかった.これらの結果より,網状赤血球ライセートのなかには,OTCの延長ペプチド部分に結合して可溶性の複合体を形成するサイトソル因子が存在することが示唆された

Publishing type：Research paper (scientific journal)  

オルニチントランスカルバミラーゼ(OTC)とOTC前駆体の性質を比較する目的で,ラットOTCを大腸菌で発現させ,沈澱画分より尿素またはグアニジン塩酸で抽出し,ほぼ均一に精製した.塩酸グアニジンで可溶化した成熟型OTC希釈後0°Cでインキュベートすることにより,比活性170 μmol/min/mg protein (native酵素の18%)まで活性を回復した.同様の条件下で,OTC前駆体は125 μmol/min/mgまで活性化された.nativeおよび再活性化OTCの沈降係数は6.2Sであるのに対し,活性化OTC前駆体は5.2Sと少し異なるが,ともに三量体を形成していることが示唆された.したがって,前駆体に附着している延長ペプチドは必ずしも前駆体のfoldingを阻害しないことが示された.精製前駆体を8M尿素で可溶化後希釈すると直ちに不溶化して沈澱するが,網状赤血球ライセート存在下では沈降係数6～11Sの可溶性複合体を形成した.成熟OTCではこのような複合体の形成は見られない.この複合体形成は精製前駆体および化学合成したOTCの延長ペプチドによって阻害されたが,成熟型OTCでは阻害されなかった.これらの結果より,網状赤血球ライセートのなかには,OTCの延長ペプチド部分に結合して可溶性の複合体を形成するサイトソル因子が存在することが示唆された

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Proteasomes are multicatalytic proteinase complexes consisting of a set of non-identical polypeptide subunits. A cDNA for component C5 of rat proteasomes was isolated by screening a Reuber H4TG hepatoma cell cDNA library using synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The polypeptide deduced from the open reading frame consisted of 240 amino acid residues with a calculated molecular weight of 26479. Computer analysis revealed little similarity of C5 to other proteins reported so far. The primary structure of C5 showed partial sequence homology to that of another component C3, but no regions of homology with the sequence of component C2. Thus C5 is concluded to be a new type of subunit of the proteasome complex. © 1990.

DOI： 10.1016/0014-5793(90)80773-C

PubMed

Publishing type：Research paper (scientific journal)  

Proteasomes are multicatalytic proteinase complexes consisting of a set of non-identical polypeptide subunits. A cDNA for component C5 of rat proteasomes was isolated by screening a Reuber H4TG hepatoma cell cDNA library using synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The polypeptide deduced from the open reading frame consisted of 240 amino acid residues with a calculated molecular weight of 26479. Computer analysis revealed little similarity of C5 to other proteins reported so far. The primary structure of C5 showed partial sequence homology to that of another component C3, but no regions of homology with the sequence of component C2. Thus C5 is concluded to be a new type of subunit of the proteasome complex. © 1990.

DOI： 10.1016/0014-5793(90)80773-C

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/0014-5793(90)80267-M

Other URL： http://orcid.org/0000-0003-2409-6556

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/0014-5793(90)80267-M

DOI： 10.1016/0014-5793(90)80267-m

Publishing type：Research paper (scientific journal)  

DOI： 10.1021/bi00467a026

J-GLOBAL

Publishing type：Research paper (scientific journal)  

DOI： 10.1021/bi00467a026

J-GLOBAL

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

DOI： 10.1007/978-1-4757-5140-6_23

PubMed

Publishing type：Research paper (international conference proceedings)  

Publishing type：Research paper (international conference proceedings)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

日本産および米国産(Limulus polyphemus)の血球中に,抗菌性を示すタキプレシン2とポリフェムシン1,2を新たに見いだし,それらの一次構造を決定した.タキプレシン2は17アミノ酸残基から成り,先に見いだしたタキプレシン1(改名)との間では,僅かにNH2末端Lys残基がArg残基に,15番目のArg残基がLys残基に置換されている以外,同じ構造を示した.一方,米国産のポリフェムシン1と2は,18アミノ酸残基を含み,両者の違いはNH2末端から10番目が,1ではArg, 2ではLysに置換されていた.これらのペプチドは,いずれもC末端Arg-α-amideを有し,かつ強塩基性のものであった.ジスルフィド結合は2個所あり,その架橋結合はポリフェムシン1を用いて決定したのみであるが,タキプレシン1と同様であった.抗菌性については,タキプレシン1と同様,グラム陰性菌と陽性菌に対して強い成長阻止活性を示し,新たに抗カビ活性のあることを見いだした

Publishing type：Research paper (scientific journal)  

日本産および米国産(Limulus polyphemus)の血球中に,抗菌性を示すタキプレシン2とポリフェムシン1,2を新たに見いだし,それらの一次構造を決定した.タキプレシン2は17アミノ酸残基から成り,先に見いだしたタキプレシン1(改名)との間では,僅かにNH2末端Lys残基がArg残基に,15番目のArg残基がLys残基に置換されている以外,同じ構造を示した.一方,米国産のポリフェムシン1と2は,18アミノ酸残基を含み,両者の違いはNH2末端から10番目が,1ではArg, 2ではLysに置換されていた.これらのペプチドは,いずれもC末端Arg-α-amideを有し,かつ強塩基性のものであった.ジスルフィド結合は2個所あり,その架橋結合はポリフェムシン1を用いて決定したのみであるが,タキプレシン1と同様であった.抗菌性については,タキプレシン1と同様,グラム陰性菌と陽性菌に対して強い成長阻止活性を示し,新たに抗カビ活性のあることを見いだした

Publishing type：Research paper (scientific journal)  

日本産および米国産(Limulus polyphemus)の血球中に,抗菌性を示すタキプレシン2とポリフェムシン1,2を新たに見いだし,それらの一次構造を決定した.タキプレシン2は17アミノ酸残基から成り,先に見いだしたタキプレシン1(改名)との間では,僅かにNH2末端Lys残基がArg残基に,15番目のArg残基がLys残基に置換されている以外,同じ構造を示した.一方,米国産のポリフェムシン1と2は,18アミノ酸残基を含み,両者の違いはNH2末端から10番目が,1ではArg, 2ではLysに置換されていた.これらのペプチドは,いずれもC末端Arg-α-amideを有し,かつ強塩基性のものであった.ジスルフィド結合は2個所あり,その架橋結合はポリフェムシン1を用いて決定したのみであるが,タキプレシン1と同様であった.抗菌性については,タキプレシン1と同様,グラム陰性菌と陽性菌に対して強い成長阻止活性を示し,新たに抗カビ活性のあることを見いだした

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Tachyplesin is an antimicrobial peptide recently found in the acid extract of hemocytes from the Japanese horseshoe crab (Tachypleus tridentatus) [Nakamura, T. et al. (1988) J. Biol Chem. 263, 16709-16713]. In our continuing studies on the peptide, we have found an isopeptide, tachyplesin II, and also polyphemusins I and II in hemocytes of the American horseshoe crab (Limulus polyphemus). The complete primary structures of these peptides, which are very similar to that of the previously isolated peptide, now named tachyplesin I, were determined to be as follows:Polyphemusin I NH2-R-R-W-C-F-R-V-C-Y-R-G-F-C-Y-R-K-C-R-CONH2Polyphemusin II NH2-R-R-W-C-F-R-V-C-Y-K-G-F-C-Y-R-K-C-R-CONH2Tachyplesin II NH2-R-W-C-F-R-V-C-Y-R-G-I- C-Y-R-K-C-R-CONH2The isopeptide, tachyplesin II, consists of 17 residues with a COOH-terminal arginine a-amide. On the other hand, both polyphemusins I and II were found to contain 18 residues due to an additional Arg residue at the NH2-terminal end as well as a COOH-terminal arginine α-amide. The disulfide linkages for polyphemusin I consisted of two bridges between Cys-4 and Cys-17 and between Cys-8 and Cys-13, which was identical to in the case of tachyplesin I. Moreover, all of these peptides inhibited the growth of not only Gram-negative and -positive bacteria but also fungi, such as Candida albicans M9. Furthermore, complex formation between these peptides and bacterial lipopolysaccharides was also observed in a double diffusion test. These results suggest that tachyplesins and poly-phemusins are probably located in the hemocyte membrane, where they act on antimi-crobial peptides as a self-defense mechanism in the horseshoe crab against invading microorganisms. © 1989 BY THE JOURNAL OF BIOCHEMISTRY.

DOI： 10.1093/oxfordjournals.jbchem.a122913

PubMed

Publishing type：Research paper (scientific journal)  

Tachyplesin I, a 17-residue peptide amide with two disulfide bridges isolated from an acid extract of horseshoe crab hemocyte debris, was synthesized by the 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase method followed by two steps of deprotection and subsequent air-oxidation. The thioanisole-mediated deprotection with 1 M trifluoromethanesulfonic acid was first employed to cleave the peptide amide from the resin and, at the same time, to deprotect the side chain protecting groups employed, except for the S-Acm (acetamidomethyl) group. The 4 Acm groups attached were next cleaved by silver trifluoromethanesulfonate. In addition, two related peptides, tachyplesin II and polyphemusin I, were similarly synthesized. Synthetic tachyplesin I inhibited the lipopolysaccharide-mediated activation of clotting factor C to the same extent as did the corresponding natural peptide. The relative potencies of synthetic tachyplesin II and synthetic polyphemusin I with respect to natural tachyplesin I (taken as 1) were 2.1 and 0.61, respectively. © 1989, The Pharmaceutical Society of Japan. All rights reserved.

DOI： 10.1248/cpb.37.2661

Publishing type：Research paper (scientific journal)  

Tachyplesin is an antimicrobial peptide recently found in the acid extract of hemocytes from the Japanese horseshoe crab (<I>Tachypleus tridentatus</I>)[Nakamura, T. et al.(1988) <I>J. Biol. Chem</I>. 263, 16709-16713]. In our continuing studies on the peptide, we have found an isopeptide, tachyplesin II, and also polyphemusins I and II in hemocytes of the American horseshoe crab (<I>Limulus polyphemus</I>). The complete primary structures of these peptides, which are very similar to that of the previously isolated peptide, now named tachyplesin I, were determined to be as follows:<BR>Polyphemusin I NH<I>2</I>-R-R-W-C-F-R-V-C-Y-R-G-F-C-Y-R-K-C-R-CONH<I>2</I><BR>Polyphemusin II NH<I>2</I>-R-R-W-C-F-R-V-C-Y-K-G-F-C-Y-R-K-C-R-CONH<I>2</I><BR>Tachyplesin II NH<I>2</I>-R-W-C-F-R-V-C-Y-R-G-I- C-Y-R-K-C-R-CONH<I>2</I><BR>The isopeptide, tachyplesin II, consists of 17 residues with a COOH-terminal arginine α-amide. On the other hand, both polyphemusins I and II were found to contain 18 residues due to an additional Arg residue at the NH, -terminal end as well as a COOH-terminal arginine α-amide. The disulfide linkages for polyphemusin I consisted of two bridges between Cys-4 and Cys-17 and between Cys-8 and Cys-13, which was identical to in the case of tachyplesin I. Moreover, all of these peptides inhibited the growth of not only Gramnegative and-positive bacteria but also fungi, such as <I>Candida albicans</I> M9. Furthermore, complex formation between these peptides and bacterial lipopolysaccharides was also observed in a double diffusion test. These results suggest that tachyplesins and polyphemusins are probably located in the hemocyte membrane, where they act on antimicrobial peptides as a self-defense mechanism in the horseshoe crab against invading microorganisms.

CiNii Article

Publishing type：Research paper (scientific journal)  

Tachyplesin is an antimicrobial peptide recently found in the acid extract of hemocytes from the Japanese horseshoe crab (Tachypleus tridentatus) [Nakamura, T. et al. (1988) J. Biol Chem. 263, 16709-16713]. In our continuing studies on the peptide, we have found an isopeptide, tachyplesin II, and also polyphemusins I and II in hemocytes of the American horseshoe crab (Limulus polyphemus). The complete primary structures of these peptides, which are very similar to that of the previously isolated peptide, now named tachyplesin I, were determined to be as follows:Polyphemusin I NH2-R-R-W-C-F-R-V-C-Y-R-G-F-C-Y-R-K-C-R-CONH2Polyphemusin II NH2-R-R-W-C-F-R-V-C-Y-K-G-F-C-Y-R-K-C-R-CONH2Tachyplesin II NH2-R-W-C-F-R-V-C-Y-R-G-I- C-Y-R-K-C-R-CONH2The isopeptide, tachyplesin II, consists of 17 residues with a COOH-terminal arginine a-amide. On the other hand, both polyphemusins I and II were found to contain 18 residues due to an additional Arg residue at the NH2-terminal end as well as a COOH-terminal arginine α-amide. The disulfide linkages for polyphemusin I consisted of two bridges between Cys-4 and Cys-17 and between Cys-8 and Cys-13, which was identical to in the case of tachyplesin I. Moreover, all of these peptides inhibited the growth of not only Gram-negative and -positive bacteria but also fungi, such as Candida albicans M9. Furthermore, complex formation between these peptides and bacterial lipopolysaccharides was also observed in a double diffusion test. These results suggest that tachyplesins and poly-phemusins are probably located in the hemocyte membrane, where they act on antimi-crobial peptides as a self-defense mechanism in the horseshoe crab against invading microorganisms. © 1989 BY THE JOURNAL OF BIOCHEMISTRY.

DOI： 10.1093/oxfordjournals.jbchem.a122913

PubMed

Publishing type：Research paper (scientific journal)  

Tachyplesin is an antimicrobial peptide recently found in the acid extract of hemocytes from the Japanese horseshoe crab (<I>Tachypleus tridentatus</I>)[Nakamura, T. et al.(1988) <I>J. Biol. Chem</I>. 263, 16709-16713]. In our continuing studies on the peptide, we have found an isopeptide, tachyplesin II, and also polyphemusins I and II in hemocytes of the American horseshoe crab (<I>Limulus polyphemus</I>). The complete primary structures of these peptides, which are very similar to that of the previously isolated peptide, now named tachyplesin I, were determined to be as follows:<BR>Polyphemusin I NH<I>2</I>-R-R-W-C-F-R-V-C-Y-R-G-F-C-Y-R-K-C-R-CONH<I>2</I><BR>Polyphemusin II NH<I>2</I>-R-R-W-C-F-R-V-C-Y-K-G-F-C-Y-R-K-C-R-CONH<I>2</I><BR>Tachyplesin II NH<I>2</I>-R-W-C-F-R-V-C-Y-R-G-I- C-Y-R-K-C-R-CONH<I>2</I><BR>The isopeptide, tachyplesin II, consists of 17 residues with a COOH-terminal arginine α-amide. On the other hand, both polyphemusins I and II were found to contain 18 residues due to an additional Arg residue at the NH, -terminal end as well as a COOH-terminal arginine α-amide. The disulfide linkages for polyphemusin I consisted of two bridges between Cys-4 and Cys-17 and between Cys-8 and Cys-13, which was identical to in the case of tachyplesin I. Moreover, all of these peptides inhibited the growth of not only Gramnegative and-positive bacteria but also fungi, such as <I>Candida albicans</I> M9. Furthermore, complex formation between these peptides and bacterial lipopolysaccharides was also observed in a double diffusion test. These results suggest that tachyplesins and polyphemusins are probably located in the hemocyte membrane, where they act on antimicrobial peptides as a self-defense mechanism in the horseshoe crab against invading microorganisms.

CiNii Article

Publishing type：Research paper (scientific journal)  

Tachyplesin I, a 17-residue peptide amide with two disulfide bridges isolated from an acid extract of horseshoe crab hemocyte debris, was synthesized by the 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase method followed by two steps of deprotection and subsequent air-oxidation. The thioanisole-mediated deprotection with 1 M trifluoromethanesulfonic acid was first employed to cleave the peptide amide from the resin and, at the same time, to deprotect the side chain protecting groups employed, except for the S-Acm (acetamidomethyl) group. The 4 Acm groups attached were next cleaved by silver trifluoromethanesulfonate. In addition, two related peptides, tachyplesin II and polyphemusin I, were similarly synthesized. Synthetic tachyplesin I inhibited the lipopolysaccharide-mediated activation of clotting factor C to the same extent as did the corresponding natural peptide. The relative potencies of synthetic tachyplesin II and synthetic polyphemusin I with respect to natural tachyplesin I (taken as 1) were 2.1 and 0.61, respectively. © 1989, The Pharmaceutical Society of Japan. All rights reserved.

DOI： 10.1248/cpb.37.2661

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Publishing type：Research paper (scientific journal)  

Total pages：228   Book type：Scholarly book Participation form：First Author

Book type：Scholarly book Participation form：First Author

Responsible for pages：378-388   Book type：Scholarly book Participation form：First Author

Responsible for pages：336-345   Book type：Scholarly book Participation form：Second Author

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

J-GLOBAL

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J-GLOBAL

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

ユビキチンは、タンパク質をプロテアソーム分解へ導く標識として発見されたが、ユビキチン分子間やユビキチン-基質間で多様に連結することで多彩な細胞機能を制御することが明らかになり、ユビキチンコード(ユビキチン暗号)と称されるようになった。Linear ubiquitin chain assembly complex(LUBAC)は、ユビキチンのN末端Met1を介する直鎖状ユビキチン鎖を生成する唯一のユビキチンリガーゼで、炎症・免疫制御に重要なnuclear factor-κB(NF-κB)シグナルを活性化する。また、直鎖状ユビキチン鎖を分解する脱ユビキチン化酵素や結合タンパク質も同定され、LUBAC制御がになう生理機能や破綻が引き起こす疾患の解明、創薬をめざした阻害剤開発が進められている。(著者抄録)

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J-GLOBAL

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J-GLOBAL

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J-GLOBAL

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Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

脱ユビキチン化酵素(DUB)は,タンパク質分解やシグナル伝達の活性化などのユビキチン化修飾を介した細胞応答を負に制御する重要な因子として注目されている.また,DUBの高発現や異常な活性化が,がんや神経疾患などを誘導することが明らかとなっており,創薬ターゲットとしても注目されている.我々はコムギ無細胞系を用いてヒトのDUBの約8割を網羅する83種類の組換えタンパク質を合成し,DUBタンパク質アレイを作製した.このDUBタンパク質アレイについて,8種類の重合様式の二量体ユビキチンを基質として,76種類のDUBについて活性を検出し,そのユビキチン鎖特異性を明らかにした.さらに,このDUBタンパク質アレイを応用することで,DUBを阻害する化合物のDUB特異性評価パネルを作製した.(著者抄録)

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

ユビキチンのN末端を介して形成される非定型型の直鎖状ユビキチン鎖は,炎症・免疫シグナル応答や細胞死を制御し,その生成を特異的に担うLUBACユビキチンリガーゼの機能不全は多くの疾患発症に関わる.筆者らは家族性筋萎縮性側索硬化症(ALS)の原因遺伝子の一つであるオプチニューリン(OPTN)やアルツハイマー病タウタンパク質の細胞機能解析から,LUBACによる直鎖状ユビキチン鎖生成が異常タンパク質封入体形成,神経炎症・細胞死の亢進に寄与することを見いだした.さらに,新規治療薬創出を最終目的としてLUBACに対する阻害剤の探索を行い,細胞毒性が低くLUBAC特異性の高い新規化合物(HOIPINs)を見いだした.本稿では,筆者らの研究を中心に国内外の動向を交えて,これらの知見を紹介したい.(著者抄録)

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

脱ユビキチン化酵素(DUB)は,タンパク質分解やシグナル伝達の活性化などのユビキチン化修飾を介した細胞応答を負に制御する重要な因子として注目されている.また,DUBの高発現や異常な活性化が,がんや神経疾患などを誘導することが明らかとなっており,創薬ターゲットとしても注目されている.我々はコムギ無細胞系を用いてヒトのDUBの約8割を網羅する83種類の組換えタンパク質を合成し,DUBタンパク質アレイを作製した.このDUBタンパク質アレイについて,8種類の重合様式の二量体ユビキチンを基質として,76種類のDUBについて活性を検出し,そのユビキチン鎖特異性を明らかにした.さらに,このDUBタンパク質アレイを応用することで,DUBを阻害する化合物のDUB特異性評価パネルを作製した.(著者抄録)

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

ユビキチンのN末端を介して形成される非定型型の直鎖状ユビキチン鎖は,炎症・免疫シグナル応答や細胞死を制御し,その生成を特異的に担うLUBACユビキチンリガーゼの機能不全は多くの疾患発症に関わる.筆者らは家族性筋萎縮性側索硬化症(ALS)の原因遺伝子の一つであるオプチニューリン(OPTN)やアルツハイマー病タウタンパク質の細胞機能解析から,LUBACによる直鎖状ユビキチン鎖生成が異常タンパク質封入体形成,神経炎症・細胞死の亢進に寄与することを見いだした.さらに,新規治療薬創出を最終目的としてLUBACに対する阻害剤の探索を行い,細胞毒性が低くLUBAC特異性の高い新規化合物(HOIPINs)を見いだした.本稿では,筆者らの研究を中心に国内外の動向を交えて,これらの知見を紹介したい.(著者抄録)

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Kind of work：Single Work  

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

J-GLOBAL

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Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Kind of work：Single Work  

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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J-GLOBAL

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J-GLOBAL

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脱ユビキチン化酵素(DUB)は連結したユビキチンのC末端を加水分解するプロテアーゼで、ヒトでは約100種存在する。DUBはシステインプロテアーゼのUSP、UCH、ジョセフィン、OTU、MINDYファミリーと金属プロテアーゼのJAMM/MPN+ファミリーという6つのファミリーに分類され、ユビキチン修飾が制御する多彩な細胞機能変換に対して拮抗的に働き、フリーのユビキチン量を確保する。したがって、DUBの遺伝子異常はユビキチンシグナルの制御不全やさまざまな疾患を発症させる。本稿ではヒトDUB酵素群を俯瞰的に概説するとともに、炎症・免疫制御をつかさどり、遺伝子異常が疾患を引き起こすDUBとして知られるCYLD、A20、OTULINを紹介する。さらに、近年注目されているオートファジー制御に関わるDUBやDUBを標的とした最新の創薬研究を解説する。(著者抄録)

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

脱ユビキチン化酵素(DUB)は連結したユビキチンのC末端を加水分解するプロテアーゼで、ヒトでは約100種存在する。DUBはシステインプロテアーゼのUSP、UCH、ジョセフィン、OTU、MINDYファミリーと金属プロテアーゼのJAMM/MPN+ファミリーという6つのファミリーに分類され、ユビキチン修飾が制御する多彩な細胞機能変換に対して拮抗的に働き、フリーのユビキチン量を確保する。したがって、DUBの遺伝子異常はユビキチンシグナルの制御不全やさまざまな疾患を発症させる。本稿ではヒトDUB酵素群を俯瞰的に概説するとともに、炎症・免疫制御をつかさどり、遺伝子異常が疾患を引き起こすDUBとして知られるCYLD、A20、OTULINを紹介する。さらに、近年注目されているオートファジー制御に関わるDUBやDUBを標的とした最新の創薬研究を解説する。(著者抄録)

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

脱ユビキチン化酵素(DUB)は連結したユビキチンのC末端を加水分解するプロテアーゼで、ヒトでは約100種存在する。DUBはシステインプロテアーゼのUSP、UCH、ジョセフィン、OTU、MINDYファミリーと金属プロテアーゼのJAMM/MPN+ファミリーという6つのファミリーに分類され、ユビキチン修飾が制御する多彩な細胞機能変換に対して拮抗的に働き、フリーのユビキチン量を確保する。したがって、DUBの遺伝子異常はユビキチンシグナルの制御不全やさまざまな疾患を発症させる。本稿ではヒトDUB酵素群を俯瞰的に概説するとともに、炎症・免疫制御をつかさどり、遺伝子異常が疾患を引き起こすDUBとして知られるCYLD、A20、OTULINを紹介する。さらに、近年注目されているオートファジー制御に関わるDUBやDUBを標的とした最新の創薬研究を解説する。(著者抄録)

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Kind of work：Joint Work  

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Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

J-GLOBAL

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Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Kind of work：Single Work  

タンパク質のユビキチン修飾は、プロテアソーム分解に導く標識として見出されたが、その後の研究から細胞内輸送、DNA修復、シグナル伝達など多彩な生理機能を制御することが明らかになってきた。我々は、HOIL-1L、HOIP、SHARPINという3サブユニットからなる複合体型ユビキチンリガーゼ(LUBAC)を見出し、LUBACがユビキチンのN末端Met1を介する「直鎖状ユビキチン鎖」という全く新しいタイプのユビキチン連結鎖を特異的に生成することで細胞内シグナル伝達のうちNF-κB経路を制御することを見出した。NF-κBは炎症応答や自然・獲得免疫制御において中心的な働きを司る転写因子で、その活性化経路の破綻は多くの癌、自己免疫疾患、炎症性疾患、生活習慣病、神経変性疾患発症に関わる。また、LUBACによって生成される直鎖状ユビキチン鎖に特異的に結合するドメインをもつタンパク質は、NF-κB活性調節に寄与する。一例として我々は、直鎖状ユビキチン鎖結合ドメイン(UBANドメイン)を有するオプチニューリン(optineurin)がNF-κB活性化経路とアポトーシスを抑制し、そのユビキチン鎖結合性の破綻が筋萎縮性側索硬化症(ALS)発症に深く関わることを突き止めた。(著者抄録)

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J-GLOBAL

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タンパク質のユビキチン修飾は、プロテアソーム分解に導く標識として見出されたが、その後の研究から細胞内輸送、DNA修復、シグナル伝達など多彩な生理機能を制御することが明らかになってきた。我々は、HOIL-1L、HOIP、SHARPINという3サブユニットからなる複合体型ユビキチンリガーゼ(LUBAC)を見出し、LUBACがユビキチンのN末端Met1を介する「直鎖状ユビキチン鎖」という全く新しいタイプのユビキチン連結鎖を特異的に生成することで細胞内シグナル伝達のうちNF-κB経路を制御することを見出した。NF-κBは炎症応答や自然・獲得免疫制御において中心的な働きを司る転写因子で、その活性化経路の破綻は多くの癌、自己免疫疾患、炎症性疾患、生活習慣病、神経変性疾患発症に関わる。また、LUBACによって生成される直鎖状ユビキチン鎖に特異的に結合するドメインをもつタンパク質は、NF-κB活性調節に寄与する。一例として我々は、直鎖状ユビキチン鎖結合ドメイン(UBANドメイン)を有するオプチニューリン(optineurin)がNF-κB活性化経路とアポトーシスを抑制し、そのユビキチン鎖結合性の破綻が筋萎縮性側索硬化症(ALS)発症に深く関わることを突き止めた。(著者抄録)

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

タンパク質のユビキチン修飾は、プロテアソーム分解に導く標識として見出されたが、その後の研究から細胞内輸送、DNA修復、シグナル伝達など多彩な生理機能を制御することが明らかになってきた。我々は、HOIL-1L、HOIP、SHARPINという3サブユニットからなる複合体型ユビキチンリガーゼ(LUBAC)を見出し、LUBACがユビキチンのN末端Met1を介する「直鎖状ユビキチン鎖」という全く新しいタイプのユビキチン連結鎖を特異的に生成することで細胞内シグナル伝達のうちNF-κB経路を制御することを見出した。NF-κBは炎症応答や自然・獲得免疫制御において中心的な働きを司る転写因子で、その活性化経路の破綻は多くの癌、自己免疫疾患、炎症性疾患、生活習慣病、神経変性疾患発症に関わる。また、LUBACによって生成される直鎖状ユビキチン鎖に特異的に結合するドメインをもつタンパク質は、NF-κB活性調節に寄与する。一例として我々は、直鎖状ユビキチン鎖結合ドメイン(UBANドメイン)を有するオプチニューリン(optineurin)がNF-κB活性化経路とアポトーシスを抑制し、そのユビキチン鎖結合性の破綻が筋萎縮性側索硬化症(ALS)発症に深く関わることを突き止めた。(著者抄録)

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J-GLOBAL

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J-GLOBAL

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CiNii Article

Other URL： http://search.jamas.or.jp/link/ui/2016004977

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Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Kind of work：Single Work  

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

CiNii Article

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Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

DOI： 10.1271/kagakutoseibutsu.52.716

CiNii Article

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Publishing type：Research paper, summary (international conference)  

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J-GLOBAL

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J-GLOBAL

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NF-κB経路は免疫や炎症応答に重要なシグナル伝達経路で,ユビキチン化などの翻訳後修飾によって時空間特異的に制御される.本稿では,LUBACユビキチンリガーゼ複合体が生成する新規「直鎖状ポリユビキチン鎖」の生成機構とNF-κB活性化における役割,これに拮抗的に働く脱ユビキチン化酵素について解説する.さらに,LUBAC変異や直鎖状ユビキチン産生不全が関与する多様な疾患について最新の知見を紹介する.(著者抄録)

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

NF-κB経路は免疫や炎症応答に重要なシグナル伝達経路で,ユビキチン化などの翻訳後修飾によって時空間特異的に制御される.本稿では,LUBACユビキチンリガーゼ複合体が生成する新規「直鎖状ポリユビキチン鎖」の生成機構とNF-κB活性化における役割,これに拮抗的に働く脱ユビキチン化酵素について解説する.さらに,LUBAC変異や直鎖状ユビキチン産生不全が関与する多様な疾患について最新の知見を紹介する.(著者抄録)

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Kind of work：Single Work  

NF-κB(nuclear factor-κB)経路は、免疫制御や炎症応答に中心的なシグナル伝達経路で、リン酸化やユビキチン化などの翻訳後修飾がその時空間特異的なシグナル制御に深く関与する。筆者らはユビキチンのN末端を介する新規「直鎖状ポリユビキチン鎖」を生成するユビキチンリガーゼ(LUBAC)を発見し、LUBACがNF-κB制御に必須であることを明らかにした。さらに、LUBACを介するNF-κB活性化を抑制する脱ユビキチン化酵素としてA20を同定し、A20は直鎖状ユビキチンに結合することでNF-κB活性を調節していること、その制御不全が悪性リンパ腫惹起に連関することを見いだした。本稿では、拡大する直鎖状ポリユビキチン鎖生成によるNF-κB制御の生理的意義とその破綻による疾患との関連について紹介する。(著者抄録)

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

NF-κB(nuclear factor-κB)経路は、免疫制御や炎症応答に中心的なシグナル伝達経路で、リン酸化やユビキチン化などの翻訳後修飾がその時空間特異的なシグナル制御に深く関与する。筆者らはユビキチンのN末端を介する新規「直鎖状ポリユビキチン鎖」を生成するユビキチンリガーゼ(LUBAC)を発見し、LUBACがNF-κB制御に必須であることを明らかにした。さらに、LUBACを介するNF-κB活性化を抑制する脱ユビキチン化酵素としてA20を同定し、A20は直鎖状ユビキチンに結合することでNF-κB活性を調節していること、その制御不全が悪性リンパ腫惹起に連関することを見いだした。本稿では、拡大する直鎖状ポリユビキチン鎖生成によるNF-κB制御の生理的意義とその破綻による疾患との関連について紹介する。(著者抄録)

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

NF-κB(nuclear factor-κB)経路は、免疫制御や炎症応答に中心的なシグナル伝達経路で、リン酸化やユビキチン化などの翻訳後修飾がその時空間特異的なシグナル制御に深く関与する。筆者らはユビキチンのN末端を介する新規「直鎖状ポリユビキチン鎖」を生成するユビキチンリガーゼ(LUBAC)を発見し、LUBACがNF-κB制御に必須であることを明らかにした。さらに、LUBACを介するNF-κB活性化を抑制する脱ユビキチン化酵素としてA20を同定し、A20は直鎖状ユビキチンに結合することでNF-κB活性を調節していること、その制御不全が悪性リンパ腫惹起に連関することを見いだした。本稿では、拡大する直鎖状ポリユビキチン鎖生成によるNF-κB制御の生理的意義とその破綻による疾患との関連について紹介する。(著者抄録)

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Kind of work：Single Work  

NF-κB経路は炎症応答や自然・獲得免疫制御にきわめて重要なシグナル伝達経路で、その機能不全は癌、慢性炎症性疾患、自己免疫疾患などの多くの病態発症に深く関連する。NF-κB経路では、キナーゼによるリン酸化とともにLys48、Lys63、Lys11、直鎖状など多様なポリユビキチン鎖形成が重要な役割を果たす。本稿では筆者らが見出した直鎖状ポリユビキチン鎖を生成するLUBACユビキチンリガーゼ複合体の生理機能を中心に、NF-κB経路の多様なユビキチン修飾による制御と病態との連関を解説する。(著者抄録)