論文 - 渡部　匡史

A LANA peptide inhibits tumor growth by inducing CHD4 protein cleavage and triggers cell death.

Hiroki Miura, Kang-Hsin Wang, Tomoki Inagaki, Frank Chuang, Michiko Shimoda, Chie Izumiya, Tadashi Watanabe, Ryan R Davis, Clifford G Tepper, Somayeh Komaki, Ken-Ichi Nakajima, Ashish Kumar, Yoshihiro Izumiya

Cell chemical biology     2024年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes a latent infection, and viral genes are poised to be transcribed in the latent chromatin. In the poised chromatins, KSHV latency-associated nuclear antigen (LANA) interacts with cellular chromodomain-helicase-DNA-binding protein 4 (CHD4) and inhibits viral promoter activation. CHD4 is known to regulate cell differentiation by preventing enhancers from activating promoters. Here, we identified a putative CHD4 inhibitor peptide (VGN73) from the LANA sequence corresponding to the LANA-CHD4 interaction surface. The VGN73 interacts with CHD4 at its PHD domain with a dissociation constant (KD) of 14 nM. Pre-treatment with VGN73 enhanced monocyte differentiation into macrophages and globally altered the repertoire of activated genes in U937 cells. Furthermore, the introduction of the peptide into the cancer cells induced caspase-mediated CHD4 cleavage, triggered cell death, and inhibited tumor growth in a xenograft mouse model. The VGN73 may facilitate cell differentiation therapy.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Conserved cysteine residues in Kaposi's sarcoma herpesvirus ORF34 are necessary for viral production and viral pre-initiation complex formation.

Tadashi Watanabe, Aidan McGraw, Kedhar Narayan, Hasset Tibebe, Kazushi Kuriyama, Mayu Nishimura, Taisuke Izumi, Masahiro Fujimuro, Shinji Ohno

Journal of virology     e0100024   2024年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

UNLABELLED: Kaposi's sarcoma herpesvirus (KSHV) ORF34 plays a significant role as a component of the viral pre-initiation complex (vPIC), which is indispensable for late gene expression across beta- and gammaherpesviruses. Although the key role of ORF34 within the vPIC and its function as a hub protein have been recognized, further clarification regarding its specific contribution to vPIC functionality and interactions with other components is required. This study employed a deep learning algorithm-assisted structural model of ORF34, revealing highly conserved amino acid residues across human beta- and gammaherpesviruses localized in structured domains. Thus, we engineered ORF34 alanine-scanning mutants by substituting conserved residues with alanine. These mutants were evaluated for their ability to interact with other vPIC factors and restore viral production in cells harboring the ORF34-deficient KSHV-BAC. Our experimental results highlight the crucial role of the four cysteine residues conserved in ORF34: a tetrahedral arrangement consisting of a pair of C-Xn-C consensus motifs. This suggests the potential incorporation of metal cations in interacting with ORF24 and ORF66 vPIC components, facilitating late gene transcription, and promoting overall virus production by capturing metal cations. In summary, our findings underline the essential role of conserved cysteines in KSHV ORF34 for effective vPIC assembly and viral replication, thereby enhancing our understanding of the complex interplay between the vPIC components. IMPORTANCE: The initiation of late gene transcription is universally conserved across the beta- and gammaherpesvirus families. This process employs a viral pre-initiation complex (vPIC), which is analogous to a cellular PIC. Although KSHV ORF34 is a critical factor for viral replication and is a component of the vPIC, the specifics of vPIC formation and the essential domains crucial for its function remain unclear. Structural predictions suggest that the four conserved cysteines (C170, C175, C256, and C259) form a tetrahedron that coordinates the metal cation. We investigated the role of these conserved amino acids in interactions with other vPIC components, late gene expression, and virus production to demonstrate for the first time that these cysteines are pivotal for such functions. This discovery not only deepens our comprehensive understanding of ORF34 and vPIC dynamics but also lays the groundwork for more detailed studies on herpesvirus replication mechanisms in future research.

• この論文にアクセスする

  DOI 学術リポジトリ
• 関連情報を調べる
  PubMed

Analysis of the interaction between the ORF42 and ORF55 proteins encoded by Kaposi’s sarcoma-associated herpesvirus

Kazushi Kuriyama, Tadashi Watanabe, Shinji Ohno

Archives of Virology ( Springer Science and Business Media LLC )  169 ( 5 )   2024年04月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

• この論文にアクセスする

  DOI

Sofosbuvir Suppresses the Genome Replication of DENV1 in Human Hepatic Huh7 Cells

Madoka Kurosawa, Fumihiro Kato, Takayuki Hishiki, Saori Ito, Hiroki Fujisawa, Tatsuo Yamaguchi, Misato Moriguchi, Kohei Hosokawa, Tadashi Watanabe, Noriko Saito-Tarashima, Noriaki Minakawa, Masahiro Fujimuro

International Journal of Molecular Sciences ( MDPI AG )  25 ( 4 ) 2022 - 2022   2024年02月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Dengue virus (DENV) causes dengue fever and dengue hemorrhagic fever, and DENV infection kills 20,000 people annually worldwide. Therefore, the development of anti-DENV drugs is urgently needed. Sofosbuvir (SOF) is an effective drug for HCV-related diseases, and its triphosphorylated metabolite inhibits viral RNA synthesis by the RNA-dependent RNA polymerase (RdRp) of HCV. (2′R)-2′-Deoxy-2′-fluoro-2′-methyluridine (FMeU) is the dephosphorylated metabolite produced from SOF. The effects of SOF and FMeU on DENV1 replication were analyzed using two DENV1 replicon-based methods that we previously established. First, a replicon-harboring cell assay showed that DENV1 replicon replication in human hepatic Huh7 cells was decreased by SOF but not by FMeU. Second, a transient replicon assay showed that DENV1 replicon replication in Huh7 cells was decreased by SOF; however, in hamster kidney BHK-21 cells, it was not suppressed by SOF. Additionally, the replicon replication in Huh7 and BHK-21 cells was not affected by FMeU. Moreover, we assessed the effects of SOF on infectious DENV1 production. SOF suppressed infectious DENV1 production in Huh7 cells but not in monkey kidney Vero cells. To examine the substrate recognition of the HCV and DENV1 RdRps, the complex conformation of SOF-containing DENV1 RdRp or HCV RdRp was predicted using AlphaFold 2. These results indicate that SOF may be used as a treatment for DENV1 infection.

• この論文にアクセスする

  DOI

Kaposi's Sarcoma-Associated Herpesvirus ORF67.5 Functions as a Component of the Terminase Complex.

Yuki Iwaisako, Tadashi Watanabe, Youichi Suzuki, Takashi Nakano, Masahiro Fujimuro

Journal of virology   97 ( 6 ) e0047523   2023年06月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA (dsDNA) gammaherpesvirus with a poorly characterized lytic replication cycle. However, the lytic replication cycle of the alpha- and betaherpesviruses are well characterized. During lytic infection of alpha- and betaherpesviruses, the viral genome is replicated as a precursor form, which contains tandem genomes linked via terminal repeats (TRs). One genomic unit of the precursor form is packaged into a capsid and is cleaved at the TR by the terminase complex. While the alpha- and betaherpesvirus terminases are well characterized, the KSHV terminase remains poorly understood. KSHV open reading frame 7 (ORF7), ORF29, and ORF67.5 are presumed to be components of the terminase complex based on their homology to other terminase proteins. We previously reported that ORF7-deficient KSHV formed numerous immature soccer ball-like capsids and failed to cleave the TRs. ORF7 interacted with ORF29 and ORF67.5; however, ORF29 and ORF67.5 did not interact with each other. While these results suggested that ORF7 is important for KSHV terminase function and capsid formation, the function of ORF67.5 was completely unknown. Therefore, to analyze the function of ORF67.5, we constructed ORF67.5-deficient BAC16. ORF67.5-deficient KSHV failed to produce infectious virus and cleave the TRs, and numerous soccer ball-like capsids were observed in ORF67.5-deficient KSHV-harboring cells. Furthermore, ORF67.5 promoted the interaction between ORF7 and ORF29, and ORF29 increased the interaction between ORF67.5 and ORF7. Thus, our data indicated that ORF67.5 functions as a component of the KSHV terminase complex by contributing to TR cleavage, terminase complex formation, capsid formation, and virus production. IMPORTANCE Although the formation and function of the alpha- and betaherpesvirus terminase complexes are well understood, the Kaposi's sarcoma-associated herpesvirus (KSHV) terminase complex is still largely uncharacterized. This complex presumably contains KSHV open reading frame 7 (ORF7), ORF29, and ORF67.5. We were the first to report the presence of soccer ball-like capsids in ORF7-deficient KSHV-harboring lytic-induced cells. Here, we demonstrated that ORF67.5-deficient KSHV also formed soccer ball-like capsids in lytic-induced cells. Moreover, ORF67.5 was required for terminal repeat (TR) cleavage, infectious virus production, and enhancement of the interaction between ORF7 and ORF29. ORF67.5 has several highly conserved regions among its human herpesviral homologs. These regions were necessary for virus production and for the interaction of ORF67.5 with ORF7, which was supported by the artificial intelligence (AI)-predicted structure model. Importantly, our results provide the first evidence showing that ORF67.5 is essential for terminase complex formation and TR cleavage.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Nigericin Induces Apoptosis in Primary Effusion Lymphoma Cells by Mitochondrial Membrane Hyperpolarization and β-Catenin Destabilization.

Haruka Umeyama, Zenpei Shigemi, Yusuke Baba, Naoko Hara, Tadashi Watanabe, Masahiro Fujimuro

Anticancer research   43 ( 6 ) 2455 - 2465   2023年06月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

BACKGROUND/AIM: Primary effusion lymphoma (PEL) is classified as a rare non-Hodgkin's B-cell lymphoma that is caused by Kaposi's sarcoma-associated herpesvirus (KSHV); PEL cells are latently infected with KSHV. PEL is frequently resistant to conventional chemotherapies. Therefore, the development of novel therapeutic agents is urgently required. Nigericin, a H+ and K+ ionophore, possesses unique pharmacological effects. However, the effects of nigericin on PEL cells remain unknown. MATERIALS AND METHODS: We examined the cytotoxic effects of the K+ ionophores, nigericin, nonactin, and valinomycin, on various B-lymphoma cells including PEL. We also evaluated ionophore-induced changes in signaling pathways involved in KSHV-induced oncogenesis. Moreover, the effects of nigericin on mitochondrial membrane potential and viral reactivation in PEL were analyzed. RESULTS: Although the three tested ionophores inhibited the proliferation of several B-lymphoma cell lines, nigericin inhibited the proliferation of PEL cells compared to KSHV-negative cells. In PEL cells, nigericin disrupted the mitochondrial membrane potential and caused the release of cytochrome c, which triggered caspase-9-mediated apoptosis. Nigericin also induced both an increase in phosphorylated p38 MAPK and proteasomal degradation of β-catenin. Combination treatment of nigericin with the p38 MAPK inhibitor SB203580 potentiated the cytotoxic effects towards PEL cells, compared to either compound alone. Meanwhile, nigericin did not influence viral replication in PEL cells. CONCLUSION: Nigericin induces apoptosis in PEL cells by mitochondrial dysfunction and down-regulation of Wnt/β-catenin signaling. Thus, nigericin is a novel drug candidate for treating PEL without the risk of de novo KSHV infection.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Kaposi's Sarcoma-Associated Herpesvirus ORF21 Enhances the Phosphorylation of MEK and the Infectivity of Progeny Virus.

Tatsuo Yamaguchi, Tadashi Watanabe, Yuki Iwaisako, Masahiro Fujimuro

International journal of molecular sciences   24 ( 2 )   2023年01月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, is the causative agent of Kaposi's sarcoma, Castleman's disease, and primary effusion lymphoma. Although the functions of the viral thymidine kinases (vTK) of herpes simplex virus-1/2 are well understood, that of KSHV ORF21 (an ortholog of vTK) is largely unknown. Here, we investigated the role of ORF21 in lytic replication and infection by generating two ORF21-mutated KSHV BAC clones: ORF21-kinase activity deficient KSHV (21KD) and stop codon-induced ORF21-deleted KSHV (21del). The results showed that both ORF21 mutations did not affect viral genome replication, lytic gene transcription, or the production of viral genome-encapsidated particles. The ORF21 molecule-dependent function, other than the kinase function of ORF21, was involved in the infectivity of the progeny virus. ORF21 was expressed 36 h after the induction of lytic replication, and endogenously expressed ORF21 was localized in the whole cytoplasm. Moreover, ORF21 upregulated the MEK phosphorylation and anchorage-independent cell growth. The inhibition of MEK signaling by U0126 in recipient target cells suppressed the number of progeny virus-infected cells. These suggest that ORF21 transmitted as a tegument protein in the progeny virus enhances the new infection through MEK up-regulation in the recipient cell. Our findings indicate that ORF21 plays key roles in the infection of KSHV through the manipulation of the cellular function.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

The Contribution of Kaposi's Sarcoma-Associated Herpesvirus ORF7 and Its Zinc-Finger Motif to Viral Genome Cleavage and Capsid Formation.

Yuki Iwaisako, Tadashi Watanabe, Manami Futo, Rimiko Okabe, Yuichi Sekine, Youichi Suzuki, Takashi Nakano, Masahiro Fujimuro

Journal of virology     e0068422   2022年09月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

During Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection, lytic-related proteins are synthesized, viral genomes are replicated as a tandemly repeated form, and subsequently, capsids are assembled. The herpesvirus terminase complex is proposed to package an appropriate genome unit into an immature capsid, by cleavage of terminal repeats (TRs) flanking tandemly linked viral genomes. Although the mechanism of capsid formation in alpha- and betaherpesviruses are well-studied, in KSHV, it remains largely unknown. It has been proposed that KSHV ORF7 is a terminase subunit, and ORF7 harbors a zinc-finger motif, which is conserved among other herpesviral terminases. However, the biological significance of ORF7 is unknown. We previously reported that KSHV ORF17 is essential for the cleavage of inner scaffold proteins in capsid maturation, and ORF17 knockout (KO) induced capsid formation arrest between the procapsid and B-capsid stages. However, it remains unknown if ORF7-mediated viral DNA cleavage occurs before or after ORF17-mediated scaffold collapse. We analyzed the role of ORF7 during capsid formation using ORF7-KO-, ORF7&17-double-KO (DKO)-, and ORF7-zinc-finger motif mutant-KSHVs. We found that ORF7 acted after ORF17 in the capsid formation process, and ORF7-KO-KSHV produced incomplete capsids harboring nonspherical internal structures, which resembled soccer balls. This soccer ball-like capsid was formed after ORF17-mediated B-capsid formation. Moreover, ORF7-KO- and zinc-finger motif KO-KSHV failed to appropriately cleave the TR on replicated genome and had a defect in virion production. Interestingly, ORF17 function was also necessary for TR cleavage. Thus, our data revealed ORF7 contributes to terminase-mediated viral genome cleavage and capsid formation. IMPORTANCE In herpesviral capsid formation, the viral terminase complex cleaves the TR sites on newly synthesized tandemly repeating genomes and inserts an appropriate genomic unit into an immature capsid. Herpes simplex virus 1 (HSV-1) UL28 is a subunit of the terminase complex that cleaves the replicated viral genome. However, the physiological importance of the UL28 homolog, KSHV ORF7, remains poorly understood. Here, using several ORF7-deficient KSHVs, we found that ORF7 acted after ORF17-mediated scaffold collapse in the capsid maturation process. Moreover, ORF7 and its zinc-finger motif were essential for both cleavage of TR sites on the KSHV genome and virus production. ORF7-deficient KSHVs produced incomplete capsids that resembled a soccer ball. To our knowledge, this is the first report showing ORF7-KO-induced soccer ball-like capsids production and ORF7 function in the KSHV capsid assembly process. Our findings provide insights into the role of ORF7 in KSHV capsid formation.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Biomolecular Fluorescence Complementation Profiling and Artificial Intelligence Structure Prediction of the Kaposi's Sarcoma-Associated Herpesvirus ORF18 and ORF30 Interaction.

Yoshiko Maeda, Tadashi Watanabe, Taisuke Izumi, Kazushi Kuriyama, Shinji Ohno, Masahiro Fujimuro

International journal of molecular sciences   23 ( 17 )   2022年08月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. During KSHV lytic infection, lytic-related genes, categorized as immediate-early, early, and late genes, are expressed in a temporal manner. The transcription of late genes requires the virus-specific pre-initiation complex (vPIC), which consists of viral transcription factors. However, the protein-protein interactions of the vPIC factors have not been completely elucidated. KSHV ORF18 is one of the vPIC factors, and its interaction with other viral proteins has not been sufficiently revealed. In order to clarify these issues, we analyzed the interaction between ORF18 and another vPIC factor, ORF30, in living cells using the bimolecular fluorescence complementation (BiFC) assay. We identified four amino-acid residues (Leu29, Glu36, His41, and Trp170) of ORF18 that were responsible for its interaction with ORF30. Pull-down assays also showed that these four residues were required for the ORF18-ORF30 interaction. The artificial intelligence (AI) system AlphaFold2 predicted that the identified four residues are localized on the surface of ORF18 and are in proximity to each other. Thus, our AI-predicted model supports the importance of the four residues for binding ORF18 to ORF30. These results indicated that wet experiments in combination with AI may enhance the structural characterization of vPIC protein-protein interactions.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

A comprehensive list of the Bunyavirales replication promoters reveals a unique promoter structure in Nairoviridae differing from other virus families.

Yutaro Neriya, Shohei Kojima, Arata Sakiyama, Mai Kishimoto, Takao Iketani, Tadashi Watanabe, Yuichi Abe, Hiroshi Shimoda, Keisuke Nakagawa, Takaaki Koma, Yusuke Matsumoto

Scientific reports   12 ( 1 ) 13560 - 13560   2022年08月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Members of the order Bunyavirales infect a wide variety of host species, including plants, animals and humans, and pose a threat to public health. Major families in this order have tri-segmented negative-sense RNA genomes, the 5' and 3' ends of which form complementary strands that serve as a replication promoter. Elucidation of the mechanisms by which viral polymerases recognize the promoter to initiate RNA synthesis is important for understanding viral replication and pathogenesis, and developing antivirals. A list of replication promoter configuration patterns may provide details on the differences in the replication mechanisms among bunyaviruses. By using public sequence data of all known bunyavirus species, we constructed a comprehensive list of the replication promoters comprising 40 nucleotides in both the 5' and 3' ends of the genome that form a specific complementary strand. Among tri-segmented bunyaviruses, members of the family Nairoviridae, including the highly pathogenic Crimean-Congo hemorrhagic fever virus, have evolved a GC-rich promoter structure differing from that of other families. The unique promoter structure might be related to the large genome size of the family Nairoviridae among bunyaviruses, and the large genome architecture might confer pathogenic advantages. The promoter list provided in this report is useful for predicting the virus family-specific replication mechanisms of bunyaviruses.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

A pyridinium‑type fullerene derivative suppresses primary effusion lymphoma cell viability via the downregulation of the Wnt signaling pathway through the destabilization of β‑catenin.

Ayano Kadota, Misato Moriguchi, Tadashi Watanabe, Yuichi Sekine, Shigeo Nakamura, Takumi Yasuno, Tomoyuki Ohe, Tadahiko Mashino, Masahiro Fujimuro

Oncology reports   47 ( 3 )   2022年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Primary effusion lymphoma (PEL) is defined as a rare subtype of non‑Hodgkin's B cell lymphoma, which is caused by Kaposi's sarcoma‑associated herpesvirus (KSHV) in immunosuppressed patients. PEL is an aggressive type of lymphoma and is frequently resistant to conventional chemotherapeutics. Therefore, the discovery of novel drug candidates for the treatment of PEL is of utmost importance. In order to discover potential novel anti‑tumor compounds against PEL, the authors previously developed a pyrrolidinium‑type fullerene derivative, 1,1,1',1'‑tetramethyl [60]fullerenodipyrrolidinium diiodide (derivative #1), which induced the apoptosis of PEL cells via caspase‑9 activation. In the present study, the growth inhibitory effects of pyrrolidinium‑type (derivatives #1 and #2), pyridinium‑type (derivatives #3 and #5 to #9) and anilinium‑type fullerene derivatives (derivative #4) against PEL cells were evaluated. This analysis revealed a pyridinium‑type derivative (derivative #5; 3‑​5'‑(etho xycarbonyl)‑1',5'‑dihydro‑2'H‑[5,6]fullereno‑C60‑Ih‑[1,9‑c]pyrrol‑2'‑yl]‑1‑methylpyridinium iodide), which exhibited antitumor activity against PEL cells via the downregulation of Wnt/β‑catenin signaling. Derivative #5 suppressed the viability of KSHV‑infected PEL cells compared with KSHV‑uninfected B‑lymphoma cells. Furthermore, derivative #5 induced the destabilization of β‑catenin and suppressed β‑catenin‑TCF4 transcriptional activity in PEL cells. It is known that the constitutive activation of Wnt/β‑catenin signaling is essential for the growth of KSHV‑infected cells. The Wnt/β‑catenin activation in KSHV‑infected cells is mediated by KSHV latency‑associated nuclear antigen (LANA). The data demonstrated that derivative #5 increased β‑catenin phosphorylation, which resulted in β‑catenin polyubiquitination and subsequent degradation. Thus, derivative #5 overcame LANA‑mediated β‑catenin stabilization. Furthermore, the administration of derivative #5 suppressed the development of PEL cells in the ascites of SCID mice with tumor xenografts derived from PEL cells. On the whole, these findings provide evidence that the pyridinium‑type fullerene derivative #5 exhibits antitumor activity against PEL cells in vitro and in vivo. Thus, derivative #5 may be utilized as a novel therapeutic agent for the treatment of PEL.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Human hepatitis B virus-derived virus-like particle as a drug and DNA delivery carrier.

Chiho Sakai, Kohei Hosokawa, Tadashi Watanabe, Youichi Suzuki, Takashi Nakano, Keiji Ueda, Masahiro Fujimuro

Biochemical and biophysical research communications   581   103 - 109   2021年12月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

The controlled release of medications using nanoparticle-based drug delivery carriers is a promising method to increase the efficacy of pharmacotherapy and gene therapy. One critical issue that needs to be overcome with these drug delivery carriers is their target specificity. We focused on the cell tropism of a virus to solve this issue, i.e., we attempted to apply hepatitis B virus-like particle (HBV-VLP) as a novel hepatic cell-selective carrier for medication and DNA. To prepare HBV-VLP, 293T cells were transfected with expression plasmids carrying HBV envelope surface proteins, large envelope protein (L), and small envelope protein (S). After 72 h post-transfection, VLP-containing culture supernatants were harvested, and HBV-VLP was labeled with red fluorescent dye (DiI) and was purified by sucrose gradient ultracentrifugation. An anticancer drugs (geldanamycin or doxorubicin) and GFP-expressing plasmid DNA were incorporated into HBV-VLP, and medication- and plasmid DNA-loaded VLPs were prepared. We evaluated their delivery capabilities into hepatocytes, other organ-derived cells, and hepatocytes expressing sodium taurocholate cotransporting polypeptide (NTCP), which functions as the cellular receptor for HBV by binding to HBV L protein. HBV-VLP selectively delivered both anticancer drugs and plasmid DNA not into HepG2, Huh7, and other organ cells but into HepG2 cells expressing NTCP. In summary, we developed a novel delivery nanocarrier using HBV-VLP that could be used as a hepatitis selective drug- and DNA-carrier for cancer treatment and gene therapy.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Pax5 mediates the transcriptional activation of the CD81 gene.

Kohei Hosokawa, Hanako Ishimaru, Tadashi Watanabe, Masahiro Fujimuro

Scientific reports   11 ( 1 ) 22919 - 22919   2021年11月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

CD81 is an integral membrane protein of the tetraspanin family and forms complexes with a variety of other cell surface membrane proteins. CD81 is involved in cell migration and B cell activation. However, the mechanism of the transcriptional regulation of the CD81 gene remains unclear. Here, we revealed that CD81 transcriptional activation was required for binding of the transcription factor Pax5 at the Pax5-binding sequence (-54)GCGGGAC(-48) located upstream of the transcriptional start site (TSS) of the CD81 gene. The reporter assay showed that the DNA sequence between - 130 and - 39 bp upstream of the TSS of the CD81 gene had promoter activity for CD81 transcription. The DNA sequence between - 130 and - 39 bp upstream of TSS of CD81 harbors two potential Pax5-binding sequences (-87)GCGTGAG(-81) and (-54)GCGGGAC(-48). Reporter, electrophoresis mobility shift, and chromatin immunoprecipitation (ChIP) assays disclosed that Pax5 bound to the (-54)GCGGGAC(-48) in the promoter region of the CD81 gene in order to activate CD81 transcription. Pax5 overexpression increased the expression level of CD81 protein, while the Pax5-knockdown by shRNA decreased CD81 expression. Moreover, we found that the expression level of CD81 was positively correlated with Pax5 expression in human tumor cell lines. Because CD81 was reported to be involved in cell migration, we evaluated the effects of Pax5 overexpression by wound healing and transwell assays. The data showed that overexpression of either Pax5 or CD81 promoted the epithelial cell migration. Thus, our findings provide insights into the transcriptional mechanism of the CD81 gene through transcription factor Pax5.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Kaposi's Sarcoma-Associated Herpesvirus ORF7 Is Essential for Virus Production.

Iwaisako Y, Watanabe T, Hanajiri M, Sekine Y, Fujimuro M

Microorganisms   9 ( 6 )   2021年05月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Kaposi's sarcoma-associated herpesvirus ORF17 plays a key role in capsid maturation.

Tsurumi S, Watanabe T, Iwaisako Y, Suzuki Y, Nakano T, Fujimuro M

Virology ( Virology )  558   76 - 85   2021年02月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

The FAT10 post-translational modification is involved in the lytic replication of Kaposi's sarcoma-associated herpesvirus.

Sugimoto A, Abe Y, Watanabe T, Hosokawa K, Adachi J, Tomonaga T, Iwatani Y, Murata T, Fujimuro M

Journal of virology ( Journal of Virology )  95 ( 10 )   2021年02月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

During Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication, host cell functions including protein expression and post-translational modification pathways are dysregulated by KSHV to promote virus production. Here, we attempted to identify key proteins for KSHV lytic replication by profiling protein expression in the latent and lytic phases using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteomic analysis, immunoblotting, and quantitative PCR demonstrated that antigen-F (HLA-F) adjacent transcript 10 (FAT10) and UBE1L2 (also known as ubiquitin-like modifier-activating enzyme 6, UBA6) were upregulated during lytic replication. FAT10 is a ubiquitin-like protein (UBL). UBE1L2 is the FAT10-activating enzyme (E1), which is essential for FAT10 modification (FAT10ylation). FAT10ylated proteins were immediately expressed after lytic induction and increased over time during lytic replication. Knockout of UBE1L2 suppressed KSHV production but not KSHV DNA synthesis. In order to isolate FAT10ylated proteins during KSHV lytic replication, we conducted immunoprecipitations using anti-FAT10 antibody and Ni-NTA chromatography of exogenously expressed His-tagged FAT10 from cells undergoing latent or lytic replication. LC-MS/MS was performed to identify FAT10ylated proteins. We identified KSHV ORF59 and ORF61 as FAT10ylation substrates. Our study revealed that the UBE1L2-FAT10 system is upregulated during KSHV lytic replication, and it contributes to viral propagation.ImportanceUbiquitin and UBL post-translational modifications, including FAT10, are utilized and dysregulated by viruses for achievement of effective infection and virion production. The UBE1L2-FAT10 system catalyzes FAT10ylation, where one or more FAT10 molecules are covalently linked to a substrate. FAT10ylation is catalyzed by the sequential actions of E1 (activation enzyme), E2 (conjugation enzyme), and E3 (ligase) enzymes. The E1 enzyme for FAT10ylation is UBE1L2, which activates FAT10 and transfers it to E2/USE1. FAT10ylation regulates the cell cycle, IFN signaling, and protein degradation; however, its primary biological function remains unknown. Here, we revealed that KSHV lytic replication induces UBE1L2 expression and production of FAT10ylated proteins including KSHV lytic proteins. Moreover, UBE1L2 knockout suppressed virus production during the lytic cycle. This is the first report demonstrating the contribution of the UBE1L2-FAT10 system to KSHV lytic replication. Our findings provide insight into the physiological function(s) of novel post-translational modifications in KSHV lytic replication.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Herpes simplex virus 1 infection induces ubiquitination of UBE1a.

Ikeda M, Watanabe T, Ito A, Fujimuro M

The Biochemical journal ( Biochemical Journal )  478 ( 1 ) 261 - 279   2021年01月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Herpes simplex virus 1 (HSV-1) is a human DNA virus that causes cold sores, keratitis, meningitis, and encephalitis. Ubiquitination is a post-translational protein modification essential for regulation of cellular events, such as proteasomal degradation, signal transduction, and protein trafficking. The process is also involved in events for establishing viral infection and replication. The first step in ubiquitination involves ubiquitin (Ub) binding with Ub-activating enzyme (E1, also termed UBE1) via a thioester linkage. Our results show that HSV-1 infection alters protein ubiquitination pattern in host cells, as evidenced by MS spectra and co-immunoprecipitation assays. HSV-1 induced ubiquitination of UBE1a isoform via an isopeptide bond with Lys604. Moreover, we show that ubiquitination of K604 in UBE1a enhances UBE1a activity; that is, the activity of ubiquitin-transfer to E2 enzyme. Subsequently, we investigated the functional role of UBE1a and ubiquitination of K604 in UBE1a. We found that UBE1-knockdown increased HSV-1 DNA replication and viral production. Furthermore, overexpression of UBE1a, but not a UBE1a K604A mutant, suppressed viral replication. Furthermore, we found that UBE1a and ubiquitination at K604 in UBE1a retarded expression of HSV-1 major capsid protein, ICP5. Our findings show that UBE1a functions as an antiviral factor that becomes activated upon ubiquitination at Lys604.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

MG132 exerts anti-viral activity against HSV-1 by overcoming virus-mediated suppression of the ERK signaling pathway.

Ishimaru H, Hosokawa K, Sugimoto A, Tanaka R, Watanabe T, Fujimuro M

Scientific reports   10 ( 1 ) 6671 - 6671   2020年04月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Herpes simplex virus 1 (HSV-1) causes a number of clinical manifestations including cold sores, keratitis, meningitis and encephalitis. Although current drugs are available to treat HSV-1 infection, they can cause side effects such as nephrotoxicity. Moreover, owing to the emergence of drug-resistant HSV-1 strains, new anti-HSV-1 compounds are needed. Because many viruses exploit cellular host proteases and encode their own viral proteases for survival, we investigated the inhibitory effects of a panel of protease inhibitors (TLCK, TPCK, E64, bortezomib, or MG132) on HSV-1 replication and several host cell signaling pathways. We found that HSV-1 infection suppressed c-Raf-MEK1/2-ERK1/2-p90RSK signaling in host cells, which facilitated viral replication. The mechanism by which HSV-1 inhibited ERK signaling was mediated through the polyubiquitination and proteasomal degradation of Ras-guanine nucleotide-releasing factor 2 (Ras-GRF2). Importantly, the proteasome inhibitor MG132 inhibited HSV-1 replication by reversing ERK suppression in infected cells, inhibiting lytic genes (ICP5, ICP27 and UL42) expression, and overcoming the downregulation of Ras-GRF2. These results indicate that the suppression of ERK signaling via proteasomal degradation of Ras-GRF2 is necessary for HSV-1 infection and replication. Given that ERK activation by MG132 exhibits anti-HSV-1 activity, these results suggest that the proteasome inhibitor could serve as a novel therapeutic agent against HSV-1 infection.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

The Lysosome Pathway Degrades CD81 on the Cell Surface by Poly-ubiquitination and Clathrin-Mediated Endocytosis.

Hosokawa K, Ishimaru H, Watanabe T, Fujimuro M

Biological & pharmaceutical bulletin ( 公益社団法人 日本薬学会 )  43 ( 3 ) 540 - 545   2020年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

<p>CD81 is a highly conserved four-transmembrane protein in mammals and widely expressed on many tissues. It belongs to the tetraspanin family and forms complexes with various cell surface membrane proteins. It also functions in cell migration and B-cell activation, which is induced by CD81 complexing with CD19, CD21 and the B-cell receptor. Thus, CD81 is thought to play a key role in regulating cell function and fate. However, little is known about the degradation mechanism of CD81. Here we found that CD81 on the plasma membrane is degraded by the lysosome pathway <i>via</i> endocytosis. The expression levels of CD81 in HEK293T cells treated with a proteasome inhibitor (lactacystin) and lysosome inhibitors (chloroquine and bafilomycin A1) were analyzed by flow cytometry. The expression of CD81 on the cell surface was increased by the lysosome inhibitors, but not lactacystin. A pulldown assay revealed that CD81 was conjugated with a K63- and K29-linked poly-ubiquitin chain before its degradation, and the poly-ubiquitination site was Lys8 at the N-terminal intracellular domain of CD81. Furthermore, mutant CD81, in which Lys8 was substituted with alanine (Ala), extended the CD81 half-life compared with wildtype. CD81 was mainly localized on the plasma membrane in normal cells, but also co-localized with lysosomal LAMP1 and early endosomal EEA1 in chloroquine-treated cells. Furthermore, a clathrin-mediated endocytosis inhibitor, chlorpromazine, stabilized CD81 expression on the cell surface. Hence, we demonstrated that CD81 is internalized by clathrin-mediated endocytosis and subsequently degraded <i>via</i> a lysosome pathway requiring the K63- and K29-linked poly-ubiquitination of CD81.</p>

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Kaposi's Sarcoma-Associated Herpesvirus ORF66 Is Essential for Late Gene Expression and Virus Production via Interaction with ORF34.

Watanabe T, Nishimura M, Izumi T, Kuriyama K, Iwaisako Y, Hosokawa K, Takaori-Kondo A, Fujimuro M

Journal of virology   94 ( 2 )   2020年01月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Synthesis and biological evaluation of novel imidazole nucleosides as potential anti-dengue virus agents.

Okano Y, Saito-Tarashima N, Kurosawa M, Iwabu A, Ota M, Watanabe T, Kato F, Hishiki T, Fujimuro M, Minakawa N

Bioorganic & medicinal chemistry   27 ( 11 ) 2181 - 2186   2019年06月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

In this work, we developed imidazole nucleoside derivatives with anti-dengue virus (DENV) activity was examined. First, compounds in a nucleosides library were screened to find lead compounds which inhibit replication of DENV. As a result, 5-ethynyl-(1-β-d-ribofuranosyl)imidazole-4-carboxamide (1; EICAR) and its 4-carbonitrile derivative EICNR (2) were selected as promising antiviral compounds. However, both of them also exhibited cytotoxicity. In order to develop an effective and less toxic compound, 4'-thio and 4'-seleno derivatives of EICAR and EICNR 3-6 were prepared. The resulting 4'-thioEICAR and 4'-thioEICNR showed inhibitory effect on DENV replication without cytotoxicity as potent as ribavirin, a positive control.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Sulforaphane Exhibits Cytotoxic Effects against Primary Effusion Lymphoma Cells by Suppressing p38MAPK and AKT Phosphorylation.

Ishiura Y, Ishimaru H, Watanabe T, Fujimuro M

Biological & pharmaceutical bulletin   42 ( 12 ) 2109 - 2112   2019年 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Primary effusion lymphoma (PEL) is a rare subtype of non-Hodgkin's B-cell lymphoma and is caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. PEL is an aggressive lymphoma and is frequently resistant to conventional chemotherapies. Sulforaphane (SFN), a natural compound found in cruciferous vegetables and broccoli sprouts, modulates signaling pathways and epigenetic gene expression. However, the anti-proliferative effects of SFN on PEL cells and the underlying mechanisms have not been identified. Here, we found that SFN decreased the viability of KSHV-infected PEL cells compared to KSHV-uninfected B-lymphoma cells. The anti-proliferative effects of SFN on PEL cells were mediated by apoptosis with activating caspases. In addition, SFN inhibited the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and AKT in PEL cells. We also showed that p38MAPK and AKT inhibitors reduced PEL cell growth. The constitutive and/or transient activation of p38MAPK and AKT signaling are necessary for the survival and proliferation of PEL cells. Our data and previous literature indicate that SFN represses the phosphorylation of p38MAPK and AKT, which results in PEL cell apoptosis. Moreover, we investigated whether MG132 or sangivamycin (Sangi) in combination with SFN potentiated the cytotoxic effects of SFN on PEL cells. Compared to treatment with SFN alone, the addition of MG132 or Sangi enhanced the cytotoxic activity of SFN in a synergistic manner. In conclusion, the anti-proliferative effects of SFN indicate its potential as a new substance for the treatment of PEL.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

No enhancing effects of plasmid-specific histone acetyltransferase recruitment system on transgene expression in vivo.

Suzuki T, Wakao Y, Watanabe T, Hori M, Ikeda Y, Tsuchiya H, Kogure K, Harada-Shiba M, Fujimuro M, Kamiya H

Nucleosides, nucleotides & nucleic acids   38 ( 12 ) 942 - 949   2019年 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Altered levels of histone acetylation are associated with changes in chromosomal gene expression. Thus, the specific acetylation of histones bound to plasmid DNA might increase transgene expression. Previously, the expression of the histone acetyltransferase domain of CREB-binding protein fused to the sequence-dependent DNA binding domain of GAL4 (GAL4-HAT) successfully improved reporter gene expression in cultured cells [ 123, 277-280 (2017)]. In this study, the same approach was applied for transgene expression in mice. The activator and reporter plasmid DNAs bearing the genes for GAL4-HAT and luciferase, respectively, were co-administered into the mouse liver by hydrodynamics-based tail vein injection, and the luciferase activity in serum was measured for two weeks. Unexpectedly, the co-injection of the GAL4-HAT and luciferase plasmid DNAs seemed to decrease, rather than increase, luciferase expression. Moreover, the co-injection apparently reduced the amount of luciferase DNA in the liver. These results indicated that this system is ineffective in vivo and suggested the exclusion of hepatic cells expressing GAL4-HAT.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Capsaicin Induces ATF4 Translation with Upregulation of CHOP, GADD34 and PUMA.

Moriguchi M, Watanabe T, Fujimuro M

Biological & pharmaceutical bulletin   42 ( 8 ) 1428 - 1432   2019年 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Capsaicin Induces Apoptosis in KSHV-Positive Primary Effusion Lymphoma by Suppressing ERK and p38 MAPK Signaling and IL-6 Expression.

Moriguchi M, Watanabe T, Kadota A, Fujimuro M

Frontiers in oncology   9   83 - 83   2019年 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Primary effusion lymphoma (PEL) is defined as a rare subtype of non-Hodgkin's B-cell lymphoma which is caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. PEL is an aggressive lymphoma and is frequently resistant to conventional chemotherapies. Therefore, it is critical to investigate novel therapeutic options for PEL. Capsaicin is a pungent component of chili pepper and possesses unique pharmacological effects, such as pain relief, anti-microbial and anti-cancer properties. Here, we demonstrate that capsaicin markedly inhibited the growth of KSHV latently infected PEL cells by inhibiting ERK, p38 MAPK and expression hIL-6, which are known to contribute to PEL growth and survival. The underlying mechanism of action by capsaicin was through the inhibition of ERK and p38 MAPK phosphorylation and signaling that affected hIL-6 expression. As a result, capsaicin induced apoptosis in PEL cells in a caspase-9 dependent manner. In line with these results, ERK (U0126) and p38 MAPK (SB203580) specific signaling inhibitors suppressed hIL-6 expression and attenuated cell growth in PEL cells. Furthermore, the addition of hIL-6 neutralizing antibody to culture medium suppressed the growth of PEL cells. We also demonstrate that capsaicin suppressed PEL cell growth in the absence of nascent viral replication. Finally, we confirmed ex vivo treatment of capsaicin attenuated PEL development in SCID mice. Taken together, capsaicin could represent a lead compound for PEL therapy without the risk of de novo KSHV infection.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Arctigenin induces the apoptosis of primary effusion lymphoma cells under conditions of glucose deprivation.

Baba Y, Shigemi Z, Hara N, Moriguchi M, Ikeda M, Watanabe T, Fujimuro M

International journal of oncology   52 ( 2 ) 505 - 517   2018年02月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

KSHV episomes reveal dynamic chromatin loop formation with domain-specific gene regulation.

Campbell M, Watanabe T, Nakano K, Davis RR, Lyu Y, Tepper CG, Durbin-Johnson B, Fujimuro M, Izumiya Y

Nature communications   9 ( 1 ) 49   2018年01月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Methylseleninic acid and sodium selenite induce severe ER stress and subsequent apoptosis through UPR activation in PEL cells.

Shigemi Z, Manabe K, Hara N, Baba Y, Hosokawa K, Kagawa H, Watanabe T, Fujimuro M

Chemico-biological interactions ( ELSEVIER IRELAND LTD )  266   28 - 37   2017年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Selenium compounds such as methylseleninic acid (MSA) and sodium selenite (SS) have been widely evaluated as potential anti-cancer agents in the clinical setting. Primary effusion lymphoma (PEL) is a non-Hodgkin's B-cell lymphoma, associated with immunosuppressed individuals, such as post-transplant or AIDS patients. Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of PEL and Kaposi's sarcoma. Here, we found that MSA and SS markedly inhibited the growth of PEL cells compared with KSHV-uninfected B cells. MSA and SS caused ER stress, inducing the unfolded protein response (UPR) pathway in PEL cells that resulted in pro-apoptotic UPR, and finally apoptosis. The expression of UPR-related molecules (GRP78 and GADD34) and pro-apoptotic UPR molecules (CHOP, Bim, or Puma) were augmented in PEL cells treated with MSA or SS. In addition, these compounds induced the activation of caspase-4, an ER stress specific caspase, as well as caspase-3,-7, and -9 in PEL cells. We confirmed that thapsigargin which is an inducer of ER stress, dramatically decreased the viability of PEL cells, compared with KSHV-uninfected Ramos cells. We also investigated whether MSA or SS caused oxidization of cellular proteins in PEL cells. MSA and SS increased the levels of oxidative proteins in PEL cells, and the anti-oxidant agent (N-acetyl-L-cysteine) restored cell viability and suppressed caspase-7 activation in PEL cells treated with MSA or SS. Finally, we confirmed that MSA and SS induced neither lytic replication nor viral production in PEL cells. Taken together, MSA and SS could serve as lead compounds for the development of novel and effective drugs against PEL without the risk of de novo KSHV production. (C) 2017 Elsevier B.V. All rights reserved.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Kaposi's sarcoma-associated herpesvirus ORF34 is essential for late gene expression and virus production.

Nishimura M, Watanabe T, Yagi S, Yamanaka T, Fujimuro M

Scientific reports ( NATURE PUBLISHING GROUP )  7 ( 1 ) 329   2017年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. KSHV establishes a life-long infection in its host and alternates between a latent and lytic infection state. During lytic infection, lytic-related genes are expressed in a temporal manner and categorized as immediate early, early, and late gene transcripts. ORF34 is an early-late gene that interacts with several viral transcription-associated factors, however its physiological importance remains poorly understood. Here, we investigated the role of ORF34 during KSHV infection by generating ORF34-deficient KSHV, using a bacterial artificial chromosome system. Our results reveal that ORF34-deficient KSHV exhibited significantly attenuated late gene expression and viral production but did not affect viral DNA replication. ORF34 interacted with transcription factors ORF18, ORF24, ORF31, and ORF66, and a novel ORF34-interaction partner, ORF23. The C-terminal region of ORF34 was important for interaction with ORF24 and viral production. Our data support a model, in which ORF34 serves as a hub for recruiting a viral transcription complex to ORF24 to promote late viral gene expression.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Asymptomatic malaria, growth status, and anaemia among children in Lao People's Democratic Republic: a cross-sectional study.

Akiyama T, Pongvongsa T, Phrommala S, Taniguchi T, Inamine Y, Takeuchi R, Watanabe T, Nishimoto F, Moji K, Kano S, Watanabe H, Kobayashi J

Malaria journal ( BIOMED CENTRAL LTD )  15 ( 1 ) 499 - 8   2016年10月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Background: Asymptomatic malaria can be observed in both stable endemic areas and unstable transmission areas. However, although much attention has been given to acute malaria infections, relatively little attention has been paid to asymptomatic malaria. Nonetheless, because the asymptomatic host serves as a reservoir for the malaria parasite, asymptomatic malaria is now recognized as an important obstacle to malaria elimination. Asymptomatic malaria is also associated with anaemia, a global public health problem with serious consequences on human health as well as social and economic development. In Lao People's Democratic Republic (Lao PDR), malaria, anaemia, and malnutrition are serious public health concerns. However, few studies have focused on the relationship between these variables. Therefore, this study investigated the relationship between asymptomatic malaria, growth status, and the prevalence of anaemia among children aged 120 months old or younger in rural villages in Lao PDR. Methods: In December 2010 and March 2011, data were collected from five villages in Savannakhet province. Anthropometric measurements, blood samples, and malaria rapid diagnostic tests were conducted. The presence of malaria was confirmed with polymerase chain reaction assays for Plasmodium falciparum. Underweight status, stunting, and anaemia were defined according to World Health Organization standards. Results: The mean age of participants (n = 319) was 88.3 months old (Standard Deviation: 20.6, ranged from 30-119 months old), and 20 participants (6.3 %) had an asymptomatic malaria infection, 92 (28.8 %) were anaemic, 123 (38.6 %) were underweight, and 137 (42.9 %) were stunted. Stunted children were more likely to be infected with asymptomatic malaria [odds ratio (OR) 3.34, 95 % confidence interval (CI) 1.25-8.93], and asymptomatic malaria was associated with anaemia [OR 5.17, 95 % CI 1.99-13.43]. Conclusions: These results suggest a significant association between asymptomatic malaria and anaemia in children. Furthermore, stunted children were more likely to have lower Hb levels and to be infected with asymptomatic malaria than children without stunting. However, further studies examining the impact of asymptomatic malaria infection on children's nutritional and development status are necessary.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

The dynamics of histone H2A ubiquitination in HeLa cells exposed to rapamycin, ethanol, hydroxyurea, ER stress, heat shock and DNA damage.

Nakata S, Watanabe T, Nakagawa K, Takeda H, Ito A, Fujimuro M

Biochemical and biophysical research communications ( ACADEMIC PRESS INC ELSEVIER SCIENCE )  472 ( 1 ) 46 - 52   2016年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Polyubiquitination plays key roles in proteasome-dependent and independent cellular events, whereas monoubiquitination is involved in gene expression, DNA repair, protein protein interaction, and protein trafficking. We previously developed an FK2 antibody, which specifically recognizes poly-Ub moieties but not free Ub. To elucidate the role of Ub conjugation in response to cellular stress, we used FK2 to investigate whether chemical stress (rapamycin, ethanol, or hydroxyurea), ER stress (thapsigargin or tunicamycin), heat shock or DNA damage (H2O2 or methyl methanesulfonate) affect the formation of Ub conjugates including histone H2A (hH2A) ubiquitination. First, we found that all forms of stress tested increased poly-ubiquitinated proteins in HeLa cells. Furthermore, rapamycin and hydroxyurea treatment, and ER stress increased ubiquitination of hH2A, while methyl methanesulfonate (MMS) treatment induced deubiquitination of hH2A. The ethanol and H2O2 treatments, and heat shock transiently induced hH2A de-ubiquitination, although deubiquitinated hH2A were ubiquitinated again by subsequent cultivation. We also revealed that FK2 reacts with not only polyubiquitinated proteins but also monoubiquitinated hH2A. With the exception of MMS, all forms of stress tested increased the acetylation of K5-hH2A, K9-hH3 and K8-hH4 in addition to ubiquitination. K118 and 1(119 of hH2A were ubiquitinated in cells under normal conditions, and 1(119 was the major ubiquitination site. The MMS-treatment and heat shock induced the deubiquitination of both K118 and K119-histone H2A. Interestingly, MMS treatment did not affect cell HeLa cell viability expressing double-mutant hH2A (KK118,119AA-hH2A), while heat shock slightly but significantly decreased viability of double-mutant hH2A expressing cells, indicating that ubiquitination of both sites associates with recovery from heat shock but not MMS treatment. Thus, we characterized FK2 reactivity and demonstrated that various stresses alter the ubiquitination status, particularly ubiquitinated hH2A and with histone acetylation, and highlight the physiological importance of hH2A ubiquitination after exposure to stress stimuli. (C) 2016 Elsevier Inc. All rights reserved.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Effects of ER stress on unfolded protein responses, cell survival, and viral replication in primary effusion lymphoma.

Shigemi Z, Baba Y, Hara N, Matsuhiro J, Kagawa H, Watanabe T, Fujimuro M

Biochemical and biophysical research communications ( ACADEMIC PRESS INC ELSEVIER SCIENCE )  469 ( 3 ) 565 - 72   2016年01月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

　概要を見る

Primary effusion lymphoma (PEL), a subtype of non-Hodgkin's B-lymphoma, is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. Endoplasmic reticulum (ER) stress induces activation of the unfolded protein response (UPR), which induces expression of ER chaperones, which in turn decrease ER stress, leading to ER homeostasis. The UPR is necessary for not only ER homeostasis but also persistent infection by, and replication of, many viruses. However, the precise roles and regulation of the UPR in KSHV infection remain poorly understood. Here, we found that IRE1 alpha and PERK were significantly downregulated in PEL cells cultured under normal conditions, compared with KSHV-uninfected B-lymphoma cells. IRE1 alpha and PERK mRNA levels were decreased in PEL cells, and KSHV-encoded LANA and v-cyclin D led to suppressed IRE1 alpha transcription. Thapsigargin-induced ER stress activated the UPR and increased the mRNA levels of UPR-related molecules, including IRE1 alpha and PERK, in PEL cells. However the IRE1 alpha and PERK mRNA levels in PEL cells were lower than those in KSHV-uninfected cells. Furthermore, ER stress induced by brefeldin A and thapsigargin dramatically reduced the viability of PEL cells, compared with KSHV-uninfected cells, and induced apoptosis of PEL cells via the pro-apoptotic UPR through expression of CHOP and activation of caspase-9. In addition to the pro-apoptotic UPR, thapsigargin-induced ER stress enhanced transcription of lytic genes, including RTA, K-bZIP and K8.1, and viral production in PEL cells resulted in induction of the lytic cycle. Thus, we demonstrated downregulation of IRE1 alpha and PERK in PEL cells, transcriptional suppression of IRE1 alpha by LANA and v-cyclin D, apoptosis induction in PEL cells by ER stress, and potentiation of lytic replication by ER stress. (C) 2015 Elsevier Inc. All rights reserved.

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Diallyl trisulfide induces apoptosis by suppressing NF-κB signaling through destabilization of TRAF6 in primary effusion lymphoma.

Shigemi Z, Furukawa Y, Hosokawa K, Minami S, Matsuhiro J, Nakata S, Watanabe T, Kagawa H, Nakagawa K, Takeda H, Fujimuro M

International journal of oncology   48 ( 1 ) 293 - 304   2016年01月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Pyrrolidinium fullerene induces apoptosis by activation of procaspase-9 via suppression of Akt in primary effusion lymphoma.

Watanabe T, Nakamura S, Ono T, Ui S, Yagi S, Kagawa H, Watanabe H, Ohe T, Mashino T, Fujimuro M

Biochemical and biophysical research communications   451 ( 1 ) 93 - 100   2014年08月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Sangivamycin induces apoptosis by suppressing Erk signaling in primary effusion lymphoma cells.

Wakao K, Watanabe T, Takadama T, Ui S, Shigemi Z, Kagawa H, Higashi C, Ohga R, Taira T, Fujimuro M

Biochemical and biophysical research communications   444 ( 2 ) 135 - 40   2014年02月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

The hematopoietic cell-specific Rho GTPase inhibitor ARHGDIB/D4GDI limits HIV type 1 replication

Watanabe T., Urano E., Miyauchi K., Ichikawa R., Hamatake M., Misawa N., Sato K., Ebina H., Koyanagi Y., Komano J.

AIDS Research and Human Retroviruses ( Mary Ann Liebert, Inc., publishers )  28 ( 8 ) 913 - 922   2012年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

• この論文にアクセスする

  DOI

Hyrtioreticulins A-E, indole alkaloids inhibiting the ubiquitin-activating enzyme, from the marine sponge Hyrtios reticulatus.

Yamanokuchi R, Imada K, Miyazaki M, Kato H, Watanabe T, Fujimuro M, Saeki Y, Yoshinaga S, Terasawa H, Iwasaki N, Rotinsulu H, Losung F, Mangindaan RE, Namikoshi M, de Voogd NJ, Yokosawa H, Tsukamoto S

Bioorganic & medicinal chemistry   20 ( 14 ) 4437 - 42   2012年07月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

• この論文にアクセスする

  DOI
• 関連情報を調べる
  PubMed

Physical and functional interactions between Daxx and STAT3

Muromoto R., Nakao K., Watanabe T., Sato N., Sekine Y., Sugiyama K., Oritani K., Shimoda K., Matsuda T.

Oncogene ( Springer Nature )  25 ( 14 ) 2131 - 2136   2006年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

• この論文にアクセスする

  DOI

Phosphorylation of threonine-265 in Zipper-interacting protein kinase plays an important role in its activity and is induced by IL-6 family cytokines

Sato N., Kamada N., Muromoto R., Kawai T., Sugiyama K., Watanabe T., Imoto S., Sekine Y., Ohbayashi N., Ishida M., Akira S., Matsuda T.

Immunology Letters ( Elsevier )  25   127 - 134   2006年03月 [ 査読有り ]

掲載種別: 研究論文（学術雑誌）

• この論文にアクセスする

  DOI