A measles virus selectively blind to signalinglymphocytic activation molecule shows anti-tumor activity against lung cancer cells.

Fujiyuki, T., Yoneda, M., Amagai, Y., Obayashi,K., Ikeda, F., Shoji, K., Murakami, Y1., Sato, H.and Kai, C.: 1Division of Molecular Pathology, TheInstitute of Medical Science, The University of To-kyo.

Lung cancer cells, particularly those of non-small-cell lung cancer, are known to express Nectin-4. Wepreviously generated a recombinant measles virus(MV) that uses Nectin-4 as its receptor but cannotbind its original principal receptor, signaling lym-phocyte activation molecule (SLAM). This virus(rMV-SLAMblind) infects and kills breast cancercells in vitro and in a subcutaneous xenograftmodel. However, it has yet to be determinedwhether rMV-SLAMblind is effective against othercancer types and in other tumor models that moreclosely represent disease. In this study, we ana-lyzed the anti-tumor activity of this virus towardslung cancer cells using a modified variant that en-codes green fluorescent protein (rMV-EGFP-SLAM-blind). We found that rMV-EGFP-SLAMblind effi-ciently infected nine, human, lung cancer cell lines,

and its infection resulted in reduced cell viability ofsix cell lines. Administration of the virus into sub-cutaneous tumors of xenotransplanted mice sup-pressed tumor growth. In addition, rMV-EGFP-SLAMblind could target scattered tumor massesgrown in the lungs of xenotransplanted mice. Theseresults suggest that rMV-SLAMblind is oncolyticfor lung cancer and that it represents a promisingtool for the treatment of this disease.

Measles virus infection inactivates cellular pro-tein phosphatase 5 with consequent suppres-sion of Sp1 and c-Myc activities.

Sato, H., Yoneda, M., Honma, R.2, Ikeda, F.,Watanabe, S2. and Kai, C.: 2Clinical Informatics,Tokyo Medical and Dental University.

MV causes several unique syndromes, includingtransient immunosuppression. To clarify the cellu-lar responses to MV infection, we previously ana-lyzed a MV-infected epithelial cell line and a lym-phoid cell line by microarray and showed that theexpression of numerous genes was up- or down-regulated in the epithelial cells. In particular, therewas a characteristic comprehensive downregulationof housekeeping genes during late stage infection.

Laboratory Animal Research Center実験動物研究施設

Professor Chieko Kai, D.V.M., Ph.DAssociate Professor Misako Yoneda, D.V.M., Ph.DAssistant Professor Hiroki Sato, Ph.DAssistant Professor Tomoko Fujiyuki, Ph.D

教 授 農学博士 甲 斐 知恵子准教授 農学博士 米 田 美佐子助 教 理学博士 佐 藤 宏 樹助 教 薬学博士 藤 幸 知 子

Our major research interests are to elucidate molecular mechanisms of patho-genicity and species specificity of negative and single strand RNA viruses(Mononegavirales), and to control viral diseases. For these purposes, we arestudying virus replication and identifying viral and host factors important for the ex-pression of pathogenicity using a novel reverse genetics technique. We are alsodeveloping new virus vaccines and virus vectors by genetic engineering. In theanimal research center, more than 30,000 mice, mainly transgenic or knockout,are kept for research of IMSUT, and the technical staff support their breeding, fro-zen storage of eggs and microbiological cleaning.

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To identify the mechanism underlying this phe-nomenon, we examined the phosphorylation statusof transcription factors and kinase/phosphatase ac-tivities in epithelial cells after infection. MV infec-tion inactivated cellular protein phosphatase 5 (PP5) that consequently inactivated DNA-dependentprotein kinase, which reduced Sp1 phosphorylationlevels, and c-Myc degradation, both of whichdownregulated the expression of many housekeep-ing genes. In addition, intracellular accumulation ofviral nucleocapsid inactivated PP5 and subsequentdownstream responses. These findings demonstratea novel strategy of MV during infection, whichcauses the collapse of host cellular functions.

Transcribed enhancers lead waves of coordi-nated transcription in transitioning mammaliancells.

Arner, E.3, The FANTOM Consortium (Kai, C.,Sato, H., Yoneda, M. et al).: 3RIKEN Omics Sci-

ence Center.

Although it is generally accepted that cellular dif-ferentiation requires changes to transcriptional net-works, dynamic regulation of promoters and en-hancers at specific sets of genes has not been previ-ously studied en masse. Exploiting the fact that ac-tive promoters and enhancers are transcribed, wesimultaneously measured their activity in 19 humanand 14 mouse time courses covering a wide rangeof cell types and biological stimuli. EnhancerRNAs, then messenger RNAs encoding transcrip-tion factors, dominated the earliest responses. Bind-ing sites for key lineage transcription factors weresimultaneously overrepresented in enhancers andpromoters active in each cellular system. Our datasupport a highly generalizable model in which en-hancer transcription is the earliest event in succes-sive waves of transcriptional change during cellulardifferentiation or activation.

Publications

Arner, E., et al. (Kai C., Sato, H.. in 104 authors.).Transcribed enhancers lead waves of coordinatedtranscription in transitioning mammalian cells.Science 347, 1010-1013, 2015.

Fujiyuki, T., Yoneda, M., Amagai, Y., Obayashi, K.,Ikeda, F., Shoji, K., Murakami, Y., Sato, H. andKai, C. A measles virus selectively blind to sig-naling lymphocytic activation molecule showsanti-tumor activity against lung cancer cells. On-cotarget , 6(28): 24895-903, 2015.

Liang, C., The FANTOM Consortium, et al. (Kai C.,Sato, H. in 260 authors.) The statistical geometryof transcriptome divergence in cell-type evolutionand cancer. Nat Commun. 6: 6066, 2015.

Miura, R., Kooriyama, T., Yoneda, M., Takenaka,A., Doki, M., Goto, Y., Sanjoba, C., Endo, Y., Fuji-yuki, T., Sugai, A., Tsukiyama-Kohara, K., Matsu-moto, Y., Sato, H. and Kai, C. Efficacy of recom-

binant distemper virus expressing LeishmaniaAntigen against Leishmania challenge in dogs.PLoS Negl Trop Dis 9(7): e003914, 2015.

Yoshihara, M., The FANTOM consortium, et al.(Kai C., Sato, H. in 258 authors.) Discovery ofmolecular markers to discriminate corneal endo-thelial cells in the human body. PLoS One 10(3): e0117581, 2015.

Sato, H., Yoneda, M., Honma, R., Ikeda, F., Watan-abe, S. and Kai, C. Measles virus infection inacti-vates cellular protein phosphatase 5 with conse-quent suppression of Sp1 and c-Myc activities. JVirol , 89(19), 9709-9718, 2015.

Young, RS., The Fantom Consortium, et al. (Kai C.,Sato, H.. in 101 authors.) The frequent evolution-ary birth and death of functional promoters inmouse and human. Genome Research , 25(10): 1546-57, 2015.

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1. Research on the Habu control

Shosaku Hattori, Takeshi Kuraishi, ShinichiYokota, Motonori Ohno1, Naoko Oda-Ueda2, Taka-hito Chijiwa1, Aichi Yoshida3, Yoshihiro Hayashi4,and Tomohisa Ogawa5: 1Department of AppliedLife Science, Faculty of Bioscience, Sojo Univer-sity, 2Department of Biochemistry, Faculty ofPharmaceutical Science, Sojo University, 3Schoolof Health Science, Faculty of Medicine, KagoshimaUniversity, 4National Museum of Nature and Sci-ence, Tokyo, 5Faculty of Agriculture, Tohoku Uni-versity

Snake bites by the venomous snake Habu, Proto-bothrops flavoviridis, have been reported annuallyabout 60 cases in the population of 100,000 in theAmami Islands. Moreover, there is no indicationthat the population of the Habu itself has de-creased, despite a campaign for capture of snakesby the Kagoshima Prefectural Government. Rat-baited box traps have been introduced to catch thesnakes and found to be quite effective. However,maintenance of live rats requires man power andits cost is expensive. Therefore, our effort has beenfocused on the development of attractant for Habu.The attractant extracted from rats seems ineffectiveif compared with use of live rats.It was known that the Habu survived the injec-

tion of the Habu venom since early times, becausesome proteins in the serum of the Habu blood com-bine to the elements of the Habu venom. The re-search of these binding proteins has been initiatedwith an objective of clinical trials. Phospholipase A2

and its isozymes isolated from Habu venom havemyonecrotic activity and hemorrhagic activity, andmetal protease has hemorahagic activity. The bind-ing proteins isolated from serum of Habu inhibitmyonecrotic activity of phospholipase A2 and itsisozymes. We found that protein-HSF and peptide-AHP isolated from the Habu serum effectively con-trol the hemorrhage caused by venom of the Habu,Ovophis okinavensis, Agkistrodon blomhoffi brevicaudus,Calloselasma rhodostoma, Bitis arietans, Bothrops asper,and, Trimeresurus stejnegeri .Further, a statistics analysis and the simulation

were done with the snakes captured by the Govern-ment, and the analysis of population dynamics ofHabu was attempted. As a result of investigatingthe individual measurement data of the capturedHabu over 9 years, we were able to obtain the gen-erous age composition of the Habu. From analyzingof the age pyramid of the Habu and the result ofquestionnaire surveys for the inhabitant in theAmami-oshima Island, the total population of theHabu which lives in this island was estimated atabout 80,000. By the analysis of the measured dataof last nine years, the snake sizes were miniatur-

Amami Laboratory of Injurious Animals奄美病害動物研究施設

Professor Chieko Kai, D.V.M., Ph.D.Assistant Professor Shinichi Yokota, D.V.M., Ph.D.

教 授 農学博士 甲 斐 知恵子助 教 人間科学博士 横 田 伸 一

The Amami Laboratory of Injurious Animals was established in 1965 at Setouchi-cho in Amami-oshima Island in order to study on endemic diseases involvingparasite, arthropods, and venomous snakes in the tropics or subtropics.The Amami-oshima Island belongs to the Nansei (Southwest) Islands and thefauna is quite different from that in other islands of Japan. Since establishment ofthe laboratory, trials have been carried out to utilize small mammals found uniquein the Amami islands as experimental animals in addition to studies on preventionof Habu bites. As well known, successful eradication of filariasis from this island isone of the monumental works of the laboratory. Our present works are as follows:

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ized, and the population of young snakes de-creased. According to these investigations, thepopulation of the Habu is expected to decrease inthe near future.These studies are supported by grants from the

Ministry of Land, Infrastructure and Transport andthe Kagoshima Prefectural Government.

2. Comparative analysis of interisland variega-tion of venom [Lys49]phospholipase A2 iso-zyme genes in Protobothrops genus snakes

Hitomi Nakamura6, Tatsuo Murakami7, ShosakuHattori, Yoshiyuki Sakaki8, Takatoshi Ohkuri6,Takahito Chijiwa, Motonori Ohno and NaokoOda-Ueda: 6Department of Biochemistry, Facultyof Pharmaceutical science, 7Department of AppliedLife Science, Faculty of Bioscience, Sojo Univer-sity, 8RIKEN Genomic Sciences center

Snake venoms contain phospholipase A2 (PLA2)isoforms as major toxic components. In especially, itis known that the myotoxic activity of venom PLA2

is characterized by a Asp to Lys substitution ofresidue 49 ([Asp49]PLA2 and [Lys49]PLA2). We com-paratively analyzed the PLA2 isozymes betweenProtobothrops tokarensis (Takarajima and Kodakara-jima) and P. flavoviridis (mainly Amami-Oshima,Tokunoshima, and Okinawa). Three [Asp49]PLA2sand one [Lys49]PLA2, named BPI, were identifiedfrom P. tokarensis (Kodakarajima island) venom.The cDNAs encoding three [Asp49]PLA2s and BPIand the gene encoding BPI were cloned and se-quenced. P. flavoviridis (Amami-Oshima island)venom have three [Lys49]PLA2s, thus showing inter-island variegation in their numbers. PtBPI gene hasLINE-1 fragment inserted into its second intron,thus duplication of PtBPI gene appears to be re-stricted. The interisland variegation of venom[Asp49]PLA2 isozyme genes in Protobothrops genussnakes in the southwestern islands of Japan is dis-cussed.

3. Reproduction of squirrel monkeys and owlmonekys.

Shosaku Hattori, Takeshi Kuraishi, ShinichiYokota, Kumiko Ikeda, Hazuki Yoshimura andChieko Kai

The squirrel monkey (Saimiri boliviensis) and theowl monkey (Aotus lemurinus griseimenbra) werewidely distributed in the tropical rainforest in Cen-tral and South America. The advantage of usingboth species for medical researches resides in itssmall size and gentle behavior. In this laboratory,squirrel monkeys have a breeding season betweenwinter and early spring. They are polygamy. Theirpuberty is 3-4 years old in females and 4-5 years

old in males. Their gestation period is about 150days. In contrast, the owl monkey is annual breed-ing animals. They are monogamy. Their puberty is3 years old for both sex. Their gestation period isabout 130 days. Nine newborns were given in re-productive groups of squirrel monkeys in 2015.Four of 9 newborns were nursed by laboratorystaffs because of neglect of their mothers. On theother hand, 2 newborns were given in 3 female owlmonkeys in 2015.

4. Intracytoplasmic sperm injection into oocytesmatured in vitro and early embryonic devel-opment in the owl monkey (Aotus lemurinus)

Ken Takeshi Kusakabe9, Takeshi Kuraishi, Sho-saku Hattori, Yasuo Kiso9, Chieko Kai, MidoriYoshizawa10: 9Joint Faculty of Veterinary Science,Yamaguchi University, 10Graduate School of Agri-cultural Science, Utsunomiya University

As mentioned above, breeding of owl monkeyhas been achieved at Amami laboratory of injuriousanimals, the number of animals produced has beensmall. We explored the possibility of employing in-tracytoplasmic sperm injection (ICSI), involving oo-cytes and sperm of owl monkeys. First, we esti-mated the morphology and normal parameters ofowl monkey sperm and oocytes. As a result, be-cause the motility and viability of owl monkeysperm are very poor, i.e. similar to those in somecases of male infertility in humans, embryo produc-tion by conventional in vitro fertilization is difficultin owl monkeys. Therefore, we decided that ICSI isrequired to produce embryos in vitro, and tried. Asa result, we were able to produce two owl monkeyembryos using ICSI of oocytes that matured to MIIstage. Both embryos reached the 10-cell stage at 98h after ICSI and showed signs of compaction, butfailed to cleave further.

5. Pathological analysis of aquatic mycobacteria(Mycobacterium marinum) infection in squir-rel monkey (Saimiri boliviensis)

Shinichi Yokota, Shosaku Hattori, ShiomiYoshida11, Takayuki Wada12, Takeshi Kuraishi13,and Chieko Kai: 11National Hospital OrganizationKinki Chuo Chest Medical Center, 12Nagasaki Uni-versity Graduate School of Biomedical Sciences,13Laboratory of Veterinary Pathology, Departmentof Veterinary Medicine, Gifu University

Mycobacterium marinum is an atypical Mycobacte-rium species, which causes opportunistic infectionsin humans by excessive exposure to an aquatic en-vironment or marine animals. To determine thepossibility for cross-species M. marinum transmis-sion, we administrated M. marinum isolated from

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Japanese Forest Green Tree Frogs (Rhacophorus ar-boreus) and from a human patient into squirrelmonkeys, and investigated the clinical conditions.The monkey inoculated with M. marinum isolatedfrom patient had local swelling at the injection site,and M. marinum was found in the axillary lymphnodes 4 weeks after the inoculation. On the otherhand, the monkey inoculated with M. marinum iso-lated from Japanese Forest Green Tree Frogs didnot show any clinical signs. The histopathologicalanalysis is currently under consideration.

6. Establishment of the DFAT cells and applica-tion to cell sheet transplant in squirrel mon-key (Saimiri boliviensis)

Shinichi Yokota, Takeshi Kuraishi, Shosaku Hat-tori, Akane Tanaka14, Hiroshi Matsuda14, andChieko Kai: 14Division of animal life science, Insti-tute of agriculture, Tokyo University of Agricul-

ture and Technology

Dedifferentiated fat cells (DFAT cells) are seemedto be a good candidate source of adult stem cells inregenerative medicine, because these cells exhibitmultilineage potential as adipose tissue-derivedstromal cells (ADSCs). We isolated squirrel monkeyDFAT cells from a small amount of adipose tissue,and confirmed adipogenesis, osteogenic and chon-drogenic differentiation in vitro. Furthermore, theDFAT cells derived from three different squirrelmonkeys were expanded to sufficient numbers fortransplantation as cell sheets. Before clinical appli-cation and transplantation of MSC-derived cells, thepreclinical safety and efficacy studies should bepreferably carried out in a non-human primate ani-mal model, because mouse stem cells have provento show great differences from the human. We areplan to apply to some wound healing model insquirrel monkey.

Publications

Watanabe, H., Matsumoto, T., Nishi, M., Kusakabe,KT., Kuraishi, T., Hattori, S., Matsumoto, H.,Fukui, E., Kuwahata, A., Ochi, M., Kiso, Y., Kai,C., Yoshizawa, M. Intracytoplasmic sperm injec-tion into oocytes matured in vitro and early em-bryonic development in the owl monkey (Aotuslemurinus). Reprod Med Biol. 10: 1007/s12522-015-0229-1, 2015.

Nakazato, C., Yoshizawa, M., Isobe, K., Kusakabe,KT., Kuraishi, T., Hattori, S., Matsumoto, H.,Fukui, E., Kuwahata, A., Ochi, M., Kiso, Y., Kai,C. Morphological characterization of spermatozoaof the night mokey. J Mamm Ova Res. 32: 37-40,2015.

Matsumoto, T., Isobe, K., Kusakabe, KT., Kuraishi,T., Hattori, S., Nakazato, C., Matsumoto, H.,Fukui, E., Kuwahata, A., Ochi, M., Kiso, Y., Kai,C. Morphological characterization and in vitro

maturation of follicular oocytes from the owlmonkey (Aotus Lemurinus). J Mamm Ova Res.;32: 103-108, 2015.

Yamaguchi, K., Chijiwa, T., Yamamura, T., Ikeda,N., Yatsui, T., Hayama, S., Hattori, S., Oda-Ueda,N., Ohno, M. Interisland variegation of venom[Lys(49)]phospholipase A2 isozyme genes in Pro-tobothrops genus snakes in the southwestern is-lands of Japan. Toxins. 107: 210-216, 2015.服部正策．徳之島手々地区でのネットフェンスとハブの捕獲調査．平成２６年度ハブとの共存に関わる総合調査事業報告書（鹿児島県）．pp.１０―１４，２０１５．倉石武，服部正策，朝沼榎．ハブ咬傷者血清中のハブ毒特異的IgE抗体の測定．平成２６年度奄美ハブ毒免疫機序応用研究報告書．（鹿児島県）．pp.５２―５３，２０１４．

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The purposes of our laboratory are concernedabout not only research but also support for all re-searchers in this institute. Our supporting activity isinvolved in advising service on gene-manipulationexperiments and on biohazards under the safetyguidelines and laws. For the research part, we in-tend to develop novel methods or new experimen-tal systems leading in the field of gene expressionand its regulation. We are concentrating mainly ondeveloping efficient adenovirus expression vectorsuseful for various fields including gene therapy.We are maintaining more than ten collaborationswithin and outside of this institute. In these col-laborations, we can supply adenovirus vectors(AdVs) enabling strictly regulated gene expressionand helper-dependent AdVs (HD AdVs) of high ca-pacity up to 30 kilobases (kb). We have establisheda unique system producing HD AdVs using 293hde12 cells. Our system is probably superior to cur-rently available system, because in the latter HDAdVs are produced using cell lines expressing Cre,which is slightly toxic to cells when expressed in alarge amount.Previously we developed a system for construc-

tion of E1-deleted AdV, also called first-generation(FG) AdVs, using a full-length viral genome withintact viral termini (Fukuda. et al ., Microbiol. Im-munol. 50: 643-654, 2006). This cassette is availablefrom Takara Bio and Nippon Gene. We have alsodeveloped a method for ON/OFF switching of gene

expression in mammalian cells using a combinationof adenovirus vector and Cre/loxP system (Kanegaeet al ., Nucleic Acids Res. 23: 3816-3821, 1995;Kanegae et al ., Gene 181: 207-212, 1996) as well asFLP/frt system (Nakano et al ., Nucleic Acids Res.29: e40, 2001; Kondo et al ., Nucleic Acids Res. 31: e76, 2003; Kondo et al ., Microbiol. Immunol., 50: 831-843, 2006; Kondo et al., J. Molec. Biol., 2009). Thesemethods continuously promote studies of variousfields of molecular biology and medicine.There are two remarkable advances from our

laboratory. We succeeded in developing new-gen-eration AdVs that may replace current FG AdVs.The most important problem of AdV is severe im-mune responses in vivo. Firstly, we have identifiedadenovirus pIX gene as a main cause of inflamma-tion: pIX gene is abnormally activated in AdV.Then we developed AdVs that do not express pIXprotein. Transgene expression was lasted for sixmonths in this new AdV (Nakai et al ., Hum. GeneTher. 18: 925-936, 2007). The AdV is now called the"low-inflammatory AdVs". For example, Cre-ex-pressing AdV, AxCANCre, is replaced by the low-inflammatory Cre-expressing AdVs, AxEFNCre(Chiyo et al ., Virus Res. 160: 89-97, 2011). Secondly,we have established a method for efficient produc-tion of AdVs lacking the genes of virus-associated(VA) RNAs that disturb cellular RNAi machinery(Maekawa et al ., Sci. Rep. 2013) using 293hde12 cellline producing a large amount of the codon-human-

Laboratory of Molecular Genetics遺伝子解析施設

Professor Izumu Saito, M.D., D.M.Sc.Assistant Professor Saki Kondo, D.M.Sc.Assistant Professor Tomoko Nakanishi, D.P.

教 授 医学博士 斎 藤 泉助 教 医学博士 近 藤 小 貴助 教 薬学博士 中 西 友 子

This laboratory has three main activities: development and supply of novel ade-novirus vectors useful for studies in various fields including gene therapy, studyabout hepatitis B virus and supporting the researchers by advising on recombinantDNA technology and on biohazards under the safety guidelines.

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ized FLPe (hFLPe) recombinase (Kondo et al. , J.Molec. Biol., 2009). The VA-deleted AdVs possiblysubstitute for current FG AdVs.The research activities in 2015 are shown below.

They include the studies of hepatitis B virus (HBV)and the application of AdVs in the field of genomeediting.

1. Efficient production of the replicating HBVgenome using "HBV-AdV system"

Mariko Suzuki, Saki Kondo, Manabu Yamasaki1,Masakatsu Shibasaki1, Yumi Kanegae, and IzumuSaito: 1Laboratory of Basic Biology, Institute ofMicrobial Chemistry (BIKAKEN), Tokyo

In most studies of HBV, both the transfection of1.2 copy-HBV genome plasmids or the cell lines inwhich the HBV genome is integrated, have beenused to elucidate the HBV replication mechanism.Previously, we reported a new method to detect theHBV genome replication using AdV, which namedHBV-AdV system. Since AdV DNA doesn't remainon the cell surface (Pei et al., BBRC, 2012), the repli-cated HBV genomes are easily and precisely de-tected when during the use of the HBV-AdV sys-tem.We applied the HBV-AdV system to HepG2 cells

and human primary hepatocytes. In the HepG2cells, where AdV showed 24-fold higher transduc-tion efficiency than plasmid transfection, the HBV-AdV system was much superior to the plasmidtransfection to detect replicating HBV genome andthe produced products. Notably, the application ofthe HBV-AdV system to the primary hepatocyteswas very efficient; it is valuable because, so far, theprimary hepatocytes can efficiently be infected onlyHBV particles derived from sera from HBV carrier.Moreover, Because our HBV-AdV system abun-dantly provided the HBV pregenomic RNA(pgRNA) using CMV promoter, the HBV-AdV sys-tem enabled us to detect the replicating HBVgenome only 4 days post-infection of AdV. More-over, using HBV-AdV system the behaviors of theartificially-mutated HBV genomes can be investi-gated in various cell lines. Thus, the HBV-AdV sys-

tem could widely be applied to the study of HBVreplication and also to the screening of anti-HBVdrug.

2. Multiplex guide RNAs of CRISPR/Cas9 systemexpressed from adenovirus vector efficientlydisrupted the HBV genome

Aya Maekawa, Saki Kondo, Mariko Suzuki, YumiKanegae, and Izumu Saito

HBV infection increases the risk of developingliver cirrhosis and hepatocellular carcinoma. Cur-rent therapeutic treatments for HBV infectionmostly use interferon alpha (IFN-) and the nucleo-tide analogs such as lamivudine. However, thesetreatments cannot effectively eliminate the cova-lently closed circular DNA (cccDNA), which pre-sents in the nuclei of the infected hepatocytes andcontinues to serve as the template for the pre-genomic RNA (pg RNA). Consequently, completeclearance of HBV is difficult.As a novel tool for genome editing, the CRISPR/

Cas9 system directed by programable guide RNA(gRNA) to the target DNA is expected to offer an-tiviral activities against HBV. In the application ofthe CRISPR/Cas9 system to disrupt the HBVcccDNA, multiplex targeting could be superior to asingle targeting for cccDNA disruption. For thisreason we ligated in tandem the multiplex expres-sion cassettes of the gRNA and were inserted intothe AdV genome in order to express gRNAs simul-taneously in a single cell. However, it is difficult toconstruct the tandemly-ligated expression cassettesof the gRNAs because of homologous recombina-tion (HR) and subsequent deletion among the re-peated sequences.We succeeded in construction of AdVs expressing

six multiplex gRNAs targeting the HBV genome.The expressed gRNAs together with Cas9-produc-ing AdVs efficiently directed cleavages and cleav-age-mediated mutations occurred in the cccDNA.These results suggest that AdV expressing multi-plex gRNA targeting HBV genome might be usedas an effective tool to treat chronic HBV infection.

Publications

Suzuki, T., Kikuguchi, C., Sharma, S., Sasaki, T.,Tokumasu, M., Adachi, S., Natsume, T., Kanegae,Y., and Yamamoto, T. CNOT3 suppression pro-motes necroptosis by stabilizing mRNAs for celldeath-inducing proteins. Sci. Rep., 5: 14779, 2015.

Suzuki, M., Kondo, S., Pei, Z., Maekawa, A., Saito,I., and Kanegae, Y. Preferable sites and orienta-tions of transgene inserted in the adenovirus vec-tor genome: the E3 site may be unfavorable for

transgene position. Gene Ther., 22(5): 421-429,2015.

Suzuki, M., Kondo, S., Yamazaki, M., Saito, I., Shi-bazaki, M., and Kanegae, Y. Efficient productionof the replicating HBV genome using "HBV-AdVsystem". 2015 International Meeting of MolecularBiology of Hepatitis B Viruses. Dolce BadNauheim, Germany.

Maekawa, A., Kondo, S., Suzuki, M., Saito, I., and

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Kanegae, Y. Multiplex guide RNAs targetingHBV genome expressed using adenovirus vectorimprove the efficiency of CRISPR/Cas9 system.

2015 International Meeting of Molecular Biologyof Hepatitis B Viruses. Dolce Bad Nauheim, Ger-many.

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<Group I>1. Global characterization of the proteome andphosphoproteome in human glioblastoma in-itiating cells by high-resolution mass spec-trometry

Hiroko Kozuka-Hata, Tomoko Hiroki, Ryo Ko-yama-Nasu1, Kouhei Tsumoto, Jun-ichiro Inoue,Tetsu Akiyama1 and Masaaki Oyama: 1Laboratoryof Molecular and Genetic Information, Institute ofMolecular and Cellular Biosciences, University ofTokyo

Glioblastoma is one of the most common and ag-gressive brain tumors with the median survival oftwelve months after diagnosis. Despite extensivestudies of this malignant tumor, the outcomes ofthe treatment have not significantly improved overthe past decade. To elucidate the underling mecha-nisms of its tumorigenicity, we performed parallelanalyses of the comprehensive proteome and phos-phoproteome in glioblastoma initiating cells thatare widely recognized as key players in showingresistance to chemotherapy and radiation. Usinghigh-resolution nanoflow LC-MS/MS (LTQ OrbitrapVelos) in combination with GELFREETM8100 frac-tionation system, we identified a total of 8,856 pro-teins and 6,073 phosphopeptides, respectively. Global

protein network analysis revealed that the mole-cules belonging to ribosome, spliceosome and pro-teasome machineries were highly enriched at theproteome level. Our in-depth phosphoproteomeanalysis based on two fragmentation methodologiesof CID and HCD detected various phosphorylationsites on neural stem cell markers such as nestin andvimentin, leading to identification of thirty-sixphosphorylation sites including eleven novel sitesof nestin protein. The SILAC-based quantitativeanalysis showed that 516 up-regulated and 275down-regulated phosphorylation sites upon epider-mal growth factor stimulation. Interestingly, thephosphorylation status of the molecules related tomTOR signaling pathway was dynamically changedupon EGF stimulation. More intriguingly, we alsoidentified some novel phosphopeptides encoded bythe undefined sequence regions of the human tran-scripts, which could be regulated upon externalstimulation in glioblastoma initiating cells. Our re-sult unveils an expanded diversity of the regulatoryphophoproteome defined by the human transcrip-tome.

2. System-wide perturbation of the proteomeand phosphoproteome dynamics in glioblas-toma stem cells through mTOR signaling ini-hibition

Medical Proteomics Laboratory疾患プロテオミクスラボラトリー

Professor Jun-ichiro Inoue, Ph.D.Professor Kouhei Tsumoto, Ph.D.Associate Professor Masaaki Oyama, Ph.D.Assistant Professor Hiroshi Sagara, Ph.D.Assistant Professor Satoru Nagatoishi, Ph.D.

教 授 薬学博士 井 上 純一郎教 授 工学博士 津 本 浩 平准教授 医学博士 尾 山 大 明助 教 医学博士 相 良 洋助 教 生命科学博士 長門石 曉

The mission of our laboratory is to develop advanced technologies for integrativeproteomic analyses from a physicochemical, structural and systems biology pointof view. Currently, we mainly focus on functional protein-protein interaction net-works related to a variety of diseases including cancer and infection. We are alsoengaged in collaborative researches regarding mass spectrometry and electronmicroscopy, which have made a substantial contribution to many scientificachievements.

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Hiroko Kozuka-Hata, Tomoko Hiroki, Ryo Ko-yama-Nasu1, Kouhei Tsumoto, Jun-ichiro Inoue,Tetsu Akiyama1 and Masaaki Oyama.

As glioblastoma is the most common and aggres-sive brain tumor with poor prognosis, systematicelucidation of signaling networks causally linked tothe tumorigenesis is very crucial for developingmore effective treatments for this intractable cancer.In our previous study, we applied a high-resolutionmass spectrometry-based proteomics technology incombination with SILAC quantitative methods tounderstand EGF-dependent phosphoproteome dy-namics in patient-derived glioblastoma stem cells.We demonstrated that the phosphorylation levels ofthe representative mTOR signaling molecules suchas RPS6 and PRAS40 were dramatically up-regu-lated upon EGF stimulation. As EGFR signaling hasbeen reported to play a pivotal role in regulatingthe maintenance of cancer stem cells, we next car-ried out mTOR inhibitor-dependent signaling per-turbations to unravel stemness-related pathways atthe network level.In the present study, we identified a total of

3,726 proteins including 49 up-regulated and 436down-regulated factors by Torin 1 treatment. Inter-estingly, we found that one of the well-known can-cer stem cell markers was significantly down-regu-lated through mTOR signaling inhibition. Our in-depth phosphoproteome analysis also led to identi-fication of 6,250 unique phosphopeptides derivedfrom 2,221 proteins and unveiled a variety of dy-namic changes regarding phosphorylation levels ofcancer and neural stem cell markers in a compre-hensive manner. The integrative view of the mTORinhibitor-dependent proteome and phosphoproteomedynamics in glioblastoma stem cells presents uswith further prospects towards understanding pre-viously unrecognized regulations at the system level.

3. Integrative network analysis based on high-accuracy quantitative phosphoproteomics re-veals a novel regulator of glioblastoma stemcell differentiation

Yuta Narushima, Hiroko Kozuka-Hata, Ryo Ko-yama-Nasu1, Kouhei Tsumoto, Jun-ichiro Inoue,Tetsu Akiyama1 and Masaaki Oyama.

Glioblastoma is one of the most malignant braintumors with poor prognosis and their developmentand progression are known to be driven byglioblastoma stem cells. Although glioblastoma stemcells lose their stem cell properties contributing tohigh tumorigenicity and resistance to the currentchemo- and radio-therapies during the course ofdifferentiation, little is known about the molecularmechanisms regulating signaling alteration in rela-tion to stemness maintenance and differentiation. In

order to elucidate the global phosphorylation-re-lated signaling events, we performed a SILAC-based quantitative phosphoproteome analysis of se-rum-induced differentiation in glioblastoma stemcells established from the tumor tissues of the pa-tient.Among a total of 2,691 phosphorylation sites on

1,584 proteins identified in our analysis, 731 phos-phorylation sites on 415 proteins were regulatedthrough the differentiation. The integrative compu-tational analyses based on the quantified phospho-proteome data revealed the relevant changes ofphosphorylation levels regarding the proteins asso-ciated with cytoskeleton reorganization such as Rhofamily GTPase and Intermediate filament signaling,in addition to transforming growth factor-beta re-ceptor type-2 (TGFBR2) as a prominent upstreamregulator involved in the serum-induced phospho-proteome regulation. The functional association ofTGFBR2 with glioblastoma stem cell differentiationwas experimentally validated through signalingperturbation using the corresponding inhibitors,which indicated that TGFBR2 could play an impor-tant role as a novel cell fate determinant inglioblastoma stem cell regulation.

4. Integrative analysis of phosphoproteome andtranscriptome dynamics defines drug-resis-tance properties of breast cancer

Masaaki Oyama, Takeshi Nagashima2, HirokoKozuka-Hata, Noriko Yumoto2, Yuichi Shiraishi2,Kazuhiro Ikeda3, Yoko Kuroki2, Noriko Gotoh4,Satoshi Inoue3, Hiroaki Kitano5 and MarikoOkada-Hatakeyama2: 2RIKEN, 3Research Centerfor Genomic Medicine, Saitama Medical Univer-sity, 4Division of Systems Biomedical Technology,IMSUT, 5Sony Computer Science Laboratories,Inc.

Signal transduction system, in orchestration withsubsequent transcriptional regulation, widely regu-lates complex biological events such as cell prolif-eration and differentiation. Therefore, a comprehen-sive and fine description of their dynamic behaviorprovides a fundamental platform for systematicallyanalyzing the regulatory mechanisms that result ineach biological effect. Here we developed an inte-grated framework for time-resolved description ofphosphoproteome and transcriptome dynamics basedon the SILAC-nanoLC-MS and GeneChip system. Inthis study, we analyzed cellular information net-works mediated by estrogen receptor/ErbB2 path-ways, which have long been implicated in drug re-sponse of breast cancer. Through shotgun identifi-cation and quantification of phosphorylated mole-cules in breast cancer MCF-7 cells, we obtained aglobal view of the dynamics regarding breast can-cer-related signaling networks upon estrogen (E2)

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or heregulin (HRG) stimulation. Comparative analy-sis of wild-type and tamoxifen-resistant MCF-7 cellsrevealed altered behaviors of signaling hub dynam-ics, indicating distinct signaling network propertiesbetween these two cell types. Pathway and motifactivity analyses using the transcriptome data sug-gested that deregulated activation of GSK3 andMAPK1/3 signaling might be associated with al-tered activation of CREB and AP-1 transcriptionfactors in tamoxifen-resistant MCF-7 cells. Thus,our integrative analysis of phosphoproteome andtranscriptome in human breast cancer cells revealeddistinct signal-transcription programs in tamoxifen-sensitive and insensitive tumor cells, which poten-tially defines drug-resistance properties against ta-moxifen.

5. System-level analysis of CagA-dependent sig-naling network dynamics by Helicobacter py-lori infection

Hiroko Kozuka-Hata, Masato Suzuki6, KotaroKiga6, Shinya Tasaki, Jun-ichiro Inoue, TadashiYamamoto7, Chihiro Sasakawa6 and MasaakiOyama: 6Division of Bacterial Infection, Depart-ment of Microbiology and Immunology, IMSUT,7Division of Oncology, Department of Cancer Biol-ogy, IMSUT

The signal transduction system within a cellregulates complex biological events in response tobacterial infection. The previous analyses of cell sig-naling in Helicobacter pylori-infected gastric epithe-lial cells have revealed that CagA, a major viru-lence factor of Helicobacter pylori, is delivered intocells via the type IV secretion system and perturbssignaling networks through the interaction with thekey signaling molecules such as SHP-2, Grb2, Crk/Crk-L, Csk, Met, and ZO-1. Although the biologicalactivity of tyrosine-phosphorylated CagA has inten-sively been studied, system-wide effects of the viru-lence factor on cellular signaling have yet to beanalyzed. Here we performed time-resolved analy-ses of phosphoproteome and CagA-interactome inhuman gastric AGS cells by CagA-positive/negativeHelicobacter pylori infection. Our highly sensitivenanoLC-MS/MS analyses in combination with theStable Isotope Labeling by Amino acids in Cell cul-ture (SILAC) technology defined CagA-dependentperturbation of signaling dynamics along with asubset of CagA-associated possible modulators on anetwork-wide scale. Our result indicated that theactivation level of the phosphotyrosine-related sig-naling molecules in AGS cells was suppressed over-all in the presence of CagA during Helicobacter py-lori infection. As Helicobacter pylori infection playspivotal roles in the progression of gastric diseasesincluding carcinogenesis, a comprehensive and finedescription of the signaling dynamics would serve

as a fundamental platform to theoretically explorefor the potential drug targets through analyzing theregulatory mechanisms at the system-level.

6. Mass spectrometry-based annotation of thehuman short ORFeome

Masaaki Oyama, Hiroko Kozuka-Hata, SumioSugano8, Tadashi Yamamoto7 and Jun-ichiro Ino-ue: 8Laboratory of Functional Genomics, Depart-ment of Medical Genome Sciences, GraduateSchool of Frontier Sciences, The University of To-kyo

In parallel with the human genome projects, hu-man full-length cDNA data has also been inten-sively accumulated. Large-scale analysis of their 5'-UTRs revealed that about half of these had a shortORF upstream of the coding region. Experimentalverification as to whether such upstream ORFs aretranslated is essential to reconsider the generality ofthe classical scanning mechanism for initiation oftranslation and define the real outline of the humanproteome. Our previous proteomics analysis ofsmall proteins expressed in human K562 cells pro-vided the first direct evidence of translation of up-stream ORFs in human full-length cDNAs (Oyamaet al., Genome Res, 14: 2048-2052, 2004). In order tograsp an expanded landscape of the human shortORFeome, we have performed an in-depth pro-teomics analysis of human K562 and HEK293 cellsusing a two-dimensional nanoLC-MS/MS system.The results led to the identification of eight protein-coding regions besides 197 small proteins with atheoretical mass less than 20 kDa that were alreadyannotated coding sequences in the curated mRNAdatabase. In addition to the upstream ORFs in thepresumed 5'-untranslated regions of mRNAs, bioin-formatics analysis based on accumulated 5'-endcDNA sequence data provided evidence of novelshort coding regions that were likely to be trans-lated from the upstream non-AUG start site or fromthe new short transcript variants generated by utili-zation of downstream alternative promoters. Pro-tein expression analysis of the GRINL1A gene re-vealed that translation from the most upstreamstart site occurred on the minor alternative splicingtranscript, whereas this initiation site was not util-ized on the major mRNA, resulting in translation ofthe downstream ORF from the second initiation co-don. These findings reveal a novel post-transcrip-tional system that can augment the human pro-teome via the alternative use of diverse translationstart sites coupled with transcriptional regulationthrough alternative promoters or splicing, leadingto increased complexity of short protein-coding re-gions defined by the human transcriptome (Oyamaet al., Mol Cell Proteomics, 6: 1000-1006, 2007).

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<Group II>

AIM: Life, as we understand it, requires of a con-certed and complex set of interactions between dif-ferent biological molecules, such as DNA, RNA,proteins, lipids, and carbohydrates. We sought tounderstand the nature of these interactions at themolecular and energetic level. Our dissecting toolsare applied to study a broad range of biologicalphenomena, and to develop the next generation oftherapeutic antibodies in the era of Bio-better andBio-superior.

1. Structural features of interfacial tyrosine resi-due in ROBO1 fibronectin domain-antibodycomplex: Crystallographic, thermodynamic, andmolecular dynamic analyses

Nakayama T, Mizohata E, Yamashita T, Nagato-ishi S, Nakakido M, Iwanari H, Mochizuki Y,Kado Y, Yokota Y, Satoh R, Tsumoto K, FujitaniH, Kodama T, Hamakubo T, Inoue T.

ROBO1, fibronectin Type-III domain (Fn)-contain-ing protein, is a novel immunotherapeutic target forhepatocellular carcinoma in humans. The crystalstructure of the antigen-binding fragment (Fab) of B2212A, the monoclonal antibody against the thirdFn domain (Fn3) of ROBO1, was determined inpursuit of antibody drug for hepatocellular carci-noma. This effort was conducted in the presence orabsence of the antigen, with the chemical featuresbeing investigated by determining the affinity ofthe antibody using molecular dynamics (MD) andthermodynamics. The structural comparison ofB2212A Fab between the complex and the free formrevealed that the interfacial Tyr(L) 50 (superscriptsL, H, and F stand for the residues in the lightchain, heavy chain, and Fn3, respectively) playedimportant roles in Fn3 recognition. That is, the aro-matic ring of Tyr(L) 50 pivoted toward Phe(F) 68,forming a CH/ interaction and a new hydrogenbond with the carbonyl O atom of Phe(F) 68. MDsimulations predicted that the Tyr(L) 50-Phe(F) 68interaction almost entirely dominated Fab-Fn3 bind-ing, and Ala-substitution of Tyr(L) 50 led to a re-duced binding of the resultant complex. On thecontrary, isothermal titration calorimetry experi-ments underscored that Ala-substitution of Tyr(L)50 caused an increase of the binding enthalpy be-tween B2212A and Fn3, but importantly, it inducedan increase of the binding entropy, resulting in asuppression of loss in the Gibbs free energy in to-tal. These results suggest that mutation analysisconsidering the binding entropy as well as thebinding enthalpy will aid in the development ofnovel antibody drugs for hepatocellular carcinoma.

2. pH responsiveness of fibrous assemblies ofrepeat-sequence amphipathic -helix polypep-tides

Takei T, Tsumoto K, Okonogi A, Kimura A, Ko-jima S, Yazaki K, Takei T, Ueda T, Miura K.

We reported previously that our designed polypep-tide 3 (21 residues), which has three repeats of aseven-amino-acid sequence (LETLAKA)3, forms notonly an amphipathic -helix structure but also longfibrous assemblies in aqueous solution. To addressthe relationship between the electrical states of thepolypeptide and its -helix and fibrous assemblyformation, we characterized mutated polypeptidesin which charged amino acid residues of 3 werereplaced with Ser. We prepared the followingpolypeptides: 2S3 (LSTLAKA)3, in which all Gluresidues were replaced with Ser residues; 6S3(LETLASA)3, in which all Lys residues were re-placed with Ser; and 2S6S3 (LSTLASA)3; in whichall Glu and Lys residues were replaced with Ser. In0.1M KCl, 2S3 formed an -helix under basic con-ditions and 6S3 formed an -helix under acid con-ditions. In 1M KCl, they both formed -helices un-der a wide pH range. In addition, 2S3 and 6S3formed fibrous assemblies under the same bufferconditions in which they formed -helices. -Helixand fibrous assembly formation by these polypep-tides was reversible in a pH-dependent manner. Incontrast, 2S6S3 formed an -helix under basic con-ditions in 1M KCl. Taken together, these findingsreveal that the charge states of the charged aminoacid residues and the charge state of the Leu resi-due located at the terminus play an important rolein -helix formation.

3. Structural basis for self-assembly of a cyto-lytic pore lined by protein and lipid

Tanaka K, Caaveiro JM, Morante K, González-Mañas JM, Tsumoto K.

Pore-forming toxins (PFT) are water-soluble pro-teins that possess the remarkable ability to self-as-semble on the membrane of target cells, where theyform pores causing cell damage. Here, we elucidatethe mechanism of action of the haemolytic proteinfragaceatoxin C (FraC), a -barrel PFT, by deter-mining the crystal structures of FraC at four differ-ent stages of the lytic mechanism, namely thewater-soluble state, the monomeric lipid-boundform, an assembly intermediate and the fully as-sembled transmembrane pore. The structure of thetransmembrane pore exhibits a unique architecturecomposed of both protein and lipids, with some ofthe lipids lining the pore wall, acting as assemblycofactors. The pore also exhibits lateral fenestra-tions that expose the hydrophobic core of the mem-

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brane to the aqueous environment. The incorpora-tion of lipids from the target membrane within thestructure of the pore provides a membrane-specifictrigger for the activation of a haemolytic toxin.

4. Click conjugation of a binuclear terbium(III)complex for real-time detection of tyrosinephosphorylation

Akiba H, Sumaoka J, Tsumoto K, Komiyama M.

Phosphorylation of proteins is closely associatedwith various diseases, and, therefore, its detectionis vitally important in molecular biology and drugdiscovery. Previously, we developed a binuclearTb(III) complex, which emits notable luminescenceonly in the presence of phosphotyrosine. In thisstudy, we conjugated a newly synthesized binuclearTb(III) complex to substrate peptides by using clickchemistry. Using these conjugates, we were able todetect tyrosine phosphorylation in real time. Theseconjugates were superior to nonconjugated Tb(III)complexes for the detection of tyrosine phospho-rylation, especially when the substrate peptidesused were positively charged. Luminescence inten-sity upon phosphorylation was enhanced 10-fold,making the luminescence intensity of this systemone of the largest among lanthanide luminescence-based systems. We also determined Michaelis-Men-ten parameters for the phosphorylation of variouskinase/peptide combinations and quantitatively ana-lyzed the effects of mutations in the peptide sub-strates. Furthermore, we successfully monitored theinhibition of enzymatic phosphorylation by inhibi-tors in real time. Advantageously, this system de-tects only the phosphorylation of tyrosine (phos-phorylated serine and threonine are virtually silent)and is applicable to versatile peptide substrates.Our study thus demonstrates the applicability ofthis system for the analysis of kinase activity,which could lead to drug discovery.

5. A pore-forming toxin requires a specific resi-due for its activity in membranes with particu-lar physicochemical properties

Morante K, Caaveiro JM, Tanaka K, González-Mañas JM, Tsumoto K.

The physicochemical landscape of the bilayermodulates membrane protein function. Actinoporinsare a family of potent hemolytic proteins from seaanemones acting at the membrane level. This familyof cytolysins preferentially binds to target mem-branes containing sphingomyelin, where they formlytic pores giving rise to cell death. Although thecytolytic activity of the actinoporin fragaceatoxin C(FraC) is sensitive to vesicles made of various lipidcompositions, it is far from clear how this toxin ad-

justs its mechanism of action to a broad range ofphysiochemical landscapes. Herein, we show thatthe conserved residue Phe-16 of FraC is critical forpore formation in cholesterol-rich membranes suchas those of red blood cells. The interaction of apanel of muteins of Phe-16 with model membranescomposed of raft-like lipid domains is inactivatedin cholesterol-rich membranes but not in choles-terol-depleted membranes. These results indicatethat actinoporins recognize different membrane en-vironments, resulting in a wider repertoire of sus-ceptible target membranes (and preys) for seaanemones. In addition, this study has unveiledpromising candidates for the development of pro-tein-based biosensors highly sensitive to the con-centration of cholesterol within the membrane.

6. The Atomic Structure of the HIV-1 gp41 Trans-membrane Domain and Its Connection to theImmunogenic Membrane-proximal ExternalRegion

Apellániz B, Rujas E, Serrano S, Morante K, Tsu-moto K, Caaveiro JM, Jiménez MÁ, Nieva JL.

The membrane-proximal external region (MPER)C-terminal segment and the transmembrane do-main (TMD) of gp41 are involved in HIV-1 enve-lope glycoprotein-mediated fusion and modulationof immune responses during viral infection. How-ever, the atomic structure of this functional regionremains unsolved. Here, based on the high resolu-tion NMR data obtained for peptides spanning theC-terminal segment of MPER and the TMD, we re-port two main findings: (i) the conformational vari-ability of the TMD helix at a membrane-buried po-sition; and (ii) the existence of an uninterrupted -helix spanning MPER and the N-terminal region ofthe TMD. Thus, our structural data provide evi-dence for the bipartite organization of TMD pre-dicted by previous molecular dynamics simulationsand functional studies, but they do not support thebreaking of the helix at Lys-683, as was suggestedby some models to mark the initiation of the TMDanchor. Antibody binding energetics examined withisothermal titration calorimetry and humoral re-sponses elicited in rabbits by peptide-based vac-cines further support the relevance of a continuousMPER-TMD helix for immune recognition. We con-clude that the transmembrane anchor of HIV-1 en-velope is composed of two distinct subdomains: 1)an immunogenic helix at the N terminus also in-volved in promoting membrane fusion; and 2) animmunosuppressive helix at the C terminus, whichmight also contribute to the late stages of the fusionprocess. The unprecedented high resolution struc-tural data reported here may guide future vaccineand inhibitor developments.

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7. Protein stabilization by an amphiphilic shortmonodisperse oligo(ethylene glycol)

Sadhukhan N, Muraoka T, Ui M, Nagatoishi S,Tsumoto K, Kinbara K.

A short, monodisperse additive (octa(ethyleneglycol) monophenyl ether) functions to suppress ag-gregation of thermally and chemically denaturedlysozyme. Control studies with shorter and non-amphiphilic derivatives revealed that the am-phiphilic structure is essential, and octa(ethyleneglycol) is nearly the minimum chain length for am-phiphilic poly(ethylene glycol)s to stabilize pro-teins.

8. Structural basis for binding of human IgG1 toits high-affinity human receptor FcRI

Kiyoshi M, Caaveiro JM, Kawai T, Tashiro S, IdeT, Asaoka Y, Hatayama K, Tsumoto K.

Cell-surface Fc receptors mediate innate andadaptive immune responses. Human Fc receptor I(hFcRI) binds IgGs with high affinity and is theonly Fc receptor that can effectively capture mono-meric IgGs. However, the molecular basis ofhFcRI's interaction with Fc has not been deter-mined, limiting our understanding of this majorimmune receptor. Here we report the crystal struc-ture of a complex between hFcRI and human Fc, at1.80 A°resolution, revealing an unique hydrophobicpocket at the surface of hFcRI perfectly suited forresidue Leu235 of Fc, which explains the high affin-ity of this complex. Structural, kinetic and thermo-dynamic data demonstrate that the binding mecha-nism is governed by a combination of non-covalentinteractions, bridging water molecules and the dy-namic features of Fc. In addition, the hinge regionof hFcRI-bound Fc adopts a straight conformation,potentially orienting the Fab moiety. These findingswill stimulate the development of novel therapeuticstrategies involving hFcRI.

9. Thermodynamics of antibody-antigen interac-tion revealed by mutation analysis of anti-body variable regions

Akiba H, Tsumoto K.

Antibodies (immunoglobulins) bind specificmolecules (i.e. antigens) with high affinity andspecificity. In order to understand their mecha-nisms of recognition, interaction analysis based onthermodynamic and kinetic parameters, as well asstructure determination is crucial. In this review,we focus on mutational analysis which gives infor-mation about the role of each amino acid residue inantibody-antigen interaction. Taking anti-hen egg

lysozyme antibodies and several anti-small mole-cule antibodies, the energetic contribution of hot-spot and non-hot-spot residues is discussed interms of thermodynamics. Here, thermodynamicsof the contribution from aromatic, charged and hy-drogen bond-forming amino acids are discussed,and their different characteristics have been eluci-dated. The information gives fundamental under-standing of the antibody-antigen interaction. Fur-thermore, the consequences of antibody engineeringare analysed from thermodynamic viewpoints: hu-manization to reduce immunogenicity and rationaldesign to improve affinity. Amino acid residuesoutside hot-spots in the interface play importantroles in these cases, and thus thermodynamic andkinetic parameters give much information about theantigen recognition. Thermodynamic analysis ofmutant antibodies thus should lead to advancedstrategies to design and select antibodies with highaffinity.

10. Osteomodulin regulates diameter and altersshape of collagen fibrils

Tashima T, Nagatoishi S, Sagara H, Ohnuma S,Tsumoto K.

Osteomodulin (OMD) is a member of the smallleucine-rich repeat proteoglycan family, which is in-volved in the organization of the extracellular ma-trix. OMD is located in bone tissue and is report-edly important for bone mineralization. However,the details of OMD function in bone formation arepoorly understood. Using the baculovirus expres-sion system, we produced recombinant humanOMD and analyzed its interaction with type I colla-gen, which is abundant in bone. In this result,OMD directly interacted with purified immobilizedcollagen and OMD suppressed collagen fibril for-mation in a turbidity assay. Morphological analysisof collagen in the presence or absence of OMDdemonstrated that OMD reduces the diameter andchanges the shape of collagen fibrils. We concludethat OMD regulates the extracellular matrix duringbone formation.

11. Differential binding of prohibitin-2 to estro-gen receptor and to drug-resistant ERmutants

Chigira T, Nagatoishi S, Tsumoto K.

Endocrine resistance is one of the most challeng-ing problems in estrogen receptor alpha (ER)-posi-tive breast cancer. The transcriptional activity ofER is controlled by several coregulators, includingprohibitin-2 (PHB2). Because of its ability to repressthe transcriptional activity of activated ER, PHB2is a promising antiproliferative agent. In this study,

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were analyzed the interaction of PHB2 with ERand three mutants (Y537S, D538G, and E380Q) thatare frequently associated with a lack of sensitivityto hormonal treatments, to help advance noveldrug discovery. PHB2 bound to ER wild-type(WT), Y537S, and D538G, but did not bind toE380Q. The binding thermodynamics of Y537S andD538G to PHB2 were favorably altered entropicallycompared with those of WT to PHB2. Our resultsshow that PHB2 binds to the ligand binding do-main of ER with a conformational change in thehelix 12 of ER.

12. Functional characterization of Val60, a keyresidue involved in the membrane-oligomeri-zation of fragaceatoxin C, an actinoporinfrom Actinia fragacea

Morante K, Caaveiro JM, Viguera AR, TsumotoK, González-Mañas JM.

Actinoporins are pore-forming toxins producedby different sea anemones that self-assemble withinthe membranes of their target cells and compromisetheir function as a permeability barrier. The re-cently published three-dimensional structures oftwo oligomeric complexes formed by fragaceatoxinC point to Val60 as a key residue involved in theoligomerization of the functional pore. To gain in-sight into the mechanism of toxin oligomerization,different point mutations have been introduced atthis position. Functional characterization of themuteins suggests that Val60 represents a hot-spotwhere the introduction of mutations hinders pro-tein assembly and reduces the overall affinity formembranes.

13. Thermodynamic characterization of the inter-action between the human Y-box bindingprotein YB-1 and nucleic acids

Tanabe Y, Nagatoishi S, Tsumoto K.

Y-box binding protein 1 (YB-1) binds to bothRNA and DNA to control transcription and transla-tion for the regulation of various cellular systems.YB-1 is overexpressed in some cancer cells and is apotential target for treatment of cancer. Herein, wedescribe isothermal titration calorimetry analyses ofthe interaction between a number of recombinantYB-1 domains and nucleic acids to identify theRNA and DNA binding sites and their bindingmechanisms. These results demonstrated that the C-terminal domain of the protein interacts with sin-gle-stranded DNA and RNA by exothermic and en-dothermic reactions, respectively. The highly con-served cold-shock domain (CSD) also bound to sin-gle-stranded RNA and DNA by exothermic and en-dothermic reactions, respectively. The specific bind-

ing manner for RNA is in the CSD, whereas DNAbinds with the most affinity to the C-terminal re-gion (amino acids 130-219). We found further thatthe C-terminal region (amino acids 220-324) regu-lates the binding stoichiometry of RNA. Thesequantitative thermodynamic results provide a pre-liminary indication on the molecular mechanism ofbinding of the multifunctional protein YB-1 to nu-cleic acids to regulate its biological function.

14. Structural and Thermodynamic Basis of Epi-tope Binding by Neutralizing and Nonneu-tralizing Forms of the Anti-HIV-1 Antibody4E10

Rujas E, Gulzar N, Morante K, Tsumoto K, ScottJK, Nieva JL, Caaveiro JM.

The 4E10 antibody recognizes the membrane-proximal external region (MPER) of the HIV-1 Envglycoprotein gp41 transmembrane subunit, exhibit-ing one of the broadest neutralizing activitiesknown to date. The neutralizing activity of 4E10 re-quires solvent-exposed hydrophobic residues at theapex of the complementarity-determining region(CDR) H3 loop, but the molecular basis for this re-quirement has not been clarified. Here, we reportthe cocrystal structures and the energetic parame-ters of binding of a peptide bearing the 4E10-epi-tope sequence (4E10ep) to nonneutralizing versionsof the 4E10 Fab. Nonneutralizing Fabs were ob-tained by shortening and decreasing the hydropho-bicity of the CDR-H3 loop (termed Loop) or bysubstituting the two tryptophan residues of theCDR-H3 apex with Asp residues (termed WDWD),which also decreases hydrophobicity but preservesthe length of the loop. The analysis was comple-mented by the first crystal structure of the 4E10 Fabin its ligand-free state. Collectively, the data ruledout major conformational changes of CDR-H3 atany stage during the binding process (equilibriumor transition state). Although these mutations didnot impact the affinity of wild-type Fab for the 4E10ep in solution, the two nonneutralizing versionsof 4E10 were deficient in binding to MPER insertedin the plasma membrane (mimicking the environ-ment faced by the antibody in vivo). The conclu-sions of our structure-function analysis strengthenthe idea that to exert effective neutralization, thehydrophobic apex of the solvent-exposed CDR-H3loop must recognize an antigenic structure morecomplex than just the linear -helical epitope andlikely constrained by the viral membrane lipids.IMPORTANCE:The broadly neutralizing anti-HIV-1 4E10 anti-

body blocks infection caused by nearly all viralstrains and isolates examined thus far. However,4E10 (or 4E10-like) antibodies are rarely found inHIV-1-infected individuals or elicited through vac-

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cination. Impediments to the design of successful4E10 immunogens are partly attributed to an in-complete understanding of the structural and bind-ing characteristics of this class of antibodies. Sincethe broadly neutralizing activity of 4E10 is abro-gated by mutations of the tip of the CDR-H3, weinvestigated their impact on binding of the MPER-epitope at the atomic and energetic levels. We con-clude that the difference between neutralizing andnonneutralizing antibodies of 4E10 is neither struc-tural nor energetic but is related to the capacity torecognize the HIV-1 gp41 epitope inserted in bio-logical membranes. Our findings strengthen theidea that to elicit similar neutralizing antibodies,the suitable MPER vaccine must be "delivered" in amembrane environment.

15. Discovery and characterization of naturaltropolones as inhibitors of the antibacterialtarget CapF from Staphylococcus aureus

Nakano K, Chigira T, Miyafusa T, Nagatoishi S,Caaveiro JM, Tsumoto K.

The rapid spread of antibiotic-resistance amongpathogenic bacteria poses a serious risk for publichealth. The search for novel therapeutic strategiesand antimicrobial compounds is needed to amelio-rate this menace. The bifunctional metalloenzymeCapF is an antibacterial target produced by certainpathogenic bacteria essential in the biosyntheticroute of capsular polysaccharide, a mucous layer onthe surface of bacterium that facilitates immuneevasion and infection. We report the first inhibitorof CapF from Staphylococcus aureus, which wasidentified by employing fragment-based method-ologies. The hit compound 3-isopropenyl-tropoloneinhibits the first reaction catalyzed by CapF, dis-rupting the synthesis of a key precursor of capsularpolysaccharide. Isothermal titration calorimetry dem-onstrates that 3-isopropenyl-tropolone binds tightly(KD = 27±7M) to the cupin domain of CapF. Inaddition, the crystal structure of the enzyme-inhibi-tor complex shows that the compound engages theessential Zn(2＋) ion necessary for the first reactioncatalyzed by the enzyme, explaining its inhibitoryeffect. Moreover, the tropolone compound alters thecoordination sphere of the metal, leading to theoverall destabilization of the enzyme. We propose3-isopropenyl-tropolone as a precursor to developstronger inhibitors for this family of enzymes to im-pair the synthesis of capsular polysaccharide inStaphylococcus aureus.

16. Structural analysis of Fc/FcR complexes: ablueprint for antibody design

Caaveiro JM, Kiyoshi M, Tsumoto K.

The number of studies and the quality of thestructural data of Fc receptors (FcRs) has rapidlyincreased in the last few years. Upon critical exami-nation of the literature, we have extracted generalconclusions that could explain differences in affinityand selectivity of FcRs for immunoglobulin G(IgG) based on structural considerations. FcRs em-ploy a little conserved asymmetric surface of do-main D2 composed of two distinct subsites to rec-ognize the well-conserved lower hinge region ofIgG1-Fc. The extent of the contact interface with theantibody in subsite 1 of the receptor (but not insubsite 2), the geometrical complementarity be-tween antibody and receptor, and the number ofpolar interactions contribute decisively towardstrengthening the binding affinity of the antibodyfor the receptor. In addition, the uncertain role ofthe N-linked glycan of IgG for the binding and ef-fector responses elicited by FcRs is discussed. Theavailable data suggest that not only the non-cova-lent interactions between IgG and FcRs but alsotheir dynamic features are essential for the immuneresponse elicited through these receptors. We be-lieve that the integration of structural, thermody-namic, and kinetic data will be critical for the de-sign and validation of the next generation of thera-peutic antibodies with enhanced effector capabili-ties.

17. Bidirectional Transformation of a Metamor-phic Protein between the Water-Soluble andTransmembrane Native States

Tanaka K, Caaveiro JM, Tsumoto K.

The bidirectional transformation of a protein be-tween its native water-soluble and integral trans-membrane conformations is demonstrated for FraC,a hemolytic protein of the family of pore-formingtoxins. In the presence of biological membranes, thewater-soluble conformation of FraC undergoes a re-markable structural reorganization generating cyto-lytic transmembrane nanopores conducive to celldeath. So far, the reverse transformation from thenative transmembrane conformation to the nativewater-soluble conformation has not been reported.We describe the use of detergents with differentphysicochemical properties to achieve the spontane-ous conversion of transmembrane pores of FraCback into the initial water-soluble state. Thermody-namic and kinetic stability data suggest that spe-cific detergents cause an asymmetric change in theenergy landscape of the protein, allowing the bidi-rectional transformation of a membrane protein.

18. Epiregulin Recognition Mechanisms by Anti-Epiregulin Antibody 9E5: Structural, Func-tional and Molecular Dynamics SimulationAnalyses

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Kado Y, Mizohata E, Nagatoishi S, Iijima M,Shinoda K, Miyafusa T, Nakayama T, YoshizumiT, Sugiyama A, Kawamura T, Lee YH, Matsu-mura H, Doi H, Fujitani H, Kodama T, ShibasakiY, Tsumoto K, Inoue T.

Epiregulin (EPR) is a ligand of the epidermalgrowth factor (EGF) family that upon binding to itsepidermal growth factor receptor (EGFR) stimulatesproliferative signaling, especially in colon cancercells. Here, we describe the three-dimensional struc-ture of the EPR antibody (the 9E5(Fab) fragment) inthe presence and absence of EPR. Among the sixcomplementarity-determining regions (CDRs), CDR1-3 in the light chain and CDR2 in the heavy chainpredominantly recognize EPR. In particular, CDR3in the heavy chain dramatically moves with cis-trans isomerization of Pro103. A molecular dynam-ics simulation and mutational analyses revealedthat Arg40 in EPR is a key residue for the specificbinding of 9E5 IgG. From ITC analysis, the disso-ciation constant is determined to be 6.5 nM. Surfaceplasmon resonance analysis revealed that the disso-ciation rate of 9E5 IgG is extremely slow. The su-perimposed structure of 9E5(Fab)-EPR on theknown complex structure of EGF-EGFR showedthat the 9E5(Fab) paratope overlaps with Domains Iand III on the EGFR, which reveals that the 9E5(Fab)-EPR complex could not bind to the EGFR.The 9E5 antibody will also be useful in medicine asa neutralizing antibody specific for colon cancer.

19. Rapid Heme Transfer Reactions betweenNEAr Transporter Domains of Staphylococ-cus aureus: A Theoretical Study Using QM/MM and MD Simulations

Moriwaki Y, Terada T, Tsumoto K, Shimizu K.

In vertebrates, most iron is present as heme or ischelated by proteins. Thus, Gram-positive patho-gens such as Staphylococcus aureus have evolvedan iron-regulated surface determinant (Isd) systemthat transports heme across thick cell walls into thecytoplasm. Recent studies have demonstrated thatheme is rapidly transferred between the NEArTransporter (NEAT) domains of the Isd system, de-spite its high affinity toward each domain, suggest-ing the presence of an intermediate NEAT•heme•NEAT complex. In the present study, we performedshort restrained molecular dynamics (MD) simula-tions to dock the acceptor NEAT domain to the do-nor NEAT•heme complex and obtained modelswhere the two NEAT domains were arranged withtwo-fold pseudo symmetry around the heme mole-cule. After turning off the restraints, complex struc-tures were stably maintained during subsequentunrestrained MD simulations, except for the hydro-gen bond between the propionate group of the

heme molecule and the donor NEAT domain, po-tentially facilitating the transition of heme from thedonor to the acceptor. Subsequent structural opti-mization using the quantum mechanics/molecularmechanics (QM/MM) method showed that two ty-rosine residues, one from each NEAT domain, weresimultaneously coordinated to the ferric heme ironin the intermediate complex only if they were de-protonated. Based on these results, we propose areaction scheme for heme transfer between NEATdomains.

<Group III>

1. Development of new methods for analyzingthe neural circuits in the retina

Neural circuits in the central nervous system arethe basis of various high-order brain functions. It isalso true in case of retina. In the retina, six mainclasses of neural cells connect systematically tomake up complex neural circuits. Characteristics ofthe neural cell functions have been examined pre-cisely by the electrophysiological methods andmodels of cell connectivity have been proposed. Butmorphological studies of the actual neural connec-tion, which constitute the physiological propertiesof retinal neurons, are not enough. Until recently tocollect ultrathin serial sections and observe in trans-mission electron microscope (TEM) were the onlyway to reveal the connectivity of actual neural cellsmorphologically. But the technical difficulties dis-couraged us from such a troublesome studies. Re-cent progress in scanning electron microscope(SEM) equipment allowed us to develop a newmethod to observe ultrathin TEM sections in SEM.Samples were specifically treated to enhance elec-tron contrast and serial thin TEM sections were col-lected on the smooth conductive matrix. By usingSEM to observe thin TEM sections, it became possi-ble to analyze much wider areas than by usingTEM. We have analyzed about 500 serial thin sec-tions of zebrafish retinal outer plexiform layer bythis method and succeeded in tracing thin proc-esses of bipolar cells into the photoreceptor termi-nal. In the course of this study, it became obviousthat thinner and larger numbers of section areneeded to reveal the accurate connectivity espe-cially for thinner processes. This year, we devel-oped a new equipment to collect huge number ofserial sections stably and efficiently. By using thisequipment, more than 1000 serial sections of lessthan 30nm thickness were successfully collectedand have been analyzing.In parallel, methods to observe thick sections,

which are difficult to observe because of their lessconductivity, were developed and tested for use incounting actual cell number in tissues. In anotherwork, 515 serial thin sections of a whole hepatic

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cell were observed with the same method and re-constituted as a model for simulating the transduc-tion of NF-kappa B signal in the real cell (ref.Ichikawa1 et al) : 1Division of Mathematical Oncol-ogy.

2. Collaborative and supportive works as elec-tron microscope core-laboratory

This group is also engaged in collaborative re-searches using electron microscope. We offer sup-ports for the research projects those need electronmicroscopic analysis. The services available in thisgroup are the conventional thin section transmis-sion electron microscopy, immuno-electron micros-copy, negative staining techniques and scanningelectron microscopy. By using these individualtechnique or combination of some of these we canoffer direct visual evidence that cannot be acquiredby other methods. This year, 23 projects in 13 labo-ratories were performed as core-laboratory works.

a. Thin section transmission electron micros-copy

Thin section transmission electron microscopy isthe most widely used technique to observe the in-ner structure of cells and tissues. In this method,samples are fixed and embedded in epoxy resin,thin sections with about 70nm thickness are cut andobserved in the electron microscope. In case of im-muno-electron microscopy, thin sections are ob-tained by similar procedure, and the antigen epi-topes exposed on the surface of the sections aremarked by sequentially reacted with appropriateprimary antibodies and colloidal gold labeled sec-ondary antibodies. This year, thin section electronmicroscopy combined with immuno-electron mi-croscopy were used in many collaborative works.

a-1. Ultrastructural analysis of entry and assem-bly of Herpes Simplex Virus

We have been performing several studies withresearch groups in Dr. Kawaguchi2's laboratory: 2Di-vision of Molecular Virology, Department of Micro-biology and Immunology, regarding the infection/replication processes of herpes simplex virus (HSV).This year, thin section electron microscopy wasused in 11 projects to analyze the function of viralproteins in trans-nuclear membrane processes of thenewly forming viruses. Among these woks, investi-gatin of the type-specific differences betweenHSSV-1 and HSV-2 Us3 protein kinases (ref.Shindo2 et al) and the work regarding the functionof HSV-1 CD98 and beta-1 integrin in the denvelop-ment proces (ref. Hirohata2 et al) were published.

a-2. Morphological analysis of the mouse Panethcells and M cells

We have been performing several studies alsowith research groups in Dr. Kiyono3's laboratory:3Division of Mucosal Immunology, Department ofMicrobiology and Immunology. This year, thin sec-tion transmission electron microscopy was used toanalyze the change of Paneth cell structures in acertain gene knock out mouse. In another work, thestructural changes of M cells were also analyzedelectron microscopically.Some other collaborative research works using

thin section electron microscopy and/or immuno-electron microscopy were performed with Dr.Noda4 et al in 4Division of Virology, Department ofMicrobiology, regarding the structure of the influ-enza viruses and ebola virus, Dr. Yasuda5's group,in 5Department of Integrated Biosciences, GraduateSchool of Frontier Sciences regarding the fine struc-ture of the developing central nervous system ofMedaka fish (ref. Yasuda5 et al).

b. Negative staining techniques

Negative staining techniques are simple and quickmethod to observe the morphology of the macromolecules. In the collaborative work with Dr.Noda4 et al., this technique combined with thin sec-tion electron microscopy was used to analyze themorphology of the influenza virus ribonucleopro-tein complex. The negative staining techniqueswere also used in some works to analyze the struc-ture of the purified proteins and the proteins inte-grated in the plasma membrane. This method con-vined with scanning electron microscopy was usedalso to analyze the function of a protein during thein vitro formation of collagen fibers in collaborationwith Dr. Tashima6 et al in 6Medical ProteomicsLaboratory ( ref. Tashima6 et al).

c. Conventional scanning electron microscopy

Conventional scanning electron microscopy is atechnique used to examine the surface structure ofthe cells, tissues or other non-biological materials.The collaborative work using scanning electron mi-croscopy convined with negative staining methodwas performed with Dr. Tashima6 as mentionedabove (ref. Tashima6 et al). Scanning electron mi-croscopy was also used to analyze the morphologi-cal changes of cultured macrophages and non-bio-logical materials as a collaborative work with Dr.Cheng7 in 7Olympus Co.

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Publications

<Group I>Hirohata Y, Kato A, Oyama M, Kozuka-Hata H,Koyanagi N, Arii J, and Kawaguchi Y. Interac-tome analysis of herpes simplex virus 1 envelopeglycoprotein H. Microbiol Immunol, 59: 331-337,2015.

Kobayashi R, Kato A, Oda S, Koyanagi N, OyamaM, Kozuka-Hata H, Arii J, and Kawaguchi Y.Function of the Herpes Simplex Virus 1 SmallCapsid Protein VP26 Is Regulated by Phospho-rylation at a Specific Site. J Virol, 89: 6141-6147,2015.

Hirohata Y, Arii J, Liu Z, Shindo K, Oyama M, Ko-zuka-Hata H, Sagara H, Kato A, and KawaguchiY. Herpes Simplex Virus 1 Recruits CD98 HeavyChain and 1 Integrin to the Nuclear Membranefor Viral De-Envelopment. J Virol, 89: 7799-7812,2015.

Liu Z, Kato A, Oyama M, Kozuka-Hata H, Arii J,and Kawaguchi Y. Role of Host Cell p32 in Her-pes Simplex Virus 1 De-Envelopment during Vi-ral Nuclear Egress. J Virol, 89: 8982-8998, 2015.

Inoue D, Nishimura K, Kozuka-Hata H, Oyama M,and Kitamura T. The stability of epigenetic factorASXL1 is regulated through ubiquitination andUSP7-mediated deubiquitination. Leukemia, 29:2257-2260, 2015.

Sato Y, Kato A, Arii J, Koyanagi N, Kozuka-HataH, Oyama M, and Kawaguchi Y. Ubiquitin-spe-cific protease 9X in host cells interacts with her-pes simplex virus 1 ICP0. J Vet Med Sci, in press.

Narushima Y, Kozuka-Hata H, Koyama-Nasu R,Tsumoto K, Inoue JI, Akiyama T, and Oyama M.Integrative Network Analysis Combined withQuantitative Phosphoproteomics Reveals TGFBR2as a Novel Regulator of Glioblastoma Stem CellProperties. Mol Cell Proteomics, in press.

Sato Y, Kato A, Maruzuru Y, Oyama M, Kozuka-Hata H, Arii J, and Kawaguchi Y. Cellular Tran-scriptional Coactivator RanBP10 and Herpes Sim-plex Virus 1 ICP0 Interact and SynergisticallyPromote Viral Gene Expression and Replication. JVirol, in press.

Taniue K, Kurimoto A, Sugimasa H, Nasu E,Takeda Y, Iwasaki K, Nagashima T, Okada-Hatakeyama M, Oyama M, Kozuka-Hata H,Hiyoshi M, Kitayama J, Negishi L, Kawasaki Y,and Akiyama T. Long noncoding RNA UPATpromotes colon tumorigenesis by inhibiting deg-radation of UHRF1. Proc Natl Acad Sci USA, inpress.

Nishimura A, Yamamoto K, Oyama M, Kozuka-Hata H, Saito H, and Tatebayashi K. A scaffoldprotein Ahk1 that associates with Hkr1, Sho1, Ste11 and Pbs2 inhibits cross-talk signaling from theHkr1 osmosensor to the Kss1 MAPK. Mol CellBiol, in press.

Homma MK, Shibata T, Suzuki T, Ogura M, Ko-zuka-Hata H, Oyama M, and Homma Y. Role forprotein kinase CK2 on cell proliferation: Assess-ing CK2 complex components in the nucleus dur-ing the cell cycle progression. In Protein KinaseCK2 Cellular Function in Normal and Disease States(ed. Khalil Ahmed, Olaf-Georg Issinger, andRyszard Szyszka), Springer, 197-226, 2015.

Kozuka-Hata H and Oyama M. Phosphoproteom-ics-based network analysis of cancer cell signal-ing systems. In Protein Modifications in PathogenicDysregulation of Signaling (ed. Jun-ichiro Inoueand Mutsuhiro Takekawa), Springer, 3-15, 2015.

<Group II>Nakayama T, Mizohata E, Yamashita T, NagatoishiS, Nakakido M, Iwanari H, Mochizuki Y, Kado Y,Yokota Y, Satoh R, Tsumoto K, Fujitani H, Ko-dama T, Hamakubo T, and Inoue T. Structuralfeatures of interfacial tyrosine residue in ROBO1fibronectin domain-antibody complex: Crystal-lographic, thermodynamic, and molecular dy-namic analyses. Protein Sci, 24: 328-340, 2015.

Takei T, Tsumoto K, Okonogi A, Kimura A, KojimaS, Yazaki K, Takei T, Ueda T, and Miura KI. pHresponsiveness of fibrous assemblies of repeat-se-quence amphipathic -helix polypeptides. ProteinSci, 24: 883-894, 2015.

Tanaka K, Caaveiro JM, Morante K, Gonzalez-Manas JM, and Tsumoto K. Structural basis forself-assembly of a cytolytic pore lined by proteinand lipid. Nature Commun, 6: 6337, 2015.

Akiba H, Sumaoka J, Tsumoto K, and KomiyamaM. Click-conjugation of binuclear terbium(III)complex for real-time detection of tyrosine phos-phorylation. Anal Chem, 87: 3834-3840, 2015.

Morante K, Caaveiro JM, Tanaka K, Gonzalez-Manas JM, and Tsumoto K. A Pore-FormingToxin Requires a Specific Residue for its Activityin Membranes with Particular PhysicochemicalProperties. J Biol Chem, 290: 10850-10861, 2015.

Apellaniz B, Rujas E, Serrano S, Morante K, Tsu-moto K, Caaveiro JM, Jimenez MA, and Nieva JL.The Atomic Structure of the HIV-1 gp41 Trans-membrane Domain and Its Connection to the Im-munogenic Membrane-proximal External Region.J Biol Chem, 290: 12999-13015, 2015.

Sadhukhan N, Muraoka T, Ui M, Nagatoishi S, Tsu-moto K, and Kinbara K. Protein stabilization byan amphiphilic short monodisperse oligo(ethyl-ene glycol). Chem Commun (Camb), 51: 8457-8460, 2015.

Akiba H and Tsumoto K. Thermodynamics of anti-body-antigen interaction revealed by mutationanalysis of antibody variable regions. J Biochem,158: 1-13, 2015.

Kiyoshi M, Caaveiro JM, Kawai T, Tashiro S, Ide T,

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Asaoka Y, Hatayama K, and Tsumoto K. Struc-tural basis for binding of human IgG1 to its high-affinity human receptor FcRI. Nature Commun,6: 6866, 2015.

Tashima T, Nagatoishi S, Sagara H, Ohnuma SI,and Tsumoto K. Osteomodulin regulates diame-ter and alters shape of collagen fibrils. BiochemBiophys Res Commun, 463: 292-296, 2015.

Chigira T, Nagatoishi S, and Tsumoto K. Differen-tial binding of prohibitin-2 to estrogen receptor and to drug-resistant ER mutants. Biochem Bio-phys Res Commun, 463: 726-731, 2015.

Morante K, Caaveiro JM, Viguera AR, Tsumoto K,and González-Mañas JM. Functional characteriza-tion of Val60, a key residue involved in the mem-brane-oligomerization of fragaceatoxin C, anactinoporin from Actinia fragacea. FEBS Lett, 589:1840-1846, 2015.

Tanabe Y, Nagatoishi S, and Tsumoto K. Thermo-dynamic characterization of the interaction be-tween the human Y-box binding protein YB-1and nucleic acids. Mol Biosyst, 11: 2441-2418,2015.

Rujas E, Gulzar N, Morante K, Tsumoto K, Scott JK,Nieva JL, and Caaveiro JM. Structural and Ther-modynamic Basis of Epitope Binding by Neutral-izing and Nonneutralizing Forms of the Anti-HIV-1 Antibody 4E10. J Virol, 89: 11975-11989,2015.

Nakano K, Chigira T, Miyafusa T, Nagatoishi S,Caaveiro JM, and Tsumoto K. Discovery andcharacterization of natural tropolones as inhibi-tors of the antibacterial target CapF from Staphy-lococcus aureus. Sci Rep, 5: 15337, 2015.

Caaveiro JM, Kiyoshi M, and Tsumoto K. Structuralanalysis of Fc/FcR complexes: a blueprint for an-tibody design. Immunol Rev, 268: 201-221, 2015.

Bai Y, Tashiro S, Nagatoishi S, Suzuki T, Yan D,Liu R, Tsumoto K, Bartlam M, and Yamamoto T.Structural basis for inhibition of the Tob-CNOT7interaction by a fragment screening approach.Protein Cell, 6: 924-928, 2015.

Tanaka K, Caaveiro JM, and Tsumoto K. Bidirec-tional Transformation of a Metamorphic Proteinbetween the Water-Soluble and TransmembraneNative States. Biochemistry, 54: 6863-6866, 2015.

Suzuki T, Yamaguchi H, Kikusato M, HashizumeO, Nagatoishi S, Matsuo A, Sato T, Kudo T, Mat-suhashi T, Murayama K, Ohba Y, Watanabe S,Kanno SI, Minaki D, Saigusa D, Shinbo H, MoriN, Yuri A, Yokoro M, Mishima E, Shima H, Aki-yama Y, Takeuchi Y, Kikuchi K, Toyohara T,Suzuki C, Ichimura T, Anzai JI, Kohzuki M,Mano N, Kure S, Yanagisawa T, Tomioka Y, To-hyomizu M, Tsumoto K, Nakada K, BonventreJV, Ito S, Osaka H, Hayashi KI, and Abe T. Mito-

chonic Acid 5 Binds Mitochondria and Amelio-rates Renal Tubular and Cardiac Myocyte Dam-age. J Am Soc Nephrol, in press.

Kado Y, Mizohata E, Nagatoishi S, Iijima M,Shinoda K, Miyafusa T, Nakayama T, YoshizumiT, Sugiyama A, Kawamura T, Lee YH, Matsu-mura H, Doi H, Fujitani H, Kodama T, ShibasakiY, Tsumoto K, and Inoue T. Epiregulin Recogni-tion Mechanisms by Anti-Epiregulin Antibody9E5: Structural, Functional and Molecular Dy-namics Simulation Analyses. J Biol Chem, inpress.

Moriwaki Y, Terada T, Tsumoto K, and Shimizu K.Rapid Heme Transfer Reactions between NEArTransporter Domains of Staphylococcus aureus:A Theoretical Study Using QM/MM and MDSimulations. PLoS One, 10: e0145125, 2015.

<Group III>Ichikawa K, Ohshima D, and Sagara H. Regulationof signal transduction by spatial parameters: acase in NF-B oscillation. IET Syst Biol, 9: 41-51,2015.

Shindo K, Kato A, Koyanagi N, Sagara H, Arii J,and Kawaguchi Y. Characterization of a HerpesSimplex Virus 1 (HSV-1) Chimera in Which theUs3 Protein Kinase Gene Is Replaced with theHSV-2 Us3 Gene. J Virol, 90: 457-473, 2015.

Ino D, Sagara H, Suzuki J, Kanemaru K, Okubo Y,and Iino M. Neuronal Regulation of SchwannCell Mitochondrial Ca(2＋) Signaling duringMyelination. Cell Rep, 12: 1951-1959, 2015.

Yasuda T, Oda S, Hibi Y, Satoh S, Nagata K, Hira-kawa K, Kutsuna N, Sagara H, and Mitani H.Embryonic Medaka Model of Microglia in theDeveloping CNS Allowing In Vivo Analysis ofTheir Spatiotemporal Recruitment in Response toIrradiation. PLoS One, 10: e0127325, 2015.

Tashima T, Nagatoishi S, Sagara H, Ohnuma S, andTsumoto K. Osteomodulin regulates diameterand alters shape of collagen fibrils. Biochem Bio-phys Res Commun, 463: 292-296, 2015.

Hirohata Y, Arii J, Liu Z, Shindo K, Oyama M, Ko-zuka-Hata H, Sagara H, Kato A, and KawaguchiY. Herpes Simplex Virus 1 Recruits CD98 HeavyChain and 1 Integrin to the Nuclear Membranefor Viral De-Envelopment. J Virol, 89: 7799-7812,2015.

Yamamoto S, Niida S, Azuma E, Yanagibashi T,Muramatsu M, Huang TT, Sagara H, Higaki S,Ikutani M, Nagai Y, Takatsu K, Miyazaki K,Hamashima T, Mori H, MatsudaN, Ishii Y, andSasahara M. Inflammation-induced endothelialcell-derivedextracellular vesicles modulate thecellular status of pericytes. Sci Rep, 5: 8505, 2015.

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