日本農芸化学会主催
Japan Society for Bioscience, Biotechnology, and Agrochemistry

第３４回 化学と生物シンポジウム
The 34th JSBBA Symposium on Chemistry and Biology

Unconventional Pathway of Protein Secretionand Internalization:
Mystery across Cell Membranes
タンパク質の分泌と細胞内導入の非定型的経路～ 細胞膜透過のミステリー ～2008 年 9 月 27 日（土）名古屋大学野依記念学術交流館
September 27, 2008 (Saturday)
Noyori Conference Hall, Nagoya UniversityJapan

Speakers

Dr. Walter Nickel

Dr. Akitada Ichinose

Dr. Marco E. Bianchi

Dr. Yohei Hirai

Dr. Alain Prochiantz

Dr. Shiroh Futaki

Registration (12:30-13:00)

Program
13:00

Opening remark: Sakayu Shimizu (Vice President of JSBBA)

Chair: Masatoshi Maki (Nagoya University, Japan)
13:05

Walter Nickel (Heidelberg University Biochemistry Center, Germany)
Overview - The mystery of unconventional protein secretion: How does
fibroblast growth factor 2 get transported into the extracellular space?

13:45

Akitada Ichinose (Yamagata University School of Medicine, Japan)
Moving outside: Translocation of a ‘cytosolic’ transglutaminase, the A subunitof coagulation factor XIII.

14:20

Marco E. Bianchi (San Raffaele Scientific Institute, Milan, Italy)
Extracellular function of high mobility group box I protein (HMGB1)
secreted from necrotic and non-necrotic cells.

Coffee Break (14:55-15:15)
Chair: Walter Nickel (Heidelberg University Biochemistry Center, Germany)
15:15

Yohei Hirai (Institute for Frontier Medical Science, Kyoto University, Japan)
Extracellular function of epimorphin/syntaxin2 as a morphogenic protein.

15:50

Alain Prochiantz (Ecole normale supérieure, Paris, France)
Secretion and internalization of homeoprotein transcription factors as direct
signaling proteins.

16:25

Shiroh Futaki (Institute for Chemical Research, Kyoto University, Japan)
Internalization mechanism of cell penetrating peptides.

17:00

Closing remark: Akira Isogai (President of JSBBA)-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-oFree Discussion over Snacks and Drinks(17:10 ~ 18:40)-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-o-oo-o-o-o-o-o-o-o-o-oTHE MYSTERY OF UNCONVENTIONAL PROTEIN SECRETION: HOW DOESFIBROBLAST GROWTH FACTOR 2 GET TRANSPORTED INTO THE
EXTRACELLULAR SPACE?

Walter Nickel
Heidelberg University Biochemistry Center, Germany
walter.nickel@bzh.uni-heidelberg.de
A number of secretory proteins are released from cells by mechanisms that are distinct from theclassical ER/Golgi-mediated secretory pathway. Recent studies revealed two critical requirements
for the secretion of one such protein, fibroblast growth
factor 2 (FGF-2). FGF-2 secretion is initiated by its
recruitment to the cytoplasmic leaflet of plasma
membranes mediated by the phosphoinositide PI(4,5)P2.
Based on mutational studies and RNAi silencing of PIP
kinases, it was shown that this interaction is required for
efficient FGF-2 secretion. A second interaction that is
critical for FGF-2 transport into the extracellular space ismediated by cell surface heparan sulfate proteoglycans(HSPGs). This has been shown by FGF-2 variant forms
that are both deficient for binding to heparan sulfates andfor secretion. In a complementary approach cell lines that
do not express functional HSPGs were demonstrated not
to secrete the wild-type form of FGF-2. Additionally,
based on affinity-purified plasma membrane vesicles withinverted topology, FGF-2 was shown to be secreted bydirect translocation across the plasma membrane in anATP- and membrane potential-independent manner. I Fig. 1: A current model of thepropose that FGF-2 membrane translocation is a molecular mechanism of FGF-2
diffusion-controlled process that is initiated by FGF-2 secretion.
binding to the phosphoinositide PI(4,5)P2. Cell surface HSPGs are functioning as an extracellularmolecular trap that drives directional transport of FGF-2 across the plasma membrane. As illustratedin Fig. 1, these findings resulted in a current working hypothesis on how FGF-2 physically traverses
the plasma membrane.

Moving Outside: Translocation of a ‘Cytosolic’ Transglutaminase, the A Subunitof Coagulation Factor XIII
Ichinose A, Kasahara K, Kaetsu H, and Souri M
Yamagata University School of Medicine, Japanaichinos@med.id.yamagata-u.ac.jp
Factor XIII (FXIII) is a plasma Transglutaminase circulating in blood as a heterotetramer consistingof two catalytic A subunits (FXIII-A) and two non-catalytic B subunits (FXIII-B). FXIII-A mainly
exists in plasma and the cytoplasma of megakaryocytes/platelets, monocytes/macrophages, andosteoblasts/chondrocytes, etc. When we cloned the cDNA and gene for FXIII-A, we first noticed
that there was no signal peptide for secretion; nor could we find any internal signal. The results ofthe expression of FXIII-A and/or FXIII-B in a heterologous mammalian cell system suggested thatFXIII-A is not secreted through the conventional secretory pathway, but is released, at least in vitro,from the cells by cell damage or apoptosis. However, it has been demonstrated that FXIII-A isexpressed on the cell surfaces of human monocytes and macrophages (Kradin et al, 1987; Conklinget al. 1989); moreover the cell surface expression of FXIII-A was augmented by macrophageactivators. It was also reported that in cultured mouse osteoblasts and chondrocytes FXIII-A isproduced as a small processed form (Al-Jallad et al, 2006; Nakano et al, 2007); ascorbic-acidtreatment dramatically stimulated the externalization of FXIII-A to the cell surface and to theextracellular matrix. To explore the possible intracellular function(s) and mechanism ofextracellular release, we investigated the intracellular localization of FXIII-A using amegakaryocytic cell line, MEG-01. In the endogenously synthesizing cells, three typical
localizations of FXIII-A were observed: 1) a diffuse pattern in the cytoplasm; 2) a filamentous
structure around the nucleus; and 3) a diffuse pattern in the nucleus. Intracellular FXIII-A partly
co-localized and interacted with an intermediate filament protein, vimentin. Furthermore, FXIII-A
was also found in the lipid raft fraction (raft) of the plasma membrane, the amount of which varied
under culture conditions. FXIII-A had also been detected on the surface of bovine and humanplatelets. When human washed platelets were activated by thrombin, fibrinogen in non-raft fractionstransformed into fibrin and translocated to the raft on the platelet surface. It is important to note that
this fibrinogen/fibrin translocation was completely blocked by the addition of cystamine, a potent
Transglutaminase inhibitor including activated FXIII (FXIIIa). Moreover, a fibrinogen mutant ofQ398A&Q399A, where both the γ－γ cross-linking sites by FXIIIa were destroyed, did not move tothe raft, suggesting that FXIII-A is essential for fibrin translocation to the surface. These findings
may facilitate our understanding of extracellular release mechanisms and intracellular functions ofFXIII-A in the cellular events of megakaryocytes/platelets.

Extracellular function of high mobility group box I protein (HMGB1) secreted
from necrotic and non-necrotic cells.
Marco E. Bianchi
San Raffaele Scientific Institute, Milan, Italy
bianchi.marco@hsr.it
Tissue damage occurs often in the life of mammals and is usually repaired. Dying cells are swiftly
phagocytosed, but before disappearing, they alert surrounding cells to activate homeostaticprograms. They release signals that recruit inflammatory cells to the site of injury, promote cell
migration and cell division to replace dead cells, and activate the immune system in anticipation ofmicrobial invasion. Many of these events involve high-mobility group box 1 protein (HMGB1), a
nuclear protein that is released passively when necrotic cells lose the integrity of their membranes.
HMGB1 behaves as a trigger of inflammation, attracting inflammatory cells, and of tissue repair,recruiting stem cells and promoting their proliferation. Moreover, HMGB1 activates dendritic cells(DCs) and promotes their functional maturation and their response to lymph node chemokines.
Activated leukocytes actively secrete HMGB1 in the microenvironment. Thus, HMGB1 acts in an
autocrine/paracrine fashion and sustains long-term repair and defense programs. DCs secrete
HMGB1 several hours after contact with the first maturation stimulus; HMGB1 secretion is critical
for their ability to reach the lymph nodes, to sustain the proliferation of antigen-specific T cells, toprevent their activation-dependent apoptosis, and to promote their polarization towards a T-helper 1phenotype. These immune responses will also be directed against self-antigens that DCs process atthe time of injury and can lead to autoimmunity.
References
Palumbo R, Galvez BG, Pusterla T, De Marchis F, Cossu G, Marcu KB and Bianchi ME (2007) Cells
migrating to sites of tissue damage in response to the danger signal HMGB1 require NF-κB activation.
J Cell Biol, 179(1):33-40.
Mollica L, De Marchis F, Spitaleri A, Dallacosta C, Pennacchini D, Zamai M, Agresti A, Trisciuoglio L,
Musco G and Bianchi ME (2007) Glycyrrhizin binds to High Mobility Group Box 1 protein (HMGB1)
and inhibits its cytokine activities. Chem Biol 14: 431-41
Bosisio D, Marazzi I, Agresti A, Shimizu N, Bianchi ME* and Natoli G* (2006) A hyper-dynamic
equilibrium between promoter-bound and nucleoplasmic dimers controls NF-κB-dependent geneactivity. EMBO J 25: 798-810 (*co-correspponding)Agresti A, Scaffidi P, Riva A, Caiolfa VR and Bianchi ME (2005) GR and HMGB1 interact only within
chromatin and influence each other’s residence time. Mol Cell 18:109-121
Rovere-Querini P, Capobianco A, Scaffidi P, Valentinis B, Catalanotti F, Giazzon M, Dumitriu IE, Müller S,Iannacone M, Traversari C, Bianchi ME and Manfredi AA (2004) HMGB1 is an endogenous immune
adjuvant released by necrotic cells. EMBO Rep 5: 825-30
Palumbo R, Sampaolesi M, De Marchis F, Tonlorenzi R, Colombetti S, Mondino A, Cossu G and Bianchi
ME. (2004) Extracellular HMGB1, a signal of tissue damage, induces mesoangioblast migration andproliferation. J Cell Biol 164: 441-9
Bonaldi T, Talamo F, Scaffidi P, Ferrera D, Porto A, Bachi A, Rubartelli A, Agresti A and Bianchi ME(2003) Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion.
EMBO J 22: 5551-60
Scaffidi P, Misteli T and Bianchi ME (2002) Release of chromatin protein HMGB1 by necrotic cells triggers
inflammation. Nature 418: 191-5

Extracellular function of epimorphin/syntaxin2 as a morphogenic protein
Yohei Hirai
Institute for Frontier Medical Sciences, Kyoto University, Japanhirai-yohei@frontier.kyoto-u.ac.jp
Elucidation of the processes involved in organogenesis requires a detailed understanding ofcomplex spatio-temporal actions of morphoregulatory molecules. The morphogen epimorphin has
been shown to function extracellularly in a wide variety of organ developmental processes, although
this molecule lacks signal peptide and the major population exists in the cytosol as the vesicle
fusion mediator syntaxin2. Here, I show the molecular mechanism underlying epimorphin’s
temporal extracellular localization, secretion and the following morphoregulatory action for thetarget epithelial cells. The SNARE domain of epimorphin, which is essential for the cytoplasmic
vesicular fusion appeared to play a key role also in the membrane translocation of this molecule.
The SNARE domain of epimorphin associates to phosphatidylserine-bound annexin II andsynaptotagmin, and the flipping of phosphatidylserine caused by physiological stimuli leads toextracellular projection of the protein complex, which bears the similarity to the non-classicalsecretion mechanism of another leaderless protein FGF1. Epimorphin is then cleaved atGlu245-His246 andtheC-terminallytruncated form isreleased
towardαv-integrin receptors
on the target cells toactivate
focal
adhesion kinase for
their morphogenesis.
Thesefindings
providekey
information about theaction of epimorphinin the extracellularmilieu and shed lighton the sophisticatedregulatory mechanism
intissuemorphogenesis by aprotein of double life.

Secretion and internalization of homeoprotein transcription factors
as direct signaling proteins
Alain Prochiantz(Collège de France)
UMR CNRS/ENS/CDF 8542 Développement et Evolution du Système Nerveux
Ecole normale supérieure, 46 rue d’Ulm, 75005 Paris, FranceAlain.Prochiantz@ens.fr
Homeogenes encode homeoprotein transcription factors with important developmental and adultfunctions. Indeed, they are key players within genetic networks that lay out the body plan andregulate morphological and physiological phenomena at the cellular and multi-cellular levels. In theearly 1990s, we observed that homeoproteins are internalized by live cells and gain direct accesstheir cytoplasm and nucleus. This finding has led to the identification of a small peptidic domain
capable of translocating across biological membranes, thus opening the field of transduction
peptides. These initial studies have allowed us and others to establish that homeoproteins shareactivities that extend beyond transcription to include translation regulation and intercellular transfer.
Beyond the mechanistic and fascinating problem of how to get in (and out), it is likely that thisnovel signaling pathway serves important physiological functions. Based on recent experiments we
propose that homeoprotein transfer may be at the origin of homeogenetic extension, boundary
formation in developing organisms, and signaling both during development and in the adult. In thecontext of the nervous system, models will be presented that illustrate how homeoprotein transfer
participates in the development of the eye anlagen, the establishment of retino-tectal maps and theregulation of the critical period for binocular vision.
Selected readings
D. Derossi, G. Chassaing and A. Prochiantz. (1998). Trojan peptides : the penetratin system for intracellulardelivery. Trends in Cell Biol., 8, 84-87.
A. Prochiantz (2000). Messenger proteins : homeoproteins, TAT and others. Curr. Opin. Cell Biol., 12,400-406.
A. Prochiantz and A. Joliot (2003). Can transcription factors function as cell-cell signaling molecules?
Nature reviews, Molecular Cell Biology, 4, 814-818.
A. Joliot & A. Prochiantz (2004). Transduction peptides, from technology to physiology. Nature Cell Biology,
6, 189-196.
I. Brunet, C. Weinl, M. Piper, A. Trembleau, M. Volovitch, B. Harris, A. Prochiantz*, & C. Holt*. (2005).
Engrailed-2 guides retinal axons. Nature, 438, 94-98.
B. Lesaffre, A. Joliot, A. Prochiantz & M. Volovitch. (2007). Direct non-cell autonomous Pax6 activity
regulates eye development in the zebrafish. Neural Development, 2, 2.
I. Brunet, A. Di Nardo, L. Sonnier, M. Beurdeley & A. Prochiantz. Shaping neural pathways with messenger
homeoproteins (2007). Trends in Neurosciences, 30, 260-267.
D. Holcman, V. Kasatkin & A. Prochiantz. (2007). Modeling homeoprotein intercellular transfer unveils aparsimonious mechaniss for gradient and boundary formation in early brain development. J. Theor. Biol., 249,503-517.
S. Sugiyama, A. Di Nardo, S. Aizawa, I. Matsuo, M. Volovitch, A. Prochiantz* & TK Hensch*. (2008).
Experience-dependent transport of Otx2 homeoprotein in the visual pathway activates postnatal corticalplasticity. Cell. In press.

Internalization mechanism of cell penetrating peptides
Shiroh Futaki
Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
futaki@scl.kyoto-u.ac.jp
Arginine-rich peptides, including octaarginine (R8) and HIV-1 Tat peptides, belong to one of themajor classes of cell-permeable peptides which deliver various proteins and macromolecules to cells.
Involvement of the endocytic pathways has recently been demonstrated in the cellular uptake of thesepeptides. We have shown that macropinocytosis is one of the major pathways for cellular uptake and thatorganization of the F-actin together with activation of the Rac protein accompanies this process. Significant
suppression of macropinocytic uptake and F-actin organization was observed in proteoglycan-deficient CHO
cells. These results suggest that efficient internalization of the Tat and other arginine-rich peptides may beinduced via interaction with membrane-associated proteoglycans by (i) concentration onto cell surfacefollowed by (ii) stimulated uptake by macropinocytosis stimulated by the interaction of peptides withproteoglycans [1,2].
On the other hand, we have examined the methods of internalization of oligoarginines having
different numbers of arginines (Rn, n=8, 12, 16). At relatively low concentrations, microscopic observationof the R12 and R16 peptides predominantly yielded endosome-like puctate signals, while upon raising thepeptide concentration, the fractions labeling the cytosol and the amount of the internalized peptides increased
dramatically [3]. Interestingly, this cytosolic peptide influx entry does not occur throughout the cell but
initiates at defined locations. Detailed real-time observation of the R12 influx into cells performed in thisstudy showed that the peptide influx accompanies the formation of unique ‘particle-like’ structures on theplasma membranes.

[1] I. Nakase et al., Biochemistry 46, 492-501 (2007)[2] I. Nakase et al., Adv. Drug. Deliv. Rev. 60, 598-607 (2008)[3] M. Kosuge et al., Bioconjug. Chem. 19, 656-664 (2008)名古屋大学野依記念学術交流会館への道案内Access map to Noyori Conference Hall

交通案内
JR名古屋駅・名鉄新名古屋駅・近鉄名古屋駅からの場合 … 地下鉄東山線藤が丘行きに乗車し、本山駅で地下鉄名城線右回りに乗り換え、名古屋大学駅下車。所要時間約30分(乗換を含む)
JR金山駅・名鉄金山駅からの場合 … 地下鉄名城線左回りに乗車し、名古屋大学駅下車。
所要時間約25分
地下鉄名古屋大学駅２番出口より徒歩約４分 Nogoya University subway station, Exit No. 2,
about 4 min walk
http://www.nagoya-u.ac.jp/camp/map_higashiyama/
野依記念学術交流館 （Tel: 052-788-6121）

問合せ先：日本農芸化学会学術活動強化委員会 シンポジウム世話人
牧 正敏 (Masatoshi Maki) 〒464-8601名古屋市千種区不老町
名古屋大学大学院生命農学研究科 応用分子生命科学専攻
Tel: 052-789-4088、E-mail:mmaki@agr.nagoya-u.ac.jp

日本農芸化学会 http://www.jsbba.or.jp/; http://www.jsbba.or.jp/e/index_e.html