jsv.umin.jpjsv.umin.jp/camp/15th/15th_slide2.pdfInsulin tolerance test CoreTg WT FDD Õ n i`lczo...

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Transcript of jsv.umin.jpjsv.umin.jp/camp/15th/15th_slide2.pdfInsulin tolerance test CoreTg WT FDD Õ n i`lczo...

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FDD wt

FLAG-Core

SPPKO cells YDD LGLGD

C

NHA

265219

DD

WT

SPPKO + SPP-HA

WT -

-FLAG-Core

-GFP

-Actin

IRES

GFPcoreFLAG

- FLAG-Core

- GFP

- Actin

- FLAG-Core(long exposure)

#1 #4 #2 #7WT SPPKO

Clone No

i z

WT or SPPKO cells

WT0 15 30 45 60 120

SPPKO

Core

GFP

Actin

0 15 30 45 60 (min)120Cycloheximide

ProteasomeLysosomeinhibitors

SPPKO cells

FLAG-Core

GFPCoreFLAG

FLAG-Core

GFPCoreFLAG

- 0.2 0.4 5 105 10 5 10EPO LAC ALLN MG132

GFP

Actin

Core

i

Core

GFP

Actin

p62

E64d PEP

BAF

SPP+ - + - + -

NT

i

FRS LT L P LU I ((.

9.5D

7 P

D5 0g

A SS RS P LU I ((+F b TS P LU I ((1

%:UL %7 P

% :D

%9.5D

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%5N SW

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%%

%%%

FDD D5 0 9.5D G?C

9.5D D5 0e pw z u

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0%% % %FDD?C

FDDGE708?C

FDDGE70?CGE70%<5

: 5 %7 P%<5

GE70%<510%. %

% :D0%

%5N SW

% G LU N P5W S%: 5 #

0%

GE70w9+ i t

N P: <5

N P:

:D

<5

FDD

E9F

:DN P: <5

SEB59E 9+ US L P

EB5S N PPWSW

GE70

WRSMS S WGE70

i

FDD<7I t

-%

%

+%

%

.%U )(#

% FDD

3 )( 3 )(% FDD

FDD?C )

FDD?C +

IS LU S P ::H V #

<7I

FDD?C

G

FDD G

FDD

gg

4

SPPによるコア蛋白質の切断が肝疾患に及ぼす影響

7 PG l7 PG FDD % l

7 PG FDD %7 PG

+1%

. %

1/%

%5N SW

FDD

?8L ?8L0%

%N P

+0%%5N SW

7 PGFDD %7 PGJG

SPP+/+ SPP+/- SPP-/-

E13.5 13 21 11E18.5 22 22 2

Postnatal(~2wk) 19 40 0

FDDw

0

20

40

60

80

100

120

0 20 40 60 80

7 PG

JG

7 PG FDD %

FDD %DULVL

UN

P#

VSW#

FDD w

7 PG FDD %

­ C

FDD %

7 PGJG

FDD

< R/ NPUU

:

:

:D

:D

7 P

7 PE9F

­ i

VVL P N P

FDD

FDD

% 5N SW

% :D

?8L

0 %

+0 %

0 %

% 7 P5W S%: 5 #

RO-0492907LY-411575KDa

28 -

38 -

28 -

YO-01027

- Actin

- GFP

- Core(Anti-FLAG)

KDa

28 -

38 -

28 -

Semagacestat

- Actin

- GFP

- Core(Anti-FLAG)

Compound E Avagacestat

Compound 34 DAPT MK-0752

- Actin

- GFP

- Core(Anti-FLAG)

KDa

28 -

38 -

28 -

Presenilin1

Plasmamembrane

C

N

N C

SPP

ERmembrane

C

N

N

C

e% i FDD xx u wt

GT1 (J1)

GT1 (TN) GT2 (J6) GT2 (J8)

GT3 (S52) GT4 (ED43) GT5 (SA13) GT6 (HK6a) GT7 (QC69)

GT2 (JFH1)

- Actin

- GFP

- Actin

- GFP

KDa

28 -

38 -

28 -

KDa28 -

38 -

28 -

GT1 (H77)

1aTN2b

4a

KDa

28 -

38 -

28 -

YO-01027

- Actin

- GFP

Huh7Various genotypes of HCV core

KC%()( / <7I

- Core (Anti-FLAG)

- Core (Anti-FLAG)

- Core (Anti-FLAG)

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

0.05 0.5 5 50

(log10)

(µM)

LY-411575

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

0.05 0.5 5 50

(log10)

(µM)

YO-01027

SPP阻害剤では耐性ウイルスは出現しにくい

Huh7.5.1 cells

passage

LY-411575YO-01027

orTPV

… X 5 ~20 times

1.E+01

1.E+02

1.E+03

1.E+04

1.E+05

1.E+06

0.0005 0.005 0.05 0.5

(log10)

(µM)

Telaprevir

Viru

s tit

er (F

FU/m

l) **

*

HCV

LY-411575YO-01027

orTPV

P-5No treatment

Evaluation ofdrug resistance

P-20No treatment

P-20No treatment

FDDw

0.0

0.4

0.8

1.2

1.6

Rela

tive

expr

essi

on(C

ore

/bet

a ac

tin)

N.D

4d 8d 12d 16dWT CoreTg LY-411575

Core mRNA in Liver

WT CoreTg 4 d 8 d 12 dLY-411575

-Actin

-Core

LY-411575 (10mg/kg) for 15 days

CoreTg

LY-411575 (10mg/kg) for 15 days

CoreTg

0

20

40

60

80

100

120

0 20 40 60 80 (min)

Plas

ma

gluc

ose

(%)

InsulinInsulin tolerance test

CoreTg

WT

FDD Gg w x

Oil Red O staining

Vehicle LY-411575

CoreTg+LY-411575

γ-secretase inhibitors with SPP inhibitionLY-411575 RO-0492907YO-01027

FDD v y w xwt h

LY-450139

DAPT

Avagacestat

MK-0752

Compound E

Compound 34

5 Y )1

5 Y .-

GA)

GA

GA+GA

GA-

GA.

GA/

GA0

GA1

5 Y .-

5 Y )1

VSWLU

7a U

1(d

ig FDD

ILU +m UP#

DRP)1 mG Y#

5UL -1m P #

5 Y )15 Y .-

KC%()( /

< R/ FDD?C< R/JGI +:)1 J5 -1c

% 5N SW

% :D

% 7 P5W S%: 5 #

?8L

0 %

+0 %

0 %

� ���� KC%()( /

RFDD. %

1/ %

% 5N SW+0 %

FDD?C< R/JG I + :)1 J5 -1c?8L

% 5N SW

% :D

% 7 P5W S%: 5 #

?8L

0 %

+0 %

0 %

I +JG :)1 J 5 -1

FDD?C< R/

KC%()( / FDD ILU +

: -0

GA/

GA.

ILU +

FDD

D P PWSUSW)

DRP -0

FDD

> SPP – EANNFAMLGLGDVV> PS1 – PEERGVKLGLGDFI

258

*****

FDD e% i DF)#

Ua -0

D P PWSUSW)

% :D

% 7 P5W S%: 5 #

% 5N SW

: -0JGJG : -07 VY WO 9

?8L

0 %

+0 %

0 %

KC%()( /7 VY WO 9 FDD : -0

2FDD

まとめ

F258

V223D219

D265

SPP

i

TRC8

5SbL L LWO CTLV P LU BL 7 VV W ().#

<S LW LWO CTLV P LU DB5F ()/#

Acknowledgement

Yoshiharu Matsuura

SayakaAizawa

JunkiHirano

TatsuyaSuzuki

Yukari Sugiyama

ShinjiKusakabe

MakotoTokunaga

YukaMiyata

The University of TokyoKazuhiko Koike

Yamanashi UniversityKohji Moriishi

Kobe UniversityIkuo Shoji

NIBIOMasahiro Oka, Yoichi Miyamoto

Hamamatsu UniversityTetsuro Suzuki

NIIDRyosuke Suzuki, Hideki Aizaki, Takaji Wakita, Tatsuo Miyamura

NCGHMSachiyo Yoshio, Tatsuya Kanto

WEHISeong Lin Khaw, Guillaume LesseneJames Vince, David CS Huang

ImmunoparasitologyMiwa Sasai, Masahiro Yamamoto

iFReC, Osaka UniversityBiochemistryShigekazu NagataHost defense

Takashi Satoh, Shizuo Akira

RIMD, Osaka University, Molecular Virology

Experimantal Genome ResearchMasahito Ikawa