ࢱࣥࣃࢡ㉁Ⓨ⌧ࡢホ౯ࡣࠊWestern blotἲࢆ⏝࠸ࡓࠋྛ⸆≀ฎ⨨ᚋࡢึ௦ᇵ 㣴ᾏ㤿⚄⤒⣽⬊ࡽsampleࢆㄪ〇ࡋࠊWestern blotࢆ⾜ࡗࡓࠋ᪉ἲࡘ࠸࡚ࡣࠊ
➨2❶‽ࡌ࡚⾜ࡗࡓࠋ
5. Calpainάᛶࡢ ᐃ
Calpain άᛶࡢ ᐃࡣࠊࢫࢡࢩࢽࣝ calpain ᇶ㉁ࢆ⏝ࡋࡓ Calpain-Glo™
Protease Assay kit (Promega)ࢆ⏝࠸࡚ ᐃࡋࡓࠋCalpain-Glo™ Reagent 50 µLࢩ ࢫࢸࣥ㛵㐃ྜ≀ࠊcalpain 㜼ᐖ⸆࠾ࡼࡧ Ca2+ࡢ⤊⃰ᗘࡀ-calpain ࡣ 1Mࠊ m-calpainࡣ1mM ࡞ࡿࡼ࠺CaCl2ࢆຍ࠼ࡓࢧࣥࣉࣝ50 µLࢆΰྜࡋࡓࠋ10 ศᚋࠊ࣑ࣝࣀ࣓࣮ࢱ࣮(FlexStation3, Molecular Devices)ࡼࡾⓎගᙉᗘࢆ ᐃ ࡋࡓࠋྛ⸆≀ฎ⨨ࡼࡿ ᐃ್ࡽ⸆≀↓ฎ⨨ࡢ ᐃ್ࢆᘬ࠸ࡓ್ࢆ⏝࠸ࠊ⁐
፹ࢆcontrolࡋ࡚ࠊcontrolᑐࡍࡿྜ࡛⾲ࡋࡓࠋCurve fittingࡣࠊGraphPad Prismࡢsigmoidal dose-response (variable slope)ࢆ⏝ࡋࠊ㓝⣲άᛶࢆ50%㜼ᐖࡍ
ࡿࡢᚲせ࡞⃰ᗘ (IC50)ࢆィ⟬ࡋࡓࠋ
6.⤫ィᏛⓗฎ⌮
ࡍ࡚ࡢ್ࡣᖹᆒ್ ± ᶆ‽ㄗᕪ࡛♧ࡋࡓࠋ᭷ពᕪ᳨ᐃࡣ୍ඖ㓄⨨ศᩓศᯒ ἲ(one-way ANOVA)࡛ゎᯒᚋࠊTukey,s multiple testࢆ⏝࠸ࡓࠋ
35
3-3 ⤖ ⤖ᯝ
1.ึ௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊࠾ࡅࡿᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛᑐࡍࡿ SAC࠾
࠾ࡧࢩࢫࢸࣥ㛵㐃ྜ≀ࡢຠᯝ
ᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛᑐࡍࡿ SAC ࡢ⚄⤒⣽⬊ಖㆤస⏝ࡢ࣓࢝ࢽࢬ࣒
ࢆ᫂ࡽࡍࡿࡓࡵࠊSAC ᵓ㐀ⓗ㢮ఝᛶࡀ㧗࠸ L-cysteine (CYS)࠾ࡼࡧ N-acetylcysteine (NAC) (Fig. 10A)ࡀTMㄏⓎ⣽⬊Ṛཬࡰࡍᙳ㡪ࡘ࠸࡚ẚ㍑᳨
ウࡋࡓࠋCYS ࠾ࡼࡧ NAC ࡣ 1 mM ࡢ⃰ᗘ࠾࠸࡚ࠊperoxynitrite (66)ࡸ
4-hydroxynonenal (67)ࡼࡿ㓟ⓗࢫࢺࣞࢫㄏⓎ㞀ᐖࡽࠊ⚄⤒ಖㆤస⏝ࢆ♧ࡍ ࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋ ࡑࡇ࡛ࠊᮏ◊✲࠾࠸࡚ࡶࠊ1 mM ࡢ SACࠊCYSࠊ NAC ࢆ⏝࠸᳨࡚ウࢆ⾜ࡗࡓࡇࢁࠊSAC ࡣ᭷ព࡞ TM ㄏⓎ⣽⬊Ṛᢚไస⏝ࢆ
♧ࡋࠊTMᭀ㟢ࡼࡾ47%ࡲ࡛పୗࡋࡓ⏕Ꮡ⋡ࢆ 76%ࡲ࡛ᅇࡋࡓࡀࠊCYS
࠾ࡼࡧNACࡣࠊ⣽⬊Ṛᢚไస⏝ࢆ♧ࡉ࡞ࡗࡓ(Fig. 10B)ࠋ
SAC
L-cysteine (CYS)
N-acetylcysteine (NAC)
CYS NAC
SAC
1 mM
(% of control)
Cell survival
TM (-) TM 10 g/mL
** *
-(A) (B)
2. ึ௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊࠾ࡅࡿ TM ㄏⓎ calpainάᛶᑐࡍࡿ SAC ࠾ࡼࡧ ࢩࢫࢸࣥ㛵㐃ྜ≀ࡢຠᯝ
ึ௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊ࡢTMᭀ㟢ᚋࡢcalpainάᛶᑐࡍࡿSAC࠾ࡼࡧࢩࢫ
Fig. 10. Effects of cysteine derivatives on TM-induced neuronal cell death in cultured rat hippocampal neurons (HPNs). (A) Chemical structure of SAC and cysteine derivatives. (B) SAC (1 mM), CYS (1 mM), and NAC (1 mM) were added simultaneously with 10 g/mL TM for 24 h. The viability of HPNs was assessed by the MTT reduction assay. Values are expressed as the relative percentages of surviving cells. Each value represents the mean ± S.E.M. for four different experiments. *P < 0.01,
**P < 0.001.㻌
36
ࢸࣥ㛵㐃ྜ≀ࡢຠᯝࢆ᳨ウࡋࡓࠋCalpain ࡢάᛶࡢᣦᶆࡋ࡚ࠊcalpain
ࡼࡾศゎࡉࢀῶᑡࡍࡿfull-length -spectrin (240 kDa)ࢆᐃ㔞ⓗゎᯒࡋࡓࠋึ
௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊ 24 㛫 TM 10 g/mL ࢆᭀ㟢ࡋࡓࡇࢁࠊfull-length
-spectrinࡢⓎ⌧ࡣ63%ῶᑡࡋࡓࡀࠊSAC 1 mMࡢే⏝ฎ⨨ࡼࡾࠊࡑࡢῶᑡ
ࡣ᭷ពᢚไࡉࢀࠊⓎ⌧ࣞ࣋ࣝࡣ90%ࡲ࡛ᅇࡋࡓ (Fig. 11)ࠋࡋࡋࠊCYS NACࡣࠊSACࡢࡼ࠺࡞full-length -spectrinࡢⓎ⌧ῶᑡࡢᢚไࢆ♧ࡉ࡞ࡗࡓ (Fig. 11)ࠋ
-actin
-spectrin 240 kDa
42 kDa control
TM (10g/mL) +SAC +CYS +NAC
-spectrin / -actin (% of control)
control +SAC +CYS +NAC
** *
-TM (10g/mL) (A)
(B)
3. Calpeptin࠾࠾ࡼࡧࢩࢫࢸࣥ㛵㐃ྜ≀ࡢ-calpainࠊm-calpainᑐࡍࡿຠᯝ SAC ࡀึ௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊࠾࠸࡚TM ࡼࡾㄏⓎࡉࢀࡿcalpain ࡢάᛶ
ࢆᢚไࡍࡿࡇࡀ♧ࡉࢀࡓࡇࡽࠊ in vitroࡢcalpainάᛶ ᐃ⣔ࢆ⏝࠸࡚
SAC ࠾ࡼࡧࢩࢫࢸࣥ㛵㐃ྜ≀ࡀ┤᥋ calpain άᛶస⏝ࢆཬࡰࡍྰ
ࡘ࠸᳨࡚ウࡋࡓࠋࡣࡌࡵࠊ᪤▱ࡢcalpain㜼ᐖ⸆࡛࠶ࡿcalpeptin (68)calpain ࡢྜᡂᇶ㉁ (Suc-LLVY-Glo)ࢆ⏝ࡋ࡚ࠊin vitro ࠾ࡅࡿࣜࢥࣥࣅࢼࣥࢺࡢ -calpainm-calpainᑐࡍࡿcalpainάᛶ ᐃ⣔ࡢ☜❧ࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝࠊ calpeptinࡣ-calpainάᛶ࠾ࡼࡧm-calpainάᛶࢆ⃰ᗘ౫Ꮡⓗ㜼ᐖࡋࠊࡑࢀࡽࡢ IC50್ (IC50 = 0.064 nM࠾ࡼࡧIC50 = 0.422 M)ࡣࠊ᪤ሗࡢIC50್ (69)ྠ⛬ᗘ
Fig. 11. Effects of cysteine derivatives on TM-induced -spectrin degradation in HPNs.
(A) The expression levels of full-length -spectrin (240 kDa) were investigated by Western blotting after exposure to TM (10 g/mL) in the absence or presence of SAC (1 mM), CYS (1 mM), and NAC (1 mM) for 24 h.
Anti--actin antibody was used as an internal control. (B) Amounts of full-length -spectrin were assessed by densitometric analysis, and the relative level of each band was calculated as described in Materials and Methods. Each value represents the mean ± S.E.M. for four different experiments. *P < 0.05, **P < 0.001.㻌
37
ࡢ್ࢆ♧ࡋࡓ (Fig. 12A, B)ࠋࡲࡓࠊcalpeptinࡣ-calpainࢆ10 nM࡛ࠊm-calpain
ࢆ10 M࡛ᢚไࡋࡓ (Fig. 12A, B)ࠋḟࠊᮏ ᐃ⣔ࢆ⏝࠸࡚ࠊSAC࠾ࡼ
ࡧࢩࢫࢸࣥ㛵㐃ྜ≀ࡀcalpainάᛶཬࡰࡍᙳ㡪ࡘ࠸᳨࡚ウࢆ⾜ࡗࡓࠋࡑ ࡢ⤖ᯝࠊSACࡣ-calpainm-calpainάᛶࢆࡶ⃰ᗘ౫Ꮡⓗ㜼ᐖࡋࡓ (Fig.
12C, D)ࠋ-CalpainάᛶᑐࡍࡿSACࡢ㜼ᐖࡣࠊm-calpainࡢάᛶ㜼ᐖࡼࡾࡶప
⃰ᗘഃ࠾࠸࡚ㄆࡵࡽࢀࠊ-calpain࡛ࡣ10 nM௨ୖࠊm-calpain࡛ࡣ1 mM௨ୖ
ࡢ⃰ᗘ࡛᭷ព࡞㜼ᐖຠᯝࢆ♧ࡋࡓ (Fig. 12C, D)ࠋࡲࡓࠊSACࡣcalpeptinࡣ␗
࡞ࡾࠊ㧗⃰ᗘ࠾࠸࡚ࡶ-calpainࠊm-calpainάᛶࢆࡣᢚไࡏࡎࠊ⏝ࡋ ࡓ⃰ᗘ⠊ᅖ࠾࠸࡚ࠊ-calpain άᛶࡣ 45%ࠊm-calpain ࡣ 63%పୗࡉࡏࡿ
␃ࡲࡗࡓࠋCYSNACࡣ⣽⬊Ṛᢚไస⏝ྠᵝ-calpainm-calpainάᛶ
ᙳ㡪ࢆཬࡰࡉ࡞ࡗࡓ (Fig. 12C, D)ࠋ
Log [M] Log [M]
-calpain activity (% of control)
Log [M] Log [M]
**
**
****
**
* **
***
(A) (B)
(C) (D)
-calpain activity (% of control) m-calpain activity (% of control)m-calpain activity (% of control)
*
Fig. 12. Effects of calpeptin and cysteine derivatives on -calpain and m-calpain activity. Inhibition curves of -calpain (A) and m-calpain (B) with varying concentrations of calpeptin were created by curve fitting as described in Materials and Methods. Similarly, inhibition curves of -calpain (C) and m-calpain (D) with varying concentrations of SAC, CYS and NAC were also created. Each value represents mean ± S.E.M. for three (A, B) and four (C, D) different experiments. *P < 0.05, **P <
0.01, ***P < 0.001 as compared with control (vehicle).㻌
38
4. -Calpain ᑐࡍࡿSACALLN࠾ࡼࡧPD150606ࡢే⏝ࡼࡿຠᯝ
SACࡀin vitroࡢ ᐃ⣔࠾࠸࡚calpainᑐࡋ┤᥋㜼ᐖຠᯝࢆ♧ࡍࡇࡽࠊ
ศᏊෆࡢࡢ㡿ᇦࢆస⏝Ⅼࡋ࡚࠸ࡿࢆ᫂ࡽࡍࡿࡓࡵࠊcalpainࡢάᛶ
୰ᚰస⏝ࡍࡿ㜼ᐖ⸆࡛࠶ࡿALLN (68)࠾ࡼࡧCa2+⤖ྜ㒊స⏝ࡍࡿ㜼ᐖ⸆
࡛࠶ࡿPD150606 (70)ࡢస⏝ࢆSACࡢᏑྰ࠾࠸࡚ẚ㍑᳨ウࡋࡓࠋALLNࡣࠊ
-calpainάᛶࢆ⃰ᗘ౫Ꮡⓗ㜼ᐖࡋ (IC50 = 0.01 nM)ࠊ1 nM࠾࠸࡚-calpain άᛶࢆ㜼ᐖࡋࡓ (Fig. 13A)ࠋ᭱ึప⃰ᗘࡢALLNࡀ♧ࡍ-calpain㜼ᐖ స⏝ᑐࡍࡿ SAC ࡢే⏝ࡼࡿᙳ㡪ࢆ᳨ウࡋࡓࠋ-Calpain άᛶࡣࠊALLN ༢
⊂ (5 pM)࡛ࡣ75%ࠊSAC༢⊂ (1 M)࡛ࡣ76%ࢆ♧ࡋࠊALLN (5 pM)SAC (1 M)ࡢే⏝࡛ࡣ57%పୗࡋࠊ-calpain㜼ᐖస⏝ࡢቑᙉࡀㄆࡵࡽࢀࡓ (Fig. 13B)ࠋ ࡉࡽࠊ㧗⃰ᗘࡢSAC (1 mM)ALLN (5 pM)ࡢే⏝࠾࠸࡚ࡶࠊే⏝ࡼࡿ
-calpain㜼ᐖస⏝ࡢቑᙉࡀㄆࡵࡽࢀࡓ (Fig. 13B)ࠋࡲࡓࠊ-calpain ࢆ᭱㜼 ᐖࡍࡿ⃰ᗘࡢALLN (1 nM)ࡣࠊ-calpainάᛶࢆ6%పୗࡉࡏࡓࡀࠊSAC (1 mM)
ࡢే⏝࡛ࡣࠊALLN༢⊂ẚ㍑ࡋ࡚-calpain㜼ᐖస⏝ኚࡣㄆࡵࡽࢀ࡞
ࡗࡓ (Fig. 13C)ࠋPD150606ࡣࠊ-calpainάᛶࢆ⃰ᗘ౫Ꮡⓗ㜼ᐖࡋ (IC50 = 20.85 M)ࠊ100 M࠾࠸࡚-calpainάᛶࢆ㜼ᐖࡋࡓ (Fig. 14A)ࠋ᭱ึప⃰
ᗘࡢPD150606ࡀ♧ࡍ-calpain㜼ᐖస⏝ᑐࡍࡿSACࡢే⏝ࡼࡿᙳ㡪ࢆ᳨ウ
ࡋࡓࠋ-CalpainάᛶࡣࠊPD150606༢⊂ (25 M)࡛ࡣ 73%ࠊSAC༢⊂ (1 M)
࡛ࡣ82%ࢆ♧ࡋࡓࡀࠊALLNࡣ␗࡞ࡾࠊPD150606 (25 M)SAC (1 M)ࡢే
⏝࡛ࡣ80%ࢆ♧ࡋࠊే⏝ࡼࡿ㜼ᐖస⏝ࡢኚࡣㄆࡵࡽࢀ࡞ࡗࡓࠋࡲࡓࠊ㧗
⃰ᗘࡢSAC (1 mM)PD150606 (25 M)ࡢే⏝࡛ࡶࠊ-calpain㜼ᐖస⏝ኚ
ࡣㄆࡵࡽࢀ࡞ࡗࡓ (Fig. 14B)ࠋ-Calpainࢆ᭱㜼ᐖࡍࡿ⃰ᗘࡢPD150606 (100 M)ࡣࠊ-calpainάᛶࢆ9%పୗࡉࡏࡓࠋPD150606 (100 M)SAC (1 mM)ࡢే⏝ࡣࠊ-calpain άᛶࢆ 28%ࡲ࡛ᅇࡉࡏࠊPD150606 ༢⊂ࡢ-calpain 㜼ᐖస⏝ࢆ᭷ពᢚไࡋࡓ (Fig. 14C)ࠋ
39
-calpain activity (% of control)
Log [M]
****
# # *
# #
#
-calpain activity (% of control)
SAC ALLN
control
-1 M
- 5 pM 5 pM
-- 5 pM
1 M 1 mM 1 mM
***
-calpain activity (% of control)
******
control
ALLN
SAC
-1 mM 1 mM
-1 nM
(A) (B) (C)
1 nM
-calpain activity (% of control)
Log [M]
# #
##
### ###
-calpain activity (% of control)
SAC PD150606
-1 M 1 M 1 mM 1 mM
-25 M 25 M 25 M
control
(A) (B) (C)
-calpain activity (% of control)
****
control *
PD150606 SAC
-1 mM 1 mM
-100 M -100 M
5. -Calpain ᑐࡍࡿSACcalpastatinࡢే⏝ࡼࡿຠᯝ
⏕ య ෆ ࡣ ࠊ 㐣 ࡞ calpain ࡢ ά ᛶ ࡀ ㉳ ࡁ ࡓ ሙ ྜ ࡢ ㄪ ⠇ ᅉ Ꮚ ࡋ ࡚ calpastatinࡀᏑᅾࡋ࡚࠸ࡿࠋࡋࡋࠊAD࠾࠸࡚ࡣcalpastatinࡢⓎ⌧ࡀῶᑡࡍ
Fig. 13. Effects of SAC on inhibition of -calpain by ALLN. (A) Inhibition curve for -calpain with varying concentrations of ALLN was created by curve fitting as described in Materials and Methods.
(B) ALLN (5 pM) with or without SAC (1 M or 1 mM) incubated with 100 nM -calpain. (C) ALLN (1 nM) with or without SAC (1 mM) incubated with 100 nM -calpain. The calpain activity was examined using an in vitro assay system as described in Materials and Methods. Each value represents the mean ± S.E.M. for three (A) or four (B, C) different experiments. #P < 0.001 as compared with control (vehicle), *P < 0.05, **P < 0.01, ***P < 0.001.㻌
Fig. 14. Effects of SAC on inhibition of -calpain by PD150606. (A) Inhibition curve of -calpain with varying concentrations of PD150606 was created by curve fitting as described in Materials and Methods. (B) PD150606 (25 M) with or without SAC (1 M or 1 mM) incubated with 100 nM -calpain. (C) PD150606 (100 M) with or without SAC (1 mM) incubated with 100 nM -calpain.
The calpain activity was examined using an in vitro assay system as described in Materials and Methods. Each value represents the mean ± S.E.M. for four different experiments. #P < 0.05, ##P <
0.01, ###P < 0.001 as compared with control (vehicle), *P < 0.05, **P < 0.001.㻌
40
ࡿࡇ࡛ࠊcalpainࡢ㐣࡞άᛶࡀㄏⓎࡉࢀࠊࡇࡢάᛶࡀ㞀ᐖⓎ⏕ࡢ୍ᅉ
࡞ࡗ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ (63)ࠋࡑࡇ࡛ࠊcalpastatin ࡢ-calpain 㜼ᐖస
⏝ཬࡰࡍSACࡢᙳ㡪ࡘ࠸᳨࡚ウࡋࡓࠋCalpastatinࡣࠊ-calpainάᛶࢆ⏝㔞 ౫Ꮡⓗ㜼ᐖࡋ (Fig. 15A)ࠊSACྠᵝࠊ㧗⃰ᗘ࠾࠸࡚ࡶ-calpainάᛶࢆ
ࡣ㜼ᐖࡏࡎࠊ࡛᭱85%ࡢ㜼ᐖస⏝ࢆ♧ࡋࡓ (Fig. 15A)ࠋḟࠊప⃰ᗘ ࡢcalpastatinࡀ♧ࡍ-calpain㜼ᐖస⏝ᑐࡍࡿSACࡢే⏝ࡼࡿᙳ㡪ࢆ᳨ウࡋ ࡓࠋ-Calpainάᛶࡣࠊcalpastatin༢⊂ (100 pM)࡛ࡣ76%ࠊSAC༢⊂ (1 M)࡛
ࡣ77%ࢆ♧ࡋࠊcalpastatin (100 pM) SAC (1 M)ࡢే⏝࡛ࡣ59%పୗࡋࠊ
calpastatin༢⊂ẚ㍑ࡋ࡚㜼ᐖస⏝ࡢቑᙉࡀㄆࡵࡽࢀࡓ (Fig. 15B)ࠋࡉࡽࠊ㧗
⃰ᗘࡢSAC (1 mM)calpastatin (100 pM)ࡢే⏝࡛ࡣࠊ-calpainάᛶࡀ44%ప
ୗࡋࠊ㜼ᐖస⏝ࡢቑᙉࡀㄆࡵࡽࢀࡓ (Fig. 15B)ࠋࡲࡓࠊ-calpainࢆ᭱㜼ᐖ ࡍࡿ⃰ᗘࡢcalpastatin (100 nM)ࡣࠊ-calpainάᛶࢆ15%పୗࡉࡏࡓࡀࠊSAC (1
mM)ే⏝ࡋ࡚ࡶ calpastatin ༢⊂ẚ㍑ࡋ࡚㜼ᐖస⏝ኚࡣㄆࡵࡽࢀ࡞ࡗ
ࡓ (Fig. 15C)ࠋ
-calpain activity (% of control)
Log [M]
****
# # *
# #
#
control
-calpain activity (% of control)
calpastatin
SAC
-1 M
-
-1 mM 1 M 1 mM
100 pM 100 pM 100 pM
-*** ******
-calpain activity (% of control) control
calpastatin
SAC - 1 mM
-1 mM
100 nM 100 nM
-(A) (B) (C)
Fig. 15. Effects of SAC on inhibition of -calpain by calpastatin. (A) Inhibition curve of -calpain with varying concentrations of calpastatin was created by curve fitting as described in Materials and Methods. (B) Calpastatin (100 pM) with or without SAC (1 M or 1 mM) incubated with 100 nM -calpain. (C) Calpastatin (100 nM) with or without SAC (1 mM) incubated with 100 nM -calpain.
The calpain activity was examined using an in vitro assay system as described in Materials and Methods. Each value represents the mean ± S.E.M. for three (A), four (C) and five (B) different experiments. #P < 0.001 as compared with control (vehicle), *P < 0.05, **P < 0.01, ***P < 0.001.㻌
41
3-4 ⪃ ⪃ᐹ
ᮏ❶࡛ࡣࠊᾏ㤿ึ௦ᇵ㣴⣔࠾ࡼࡧ in vitro ࡢ calpain άᛶ ᐃ⣔ࢆ⏝࠸࡚ࠊ
calpain ᑐࡍࡿ SAC ࡢస⏝ࢆ୰ᚰ᳨ウࢆ⾜ࡗࡓࠋ㏆ᖺࠊ⊂❧ࡋࡓ⚄⤒⣽⬊
ṚㄏⓎᶵᵓ⪃࠼ࡽࢀ࡚࠸ࡓᑠ⬊య࣑ࢺࢥࣥࢻࣜࡢ㛫ᐦ᥋࡞㛵ಀࡀ࠶ࡿ
ࡇࡀ♧ࡉࢀ (71)ࠊᑠ⬊యࢫࢺࣞࢫㄏᑟ⸆࡛࠶ࡿthapsigarginࡸbefeldin Aࡣ࣐
࢘ࢫᾏ㤿⚄⤒⣽⬊⏤᮶ࡢHT22⣽⬊࠾࠸࡚ࠊάᛶ㓟⣲✀ࡢୖ᪼ࢆᘬࡁ㉳ࡇࡋࠊ
⣽⬊ṚࢆㄏⓎࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ (72)ࠋࡇࢀࡽࡢ⤖ᯝࡼࡾࠊTM ㄏⓎ⣽
⬊Ṛ࠾ࡅࡿ SAC ࡢ⚄⤒ಖㆤస⏝ࡢ୍ࡘࡋ࡚ࠊSAC ࡢᢠ㓟స⏝ࡀ㛵ࡍ
ࡿྍ⬟ᛶࡶṧࡉࢀ࡚࠸ࡿࠋࡑࡇ࡛ࠊSACࡢᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛᢚไస⏝
ࢆࡉࡽ᫂ࡽࡍࡿࡓࡵࠊึ௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊࠾࠸࡚ࠊSACᵓ㐀ⓗ
㢮ఝᛶࡀ㧗࠸CYS࠾ࡼࡧNACࡀTMㄏⓎ⣽⬊Ṛཬࡰࡍᙳ㡪ࡘ࠸᳨࡚ウࡋ ࡓࠋCYSࡸNACࡣᢠ㓟స⏝ࢆ᭷ࡍࡿࡓࡵࠊperoxynitrite (66)ࡸ4-hydroxynonenal (67)ࡼࡿ㓟ࢫࢺࣞࢫㄏⓎ⚄⤒⣽⬊Ṛࡽ⚄⤒ಖㆤస⏝ࢆ♧ࡍࡇࡀሗ࿌ࡉ
ࢀ࡚࠸ࡿࠋࡋࡋ࡞ࡀࡽࠊCYS࠾ࡼࡧNACࡣTM ㄏⓎ⣽⬊Ṛᑐࡋ࡚ಖㆤస
⏝ࢆ♧ࡉ࡞ࡗࡓ (Fig. 10B)ࠋᮏ◊✲࡛⏝ࡋࡓTMࡣࠊᑠ⬊యࡢN-glycosylation 㜼ᐖࡼࡾ┤᥋ᵓ㐀␗ᖖࢱࣥࣃࢡ㉁ࢆ✚ࡍࡿࡇ࡛ᑠ⬊యࢫࢺࣞࢫࢆᘬࡁ㉳
ࡇࡋ࡚⣽⬊ṚࢆㄏⓎࡍࡿࡓࡵࠊ㓟ࢫࢺࣞࢫࡢ㛵ࡀᑡ࡞࠸ࡇࡀሗ࿌ࡉࢀ࡚
࠾ࡾ (73)ࠊࡇࡢࡓࡵ CYSࠊNAC ࡼࡿಖㆤస⏝ࡣㄆࡵࡽࢀ࡞ࡗࡓࡶࡢ⪃
࠼ࡽࢀࡿࠋࡲࡓࠊTM ㄏⓎ⣽⬊Ṛᢚไస⏝ࡀ SAC ࡢࡳ࡛ㄆࡵࡽࢀࡓࡇࡼࡾࠊ ᾏ㤿⚄⤒⣽⬊࠾ࡅࡿSACࡢ⚄⤒ಖㆤస⏝ࡣࠊ㓟ࢫࢺࣞࢫᑐࡋ࡚࡛ࡣ࡞ࡃࠊ ᑠ⬊యࢫࢺࣞࢫ㛵㐃ࡋࡓ࢝ࢫࢣ࣮ࢻࢆᢚไࡍࡿࡇ㉳ᅉࡋ࡚࠸ࡿྍ⬟ᛶࡀ
♧ࡉࢀࡓࠋ
㏆ᖺࠊ-calpain ࡣADᝈ⪅⬻ (57)ࡸAPP ࢺࣛࣥࢫࢪ࢙ࢽࢵࢡ࣐࢘ࢫ⬻ (58)
࠾࠸࡚ࠊάᛶࡀஹ㐍ࡋ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡉࡽࠊࣄࢺ⚄⤒ⱆ⭘
⣽⬊SH-SY5Y⣽⬊࠾ࡅࡿAㄏⓎ⣽⬊Ṛᑐࡋ࡚ࠊ-calpain㑅ᢥᛶࡢ㧗࠸
㜼ᐖ⸆conjugated linoleic acidࡢ᭷⏝ᛶࡀሗ࿌ࡉࢀ࡚࠸ࡿ (74)ࠋࡲࡓࠊPD150606
42
ࡣ㏆ᒀ⣽⟶ୖ⓶⣽⬊LLC-PK1⣽⬊ (75)ࠊALLNࡣ࣐࢘ࢫ⥺⥔ⱆ⣽⬊NIH3T3
⣽⬊ (76)࠾࠸࡚ࠊTM ㄏⓎ⣽⬊Ṛࢆᢚไࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠾ࡾࠊTM ㄏ
Ⓨ⣽⬊Ṛࡣ calpain ࡢάᛶࡀ㛵ࡋࠊcalpain άᛶࡢ㜼ᐖࡣ⣽⬊ಖㆤస⏝ࢆ
♧ࡍࡇࡀ♧၀ࡉࢀ࡚࠸ࡿࠋᮏ◊✲࠾࠸࡚ࡶᇵ㣴ᾏ㤿ษ∦ࡢTMᭀ㟢ࡼࡾࠊ
calpain ࡢάᛶࢆㄏⓎࡋࠊSAC ࡣࡑࡢάᛶࢆᢚไࡋࡓ (Fig. 9A)ࠋࡑࡇ࡛ࠊ
ึ௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊࠾࠸࡚ࠊSAC ࡀTM ᭀ㟢ࡼࡿcalpain ࡢάᛶཬࡰ
ࡍᙳ㡪ࡘ࠸᳨࡚ウࡋࡓࡇࢁࠊTMᭀ㟢ࡼࡾfull-length -spectrinࡢⓎ⌧ῶ ᑡࡀㄆࡵࡽࢀࠊSACࡣࡇࡢfull-length -spectrinࡢⓎ⌧ῶᑡࢆᢚไࡋࡓࡀࠊ⣽⬊
ṚᑐࡍࡿሙྜྠᵝCYS ࠾ࡼࡧNAC ࡣᙳ㡪ࢆཬࡰࡉ࡞ࡗࡓ (Fig. 11)ࠋ ௨ୖࡢ⤖ᯝࡼࡾࠊึ௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊࠾࠸࡚ࡶࠊSAC ࡀ calpain ࡢάᛶ
ࢆᢚไࡍࡿࡇࡀ♧၀ࡉࢀࡓࠋࡑࡇ࡛ࠊSAC ࡀ calpain άᛶཬࡰࡍస⏝ࡘ
࠸࡚ࠊin vitro ࡢ calpain άᛶ ᐃ⣔ࢆ⏝࠸᳨࡚ウࡋࡓࠋSAC ࡣ⃰ᗘ౫Ꮡⓗ
-calpain࠾ࡼࡧm-calpainάᛶࢆ㜼ᐖࡋࡓࡀࠊcalpeptinࡢᙉຊ࡞㜼ᐖຠᯝࡣ␗
࡞ࡾࠊ㧗⃰ᗘ࡛ࡶ㜼ᐖࡢ⛬ᗘࡣᙅࡃࠊ✜ࡸ࡞㜼ᐖస⏝ࢆ♧ࡋࡓ (Fig. 12C, D)ࠋ
୍᪉ࠊCYSࡸNACࡣ-calpainࠊm-calpainάᛶᙳ㡪ࢆཬࡰࡉ࡞ࡗࡓ (Fig. 12C,
D)ࠋࡇࢀࡽࡢ⤖ᯝࡼࡾࠊSAC ࡣ-࠾ࡼࡧ m-calpain άᛶࢆ┤᥋㜼ᐖࡍࡿࡇ࡛
ᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛࢆᢚไࡍࡿྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋࡲࡓࠊCYS࠾ࡼࡧ NAC ࡀࠊ-calpainࠊm-calpain ᙳ㡪ࢆཬࡰࡉ࡞ࡗࡓࡇࡼࡾࠊcalpain 㜼ᐖ స⏝ࡣࠊSHᇶࡣ㛵ࡋ࡞࠸ࡇࡀ♧၀ࡉࢀࡓࠋ
ḟࠊSAC ࡢ-calpain 㜼ᐖస⏝ࡀ calpain ࡢࡢ㒊స⏝ࡋ࡚࠸ࡿࡢ᳨
ウࡍࡿࡓࡵࠊcalpainࡢάᛶ୰ᚰస⏝ࡍࡿALLN (68)ࡲࡓࡣCa2+⤖ྜ㒊
స⏝ࡍࡿPD150606 (70)SACࡢే⏝ࡀ-calpainάᛶཬࡰࡍᙳ㡪ࡘ࠸࡚
᳨ウࢆ⾜ࡗࡓࠋప⃰ᗘࡢྛ㜼ᐖ⸆SACࡢే⏝ࡼࡿᙳ㡪ࢆ᳨ウࡋࡓ⤖ᯝࠊ ALLNࡢ-calpain㜼ᐖస⏝ࡣࠊSACࡢే⏝ࡼࡾ᭷ពቑᙉࡉࢀࡓࡀ (Fig. 13B)ࠊ
PD150606ࡢ㜼ᐖస⏝ࡣࠊSACࡢᙳ㡪ࢆཷࡅ࡞ࡗࡓ (Fig. 14B)ࠋࡲࡓࠊ᭱㜼
ᐖస⏝ࢆ♧ࡍ⃰ᗘࡢྛ㜼ᐖ⸆ SAC ࡢే⏝ࡼࡿᙳ㡪ࢆ᳨ウࡋࡓࡇࢁࠊ
43
ALLN ࡢ㜼ᐖస⏝ࡣᙳ㡪ࢆཬࡰࡉ࡞ࡗࡓࡀ (Fig. 13C)ࠊPD150606 ࡢ -calpain㜼ᐖస⏝ࢆ᭷ពᢚไࡋࡓ (Fig. 14C)ࠋ௨ୖࡢ⤖ᯝࡽࠊSACࡣcalpain ࡢCa2+⤖ྜ㒊స⏝ࡋࠊcalpain㜼ᐖస⏝ࢆ♧ࡍྍ⬟ᛶࡀ♧၀ࡉࢀࡓࠋࡇࢀࡲ
࡛ࠊcalpeptinࡢࡼ࠺࡞άᛶ୰ᚰస⏝ࡍࡿ calpain㜼ᐖ⸆ࡣ calpainࢯࣇ
࢛࣮࣒㛫ࡢ㑅ᢥᛶḞࡃࡶࡢࡢ (69)ࠊCa2+⤖ྜ㒊స⏝ࡍࡿ PD151746 ࡣ
m-calpainẚ㍑ࡋࠊ-calpain㑅ᢥᛶࡢ㧗࠸㜼ᐖ⸆࡛࠶ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸
ࡿ (70)ࠋᮏ◊✲࠾࠸࡚ࠊSACࡢ-calpainάᛶᑐࡍࡿ᭷ព࡞㜼ᐖస⏝ࡣ10 nM ௨ୖ࡛ㄆࡵࡽࢀࡓࡢᑐࡋ࡚ࠊm-calpainᑐࡍࡿ㜼ᐖస⏝ࡣ1 mM௨ୖ࡛ㄆࡵ
ࡽࢀࠊSACࡣm-calpainẚ㍑ࡋ࡚-calpain㧗࠸㑅ᢥᛶࢆ♧ࡋࡓࡀ (Fig. 12C,
D)ࠊࡇࡢࡇࡶ SAC ࡀ Ca2+⤖ྜ㒊స⏝ࡍࡿྍ⬟ᛶࢆᙉࡃᨭᣢࡋ࡚࠸ࡿ
⪃࠼ࡿࠋ
㏆ᖺࠊADᝈ⪅⬻ (63)ࡸADែࣔࢹ࣐ࣝ࢘ࢫ⬻(58)࡛ࡣࠊcalpastatinࡢⓎ⌧
ࣞ࣋ࣝࡀῶᑡࡋ࡚࠸ࡿࡇሗ࿌ࡉࢀ࡚࠸ࡿࠋCalpastatin ࡣࠊCa2+ࡼࡗ࡚άᛶ
ࡉࢀࡓcalpain⤖ྜࡍࡿࡇࡀ♧ࡉࢀ࡚࠾ࡾ (77)ࠊcalpainࡢ㡿ᇦIࠊIIࡢ㛫
࠶ࡿάᛶ୰ᚰ㏆ഐ (77) Ca2+⤖ྜ㒊 (78)ࡢ୧᪉స⏝ࡋ࡚ calpainࢆ㜼ᐖ ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࡀࠊ⌧ᅾࡲ࡛ࠊcalpastatin ࡼࡿcalpain㜼ᐖᶵᵓࡢ ヲ⣽ࡣ᫂࡞ࡲࡲ࡛࠶ࡿࠋSACࡣࠊప⃰ᗘࡢcalpastatinࡀ♧ࡍ-calpain㜼ᐖస
⏝ࢆቑᙉࡋࡓࡀࠊ㧗⃰ᗘࡢcalpastatin ࡼࡿ᭱ࡢ-calpain㜼ᐖస⏝ᑐࡋ࡚
ࡣᙳ㡪ࢆཬࡰࡉ࡞ࡗࡓ (Fig. 15B, C)ࠋࡋࡓࡀࡗ࡚ࠊSACࡣcalpastatinࡢస⏝
ࡀ༑ศ࡛ࡣ࡞࠸ࡣቑᙉࡍࡿࡀࠊcalpastatin ࡀ༑ศᶵ⬟ࡋ࡚࠸ࡿሙྜࡣ ᙳ㡪ࡋ࡞࠸ࡇࡀ♧၀ࡉࢀࡓࠋ
Calpainࡢάᛶ␗ᖖࡣࠊADࡔࡅ࡛࡞ࡃࠊ⬻⾑ࡸⓑෆ㞀࡞ᵝࠎ࡞ᝈ
῝ࡃ㛵ࡋ࡚࠸ࡿࡇࡀ♧၀ࡉࢀ࡚࠸ࡿ (62)ࠋࡑࡢࡓࡵࠊcalpainࢆᶆⓗࡋࡓ
⒪⸆ࡢ㛤Ⓨࡀ㐍ࡵࡽࢀ࡚࠸ࡿࡶࡢࡢࠊࡇࢀࡲ࡛ᐇ⏝ࡣ⮳ࡗࡓࡣⓙ↓࡛
࠶ࡿࠋࡑࡢ୍ᅉࡋ࡚ࠊcalpainࡣ⏕⥔ᣢᚲ㡲࡞㓝⣲࡛࠶ࡿࡓࡵࠊᚲせ௨ୖ
ᢚไࡍࡿ㔜࡞స⏝ࢆ⏕ࡴྍ⬟ᛶࡀᣲࡆࡽࢀࡿࠋ࠼ࡤࠊྍ㏫ⓗ
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calpain㜼ᐖ⸆࡛࠶ࡿbenzyloxycarbonyl-Leu-Leu-Tyr diazomethyl ketone ࡣࠊࣄࢺ 㣗㐨ୖ⓶⣽⬊ࡢTE2⣽⬊ࡸࣄࢺᏊᐑ⒴⏤᮶ࡢC-33A⣽⬊࠾࠸࡚ࠊ⣽⬊ቑṪ㜼 ᐖࢆᘬࡁ㉳ࡇࡍࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ (79)ࠋࡇࢀࡽ␗࡞ࡾࠊྍ㏫ⓗ࡞calpain 㜼ᐖస⏝ࢆ♧ࡍcalpain㜼ᐖ⸆XIࡣࠊ▷㛫ࡢᢞ࠾࠸࡚⥙⭷ኚᛶ࣐࢘ࢫ
ࡽసᡂࡋࡓ⥙⭷እ᳜∦࠾ࡅࡿගཷᐜయ⣽⬊Ṛᑐࡋ࡚ࠊಖㆤຠᯝࢆ♧ࡍࡇ
ࡀሗ࿌ࡉࢀ࡚࠸ࡿ (80)ࠋࡋࡋࠊcalpain 㜼ᐖ⸆ XI ࡢᩘ᪥㛫ࡢ㐃⥆ᢞࡣࠊ TdT-mediated dUTP nick end labeling (TUNEL)㝧ᛶ⣽⬊ࡢⴭࡋ࠸ቑຍࢆᘬࡁ㉳ࡇ ࡍ [80]ࠋࡇࢀᑐࡋ࡚ࠊෆᅾᛶcalpain㜼ᐖࢱࣥࣃࢡ㉁࡛࠶ࡿcalpastatinࡣ▷ᮇ 㛫࠾ࡼࡧ㛗ᮇ㛫ࡢᢞ࠾࠸࡚ࡶࠊ⥙⭷ኚᛶ࣐࢘ࢫࡢ⥙⭷እ᳜∦࠾ࡅࡿගཷ
ᐜయ⣽⬊Ṛࢆಖㆤࡍࡿࡇࡀ♧ࡉࢀ࡚࠸ࡿ (80)ࠋࡇࢀࡽࡢሗ࿌ࡽࠊcalpain㜼 ᐖࡀ⣽⬊ࡢ⏕Ꮡཬࡰࡍᙳ㡪ࡣࠊ⣽⬊ࡢ✀㢮ࡸ㜼ᐖᣢ⥆㛫ࡼࡾኚࡋࠊ
calpainࡢ㛗ᮇ࠾ࡼࡧ㐣࡞㜼ᐖࡣࠊ⣽⬊ࡢ⏕Ꮡᝏᙳ㡪ࢆཬࡰࡍࡇࡀ♧၀ࡉ
ࢀࠊ⒪⸆ࡋ࡚ࡣ⦆࡞స⏝ࡢࡶࡢࡀⰋ࠸⪃࠼ࡽࢀࡿࠋᮏcalpainάᛶ ᐃ
⣔࠾࠸࡚ࠊ㧗⃰ᗘࡢ ALLNࠊPD150606 ࡣࠊ-calpain ࢆ㜼ᐖࡋࡓࡀࠊ SAC ࡣࠊcalpastatinྠᵝࠊ㧗⃰ᗘ࠾࠸࡚ࡶ-calpainάᛶࢆࡣ㜼ᐖ ࡋ࡞ࡗࡓࠋࡋࡓࡀࡗ࡚ࠊSACࡣࠊᙉຊ࡞ྜᡂcalpain㜼ᐖ⸆␗࡞ࡾࠊcalpastatin ࡀ༑ศᶵ⬟ࡋ࡚࠸࡞࠸ሙྜࡣࠊࡑࡢᶵ⬟ࢆ⿵࠺ࡇࡢ࡛ࡁࡿ ⦆ᚎ࡞
calpain 㜼ᐖస⏝ࢆ♧ࡍ⪃࠼ࡽࢀࠊAD ⒪⸆ࡋ࡚ࡶ᭷⏝࡛࠶ࡿ⪃࠼ࡽࢀ
ࡓࠋࡲࡓࠊࡇࢀࡽࡢస⏝ࡀᑠ⬊యࢫࢺࣞࢫㄏⓎᾏ㤿⚄⤒⣽⬊Ṛಖㆤస⏝ࢆ♧
ࡍ⪃࠼ࡿࠋࡋࡋࠊSACࡼࡿcalpastatinࡢቑᙉస⏝ࡘ࠸࡚ࡣࠊࡲࡔ᫂
࡞Ⅼࡀṧࡗ࡚࠾ࡾࠊᚋࡉࡽ࡞ࡿ᳨ウࡀᚲせ࡛࠶ࡿ⪃࠼ࡿࠋ
45
3-5 ᑠ ᑠᣓ
1. ึ௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊࠾࠸࡚ࠊSACࡣTMㄏⓎ⣽⬊Ṛࢆᢚไࡋࡓࠋࡋ
ࡋࠊCYS࠾ࡼࡧNACࡣᢚไస⏝ࡣㄆࡵࡽࢀ࡞ࡗࡓࠋ
2. ึ௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊࠾࠸࡚ࠊSACࡣTMฎ⨨ࡼࡿfull-length -spectrin ࡢⓎ⌧㔞ࡢῶᑡࢆᢚไࡋࡓࡀࠊCYS ࠾ࡼࡧ NAC ᢚไస⏝ࡣㄆࡵࡽࢀ࡞
ࡗࡓࠋ
3. In vitroࡢcalpainάᛶ ᐃ⣔࠾࠸࡚ࠊSACࡣ-calpain࠾ࡼࡧm-calpainࡢ άᛶࢆ⃰ᗘ౫Ꮡⓗ㜼ᐖࡋࡓࠋࡑࡢస⏝ࡣcalpeptinẚ㍑ࡋ࡚ᙅࡃࠊ㧗
⃰ᗘ࠾࠸࡚ࡶcalpainάᛶࢆࡣ㜼ᐖࡋ࡞ࡗࡓࠋ
4. ALLN SAC ࡢే⏝࠾࠸࡚ࠊSAC ࡣప⃰ᗘࡢ ALLNࡀ♧ࡍ-calpain㜼 ᐖస⏝ࢆቑᙉࡋࡓࠋࡲࡓࠊ-calpainࡢ᭱㜼ᐖస⏝ࢆ♧ࡍ⃰ᗘࡢALLNࡢ 㜼ᐖస⏝ᙳ㡪ࢆཬࡰࡉ࡞ࡗࡓࠋ
5. PD150606 SAC ࡢే⏝࠾࠸࡚ࠊSAC ࡣప⃰ᗘࡢ PD150606 ࡀ♧ࡍ
-calpain 㜼ᐖస⏝ᙳ㡪ࢆཬࡰࡉ࡞ࡗࡓࠋࡋࡋࠊ-calpainࡢ᭱㜼ᐖ
స⏝ࢆ♧ࡍ⃰ᗘࡢPD150606ࡢ㜼ᐖస⏝ᑐࡋ࡚ࡣᢚไస⏝ࢆ♧ࡋࡓࠋ
6. Calpastatin SAC ࡢే⏝࠾࠸࡚ࠊSAC ࡣప⃰ᗘࡢ calpastatin ࡀ♧ࡍ
-calpain 㜼ᐖస⏝ࢆቑᙉࡋࡓࠋࡲࡓࠊ-calpain ࡢ᭱㜼ᐖస⏝ࢆ♧ࡍ⃰ᗘ
ࡢcalpastatinࡢ㜼ᐖస⏝ᙳ㡪ࢆཬࡰࡉ࡞ࡗࡓࠋ
௨ୖࡢ⤖ᯝࡼࡾࠊึ௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊࠾࠸࡚ࠊSACࡣᑠ⬊యࢫࢺࣞࢫㄏ
Ⓨ⣽⬊Ṛࢆ㢧ⴭᢚไࡋࡓࠋࡑࡢ⚄⤒ಖㆤస⏝ࡣᢠ㓟స⏝ࡣ㛵ࡏࡎࠊ
46
calpainࡢCa2+⤖ྜ㒊ࡢ┦స⏝ࢆᘬࡁ㉳ࡇࡋࠊcalpainάᛶࢆ┤᥋ⓗࡘ⦆
ࡸ㜼ᐖࡍࡿࡇࡀࠊᑡ࡞ࡃࡶ୍㒊㛵ࡍࡿࡇࡀ♧၀ࡉࢀࡓࠋ
47
➨ 4 ❶
⥲ᣓ
48
ᮏ◊✲࠾࠸࡚ࡣࠊࡲࡎ๓༙࠾࠸࡚ࠊᾏ㤿⚄⤒⣽⬊࠾ࡅࡿ amyloid
-peptide (Aࡢ⣽⬊ṚㄏⓎ࣓࢝ࢽࢬ࣒ࢆ᫂ࡽࡍࡿࡓࡵࠊAࡀᑠ⬊యࢫࢺࣞ
ࢫㄏⓎ⣽⬊Ṛཬࡰࡍᙳ㡪ࡑࡢᶵᗎࢆᇵ㣴ᾏ㤿ษ∦࠾࠸᳨࡚ウࡋࡓࠋ
A25-35ࡣࠊtunicamycin (TM)ࡼࡿᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛࢆ㢧ⴭቑᙉࡍࡿ
ࡇࠊࡲࡓࠊࡑࡢ⣽⬊Ṛቑᙉࡣᾏ㤿ࡢ㡿ᇦ≉␗ᛶࡀ࠶ࡾࠊLᆺ Ca2+ࢳࣕࢿࣝ
ࢆࡋࡓ⣽⬊ෆ Ca2+⃰ᗘࡢୖ᪼ࡼࡾࠊcalpain ࡢάᛶࢆᘬࡁ㉳ࡇࡋࠊ
caspase-12 ࢆࡋ࡚ࠊ⚄⤒⣽⬊Ṛࢆቑᙉࡍࡿࡇࢆ᫂ࡽࡋࡓࠋᮏ◊✲ࡼ
ࡾࠊAࡢ✚ࡶࠊ⪁➼ࡼࡾᑠ⬊యࢫࢺࣞࢫࡀቑຍࡍࡿࡼ࠺࡞᮲௳
࠾࠸࡚ࠊAࡀᑠ⬊యࢫࢺࣞࢫࡼࡿ⚄⤒⣽⬊Ṛࢆቑᙉࡍࡿᶵᵓࡀࠊᑡ࡞ࡃࡶ
୍㒊ࠊࣝࢶࣁ࣐࣮ (AD)ࢆⓎࡉࡏࡿཎᅉ࡛࠶ࡿྍ⬟ᛶࢆ♧ࡋࡓࠋࡲࡓࠊ S-allyl-L-cysteine (SAC)ࡢ⚄⤒ಖㆤస⏝ࢆ᳨ウࡋࠊA25-35ࡢTMㄏⓎ⣽⬊Ṛቑᙉ
ࢆ㢧ⴭᢚไࡍࡿࡇࠊ࠾ࡼࡧࠊࡑࡢ⣽⬊ಖㆤస⏝ࡣSACࡢcalpainάᛶ㜼 ᐖస⏝ࡀ㛵ࡍࡿࡇࢆ♧ࡋࡓࠋ
ᚋ༙࠾࠸࡚ࡣࠊ๓༙ࡢᇵ㣴ᾏ㤿ษ∦࠾ࡅࡿ▱ぢࢆࡶࠊSACࡢᑠ⬊య ࢫࢺࣞࢫㄏⓎ⣽⬊Ṛᑐࡍࡿಖㆤస⏝ࡢᶵᗎࢆ᫂ࡽࡍࡿ┠ⓗ࡛ࠊᾏ㤿⚄⤒
⣽⬊ࡢึ௦ᇵ㣴⣔࠾ࡼࡧin vitroࡢcalpainάᛶ ᐃ⣔ࢆ⏝࠸࡚ࠊcalpainᑐࡍ
ࡿSACࡢస⏝ࡘ࠸࡚ヲ⣽᳨ウࡋࡓࠋึ௦ᇵ㣴ᾏ㤿⚄⤒⣽⬊࠾࠸࡚ࠊSAC ࡣᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛࢆ㢧ⴭᢚไࡋࠊࡑࡢ⚄⤒ಖㆤస⏝ࡣᢠ㓟స
⏝ࡣ㛵ࡏࡎࠊᑠ⬊యࢫࢺࣞࢫ࡛ஹ㐍ࡋࡓcalpainάᛶࡢᢚไࡀ㛵ࡍࡿࡇࢆ
᫂ࡽࡋࡓࠋࡲࡓࠊin vitroࡢcalpainάᛶ ᐃ⣔࠾࠸࡚ࠊSACࡣcalpainࡢ Ca2+⤖ྜ㒊ࡢ┦స⏝ࡼࡾ-calpainάᛶࢆ⦆ࡸ㜼ᐖࡋࠊࡇࡢ⦆࡞
㜼ᐖస⏝ࡀᑠ⬊యࢫࢺࣞࢫㄏⓎ⣽⬊Ṛᑐࡍࡿಖㆤస⏝㛵ࡍࡿࡇࢆ♧ࡋ ࡓࠋࡇࢀࡽࡢ▱ぢࡢᴫ␎ࢆFig. 16♧ࡋࡓࠋSACࡣcalpainࢆᶆⓗࡋ࡚⚄⤒
ಖㆤస⏝ࢆ♧ࡍࡇࡽࠊ᪂つAD⒪⸆ࡋ࡚ࡢྍ⬟ᛶࢆ᭷ࡋ࡚࠸ࡿ⪃࠼
ࡽࢀࡓࠋ
49
caspase-12
Ca2+
calpain
⚄⤒⣽⬊Ṛ
A
Ca2+
caspase-3
Ca2+⤖ྜ㒊
άᛶ୰ᚰ
SAC
LᆺCa2+䝏䝱䝛䝹 ᑠ⬊య ᑠ⬊య䝇䝖䝺䝇
CHOP
Ca2+
Fig. 16. A schematic diagram showing the mecanisms of A potentiates ER stress-induced neuronal cell death and neuroprotective effect of SAC in hippocampus neuron.
50
ཧ
ཧ⪃ᩥ⊩
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