新規高感度定量法を用いた小胞体ストレス応答性因子 mesencephalic astrocyte-derived neurotrophic factor とその paralog の性状解析

Title 新規高感度定量法を用いた小胞体ストレス応答性因子mesencephalic astrocyte-derived neurotrophic factor とその paralog の性状解析( 本文(Fulltext) ) Author(s) 則定, 純平 Report No.(Doctoral Degree) 博士(薬科学) 連創博甲第41号 Issue Date 2018-03-25 Type 博士論文 Version ETD URL http://hdl.handle.net/20.500.12099/75254 ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。

            2018 ๎ᐃ ⣧ᖹ㻌 ᪂つ㧗ឤᗘᐃ㔞ἲ䜢⏝䛔䛯ᑠ⬊య䝇䝖䝺䝇ᛂ⟅ᛶᅉᏊ㻌mesencephalic

astrocyte-derived neurotrophic factor 䛸䛭䛾 paralog 䛾ᛶ≧ゎᯒ

┠ ┠ḟ ┠ḟ... 2 -␎ㄒ... 3 -➨୍❶ ᗎㄽ... 5 11. ᑠ⬊య䛸㍺㏦... 5 12. ᑠ⬊య䝇䝖䝺䝇... 8

13. Mesencephalic astrocytederived neurotrophic factor (MANF)... 11

14. Cerebral dopamine neurotrophic factor (CDNF) ... 12

15. ᮏ◊✲䛾┠ⓗ... 14 -➨஧❶ MANF ศἪ䛻ᑐ䛩䜛㧗ឤᗘᐃ㔞ἲ䛾ᵓ⠏ ... 15 21. せ⣙ ... 15 22. ᮦᩱ䛸᪉ἲ ... 16 23. ⤖ᯝ ... 19 24. ⪃ᐹ ... 33

-➨୕❶ NanoLuc Luciferase Assay 䜢⏝䛔䛯 MANF 䛸 CDNF 䛾 ศἪไᚚ䛻㛵䛩䜛 ẚ㍑᳨ウ... 37 31. せ⣙ ... 37 32. ᮦᩱ䛸᪉ἲ ... 38 33. ⤖ᯝ ... 40 34. ⪃ᐹ ... 51 -➨ᅄ❶ ᑠ⬊య䝇䝖䝺䝇ᛂ⟅ᛶᅉᏊ CRELD2 䛾ศἪ䛻ᑐ䛩䜛 MANF ཬ䜃 CDNF 䛾ᙳ㡪 䛻㛵䛩䜛ẚ㍑᳨ウ... 53 41. せ⣙ ... 53 42. ᮦᩱ䛸᪉ἲ ... 54 43. ⤖ᯝ ... 56 44. ⪃ᐹ ... 64 -➨஬❶ ⥲ᣓ... 67 -ㅰ㎡... 70 ཧ⪃ᩥ⊩... 71

-␎ ␎ㄒ

amino acid response element (AARE) activating transcription factor 4 (ATF4) activating transcription facor 6 (ATF6) amyloid E (AE)

arginine-rich protein mutated in early stage tumors (Armet) complementary DNA (cDNA)

cerebral dopamine neurotrophic factor (CDNF) cysteine-rich with EGF-like domains 2 (CRELD2) C terminal amino acid deleted type (¨C)

dithiothreitol (DTT)

ethylenediaminetetraacetic acid (EDTA)

eukaryotic translation initiation factor 2D (eIF2D) endoplasmic reticulum (ER)

ER associated degradation (ERAD) ER stress response element (ERSE) fetal bovine serum (FBS)

glyceraldehyde 3-phosphate dehydrogenase (G3PDH)

growth arrest and DNA damage-inducible protein 34 (GADD34) growth arrest and DNA damage-inducible protein 153 (GADD153) green fluorescent protein (GFP)

glucose regulated protein 78 kDa (GRP78) glucose regulated protein 94 kDa (GRP94) human embryonic kidney cells 293 (HEK293)

homocysteine-induced endoplasmic reticulum protein (Herp) inositol-requiring enzyme 1 (IRE1)

lipopolysaccharide (LPS)

mesencephalic astrocyte-derived neurotrophic factor (MANF) NanoLuc (NL)

NanoLuc Luciferase Assay (NL assay) phosphate buffered saline (PBS) polymerase chain reaction (PCR) protein disulfide isomerase (PDI)

polyvinylidene difluoride (PVDF) reverse transcription PCR (RT-PCR) serine protease site-1 (S1P)

serine protease site-2 (S2P) sodium dodecyl sulfate (SDS) serum free (SF)

serum-glucose free (SGF) signal-recognition particle (SRP) thapsigargin (Tg)

tunicamycin (Tm)

unfolded protein response (UPR) upstream open reading frame (uORF) X box-binding protein-1 (XBP1)

western blotting (WB) wild-type (wt)

➨ ➨୍❶ ᗎㄽ 1-1. ᑠ⬊య䛸㍺㏦ 䝠䝖䛿⣙ 60 ඙ಶ䛾⣽⬊䛛䜙ᡂ䜚❧䛳䛶䛔䜛䚹䛭䜜䜙䛾⣽⬊䛿䚸䛔䛟䛴䛛䛾䜎䛸䜎䜚 䜢ᙧᡂ䛧䚸䜎䛸䜎䜚ẖ䛻≉ᚩⓗ䛺ᶵ⬟䜢Ⓨ⌧䛥䛫䜛䛣䛸䛷䚸ಶయ䛾⏕Ꮡ䞉ᡂ㛗䜢ㄪ⠇䛧 䛶䛔䜛䚹䛭䛾䜎䛸䜎䜚䜢⮚ჾ䛸䛔䛔䚸ྛ⮚ჾ୰䛾⣽⬊䛿ᩘከ䛟䛾䝍䞁䝟䜽㉁䜢ྜᡂ䛧䚸 ⮚ჾ≉␗ⓗ䛺ᶵ⬟䜢⥔ᣢ䛧䛶䛔䜛䚹䜎䛯䚸ྛ⮚ჾ䛜༠ㄪⓗ䛻ാ䛟䛣䛸䛻䜘䜚䚸䝠䝖ಶయ 䛾೺ᗣ䛜ಖ䛯䜜䛶䛔䜛䚹⣽⬊䜒䜎䛯⣽⬊ෆᑠჾᐁ䛾㞟ྜయ䛷䛒䜚䚸䛭䜜䜙䛾༠ㄪᛶ 䛻䜘䜚⣽⬊ᶵ⬟䞉⏕Ꮡ䛜⥔ᣢ䛥䜜䛶䛔䜛䚹䛭䛾୍䛴䛷䛒䜛ᑠ⬊య (endoplasmic reticulum, ER) 䛷䛿䚸⣽⬊䛜ྜᡂ䛩䜛䝍䞁䝟䜽㉁䛾ෆ䚸⣙ 30% 䛾䝍䞁䝟䜽㉁䛜ᑠ ⬊యෆ⭍䜈䛸㍺㏦䛥䜜䜛䚹䛭䜜䜙䛾䝍䞁䝟䜽㉁䛿ᑠ⬊యศᏊ䝅䝱䝨䝻䞁➼䛻䜘䜚㐺ษ 䛻ᢡ䜚䛯䛯䜎䜜䛯ᚋ䚸䝂䝹䝆య䜢௓䛧䛶᭱⤊┠ⓗᆅ䜈䛸㍺㏦䛥䜜䜛 [1, 2, 3]䚹䝍䞁䝟 䜽㉁䛻䛿≉ᐃ䛾⣽⬊ෆᑠჾᐁ䛻ᒁᅾ䛩䜛䜒䛾䛜Ꮡᅾ䛧䛶䛔䜛䚹୍⯡ⓗ䛻䚸䛭䜜䜙䝍䞁 䝟䜽㉁䛿≉ᚩⓗ䛺䜰䝭䝜㓟㓄ิ (䝅䜾䝘䝹䝨䝥䝏䝗) 䜢᭷䛧䛶䛚䜚䚸ᑠ⬊య䜈䛸㐠䜀 䜜䜛䝍䞁䝟䜽㉁䛻䛿 N ᮎ➃䛻䝅䜾䝘䝹䝨䝥䝏䝗䛜Ꮡᅾ䛧䛶䛔䜛䚹䜎䛯ᑠ⬊య䛻ᒁᅾ

䛩䜛䝍䞁䝟䜽㉁䛿 C ᮎ➃䛻ᑠ⬊యᒁᅾ䝅䜾䝘䝹 (canonical ER retrieval signal;

Lys-Asp-Glu-Leu, KDEL) 㓄ิ䜢᭷䛧䛶䛔䜛ሙྜ䛜ከ䛔 (ᅗ 1) [3, 4]䚹N ᮎ➃䝅䜾

䝘䝹䝨䝥䝏䝗䛿ᑠ⬊య⭷ୖ䛻Ꮡᅾ䛩䜛䝍䞁䝟䜽㉁㍺㏦య (protein translocator) 䛸

఍ྜ䛧䚸⩻ヂ㏵୰䛾䝸䝪䝋䞊䝮䜢ᑠ⬊య㏆ഐ䛻ᅛᐃ䛩䜛䛸ඹ䛻䚸ྜᡂ䛥䜜䛯䝫䝸䝨䝥 䝏䝗㙐䜢ᑠ⬊యෆ⭍䜈䛸⛣⾜䛥䛫䜛䚹䝫䝸䝨䝥䝏䝗㙐䛜㐺ษ䛻ᢡ䜚䛯䛯䜎䜜䛯ᚋ䚸䝅 䜾䝘䝹䝨䝥䝏䝗ศゎ㓝⣲䛻䜘䜚䝅䜾䝘䝹䝨䝥䝏䝗䛿᪂つ䝍䞁䝟䜽㉁䛛䜙ษ᩿䛥䜜䜛 [5]䚹䛭䛾ᚋ䚸glucose regulated protein, 78 kDa (GRP78)䚸glucose regulated protein, 94 kDa (GRP94) ➼䛾ᑠ⬊యศᏊ䝅䝱䝨䝻䞁䛻䜘䜛ᢡ䜚䛯䛯䜏䚸protein disulfide isomerases (PDI) 䛻䜘䜛䝆䝇䝹䝣䜱䝗⤖ྜ䛾ᙧᡂ䛺䛹䛾⩻ヂᚋಟ㣭䛜䛚䛣

䛺䜟䜜䜛䚹䛭䜜䜙䛻䜘䜚㐺ษ䛻ᢡ䜚䛯䛯䜎䜜䛯᪂つ䝍䞁䝟䜽㉁䛿䚸COP-II ᑠ⬊䜢௓䛧

䛶䚸䝂䝹䝆య䜈䛸㍺㏦䛥䜜䜛 [6]䚹䛭䛾㝿䚸C ᮎ➃䛻 KDEL 㓄ิ䜢᭷䛩䜛 GRP78

䛺䛹䛾䝍䞁䝟䜽㉁䛿䚸cis-䝂䝹䝆యୖ䛻Ꮡᅾ䛩䜛 KDEL ཷᐜయ䛻䜘䜚ㄆ㆑䛥䜜䚸

COP-I ᑠ⬊䜢௓䛧䛶ᑠ⬊య䜈䛸㏫⾜㍺㏦䛥䜜䜛䛣䛸䛻䜘䜚䚸ᑠ⬊య䛻ᒁᅾ䛩䜛 (ᅗ 2) [7, 8]䚹

ᅗ

ᅗ 1. ඾ᆺⓗ䛺䝅䜾䝘䝹䝨䝥䝏䝗䛾୍ぴ

඾ᆺⓗ䛺䝅䜾䝘䝹䝨䝥䝏䝗㓄ิ䜢䜰䝭䝜㓟୍ᩥᏐ⾲グ䛻䛶♧䛧䛯䚹+H3N- 䛿䝍䞁䝟䜽㉁䛾

N ᮎ➃䜢♧䛧䚸-COO- _{䛿䝍䞁䝟䜽㉁䛾 C ᮎ➃䜢♧䛩 (Alberts B, Johnson A, Lewis J,}

Morgan D, Raff M, Roberts K, et al. Molecular Biology of THE CELL. 6th ed. New York: Garland Science; 2014 䜘䜚୍㒊ᨵኚ)䚹

(b) ᅗ ᅗ 2. ᑠ⬊య䛻䛚䛡䜛䝍䞁䝟䜽㉁ྜᡂཬ䜃ศἪ⤒㊰䛾ᶍᘧᅗ (a) ᑠ⬊యෆ⭍䜈䛾䝫䝸䝨䝥䝏䝗㙐䛾ఙ㛗ཬ䜃⛣⾜䛾ᶍᘧᅗ䚹 ⣽⬊㉁୰䛻Ꮡᅾ䛩䜛ᑠ⬊య䝅䜾䝘䝹䝨䝥䝏䝗䛸䝸䝪䝋䞊䝮䛿䚸䝅䜾䝘䝹ㄆ㆑⢏Ꮚ (signal-recognition particle, SRP) 䛸఍ྜ䛧䚸SRP-䝸䝪䝋䞊䝮」ྜయ䜢ᙧᡂ䛩䜛䚹䛭䛾㝿䚸 ୍᫬ⓗ䛻⩻ヂ䛿೵Ṇ䛩䜛䚹䛭䛾ᚋ䚸SRP-䝸䝪䝋䞊䝮」ྜయ䛿䚸ᑠ⬊య⭷ୖ䛻䛒䜛 SRP ཷ ᐜయ䛻䜘䛳䛶䚸ᑠ⬊య⭷㏆ഐ䛻ಀ␃䛥䜜䜛䚹ḟ䛔䛷䚸䝸䝪䝋䞊䝮ཬ䜃ᑠ⬊య䝅䜾䝘䝹䝨䝥䝏䝗 䛿 SRP 䛛䜙஋㞳䛧䚸䝍䞁䝟䜽㉁㍺㏦య䛸⤖ྜ䛩䜛䚹䛭䜜䛻䜘䜚⩻ヂ䛿෌㛤䛥䜜䚸䝫䝸䝨䝥䝏 䝗㙐䛜ᑠ⬊యෆ⭍䜈䛸㍺㏦䛥䜜䜛䚹⩻ヂ䛜᏶஢䛧䚸ᑠ⬊యศᏊ䝅䝱䝨䝻䞁䛻䜘䜚㐺ษ䛻ᢡ䜚 䛯䛯䜎䜜䛯᪂つ䝍䞁䝟䜽㉁䛿䚸䝅䜾䝘䝹䝨䝥䝏䝗ศゎ㓝⣲䛻䜘䜚䚸䝅䜾䝘䝹䝨䝥䝏䝗㒊఩䛜ษ ᩿䛥䜜䜛䚹୍᪉䚸䝸䝪䝋䞊䝮䛸஋㞳䛧䛯 SRP 䛿 GTP ຍỈศゎ䜢௓䛧䛯ᙧ≧ኚ໬䛻䜘䜚ཷ ᐜయ䛸㞳䜜䜛 (Alberts B, Johnson A, Lewis J, Morgan D, Raff M, Roberts K, et al. Molecular Biology of THE CELL. 6th ed. New York: Garland Science; 2014 䜘䜚୍㒊ᨵ ኚ)䚹 (b) ศἪ䝍䞁䝟䜽㉁䚸⭷䝍䞁䝟䜽㉁ཬ䜃ᑠ⬊యᒁᅾ䝍䞁䝟䜽㉁䛾㍺㏦⤒㊰䛾ᶍᘧᅗ䚹 ᑠ⬊యᒁᅾ䝅䜾䝘䝹㓄ิ (KDEL 㓄ิ) 䜢᭷䛧䛶䛔䛺䛔䝍䞁䝟䜽㉁ (ⲔⰍ䞉⳻ᙧ) 䛿䚸 COP-II ᑠ⬊䛻䜘䜛㍺㏦⤒㊰䛻䜘䛳䛶䚸䝂䝹䝆య䜢௓䛧䛶⣽⬊እ䜈䛸ศἪ䛥䜜䜛䛛䚸ⱝ䛧䛟䛿 ⣽⬊⭷䜈䛸㐠䜀䜜䜛䚹୍᪉䚸KDEL 㓄ิ䜢᭷䛧䛶䛔䜛䝍䞁䝟䜽㉁ (㯤Ⰽ䞉୸ᆺ) 䛿䚸COP-II ᑠ⬊䛻䜘䛳䛶䝂䝹䝆య䜈䛸㐠䜀䜜䛯ᚋ䚸cis-䝂䝹䝆⭷ୖ䛾 KDEL ཷᐜయ䛻䜘䜚ㄆ㆑䛥䜜䚸 COP-I ᑠ⬊䜢௓䛧䛶ᑠ⬊య䜈䛸㏫⾜㍺㏦䛥䜜䜛䛣䛸䛷䚸ᑠ⬊య䛻෌䜃ᒁᅾ䛩䜛䚹

1-2. ᑠᑠ⬊య䝇䝖䝺䝇

ᑠ⬊య䛷䛿䚸ྜᡂ䛥䜜䛯⭷䝍䞁䝟䜽㉁䞉ศἪ䝍䞁䝟䜽㉁䜢㐺ษ䛻ᢡ䜚䛯䛯䜏䚸䝂䝹 䝆య䜈䛸㍺㏦䛧䛶䛔䜛䚹䛭䛾⤒㊰䛻䛚䛔䛶䚸㐺ษ䛻ᢡ䜚䛯䛯䜎䜜䛺䛛䛳䛯䝍䞁䝟䜽㉁ 䛿䚸ᑠ⬊య䛛䜙⣽⬊㉁䜈䛸㍺㏦䛥䜜䚸䝴䝡䜻䝏䞁䞉䝥䝻䝔䜰䝋䞊䝮⣔䛻䛶ศゎ䛥䜜䜛䚹

䛣䛾ᶵᵓ䜢ᑠ⬊య㛵㐃ศゎ (ER associated degradation, ERAD) 䛸࿧䜃䚸ᢡ䜚␚

䜏୙඲䝍䞁䝟䜽㉁䛾㝖ཤ䜢㏻䛨䛶䚸ᑠ⬊యෆ䛾䝍䞁䝟䜽㉁ရ㉁ᜏᖖᛶ䛻ᐤ୚䛧䛶䛔

䜛 [9]䚹ᰤ㣴㣚㣹䚸⹫⾑➼䛻䜘䜚䚸ᑠ⬊య䛾ෆ㒊⎔ቃ䛜ᝏ໬䛩䜛䛸ᑠ⬊యෆ䛾䝍䞁䝟

䜽㉁ရ㉁ᜏᖖᛶ䛜ᔂ䜜䚸ᢡ䜚␚䜏୙඲䝍䞁䝟䜽㉁䛜ᑠ⬊యෆ⭍䛻⵳✚䛩䜛䚹䛣䛾≧

ែ䜢ᑠ⬊య䝇䝖䝺䝇 (ER stress) 䛸䛔䛔䚸⣽⬊䛿䛣䛾䝇䝖䝺䝇䜢ῶᙅ䛩䜛䛯䜑䛻䚸ᢡ

䜚␚䜏୙඲䝍䞁䝟䜽㉁ᛂ⟅ (unfolded protein response, UPR) 䜢ច㉳䛩䜛 (ᅗ 3)

[10]䚹

UPR 䛿䚸ᑠ⬊య⭷ୖ䛻Ꮡᅾ䛩䜛 3 䛴䛾୺せ䛺䝇䝖䝺䝇䝉䞁䝃䞊 PKR-like endoplasmic reticulum kinase (PERK) [11]䚸inositol-requiring enzyme 1 (IRE1) [12], activating transcription factor 6 (ATF6) [13, 14]䚸䜢㉳Ⅼ䛸䛩䜛䝇䝖䝺䝇ᛂ⟅⤒ ㊰䛻䜘䜚ᵓᡂ䛥䜜䛶䛔䜛䚹㏻ᖖ䚸䛭䜜䜙䝇䝖䝺䝇䝉䞁䝃䞊䛻䛿ᑠ⬊యศᏊ䝅䝱䝨䝻䞁 GRP78 䛜఍ྜ䛧䛶䛚䜚䚸UPR ⤒㊰䛿୙άᛶ䛻ಖ䛯䜜䛶䛔䜛䛜䚸ᢡ䜚␚䜏୙඲䝍䞁

䝟䜽㉁䛾⵳✚䛻క䛔 GRP78 䛜䝇䝖䝺䝇䝉䞁䝃䞊䛛䜙஋㞳䛩䜛䛸䚸䛭䜜䜙䝇䝖䝺䝇䝉

䞁䝃䞊䛿άᛶ໬䛥䜜䚸௨ୗ䛻♧䛩䜘䛖䛺 UPR ⤒㊰䛜ច㉳䛥䜜䜛 (ᅗ 3) [15-17]䚹

PERK ⤒㊰䛷䛿䚸஧㔞య໬䛧⮬ᕫ䝸䞁㓟໬䛻䜘䜚άᛶ໬䛥䜜䛯 PERK 䛜⩻ヂㄪ

⠇ᅉᏊ HXNDU\RWLF WUDQVODWLRQ LQLWLDWLRQ IDFWRU Į H,)Į) 䜢䝸䞁㓟໬䛩䜛䚹䛣䜜䛻

䜘䜚䝸䝪䝋䞊䝮㛤ጞ」ྜయ䛾ᙧᡂ䛜ᢚไ䛥䜜䚸㛤ጞ䝁䝗䞁䛾ㄆ㆑䛜㜼ᐖ䛥䜜䜛䚹ᚑ䛳 䛶䚸⣽⬊඲య䛷䛾䝍䞁䝟䜽㉁䛾⩻ヂ䛜ᢚไ䛥䜜䚸ᢡ䜚␚䜏୙඲䝍䞁䝟䜽㉁䛾ᑠ⬊య

䜈䛾䛥䜙䛺䜛⵳✚䛜ᢚ䛘䜙䜜䜛 [11, 18]䚹ER 䝇䝖䝺䝇ᛂ⟅ᛶ㌿෗ㄪ⠇ᅉᏊ䛾୍䛴

activating transcription factor 4 (ATF4) 䛿䚸ᢚไᛶ䛾ୖὶ䜸䞊䝥䞁䝸䞊䝕䜱䞁䜾䝣䝺

䞊䝮 (uORF) 䜢᭷䛧䛶䛔䜛䚹䛣䛾䛯䜑䚸␗ᖖ䛜䛺䛔᫬䛿䚸ATF4 䛾⩻ヂ䛜ᢚไ䛥䜜

䛶䛔䜛䛜䚸ᑠ⬊య䝇䝖䝺䝇᫬䛻䛿䚸uORF 䛾㛤ጞ䝁䝗䞁䛜ㄞ䜏㣕䜀䛥䜜䜛䛺䛹䛾⌮⏤

䛻䜘䜚䚸ATF4 䛾⩻ヂ䛿ಁ㐍䛥䜜䜛 [19, 20]䚹䛣䜜䛻䜘䜚䚸growth arrest and DNA

damage-inducible protein 34 (GADD34) 䚸 growth arrest and DNA damage-inducible protein 153 (GADD153) 䜢ྵ䜐ᶆⓗᅉᏊ䛾㌿෗䛜ㄏᑟ䛥䜜䜛䚹

H,)Į 䝸䞁㓟໬䛻క䛖⣽⬊඲య䛷䛾⩻ヂᢚไ䛿⣽⬊⏕Ꮡ䛻᭷฼䛻ാ䛟䛜䚸㛗ᮇⓗ䛺 ⩻ヂ㜼ᐖ䛿䜸䞊䝖䝣䜯䝆䞊䜢ㄏⓎ䛧䚸⣽⬊Ṛ䛾ཎᅉ䛸䛺䜛䚹䜎䛯䚸GADD153 䛿⣽⬊ ࿘ᮇ೵Ṇ䜔䜰䝫䝖䞊䝅䝇䛻㛵䜟䜛ከ䛟䛾㑇ఏᏊ䛾Ⓨ⌧䜢ㄪ⠇䛧䚸⣽⬊Ṛ䜢ㄏᑟ䛩䜛䛣 䛸䛜ሗ࿌䛥䜜䛶䛔䜛䚹 GADD34 䛿 eIF2Į 䛾⬺䝸䞁㓟໬䜢௓䛧䛶䚸䝇䝖䝺䝇ㄏᑟᛶ 㑇ఏᏊⓎ⌧䜢䝣䜱䞊䝗䝞䝑䜽㜼ᐖ䛧䛶䛔䜛 [21-25]䚹 IRE1 䛿䚸GRP78 䛾஋㞳䛻క䛔䚸஧㔞య໬䚸⮬ᕫ䝸䞁㓟໬䛩䜛䛣䛸䛷άᛶ໬䛥䜜䚸

㌿෗ㄪ⠇ᅉᏊ X-box binding protein 1 (XBP1) 䛾䝇䝥䝷䜲䝅䞁䜾䜢⾜䛖䚹XBP1 䛿 ㏻ᖖ䚸䝇䝥䝷䜲䝅䞁䜾䛥䜜䛶䛔䛺䛔 unspliced XBP1 (uXBP1) 䛸䛧䛶Ꮡᅾ䛧䛶䛔䜛䚹 ୍᪉䛷䚸䝇䝥䝷䜲䝅䞁䜾䛥䜜䛯 XBP1 (sXBP1) 䛿᰾ෆ䛻⛣⾜䛧䚸ER 䝇䝖䝺䝇㛵㐃 ᅉᏊ䛾䝥䝻䝰䞊䝍䞊㓄ิ䛻⤖ྜ䛩䜛䛣䛸䛷䚸䛭䜜䜙ᅉᏊ䛾㌿෗䜢ಁ㐍䛩䜛䚹IRE1 䛿䚸 ERAD 㛵㐃ᅉᏊ䛾Ⓨ⌧ୖ᪼䜢ᘬ䛝㉳䛣䛩䛣䛸䛻䜘䜚䚸ᑠ⬊య䝇䝖䝺䝇䛾⦆࿴䜢䛥䛫䚸 ᑠ⬊యෆ䝍䞁䝟䜽㉁ಖᣢ䛻ᐤ୚䛧䛶䛔䜛 [10, 26]䚹 ATF6 䛿䚸␗ᖖ䛜䛺䛡䜜䜀䚸䝆䝇䝹䝣䜱䝗⤖ྜ䛻䜘䜛஧㔞య䚸ⱝ䛧䛟䛿䚸䝆䝇䝹䝣䜱䝗 ⤖ྜ䜢ෆໟ䛩䜛㓟໬ᆺ༢㔞య䛸䛧䛶Ꮡᅾ䛧䛶䛔䜛䚹ᑠ⬊య䝇䝖䝺䝇᫬䛻䛿ᑠ⬊యෆ ⭍䛷䛾㓟໬䞉㑏ඖ≧ែ䛾ᆒ⾮䛜◚䜜䚸ATF6 䛿 2 䛴䛾䝏䜸䞊䝹ᇶ䜢᭷䛩䜛㑏ඖᆺ ༢㔞య䛸䛺䜛䚹䛥䜙䛻䚸GRP78 䛜஋㞳䛧䛯ᚋ䚸ER 䛛䜙䝂䝹䝆య䛻⛣⾜䛧䛯㑏ඖᆺ

༢㔞య ATF6 䛿䚸serine protease site-1 (S1P) 䛸 serine protease site-2 (S2P)

䛻䜘䜚⭷㏆ഐ䛷䛾ษ᩿䜢ཷ䛡䚸άᛶ໬ᆺ ATF6 (cleaved ATF6) 䛸䛺䜛 [27]䚹ḟ䛔

䛷䚸cleaved ATF6 䛿᰾䛻⛣⾜ᚋ䚸GRP78 䛺䛹䛾ศᏊ䝅䝱䝨䝻䞁㑇ఏᏊ䛾㌿෗䜢

ㄏᑟ䛩䜛䚹䜎䛯䚸homocysteine-induced endoplasmic reticulum protein (Herp) 䛺

䛹䛾 ERAD 㛵㐃ᅉᏊ䛾㌿෗䜒ㄏᑟ䛩䜛䛣䛸䛷䚸ᢡ䜚䛯䛯䜏୙඲䝍䞁䝟䜽㉁䛾㝖ཤ䜢

ಁ㐍䛥䛫䜛 [9, 10, 28, 29]䚹

UPR 䛻䜘䛳䛶ㄏᑟ䛥䜜䜛㑇ఏᏊⓎ⌧䛿䚸䝥䝻䝰䞊䝍䞊ୖ䛾≉␗ⓗ䛺㓄ิ䛻㌿෗

ㄪ⠇ᅉᏊ䛜⤖ྜ䛩䜛䛣䛸䛷ᘬ䛝㉳䛣䛥䜜䜛䚹GADD153 㑇ఏᏊ䛿䝥䝻䝰䞊䝍䞊ୖ䛻

amino acid response element (AARE; ATTGCATCA) 䚸 ER stress response element (ERSE; CCAAT-N9-CCACG) 㓄ิ䜢ᣢ䛱䚸䛭䜜䛮䜜 ATF4䚸cleaved ATF6 䛜⤖ྜ䛩䜛䛣䛸䛷䚸䛭䛾㌿෗䛿ㄏᑟ䛥䜜䜛䛣䛸䛜ሗ࿌䛥䜜䛶䛔䜛 [30]䚹䜎䛯䚸

GRP78 䛺䛹䛾ศᏊ䝅䝱䝨䝻䞁㑇ఏᏊ䛾䝥䝻䝰䞊䝍䞊ୖ䛻䛿 ERSE 㓄ิ䛜Ꮡᅾ䛩

䜛䛣䛸䛜ከ䛟䚸cleaved ATF6 䛾⤖ྜ䛻䜘䜚㌿෗䛜㛤ጞ䛥䜜䜛 [28]䚹୍᪉䚸ERAD 㛵

㐃 㑇 ఏ Ꮚ 䛾 ୍ 䛴 䛷 䛒 䜛 Herp 䛾 䝥 䝻 䝰 䞊 䝍 䞊 ୖ 䛻 䛿 䚸 AARE 䚸 ERSE-II

(ATTGG-N-CCACG) 㓄 ิ 䛜 Ꮡ ᅾ 䛧 䚸 䛭 䜜 䛮 䜜 ATF4 䚸 cleaved ATF6 䜎 䛯 䛿 sXBP1 䛜⤖ྜ䛩䜛䛣䛸䛷䚸Herp 㑇ఏᏊ䛾㌿෗䛜ㄏᑟ䛥䜜䜛[31, 32]䚹

ᅗ ᅗ 3. ᢡ䜚␚䜏୙඲䝍䞁䝟䜽㉁ᛂ⟅ (UPR) 䛾ᴫ␎ᅗ ᑠ⬊య䝇䝖䝺䝇䝉䞁䝃䞊 PERK䚸IRE1䚸ATF6 䛿㠀䝇䝖䝺䝇ୗ䛷䛿 GRP78 䛸఍ྜ䛧䛶䛚 䜚䚸୙άᛶ໬≧ែ䛻䛒䜛 (ᅗ ྑ➃)䚹䛧䛛䛧䚸ᢡ䜚␚䜏୙඲䝍䞁䝟䜽㉁䛾⵳✚䛻క䛔䚸 GRP78 䛜㐟㞳䛩䜛䛸䚸䝇䝖䝺䝇䝉䞁䝃䞊䛿άᛶ໬䛧䚸䛭䜜䛮䜜䛾⤒㊰䜢άᛶ໬䛥䛫䜛䚹UPR 䛾άᛶ໬䛻䜘䜚䚸᪂つ䝍䞁䝟䜽㉁䛾⩻ヂ೵Ṇ䚸ศᏊ䝅䝱䝨䝻䞁䛾Ⓨ⌧ㄏᑟཬ䜃䚸ᑠ⬊య㛵㐃 ศゎ (ERAD) 䛾άᛶ໬䛜⾜䜟䜜䚸ᑠ⬊య䝇䝖䝺䝇䛾ゎᾘ䜢ᅗ䜛䚹䛧䛛䛧䚸㛗ᮇⓗ䛺 UPR 䛾άᛶ໬䛿 GADD153 䛾Ⓨ⌧ㄏᑟ䛺䛹䜢㏻䛧䛶⣽⬊Ṛ䜢ㄏᑟ䛩䜛䚹

1-3. Mesencephalic astrocyte-derived neurotrophic factor (MANF)

Mesencephalic astrocyte-derived neurotrophic factor (MANF) 䛿ᙜึ䚸

arginine-rich protein mutated in early stage tumors (Armet) 䛸ྡ䛵䛡䜙䜜䚸⭘⒆⣽

⬊䛻䛚䛔䛶㧗㢖ᗘ䛻ኚ␗䜢᭷䛧䛶䛔䜛䝍䞁䝟䜽㉁䛷䛒䜛䛣䛸䛜ሗ࿌䛥䜜䛶䛔䛯 [33,

34]䚹䛭䛾ᚋ䚸2003 ᖺ䛻 Petrova 䜙䛻䜘䜚䚸䝷䝑䝖୰⬻ I ᆺ䜰䝇䝖䝻䝃䜲䝖⣽⬊䛾ᇵ 㣴ᾮ୰䛛䜙༢㞳䛥䜜䛯䝗䞊䝟䝭䞁సືᛶ䝙䝳䞊䝻䞁䜢㑅ᢥⓗ䛻ಖㆤ䛩䜛⚄⤒ᰤ㣴ᅉ

Ꮚ MANF 䛸ྠ୍䛷䛒䜛䛣䛸䛜ሗ࿌䛥䜜䛯 [35]䚹MANF 䛿 6-hydroxydopamine

(6-OHDA) ㄏᑟᛶ䝟䞊䜻䞁䝋䞁⑓䝰䝕䝹䝷䝑䝖䛻䛚䛔䛶䚸䝗䞊䝟䝭䞁⚄⤒䜢㑅ᢥⓗ䛻 ಖㆤ䛩䜛䛣䛸䛜ሗ࿌䛥䜜䛶䛔䜛 [36]䚹䜎䛯䚸MANF mRNA 䛿⤌⧊䛻ᗈ䛟Ⓨ⌧䛧䛶䛚 䜚䚸≉䛻⮅⮚䛾䜘䛖䛺ෆศἪ⣔䛾⣽⬊䛷䛿Ⓨ⌧䝺䝧䝹䛜㧗䛔䛣䛸䜒ሗ࿌䛥䜜䛶䛔䜛 [37]䚹MANF 䝜䝑䜽䜰䜴䝖䝬䜴䝇䛿ㄌ⏕᫬䜘䜚㔜⠜䛺䜲䞁䝇䝸䞁Ḟᦆᆺ⢾ᒀ⑓䜢Ⓨ⑕ 䛧䛶䛚䜚䚸䛭䛾䝬䜴䝇䛷䛿⮅⮚ ȕ ⣽⬊䛾ቑṪ䛜ᢚไ䛥䜜䚸䜰䝫䝖䞊䝅䝇䛻䜘䜛⣽⬊ Ṛ䛾ቑຍ䛜♧䛥䜜䛶䛔䜛䚹䛥䜙䛻䚸streptozotocin ㄏᑟᛶ⢾ᒀ⑓䝬䜴䝇䛻䛚䛔䛶䚸⤌ 䜏᥮䛘ᆺ MANF ฎ⌮ཬ䜃䜴䜱䝹䝇፹௓ᛶ MANF 㐣๫Ⓨ⌧䛿 ȕ ⣽⬊䛾ቑṪಁ 㐍䚸䜰䝫䝖䞊䝅䝇䛛䜙䛾⣽⬊ಖㆤస⏝䜢♧䛧䛯 [38]䚹䜎䛯䚸ᑠ⬊య䝇䝖䝺䝇ㄏᑟᛶ⣽ ⬊Ṛ䛛䜙䝠䝖㦵⫗⭘⣽⬊ᰴ U2OS 䜢ಖㆤ䛩䜛 [39] 䛣䛸䜒ሗ࿌䛥䜜䛶䛔䜛䛣䛸䛛䜙䚸 MANF 䛾⣽⬊ಖㆤຠᯝ䛿䚸⚄⤒⣔䛾䜏䛺䜙䛪䚸ከ䛟䛾ᮎᲈ⤌⧊䛷䜒䜏䜙䜜䜛䛸⪃䛘 䜙䜜䜛䚹䛧䛛䛧䚸䛭䛾ヲ⣽䛺ศᏊᶵᵓ䜔䚸ᑠ⬊య䝇䝖䝺䝇ୗ䛻䛚䛡䜛ᙺ๭䛻䛴䛔䛶䛿 ᮍ䛰᫂䜙䛛䛻䛺䛳䛶䛔䛺䛔䚹 MANF 䛿⣙ 18 kDa 䛾ศἪᆺ䝍䞁䝟䜽㉁䛷䛒䜚䚸N ᮎ➃䛻䝅䜾䝘䝹䝨䝥䝏䝗䜢᭷ 䛧䛶䛔䜛 [35]䚹䛥䜙䛻䚸N ᮎ➃䝅䜾䝘䝹䝨䝥䝏䝗䜢ྵ䜐㡿ᇦ䜢Ḟኻ䛥䛫䜛䛸䚸MANF 䛿୙Ᏻᐃ໬䛩䜛䛣䛸䛜ሗ࿌䛥䜜䛶䛔䜛 [40]䚹୍᪉䚸C ᮎ➃䛻䛿ᑠ⬊యᒁᅾ䝍䞁䝟䜽 ㉁䛻䜏䜙䜜䜛 KDEL ᵝ䛾 RTDL 㓄ิ䜢᭷䛧䛶䛚䜚 (ᅗ 4)䚸⣽⬊እ䜈ศἪ䛥䜜䜛 ୍᪉䛷䚸⣽⬊ෆ䛻୍㒊ಖᣢ䛥䜜䜛䛣䛸䛜᫂䜙䛛䛻䛥䜜䛶䛔䜛 [41, 42]䚹GRP78 䛸䛾 ඹⓎ⌧䛻䜘䜚䚸MANF 䛾ศἪ䛿ⴭ䛧䛟ῶᑡ䛩䜛䛣䛸 [40]䚸MANF 䛿ᑠ⬊య䝇䝖䝺䝇 ᛂ⟅ᛶᅉᏊ䛷䛒䜚䚸$7)Į䚸$7)ȕ䚸XBP-1 䛻䜘䜚ERSE-II 㓄ิ䜢௓䛧䛶䚸䛭䛾㌿

෗䛜ㄏᑟ䛥䜜䜛䛣䛸䛜ሗ࿌䛥䜜䛶䛔䜛 [43-45]䚹Mizobuchi 䜙䛿䚸MANF 䛜 ERAD

䛾ᵓᡂせ⣲䛷䛒䜛 Herp 䛸ྠᵝ䛻䚸ERSE-II 㓄ิ䜢௓䛧䛶Ⓨ⌧䛜ㄏᑟ䛥䜜䜛䛣䛸䛛 䜙䚸MANF 䛜 ERAD ᶵᵓ䛻㛵ಀ䛧䛶䛔䜛䛸ᥦ᱌䛧䛶䛔䜛 [44]䚹䛧䛛䛧䛺䛜䜙䚸䛭䜜 䛻ᑐ䛩䜛᫂☜䛺⟅䛘䛿ฟ䛶䛔䛺䛔䚹 䛥䜙䛻䚸㏆ᖺ䚸ᾏ⥥⏤᮶ MANF 䜢 HEK293 䛻䛶㐣๫Ⓨ⌧䛥䛫䜛䛣䛸䛻䜘䜚䚸LPS ㄏᑟᛶ䜹䝇䝟䞊䝊 3 䛾άᛶ໬䛜ῶᑡ䛧䚸⣽⬊⏕Ꮡ⋡䛿ୖ᪼䛩䜛䛣䛸䛜ሗ࿌䛥䜜䛯 [46]䚹MANF 䛿䚸I ᆺ⢾ᒀ⑓ᗂඣᝈ⪅䛾⾑Ύ୰䛻䛚䛔䛶ከ䛟Ꮡᅾ䛧䛶䛔䜛 [47] 䛣䛸 䛛䜙䚸MANF 䛸ච␿ᶵᵓ䛸䛾㛵㐃䛜♧၀䛥䜜䛶䛔䜛䚹䛧䛛䛧䛺䛜䜙䚸䛭䛾ヲ⣽䛺ศᏊ

䛣䜜䜙䛾䛣䛸䛛䜙䚸MANF 䛾⩻ヂᚋ⣽⬊ෆ㍺㏦䜔ศἪ㐣⛬䛷䛾ㄪ⠇ᶵᵓ䜢⌮ゎ 䛧䚸⏕⌮ᶵ⬟䜢ゎ᫂䛩䜛䛣䛸䛿䚸䜰䝹䝒䝝䜲䝬䞊⑓䚸䝟䞊䜻䞁䝋䞁⑓䛺䛹䛾⚄⤒ኚᛶ ⑌ᝈ䚸୪䜃䛻䚸ᮎᲈ䛾⢾ᒀ⑓䜢䛿䛨䜑䛸䛧䛯ᑠ⬊య䝇䝖䝺䝇㛵㐃⑌ᝈ䜔ච␿⑌ᝈ䛺䛹 䛾἞⒪ἲ䛾☜❧䛻䛸䛳䛶኱䛝䛺ព࿡䜢䜒䛴䛸⪃䛘䜙䜜䜛䚹

1-4. Cerebral dopamine neurotrophic factor (CDNF)

⬨᳝ື≀䛷䛿䚸MANF 䝟䝷䝻䜾 䠄paralog䠅 䛸䛧䛶 cerebral dopamine

neurotrophic factor (CDNF) 䛜Ꮡᅾ䛩䜛 [35, 48]䚹CDNF 䛿 MANF 䛸ྠᵝ䛻䚸㐍

໬ⓗ䛻ಖᏑ䛥䜜䛯ศἪ䝍䞁䝟䜽㉁䛷䛒䜚䚸6-OHDA ㄏᑟᛶ䝟䞊䜻䞁䝋䞁⑓䝰䝕䝹䝷䝑 䝖䛻ᑐ䛩䜛⚄⤒⣽⬊ಖㆤຠᯝ䜢࿊䛩䜛䝍䞁䝟䜽㉁䛸䛧䛶ྠᐃ䛥䜜䛯 [48]䚹䛭䛾ᚋ䚸 AAV2 䜴䜲䝹䝇䝧䜽䝍䞊䛻䜘䜛 CDNF 㐣๫Ⓨ⌧䜔CDNF 䛾⬻ෆᢞ୚䛿䚸6-OHDA ㄏᑟᛶ䝟䞊䜻䞁䝋䞁⑓䝰䝕䝹䝷䝑䝖䛻䛚䛔䛶䚸୰⬻䝗䞊䝟䝭䞁సືᛶ⚄⤒⣽⬊䛻ᑐ䛩 䜛⚄⤒⣽⬊ಖㆤຠᯝ䜢♧䛩䛣䛸䛜ሗ࿌䛥䜜䛯 [48-52]䚹୍⯡ⓗ䛻䚸䝟䝷䝻䜾䛾䝍䞁䝟 䜽㉁㛫䛷䛿␗䛺䜛ᶵ⬟䜢᭷䛧䛶䛔䜛ሙྜ䛜ከ䛔䛯䜑䚸MANF 䛸 CDNF 䛾ᶵ⬟䜒஫ 䛔䛻␗䛺䜛ྍ⬟ᛶ䛜䛒䜛䚹䛧䛛䛧䚸⌧ᅾ䛾䛸䛣䜝䚸୧⪅ඹ㏻䛾ᶵ⬟䛸䛧䛶䝗䞊䝟䝭䞁⚄ ⤒䛻ᑐ䛩䜛⣽⬊ಖㆤຠᯝ䛜ሗ࿌䛥䜜䛶䛔䜛䛾䜏䛷䛒䜛䚹䜎䛯䚸୧⪅䛾䜰䝭䝜㓟㓄ิ䜒 ⣙ 59 % ୍⮴䛧䛶䛚䜚䚸㧗䛔┦ྠᛶ䜢♧䛧䛶䛔䜛 (ᅗ 4)䚹䜎䛯䚸䝅䝵䜴䝆䝵䜴䝞䜶䜢 ⏝䛔䛯ᵓ㐀ୖ䛾ẚ㍑᳨ウ䛷䛿䚸᫂☜䛺┦㐪Ⅼ䛿᫂䜙䛛䛻䛺䛳䛶䛔䛺䛔 [53]䚹୧⪅䛾 ┦㐪Ⅼ䛸䛧䛶䛿䚸CDNF 䛿䚸ᑠ⬊య䝇䝖䝺䝇䛾᭷↓䛻䛛䛛䜟䜙䛪䚸ᜏᖖⓗ䛻Ⓨ⌧䛧䛶 䛔䜛䛣䛸䛜♧䛥䜜䛶䛔䜛 [39]䚹䛥䜙䛻䚸6-OHDA ㄏᑟᛶ䝟䞊䜻䞁䝋䞁⑓䝰䝕䝹䝷䝑䝖

䛻ᑐ䛧䛶䚸CDNF䚸glial cell line-derived neurotrophic factor (GDNF)䚸MANF 䜢㛗

ᮇⓗᢞ୚䛧䛯◊✲䛷䛿䚸CDNF 䛾䜏䛜㯮㉁⦓ᐦ㒊୪䜃䛻⥺᮲య䛻䛚䛔䛶䝗䞊䝟䝭䞁 సືᛶ⚄⤒⣽⬊䛾⬺ⴠ䜢ᢚไ䛧䛯䛣䛸䛜ሗ࿌䛥䜜䛶䛔䜛 [52]䚹䛧䛛䛧䛺䛜䜙䚸䛭䛾᫂ ☜䛺స⏝ᶵᗎ䛿ゎ᫂䛥䜜䛶䛔䛺䛔䚹䛣䛾䜘䛖䛻䚸୧⪅䜢┤᥋ⓗ䛻ẚ㍑᳨ウ䛧䛯ሗ࿌ 䛿ከ䛟䛺䛔䛣䛸䛛䜙䚸MANF ཬ䜃 CDNF 䛾ᛶ≧ゎᯒ䜢㏻䛧䛶䚸୧⪅䜢┤᥋ẚ㍑䛧䚸 ┦ఝⅬ䚸┦㐪Ⅼ䜢᫂☜䛻䛩䜛䛣䛸䛿䚸ྛ䚻䛾ᶵ⬟ゎᯒ䜢኱䛝䛟㐍Ṍ䛥䛫ᚓ䜛䛸⪃䛘䜙 䜜䜛䚹

ᅗ

ᅗ 4. 䝠䝖䚸䝬䜴䝇䛾 MANF ཬ䜃 CDNF 䛾䜰䝭䝜㓟㓄ิ䛿┦ྠᛶ䛜㧗䛔䚹

* 䛿 MANF ཬ䜃 CDNF 䛷୍⮴䛧䛶䛔䜛䜰䝭䝜㓟䜢♧䛩䚹C ᮎ➃䛾⥳䛷♧䛧䛯䜰䝭䝜㓟

䛿㠀ᴟᛶഃ㙐䜰䝭䝜㓟䛷䛒䜛䚹䜎䛯䚸㯤Ⰽ䚸㉥Ⰽ䚸㟷Ⰽ䛿䛭䜜䛮䜜䚸ᴟᛶ↓㟁Ⲵഃ㙐䜰䝭䝜㓟䚸 ᴟᛶṇ㟁Ⲵ䜰䝭䝜㓟䚸ᴟᛶ㈇㟁Ⲵ䜰䝭䝜㓟䜢♧䛩䚹N ᮎ➃䛾䝅䜾䝘䝹䝨䝥䝏䝗㓄ิ䛿ୗ⥺䛷 ♧䛧䚸C ᮎ➃ KDEL ᵝ㓄ิ䛿ኴᏐ䛷♧䛧䛯 [41]䚹MANF ཬ䜃 CDNF 䛾㓄ิ䛿 NCBI 䝕䞊䝍䝧䞊䝇䜘䜚ྲྀᚓ䛧䛯 (䝠䝖 MANF: NP_006001.4䚸䝬䜴䝇 MANF: NP_083379.2䚸䝠䝖 CDNF: NP_001025125.2䚸䝬䜴䝇 CDNF: NP_808315.1)䚹

1-5. ᮏᮏ◊✲䛾┠ⓗ ER 䝇䝖䝺䝇䛻䜘䛳䛶άᛶ໬䛥䜜䜛 UPR 䛿ᗈ⠊䛺⣽⬊ෆᛂ⟅䛻㛵䜟䛳䛶䛚䜚䚸䛭 䛾◚⥢䜔㐣๫䛺άᛶ໬䛿ᵝ䚻䛺⑌ᝈ䛻⧅䛜䜛䚹䜘䛳䛶䚸UPR 䛻㛵䜟䜛㑇ఏᏊ䛾⏕ ⌮ᶵᵓ䜢▱䜛䛣䛸䛿䚸ከᵝ䛺⑌ᝈ䛾ཎᅉゎ᫂䜔἞⒪䛻䛚䛔䛶㔜せ䛷䛒䜛䚹MANF 䛿 ⚄⤒䞉㠀⚄⤒⣽⬊䛻ᑐ䛩䜛⣽⬊ಖㆤຠᯝ䜢♧䛧䛶䛔䜛䛜䚸䛭䛾ヲ⣽䛺ศᏊ䝯䜹䝙䝈 䝮䛿᫂䜙䛛䛻䛥䜜䛶䛔䛺䛔䚹 MANF 䛿 C ᮎ➃䛻 KDEL ᵝ㓄ิ䜢᭷䛧䛶䛚䜚䚸⣽⬊እ䜈䛸ศἪ䛥䜜䜛୍᪉䛷䚸 ୍㒊䛿ᑠ⬊య䛻ᒁᅾ䛧䛶䛔䜛䚹䜎䛯䚸MANF 䛿䚸GRP78 䛸䛾ඹⓎ⌧䛻䜘䜚ⴭ䛧䛟ศ Ἢ䛜ᢚไ䛥䜜䚸⣽⬊ෆ䛻⵳✚䛩䜛䛣䛸䛜᫂䜙䛛䛻䛺䛳䛶䛔䜛 [40]䚹䜘䛳䛶䚸MANF 䛾 ⏕⌮ᶵ⬟䛾ゎ᫂䛻䛿ศἪไᚚᶵᵓ䛻ᑐ䛩䜛▱ぢ䛜ᚲせ䛷䛒䜛䛸⪃䛘䜙䜜䜛䚹䛧䛛䛧 䛺䛜䜙䚸᪤Ꮡ䛾᪉ἲ䛷䛿䚸ศἪᆺ MANF 䛾᳨ฟឤᗘཬ䜃ᐃ㔞ᛶ䛻ၥ㢟䛜䛒䛳䛯䚹 䛥䜙䛻䚸ෆᅉᛶ MANF 䛾Ⓨ⌧ไᚚཬ䜃⩻ヂᚋไᚚ䛻䛿ᵝ䚻䛺ᅉᏊ䛜㛵୚䛧䛶䛔䜛 䛣䛸䛜ண 䛥䜜䛯䛯䜑䚸ᮏ◊✲䛷䛿䚸MANF ᙉไⓎ⌧䝰䝕䝹䛻䜘䜛ศἪไᚚᶵᵓ䛾 ᳨ウ䜢ヨ䜏䛯䚹䛭䛾Ⅽ䛻䚸పศᏊ㔞䝹䝅䝣䜵䝷䞊䝊 NanoLuc (NL) [54] 䜢⏝䛔䛶䚸 MANF 䛾ศἪ䜢㧗ឤᗘୟ䛴ᐃ㔞ⓗ䛻᳨ฟ䛩䜛᪉ἲ䜢ᵓ⠏䛧䛯 (➨஧❶)䚹 ḟ䛔䛷䚸ྠ䝅䝇䝔䝮䜢 CDNF 䛻䜒ᛂ⏝䛧䛯䚹MANF 䛾ศἪไᚚ䛻㛵䛧䛶䛿䚸ᙜ ◊✲ᐊ䜢ྵ䜑䚸㐣ཤ䛻ሗ࿌ [40, 55, 56] 䛜䛒䛳䛯䛜䚸CDNF 䛾ศἪไᚚ䛻㛵䛩䜛 䜒䛾䛿䛺䛛䛳䛯䚹䛭䛾䛯䜑䚸ᮏ◊✲䛷䛿䚸MANF ศἪไᚚ䛻㛵䛩䜛䛣䜜䜎䛷䛾⤖ᯝ 䜢ᇶ䛻䛧䛶䚸CDNF 䛾ศἪไᚚ䛻㛵䛩䜛᳨ウ䜢⾜䛖䛸ඹ䛻䚸MANF 䛸 CDNF 䜢┤ ᥋ẚ㍑䛧䚸୧⪅䛾┦㐪Ⅼ䞉┦ఝⅬ䛻ᑐ䛩䜛᳨ウ䜢⾜䛳䛯 (➨୕❶)䚹 䛥䜙䛻䚸ᙜ◊✲ᐊ䛷⾜䜟䜜䛯䚸ᑠ⬊య䝇䝖䝺䝇ᛂ⟅ᛶᅉᏊ cysteine-rich with

EGF-like domains 2 (CRELD2) 䛾ྠᐃ䛸ศἪไᚚᶵᵓ䛻㛵䛩䜛◊✲䛾୰䛷䚸 MANF 䛜 CRELD2 䛾ศἪ䜢ಁ㐍䛥䛫䜛䛣䛸䜢ぢฟ䛧䛯 [57- 59]䚹䛭䛣䛷䚸ᮏ◊✲

䛷䛿䚸CRELD2 䛾ศἪ䛻ᑐ䛩䜛 MANF 䛸 CDNF 䛾ຠᯝ䜢ẚ㍑᳨ウ䛧䛯 (➨ᅄ

➨ ➨஧❶ MANF ศἪ䛻ᑐ䛩䜛㧗ឤᗘᐃ㔞ἲ䛾ᵓ⠏ 2-1. せ⣙ MANF 䛿⚄⤒⣔䞉㠀⚄⤒⣔⣽⬊䛻ᑐ䛩䜛⣽⬊ಖㆤຠᯝ䛜ከᩘሗ࿌䛥䜜䛶䛔䜛 [35, 36, 38, 39, 46]䚹䛧䛛䛧䚸䛭䛾ヲ⣽䛺ศᏊᶵᵓ䛿ᮍ䛰᫂䜙䛛䛻䛺䛳䛶䛔䛺䛔䚹 MANF 䛿ᑠ⬊యศᏊ䝅䝱䝨䝻䞁 GRP78 䛸䛾ඹⓎ⌧䛻䜘䜚䚸ศἪ䛜ⴭ䛧䛟ᢚไ䛥䜜䚸 ⣽⬊ෆ䛻⵳✚䛩䜛䛣䛸䛜ሗ࿌䛥䜜䛶䛔䜛䛣䛸䛛䜙 [40]䚸MANF 䛾⏕⌮ᶵ⬟䛾ゎ᫂䛻 䛿䚸ศἪ䛺䛹䛾⩻ヂᚋไᚚᶵᵓ䛻ᑐ䛩䜛▱ぢ䛜኱䛝䛟ᙺ❧䛴䛣䛸䛜⪃䛘䜙䜜䜛䜒䛾 䛾䚸ᚑ᮶䛾䜴䜶䝇䝍䞁䝤䝻䝑䝖ἲ䛷䛿䚸ศἪ䛥䜜䛯 MANF 䜢᳨ฟ䛩䜛㝿䛾ឤᗘཬ䜃 ᐃ㔞ᛶ䛻ၥ㢟䛜䛒䛳䛯䚹䛭䛣䛷䚸ᮏ◊✲䛷䛿䚸㧗ឤᗘ䞉పศᏊ㔞䝹䝅䝣䜵䝷䞊䝊 NanoLuc (NL) 䜢⏝䛔䛶䚸MANF 䛾ศἪ䜢㧗ឤᗘ䞉ᐃ㔞ⓗ䛻ゎᯒ䛩䜛᪂つᐃ㔞ἲ (NanoLuc Luciferase Assay: NL assay) 䜢ᵓ⠏䛧䚸MANF 䛾ศἪไᚚ䛻㛵䛩䜛᳨

ウ䜢⾜䛳䛯䚹ඛ䛪䚸MANF 䛾䝅䜾䝘䝹䝨䝥䝏䝗䛾ୗὶ䛻 NanoLuc 䜢ᤄධ䛧䛯⼥ྜ

ᆺ MANF (SP-NL-MANF) 䜢స〇䛧䚸HEK293 ⣽⬊䛻䛚䛔䛶䚸⤒᫬ⓗ䛺Ⓨ⌧ཬ䜃

ศἪ㔞䛾ኚ໬䜢㔝⏕ᆺ (wt) MANF 䛸ẚ㍑䛧䛯䚹ḟ䛔䛷䚸㐣ཤ䛾▱ぢ䛻ᚑ䛔䚸ኚ␗ ᆺ Sar1(H79G) ཬ䜃 GRP78 䛸䛾ඹⓎ⌧䛻䜘䜛ศἪ㔞ኚ໬䜢 NL assay 䛻䛶ゎ ᯒ䛧䛯䚹䛭䛾⤖ᯝ䚸SP-NL-MANF 䛿 wt MANF 䛸ྠᵝ䛾ศἪไᚚ䜢ཷ䛡䜛䛣䛸䜢 ♧䛩䛸䛸䜒䛻䚸NL assay 䛿ᚑ᮶䛾䜴䜶䝇䝍䞁䝤䝻䝑䝖ἲ (WB) 䛸ẚ㍑䛧䛶䚸㧗ឤᗘୟ 䛴ᐃ㔞ⓗ䛻 MANF 䛾ศἪ䜢᳨ฟ䛷䛝䜛䛣䛸䛜ศ䛛䛳䛯䚹᭱ᚋ䛻䚸ྠἲ䜢⏝䛔䛶䚸䝷 䝑䝖䜲䞁䝇䝸䝜䞊䝬 INS-1 ⣽⬊䛻䛚䛡䜛 MANF 䛾Ⓨ⌧䞉ศἪไᚚ䛻ᑐ䛩䜛᳨ウ䜢䚸 NL assay ཬ䜃 RT-PCR ἲ䜢⏝䛔䛶⾜䛳䛯䚹ᑠ⬊య䝇䝖䝺䝇ㄏᑟ๣䝍䝥䝅䜺䝹䜼䞁 (Tg) ฎ⌮䛻䜘䜚䚸MANF mRNA Ⓨ⌧㔞䚸ཬ䜃䚸SP-NL-MANF ศἪ㔞䛿ቑຍ䛧䛯䚹

୍᪉䛷䚸ሷ໬䜹䝸䜴䝮 (KCl) ฎ⌮䛷䛿䚸MANF mRNA Ⓨ⌧㔞䛿ኚ໬䛧䛺䛔䜒䛾䛾䚸

SP-NL-MANF ศἪ㔞䛿ቑຍ䛧䛯䚹䛥䜙䛻䚸ᑠ⬊య䝇䝖䝺䝇ㄏᑟ๣䝒䝙䜹䝬䜲䝅䞁 (Tm) ฎ⌮䛷䛿䚸MANF mRNA Ⓨ⌧㔞䛿ቑຍ䛧䛯䜒䛾䛾䚸SP-NL-MANF ศἪ㔞䛻

ኚ໬䛿䛺䛛䛳䛯䚹䛣䜜䜙䛾䛣䛸䛛䜙䚸MANF 䛾ศἪไᚚᶵᵓ䛿ᑠ⬊య䝇䝖䝺䝇䛸䛿⊂

2-2. ᮦᮦᩱ䛸᪉ἲ

2-2-1. ヨ⸆

Tg䚸Tm 䛿 Sigma-Aldrich ♫䛛䜙㉎ධ䛧䛯䚹MANF ᢠయ䛿䚸Abcam ♫ཬ䜃䚸 R&D systems ♫䛛䜙㉎ධ䛧䛯䚹

2-2-2. 䝁䞁䝇䝖䝷䜽䝖䛾స〇

wt MANF 䛿 Neuro2A (N2a) ⣽⬊䛾 cDNA 䛛䜙 PCR ἲ䛻䜘䜚 ቑᖜ䛧䚸 pcDNA3.1 䝧䜽䝍䞊䜈䛸⤌䜏㎸䜣䛰䚹SP-NL-MANF 䛿䚸wt MANF 䛾䝅䜾䝘䝹䝨䝥

䝏䝗㓄ิ䛾ୗὶ䛻 NanoLuc 䜢ᤄධ䛧䚸㑇ఏᏊ᩿∦䜢 PCR ἲ䛻䛶ቑᖜ䛧䛯ᚋ䚸

pcDNA3.1 䝧䜽䝍䞊䜈䛸䝃䝤䜽䝻䞊䝙䞁䜾䛧䛯䚹wt GRP78 䛿䚸DNAFORM (RIKEN, Japan) 䛛䜙ᚓ䛯䚹C ᮎ➃ KDEL 㓄ิḞᦆᆺ ǻ& *53 䛿 wt GRP78 䜢㗪

ᆺ䛸䛧䛶䚸PCR ἲ䛻䜘䜚㑇ఏᏊ᩿∦䜢ቑᖜ䛧䚸pcDNA3.1 䝧䜽䝍䞊䜈ᤄධ䛩䜛䛣䛸䛷

స〇䛧䛯䚹Sar1 (H79G) 䛿 Liu ཬ䜃 Lippincott-Schwartz 䛛䜙ㆡΏ䛥䜜䛯䜒䛾䜢

౑⏝䛧䛯 [60]䚹KDEL ཷᐜయ 1 䛿 Ruddock 䛻䜘䜚ㆡ䜚ཷ䛡䛯 [41]䚹

2-2-3. ⣽⬊ᇵ㣴䛸⸆๣่⃭

HEK293 ⣽ ⬊ 䚸 N2a ⣽ ⬊ 䛿 䚸 8% fetal bovine serum (FBS) 䛸 1% penicillin/streptomycin (P/S) 䜢 ྵ 䜐 Dulbecco’s modified Eagle’s minimum

essential medium (D-MEM) (High Glucose, WAKO) 䜢⏝䛔䛶䚸37°C䚸5% CO2

incubator ෆ䛷ᇵ㣴䛧䛯䚹䝷䝑䝖䜲䞁䝇䝸䝜ʊ䝬 INS-1 ⣽⬊䛿䚸8% 㠀ື໬ FBS 䜢

ྵ䜐 RPMI-1640 (WAKO) 䜢⏝䛔䛶ᇵ㣴䛧䛯 [61]䚹WB 䛻⏝䛔䜛㝿䛻䛿䚸6-well

ⱝ䛧䛟䛿 12-well plate 䛻䚸NL assay 䛻⏝䛔䜛㝿䛻䛿䚸48-well ⱝ䛧䛟䛿 96-well

plate 䛻 ⣽ ⬊ 䜢 䜎 䛔 䛯 䚹 㑇 ఏ Ꮚ ᑟ ධ 䛿 䚸 Lipofectamine-PLUS Reagent (Life Technologies) 䜢⏝䛔䛶䚸ῧ௜䛾౑⏝᪉ἲ䛻ᚑ䛔䛚䛣䛺䛳䛯䚹䝷䝑䝖䜲䞁䝇䝸䝜䞊䝬 INS-1 ⣽⬊䛻䛚䛡䜛 MANF ᜏᖖⓗⓎ⌧ᰴ䛿䚸MANF 㑇ఏᏊ䜢ᑟධᚋ䚸㐺ษ䛺⃰

ᗘ䛾 G418 䛻䜘䜚䝉䝺䜽䝅䝵䞁䛩䜛䛣䛸䛻䜘䜚ᶞ❧䛧䛯䚹ᮏ◊✲䛷䛿௨ୗ䛾᮲௳䛷่

⃭ 䜢 ⾜ 䛳 䛯: ↓ ⾑ Ύ ᇵ ᆅ (serum free, SF) 䚸 ⾑ Ύ 䞉 䜾 䝹 䝁 䞊 䝇 ↓ ῧ ຍ ᇵ ᆅ

(serum-glucose free䚸SGF)䚸ሷ໬䜹䝸䜴䝮 (KCl 50 mM)䚸7J  ȝ0䚸Tm (1 ȝJP/䚹

2-2-4. NanoLuc luciferase assay (NL assay)

⣽⬊䜢 1× passive lysis buffer (Promega) 䛻䜘䛳䛶⁐ゎ䛧䚸ᅇ཰䛧䛯ヨᩱ䜢 15

min (15000 rpm䚸4°C) 㐲ᚰ䛧䛯ᚋ䚸NanoLuc assay system (Promega) 䛻䜘䜚 NanoLuc άᛶ䜢 ᐃ䛧䛯䚹ᇵ㣴ୖΎ䛿㐲ᚰฎ⌮䜢⾜䜟䛪䛻䚸┤᥋ ᐃ䛻⏝䛔䛯䚹  ᐃ್䜢䚸௨ୗ䛾ィ⟬ᘧ䛻ᇶ䛵䛝ィ⟬䛧䚸⣽⬊ෆእ䛾䝹䝅䝣䜵䝷䞊䝊άᛶ䛸䛧䛯䚹

y = x ×ܽ ܾ (y: 䝹䝅䝣䜵䝷䞊䝊άᛶ䚸x:  ᐃ್䚸a: ᅇ཰䛧䛯ᾮ㔞䚸b:  ᐃ䛻౑⏝䛧䛯ᾮ㔞) 䛥䜙䛻䚸⣽⬊ෆእ䛾䝹䝅䝣䜵䝷䞊䝊άᛶ䛾ྜィ್䛻ᑐ䛩䜛⣽⬊እ䛾䝹䝅䝣䜵䝷䞊䝊 άᛶ䛾๭ྜ䜢 % release 䛸䛧䛯䚹ᐇ㦂䛿 triplicate 䛷⾜䛔䚸䜾䝷䝣ୖ䛾್䛿άᛶ್ ±ᶆ‽೫ᕪ䛷♧䛧䛯䚹

2-2-5. Reverse transcription–polymerase chain reaction (RT-PCR)

Total RNA 䜢ᅇ཰䛩䜛䛯䜑䛻䚸TRIzol (Life Technologies) 䛻䛶⣽⬊䜢⁐ゎ䛥䛫

䛯䚹䛭䛾ᚋ䚸⣽⬊⁐ゎᾮ䛻䜽䝻䝻䝩䝹䝮䜢ຍ䛘䚸ịୖ䛻䛶 10 ศ㛫㟼⨨䛧䛯䚹10 ศ

㛫㐲ᚰ (15,000 rpm䚸4°C) 䛧䚸Ỉᒙ䜢ᅇ཰䛧䛯䚹ḟ䛔䛷䚸᰾㓟䜢䜲䝋䝥䝻䝟䝜䞊䝹ỿ

Ẋ䛥䛫䛯ᚋ䚸70% 䜶䝍䝜䞊䝹䛻䛶ỿẊ䜢Ὑί䛧䚸ỿẊ≀䜢 total RNA 䛸䛧䛶౑⏝䛧

䛯䚹TRWDO51$ȝJȝ/ 䛿䚸72°C 䛷 10 ศಖ 䛧䛯ᚋ䚸RT ཯ᛂ⁐ᾮ (10 mM

DTT䚸0.5 mM dNTP䚸 QJȝ/ UDQGRP QLQHPHUV䚸 8ȝ/ 51DVH287 /LIH Technologies)䚸8ȝ/SULPHVXSHUVFULSW,,,51DVHUHYHUVHWUDQVFULSWDVH/LIH Technologies)) 䛸ΰྜ䛧䚸42°C 䛷 60 ศಖ 䛩䜛䛣䛸䛷 cDNA 䜢ᚓ䛯䚹┠ⓗ䛸䛩

䜛㑇ఏᏊ䛿䚸PCR ཯ᛂᾮ  ȝ0 䝥䝷䜲䝬䞊䚸0.1 mM dNTP䚸  P8ȝ/ U7DT

(Taq PCR kit䚸Takara)) 䛻䜘䜚ቑᖜ䛧䛯䚹ᮏ◊✲䛷⏝䛔䛯䝥䝷䜲䝬䞊䝨䜰䛿

MANF sense primer, 5’-GTTTGTATTTCTTATCTGGG-3’ MANF antisense primer, 5’-TCTTCTTCAGCTTCTCACAG-3’ XBP1 sense primer, 5’-ACGCTTGGGAATGGACACG-3’

XBP1 antisense primer, 5’-ACTTGTCCAGAATGCCCAAAAG-3’ GADD153 sense primer, 5’-GAATAACAGCCGGAACCTGA-3’ GADD153 antisense primer, 5’-GGACGCAGGGTCAAGAGTAG-3’ GRP78 sense primer, 5’-ACCAATGACCAAAACCGCCT-3’

GRP78 antisense primer, 5’-GAGTTTGCTGATAATTGGCTGAAC-3’ GAPDH sense primer, 5’-ACCACAGTCCATGCCATCAC-3’

GAPDH antisense primer, 5’-TCCACCACCCTGTTGCTGTA-3’

䛷䛒䜛䚹඾ᆺⓗ䛺཯ᛂ᮲௳䛿 96°C 䛷 30 ⛊䚸58°C 䛷 30 ⛊䚸72°C 䛷 30 ⛊䛷

⾜䛳䛯䚹⤖ᯝ䛻䛿ྛ㑇ఏᏊ䛻㐺ษ䛺䝃䜲䜽䝹 (20-28 䝃䜲䜽䝹) 䛷ቑᖜ䛧䛯䜒䛾䜢♧

䛧䛯䚹PCR ⏘≀䛿 2.0% 䛾䜰䜺䝻䞊䝇䝀䝹䜢⏝䛔䛶㟁ẼὋື䛻䜘䛳䛶ศ㞳䛧䚸䜶䝏

䝆䜴䝮䝤䝻䝬䜲䝗䛷ྍど໬䛧䛯䚹

2-2-6. 䜴䜴䜶䝇䝍䞁䝤䝻䝑䝖ἲ (WB)

1ȝJPOOHXSHSWLQ䚸ȝJPOSHSVWDWLQ$@ 䛻䜘䜚⁐ゎ䛧䚸Bradford ἲ (BIO-RAD)

䜢⏝䛔䛶䝍䞁䝟䜽㉁⃰ᗘ䜢 ᐃ䛧䛯䚹䛭䛾ᚋ䚸➼㔞䛾 2× sodium dodecyl sulfate

(SDS)-Laemmli sample buffer [62.5 mM Tris–HCl (pH 6.8)䚸2% SDS䚸10% glycerol] 䜢ຍ䛘䚸100°C 䛷 5 ศ㛫ຍ⇕䛧䛯䚹ศἪ䛥䜜䛯䝍䞁䝟䜽㉁䜢᳨ฟ䛩䜛䛻

䛿䚸ᇵ㣴ᾮ䜢୍㒊ᅇ཰䛧䚸5 ศ㛫㐲ᚰ (3200 rpm, 4°C) 䛩䜛䛣䛸䛻䜘䜚䚸ᾋ㐟䛧䛯⣽

⬊䜢㝖ཤ䛧䛯䚹䛭䛾ᚋ䚸¼ 㔞䛾 4× SDS-Laemmli sample buffer 䛸ᠱ⃮䛧䛯䚹➼㔞

䛾⣽⬊䝩䝰䝆䝛䞊䝖ⱝ䛧䛟䛿䚸ᇵ㣴ᾮ䜢䚸12.5-15% SDS polyacrylamide gel 䛻䜘䜚

ศ㞳䛧䚸polyvinylidene difluoride membrane (PVDF ⭷) (GE Healthcare) 䛻㌿෗

䛧䛯䚹┠ⓗ䝍䞁䝟䜽㉁ 䛿䚸ᢠ MANF ᢠయ (abcam, U.K. and R&D Systems,

U.S.A.) 䜢౑⏝䛧䚸enhanced chemiluminescence (GE Healthcare) 䛻䛶᳨ฟ䛧 䛯䚹

2-2-7. ⤫⤫ィฎ⌮

ᚓ䜙䜜䛯⤖ᯝ䛿ᖹᆒ್±ᶆ‽೫ᕪ䛷⾲♧䛧䛯䚹⤫ィฎ⌮䛿䚸student’s t-test 䚸ⱝ

䛧䛟䛿䚸one-way ANOVA 䛾ᚋ䛻 Tukey’s test 䜢⾜䛔ゎᯒ䛧䛯䚹p<0.05 䜢⤫ィⓗ

2-3. ⤖⤖ᯝ 2-3-1. పศᏊ㔞䝹䝅䝣䜵䝷䞊䝊 NanoLuc 䜢⏝䛔䛯 MANF ศἪ䛻ᑐ䛩䜛 㧗ឤᗘᐃ㔞ἲ䛾ᵓ⠏ ௨๓䚸ᙜ◊✲ᐊ䛷䛿䚸HEK293 ⣽⬊䛻ᵝ䚻䛺 MANF ኚ␗య䜢㑇ఏᏊᑟධ䛧䚸 MANF ⏘⏕䛸ศἪ㔞䛻䛴䛔䛶ゎᯒ䜢䛚䛣䛺䛳䛯 [40]䚹䛧䛛䛧䚸ᇵ㣴ᾮ୰䛻ศἪ䛥䜜 䛯 MANF 䜢ゎᯒ䛩䜛ୖ䛷䚸䛔䛟䛴䛛䛾ၥ㢟䛜⪃䛘䜙䜜䛯䚹ඛ䛪䚸ศἪ䛥䜜䛯 MANF 䛜 WB 䛷䛾᳨ฟ㝈⏺䜢༑ศ䛻ୖᅇ䜛䜎䛷䛻ᇵ㣴᫬㛫䛜 12 ᫬㛫௨ୖᚲせ 䛷䛒䜛䛣䛸䚸ḟ䛻䚸ᇵ㣴⣽⬊ᩘ䛜ᑡ䛺䛔᮲௳ୗ䛷䛿 MANF 䜢᳨ฟ䛩䜛䛯䜑䛻ᇵ㣴 ᾮ䜢༑ศ䛻⃰⦰䛩䜛ᚲせ䛜䛒䜛䛣䛸䚸᭱ᚋ䛻䚸WB 䛷䛿ྠ᫬䛻ᢅ䛘䜛᳨యᩘ䛜㝈䜙 䜜䛶䛔䜛䛣䛸䛷䛒䜛䚹䛣䜜䜙䜢ඞ᭹䛧䚸ᚑ᮶䛾 WB 䜘䜚䜒ከ᳨య䜢⡆౽䚸㧗ឤᗘୟ䛴 ᐃ㔞ⓗ䛻ศἪ MANF 䜢 ᐃ䛩䜛䛯䜑䛻䚸ᮏ◊✲䛷䛿⣙ 19 kDa 䛾పศᏊ㔞䝹䝅 䝣䜵䝷䞊䝊 NanoLuc (NL) 䛻ὀ┠䛧䛯 [54]䚹NL 䛿䚸171 䜰䝭䝜㓟䛛䜙䛺䜛䝖䝀䜸䜻 䝠䜸䝗䝅䜶䝡⏤᮶䝹䝅䝣䜵䝷䞊䝊䛷䛒䜚䚸⌧ᅾ䚸㑇ఏᏊ䛾㌿෗άᛶ䜔䝍䞁䝟䜽㉁䛾⣽ ⬊ෆᒁᅾ䜢ゎᯒ䛩䜛ୖ䛷୺䛸䛧䛶⏝䛔䜙䜜䛶䛔䜛䝩䝍䝹䝹䝅䝣䜵䝷䞊䝊 (548 䜰䝭䝜

㓟) 䜔䚸enhanced green fluorescent protein (239 䜰䝭䝜㓟) 䜘䜚䜒ศᏊ㔞䛜ᑠ䛥䛔䚹

䛣䛾䛣䛸䛛䜙䚸NL ⼥ྜᆺ MANF 䛿ᚑ᮶䛾⼥ྜᆺ MANF 䛻ẚ䜉䚸䜘䜚㔝⏕ᆺ䛻㏆ 䛔ᣲື䜢♧䛩䛣䛸䛜ᮇᚅ䛥䜜䚸ୟ䛴䚸NL άᛶ䜢 ᐃ䛩䜛䛣䛸䛷䚸WB 䜘䜚䜒䚸㧗ឤᗘ 䛻⣽⬊ෆ㍺㏦䜔ศἪ䜢▷᫬㛫䛷ᐃ㔞ⓗ䛻ゎᯒ䛷䛝䚸䛥䜙䛻䚸ከ᳨య䜢୍ᗘ䛻⾜䛘䜛 䛣䛸䛜ྍ⬟䛻䛺䜛䛸⪃䛘䛯䚹 䛭䛣䛷ᮏ◊✲䛷䛿䚸MANF 䛾䝅䜾䝘䝹䝨䝥䝏䝗䛾ୗὶ䛻 NL 䜢ᤄධ䛧䛯 SP-NL-MANF 䜢స〇䛧䛯䚹MANF ศἪ䛻ᑐ䛩䜛㧗ឤᗘᐃ㔞ἲ䛾ᵓ⠏䛻䛒䛯䜚䚸㐣 ཤ䛻䛚䛣䛺䜟䜜䛯 MANF 䛻㛵䛩䜛▱ぢ䛻ᚑ䛔䚸MANF ศἪ䛻ᑐ䛩䜛᳨ウ䜢ຍ䛘 䛯䚹 2-3-2. wt MANF ཬ䜃 SP-NL-MANF ศἪ㔞䛾⤒᫬ኚ໬ ᭱ึ䛻䚸SP-NL-MANF 䛜 wt MANF 䛸ྠᵝ䛻ᇵ㣴ᾮ୰䛻ศἪ䛥䜜䜛䛛䛹䛖䛛䜢 WB ཬ䜃 NL assay 䛻䛶ゎᯒ䛧䛯䚹WB 䛻䜘䜛ゎᯒ䛾⤖ᯝ䚸SP-NL-MANF 䛿䚸⤒

᫬ⓗ䛻wt MANF 䛻㢮ఝ䛧䛯ศἪ㔞䛾ቑຍ䜢♧䛧䛯 (ᅗ 5a)䚹୍᪉䚸NL assay 䛷䛿䚸

䜘䜚ᑡ㔞䛾ヨᩱ䛷SP-NL-MANF 䛾άᛶ䜢᳨ฟ䛷䛝䛯䛣䛸䛻ຍ䛘䚸WB 䛷䛿᳨ฟ㝈

⏺䛻㏆䛛䛳䛯 4 ᫬㛫ᇵ㣴ᚋ䛾ヨᩱ䛷䜒䚸SP-NL-MANF 䛾Ꮡᅾ䜢ホ౯䛷䛝䜛༑ศ

䛺್䛜ᚓ䜙䜜䛯 (ᅗ 5b)䚹䛣䛾䛣䛸䛛䜙䚸NL assay 䛷䛿 WB 䜘䜚䜒䚸SP-NL-MANF

ᅗ

ᅗ 5. wt MANF 䛸 SP-NL-MANF 䛾⤒᫬ⓗ䛺ศἪ㔞ኚ໬

12-well plate 䛻䜎䛔䛯 HEK293 ⣽⬊䛻 wt MANF 䜎䛯䛿 SP-NL-MANF 䜢୍㐣ᛶ䛻 㑇ఏᏊᑟධ䛧䚸24 ᫬㛫ᚋ䛻ᇵᆅ䜢↓⾑Ύᇵᆅ䛻஺᥮䛧䛯䚹䛭䛾ᚋ䚸4 ᫬㛫ẖ䛻⣽⬊ཬ䜃 ᇵ㣴ᾮ䜢ᅇ཰䛧䛯䚹

(a) WB 䛻䜘䜛 wt MANF 䛸 SP-NL-MANF 䛾⤒᫬ⓗ䛺Ⓨ⌧㔞ཬ䜃ศἪ㔞䛾ゎᯒ

ୖẁ (Medium) 䛜ᇵ㣴ᾮ䛾䚸ୗẁ (Lysates) 䛜⣽⬊䝩䝰䝆䝛䞊䝖䛾ゎᯒ⤖ᯝ䛷䛒䜛䚹ᇵ 㣴ᾮཬ䜃⣽⬊䝩䝰䝆䝛䞊䝖୰䛾 SP-NL-MANF ୪䜃䛻ᇵ㣴ᾮ୰䛾 wt MANF 䛾᳨ฟ䛻䛿 ᢠ MANF ᢠయ (R&D Systems) 䜢⏝䛔䛯䚹⣽⬊䝩䝰䝆䝛䞊䝖୰䛾 wt MANF 䛾᳨ฟ䛻䛿䚸 ᢠ MANF ᢠయ (abcam) 䜢⏝䛔䛯䚹

(b) NanoLuc assay 䛻䜘䜛 wt MANF 䛸 SP-NL-MANF 䛾⤒᫬ⓗ䛺ศἪ㔞䛾ゎᯒ

(a) 䛷ᅇ཰䛧䛯ᇵ㣴ᾮ䜢⏝䛔䛶䚸NL assay 䜢⾜䛳䛯䚹ᐇ㦂᪉ἲ䛻グ㍕䛧䛯ィ⟬ᘧ䜢⏝䛔 䛶䚸⣽⬊እ䛾 NanoLuc 䝹䝅䝣䜵䝷䞊䝊άᛶ䜢ồ䜑䛯䚹

2-3-3. Sar1(H79G) 䛸䛾ඹⓎ⌧䛻䜘䜛 wt MANF ཬ䜃 SP-NL-MANF 䛾

ศἪ㔞ኚ໬

ᙜ◊✲ᐊ䛷䛿䚸MANF 䛸䚸COP-II ᑠ⬊䛾ᵓᡂ䛻ᚲ㡲䛺䝍䞁䝟䜽㉁䛷䛒䜛 Sar1

䛾ኚ␗య (Sar1(H79G)) 䛸䛾ඹⓎ⌧䛻䜘䜚䚸MANF 䛾ศἪ䛜 COP-II ᑠ⬊䜢௓䛧

COP-II ᑠ⬊䜢௓䛧䛶㍺㏦䛥䜜䛶䛔䜛䛣䛸䜢ド᫂䛩䜛䛯䜑䛻䚸ኚ␗ᆺ Sar1(H79G)

䛸ඹⓎ⌧ୗ䛻䛚䛡䜛 wt MANF 䛸 SP-NL-MANF ศἪ㔞䛾ኚ໬䜢䚸WB ཬ䜃 NL

assay 䛻䛶ゎᯒ䛧䛯䚹ᅗ 6a 䛻♧䛩䜘䛖䛻䚸wt MANF 䛷䛿㐣ཤ䛾ሗ࿌㏻䜚䚸ኚ␗ᆺ Sar1(H79G) 䛸䛾ඹⓎ⌧䛻䜘䜚䚸䛭䛾ศἪ䛜ⴭ䛧䛟ῶᑡ䛧䛶䛔䛯䚹䛥䜙䛻䚸

SP-NL-MANF 䛻䛚䛔䛶䜒䚸wt MANF 䛸ྠᵝ䛻䚸ኚ␗ᆺ Sar1(H79G) 䛸䛾ඹⓎ⌧

䛻䜘䜚䚸⼥ྜᆺMANF 䛾ศἪ䛜ⴭ䛧䛟ῶᑡ䛧䛯䚹NL assay 䛻䜘䜛ゎᯒ䛾⤖ᯝ䚸ኚ␗ ᆺ Sar1(H79G) 䛸䛾ඹⓎ⌧䛻䜘䛳䛶䚸⣽⬊እ䝹䝅䝣䜵䝷䞊䝊άᛶ䛾ⴭ䛧䛔ῶᑡ䛜ㄆ 䜑䜙䜜䛯 (ᅗ 6b)䚹௨ୖ䛾⤖ᯝ䛛䜙䚸SP-NL-MANF 䛿 wt MANF 䛸ྠᵝ䛻 COP-II ᑠ⬊䜢௓䛧䛯⤒㊰䛻䜘䜚ศἪ䛥䜜䛶䛔䜛䛣䛸䛜ศ䛛䛳䛯䚹䜎䛯䚸NL assay 䛾 ⤖ᯝ䛿 WB 䛾⤖ᯝ䜢཯ᫎ䛧䛶䛔䜛䛣䛸䛛䜙䚸⣽⬊ෆእ䛾䝹䝅䝣䜵䝷䞊䝊άᛶ䛿䚸 MANF 䛾⣽⬊ෆⓎ⌧㔞ཬ䜃ศἪ㔞䜢♧䛧䛶䛔䜛䛸⪃䛘䜙䜜䜛䚹䛧䛯䛜䛳䛶䚸䛣䜜௨

㝆䛾ᐇ㦂䛷䛿䚸MANF ⣽⬊ෆⓎ⌧㔞ཬ䜃ศἪ㔞䜢ゎᯒ䛩䜛ሙྜ䚸48-well plate 䜢

ᅗ

ᅗ 6 ኚ␗ᆺ Sar1(H79G) ඹⓎ⌧䛻䜘䜛 wt MANF 䛸 SP-NL-MANF 䛾Ⓨ⌧㔞ཬ䜃ศ

Ἢ㔞䜈䛾ᙳ㡪

12-well plate 䛻䜎䛔䛯 HEK293 ⣽⬊䛻䚸wt MANF 䜎䛯䛿 SP-NL-MANF 䛸ඹ䛻 Sar1(H79G) 䜢୍㐣ᛶ䛻㑇ఏᏊᑟධ䛧䚸24 ᫬㛫ᚋ䛻ᇵᆅ䜢↓⾑Ύᇵᆅ䛻஺᥮䛧䛯䚹䛭䛾 ᚋ䚸12 ᫬㛫ᇵ㣴䛧䚸⣽⬊ཬ䜃ᇵ㣴ᾮ䜢ᅇ཰䛧䛯䚹

(a) WB 䛻䜘䜛 wt MANF 䛸 SP-NL-MANF 䛾⣽⬊ෆⓎ⌧㔞ཬ䜃ศἪ㔞䛾ゎᯒ

ᅗ 5a 䛸ྠᵝ䛻䛧䛶䚸wt MANF䚸SP-NL-MANF 䛾᳨ฟ䜢⾜䛳䛯䚹

(b) NanoLuc luciferase assay 䛻䜘䜛 SP-NL-MANF 䛾ศἪ㔞䛾ゎᯒ

ᅗ 5b 䛸ྠᵝ䛻䛧䛶䚸 ᐃ䜢⾜䛳䛯䚹ᑦ䚸SP-NL-MANF 䛸ኚ␗ᆺ Sar1(H79G) 䜢ඹⓎ ⌧䛥䛫䛯್䜢1 䛸䛧䛶ᶆ‽໬䛧䛯䚹

2-3-4. KDEL ཷᐜయ 1 䛸䛾ඹⓎ⌧䛻䜘䜛 SP-NL-MANF 䛾ศἪ㔞ኚ໬

Henderson 䜙䛿䚸KDEL ཷᐜయ 1 䛸䛾ඹⓎ⌧䛻䜘䜚䚸MANF 䛾ศἪ䛜ῶᑡ䛩

䜛䛣䛸䜢ሗ࿌䛧䛯 [55]䚹䛭䛣䛷 SP-NL-MANF 䜢⏝䛔䛶ゎᯒ䛧䛯䛸䛣䜝䚸㐣ཤ䛾ሗ࿌

䛸୍⮴䛧䛶䚸⼥ྜᆺMANF 䛾ศἪ㔞䛿ⴭ䛧䛟ῶᑡ䛧䛯 (ᅗ 7a, b)䚹୍᪉䚸⣽⬊ෆ䛾

ᅗ

ᅗ 7. KDEL ཷᐜయ 1 䛻䜘䜛 SP-NL-MANF 䛾Ⓨ⌧㔞ཬ䜃ศἪ㔞䜈䛾ᙳ㡪

SP-NL-MANF 䛸ඹ䛻 mock 䜎䛯䛿 KDEL ཷᐜయ 1 (KDEL R1) 䛸 SP-NL-MANF 䜢 HEK293 ⣽⬊䛻ඹⓎ⌧䛥䛫䛯䚹 (a) SP-NL-MANF 䛾⥲Ⓨ⌧㔞䛻ᑐ䛩䜛ศἪẚ⋡䛾ゎᯒ ᐇ㦂᪉ἲ䛻グ㍕䛧䛯ィ⟬ᘧ䜢⏝䛔䛶䚸⣽⬊እ䛾 NanoLuc 䝹䝅䝣䜵䝷䞊䝊άᛶ䜢ồ䜑䚸 SP-NL-MANF 䛾⣽⬊እศἪ㔞䛸䛧䛯䚹 (b) ⣽⬊እ NanoLuc 䝹䝅䝣䜵䝷䞊䝊άᛶ ᐇ㦂᪉ἲ䛻グ㍕䛧䛯ィ⟬ᘧ䜢⏝䛔䛶䚸⣽⬊ෆ NanoLuc 䝹䝅䝣䜵䝷䞊䝊άᛶ䜢ồ䜑䚸 SP-NL-MANF 䛾⣽⬊ෆⓎ⌧㔞䛸䛧䛯䚹 (c) ⣽⬊ෆ NanoLuc 䝹䝅䝣䜵䝷䞊䝊άᛶ (b) 䛸 (c) 䜢 ᐃ䛧䛯ᚋ䚸ᐇ㦂᪉ἲ䛻グ㍕䛧䛯ィ⟬ᘧ䜢⏝䛔䛶䚸⥲Ⓨ⌧㔞䛻ᑐ䛩䜛ศἪ㔞 䛾๭ྜ (% release) 䜢⟬ฟ䛧䛯䚹(**; p< 0.01)䚹 2-3-5. wt GRP78䚸ǻ&*53 䛸䛾ඹⓎ⌧䛻䜘䜛 SP-NL-MANF 䛾ศἪ㔞ኚ໬ ᙜ◊✲ᐊ䛷䛿䚸ᑠ⬊యศᏊ䝅䝱䝨䝻䞁 GRP78 䛸䛾ඹⓎ⌧䛻䜘䜚䚸MANF 䛾ศ Ἢ䛜ⴭ䛧䛟ῶᑡ䛩䜛䛣䛸䜢ሗ࿌䛧䛯 [40]䚹䛭䛣䛷ᮏ䝅䝇䝔䝮䜢⏝䛔䛶䚸GRP78 䛸䛾ඹ Ⓨ⌧䛻䜘䜛 SP-NL-MANF 䛾ศἪ㔞ኚ໬䜢ゎᯒ䛧䛯䛸䛣䜝䚸㐣ཤ䛾ሗ࿌䛸୍⮴䛧䛶䚸 wt GRP78 䛸䛾ඹⓎ⌧䛻䜘䜚䚸SP-NL-MANF 䛾ศἪ䛿᭷ព䛻ῶᑡ䛧䛯 (ᅗ 8a)䚹 䜎䛯䚸wt GRP78 䛻䜘䜛 SP-NL-MANF 䛾ศἪῶᑡ䛻క䛔䚸⣽⬊ෆ䛾䝹䝅䝣䜵䝷䞊䝊 άᛶ䛜ቑຍ䛩䜛䛸ண᝿䛥䜜䛯䛜䚸䛣䜜䛻཯䛧䛶⣽⬊ෆ䝹䝅䝣䜵䝷䞊䝊άᛶ䛻䛿ኚ໬ 䛿ぢ䜙䜜䛺䛛䛳䛯 (ᅗ 8c)䚹୍᪉䚸KDEL ᵝ㓄ิ䜢Ḟᦆ䛥䛫䛯 *53ǻ& GRP78) 䛸䛾ඹⓎ⌧䛷䛿䚸wt GRP78 䛷ぢ䜙䜜䛯ศἪᢚไ䛿ῶᙅ䛧䛯 (ᅗ 8a, b)䚹 䜎䛯䚸⣽⬊ෆ SP-NL-MANF 䛿ቑຍഴྥ䛜ぢ䜙䜜䛯䜒䛾䛾䚸᭷ព䛺ኚ໬䛿ㄆ䜑䜙 䜜䛺䛛䛳䛯䚹 ௨ୖ䛾䛣䛸䛛䜙䚸SP-NL-MANF 䛾ᣲື䛿䚸䛣䜜䜎䛷䛻ሗ࿌䛥䜜䛯 wt MANF ศ Ἢ䛻䛴䛔䛶䛾▱ぢ䛸୍⮴䛧䛶䛔䜛䛣䛸䛜♧䛥䜜䛯 [40, 55]䚹䜎䛯䚸SP-NL-MANF 䜢 ⏝䛔䜛䛣䛸䛻䜘䜚䚸MANF ศἪ䜢⡆౽䚸㧗ឤᗘୟ䛴䚸ᐃ㔞ⓗ䛻ゎᯒ䛩䜛䛣䛸䜢ྍ⬟䛻 䛧䛯䚹

ᅗ

ᅗ 8. wt GRP78 䜎䛯䛿 ǻ& GRP78 䛾ඹⓎ⌧䛻䜘䜛 SP-NL-MANF 䛾Ⓨ⌧㔞ཬ䜃ศἪ

㔞䜈䛾ᙳ㡪

48-well plate 䛻䜎䛔䛯 HEK293 ⣽⬊䛻 SP-NL-MANF 䛸ඹ䛻 mock䚸wt GRP78䚸ⱝ䛧 䛟䛿䚸ǻ&*53 䜢୍㐣ᛶ䛻ᙉไⓎ⌧䛥䛫䚸24 ᫬㛫ᚋ䛻↓⾑Ύᇵᆅ (SF) 䛻஺᥮䛧䛯䚹䛭 䛾 4 ᫬㛫ᚋ䛻䚸⣽⬊䛸ᇵ㣴ᾮ䜢ᅇ཰䛧䚸䝹䝅䝣䜵䝷䞊䝊άᛶ䜢 ᐃ䛧䛯䚹** 䛸 # 䛿䚸ྛ䚻 mock 䜎䛯䛿 wt GRP78 䜢ඹⓎ⌧䛥䛫䛯⣽⬊䛻ẚ䜉䛶䚸᭷ព䛺ᕪ䛜䛒䜛䛣䛸䜢♧䛧䛶䛔䜛 (#; p < 0.05, **; p< 0.01)䚹 ᐇ㦂᪉ἲ䛻グ㍕䛧䛯ィ⟬ᘧ䜢⏝䛔䛶䚸SP-NL-MANF 䛾ศἪ㔞 (b) 䛸⣽⬊ෆⓎ⌧㔞 (c) 䜢ồ䜑䚸⥲Ⓨ⌧㔞䛻ᑐ䛩䜛ศἪ㔞䛾๭ྜ (a) 䜢⟬ฟ䛧䛯䚹

2-3-6. INS-1 SP-NL-MANF ᜏᖖⓎ⌧⣽⬊ᰴ䜢⏝䛔䛯 MANF ศἪ䛻ᑐ䛩䜛

ᇵ㣴ᾮ୰䜈䛾⾑Ύῧຍ䛾ᙳ㡪 ⢾ᒀ⑓䛷䛿䚸ȕ ⣽⬊Ṛ䛾ཎᅉ䛾୍䛴䛻ᑠ⬊య䝇䝖䝺䝇䛜ᣲ䛢䜙䜜䛶䛔䜛䚹ȕ ⣽⬊ 䛷䛿ᜏᖖⓗ䛻䜲䞁䝇䝸䞁䜢⏘⏕䛩䜛䛯䜑䛻ᑠ⬊య䛜Ⓨ㐩䛧䛶䛔䜛䛸ඹ䛻䚸ᚤᙅ䛺ᑠ⬊ య䝇䝖䝺䝇䛻䛥䜙䛥䜜䛶䛔䜛䛣䛸䛜᫂䜙䛛䛻䛺䛳䛶䛔䜛 [62, 63]䚹䜎䛯䚸ኚ␗ᆺ䜲䞁䝇䝸 䞁㑇ఏᏊ Ins2(C96Y) 䜢ᜏᖖⓗ䛻Ⓨ⌧䛩䜛䝷䝑䝖䜲䞁䝇䝸䝜䞊䝬 INS-1 ⣽⬊䛷䛿䚸 ✀䚻䛾 UPR 㑇ఏᏊ䛸ྠᵝ䛻 MANF 㑇ఏᏊ䛾Ⓨ⌧䜒ቑຍ䛧䛶䛔䜛䛣䛸䛜ሗ࿌䛥䜜 䛯 [64]䚹䛣䛾䛣䛸䛛䜙䚸MANF 䛿⢾ᒀ⑓䛻䜘䜛 ȕ ⣽⬊Ṛ䛸䛾㛵㐃䛜⪃䛘䜙䜜䛯䚹௨

ୖ䜘䜚䚸INS-1 ⣽⬊䛻䛚䛡䜛 MANF ศἪไᚚᶵᵓ䜢ゎᯒ䛩䜛䛣䛸䛿䚸MANF 䛾ᶵ

⬟ゎᯒ䛻䛴䛺䛜䜛▱ぢ䜢䜒䛯䜙䛩䛸⪃䛘䛯䚹䛭䛣䛷䚸INS-1 ⣽⬊䛻䛶 SP-NL-MANF

ᵓ䛻ᑐ䛩䜛᳨ウ䜢䛚䛣䛺䛳䛯 (ᅗ 9a)䚹 WB 䛻䜘䛳䛶䚸ᇵ㣴ᾮ୰䛻ศἪ䛥䜜䛯 MANF 䜢᳨ฟ䛩䜛㝿䛻䛿䚸⾑Ύ䝍䞁䝟䜽 ㉁䛻䜘䜛ᖸ΅䜢㜵䛠䛯䜑䛻䚸↓⾑Ύᇵᆅ䜢⏝䛔䛶ᇵ㣴䜢䛚䛣䛺䛖ᚲせ䛜䛒䛳䛯䚹䛧䛛 䛧䚸NL assay 䛷䛿䚸᳨ฟ䛾㝿䛻ᢠయ䜢ᚲせ䛸䛧䛺䛔Ⅽ䚸⾑Ύ䛾Ꮡᅾ䛿ၥ㢟䛸䛺䜙䛺 䛔䚹䛭䛾䛯䜑䚸ᚑ᮶䛾 WB 䛻䛶᳨ฟ䛩䜛䛯䜑䛾ᇵ㣴᮲௳䛷䛺䛡䜜䜀䚸⼥ྜᆺ MANF 䛿⏕యෆ䛷䛾ᛂ⟅䛻㏆䛔ᣲື䜢♧䛩䛾䛷䛿䛺䛔䛛䛸⪃䛘䛯䚹䛭䛣䛷䚸⾑Ύῧ ຍཪ䛿↓ῧຍ᮲௳ୗ䛻䛚䛡䜛 SP-NL-MANF 䛾Ⓨ⌧㔞ཬ䜃ศἪ㔞䛾ኚ໬䜢 NL assay 䛻䛶ゎᯒ䛧䛯䚹䛭䛾⤖ᯝ䚸⾑Ύ↓ῧຍ䛾᮲௳ୗ䛷䛿䚸⼥ྜᆺ MANF 䛾ศἪ 㔞䛿ⴭ䛧䛟పୗ䛧䛶䛔䜛䛣䛸䛜᫂䜙䛛䛸䛺䛳䛯 (ᅗ 9b)䚹

ᅗ

ᅗ 9. SP-NL-MANF INS-1 ᰴ䜢⏝䛔䛯⼥ྜᆺ MANF 䛾ศἪ㔞䛾⤒᫬ⓗኚ໬ (a) SP-NL-MANF INS-1 ᰴ䛻䛚䛡䜛⼥ྜᆺ MANF 䝍䞁䝟䜽㉁䛾Ⓨ⌧

6-well 䛻 mock 䜎䛯䛿 SP-NL-MANF 䛾ᜏᖖⓎ⌧ INS-1 ⣽⬊ᰴ䜢䜎䛝䚸䝉䝭䝁䞁䝣䝹 䜶䞁䝖䜎䛷ᇵ㣴䛧䚸ᇵᆅ䜢↓⾑Ύᇵᆅ䛸஺᥮䛧䛯䚹䛥䜙䛻 12 ᫬㛫ᇵ㣴ᚋ䚸⣽⬊䛸ᇵ㣴ᾮ䜢ᅇ ཰䛧䛯䚹

(b-d) ⾑ΎᏑᅾ䞉㠀Ꮡᅾୗ䛻䛚䛡䜛 MANF ศἪ㔞ኚ໬䛾ゎᯒ

48-well plate 䛻 SP-NL-MANF INS-1 ᰴ䜢䜎䛝䚸24 ᫬㛫ᚋ䛻ᇵᆅ䜢䚸↓⾑Ύᇵᆅ䚸ⱝ䛧䛟 䛿䚸㏻ᖖᇵᆅ䛻஺᥮䛧䛯䚹䛭䛾ᚋ䚸4 ᫬㛫ẖ䛻⣽⬊䛸ᇵ㣴ᾮ䜢ᅇ཰䛧䚸䝹䝅䝣䜵䝷䞊䝊άᛶ䜢  ᐃ䛧䛯䚹ྛ䚻䛾᮲௳ୗ䛻䛚䛡䜛 SP-NL-MANF ศἪ㔞䛾⥲Ⓨ⌧㔞䛻ᑐ䛩䜛๭ྜ (b)䚸ྛ ᮲௳ୗ䛻䛚䛡䜛 SP-NL-MANF 䛾ศἪ㔞 (c) 䛸⣽⬊ෆⓎ⌧㔞 (d) 䜢䚸ᐇ㦂᪉ἲ䛻グ㍕䛧 䛯ィ⟬ᘧ䜢⏝䛔䛶ồ䜑䛯䚹

2-3-7. ᑠᑠ⬊య䝇䝖䝺䝇ཬ䜃䜹䝹䝅䜴䝮䝅䜾䝘䝹䛾 MANF ศἪ䛻ཬ䜌䛩ᙳ㡪

ඛ䛪䚸N2a ⣽⬊䛸㔝⏕ᆺ INS-1 ⣽⬊䜢⏝䛔䚸䝍䝥䝅䜺䝹䜼䞁(Tg) 䛻䜘䜛ฎ⌮䜎

䛯䛿⾑Ύ↓ῧຍ (SF) 䛾᮲௳䛷ᇵ㣴䛧䚸MANF ཬ䜃ᑠ⬊య䝇䝖䝺䝇䝬䞊䜹䞊㑇ఏ

Ꮚ䛾Ⓨ⌧䜢᳨ウ䛧䛯䚹㐣ཤ䛾ሗ࿌㏻䜚䚸N2a ⣽⬊䛷䛿 Tg ฎ⌮䛻䜘䜚䚸GRP78䚸

sXBP1䚸GADD153 mRNA 䛸ඹ䛻 MANF mRNA 䛾Ⓨ⌧㔞䛜ቑຍ䛧䛶䛔䛯 [40]䚹

䛥䜙䛻䚸N2a ⣽⬊䛸ྠᵝ䛻 INS-1 ⣽⬊䛻䛚䛔䛶䜒䚸Tg ฎ⌮䛻䜘䜚 MANF 㑇ఏ

ᏊⓎ⌧䛿ୖ᪼䛧䛯䚹䛧䛛䛧䚸⾑Ύ↓ῧຍ᮲௳ୗ䛷䛿䚸ᑠ⬊య䝇䝖䝺䝇䝬䞊䜹䞊㑇ఏ Ꮚ䛸MANF 㑇ఏᏊ䛸䜒䚸mRNA Ⓨ⌧㔞䛿䜋䛸䜣䛹ኚ໬䛧䛺䛔䛣䛸䛜ศ䛛䛳䛯 (ᅗ

10a)䚹୍᪉䚸SP-NL-MANF INS-1 ᰴ䜢⏝䛔䛯 NL assay 䛷䛿䚸Tg ฎ⌮䛻䜘䜚 MANF 䛾⣽⬊ෆⓎ⌧㔞䛿ῶᑡ䛧䚸⣽⬊እศἪ㔞䛿ቑຍ䛩䜛䛣䛸䛜᫂䜙䛛䛸䛺䛳䛯䚹

䜎䛯䚸⾑Ύ↓ῧຍ᮲௳ୗ䛷䛿䚸⣽⬊ෆཬ䜃⣽⬊እ䛾 SP-NL-MANF άᛶ䛜ῶᑡ䛧

䛯 (ᅗ 10b-d)䚹䛣䛾ῶᑡ䛿䚸↓⾑Ύᇵᆅ䛻䜴䝅⾑Ύ䜰䝹䝤䝭䞁 (BSA) 䜢ῧຍ䛩䜛

ᅗ

ᅗ 10. SP-NL-MANF INS-1 ᰴ䜢⏝䛔䛯ྛ✀่⃭䛻ᑐ䛩䜛 SP-NL-MANF ศἪ㔞䛾ኚ໬ (a) Tg ฎ⌮䚸↓⾑Ύᇵᆅ䛻䜘䜛䝇䝖䝺䝇䝬䞊䜹䞊㑇ఏᏊ䛾Ⓨ⌧ኚ໬

6-well 䛻 Neuro2a ⣽⬊䜔 INS-1 ⣽⬊䜢䜎䛝䚸䛭䜜䛮䜜䝉䝭䝁䞁䝣䝹䜶䞁䝖䜎䛷ᇵ㣴䛧 䛯ᚋ䚸Tg ฎ⌮䚸ⱝ䛧䛟䛿䚸↓⾑Ύᇵᆅ (SF) ஺᥮䜢⾜䛳䛯䚹12 ᫬㛫ᇵ㣴ᚋ䚸⣽⬊䜢ᅇ཰䛧 䛯䚹ᅗ୰䛾㯮୕ゅ༳䛿 uXBP1䚸ⓑ୕ゅ༳䛿 sXBP1 䜢ព࿡䛩䜛䚹

(b-d) Tg ฎ⌮䚸↓⾑Ύᇵᆅ䛻䜘䜛 MANF Ⓨ⌧㔞ཬ䜃ศἪ㔞䛾ኚ໬

48-well plate 䛻 SP-NL-MANF INS-1 ᰴ䜢䜎䛝䚸24 ᫬㛫ᚋ䛻ᇵᆅ䜢஺᥮䛧䚸Tg ฎ⌮䚸 ⱝ䛧䛟䛿䚸↓⾑Ύᇵᆅ䛻䛶䚸4 ᫬㛫ᇵ㣴䛧䛯䚹䛭䛾ᚋ䚸⣽⬊䛸ᇵ㣴ᾮ䜢ᅇ཰䛧䚸䝹䝅䝣䜵䝷䞊 䝊άᛶ䜢 ᐃ䛧䛯䚹SP-NL-MANF 䛾⥲Ⓨ⌧㔞䛻ᑐ䛩䜛ศἪ㔞䛾๭ྜ (b)䚸MANF 䛾ศ Ἢ㔞 (c) 䛸 MANF 䛾⣽⬊ෆⓎ⌧㔞 (d) 䜢䚸ᐇ㦂᪉ἲ䛻グ㍕䛧䛯ィ⟬ᘧ䜢⏝䛔䛶ồ䜑 䛯䚹

48-well plate 䛻 SP-NL-MANF INS-1 ᰴ䜢䜎䛝䚸24 ᫬㛫ᚋ䛻ᇵᆅ䜢⾑Ύῧຍ䚸ⱝ䛧䛟 䛿䚸↓ῧຍᇵᆅ䛻஺᥮䛧䚸vehicle 䜎䛯䛿 1% BSA 䛻䛶 4 ᫬㛫ฎ⌮䛧䛯䚹䛭䛾ᚋ䚸⣽⬊䛸 ᇵ㣴ᾮ䜢ᅇ཰䛧䚸䝹䝅䝣䜵䝷䞊䝊άᛶ䜢 ᐃ䛧䛯䚹SP-NL-MANF 䛾⥲Ⓨ⌧㔞䛻ᑐ䛩䜛ศἪ 㔞䛾๭ྜ (e)䚸MANF 䛾ศἪ㔞 (f) 䛸 MANF 䛾⣽⬊ෆⓎ⌧㔞 (g) 䜢䚸ᐇ㦂᪉ἲ䛻グ㍕ 䛧䛯ィ⟬ᘧ䜢⏝䛔䛶ồ䜑䛯䚹 Tg 䛿ᑠ⬊య⭷ୖ Ca2+-ATPase 䜢㜼ᐖ䛩䜛䛣䛸䛻䜘䜚䚸ᑠ⬊య୰䛾䜹䝹䝅䜴䝮 䜲䜸䞁䜢ᯤῬ䛥䛫䚸ᑠ⬊య䝇䝖䝺䝇䜢ᘬ䛝㉳䛣䛩䚹䜹䝹䝅䜴䝮䜲䜸䞁䛿䚸䝅䝘䝥䝇ᑠ⬊ 䛜⣽⬊⭷䛻⼥ྜ䛩䜛㝿䛻ᚲせ䛺䛣䛸䛜᫂䜙䛛䛻䛥䜜䛶䛔䜛 [65-67]䚹䛣䛾䛣䛸䛛䜙䚸 Tg ฎ⌮䛻䜘䜛 MANF ศἪಁ㐍䛻ᑐ䛧䛶䚸ᑠ⬊య䝇䝖䝺䝇䛷䛿䛺䛟䚸䜹䝹䝅䜴䝮䜲䜸 䞁䛜㛵୚䛧䛶䛔䜛ྍ⬟ᛶ䛜⪃䛘䜙䜜䛯䚹䛣䛾௬ㄝ䛻⟅䛘䜛䛯䜑䚸㧗⃰ᗘሷ໬䜹䝸䜴䝮 (KCl) 䜢⏝䛔䛶 ᅗ 10 䛸ྠᵝ䛺ゎᯒ䜢䛚䛣䛺䛳䛯䚹㧗⃰ᗘ KCl ฎ⌮䛻䜘䜛⣽⬊እ 䜹䝸䜴䝮䜲䜸䞁⃰ᗘ䛾ቑຍ䛻క䛔䚸⣽⬊⾲㠃䛾⭷㟁఩䛜ୖ᪼䛧䚸⬺ศᴟ䛩䜛䚹䛭䜜 䛻䜘䜚䚸㟁఩౫Ꮡᛶ䜹䝹䝅䜴䝮䝏䝱䝛䝹䛜㛤Ꮝ䛧䚸⣽⬊ෆ䜈䛾䜹䝹䝅䜴䝮䜲䜸䞁䛾ὶ ධ䛜㉳䛣䜛䚹ᅗ 11 䛷♧䛩䜘䛖䛻䚸Tg ฎ⌮ཬ䜃 KCl ฎ⌮䛻䜘䜚䚸SP-NL-MANF 䛾

ศἪ㔞䛿ቑຍ䛧䛯䚹䛧䛛䛧䚸KCl ฎ⌮䛷䛿䚸sXBP1䚸GADD153 ཬ䜃 MANF mRNA

ᅗ

ᅗ 11. SP-NL-MANF INS-1 ᰴ䜢⏝䛔䛯ྛ✀่⃭䛻ᑐ䛩䜛 SP-NL-MANF ศἪ㔞䛾ኚ໬ (a) Tg䚸KCl ฎ⌮䛻䜘䜛 UPR 㑇ఏᏊ䛾Ⓨ⌧ኚ໬

6-well 䛻 INS-1 ⣽⬊䜢䜎䛝䚸䝉䝭䝁䞁䝣䝹䜶䞁䝖䜎䛷ᇵ㣴䛧䛯ᚋ䚸Tg ฎ⌮䚸ⱝ䛧䛟䛿䚸KCl ฎ⌮䜢12 ᫬㛫䛚䛣䛺䛔䚸⣽⬊䜢ᅇ཰䛧䛯䚹ᅗ୰䛾㯮୕ゅ༳䛿 uXBP1䚸ⓑ୕ゅ༳䛿

sXBP1 䜢ព࿡䛩䜛䚹

(b-d) Tg䚸KCl ฎ⌮䛻䜘䜛 MANF Ⓨ⌧ཬ䜃ศἪ㔞䛾ኚ໬

48-well plate 䛻 SP-NL-MANF INS-1 ᰴ䜢䜎䛝䚸24 h ᚋ䛻䚸Tg 䜎䛯䛿 KCl 䛻䛶่⃭䛧䛯䚹 4 ᫬㛫ᇵ㣴ᚋ䚸⣽⬊䛸ᇵ㣴ᾮ䜢ᅇ཰䛧䚸䝹䝅䝣䜵䝷䞊䝊άᛶ䜢 ᐃ䛧䛯䚹SP-NL-MANF 䛾䚸 ⥲Ⓨ⌧㔞䛻ᑐ䛩䜛ศἪ㔞䛾๭ྜ (b)䚸MANF 䛾ศἪ㔞 (c) 䛸 MANF 䛾⣽⬊ෆⓎ⌧㔞 (d) 䜢䚸ᐇ㦂᪉ἲ䛻グ㍕䛧䛯ィ⟬ᘧ䜢⏝䛔䛶ồ䜑䛯䚹

䛥䜙䛻䚸INS-1 ⣽⬊䜢⏝䛔䛶䚸ᑠ⬊య䝇䝖䝺䝇ㄏᑟ๣䝒䝙䜹䝬䜲䝅䞁 (Tm) ฎ⌮䜎 䛯䛿⾑Ύ䞉䜾䝹䝁䞊䝇↓ῧຍᇵᆅ (SGF) ஺᥮ᚋ䚸MANF mRNA 䛾Ⓨ⌧㔞ཬ䜃 MANF ศἪ㔞䛻䛴䛔䛶ゎᯒ䛧䛯䚹Tm 䛿 N ᆺ⢾㙐ಟ㣭䜢㜼ᐖ䛧䚸ᑠ⬊య䝇䝖䝺䝇 䜢ᘬ䛝㉳䛣䛩䛯䜑䚸Tg 䛸䛿␗䛺䜚⣽⬊ෆ䜹䝹䝅䜴䝮䜲䜸䞁⃰ᗘ䛿ኚ໬䛧䛺䛔䚹୍᪉䚸 ⾑Ύ䞉䜾䝹䝁䞊䝇↓ῧຍ䛾᮲௳ୗ䛻䛚䛔䛶䛿䜾䝹䝁䞊䝇䛾౪⤥୙㊊䛻䜘䜛ᰤ㣴㣚㣹 䛛䜙ᑠ⬊య䝇䝖䝺䝇䛾ㄏᑟ䛜ண 䛥䜜䛯䚹䛧䛛䛧䛺䛜䜙䚸SGF ฎ⌮䛷䛿䚸GADD153 㑇ఏᏊ䛾Ⓨ⌧䛿ಁ㐍䛥䜜䛯䛜䚸௚䛾ᑠ⬊య䝇䝖䝺䝇䝬䞊䜹䞊㑇ఏᏊཬ䜃 MANF 㑇 ఏᏊ䛾Ⓨ⌧䛻ᙳ㡪䛿ཬ䜌䛥䛺䛛䛳䛯 (ᅗ 12a)䚹SP-NL-MANF 䛻㛵䛧䛶䛿䚸⾑Ύ↓ ῧຍ䛾᮲௳䛸ྠᵝ䛻䚸⣽⬊ෆཬ䜃⣽⬊እ䛷䚸䛭䛾άᛶ䛿ῶᑡ䛧䛶䛔䛯䚹୍᪉䚸Tm ฎ⌮䛷䛿䚸Tg 䛸ྠᵝ䛻䚸MANF ཬ䜃 UPR 㑇ఏᏊ䛾Ⓨ⌧䛜ቑຍ䛧䛶䛔䛯 (ᅗ 12a)䚹䛧䛛䛧䛺䛜䜙䚸SP-NL-MANF 䛾ศἪ㔞䛿䚸Tg ฎ⌮᫬䛸䛿␗䛺䜚䚸ẚ㍑⩌䛸䛾 㛫䛻ኚ໬䛿ㄆ䜑䜙䜜䛺䛛䛳䛯 (ᅗ 12b-d)䚹

ᅗ

ᅗ 12. SP-NL-MANF INS-1 ᰴ䜢⏝䛔䛯ྛ✀่⃭䛻ᑐ䛩䜛 SP-NL-MANF ศἪ㔞ኚ໬ (a) Tm ฎ⌮⾑Ύ䞉䜾䝹䝁䞊䝇↓ῧຍᇵᆅ䛻䜘䜛 UPR 㑇ఏᏊ䛾Ⓨ⌧ኚ໬

6-well 䛻 INS-1 ⣽⬊䜢䜎䛝䚸䝉䝭䝁䞁䝣䝹䜶䞁䝖䜎䛷ᇵ㣴䛧䛯ᚋ䚸Tm ฎ⌮ⱝ䛧䛟䛿⾑Ύ䞉䜾 䝹䝁䞊䝇↓ῧຍᇵᆅ䛷䛾ᇵ㣴䜢 12 ᫬㛫䛚䛣䛺䛔䚸⣽⬊䜢ᅇ཰䛧䛯䚹

(b-d) Tm ฎ⌮⾑Ύ䞉䜾䝹䝁䞊䝇↓ῧຍᇵᆅ䛻䜘䜛 MANF Ⓨ⌧ཬ䜃ศἪ㔞䛾ኚ໬

48-well plate 䛻 SP-NL-MANF INS-1 ᰴ䜢䜎䛝䚸24 ᫬㛫ᚋ䛻䚸Tm ฎ⌮ⱝ䛧䛟䛿⾑Ύ䞉䜾 䝹䝁䞊䝇↓ῧຍᇵᆅ䜈䛾஺᥮䜢䛚䛣䛺䛳䛯䚹4 ᫬㛫ᇵ㣴ᚋ䚸⣽⬊䛸ᇵ㣴ᾮ䜢ᅇ཰䛧䚸䝹䝅䝣 䜵䝷䞊䝊άᛶ䜢 ᐃ䛧䛯䚹SP-NL-MANF 䛾䚸⥲Ⓨ⌧㔞䛻ᑐ䛩䜛ศἪ㔞䛾๭ྜ (b)䚸MANF 䛾ศἪ㔞 (c) 䛸 MANF 䛾⣽⬊ෆⓎ⌧㔞 (d) 䜢ᐇ㦂᪉ἲ䛻グ㍕䛧䛯ィ⟬ᘧ䜢⏝䛔䛶ồ䜑 䛯䚹

2-4. ⪃⪃ᐹ ᑠ⬊య䝇䝖䝺䝇䛸䛿䚸ᢡ䜚䛯䛯䜏୙඲䝍䞁䝟䜽㉁䛜⣽⬊ෆᑠჾᐁ䛾⢒㠃ᑠ⬊య䛻 ⵳✚䛧䛯≧ែ䜢ᣦ䛧䚸䜰䝹䝒䝝䜲䝬䞊⑓䚸䝟䞊䜻䞁䝋䞁⑓䜢䛿䛨䜑䛸䛩䜛ከ䛟䛾⚄⤒ኚ ᛶ⑌ᝈ䜔⹫⾑䚸⢾ᒀ⑓䛺䛹ᵝ䚻䛺⑌ᝈ䛾Ⓨ⑕䞉㐍⾜䛻῝䛟㛵䜟䜛䛣䛸䛜᫂䜙䛛䛻䛥䜜 䛶䛝䛶䛔䜛䚹ᑠ⬊య䝇䝖䝺䝇ୗ䛷䛿䚸ᵝ䚻䛺㑇ఏᏊ䛾Ⓨ⌧䛜ㄏᑟ䛥䜜䛶䛚䜚䚸MANF 䛿䛭䛾䛖䛱䛾䜂䛸䛴䛷䛒䜛 [21, 43-45]䚹MANF 䛿⣙ 18 kDa 䛾ศἪᛶ䝍䞁䝟䜽㉁ 䛷䛒䜚䚸䛭䛾ศἪไᚚᶵᵓ䛻ᑐ䛩䜛▱ぢ䛿䚸MANF 䛾ᶵ⬟ゎᯒ䛻䛚䛔䛶㔜せ䛷䛒䜛 䛸⪃䛘䜙䜜䜛䚹

Apostolou 䜙䛿䚸MANF 䛻ᑐ䛧䛶㧗䛔≉␗ᛶ䜢ᣢ䛴 MANF ᢠయ䜢స〇䛧䚸✀䚻

䛾⣽⬊ᰴ䛻䛚䛔䛶䚸MANF 䝍䞁䝟䜽㉁䛾Ⓨ⌧㔞䛜ᑠ⬊య䝇䝖䝺䝇䛻ᛂ⟅䛧䛶ቑຍ䛩

䜛䛣䛸䜢♧䛧䛯 [39]䚹䛧䛛䛧䛺䛜䜙䚸ෆᅉᛶ MANF 䛾ศἪ䛻ᑐ䛩䜛ศᯒ䛷䛿䚸ᇵ㣴

ᾮ䜢༑ศ䛻⃰⦰䛧䛶䜒䚸ṇ☜䛺್䜢ᚓ䜛䛣䛸䛿㞴䛧䛟䚸ศἪไᚚ䛻㛵䛧䛶᳨ウ䛩䜛䛻

䛿㔞ⓗ䛻୙༑ศ䛷䛒䜛䛣䛸䜒♧䛥䜜䛯䚹஦ᐇ䚸Apostolou 䜙䛿䚸ᑠ⬊య䝇䝖䝺䝇ୗ䛻

䛚䛡䜛 MANF 䛾ศἪ䛻䛴䛔䛶䚸6x His ᶆ㆑䛧䛯 MANF 䛻䜘䜚ゎᯒ䛧䛶䛔䜛䚹䛣䛾

䛣䛸䛛䜙䚸MANF 䛾ศἪไᚚᶵᵓ䛻ᑐ䛧䛶᳨ウ䛩䜛㝿䛿㐣๫Ⓨ⌧ MANF 䜢⏝䛔䜛 ᚲせ䛜䛒䜛䛸⪃䛘䚸ᙜ◊✲ᐊ䛷䛿䝬䜴䝇⚄⤒⣽⬊ᰴ Neuro2a ⏤᮶䛾 MANF 㑇 ఏᏊ䜢⏝䛔䛶䚸MANF 䛻ᑐ䛩䜛᳨ウ䜢䛚䛣䛺䛳䛶䛝䛯䚹 ᙜ◊✲ᐊ䜢ྵ䜐䛔䛟䛴䛛䛾◊✲ᐊ䛷䛿䚸MANF ᙉไⓎ⌧䝰䝕䝹䜢⏝䛔䜛䛣䛸䛷䚸䛭 䛾⏘⏕ཬ䜃ศἪไᚚ䛻㛵䛩䜛◊✲䜢䛚䛣䛺䛳䛶䛔䜛 [39, 40, 55, 56]䚹MANF 䛾 C ᮎ➃ KDEL ᵝ㓄ิ (-RTDL) 䛿䚸GRP78 䛾඾ᆺⓗ䛺 KDEL 㓄ิ䛸ẚ㍑䛧䛶䚸 KDEL ཷᐜయ䜈䛾⤖ྜຊ䛜ᙅ䛔䛣䛸䛜ሗ࿌䛥䜜䛶䛔䜛 [41]䚹䛣䜜䜢ཷ䛡䚸

Glembotski 䛿䚸MANF 䛾ศἪ䛜 KDEL ཷᐜయ䛻ᑐ䛩䜛 GRP78 䛸䛾➇ྜ䛻䜘

䜚ไᚚ䛥䜜䜛䛣䛸䜢ᥦၐ䛧䛯 [68]䚹ḟ䛔䛷䚸MANF ศἪไᚚ䜢᫂䜙䛛䛻䛩䜛䛯䜑䛻䚸

MANF 䛸 ER-Golgi ᶵ⬟䛻㛵䜟䜛ྛᅉᏊ䜢ඹⓎ⌧䛥䛫䛯᳨ウ䛜ᙜ◊✲ᐊ䜢䛿䛨䜑

䛸䛧䛶ᗄ䛴䛛ሗ࿌䛥䜜䛯䚹ᙜ◊✲ᐊ䛷䛿䚸MANF 䛾⏘⏕ཬ䜃ศἪไᚚ䛻㛵䛩䜛◊✲

䜢䛚䛣䛺䛖䛯䜑䛻䚸HEK293 ⣽⬊䜢⏝䛔䚸KDEL ᵝ㓄ิ䜢Ḟኻ䛥䛫䛯 MANF (¨C

MANF) 䜢ྵ䜐ྛ✀䝁䞁䝇䝖䝷䜽䝖䜢䝖䝷䞁䝇䝣䜵䜽䝅䝵䞁䛧䚸⏘⏕䞉ศἪไᚚ䛻㛵䛧䛶᳨ ウ䜢䛚䛣䛺䛳䛯䚹䛭䛾⤖ᯝ䚸MANF 䛿䚸ኚ␗ᆺ Sar1(H79G) 䜔 GRP78 䛸䛾ඹⓎ ⌧䛻䜘䜚䚸ⴭ䛧䛟ศἪ䛜ᢚไ䛥䜜䜛䛣䛸䜢ぢฟ䛧䛯 [40]䚹୍᪉䚸Glembotski 䜙䛿䚸㐣 ๫Ⓨ⌧䛥䛫䛯 MANF 䛸 GRP78 䛾ච␿ỿ㝆䛻䜘䜛ゎᯒ䛾⤖ᯝ䛛䜙䚸MANF 䛿䜹 䝹䝅䜴䝮䜲䜸䞁౫Ꮡⓗ䛻 GRP78 䛸఍ྜ䛧䚸KDEL ཷᐜయ䛻౫Ꮡⓗ䛺㍺㏦ᶵᵓ䛻 䜘䜚ศἪ䛜ไᚚ䛥䜜䛶䛔䜛䛣䛸䜢♧၀䛧䛯 [56]䚹䛩䛺䜟䛱䚸ṇᖖ䛺≧ែ䛷䛿䚸MANF 䛿䚸KDEL ཷᐜయ䜢௓䛧䛶䚸䝂䝹䝆య䛛䜙ᑠ⬊య䜈䛸㏫⾜㍺㏦䛥䜜䛯ᚋ䚸䜹䝹䝅䜴䝮 䜲䜸䞁౫Ꮡⓗ䛺 GRP78 䛸䛾఍ྜ䛻䜘䜚䚸ᑠ⬊యෆ䛻ಖᣢ䛥䜜䜛䚹୍᪉䛷䚸ᑠ⬊య

GRP78 䛸఍ྜ䛫䛪䚸KDEL ཷᐜయ䛻ᑐ䛧ぶ࿴ᛶ䛾㧗䛔௚䛾ᑠ⬊య䝍䞁䝟䜽㉁䛸➇

ྜ䛧䚸⣽⬊እ䜈䛸ศἪ䛥䜜䜛䛸㏙䜉䛶䛔䜛䚹䛥䜙䛻䚸Henderson 䜙䛿䚸GFP ⼥ྜᆺ

MANF 䜢⏝䛔䛯ᐇ㦂䛻䜘䜚䚸KDEL ཷᐜయ䛸䛾ඹⓎ⌧䛻䜘䜚䚸MANF 䛾ศἪ䛜ῶ

ᑡ䛩䜛䛣䛸䜢ሗ࿌䛧䛯 [55]䚹 䛧䛛䛧䛺䛜䜙䚸⣽⬊እ䛻ศἪ䛥䜜䛯 MANF 䜢 WB ゎᯒ䛩䜛ୖ䛷䚸䛔䛟䛴䛛䛾ၥ 㢟䛜䛒䜛䚹ඛ䛪䚸ศἪ䝍䞁䝟䜽㉁䜢᳨ฟ䛩䜛㝿䛻䚸ᇵ㣴ᾮ୰䛾⾑Ύ䝍䞁䝟䜽㉁䛿ヨᩱ 䛾⃰⦰䜢ጉᐖ䛩䜛䛣䛸䛛䜙䚸↓⾑Ύᇵᆅ䛻䛶ᇵ㣴䛩䜛ᚲせ䛜䛒䜛䛣䛸䚸ḟ䛻䚸ศἪ䛥 䜜䛯 MANF 䛜 WB 䛷䛾᳨ฟ㝈⏺䜢༑ศ䛻ୖᅇ䜛䜎䛷䛻୍ᐃ᫬㛫௨ୖᇵ㣴䛩䜛 ᚲせ䛷䛒䜛䛣䛸䚸䛥䜙䛻䚸MANF 䛜 161 䜰䝭䝜㓟䛛䜙䛺䜛䛾䛻ᑐ䛧䚸GFP 䛿 241 䜰 䝭䝜㓟䛛䜙䛺䜚኱䛝䛔䛾䛷䚸ᶵ⬟䜢ゎᯒ䛩䜛㝿䛻 MANF 䛸ྠ䛨䜘䛖䛻ᣲື䛧䛺䛔ྍ⬟ ᛶ䛜⪃䛘䜙䜜䛯䚹䛣䜜䜙䛾ၥ㢟Ⅼ䜢ゎỴ䛩䜛䛣䛸䛜䚸MANF 䛻㛵䛩䜛ศἪไᚚᶵᵓ 䛾䛥䜙䛺䜛◊✲䛻ᚲせ䛷䛒䜛䛸⪃䛘䛯䚹䛭䛣䛷䚸ᮏ◊✲䛷䛿䚸MANF 䛾⏘⏕ཬ䜃ศἪ 䛻䛴䛔䛶䚸171 䜰䝭䝜㓟䛛䜙䛺䜛పศᏊ㔞䝹䝅䝣䜵䝷䞊䝊 NanoLuc 䜢⏝䛔䚸▷᫬㛫 䛷 ᐃ䛷䛝䜛㧗ឤᗘ䛷ᐃ㔞ⓗ䛺 ᐃἲ䜢ᵓ⠏䛧䛯䚹 ඛ䛪䚸SP-NL-MANF 䛾ᣲື䜢 wt MANF 䛸ẚ㍑䛧䚸WB 䛻䜘䜚᳨ウ䛧䛯䛸䛣䜝䚸 SP-NL-MANF 䛿㔝⏕ᆺ䛸㢮ఝ䛧䛯⤒᫬ⓗ䛺ศἪ㔞ኚ໬䜢♧䛩䛣䛸䛜ศ䛛䛳䛯䚹䛥䜙 䛻NL assay 䛷䛿 WB 䛻䛚䛔䛶᳨ฟ㝈⏺௜㏆䛷䛒䛳䛯 4 ᫬㛫ᇵ㣴᮲௳ୗ䛻䛚䛔 䛶䚸༑ศ䛻ศᯒྍ⬟䛺್䜢♧䛧䛶䛔䛯䚹ḟ䛔䛷䚸ኚ␗ᆺ Sar1 (H79G) 䛸䛾ඹⓎ⌧䛻 䜘䜛ኚ໬䛻䛴䛔䛶ゎᯒ䛧䛯䚹ᙜ◊✲ᐊ䛷䛾 wt MANF 䜢⏝䛔䛯㐣ཤ䛾◊✲䛸ྠᵝ䛻䚸 SP-NL-MANF 䜒䚸ኚ␗ᆺ Sar1 (H79G) 䛸䛾ඹⓎ⌧䛻䜘䜚䚸ⴭ䛧䛟ศἪ䛜ᢚไ䛥䜜 䛯䚹䛣䛾䛣䛸䛛䜙䚸SP-NL-MANF 䛿㔝⏕ᆺ䛸ྠᵝ䛻 ᑠ⬊య䛛䜙䛾 COP-II ᑠ⬊䜢 ௓䛧䛶㍺㏦䛥䜜䛶䛔䜛䛣䛸䛜᫂䜙䛛䛻䛺䛳䛯䚹䜎䛯䚸䛣䛾⌧㇟䛿 NL assay 䛻䜘䜚䚸ᐃ 㔞ⓗ䛻ゎᯒ䛷䛝䛯䚹䛣䛾䛣䛸䛛䜙䚸NL assay 䜢⏝䛔䜛䛣䛸䛻䜘䜚䚸WB 䜘䜚䜒㎿㏿䛺 MANF ศἪ䛾ゎศᯒ䛜ྍ⬟䛻䛺䛳䛯䛣䛸䛜♧䛥䜜䛯䚹ᮏ䝅䝇䝔䝮䛿䚸MANF ศἪ䛻 ↔Ⅼ䜢ᙜ䛶䛯୍ḟ䝇䜽䝸䞊䝙䞁䜾䛻኱䛝䛟ᙺ❧䛴䜒䛾䛸⪃䛘䜙䜜䜛䚹 ḟ䛻䚸ศἪไᚚᅉᏊ䛸䛾ඹⓎ⌧䛻䜘䜛MANF 䛾⣽⬊ᑠჾᐁ䛻䛚䛡䜛⛣ື䛻ཬ䜌 䛩ᙳ㡪䜢ゎᯒ䛧䛯䚹SP-EGFP-MANF 䜢⏝䛔䛶ゎᯒ䜢䛚䛣䛺䛳䛯 Henderson 䜙䛾 ሗ࿌ [55] 䛻ᚑ䛔䚸KDEL ཷᐜయ 1 䛸䛾ඹⓎ⌧䛻䜘䜛ᙳ㡪䜢᳨ウ䛧䛯䚹

SP-NL-MANF 䛸 KDEL ཷᐜయ 1 䛸䛾ඹⓎ⌧᫬䛻䜘䜚䚸⼥ྜᆺ MANF 䛾ศἪ䛿

ⴭ䛧䛟ῶᑡ䛧䛯䚹䜎䛯䚸ᙜ◊✲ᐊ䛷䛿䛣䜜䜎䛷䛻䚸GRP78 䛸䛾ඹⓎ⌧䛻䜘䜚䚸MANF 䛾ศἪ䛜ⴭ䛧䛟ᢚไ䛥䜜䜛䛣䛸䜢ሗ࿌䛧䛯 [40]䚹SP-NL-MANF 䜒ྠᵝ䛻䚸wt GRP78 䛸䛾ඹⓎ⌧䛻䜘䜚䚸ⴭ䛧䛟ศἪ䛜ᢚไ䛥䜜䛯䚹䛧䛛䛧䛺䛜䜙䚸C ᮎ➃Ḟᦆᆺ (ǻC) GRP78 䛸䛾ඹⓎ⌧䛻䛚䛔䛶䛿䚸MANF 䛾ศἪ䛿ኚ໬䛧䛺䛛䛳䛯䚹㐣ཤ䛾ሗ ࿌䛷䛿䚸ǻC MANF 䜒 wt GRP78 䛸䛾ඹⓎ⌧䛻䜘䜚ศἪ䛜ᢚไ䛥䜜䜛䛣䛸䜢♧䛧䛯 [40]䚹䛣䜜䜙䛾▱ぢ䜘䜚䚸GRP78 䛻䜘䜛 MANF 䛾ศἪᢚไ䛻䛿䚸GRP78 䛾 C ᮎ➃ KDEL 㓄ิ䛿኱䛝䛟ᐤ୚䛧䛶䛔䜛䛜䚸MANF ⮬య䛾 RTDL 㓄ิ䛿䜋䛸䜣䛹

ᐤ୚䛧䛶䛔䛺䛔䛸⪃䛘䜙䜜䜛䚹䛣䛾䛣䛸䛛䜙䚸MANF ศἪᢚไᶵᵓ䛻䛿䚸GRP78 䛸 KDEL ཷᐜయ 1 䛾┦஫స⏝䛜㛵୚䛧䛶䛔䜛䛣䛸䛜♧၀䛥䜜䛯䚹 ኚ␗ᆺ䜲䞁䝇䝸䞁㑇ఏᏊ䜢⏝䛔䛯ゎᯒ䛛䜙䚸MANF 䛿⮅⮚ ȕ ⣽⬊䛾⏕Ꮡ䛻㛵୚ 䛧䛶䛔䜛䛸⪃䛘䜙䜜䜛 [64]䚹ᚑ䛳䛶䚸䝷䝑䝖䜲䞁䝇䝸䝜䞊䝬 INS-1 ⣽⬊䜢⏝䛔䚸MANF ศἪไᚚᶵᵓ䛻䛴䛔䛶᳨ウ䜢ຍ䛘䛯䚹ඛ䛪䚸SP-NL-MANF ᜏᖖⓎ⌧䛥䛫䛯 INS-1 ⣽⬊ᰴ䜢ᶞ❧䛧䚸⾑ΎᏑᅾ䚸㠀Ꮡᅾୗ䛻䛚䛡䜛ศἪ㔞ኚ໬䜢⤒᫬ⓗ䛻ゎᯒ䛧䛯䛸䛣 䜝䚸⾑Ύ㠀Ꮡᅾୗ䛷䛿䚸MANF ศἪ䛿ⴭ䛧䛟ప䛔್䜢♧䛧䛯䚹䛭䛣䛷䚸↓⾑Ύᇵᆅ䛻 BSA 䜢ຍ䛘䛯䛸䛣䜝䚸MANF 䛾ศἪ㔞䛜ቑຍ䛧䛯䚹㐣ཤ䛾ሗ࿌䛻䛚䛔䛶䚸N9 䝭䜽䝻 䜾䝸䜰⣽⬊䛻䛚䛔䛶䚸BSA ฎ⌮䛻䜘䜚䚸⅖⑕ᛶ䝃䜲䝖䜹䜲䞁 TNF-Į,/-ȕ 䛾ศἪ䛜 ቑຍ䛧䛯䛸䛾ሗ࿌䛜䛒䜛 [69]䚹MANF 䛾 N ᮎ➃㡿ᇦ䛿䚸⬡㉁⤖ྜ⬟䜢᭷䛩䜛䛸⪃ 䛘䜙䜜䛶䛔䜛䛣䛸䛛䜙䚸⬡㉁-BSA 」ྜయ䛻 MANF 䛜఍ྜ䛩䜛䛣䛸䛷䚸䛭䛾ศἪ䛿ಁ 㐍䛥䜜䜛ྍ⬟ᛶ䛜⪃䛘䜙䜜䜛 [42]䚹୍᪉䚸⢭〇䛧䛯⾑Ύ䜰䝹䝤䝭䞁ῧຍ䛻䜘䜚䚸ึ௦ ᇵ㣴䝭䜽䝻䜾䝸䜰䛻䛶䚸⣽⬊ෆ䜹䝹䝅䜴䝮䜲䜸䞁⃰ᗘ䛾ୖ᪼䛜ሗ࿌䛥䜜䛶䛔䜛 [70]䚹 ᚑ䛳䛶䚸ᮏ⣔䛷ぢ䜙䜜䛯BSA ฎ⌮䛻క䛖↓⾑Ύᇵᆅ୰䛷䛾 MANF ศἪ䛾ቑຍ䛻 䛿䚸⬡㉁䜻䝱䝸䝲䞊䛻䜘䜛ᘬ䛝ᢤ䛝䛷䛿䛺䛟䚸⣽⬊ෆ Ca2+ ቑຍ䛻䜘䜛㛤ཱྀᨺฟ䛾ಁ 㐍䛜㛵୚䛧䛶䛔䜛ྍ⬟ᛶ䜒⪃䛘䜙䜜䜛䚹௒ᚋ䚸MANF 䛸⬡㉁䛸䛾┦஫స⏝䛻╔┠䛧 䛯ゎᯒ䛜ᚲせ䛷䛒䜛䚹 ᑠ⬊య䝇䝖䝺䝇ㄏᑟ๣ Tm 䛻䜘䜚ฎ⌮䛧䛯䛸䛣䜝䚸MANF 㑇ఏᏊ䛿Ⓨ⌧䛜ಁ㐍䛥 䜜䛯䛻䜒䛛䛛䜟䜙䛪䚸SP-NL-MANF 䛾ศἪ䛿䜋䛸䜣䛹ኚ໬䛧䛺䛛䛳䛯䚹୍᪉䚸ᑠ⬊ య䝇䝖䝺䝇ㄏᑟ๣ Tg 䛻䜘䜚䚸SP-NL-MANF ศἪ㔞䛿ⴭ䛧䛟ቑຍ䛧䛯䚹Tg ཬ䜃 Tm 䛿䚸ඹ䛻ᑠ⬊య䝇䝖䝺䝇ㄏᑟ๣䛷䛒䜛䛜䚸⣽⬊ෆ䜹䝹䝅䜴䝮䜲䜸䞁⃰ᗘ䛻ኚ໬䜢 䜒䛯䜙䛩Ⅼ䛷ᕪ␗䛜䛒䜛䚹䛣䛾䛣䛸䛛䜙䚸MANF 䛾ศἪไᚚ䛻㛵䛧䛶䚸⣽⬊ෆ䜹䝹䝅 䜴䝮䜲䜸䞁䛜኱䛝䛟ᐤ୚䛧䛶䛔䜛䛾䛷䛿䛸⪃䛘䜙䜜䜛䚹䛥䜙䛻䚸䜹䝹䝅䜴䝮䜲䜸䞁䛾ᐤ ୚䛻䛴䛔䛶᳨ウ䛩䜉䛟䚸KCl ่⃭䜢䛚䛣䛺䛳䛯䚹㧗⃰ᗘ䛾 KCl 䛿䚸⣽⬊⭷䜢⬺ศᴟ 䛥䛫䚸㟁఩౫Ꮡᛶ䜹䝹䝅䜴䝮䝏䝱䝛䝹䛜㛤Ꮝ䛧䛶䚸⣽⬊ෆ䜹䝹䝅䜴䝮䜲䜸䞁⃰ᗘ䛜ୖ ᪼䛩䜛䚹㧗⃰ᗘ KCl ฎ⌮䛾⤖ᯝ䚸MANF 䛾ศἪ䛿 Tg ฎ⌮᫬䛸ྠᵝ䛻ቑຍ䛧䛯䚹 ୍᪉䚸MANF 㑇ఏᏊ䛾Ⓨ⌧䛻䛿ኚ໬䛜ㄆ䜑䜙䜜䛺䛛䛳䛯䛣䛸䛛䜙䚸MANF 䛾୍㐣 ⓗ䛺ศἪไᚚ䛻䛿ᑠ⬊య䝇䝖䝺䝇䛿㛵୚䛫䛪䚸⣽⬊ෆ䜹䝹䝅䜴䝮䜲䜸䞁䝅䜾䝘䝹䛜 ኱䛝䛟ᐤ୚䛩䜛䛣䛸䛜᫂䜙䛛䛻䛺䛳䛯䚹 ඛ䛻㏙䜉䛯䜘䛖䛻䚸Glembotski 䜙䛿䚸ᑠ⬊యෆ䜹䝹䝅䜴䝮䜲䜸䞁䛜ᯤῬ䛩䜛䛣䛸 䛻䜘䜚䚸GRP78 䛸 MANF 䛜஋㞳䛧䚸MANF 䛾ศἪ䛜ಁ㐍䛥䜜䜛䛣䛸䜢ሗ࿌䛧䛯 [56]䚹䛧䛛䛧䛺䛜䜙䚸ᮏ◊✲䛷䛿䚸KCl ฎ⌮䛻క䛖⣽⬊㉁୰䜹䝹䝅䜴䝮䜲䜸䞁䛾ୖ᪼ 䛻䜘䛳䛶䜒䚸እ᮶ᛶ MANF 䛾ศἪ䛿ቑຍ䛧䛶䛔䛯䚹䛣䛾䛣䛸䛛䜙䚸MANF 䛾ศἪ䛿䚸 ⣽⬊㉁୰䛾䜹䝹䝅䜴䝮䜲䜸䞁䛜㛵䜟䜛඾ᆺⓗ䛺㛤ཱྀᨺฟ䛾ᶵᵓ䛻ຍ䛘䚸ᑠ⬊యෆ䜹 䝹䝅䜴䝮䜲䜸䞁⃰ᗘ䛜㛵䜟䜛 GRP78 䛸䛾఍ྜ≧ែ䛾ኚ໬䛻䜘䜛ไᚚᶵᵓ䛾஧䛴

䛔䛶䚸↓่⃭᫬䛸่⃭䛻䜘䜛⣽⬊ෆ䜹䝹䝅䜴䝮䜲䜸䞁⃰ᗘ䛾ୖ᪼᫬䛻䛚䛡䜛୧ᶵᵓ 䛾㛵㐃ᛶ䛻䛴䛔䛶䛾᳨ウ䛩䜛ᚲせ䛷䛒䜛䚹୍᪉䛷䚸⮅⮚ ȕ ⣽⬊䛷䛿䚸㛗ᮇ䛻䜟䛯䜚 㧗⾑⢾≧ែ䛜⥅⥆䛩䜛䛣䛸䛻䜘䜚䚸䜾䝹䝁䞊䝇ㄏᑟᛶ䜲䞁䝇䝸䞁ศἪ䛿ᢚไ䛥䜜䜛䛣 䛸䛜▱䜙䜜䛶䛔䜛䚹䛥䜙䛻䚸GRP78 䜢㐣๫Ⓨ⌧䛥䛫䜛䛣䛸䛷䚸䜾䝹䝁䞊䝇ㄏᑟᛶ䛾䜲 䞁䝇䝸䞁ศἪ㔞䛿ᅇ᚟䛩䜛䛣䛸䛜ሗ࿌䛥䜜䛯 [71]䚹௒ᚋ䚸MANF 䛻㛵䛩䜛ศἪไᚚ ᶵᵓ䛜䚸䜲䞁䝇䝸䞁➼䛾௚䛾ศἪ䝍䞁䝟䜽㉁䛻䜒㐺⏝䛥䜜䜛䛛䛹䛖䛛᳨ウ䛩䜛䛸䛸䜒䛻䚸 MANF 䛜䜲䞁䝇䝸䞁䛸ྠ䛨ศἪᑠ⬊䜢௓䛧䛶ศἪ䛥䜜䛶䛔䜛ྍ⬟ᛶ䚸䜎䛯䛿䚸MANF 䛜䜲䞁䝇䝸䞁➼䛾ศἪไᚚ䛻㛵୚䛧䛶䛔䜛ྍ⬟ᛶ䛻䛴䛔䛶᳨ウ䜢ຍ䛘䜛ᚲせ䛜䛒 䜛䚹 ᮏ䝅䝇䝔䝮䜢⏝䛔䛯㧗ឤᗘ ᐃἲ䛻䜘䜚 ⣽⬊ಖㆤᅉᏊ䛷䛒䜛MANF 䛾ศἪ䜢ಁ 㐍䛥䛫䜛໬ྜ≀䛾᥈⣴䛜⡆౽䛻䛺䛳䛯䛣䛸䛛䜙䚸ᑠ⬊య䝇䝖䝺䝇㛵㐃⑌ᝈ䜔⚄⤒ኚ ᛶ⑌ᝈ➼䛾ண㜵⸆䜔἞⒪⸆䛾ྠᐃ䛻᭷⏝䛷䛒䜛䛸⪃䛘䜙䜜䜛䚹䛥䜙䛻䚸MANF 䜢㧗 ឤᗘ䛻᳨ฟ䛷䛝䜛䛣䛸䛛䜙䚸ཷᐜయ䜔⤖ྜᅉᏊ䛾᥈⣴䛻䜒᭷⏝䛷䛒䜛䛸⪃䛘䜙䜜䜛䚹

➨

➨୕❶ NanoLuc Luciferase Assay 䜢⏝䛔䛯 MANF 䛸 CDNF 䛾

ศἪไᚚ䛻㛵䛩䜛ẚ㍑᳨ウ

3-1. せ⣙

⬨᳝ື≀䛷䛿䚸MANF 䝟䝷䝻䜾䛸䛧䛶 cerebral dopamine neurotrophic factor

(CDNF) 䛜Ꮡᅾ䛩䜛 [42, 48]䚹CDNF 䛿 MANF 䛸ྠᵝ䛻 6-OHDA ㄏᑟᛶ䝟䞊 䜻䞁䝋䞁⑓䝰䝕䝹䝷䝑䝖䛻䛚䛔䛶⚄⤒⣽⬊ಖㆤຠᯝ䛜ሗ࿌䛥䜜䛶䛔䜛 [48-52]䚹୍᪉ 䛷䚸CDNF 䛿 MANF ྠᵝ䛻ศἪᛶ䝍䞁䝟䜽㉁䛷䛒䜛䛜䚸ᑠ⬊య䝇䝖䝺䝇䛻㛵䜟䜙 䛪䚸ᜏᖖⓗ䛻Ⓨ⌧䛧䛶䛔䜛䛣䛸䛜᫂䜙䛛䛻䛺䛳䛶䛔䜛 [39]䚹䝟䝷䝻䜾䛾䝍䞁䝟䜽㉁䛷 䛿஫䛔䛻␗䛺䜛ᶵ⬟䜢᭷䛧䛶䛔䜛ሙྜ䛜ከ䛔䛾䛷䚸MANF 䛸 CDNF 䛾ᶵ⬟䜒␗䛺 䜛ྍ⬟ᛶ䛜䛒䜛䚹䛧䛛䛧䛺䛜䜙䚸⌧ᅾ䛾䛸䛣䜝䚸୧⪅䛾ᶵ⬟䛻㛵䛩䜛ሗ࿌䛸䛧䛶䛿䚸ඹ 䛻䝗䞊䝟䝭䞁⚄⤒䛻ᑐ䛩䜛⣽⬊ಖㆤຠᯝ䛜ከ䛟䜢༨䜑䛶䛔䜛䚹䛥䜙䛻䚸୧⪅䜢ẚ㍑᳨ ウ䛧䛯◊✲䛜ᑡ䛺䛔䛯䜑䚸MANF 䛸 CDNF 䛾┦㐪Ⅼ䛻䛴䛔䛶䛾▱ぢ䛿䜋䛸䜣䛹䛺 䛔 [39, 52. 53]䚹ᚑ䛳䛶䚸MANF 䛸ඹ䛻 CDNF 䜢ᛶ≧ゎᯒ䛧䚸୧⪅䛾┦ఝⅬ䚸┦㐪 Ⅼ䜢᫂☜䛻䛩䜛䛣䛸䛿䚸ྛ䚻䛾ᶵ⬟ゎᯒ䛻䛚䛔䛶䚸኱䛔䛻ᙺ❧䛴䛸⪃䛘䜙䜜䜛䚹䛭䛣 䛷䚸ᮏ◊✲䛷䛿䚸➨஧❶䛷ᵓ⠏䛧䛯ᐃ㔞ἲ䜢 CDNF 䛻䜒㐺⏝䛧䚸ศἪไᚚ䛻㛵䛧䛶 MANF 䛸䛾ẚ㍑᳨ウ䜢⾜䛳䛯䚹䛭䛾⤖ᯝ䚸CDNF 䛾ศἪ䛿 MANF 䛾ሙྜ䛸ྠᵝ 䛻䚸ኚ␗ᆺ Sar1 (H79G) 䛻䜘䜚ⴭ䛧䛟ᢚไ䛥䜜䛯䚹䜎䛯䚸GRP78 䜔 KDEL ཷᐜయ 1 䛸䛾ඹⓎ⌧䛻䜘䜚䚸⣽⬊እ CDNF 䛿᭷ព䛻ῶᑡ䛧䛯䚹䛧䛛䛧䚸ୖグᅉᏊ䛻䜘䜛ศ Ἢᢚไ䛾⛬ᗘ䛿 MANF 䛸ẚ㍑䛧䛶⦆䜔䛛䛷䛒䛳䛯䚹௨ୖ䜘䜚䚸CDNF 䛿 MANF 䛸㢮ఝ䛧䛯ศἪไᚚ䜢ཷ䛡䛶䛔䜛䜒䛾䛾䚸䛭䛾⛬ᗘ䛻ᕪ䛜䛒䜛䛣䛸䛜᫂䜙䛛䛻䛺䛳䛯䚹 䛣䛾䛣䛸䛛䜙䚸MANF 䛜ᑠ⬊య䝇䝖䝺䝇䛻ᛂ⟅䛧䚸⣽⬊ෆ䛻䛶䛭䛾ᶵ⬟䜢Ⓨ᥹䛩䜛䛾 䛻ᑐ䛧䚸CDNF 䛿ᜏᖖⓗ䛻⣽⬊ෆእ䛻䛶ಖㆤᅉᏊ䛸䛧䛶䛾ᶵ⬟䜢Ⓨ᥹䛧䛶䛔䜛ྍ⬟ ᛶ䛜⪃䛘䜙䜜䜛䚹ᮏ◊✲䛻䜘䜚ᚓ䜙䜜䛯 MANF ཬ䜃 CDNF 䛾┦㐪Ⅼ䛿䚸୧⪅䛾 ᶵ⬟䜢ゎ᫂䛻ᙺ❧䛴䜒䛾䛸⪃䛘䜙䜜䜛䚹

3-2. ᮦᮦᩱ䛸᪉ἲ

3-2-1. ヨ⸆

୍ḟᢠయ䛿௨ୗ䛾䜒䛾䜢౑⏝䛧䛯䚹ᢠ actin ᢠయ (Calbiochem, U.S.A.); ᢠ

CDNF ᢠయ䚸ᢠ Flag ᢠయ (M2, Sigma-Aldrich, U.S.A.); ᢠ GRP78 ᢠయ (Cell Signaling, U.S.A.); ᢠ MANF ᢠయ (abcam, U.K. and R&D Systems, U.S.A.);

ᢠ Myc ᢠయ (Santa Cruz Biotechnology, U.S.A).

3-2-2. 䝁䞁䝇䝖䝷䜽䝖䛾స〇

wt CDNF 䛿䚸Neuro2A ⣽⬊䜘䜚ᢳฟ䛧䛯 RNA 䜢ඖ䛻ㄪ〇䛧䛯 cDNA 䛛䜙 PCR

ἲ䛻䜘䛳䛶㑇ఏᏊ᩿∦䜢ቑᖜ䛧䚸pcDNA3.1 䝧䜽䝍䞊䜈䛸⤌䜏㎸䜣䛰䚹DsRed2-䚸

EGFP-䚸Flag-epitope ⼥ྜᆺ MANF ཬ䜃 CDNF 䛿䚸䛭䜜䛮䜜䛾䝅䜾䝘䝹䝨䝥䝏

䝗㓄ิ䛾ୗὶ䛻䚸ྛ✀ tag 䜢ᤄධ䛧䚸㑇ఏᏊ᩿∦䜢 PCR ἲ䛻䛶ቑᖜ䛧䛯ᚋ䚸

pcDNA3.1 䝧 䜽 䝍 䞊 䜈 䛸 䝃 䝤 䜽 䝻 䞊 䝙 䞁 䜾 䛩 䜛 䛣 䛸 䛷 స 〇 䛧 䛯 䚹 wt GRP 䛿

DNAFORM (RIKEN, Japan) 䛛 䜙 ᚓ 䛯 䚹 Sar1 (H79G) 䛿 Liu ཬ 䜃

Lippincott-Schwartz 䛛䜙ㆡΏ䛥䜜䛯䜒䛾䜢౑⏝䛧䛯 [59]䚹KDEL ཷᐜయ 1 䛿 Ruddock 䜘䜚ㆡ䜚ཷ䛡䛯 [41]䚹

3-2-3. ⣽⬊ᇵ㣴䛸⸆๣่⃭

HEK293 ⣽⬊䛿䚸8% fetal bovine serum (FBS) 䛸 1% penicillin/streptomycin (P/S) 䜢ྵ䜐 Dulbecco’s modified Eagle’s minimum essential medium (D-MEM)

(High Glucose, WAKO) 䜢⏝䛔䛶䚸37°C䚸5% CO2 incubator ෆ䛷ᇵ㣴䛧䛯䚹WB

䜢䛚䛣䛺䛖㝿䛿䚸12-well plate 䛻⣽⬊䜢䜎䛝䚸1 ᪥ᚋ䛻ᐇ㦂䛻౑⏝䛧䛯䚹NL assay

䜢⾜䛖㝿䛿 48-well plate 䛻䜎䛝䚸24h ᚋ䛻䚸ᐇ㦂䜢䛚䛣䛺䛳䛯䚹䝖䝷䞁䝇䝣䜵䜽䝅䝵䞁

䛿 䚸Lipofectamine-PLUS Reagent (Life Technologies) ⱝ 䛧 䛟 䛿 䚸 PEI-MAX

(Polysciences, U.S.A.) 䜢⏝䛔䛶䚸ῧ௜䛾౑⏝᪉ἲ䛻ᚑ䛔䛚䛣䛺䛳䛯䚹

3-2-4. NanoLuc luciferase assay

⣽⬊䜢 1× passive lysis buffer (Promega) 䛻䜘䛳䛶⁐ゎ䛧䚸ᅇ཰䛧䛯ヨᩱ䜢 15

min (15000 rpm䚸4°C) 㐲ᚰ䛧䛯ᚋ䚸NanoLuc assay system (Promega) 䛻䜘䜚 NanoLuc άᛶ䜢 ᐃ䛧䛯䚹ᇵ㣴ᾮ䛿㐲ᚰฎ⌮䜢⾜䜟䛪䛻䚸┤᥋ ᐃ䛻⏝䛔䛯䚹  ᐃ್䜢䚸௨ୗ䛾ィ⟬ᘧ䛻ᇶ䛵䛝ィ⟬䛧䚸⣽⬊ෆእ䛾䝹䝅䝣䜵䝷䞊䝊άᛶ䛸䛧䛯䚹 y = x ×ܽ ܾ (y: 䝹䝅䝣䜵䝷䞊䝊άᛶ䚸x:  ᐃ್䚸a: ᅇ཰䛧䛯ᾮ㔞䚸b:  ᐃ䛻౑⏝䛧䛯ᾮ㔞) 䛥䜙䛻䚸⣽⬊ෆእ䛾䝹䝅䝣䜵䝷䞊䝊άᛶ䛾ྜィ್䛻ᑐ䛩䜛⣽⬊እ䛾䝹䝅䝣䜵䝷䞊䝊

άᛶ䛾๭ྜ䜢 % release 䛸䛧䛯䚹ᐇ㦂䛿 triplicate 䛷⾜䛔䚸䜾䝷䝣ୖ䛾್䛿άᛶ್ ±ᶆ‽೫ᕪ䛷♧䛧䛯䚹

3-2-5. 䜴䜴䜶䝇䝍䞁䝤䝻䝑䝖ἲ (WB)

⣽⬊䛿 ice-cold PBS 䛻䛶ᅇ཰䛧䚸homogenate buffer [20 mM Tris–HCl (pH 8.0)䚸

137 mM NaCl䚸2 mM EDTA䚸10% glycerol䚸1% TritonX-100䚸1 mM PMSF䚸10 ȝJPOOHXSHSWLQ䚸ȝJPOSHSVWDWLQ$@ 䛻䜘䜚⁐ゎ䛧䚸Bradford ἲ (BIO-RAD) 䜢

⏝䛔䛶䝍䞁䝟䜽㉁⃰ᗘ䜢 ᐃ䛧䛯䚹䛭䛾ᚋ䚸➼㔞䛾 2× sodium dodecyl sulfate

(SDS)-Laemmli sample buffer [62.5 mM Tris–HCl (pH 6.8)䚸2% SDS䚸10% glycerol] 䜢ຍ䛘䚸100°C 䛷 5 ศ㛫ຍ⇕䛧䛯䚹ศἪ䛥䜜䛯䝍䞁䝟䜽㉁䜢᳨ฟ䛩䜛䛻

䛿䚸ᇵ㣴ᾮ䜢୍㒊ᅇ཰䛧䚸5 ศ㛫㐲ᚰ (3200 rpm, 4°C) 䛩䜛䛣䛸䛻䜘䜚䚸ᾋ㐟䛧䛯⣽

⬊䜢㝖ཤ䛧䛯䚹䛭䛾ᚋ䚸¼ 㔞䛾 4× SDS-Laemmli sample buffer 䛸ᠱ⃮䛧䛯䚹➼㔞

䛾⣽⬊䝩䝰䝆䝛䇷䝖ⱝ䛧䛟䛿䚸ᇵ㣴ᾮ䜢䚸12.5-15% SDS polyacrylamide gel 䛻䜘䜚

ศ㞳䛧䚸polyvinylidene difluoride membrane (PVDF ⭷) (GE Healthcare) 䛻㌿෗

䛧䛯䚹┠ⓗ䝍䞁䝟䜽㉁䛿䚸ᢠ actin ᢠయ (Calbiochem, U.S.A.); ᢠ CDNF ᢠయ䚸

ᢠ Flag ᢠయ (M2, Sigma-Aldrich, U.S.A.); ᢠ GRP78 ᢠయ (Cell Signaling,

U.S.A.); ᢠ MANF ᢠయ (abcam, U.K. and R&D Systems, U.S.A.); ᢠ Myc ᢠ

య (Santa Cruz Biotechnology, U.S.A)䜢⏝䛔䛶䚸enhanced chemiluminescence

(GE Healthcare) 䛻䜘䜚᳨ฟ䛧䛯䚹 3-2-6. ⺯ගほᐹ COS7 ⣽⬊䜢䚸poly-D-lysine 䛷䝁䞊䝖䛧䛯䜺䝷䝇ୖ䛻᧛䛝䚸⩣᪥䚸ྛ䝁䞁䝇䝖䝷䜽䝖 (SP-EGFP-MANF䚸SP-DsRed2-MANF䚸SP-EGFP-CDNF and/or SP-DsRed2-CDNF) 䜢䝖䝷䞁䝇䝣䜵䜽䝅䝵䞁䛧䛯䚹48 ᫬㛫ᇵ㣴䛧䛯ᚋ䚸PBS 䛻䛶Ὑ ί䛧䚸4% 䝟䝷䝩䝹䝮䜰䝹䝕䝠䝗ฎ⌮䜢 15 ศ㛫䛚䛣䛺䛔䚸⣽⬊䜢ᅛᐃ໬䛧䛯䚹䛭䛾

ᚋ䚸䜹䝞䞊䝇䝸䝑䝥䜢 PermaFluor Mountant Medium (Thermo Fisher Scientific,

U.S.A.) 䛻䛶䝇䝷䜲䝗䜺䝷䝇䛻᥋╔䛧䚸⺯ග䜢ほᐹ䛧䛯(470 nm and 540 nm filters:BZ-9000; KEYENCE, Japan)䚹

3-2-7. ⤫ィฎ⌮

⤖ᯝ䛿ᖹᆒ್±ᶆ‽೫ᕪ䛷♧䛧䛯䚹ྛᐇ㦂䛿ᑡ䛺䛟䛸䜒 3 ᅇ⧞䜚㏉䛧䛚䛣䛺䛳䛯䚹

⤫ィฎ⌮䛿䚸student’s t-test 䜢⾜䛔ゎᯒ䛧䛯䚹p<0.05 䜢⤫ィⓗ䛻᭷ព䛷䛒䜛䛸䛧

3-3. ⤖⤖ᯝ 3-3-1. Flag ᶆ㆑ MANF ཬ䜃 CDNF 䛾Ⓨ⌧୪䜃䛻ศἪ㔞䛾ẚ㍑ MANF 䛾ศἪไᚚ䛻㛵䛧䛶䛿䚸ᙜ◊✲ᐊ䜢ྵ䜑䚸䛔䛟䛴䛛䛾ሗ࿌䛜䛒䜛䛜 [39, 40, 55, 56]䚸CDNF 䛻㛵䛧䛶䛾ሗ࿌౛䛿ᑡ䛺䛔 [39, 72]䚹䛭䛣䛷䚸MANF 䛸 CDNF 䛾↓่⃭᫬䛻䛚䛡䜛ศἪ㔞䜢 WB 䜢⏝䛔䛶ẚ㍑䛧䛯䚹WB 䛻䛶 MANF ཬ䜃 CDNF 䜢 ẚ ㍑ ᳨ ウ 䛩 䜛 䛯 䜑 䛻 䚸 ୧ ⪅ 䛾 䝅 䜾 䝘 䝹 䝨 䝥 䝏 䝗 㓄 ิ 䛾 ୗ ὶ 䛻 Flag-epitope 䜢ᤄධ䛧䛯䝁䞁䝇䝖䝷䜽䝖 (SP-Flag-MANF ཬ䜃 SP-Flag-CDNF)䚸୪

䜃 䛻 䚸 䝅 䜾 䝘 䝹 䝨 䝥 䝏 䝗 䜢 Ḟ ᦆ 䛥 䛫 䚸Flag-epitope 䛸⼥ྜ䛥䛫䛯䝁䞁䝇䝖䝷 䜽䝖

(Flag-MANF䚸Flag-CDNF) 䜢స〇䛧䛯䚹HEK293 ⣽⬊䛻䚸Fig. 13a 䛻グ㍕䛧䛯ྛ 䝁䞁䝇䝖䝷䜽䝖䜢䝖䝷䞁䝇䝣䜵䜽䝅䝵䞁䛧䚸ྛ䚻䛾䝍䞁䝟䜽㉁䛾⣽⬊ෆⓎ⌧㔞䛸ศἪ㔞䜢 ẚ㍑䛧䛯䚹SP-Flag-MANF 䛸 SP-Flag-CDNF 䛿㔝⏕ᆺ䛾䜒䛾䛸ྠᵝ䛻⣽⬊ෆ䛻 Ⓨ⌧䛧䛶䛚䜚䚸䛭䛾୍㒊䛿⣽⬊እ䜈䛸ศἪ䛥䜜䛶䛔䛯䚹䜎䛯䚸୧⪅䛾⣽⬊ෆ䛻䛚䛡䜛 Ⓨ⌧㔞䛸ศἪ㔞䛻኱䛝䛺ᕪ䛿䛺䛛䛳䛯䛣䛸䛛䜙䚸MANF ཬ䜃 CDNF 䛾ṇᖖ䛺≧ែ 䛻䛚䛡䜛Ⓨ⌧㔞䛸ศἪ㔞䛻䛿ᕪ␗䛿䛺䛔䛣䛸䛜ศ䛛䛳䛯䚹䛥䜙䛻䚸䝅䜾䝘䝹䝨䝥䝏䝗 䜢Ḟᦆ䛥䛫䛯 Flag-MANF 䛸 Flag-CDNF 䛿䚸⣽⬊እ䛷䛿᳨ฟ䛷䛝䛺䛛䛳䛯 䠄ᅗ 13b upper panel䠅䚹