Title
小胞体ストレス誘導性 Cation transport regulator homolog 1 の
発現制御および機能の解析( 本文(Fulltext) )
Author(s)
野村, 雄紀
Report No.(Doctoral
Degree)
博士(薬科学) 連創博甲第37号
Issue Date
2017-03-25
Type
博士論文
Version
ETD
URL
http://hdl.handle.net/20.500.12099/56207
※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。
2017
㔝ᮧ
㞝⣖
ᑠ⬊యࢫࢺࣞࢫㄏᑟᛶ
Cation transport regulator homolog 1 ࡢⓎ⌧ไᚚ
࠾ࡼࡧᶵ⬟ࡢゎᯒ
Characterization of the expression and function of an ER stress-inducible
cation transport regulator homolog 1
┠ḟ ␎ㄒ ➨୍❶ ᗎㄽ 1-1. 4 1-2. 7 1-3. 10 1-4. 12 1-5. 15 ➨❶ ER ࢫࢺࣞࢫୗ࠾ࡅࡿ Chac1 ࡢ㌿ไᚚゎᯒ࠾ࡼࡧ⩻ヂᚋࡢ ࣉࣟࢸࢯ࣮࣒ࡼࡿศゎ 2-1. 17 2-2. 18 2-2-1. 18 2-2-2. 18 2-2-3. 18 2-2-4. 19 2-2-5. 19 2-2-6. 20 2-2-7. 20 2-3. 21 2-3-1. Neuro2a ⣽⬊࠾ࡅࡿ ER ࢫࢺࣞࢫㄏᑟᛶࡢ 21 2-3-2. Neuro2a ⣽⬊࠾ࡅࡿ ER ࢫࢺࣞࢫㄏᑟᛶࡢ࣐࢘ࢫ Chac1 ࣉ࣮ࣟࣔࢱ࣮ 23 2-3-3. Neuro2a ⣽⬊࠾ࡅࡿࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ࢩࢫࢸ࣒ࢆࡋࡓ 2-4. 29 ᑠ⬊యࢫࢺࣞࢫᛂ⟅ ࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ࢩࢫࢸ࣒ ࢱࣥࣃࢡ㉁ࡢရ㉁⟶⌮ᶵᵓᝈ
Cation transport regulator homolog 1 (Chac1) ᮏ◊✲ࡢ┠ⓗ ᢒ㘓 ᮦᩱ᪉ἲ ྛ✀⸆ᢠయ ࣉࣛࢫ࣑ࢻࡢస〇 ⣽⬊ᇵ㣴⸆ฎ⌮ RT-PCR ࣝࢩࣇ࢙࣮ࣛࢮ࣏࣮ࣞࢱ࣮ࢵࢭ ࢙࢘ࢫࢱࣥࣈࣟࢵࢺἲ ⤫ィゎᯒ ⤖ᯝ άᛶゎᯒ Chac1 ⩻ヂᚋไᚚ ⪃ᐹ Chac1 mRNA Ⓨ⌧ୖ᪼ 4 1 17 28 21
➨୕❶ Chac1 ࡢ⩻ヂ࠾ࡼࡧ⩻ヂᚋࡢไᚚᶵᵓࡢゎᯒ 3-1. 32 3-2. 33 3-2-1. 33 3-2-2. 33 3-2-3. 34 3-2-4. 34 3-2-5. 35 3-2-6. 35 3-2-7. 36 3-2-8. 36 3-2-9. 36 3-3. 37 3-3-1. 3-3-2. 5’UTR ෆࡢ Kozak ᵝ㓄ิࡼࡿ Chac1 ⩻ヂୖ᪼ 2 ␒┠ࡢ࣓ࢳ࢜ࢽࣥ 3-3-3. HEK293 ⣽⬊࠾ࡅࡿ Ub ඹⓎ⌧ࡼࡿ Chac1 ࡢᏳᐃ 3-3-4. HEK293 ⣽⬊࠾ࡅࡿࣜࢪࣥࢆྵࡲ࡞࠸ኚ␗ᆺ Chac1 (K0) Ⓨ⌧ᑐࡍࡿ 3-3-5. Ub ඹⓎ⌧ࡼࡿᏳᐃࣉࣟࢸࢯ࣮࣒ࡼࡿศゎᑐࡍࡿ 3-3-6. 48 3-4. ⪃ᐹ 49 ➨ᅄ❶ ⥲ᣓ ㅰ㎡ ᘬ⏝ᩥ⊩ ᢒ㘓 ᮦᩱ᪉ἲ ྛ✀⸆ᢠయ ࣉࣛࢫ࣑ࢻࡢస〇 ⣽⬊ᇵ㣴⸆ฎ⌮ RT-PCR ࢙࢘ࢫࢱࣥࣈࣟࢵࢺἲ චỿ㝆ἲ GSH 㔞ࡢ ᐃ ࣉࣟࢸࢯ࣮࣒άᛶ ᐃ ⤫ィゎᯒ ⤖ᯝ ࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ࢩࢫࢸ࣒ࡼࡿ Chac1 Ⓨ⌧ไᚚ ࢥࢻࣥࡽ⩻ヂࡉࢀࡓపศᏊᆺ Chac1 ┤᥋ࡢ Ub Ub ඹⓎ⌧ MG132 ฎ⌮ࡢຠᯝ Chac1 N ᮎ➃㡿ᇦࡢᐤࡘ࠸࡚ 㔝⏕ᆺࠊపศᏊᆺ Chac1 Ⓨ⌧ࡀ⣽⬊ෆ GSH 㔞ཬࡰࡍᙳ㡪 32 37 40 42 44 46 54 56 57
1
␎ㄒ
amino acid (aa)
amino acid response element (AARE) ATF/CRE modifier (ACM)
apoptosis inducing factor (AIF)
N-Acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN)
asparagine synthetase (ASNS)
activating transcription factor 4 (ATF4) activating transcription facor 6 (ATF6) amyloid E (AE)
bafilomycin A1 (Baf) brefeldin A (BFA)
basic leucine zipper domain (bZIP)
CCAAT/enhancer-binding protein E (C/EBPE) cation transport regulator homolog 1 (Chac1) C-terminus of Hsc-70-interacting protein (CHIP) cycloheximide (CHX)
concanamycin A (CMA)
cAMP response element binding protein (CREB) cysteine-rich with EGF-like domains 2 (CRELD2) dimethyl sulfoxide (DMSO)
dithiothreitol (DTT)
ethylenediaminetetraacetic acid (EDTA)
eukaryotic translation initiation factor 2D (eIF2D) endoplasmic reticulum (ER)
ER associated degradation (ERAD)
extracellular signal-regulated kinase 3 (ERK3) ER stress response element (ERSE)
2
growth arrest and DNA damage-inducible protein 153 (GADD153) green fluorescent protein (GFP)
glucose regulated protein 78 (GRP78) glutathione (GSH)
human aortic endothelial cells (HAEC)
human embryonic kidney cells 293 (HEK293)
homocysteine-induced endoplasmic reticulum protein (Herp) inositol-requiring enzyme 1 (IRE1)
inhibitor of kappa B (INB)
kelch like ECH associated protein 1 (Keap1)
mesencephalic astrocyte-derived neurotrophic factor (MANF) mitogen-activated protein kinase (MAPK)
nuclear factor-kappa B (NF-NB) National Institutes of Health (NIH)
NF-E2-like basic leucine zipper transcriptional activator (Nrf2)
oxidized 1-palmitoyl-2-arachidonyl-sn-3- glycero-phosphorylcholine (ox-PAPC) aka Parkinson protein 2 (Parkin)
phosphate buffered saline (PBS) polymerase chain reaction (PCR)
PKR-like endoplasmic reticulum kinase (PERK) protein kinase A (PKA)
protein kinase C (PKC)
phorbol 12-myristate 13-acetate (PMA) phenylmethylsulfonyl fluoride (PMSF) reactive oxygen species (ROS) reverse transcription PCR(RT-PCR) serine protease site-1 (S1P)
serine protease site-2 (S2P) sodium dodecyl sulfate (SDS) serum free (SF)
3
thapsigargin (Tg) tunicamycin (Tm)
tumor necrosis factor receptor superfamily member 6B (TNFRSF6B) tribbles 3 homolog (TRIB3)
ubiquitin (Ub)
Ub conjugating enzyme (Ubc) unfolded protein response (UPR) X box-binding protein-1 (XBP1)
4
➨
➨୍❶ ᗎㄽ
1-1. ᑠ⬊యࢫࢺࣞࢫᛂ⟅ᑠ⬊య (ER; endoplasmic reticulum) ࡣ⭷ࢱࣥࣃࢡ㉁ࡸศἪࢱࣥࣃࢡ㉁ࡢ㐺ษ࡞ᢡࡾ ࡓࡓࡳ࣭⩻ヂᚋಟ㣭ࢆ⾜ࡗ࡚࠸ࡿࠋER ෆ㒊ࡢ⎔ቃᝏࡼࡗ࡚ࠊᢡࡾࡓࡓࡳࢱࣥ ࣃࢡ㉁ࡀ✚ࡋࠊER ࢫࢺࣞࢫࡀ⏕ࡌࡿࠊࢱࣥࣃࢡ㉁ࡢ⩻ヂᢚไࠊศᏊࢩࣕ࣌ࣟࣥ 㑇ఏᏊࡢ㌿ࠊᢡࡾࡓࡓࡳࢱࣥࣃࢡ㉁ࡢศゎ࡞ࢆㄏᑟࡍࡿUPR (unfolded protein response) ࡤࢀࡿᛂ⟅ࡀጞືࡍࡿࠋࡍ࡞ࢃࡕࠊUPR ࡣ 3 ࡘࡢせ࡞ࢫࢺࣞࢫᛂ ⟅⤒㊰ࡀ࠶ࡾࠊER ⭷ࢱࣥࣃࢡ㉁࡛࠶ࡿ PERK (PKR-like endoplasmic reticulum kinase)ࠊ ATF6 (activating transcription facor 6)ࠊIRE1 (inositol-requiring enzyme 1) ࡽ㛤ጞࡉ ࢀࡿ୍㐃ࡢᛂࡀᘬࡁ㉳ࡇࡉࢀࡿ (1)ࠋPERK ࡽጞࡲࡿ⤒㊰࡛ࡣࠊPERK ࡢ㔞యࠊ ⮬ᕫࣜࣥ㓟ࡼࡗ࡚ࠊࡑࡢୗὶࡢ eIF2D (eukaryotic translation initiation factor 2D)ࡀ ࣜࣥ㓟ࡉࢀࡿ (2ࠊ3)ࠋࡇࢀࡼࡾࠊయⓗ࡞ࢱࣥࣃࢡ㉁ࡢ⩻ヂࡀᢚไࡉࢀࠊER ෆ ␗ᖖࢱࣥࣃࢡ㉁ࡀࡉࡽ✚ࡋ࡞࠸ࡼ࠺ࡍࡿࠋ୍᪉ࠊࡇࡢ≧ἣୗ࡛ ER ࢫࢺࣞࢫㄏ ᑟᛶ㌿ᅉᏊࡢ୍ࡘ ATF4 (activating transcription factor 4) ࡢ⩻ヂࡣୖ᪼ࡋࠊGADD153 (growth arrest and DNA damage-inducible protein 153) ࢆࡣࡌࡵࡍࡿᵝࠎ࡞㑇ఏᏊࡢ ㌿ࡀㄏᑟࡉࢀࡿ (4ࠊ5)ࠋGADD153 ࡣ⣽⬊࿘ᮇṆࡸ࣏ࢺ࣮ࢩࢫ㛵ࢃࡿከࡃࡢ 㑇ఏᏊࡸࢱࣥࣃࢡ㉁స⏝ࡋࠊ⣽⬊Ṛࢆㄏᑟࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ (6ࠊ7)ࠋATF6 ࡽጞࡲࡿ⤒㊰࡛ࡣࠊER ࡽࢦࣝࢪయ⛣⾜ࡋࡓ ATF6 ࡣ S1P (serine protease site-1) S2P (serine protease site-2) ࡼࡿษ᩿ࢆཷࡅࠊάᛶᆺ ATF6 (cleaved ATF6) ࡀ⏕ࡌࡿࠋḟ࠸࡛ࠊcleaved ATF6 ࡣ᰾⛣⾜ࡋࠊGRP78 (glucose regulated protein 78) ࡞ࡢศᏊࢩࣕ࣌ࣟࣥ㑇ఏᏊࡢ㌿ࢆㄏᑟࡍࡿࡇ࡛ࠊᢡࡾࡓࡓࡳࢱࣥࣃࢡ㉁ࡢ ᵓᡂࢆಁ㐍ࡍࡿ (8)ࠋIRE1 ࡽጞࡲࡿ⤒㊰࡛ࡣࠊIRE1 ࡢ㔞యࠊ⮬ᕫࣜࣥ㓟ࡼ ࡗ࡚ࠊXBP1 mRNA (uXBP1) ࡀࢫࣉࣛࢩࣥࢢࡉࢀࡿ (sXBP1)ࠋḟ࠸࡛ࠊsXBP1 ࡣ᰾ ⛣⾜ࡋࠊศᏊࢩࣕ࣌ࣟࣥࡸ ER ෆࡢᢡࡾࡓࡓࡳࢱࣥࣃࢡ㉁ࢆศゎࡍࡿ ERAD (ER associated degradation) 㛵ࢃࡿ㑇ఏᏊࡢⓎ⌧ࢆㄏᑟࡍࡿࡇ࡛ࢫࢺࣞࢫࢆ⦆ ࡍࡿ (9)ࠋERAD ࡣᚋ㏙ࡍࡿࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ࢩࢫࢸ࣒ࡼࡗ࡚ᢡࡾࡓࡓࡳ ࢱࣥࣃࢡ㉁ࢆศゎࡍࡿᶵᵓ࡛࠶ࡿ (ᅗ 1-1)ࠋ
5
UPR ࡼࡗ࡚ㄏᑟࡉࢀࡿ㑇ఏᏊⓎ⌧ࡣࠊࣉ࣮ࣟࣔࢱ࣮ୖࡢ≉␗ⓗ࡞㓄ิ㌿ㄪ⠇ ᅉᏊࡀ⤖ྜࡍࡿࡇ࡛ᘬࡁ㉳ࡇࡉࢀࡿࠋGADD153 㑇ఏᏊࡣࣉ࣮ࣟࣔࢱ࣮ୖ AARE (amino acid response element; ATTGCATCA)ࠊERSE (ER stress response element; CCAAT-N9-CCACG) 㓄ิࢆᣢࡕࠊࡑࢀࡒࢀ ATF4ࠊcleaved ATF6 ࡀ⤖ྜࡍࡿࡇ࡛ࠊ ࡑࡢ㌿ࡣㄏᑟࡉࢀࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ (10)ࠋࡲࡓࠊGRP78 ࡞ࡢศᏊࢩࣕ࣌ࣟ ࣥ㑇ఏᏊࡢࣉ࣮ࣟࣔࢱ࣮ୖࡣ ERSE 㓄ิࡀᏑᅾࡍࡿࡇࡀከࡃࠊcleaved ATF6 ࡢ ⤖ྜࡼࡾ㌿ࡀ㛤ጞࡉࢀࡿ (8)ࠋ୍᪉ࠊERAD 㛵㐃㑇ఏᏊࡢ୍ࡘ࡛࠶ࡿ Herp (homocysteine-induced endoplasmic reticulum protein) ࡢࣉ࣮ࣟࣔࢱ࣮ୖࡣࠊ AAREࠊ ERSE-II (ATTGG-N-CCACG) 㓄ิࡀᏑᅾࡋࠊࡑࢀࡒࢀ ATF4ࠊcleaved ATF6 ࡲࡓࡣ sXBP1 ࡀ⤖ྜࡍࡿࡇ࡛ࠊHerp 㑇ఏᏊࡢ㌿ࡀㄏᑟࡉࢀࡿ (11ࠊ12)ࠋ
6 ᅗ1-1. ER ᜏᖖᛶࡢ◚⥢ࡼࡗ࡚సືࡍࡿ UPR ᶵᵓ ER ෆ㒊ࡢ⎔ቃᝏࡼࡗ࡚ࠊᢡࡾࡓࡓࡳࢱࣥࣃࢡ㉁ࡢ✚ࡀஹ㐍ࡍࡿࠊER ࢫࢺࣞ ࢫࡀ⏕ࡌࠊUPR ࡤࢀࡿᛂ⟅ࡀᘬࡁ㉳ࡇࡉࢀࡿࠋUPR ࡣ ER ⭷ࢱࣥࣃࢡ㉁࡛࠶ࡿ PERKࠊATF6ࠊIRE1 ࡼࡗ࡚㛤ጞࡉࢀࠊྛࠎࡢୗὶࢩࢢࢼࣝࡀάᛶࡍࡿࠋࡇࢀࡽࡢࢫࢺ ࣞࢫᛂ⟅⤒㊰࡛ࠊࢱࣥࣃࢡ㉁ࡢ⩻ヂᢚไࠊศᏊࢩࣕ࣌ࣟࣥࡢㄏᑟ࡞ࡼࡾࠊࢱࣥࣃࢡ㉁ࡢ ᢡࡾࡓࡓࡳࡸERAD ࡢάᛶࢆ㏻ࡋ࡚ࢫࢺࣞࢫᑐฎࡍࡿࠋࡋࡋࠊER ࡢࢫࢺࣞࢫ≧ ែࡀᣢ⥆ࡍࡿሙྜࡣࠊGADD153 ࡢⓎ⌧ୖ᪼࡞ࢆࡋ࡚ࠊ⣽⬊Ṛࢆㄏᑟࡍࡿࠋ
7 1-2. ࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ࢩࢫࢸ࣒ ࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ࢩࢫࢸ࣒ࡣࣘࣅ࢟ࢳࣥ (ubiquitin; Ub) ࡉࢀࡓࢱࣥࣃ ࢡ㉁ࢆ 26S ࣉࣟࢸࢯ࣮࣒ࡤࢀࡿ」ྜయࡀ≉␗ⓗศゎࡍࡿᶵᵓ࡛࠶ࡿ (13ࠊ 14)ࠋࡇࡢᶵᵓࡣ␗ᖖࢱࣥࣃࢡ㉁㝖ཤ࣭࢚ࣥࢻࢧࢺ࣮ࢩࢫ࣭ࢩࢢࢼࣝఏ㐩࣭DNA ಟ࣭ ㌿ไᚚ࡞ࡢᗈ⠊࡞⣽⬊ෆᶵᵓ㛵ࢃࡗ࡚࠸ࡿ (14-17)ࠋ
Ub ࡣ 76 ࣑ࣀ㓟 (amino acid; aa) ࡽᡂࡿࢱࣥࣃࢡ㉁࡛࠶ࡾ (ᅗ 1-2 (A))ࠊUb ࡢ ຍࡣ Ub ࡢ 76 ␒┠ࡢࢢࣜࢩࣥṧᇶࡢ࢝ࣝ࣎࢟ࢩࣝᇶᶆⓗࢱࣥࣃࢡ㉁ࡢࣜࢪࣥṧ ᇶࡢ H-࣑ࣀᇶࡢ㛫ࡢࢯ࣌ࣉࢳࢻ⤖ྜࡼࡗ࡚ᙧᡂࡉࢀࡿࠋࡲࡓࠊᶆⓗࢱࣥࣃࢡ㉁ ⤖ྜࡋࡓ Ub ࡢࣜࢪࣥṧᇶ㐟㞳 Ub ࡀࢯ࣌ࣉࢳࢻ⤖ྜࢆࡉࡽᙧᡂࡍࡿࡇ࡛ ࣏ࣜࣘࣅ࢟ࢳࣥ (polyUb) 㙐ࡀᙧᡂࡉࢀࡿࠋ୍ศᏊࡢ Ub ࡣ 7 ࡘࡢࣜࢪࣥṧᇶ (K6ࠊ K11ࠊK27ࠊK29ࠊK33ࠊK48ࠊK63) ࡀྵࡲࢀ࡚࠾ࡾࠊ୍⯡ⓗ 1 ✀㢮ࡢࣜࢪࣥṧᇶࢆ ࡋ࡚ polyUb 㙐ࡀᙧᡂࡉࢀࡿࠋUb ࡢ 48 ␒┠ࡢࣜࢪࣥṧᇶࢆࡋ࡚ Ub ࡀ㐃࡞ࡗ ࡓ K48 polyUb 㙐ࡣᶆⓗࢱࣥࣃࢡ㉁ࢆ 26S ࣉࣟࢸࢯ࣮࣒࡛ࡢศゎᑟࡃࡇࡀᗈ ࡃ▱ࡽࢀ࡚࠸ࡿࠋ୍᪉ࠊUb ࡢ 63 ␒┠ࡢࣜࢪࣥṧᇶࢆࡋ࡚ Ub ࡀ㐃࡞ࡗࡓ K63 polyUb 㙐ࡣศゎࡣ㛵ࡏࡎࠊ࢚ࣥࢻࢧࢺ࣮ࢩࢫ࣭ࢩࢢࢼࣝఏ㐩࣭DNA ಟ࡞ 㛵ࢃࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ (15ࠊ16)ࠋ ᅗ1-2. (A) ࣐࢘ࢫ Ub ୍ศᏊࡢ㓄ิ (NM_019639ࠊNCBI ࢹ࣮ࢱ࣮࣋ࢫࡼࡾྲྀᚓ) Ub ࡢ⤖ྜࡣ 3 ✀㢮ࡢ㓝⣲ࡀ㛵ࢃࡗ࡚࠸ࡿࠋࡲࡎࠊࣘࣅ࢟ࢳࣥάᛶ㓝⣲ (E1, ubiquitin activating enzyme) ࡀ ATP ࡢ AMP ࡢຍỈศゎ౫Ꮡⓗ࡞ࢹࢽࣝࢆ ࡋ࡚ࠊUb ࡢ 76 ␒┠ࡢࢢࣜࢩࣥṧᇶࡢ࢝ࣝ࣎࢟ࢩࣝᇶ E1 ࡢάᛶ୰ᚰ࡛࠶ࡿࢩࢫࢸ ࣥṧᇶࡢ㛫㧗࢚ࢿࣝࢠ࣮ࢳ࢚࢜ࢫࢸࣝ⤖ྜࢆᙧᡂࡍࡿࡇ࡛ Ub ࢆάᛶࡍࡿࠋ ḟࠊE1 ࡣࣘࣅ࢟ࢳࣥ⤖ྜ㓝⣲ (E2, ubiquitin conjugating enzyme; Ubc) ࣘࣅ࢟ࢳ
8
ࣥᵝࢻ࣓ࣥࢆࡋ࡚⤖ྜࡋࠊE2 ࡣࢳ࢚࢜ࢫࢸࣝ⤖ྜࢆࡋ࡚ࠊάᛶࡋࡓ Ub ࢆཷ ࡅྲྀࡿࠋ᭱ᚋࠊE2 ࡣࣘࣅ࢟ࢳ࣮ࣥࣜ࢞ࢮ (E3, ubiquitin ligase) ࡢゐ፹ࢻ࣓ࣥ⤖ ྜࡋࠊᶆⓗࢱࣥࣃࢡ㉁ Ub ࢆຍࡍࡿ (14ࠊᅗ 1-2 (B))ࠋE1 ࡣ࡚ࡢ┿᰾⏕≀࠾ ࠸࡚ 1 ✀㢮ࡋᏑᅾࡋ࡞࠸ࡀࠊE2 ࡣࣄࢺ࡛ࡣ⣙ 35 ✀㢮ࠊE3 ࡣ 600 ✀㢮௨ୖࡢ㑇 ఏᏊࡀྠᐃࡉࢀ࡚࠸ࡿ (18)ࠋE3 ࡣࡑࡢάᛶ୰ᚰ࡛࠶ࡿࣔࢳ࣮ࣇࡢ㐪࠸ࡽ HECT ᆺࠊ RING ᆺࠊU-box ᆺ 3 ࡘศ㢮ࡉࢀࠊᶆⓗࢱࣥࣃࢡ㉁ࡢከᵝᛶࢆᙧᡂࡋ࡚࠸ࡿ (19)ࠋ ᅗ 1-2. (B) ࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ࢩࢫࢸ࣒࠾ࡅࡿ Ub ᛂ Ub ࡣ 3 ✀㢮ࡢ㓝⣲ (E1ࠊE2ࠊE3) ࡼࡿ㐃⥆ⓗ࡞ Ub ࡢຍᛂ ࢆࡋ࡚⾜ࢃࢀࠊ᭱⤊ⓗࠊᶆⓗࢱࣥࣃࢡ㉁ Ub ࡀ⤖ྜࡍࡿࠋ (Jung T et al. The proteasomal system. Mol Aspects Med 30, 191-296 (2009) ࡼࡾ୍㒊ᨵ⦅ࡋ࡚㌿㍕)
ࡉࡽࠊUb ࡀຍࡉࢀࡿ㒊ࡶከᵝᛶࡀᏑᅾࡍࡿࠋ୍⯡ⓗ Ub ࡣᶆⓗࢱࣥࣃࢡ ㉁ࡢࣜࢪࣥṧᇶຍࡉࢀࡿࡀࠊࡑࢀ௨እࡢ㒊ࡀ Ub ࡉࢀࡿ㠀ᶆ‽ⓗ࡞ Ub
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ࡘ࠸࡚ࡶሗ࿌ࡉࢀ࡚࠸ࡿ (20)ࠋp21ࠊERK3 (extracellular signal-regulated kinase 3)ࠊ Cyclin G1 ࡣ N ᮎ➃࣓ࢳ࢜ࢽࣥṧᇶࡢ D-࣑ࣀᇶ Ub ࡀࢯ࣌ࣉࢳࢻ⤖ྜࢆᙧᡂ ࡍࡿࡇ࡛ Ub ࡉࢀࠊࣉࣟࢸࢯ࣮࣒ࡼࡿศゎࢆཷࡅࡿ (21-24)ࠋࡉࡽࠊࢩࢫࢸ ࣥࠊࢭࣜࣥࠊࢺࣞ࢜ࢽࣥࠊࢳࣟࢩࣥṧᇶࡶ Ub ࡢᶆⓗ࡛࠶ࡿࡇࡀሗ࿌ࡉࢀ࡚࠾ࡾࠊ ࢩࢫࢸࣥṧᇶࡢ Ub ࡛ࡣࢳ࢚࢜ࢫࢸࣝ⤖ྜࡀᙧᡂࡉࢀࡿ (25-28ࠊᅗ 1-2 (C))ࠋ ᅗ 1-2. (C) Ub 㒊ࡢከᵝᛶ Ub ࡣ୍⯡ⓗ Ub ࡢ 76 ␒┠ࡢࢢࣜࢩࣥṧᇶࡢ࢝ࣝ࣎࢟ࢩࣝᇶᶆ ⓗࢱࣥࣃࢡ㉁ࡢࣜࢪࣥṧᇶࡢ H-࣑ࣀᇶࡢ㛫ࡢࢯ࣌ࣉࢳࢻ⤖ྜࡼ ࡗ࡚ᙧᡂࡉࢀࡿࡀࠊࡑࢀ௨እࡢ㒊ࡀ Ub ࡉࢀࡿ㠀ᶆ‽ⓗ࡞ Ub ࡶ Ꮡᅾࡍࡿࠋࢱࣥࣃࢡ㉁ࡢ N ᮎ➃࣓ࢳ࢜ࢽࣥࠊࢩࢫࢸࣥࠊࢭࣜࣥࠊࢺࣞ ࢜ࢽࣥࠊࢳࣟࢩࣥṧᇶࡶ Ub ࡢ⤖ྜ㒊࡛࠶ࡿࠋUb ࡣࢩࢫࢸࣥṧᇶ ࡣࢳ࢚࢜ࢫࢸࣝ⤖ྜࢆᙧᡂࡋࠊࢭ࣭ࣜࣥࢺࣞ࢜ࢽ࣭ࣥࢳࣟࢩࣥṧᇶࡣ ࢯ࣌ࣉࢳࢻ⤖ྜࢆᙧᡂࡍࡿࠋ
(McDowell GS et al. Non-canonical ubiquitylation: mechanisms and consequences. Int J Biochem Cell Biol 45, 1833–1842 (2013) ࡼࡾ㌿㍕)
10 1-3. ࢱࣥࣃࢡ㉁ࡢရ㉁⟶⌮ᶵᵓᝈ UPR ࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ࢩࢫࢸ࣒ࡣࡕࡽࡶ⣽⬊ෆࢱࣥࣃࢡ㉁ࡢရ㉁⟶ ⌮ࢆᢸࡗ࡚࠾ࡾࠊERAD ࡀ ER ෆࡢᢡࡾࡓࡓࡳࢱࣥࣃࢡ㉁ࢆࣉࣟࢸࢯ࣮࣒ࡼ ࡗ࡚ศゎࡍࡿᶵᵓ࡛࠶ࡿࡇࡽࡶ୧⪅ࡣᐦ᥋㛵㐃ࡋ࡚࠸ࡿࠋࡑࡋ࡚ࠊࡇࢀࡽࡣᗈ⠊ ࡞⣽⬊ෆᶵᵓ㛵ࢃࡗ࡚࠾ࡾࠊࡑࡢ◚⥢ࡼࡗ࡚ᵝࠎ࡞ᝈࡀᘬࡁ㉳ࡇࡉࢀࡿ (29ࠊ30ࠊ ᅗ1-3)ࠋ ⚄⤒ኚᛶᝈ࡛࠶ࡿࣝࢶࣁ࣐࣮ࡸࣃ࣮࢟ࣥࢯࣥ࡞ࡣ⣽⬊ෆ␗ᖖࢱࣥࣃ ࢡ㉁ࡢ✚ࡀぢࡽࢀࠊ⣽⬊㞀ᐖࢆࡶࡓࡽࡍࡇࡀᝈࡢ୍ᅉ࡞ࡗ࡚࠸ࡿ (31ࠊ32)ࠋ ࣝࢶࣁ࣐࣮ࡣ⬻ෆࡢ࣑ࣟࢻ⪁ே⌜⚄⤒ཎ⥺⥔ኚࡢ✚ࢆ⚄⤒⌮Ꮫ ⓗ≉ᚩࡋࠊ㐍⾜ᛶࡢㄆ▱ࢆ⮫ᗋ≧ࡍࡿ (31ࠊ33)ࠋ࣑ࣟࢻ⪁ே⌜ࡢᡂ ศࡣ࣑ࣟࢻE (amyloid E; AE) ࢱࣥࣃࢡ㉁࡛࠶ࡾࠊ⚄⤒ཎ⥺⥔ኚࡢᡂศࡣᚤᑠ ⟶ࡢᵓᡂᡂศ࡛࠶ࡿࢱ࢘ࢱࣥࣃࢡ㉁࡛࠶ࡿࠋAE ࡢ㐣⏘⏕ࡸࢱ࢘ࡢࣜࣥ㓟ࡼࡗ࡚ࠊ ⣽⬊ෆࡑࢀࡽࢱࣥࣃࢡ㉁ࡢจ㞟࣭✚ࡀ㉳ࡇࡾࠊUPR άᛶ࣭ࣉࣟࢸࢯ࣮࣒άᛶ పୗࢆㄏᑟࡍࡿࡇ࡛ࠊẘᛶࢆⓎࡍࡿ⪃࠼ࡽࢀ࡚࠸ࡿ (33ࠊ34)ࠋ ࣃ࣮࢟ࣥࢯࣥࡣ୰⬻㯮㉁⦓ᐦ㒊ࡢ㑅ᢥⓗ࡞ࢻ࣮ࣃ࣑ࣥ⚄⤒⬺ⴠࢆ࡞≉ᚩࡋࠊࢻ ࣮ࣃ࣑ࣥḞஈࡼࡿ㐠ື㞀ᐖ (ᡓ࣭ᅛ⦰࣭↓ື࣭ጼໃᑕ㞀ᐖ࡞) ࢆ⮫ᗋ≧ ࡍࡿ (32)ࠋࣃ࣮࢟ࣥࢯࣥࡢཎᅉ㑇ఏᏊࡣ࠸ࡃࡘྠᐃࡉࢀ࡚࠸ࡿࠋࡑࡢ୍ࡘ࡛࠶ࡿ D-ࢩࢾࢡࣞࣥࡣ⚄⤒⣽⬊ከࡃⓎ⌧ࡋ࡚࠾ࡾࠊⅬኚ␗ࡼࡗ࡚࣑ࢫࣇ࢛࣮ࣝࢻࢆ㉳ ࡇࡋࠊ✚ࡍࡿࡇ࡛ࠊࣉࣟࢸࢯ࣮࣒ᶵ⬟ࢆྵࡴẘᛶࢆⓎࡍࡿࡇࡀ▱ࡽࢀ࡚ ࠸ࡿ (35)ࠋࡲࡓࠊE3 ࡛࠶ࡿ Parkin (aka Parkinson protein 2) ࡶࣃ࣮࢟ࣥࢯࣥࡢཎ ᅉ㑇ఏᏊࡢ୍ࡘ࡛࠶ࡾࠊࡑࡢάᛶࡀపୗࡍࡿࡇ࡛ࠊᇶ㉁ࡢ୍ࡘ࡛࠶ࡿ Pael receptor ࡀ✚ࡍࡿࠋPael receptor ࡣ ERAD ࡼࡗ࡚ศゎࡉࢀࡿࡇࡀሗ࿌ࡉࢀ࡚࠾ࡾࠊࡑࡢ 㐣࡞✚ࡀ ER ࢫࢺࣞࢫࢆᘬࡁ㉳ࡇࡋࠊẘᛶࢆⓎࡍࡿ⪃࠼ࡽࢀ࡚࠸ࡿ (32ࠊ36)ࠋ ࡲࡓࠊ⣽⬊࿘ᮇࢆไᚚࡋ࡚࠸ࡿ p53 ࡸ RB ࡞ࡢࢱࣥࣃࢡ㉁ࡶࣘࣅ࢟ࢳࣥ-ࣉࣟࢸ ࢯ࣮࣒ࢩࢫࢸ࣒ࡼࡗ࡚ศゎࡉࢀ࡚࠾ࡾࠊࡑࢀࡽࢆศゎࡍࡿ E3 ࡢ␗ᖖࡼࡾࡀࢇ ⧅ࡀࡿ (37)ࠋࡀࢇ⣽⬊ࡣప㓟⣲ࡸࢢࣝࢥ࣮ࢫḞஈ࡞ࡢࢫࢺࣞࢫ≧ែᖖࡉࡽ ࡉࢀ࡚࠾ࡾࠊࡑࡢ≧ែ㐺ᛂࡋࠊ⏕Ꮡࡍࡿࡓࡵ UPR ࢆ⏝ࡋ࡚࠸ࡿ (38)ࠋࡼࡗ࡚ࠊ UPR 㛵ࢃࡿ㑇ఏᏊࡢⓎ⌧ࢆไᚚࡍࡿࡇ࡛ࡀࢇ⣽⬊≉␗ⓗ⣽⬊Ṛࢆㄏᑟࡉࡏࡿࡇ ࡀᢠࡀࢇࡢ୍ࡘࡢᶆⓗ࡞ࡿࡶࡢ⪃࠼ࡽࢀࡿ (39)ࠋࡑࡢ୰ࡣ GRP78 ࡢศᏊ
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ࢩࣕ࣌ࣟࣥάᛶࢆᢚไࡍࡿྜ≀ࡸࣉࣟࢸࢯ࣮࣒ࢆ㜼ᐖࡍࡿࡇ࡛ UPR ࢆࡉࡽ άᛶࡉࡏࡿ⸆ࡀᣲࡆࡽࢀ࡚࠸ࡿ (40ࠊ41)ࠋ
ᅗ1-3. UPR ࣉࣟࢸࢯ࣮࣒ᶵ⬟㛵ࢃࡿᝈࡑࡢཎᅉ㑇ఏᏊ ATNF receptor super family, member 1A (TNFRSF1)ࠊtRNA nucleotidyltransferase, CCA-adding 1 (TRNT1)ࠊproteasome subunit beta type 8 (PSMB8)ࠊislet amyloid polypeptide (IAPP)ࠊamyloid precursor protein (APP)ࠊapolipoprotein E (APOE)ࠊ a disintegrin andmetalloproteinase domain 10 (ADAM10)ࠊpreselenin 1 (PS1)ࠊ preselenin 2 (PS2)ࠊleucine rich repeat kinase 2 (LRRK2)ࠊsynuclein alpha (SNCA)ࠊ PTEN-induced putative kinase 1 (PINK)ࠊoncogene DJ 1 (DJ-1)ࠊaka Parkinson protein 7 (PINK1)ࠊHTRA serine peptidase 2 (HTRA2)ࠊsuperoxidase dismutase 1 (SOD1)ࠊtransactive response DNA binding protein 1 (TDP43)ࠊprotein disulfide isomerase (PDI)ࠊhuntingtin (HTT)ࠋ
(Agyemang AF et al. Protein misfolding and dysregulated protein homeostasis in autoinflammatory diseases and beyond. Semin Immunopathol 37, 335-347 (2015) ࡼࡾ୍㒊ᨵ⦅)
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1-4. Cation transport regulator homolog 1 (Chac1)
ࣄࢺື⬦ෆ⓶⣽⬊ (human aortic endothelial cells; HAEC) ࢆࢸ࣮࣒ࣟᛶື⬦◳ ࡢ ᕢ ୰ ࡛ ቑ ຍ ࡍ ࡿ 㓟 ࣜ ࣥ ⬡ ㉁ ࡛ ࠶ ࡿ ox-PAPC (oxidized 1-palmitoyl-2- arachidonyl-sn-3-glycero-phosphorylcholine) ่࡛⃭ࡋࡓㄏᑟࡉࢀࡿ㑇ఏᏊࡋ࡚ Chac1 ࡣྠᐃࡉࢀࡓ (42)ࠋࡲࡓࠊHAECࠊࣄࢺ⫾ඣ⭈⮚⣽⬊ (human embryonic kidney cells 293; HEK293)ࠊࣄࢺᏊᐑ㢕ࡀࢇ⣽⬊ (HeLa) ࢆ N ᆺ⢾㙐ಟ㣭㜼ᐖ࡛࠶ࡿࢶࢽ࢝ ࣐ࢩࣥ (tunicamycin; Tm)ࠊ㑏ඖ࡛࠶ࡿ dithiothreitol (DTT)ࠊER ⭷ Ca2+-ATPase
㜼ᐖ࡛࠶ࡿࢱࣉࢩ࢞ࣝࢠࣥ (thapsigargin; Tg) ࡞ࡢ ER ࢫࢺࣞࢫㄏᑟ࡛ฎ⌮ࡋ ࡓࡶ Chac1 mRNA Ⓨ⌧ࡀୖ᪼ࡍࡿࡇࡀሗ࿌ࡉࢀࠊATF4 ࡼࡗ࡚ㄏᑟࡉࢀࡿ GADD153 ࡢୗὶᏑᅾࡍࡿࡇࡶ♧၀ࡉࢀࡓ (43)ࠋ⌧ᅾ࡛ࡣࠊࡢ✀ࠎࡢ ER ࢫࢺ ࣞࢫㄏᑟ่⃭ࡼࡗ࡚ࡶࠊChac1 mRNA Ⓨ⌧ୖ᪼ࡀ㉳ࡇࡿࡇࡣᡃࠎࢆྵࡵ࡚ሗ࿌ࡉ ࢀ࡚࠾ࡾ (➨❶)ࠊER ࢫࢺࣞࢫㄏᑟᛶ㑇ఏᏊࡋ࡚ㄆ㆑ࡉࢀ࡚࠸ࡿ (43-49)ࠋ Chac1 ࡢࣉ࣮ࣟࣔࢱ࣮ୖࡣ ATF4 ࡸࡑࡢ㛵㐃ࡍࡿ㌿ᅉᏊࡼࡗ࡚ㄆ㆑ࡉࢀࡿ CREB (cAMP response element binding protein) /ATFࠊAARE 㓄ิࡢࡼ࠺࡞㧗࠸┦ྠᛶ ࢆᣢࡗࡓ≉ᚩⓗ࡞㓄ิࡀྵࡲࢀ࡚࠸ࡿ (ᅗ 2-2)ࠋᙜึࠊChac1 ࡣ GADD153 ࡢୗὶ Ꮡᅾࡍࡿࡇࡀ♧၀ࡉࢀࡓࡀࠊࣉ࣮ࣟࣔࢱ࣮ゎᯒࡼࡾ ATF4 ࡀ┤᥋㛵ࡍࡿࡇࡀ ᫂ࡽ࡞ࡗ࡚࠸ࡿ (50)ࠋࡑࡢᚋࠊ➨❶࡛㏙ࡿࡼ࠺ࠊATF4 ᙉไⓎ⌧ࡼࡗ࡚ CREB/ATFࠊAARE 㓄ิࢆࡋ࡚ࠊ࣐࢘ࢫ Chac1 ࣉ࣮ࣟࣔࢱ࣮άᛶࡀୖ᪼ࡍࡿࡇࡀ ศࡗࡓࠋ⌧ᅾ࡛ࡣࠊࣄࢺ Chac1 ࣉ࣮ࣟࣔࢱ࣮ୖࡢ ATF4 ᛂ⟅ᛶࡢ㓄ิࡘ࠸࡚ࡶྠ ᐃࡉࢀ࡚࠸ࡿ (51)ࠋࡉࡽࠊChac1 ࡣஙࡀࢇࡸ༸ᕢࡀࢇࡢணᚋⰋ mRNA Ⓨ⌧ ࡀୖ᪼ࡋ࡚࠸ࡿࡇࠊࡑࢀࡽࡢ⣽⬊࡛Ⓨ⌧ࢆࣀࢵࢡࢲ࢘ࣥࡍࡿ⣽⬊㐟㉮ࡀᢚไࡉࢀࡿ ࡞ࠊࡀࢇࡢ㛵ࢃࡾࡘ࠸࡚ࡢሗ࿌ࡶࡉࢀ࡚࠸ࡿ (52)ࠋ
Chac1 mRNA ࡢⓎ⌧ไᚚ㛵ࡍࡿሗ࿌ࡣࡉࢀ࡚࠸ࡿࡀࠊChac1 ࢱࣥࣃࢡ㉁ࡢⓎ⌧ไ ᚚ㛵ࡍࡿሗ࿌ࡣ࡞࠸ࠋࡼࡗ࡚ࠊ➨❶ࠊ➨୕❶࠾࠸࡚ࠊࡑࡢヲ⣽ࢆ᳨ウࡋࡓࠋ
ࢢࣝࢱࢳ࢜ࣥ (glutathione; GSH) ࡣࢢࣝࢱ࣑ࣥ㓟࣭ࢩࢫࢸ࣭ࣥࢢࣜࢩࣥࡽ࡞ࡿࢺ ࣜ࣌ࣉࢳࢻ࡛࠶ࡾࠊ⏕యෆࡢᛂᛶᐩࡴάᛶ㓟⣲✀ (reactive oxygen species; ROS) ࡢ㝖ཤ῝ࡃ㛵ࢃࡗ࡚࠸ࡿࠋGSH ࡢ⏘⏕⣔ࡀᦆ࡞ࢃࢀࠊROS ࡀ✚ࡍࡿࡀࢇࡸ⚄ ⤒ኚᛶᝈ࡞ࡢẼ⧅ࡀࡿ (53)ࠋKumar ࡽࡣࠊChac1 ࢱࣥࣃࢡ㉁ࡢᶵ⬟ࡋ࡚ J-glutamyl cyclotransferase άᛶࢆᣢࡕࠊGSH స⏝ࡋࠊ5-oxoprorine
cysteine-13 glycine ศゎࡍࡿࡇࢆሗ࿌ࡋࡓ (54ࠊᅗ 1-4)ࠋࡑࡋ࡚ࠊࡑࡢ㓝⣲άᛶ㒊ࡣ࣐࢘ࢫ ࣄࢺ࡛ࡑࢀࡒࢀ 116 ␒┠ࠊ115 ␒┠ࡢࢢࣝࢱ࣑ࣥ㓟ṧᇶ࡛࠶ࡾࠊࡇࡢṧᇶኚ␗ࢆ ຍ࠼ࡿ㓝⣲άᛶࢆኻ࠺ (51ࠊ55)ࠋࡲࡓࠊChac1 ࡣ Notch ࡢ 1669 ␒┠ࡢࢢࣝࢱ࣑ ࣥ㓟ṧᇶࢆ⬺ࢢࣜࢩࣥࡍࡿࡇ࡛ࠊNotch ࢩࢢࢼࣝࢆᢚไࡋࠊ⚄⤒ศࢆไᚚࡍࡿࡇ ࡶሗ࿌ࡉࢀ࡚࠸ࡿ (56ࠊ57)ࠋ ௨ୖࡢࡼ࠺ࠊER ࢫࢺࣞࢫㄏᑟᛶࡢ Chac1 ࡣ GSH ไᚚࢆࡍࡿ㓟ࢫࢺࣞࢫ ࡢ㛵㐃ࡢࡳ࡞ࡽࡎࠊࡑࡢ࣮ࣜࢮάᛶࡼࡾࠊ࠸ࡃࡘࡢࢱࣥࣃࢡ㉁ࢆไᚚࡍࡿࡇ࡛ࠊ Ⓨ⏕࣭ศࢆྵࡵࡓ⏕⌮࣭ែᛂ㛵ࢃࡿ⪃࠼ࡽࢀࡿࠋ
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ᅗ1-4. GSH ⏕ྜᡂ࠾ࡅࡿ᥎ᐃୖࡢ J-glumamyl cycle Chac1 ࡢ GSH ศゎᶵᵓ (A) GSH ࡢ⏕ྜᡂࡣ⣽⬊ෆࠊ⣽⬊እ࠾ࡅࡿᵝࠎ࡞㓝⣲ࡀ㛵ࢃࡗ࡚࠸ࡿ⪃࠼ࡽࢀ࡚࠾ ࡾࠊJ-glutamyl cyclotransferase ࡣ J-glutamyl-amino acid ࢆ 5-oxoproline amino acid ศゎࡍࡿ㓝⣲࡛࠶ࡿࠋJ-glutamylcysteine synthetase (J-GCS)ࠊglutathione synthetase (GS)ࠊ J-glutamyltransferase (J-GT)ࠋ
(Kalinina EV et al. Role of glutathione, glutathione transferase, and glutaredoxin in regulation of redox-dependent processes. Biochemistry (Mosc) 79, 1562-1583 (2014) ࡼࡾ୍㒊ᨵ⦅) (B) Chac1 ࢱࣥࣃࢡ㉁ࡢ J-glutamyl cyclotransferase άᛶ୰ᚰࡣ࣐࢘ࢫࣄࢺ࡛ࡑࢀࡒࢀ 116 ␒┠ࠊ115 ␒┠ࡢࢢࣝࢱ࣑ࣥ㓟ṧᇶ࡛࠶ࡿࠋ
(Kumar A et al. Mammalian proapoptotic factor ChaC1 and its homologues function as J-glutamyl cyclotransferases acting specifically on glutathione. EMBO Rep 13, 1095-1101 (2012) ࡼࡾ୍㒊ᨵ⦅)
15 1-5. ᮏ◊✲ࡢ┠ⓗ ER ࢫࢺࣞࢫࡼࡗ࡚άᛶࡉࢀࡿ UPR ࡣᗈ⠊࡞⣽⬊ෆᛂ⟅㛵ࢃࡗ࡚࠾ࡾࠊࡑࡢ ◚⥢ࡸ㐣࡞άᛶࡣᵝࠎ࡞ᝈ⧅ࡀࡿࠋࡼࡗ࡚ࠊUPR 㛵ࢃࡿ㑇ఏᏊࡢⓎ⌧ᶵᵓ ࢆ▱ࡿࡇࡣࠊከᵝ࡞ᝈࡢཎᅉゎ᫂ࡸ⒪࠾࠸࡚㔜せ࡛࠶ࡿࠋ3 ࡘࡢせ࡞ ER ࢫ ࢺࣞࢫᛂ⟅⤒㊰࡛࠶ࡿ PERK-ATF4ࠊATF6ࠊIRE1-sXBP1 ࡘ࠸࡚ࡣⓎ⌧࣭ไᚚᶵᵓࡢ ゎᯒࡀ㐍ࢇ࡛࠸ࡿ (29)ࠋࡋࡋࠊࡑࢀࡽࡢୗὶࡢ㑇ఏᏊⓎ⌧ࡘ࠸࡚ࡣᮍࡔ༑ศゎ᫂ ࡉࢀ࡚࠾ࡽࡎࠊChac1 ࡣࡑࡢ୰࡛ࡶẚ㍑ⓗ᪂つࡢᅉᏊ࡛࠶ࡿࠋࡲࡓࠊUPR ࡼࡿ࣏ ࢺ࣮ࢩࢫㄏᑟᶵᵓࡘ࠸࡚ࡶࠊࡉࡽ࡞ࡿゎᯒࡀᚲせ࡛࠶ࡿࠋ Chac1 ࡣ ER ࢫࢺࣞࢫࢆㄏᑟࡍࡿ✀ࠎࡢ่⃭ࡼࡗ࡚ mRNA Ⓨ⌧ࡀୖ᪼ࡍࡿࡇ ࡀศࡗ࡚࠾ࡾ (42-48)ࠊࡑࡢࣉ࣮ࣟࣔࢱ࣮ୖࡣ CREB/ATFࠊAARE 㓄ิ࡞ࡢ㌿ ㄪ⠇ᅉᏊࡀ⤖ྜࡍࡿ⪃࠼ࡽࢀࡿ≉ᚩⓗ࡞㓄ิࡀᏑᅾࡍࡿࠋ3 ࡘࡢせ࡞ ER ࢫࢺ ࣞࢫᛂ⟅⤒㊰ࡢ୰࡛ࠊPERK-ATF4 ࡀ Chac1 㑇ఏᏊࡢ㌿ไᚚ㛵ࡍࡿࡇࡀ♧၀ ࡉࢀࡓ (50)ࠋࡼࡗ࡚ࠊChac1 Ⓨ⌧ᶵᵓࡘ࠸࡚ヲࡋࡃ▱ࡿࡓࡵࠊATF4 ᛂ⟅ᛶࡢ࣐ ࢘ࢫ Chac1 ࣉ࣮ࣟࣔࢱ࣮ୖࡢ㡿ᇦࡘ࠸࡚ゎᯒࢆ⾜ࡗࡓ (➨❶)ࠋ ࡲࡓࠊChac1 ࢱࣥࣃࢡ㉁㛵ࡋ࡚ࡣࠊGSH ศゎࡸ Notch ࢩࢢࢼࣝ㛵㐃ࡋࡓᶵ⬟ ࡘ࠸࡚ሗ࿌ࡉࢀ࡚࠸ࡿࡀ (53-56)ࠊChac1 ࡢ⩻ヂ࠾ࡼࡧ⩻ヂᚋㄪ⠇ᶵᵓࡘ࠸࡚ࡢሗ ࿌ࡣࢇ࡞࠸ࠋࡑࡇ࡛ࠊChac1 ࢱࣥࣃࢡ㉁Ⓨ⌧ࡢไᚚᶵᵓࡢゎ᫂ࢆ┠ⓗࡋ࡚ࠊࣘ ࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ࢩࢫࢸ࣒ Chac1 mRNA ୖᏑᅾࡍࡿ 5’㠀⩻ヂ㡿ᇦ (5’UTRࠊᅗ 1-5) ↔Ⅼࢆᙜ࡚࡚ゎᯒࡋࡓ (➨୕❶)ࠋ
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ᅗ 1-5. ࣐࢘ࢫࠊࣛࢵࢺࠊࣄࢺ㛫࡛ࡢ Chac1 mRNA ෆࡢ 5’UTR ࡢẚ㍑
࣐࢘ࢫࠊࣛࢵࢺࠊࣄࢺ㛫࡛ 5’UTR ࡣࠊ᥎ᐃୖࡢ⩻ヂ㛤ጞⅬ┤๓ࡢ Kozak ᵝ㓄ิࡶྵࡵ㧗 ᗘಖᏑࡉࢀ࡚࠸ࡿ ( * ࡀಖᏑࡉࢀ࡚࠸ࡿ㓄ิࢆ⾲ࡍ)ࠋATG (㉥) ࡀ᥎ᐃୖࡢ⩻ヂ㛤ጞⅬ࡛ ࠶ࡿࠋࣄࢺ࡛ࡣ᥎ᐃୖࡢ⩻ヂ㛤ጞⅬୖὶࡶ࠺୍ࡘ ATG (㟷) ࡀᏑᅾࡍࡿࠋ࣐࢘ࢫ (NM_026929)ࠊࣛࢵࢺ (NM_001173437)ࠊࣄࢺ (NM_024111) ࡢ Chac1 5’UTR ࡢ㓄ิࡣ ࡑࢀࡒࢀ NCBI ࢹ࣮ࢱ࣮࣋ࢫࡼࡾྲྀᚓࡋࡓࠋ
17
➨
➨❶
ER ࢫࢺࣞࢫୗ࠾ࡅࡿ Chac1 ࡢ㌿ไᚚゎᯒ࠾ࡼࡧ⩻ヂᚋࡢ
ࣉࣟࢸࢯ࣮࣒ࡼࡿศゎ
2-1. ᢒ㘓 ㏆ᖺࠊChac1 ࡀ ER ࢫࢺࣞࢫࡼࡾㄏᑟࡉࢀࡿࠊ᪂つࡢ࣏ࢺ࣮ࢩࢫಁ㐍ᅉᏊ࡛࠶ ࡿࡇࡀሗ࿌ࡉࢀࡓ (43)ࠋࡲࡓࠊ3 ࡘࡢせ࡞ ER ࢫࢺࣞࢫᛂ⟅⤒㊰ࡢ୰࡛ࠊPERK-ATF4 ࡀ Chac1 㑇ఏᏊⓎ⌧ࡢ㌿ไᚚ㛵ࡍࡿࡇࡀ♧၀ࡉࢀࡓ (50)ࠋࡋࡋࠊ Chac1 ࣉ࣮ࣟࣔࢱ࣮ࡢヲ⣽࡞ゎᯒࡘ࠸࡚ࡣࡲࡔ⾜ࢃࢀ࡚࠸࡞࠸ࠋᮏ◊✲࡛ࡣࠊER Ca2+-ATPase 㜼ᐖ࡛࠶ࡿ Tg ࡛ฎ⌮ࡋࡓ࣐࢘ࢫ⚄⤒ⱆ⣽⬊⭘ (Neuro2a) ࠾ࡅࡿChac1 mRNA Ⓨ⌧ㄏᑟࡘ࠸࡚ࠊDNA ࣞ RT-PCR ἲࢆ⏝࠸࡚ゎᯒࡋࡓࠋChac1 mRNA Ⓨ⌧ࡣࡢ ER ࢫࢺࣞࢫㄏᑟ࡛࠶ࡿ Tm ࡸ BFA (brefeldin A) ฎ⌮ࡼࡗ࡚ ࡶⴭࡋࡃㄏᑟࡉࢀࡓࠋࣝࢩࣇ࢙࣮ࣛࢮ࣏࣮ࣞࢱ࣮ࢵࢭࢆ⏝࠸ࡓ࣐࢘ࢫ Chac1 ࣉࣟ ࣮ࣔࢱ࣮άᛶࡢゎᯒ࡛ࡣࠊࣉ࣮ࣟࣔࢱ࣮ୖࡢ CREB/ATF AARE ࡤࢀࡿ 2 ࡘࡢ ≉ᚩⓗ࡞㓄ิࡀ Tm ฎ⌮ࡸ ATF4 ᙉไⓎ⌧ᑐࡋ࡚ᛂ⟅ࡍࡿࡇࡀศࡗࡓࠋChac1 ࣉ࣮ࣟࣔࢱ࣮ୖࡢ୍᪉ࡢ㓄ิᑐࡍࡿኚ␗࡛ࡣ Tm ฎ⌮ࡸ ATF4 ᙉไⓎ⌧ࡼࡿᛂ ⟅ᛶࢆ㜼ᐖࡋ࡞ࡗࡓࠋࡑࡇ࡛ࠊ୧᪉ࡢ㓄ิᑐࡋኚ␗ࢆᑟධࡋࡓࡇࢁࠊᇶ♏㌿ά ᛶ ER ࢫࢺࣞࢫ่⃭ᛂ⟅⬟ࡀⴭࡋࡃῶᑡࡋࡓࠋࡉࡽࠊ㌿ไᚚຍ࠼࡚ࠊ࣐࢘ࢫ Chac1 Ⓨ⌧ࢥࣥࢫࢺࣛࢡࢺࢆ୍㐣ⓗᙉไⓎ⌧ࡋࡓ Neuro2a ⣽⬊࠾࠸࡚ࠊChac1 ࢱࣥࣃࢡ㉁Ⓨ⌧ࢆ᳨ウࡋࡓࠋࡑࡢ⤖ᯝࠊࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ศゎ⣔㜼ᐖ࡛࠶ ࡿ MG132 Ꮡᅾୗ࡛ࡢࡳ Chac1 ࡀ᳨ฟ࡛ࡁࡓࠋ௨ୖࡼࡾࠊ⚄⤒⣽⬊⣔࠾ࡅࡿ Chac1 ࡢ㌿࠾ࡼࡧ⩻ヂᚋไᚚࡘ࠸࡚ึࡵ࡚ゎᯒࢆ⾜ࡗࡓࠋ
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2-2. ᮦᩱ᪉ἲ
2-2-1. ྛ✀⸆ᢠయ
TgࠊTmࠊBFAࠊforskolin ࡣ Sigma-Aldrich ࡼࡾ㉎ධࡋࡓࠋMG132 ࡣ Peptide Institute ࡽ㉎ධࡋࡓࠋChac1ࠊActin ᑐࡍࡿᢠయࡣࡑࢀࡒࢀ AbcamࠊCalbiochem ࡽ㉎ධ ࡋࡓࠋ
2-2-2. ࣉࣛࢫ࣑ࢻࡢస〇
࣐࢘ࢫ Chac1 ࣉ࣮ࣟࣔࢱ࣮࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺࣛࢡࢺࢆస〇ࡍࡿࡓࡵࠊNeuro2a ⣽⬊ࡽࢤࣀ࣒ DNA ࢆᢳฟࡋࠊChac1 ࣉ࣮ࣟࣔࢱ࣮㡿ᇦ (-334/+112) (ᅗ 2-2) ࢆ PCR (polymerase chain reaction) ἲࡼࡗ࡚ቑᖜࡋࡓࠋࡑࡋ࡚ࠊPCR ⏘≀ࢆ pGL3b (pGL3-Basic) ࣋ࢡࢱ࣮ (Promega) ࢡ࣮ࣟࢽࣥࢢࡋࡓࠋḞᦆᆺࠊኚ␗ᆺ Chac1 ࣉࣟ ࣮ࣔࢱ࣮ࢆྵࡴࡢ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺࣛࢡࢺࡶ PCR ἲࡼࡗ࡚స〇ࡋࡓࠋ㌿㛤ጞ Ⅼࡣ NCBI ࢹ࣮ࢱ࣮࣋ࢫ (NM_026929ࠊNM_024111) ࢆ⏝࠸࡚ᐃ⩏ࡋࡓࠋ࣐࢘ࢫ ATF4 Ⓨ⌧ࢥࣥࢫࢺࣛࢡࢺࢆస〇ࡍࡿࡓࡵࠊNeuro2a ⣽⬊⏤᮶ࡢ mRNA ࡽ RT-PCR(reverse transcription PCR) ἲࡼࡗ࡚ cDNA ࢆቑᖜࡋࠊATF4 ࢱࣥࣃࢡ㉁ࢥ࣮ ࢻ㡿ᇦࢆ pFLAG-CMV ࣋ࢡࢱ࣮ (Sigma-Aldrich) ࢡ࣮ࣟࢽࣥࢢࡋࡓࠋ࣐࢘ࢫ Chac1 㑇ఏᏊ (NM_026929) ࡣ PCR ἲࡼࡗ࡚ C57/BL6 ࣐࢘ࢫ⬻⏤᮶ࡢ mRNA ࡽ RT-PCR ࡼࡗ࡚ cDNA ࢆቑᖜࡋࠊChac1 ࢱࣥࣃࢡ㉁ࢥ࣮ࢻ㡿ᇦ (+162/+833) (ᅗ 3-1 (A)) ࢆ pcDNA3.3-1 ࣋ࢡࢱ࣮ (Life Technologies) ࢡ࣮ࣟࢽࣥࢢࡋࡓࠋ
2-2-3. ⣽⬊ᇵ㣴⸆ฎ⌮
Chac1 㑇ఏᏊゎᯒࡣ ER ࢫࢺࣞࢫᛂ⟅ࡘ࠸࡚ࡢඛ⾜ㄽᩥᚑ࠸ࠊNeuro2a ⣽⬊ࢆ ⏝࠸ࡓ (58ࠊ59)ࠋNeuro2a ⣽⬊ࡣ 8% ࢘ࢩ⫾ඣ⾑Ύ 1% ࣌ࢽࢩࣜࣥ-ࢫࢺࣞࣉࢺ࣐ ࢩࣥࢆྵࢇࡔ Dulbecco’s Modified Eagle’s Medium ࡼࡗ࡚ᇵ㣴ࡋࡓࠋࡇࡢ◊✲ ࠾ ࡅ ࡿ ྛ ࢥ ࣥ ࢫ ࢺ ࣛ ࢡ ࢺ ࡢ ࢺ ࣛ ࣥ ࢫ ࣇ ࢙ ࢡ ࢩ ࣙ ࣥ ࡣ ࣓ ࣮ ࢝ ࣮ ࡢ ㄝ ᫂ ᚑ ࠸ ࠊ Lipofectamine Plus reagent (Life Technologies) ࢆ⏝࠸࡚⾜ࡗࡓࠋNeuro2a ⣽⬊ࡣ Tg (0.1 PM)ࠊTm (2 Pg/mL)ࠊBFA (5 Pg/mL)ࠊ⾑ΎḞஈ (serum free; SF)ࠊforskolin (10 PM)ࠊ PMA (phorbol 12-myristate 13-acetate) (0.1 PM)ࠊMG132 (20 PM) ࡚グ㍕ࡢ㛫ฎ⌮ ࡋࡓࠋ
19
2-2-4. RT-PCR
TRIzol (Life Technologies) ࡚⣽⬊ࢆ⁐ゎᚋࠊࢡ࣒ࣟࣟ࣍ࣝࢆຍ࠼࡚ΰࡋࠊịୖ࡛ 10 ศ㟼⨨ࡋࡓࠋࡑࡋ࡚ࠊ15,000 rpmࠊ4Υ ࡛ 10 ศ㐲ᚰᚋࠊୖᒙࢆ୍ᐃ㔞⛣ࡋࠊࢯࣉࣟ ࣃࣀ࣮ࣝࢆຍ࠼࡚ΰࡋࡓࠋịୖ࡛ 15 ศᨺ⨨ᚋࠊ15,000 rpmࠊ4Υ ࡛ 10 ศ㐲ᚰࡋࡓࠋ ୖΎࢆ㝖ཤࡢᚋࠊ70% ࢚ࢱࣀ࣮ࣝࢆຍ࠼ࠊ8,500 rpmࠊ4Υ ࡛ 5 ศ㐲ᚰᚋࠊୖΎࢆ㝖 ཤࡋࠊỿẊ≀ࢆ RNA ࡋ࡚⏝ࡋࡓࠋ RNA ࢆ 72Υ ࡛ 10 ศಖ ࡋࡓᚋࠊ RNA (0.5 Pg/10 PL)ࠊDTT (10 mM)ࠊdNTP (0.5 mM)ࠊrandom ninemers (0.05 Pg/10 PL)ࠊ RNaseOUT (2 U/10 PL) (Life Technologies) ࠊ prime superscript III RNase reverse transcriptase (25 U/10 PL) (Life Technologies) ࢆΰྜࡋࠊ42Υ ࡛ 60 ศಖ ࡍࡿࡇ ࡛ cDNA ࡋࡓࠋ≉ᐃࡢ㑇ఏᏊⓎ⌧ࡣ௨ୗ♧ࡍࣉ࣐࣮ࣛ࣌ (1 PM)ࠊdNTP (0.1 mM)ࠊrTaq (0.25 U/10 PL) (Taq PCR kitࠊTakara) ࢆྵࡴ PCR reaction mixture ࢆ࠺ ࡇ࡛ቑᖜࡋࡓࠋࡇࡢ◊✲࡛⏝࠸ࡓࣉ࣐࣮ࣛ࣌ࡣ
Chac1 sense primer; 5’-CATAGGGGCAGCGACAAGATG-3’ࠊ Chac1 antisense primer; 5’-CTGTGTGGCAATGACCTCTTC-3’ࠊ GADD153 sense primer; 5’-GAATAACAGCCGGAACCTGA-3’ࠊ GADD153 antisense primer; 5’-GGACGCAGGGTCAAGAGTAG-3’ࠊ GRP78 sense primer; 5’-ACCAATGACCAAAACCGCCT-3’ࠊ
GRP78 antisense primer; 5’-GAGTTTGCTGATAATTGGCTGAAC-3’ࠊ G3PDH sense primer; 5’-ACCACAGTCCATGCCATCAC-3’ࠊ
G3PDH antisense primer; 5’-TCCACCACCCTGTTGCTGTA-3’
࡛࠶ࡿࠋᆺⓗ࡞ᛂ᮲௳ࡣ 96Υ ࡛ 30 ⛊ࠊ58Υ ࡛ 30 ⛊ࠊ72Υ ࡛ 30 ⛊࡛⾜ࡗ ࡓࠋ⤖ᯝࡣྛ㑇ఏᏊ㐺ษ࡞ࢧࢡࣝ (20 ࡽ 28 ࢧࢡࣝ) ࡛ቑᖜࡋࡓࡢࡶࡢࡀ♧ ࡋ࡚࠶ࡿࠋcDNA ቑᖜ⏘≀ࡣ 2.0% ࡢ࣮࢞ࣟࢫࢤࣝࢆ⏝࠸࡚㟁ẼὋືࡼࡗ࡚ศ㞳 ࡋࠊ࢚ࢳࢪ࣒࢘ࣈ࣐ࣟࢻ࡛ྍどࡋࡓࠋChac1 㑇ఏᏊࡢ┦ᑐⓗ࡞ mRNA Ⓨ⌧ࣞ࣋ࣝ ࡣ NIH imaging ࢆ⏝࠸࡚ᩘ್ࡋࠊ⸆࡛ᮍฎ⌮ࡢ⣽⬊ࡢ G3PDH(glyceraldehyde 3-phosphate dehydrogenase) ࡢ್࡛ᶆ‽ࡋࡓࡶࡢࢆ♧ࡋࡓࠋᐇ㦂ࡣ」ᩘᅇ⾜࠸ࠊ⌧ ᛶࢆ☜ㄆࡋࡓࠋ
2-2-5. ࣝࢩࣇ࢙࣮ࣛࢮ࣏࣮ࣞࢱ࣮ࢵࢭ
20
࡚ࡢ pGL4.70 (Rluc) (0.04 Pg) (Promega) ࢆ࠸ࠊ48 well ࣉ࣮ࣞࢺࡲ࠸ࡓ Neuro2a ⣽⬊ Lipofectamine Plus reagent (Life Technologies) ࢆ⏝࠸࡚ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥ ࢆ⾜ࡗࡓ (58)ࠋࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡽ 24 㛫ᚋࠊ⣽⬊ࢆ Tm (2 Pg/mL)ࠊforskolin (10 PM)ࠊPMA (0.1 PM) ࡲࡓࡣࠊ⁐፹࡛࠶ࡿ DMSO (dimethyl sulfoxide) ࡢࡳࢆྵࢇࡔ ᇵᆅ࡛ 12 㛫ฎ⌮ࡋࡓࠋࣉ࣮ࣟࣔࢱ࣮άᛶᑐࡍࡿ ATF4 ᙉไⓎ⌧ࡢຠᯝࢆゎᯒࡍ ࡿሙྜࡣࠊATF4 Ⓨ⌧ࢥࣥࢫࢺࣛࢡࢺ (0.01 Pg)ࠊࡲࡓࡣࠊ✵࣋ࢡࢱ࣮ (mock) (0.01 Pg) ࢆ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺࣛࢡࢺඹࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࠊ36 㛫ᇵ㣴ࡋࡓࠋྛ᮲ ௳࡛ᇵ㣴ࡋࡓᚋࠊ1 x passive lysis buffer (Promega) ࢆ⏝࠸࡚⣽⬊ࢆᐊ ࡛ 15 ศ㛫⁐ ゎࡋࠊ㐲ᚰᚋࠊୖΎࢆ ᐃ⏝࠸ࡓࠋྛ⣽⬊⁐ゎ≀ࡢࣉ࣮ࣟࣔࢱ࣮άᛶࡣ Dual-Luciferase assay system (Promega) ࢆ⏝࠸࡚」ᩘᅇ ᐃࡋࡓࠋࡑࢀࡒࢀࡢࣉ࣮ࣟࣔࢱ ࣮άᛶࡣྠࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࢆ⾜ࡗࡓ࣑࢘ࢩࢱࢣࣝࢩࣇ࢙࣮ࣛࢮࡢάᛶ࡛ ᶆ‽ࡋࠊ┦ᑐⓗ࡞άᛶࡋ࡚♧ࡋࡓࠋᐇ㦂ࡣ」ᩘᅇ⾜࠸ࠊ⌧ᛶࢆ☜ㄆࡋࡓࠋ
2-2-6. ࢙࢘ࢫࢱࣥࣈࣟࢵࢺἲ
⣽⬊ࡣ homogenate buffer [20 mM Tris-HCl (pH 8.0)ࠊ137 mM NaClࠊ2 mM EDTA (ethylenediaminetetraacetic acid)ࠊ10% ࢢࣜࢭ࣮ࣟࣝࠊ1% Triton X-100ࠊ1 mM PMSF (phenylmethylsulfonyl fluoride)ࠊ10 Pg/mL ࣟ࣌ࣉࢳࣥࠊ10 Pg/mL ࣉࢫࢱࢳࣥ A ] ࡛⁐ゎࡋࡓࠋྛ⣽⬊⁐ゎ≀ࢆ Bradford ἲ࡚ࠊࢱࣥࣃࢡ㔞 ᐃᚋࠊྛ⣽⬊⁐ゎ≀ࡣ SDS (sodium dodecyl sulfate) Laemmli sample buffer [62.5 mM Tris-HCl (pH 6.8)ࠊ2% SDSࠊ10% ࢢࣜࢭ࣮ࣟࣝ] ΰࡋࡓࠋ➼㔞ࡢ⣽⬊⁐ゎ≀ࢆ 12.5% ࣏ࣜࢡ࣑ࣜࣝ ࢻ㟁ẼὋືࢤࣝࢆ⏝࠸࡚ศ㞳ࡋࠊpolyvinylidene difluoride membranes (Merck Millipore) ㌿ࡋࡓࠋࡑࡋ࡚ࠊChac1 (1:1,000)ࠊActin (1:5,000) ᑐࡍࡿᢠయࢆ⏝࠸࡚ࠊenhanced chemiluminescence (GE Healthcare Bioscience) ࡼࡗ࡚┠ⓗࡢࣂࣥࢻࢆ᳨ฟࡋࡓࠋᐇ 㦂ࡣ」ᩘᅇ⾜࠸ࠊ⌧ᛶࢆ☜ㄆࡋࡓࠋ
2-2-7. ⤫ィゎᯒ
⤖ᯝࡣ♧ࡋ࡚࠶ࡿᅇᩘࡢᖹᆒ s ᶆ‽೫ᕪ࡛⾲ࡋࡓࠋ⤫ィゎᯒࡣ one way-ANOVA ࡼࡾ್ࢆゎᯒࡋࠊFischer’s PLSD ᳨ᐃࢆ⾜ࡗࡓࠋ*p < 0.05 ࡣ⤫ィⓗ᭷ពࡳ࡞ࡍࠋ
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2-3. ⤖ᯝ
2-3-1. Neuro2a ⣽⬊࠾ࡅࡿ ER ࢫࢺࣞࢫㄏᑟᛶࡢ Chac1 mRNA Ⓨ⌧ୖ᪼ ᪂つࡢ ER ࢫࢺࣞࢫㄏᑟᛶࡢ㑇ఏᏊ࡛࠶ࡿ Chac1 ࡢⓎ⌧ࡣ≉ᐃࡢ⌮Ꮫⓗ≧ἣୗ ࡛ୖ᪼ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ (43-49ࠊ52)ࠋMungrue ࡽࡣ Chac1 ࡀ ATF4 ࡢୗ ὶࡢ㑇ఏᏊ࡛࠶ࡿࡇࢆሗ࿌ࡋ࡚࠸ࡿࡀࠊER ࢫࢺࣞࢫୗ࠾ࡅࡿ㌿ไᚚࡢṇ☜࡞ᶵ ᵓࡘ࠸࡚ࡣࡲࡔゎᯒࡉࢀ࡚࠸࡞࠸ (43)ࠋ௨๓ࠊᡃࠎࡣ CRELD2 (cysteine-rich with EGF-like domains 2)ࠊMANF (mesencephalic astrocyte-derived neurotrophic factor) 㑇 ఏᏊࡢⓎ⌧ࡀ Tg ࡛ฎ⌮ࡋࡓ Neuro2a ⣽⬊࠾࠸࡚ୖ᪼ࡍࡿࡇࢆ DNA ࣞࡢ ⤖ᯝᇶ࡙࠸࡚ゎᯒࡋࡓ (58ࠊ59)ࠋࡇࡢ DNA ࣞゎᯒ࡛ࡣ Chac1 ࡶ Tg ㄏᑟᛶ 㑇ఏᏊྵࡲࢀ࡚࠸ࡓࡇࡽࠊNeuro2a ⣽⬊࠾ࡅࡿ ER ࢫࢺࣞࢫㄏᑟᛶࡢ Chac1 mRNA Ⓨ⌧ࢆ RT-PCR ἲࢆ⏝࠸࡚ゎᯒࡋࡓࠋᅗ 2-1 (A) ♧ࡍࡼ࠺ࠊTg ฎ⌮ࡣ 㛫౫Ꮡⓗ Chac1ࠊGADD153ࠊGRP78 mRNA Ⓨ⌧ୖ᪼ࢆᘬࡁ㉳ࡇࡋࡓࠋG3PDH mRNA Ⓨ⌧࡛ࣞ࣋ࣝᶆ‽ࡋࡓ Chac1 mRNA Ⓨ⌧ࡣ Tg ฎ⌮ᚋࠊ4ࠊ8ࠊ12 㛫࡛ࡑ ࢀࡒࢀ 1.5ࠊ1.8ࠊ2.3 ಸቑຍࡋࡓࠋࡢ ER ࢫࢺࣞࢫㄏᑟᛶࡢ⸆࡛࠶ࡿ TmࠊBFA ฎ ⌮࡛ࡶGADD153ࠊGRP78 mRNA ࢆྠᵝቑຍࡉࡏࠊChac1 mRNA ࡣࡑࢀࡒࢀ 1.7ࠊ 1.5 ಸୖ᪼ࡋࡓ (ᅗ 2-1 (B))ࠋࡋࡋࠊ⾑ΎḞஈ᮲௳࡛ࡣኚࡋ࡞ࡗࡓࠋ
22 ᅗ
ᅗ2-1. Neuro2a ⣽⬊࠾ࡅࡿ ER ࢫࢺࣞࢫㄏᑟᛶࡢ Chac1 mRNA Ⓨ⌧ୖ᪼
(A) Neuro2a ⣽⬊ࢆ Tg (0.1 PM) ࡚グ㍕ࡢ㛫ฎ⌮ࡋࡓࠋ(B) Neuro2a ⣽⬊ࢆ Tg (0.1 PM)ࠊTm (2 Pg/mL)ࠊBFA (5 Pg/mL)ࠊ⾑ΎḞஈ (SF)ࠊ⁐፹࡛࠶ࡿ DMSO (con) ࡛ 8 㛫 ฎ⌮ࡋࡓࠋࡑࢀࡒࢀࡢ⣽⬊ࢧࣥࣉࣝࡽᢳฟࡋࡓ RNA ࢆ⏝࠸࡚ RT-PCR ἲࢆ⾜ࡗࡓࠋ
ᅗ2-2. ࣐࢘ࢫࣄࢺ㛫࡛ࡢ Chac1 㑇ఏᏊࡢࣉ࣮ࣟࣔࢱ࣮㓄ิࡢẚ㍑㻌
࣐࢘ࢫࣄࢺ㛫࡛ಖᏑࡉࢀ࡚࠸ࡿ Chac1 㑇ఏᏊࡢࣉ࣮ࣟࣔࢱ࣮㓄ิࡣࢫࢱࣜࢫࢡ (*) ࡛ࠊ᥎ᐃୖࡢ㌿㛤ጞⅬࡣ▮༳࡛♧ࡋࡓࠋ࣐࢘ࢫ (NM_026929)ࠊࣄࢺ (NM_024111) ࡢ Chac1 㑇ఏᏊ㓄ิࡣࡑࢀࡒࢀ NCBI ࢹ࣮ࢱ࣮࣋ࢫࡼࡾྲྀᚓࡋࡓࠋ
㻌
23 2-3-2. Neuro2a ⣽⬊࠾ࡅࡿ ER ࢫࢺࣞࢫㄏᑟᛶࡢ࣐࢘ࢫ Chac1 ࣉ࣮ࣟࣔࢱ࣮ά ᛶゎᯒ ᅗ2-2 ♧ࡍࡼ࠺ࠊ࣐࢘ࢫ Chac1 ࣉ࣮ࣟࣔࢱ࣮㡿ᇦࡣࣄࢺ Chac1 ࣉ࣮ࣟࣔࢱ࣮ 㡿ᇦẚ㍑ࡋ࡚ⰋࡃಖᏑࡉࢀ࡚࠸ࡿࠋࡑࡢ㡿ᇦࡣ ATF4 ࡸࡑࡢ㛵㐃ࡍࡿ㌿ᅉᏊ ࡼࡗ࡚ㄆ㆑ࡉࢀࡿ CREB/ATFࠊAARE 㓄ิࡢࡼ࠺࡞㧗࠸┦ྠᛶࢆᣢࡗࡓ≉ᚩⓗ࡞㓄ิ ࡀྵࡲࢀ࡚࠸ࡿࠋChac1 ࣉ࣮ࣟࣔࢱ࣮άᛶไᚚ࠾ࡅࡿࡇࢀࡽࡢ㓄ิࡢᙺࢆゎᯒࡍ ࡿࡓࡵࠊ㛗ࡉࡢ␗࡞ࡿ࣐࢘ࢫ Chac1 ࣉ࣮ࣟࣔࢱ࣮㡿ᇦ࠾ࡼࡧ CREB/ATF ࡸ AARE 㓄ิኚ␗ࢆᣢࡘ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺࣛࢡࢺࢆస〇ࡋࠊNeuro2a ⣽⬊ࢺࣛࣥࢫࣇ࢙ ࢡࢩࣙࣥࡋࡓࠋࡑࡢ⤖ᯝࠊCREB/ATFࠊAARE 㓄ิࢆྵࡴ Chac1 ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺ ࣛࢡࢺ (-334/+112ࠊ-262/+112) ࡣ୧㓄ิࢆḞ࠸ࡓࡶࡢ (-136/+112) ẚ㍑ࡋ࡚ࠊࡼࡾ 㧗࠸ᇶ♏㌿άᛶࢆᣢࡕࠊTm ฎ⌮ (ᅗ 2-3 (A)) ࡸ ATF4 ᙉไⓎ⌧ (ᅗ 2-3 (B)) ᛂ ⟅ࡋࡓࠋࡲࡓࠊCREB/ATF 㓄ิࢆḞ࠸ࡓ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺࣛࢡࢺ (-210/+112) ࡛ࡣࠊ ᇶ♏㌿άᛶࡀ 50% ௨ୖῶᑡࡋࡓࡀࠊୖグࡢ 2 ࡘࡢฎ⌮ᑐࡋ࡚᭷ព࡞ᛂ⟅ࢆ♧ࡋ ࡓࠋCREB/ATF ཬࡧ AARE 㓄ิࢆḞ࠸ࡓ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺࣛࢡࢺ (-136/+112) ࡣ ᇶ♏㌿άᛶࡀࡑࢀࡽࡢ㓄ิࢆྵࡴࡶࡢ (-334/+112ࠊ-262/+112) ẚ㍑ࡋ࡚ 10 ศࡢ 1 ௨ୗ࡞ࡾࠊࡉࡽ Tm ฎ⌮ࠊATF4 ᙉไⓎ⌧ᑐࡍࡿᛂ⟅ᛶࡀᾘኻࡋࡓࠋ
24
ᅗ2-3. Neuro2a ⣽⬊࠾ࡅࡿ Tm ฎ⌮ ATF4 ᙉไⓎ⌧ࡼࡿ Chac1 ࣉ࣮ࣟࣔࢱ࣮
άᛶゎᯒ
(A) Neuro2a ⣽⬊ pGL3b ࡲࡓࡣྛ࣐࢘ࢫ Chac1 ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺࣛࢡࢺࢆࢺࣛࣥ ࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ24 㛫ᚋࠊTm (2 Pg/mL) ࡲࡓࡣ⁐፹࡛࠶ࡿ DMSO (-) ࡛ 12 㛫 ฎ⌮ࡋࠊࣉ࣮ࣟࣔࢱ࣮άᛶࢆ ᐃࡋࡓࠋ(B)Neuro2a ⣽⬊ pGL3b ࡲࡓࡣྛ࣐࢘ࢫ Chac1 ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺࣛࢡࢺ ATF4 Ⓨ⌧ࢥࣥࢫࢺࣛࢡࢺࡲࡓࡣ✵࣋ࢡࢱ࣮ (mock) ࢆඹ ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ36 㛫ᚋࠊࣉ࣮ࣟࣔࢱ࣮άᛶࢆ ᐃࡋࡓࠋ⤖ᯝࡣ␗࡞ࡿ 3 wells ࡼࡾᚓࡽࢀࡓ್ࡢᖹᆒ s ᶆ‽೫ᕪ࡛⾲ࡋࠊᮍฎ⌮ࡢ pGL3b ࣋ࢡࢱ࣮ࡢ್࡛ᶆ‽ ࡋࡓ (*p < 0.05)ࠋ
㻌
ḟ࠸࡛ࠊChac1 mRNA ࡢⓎ⌧࠾ࡅࡿ CREB/ATF 㓄ิࢆࡍࡿ㌿ไᚚࡘ࠸࡚ ࡉࡽゎᯒࡍࡿࡓࡵࠊPKA (protein kinase A)-CREB ࢝ࢫࢣ࣮ࢻάᛶ࡛࠶ࡿ forskolin ࡛ Neuro2a ⣽⬊ࢆฎ⌮ࡋࡓࠋணഛᐇ㦂ࡼࡾࠊforskolin ฎ⌮ࡣ CREB ࡢࣜࣥ 㓟ࢆㄏᑟࡋࡓࡀࠊChac1 mRNA Ⓨ⌧ㄏᑟ Chac1 ࣉ࣮ࣟࣔࢱ࣮άᛶୖ᪼ࢆᘬࡁ㉳ ࡇࡉ࡞ࡗࡓ (ᅗ 2-4 (A)ࠊ(B))ࠋࡲࡓࠊChac1 ࡢ㌿ไᚚࡘ࠸࡚ࠊPKC (protein kinase C) άᛶ࡛࠶ࡿ PMA ࡢຠᯝࡶゎᯒࡋࡓࡀࠊmRNA Ⓨ⌧ࠊࣉ࣮ࣟࣔࢱ࣮άᛶᙳ㡪 ࢆཬࡰࡉ࡞ࡗࡓ (ᅗ 2-4 (A)ࠊ(C))ࠋ
25
ᅗ 2-4. Neuro2a ⣽ ⬊ ࠾ ࡅ ࡿ Chac1 mRNA Ⓨ ⌧ ࣉ ࣟ ࣔ ࣮ ࢱ ࣮ ά ᛶ ᑐ ࡍ ࡿ ForskolinࠊPMA ฎ⌮ࡢຠᯝ
(A)Neuro2a ⣽⬊ࢆ Tm (2 Pg/mL)ࠊforskolin (F) (10 PM)ࠊPMA (0.1 PM) ࡲࡓࡣ⁐፹࡛࠶ ࡿ DMSO (con) ࡛ 12 㛫ฎ⌮ࡋࡓࠋࡑࢀࡒࢀࡢ⣽⬊ࢧࣥࣉࣝࡽᢳฟࡋࡓ RNA ࢆ⏝ ࠸࡚ RT-PCR ἲࢆ⾜ࡗࡓࠋ(B) Neuro2a ⣽⬊ pGL3b ࡲࡓࡣྛ࣐࢘ࢫ Chac1 ࣏࣮ࣞࢱ ࣮ࢥࣥࢫࢺࣛࢡࢺࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ24 㛫ᚋࠊforskolin (F) (10 PM)ࠊTm (2 Pg/mL) ࡲࡓࡣ DMSO (-) ࡛ 12 㛫ฎ⌮ࡋࠊࣉ࣮ࣟࣔࢱ࣮άᛶࢆ ᐃࡋࡓࠋ(C) Neuro2a ⣽⬊ pGL3b ࡲࡓࡣྛ࣐࢘ࢫ Chac1 ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺࣛࢡࢺࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙ ࣥࡋࡓࠋ24 㛫ᚋࠊPMA (0.1 PM) ࡲࡓࡣ DMSO (-) ࡛ 12 㛫ฎ⌮ࡋࠊࣉ࣮ࣟࣔࢱ࣮ά ᛶࢆ ᐃࡋࡓࠋ⤖ᯝࡣ␗࡞ࡿ 3 wells ࡼࡾᚓࡽࢀࡓ್ࡢᖹᆒ s ᶆ‽೫ᕪ࡛⾲ࡋࠊᮍฎ⌮ࡢ pGL3b ࣋ࢡࢱ࣮ࡢ್࡛ᶆ‽ࡋࡓ (*p < 0.05)ࠋ
26 ER ࢫࢺࣞࢫㄏᑟᛶࡢ Chac1 Ⓨ⌧࠾ࡅࡿ CREB/ATFࠊAARE 㓄ิࡢᙺࡘ࠸ ࡚ࡼࡾヲࡋࡃ⌮ゎࡍࡿࡓࡵࠊ୧㓄ิኚ␗ࢆྵࡴ Chac1 ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺࣛࢡࢺ ࢆ⏝࠸࡚ࣉ࣮ࣟࣔࢱ࣮άᛶࢆゎᯒࡋࡓࠋᅗ2-5 (A) ♧ࡍࡼ࠺ࠊኚ␗ᆺ࣏࣮ࣞࢱ࣮ࢥ ࣥࢫࢺࣛࢡࢺ (-262/+112 m1ࠊm2) ࡣ㔝⏕ᆺࡢࡶࡢ (-262/+112) ẚ㍑ࡋ࡚ᇶ♏㌿ άᛶࡀపࡗࡓࠋࡋࡋࠊࡇࢀࡽࡢኚ␗ᆺ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺࣛࢡࢺࡣ Tm ฎ⌮ࡸ ATF4 ᙉไⓎ⌧ᑐࡋࠊ᭷ពᛂ⟅ࡋࡓ (ᅗ 2-5 (B)ࠊ(C))ࠋ୧㓄ิኚ␗ᆺ࣏࣮ࣞࢱ࣮ࢥ ࣥࢫࢺࣛࢡࢺ (-262/+112 m3) ࡛ࡣ୧㓄ิࢆḞ࠸ࡓࡶࡢ (-136/+112) ྠᵝ่⃭ ᑐࡍࡿᛂ⟅ᛶࡀᾘኻࡋ࡚࠸ࡓࠋࡲࡓࠊAARE 㓄ิࡢࡳࢆྵࡴ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺࣛࢡࢺ ࡢ AARE 㓄ิᑐࡍࡿኚ␗ (-210/+112 m1ࠊm2ࠊm3) ࡣᇶ♏㌿άᛶࢆῶᑡࡉࡏࠊTm ฎ⌮ࡸ ATF4 ᙉไⓎ⌧ᛂ⟅ࡋ࡞ࡃ࡞ࡗࡓ (ᅗ 2-5 (D)ࠊ(E)ࠊ(F))ࠋ
27 ᅗ
ᅗ2-5. Tm ฎ⌮ࠊATF4 ᙉไⓎ⌧ᑐࡍࡿኚ␗ᆺ࣐࢘ࢫ Chac1 ࣉ࣮ࣟࣔࢱ࣮ࡢᛂ⟅ᛶ
ࡢゎᯒ
㻌
(A) ࣐࢘ࢫ Chac1 ࣉ࣮ࣟࣔࢱ࣮ෆࡢ CREB/ATFࠊAARE 㓄ิ (ᩥᏐ) ࠾ࡼࡧࡑࡢኚ␗ (ᑠᩥᏐ) ࢆ♧ࡍࠋ(B) Neuro2a ⣽⬊ pGL3b ࡲࡓࡣྛ࣐࢘ࢫ Chac1 ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺ ࣛࢡࢺࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ24 㛫ᚋࠊTm (2 Pg/mL) ࡲࡓࡣ⁐፹࡛࠶ࡿ DMSO (-) ࡛ 12 㛫ฎ⌮ࡋࠊࣉ࣮ࣟࣔࢱ࣮άᛶࢆ ᐃࡋࡓࠋ(C) Neuro2a ⣽⬊ pGL3b ࡲࡓࡣ ྛ࣐࢘ࢫ Chac1 ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺࣛࢡࢺ ATF4 Ⓨ⌧ࢥࣥࢫࢺࣛࢡࢺࡲࡓࡣ✵࣋ࢡࢱ ࣮ (mock) ࢆඹࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ36 㛫ᚋࠊࣉ࣮ࣟࣔࢱ࣮άᛶࢆ ᐃࡋࡓࠋ (D) ࣐࢘ࢫ Chac1 ࣉ࣮ࣟࣔࢱ࣮ෆࡢ AARE 㓄ิ (ᩥᏐ) ࠾ࡼࡧࡑࡢኚ␗ (ᑠᩥᏐ) ࢆ ♧ࡍࠋ(E) Neuro2a ⣽⬊ pGL3b ࡲࡓࡣྛ࣐࢘ࢫ Chac1 ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺࣛࢡࢺࢆࢺ ࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ24 㛫ᚋࠊTm (2 Pg/mL) ࡲࡓࡣ⁐፹࡛࠶ࡿ DMSO (-) ࡛ 12 㛫ฎ⌮ࡋࠊࣉ࣮ࣟࣔࢱ࣮άᛶࢆ ᐃࡋࡓࠋ(F) Neuro2a ⣽⬊ pGL3b ࡲࡓࡣྛ࣐࢘ࢫ Chac1 ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺࣛࢡࢺ ATF4 Ⓨ⌧ࢥࣥࢫࢺࣛࢡࢺࡲࡓࡣ✵࣋ࢡࢱ࣮ (mock) ࢆඹࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ36 㛫ᚋࠊࣉ࣮ࣟࣔࢱ࣮άᛶࢆ ᐃࡋࡓࠋ⤖ᯝࡣ␗࡞ ࡿ 3 wells ࡼࡾᚓࡽࢀࡓ್ࡢᖹᆒ s ᶆ‽೫ᕪ࡛⾲ࡋࠊᮍฎ⌮ࡢ pGL3b ࣋ࢡࢱ࣮ࡢ್࡛ ᶆ‽ࡋࡓ (*p < 0.05)ࠋ
㻌
28 2-3-3. Neuro2a ⣽⬊࠾ࡅࡿࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ࢩࢫࢸ࣒ࢆࡋࡓ Chac1 ⩻ヂᚋไᚚ ࣐࢘ࢫ Chac1 㑇ఏᏊࡢ㌿ไᚚゎᯒຍ࠼࡚ࠊ࣐࢘ࢫ Chac1 ࢱࣥࣃࢡ㉁Ⓨ⌧ࡘ ࠸࡚ゎᯒࢆ⾜ࡗࡓࠋER ࢫࢺࣞࢫㄏᑟ (TgࠊTm) ฎ⌮ࡋࡓ Neuro2a ⣽⬊࠾ࡅࡿෆ ᅉᛶ Chac1 ࢱࣥࣃࢡ㉁ࡣ᳨ฟ࡛ࡁ࡞ࡗࡓࡓࡵࠊChac1 ࢆᙉไⓎ⌧ࡍࡿࡇ࡛ゎᯒ ࢆ㐍ࡵࡓࠋෆᅉᛶ Chac1 mRNA ࡣ TgࠊTm ฎ⌮࡛ኚࡋࡓࡀࠊMG132 ฎ⌮࡛ࡣⴭ ࡋ࠸ᙳ㡪ࢆཷࡅ࡞ࡗࡓ (ᅗ 2-6 (A))ࠋ୍᪉ࠊChac1 ࡣ Chac1 Ⓨ⌧ࢥࣥࢫࢺࣛࢡࢺࢆ ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋ࡚ࡽ 24 㛫ᚋࠊࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ศゎ⣔㜼ᐖ ࡛࠶ࡿ MG132 ࡛ 12 㛫ฎ⌮ࡋࡓ⣽⬊࠾࠸᳨࡚ฟࡉࢀࡓ (ᅗ 2-6 (B))ࠋ ᅗ ᅗ2-6. Neuro2a ⣽⬊࠾ࡅࡿ MG132 ฎ⌮ࡼࡿ Chac1 ࢱࣥࣃࢡ㉁Ⓨ⌧ୖ᪼
㻌
(A)Neuro2a ⣽⬊ࢆ Tg (0.1 PM)ࠊTm (2 Pg/mL)ࠊMG132 (MG) (20 PM) ࡲࡓࡣ⁐፹࡛࠶ࡿ DMSO (con) ࡛ 12 㛫ฎ⌮ࡋࡓࠋࡑࢀࡒࢀࡢ⣽⬊ࢧࣥࣉࣝࡽᢳฟࡋࡓ RNA ࢆ⏝࠸ ࡚ RT-PCR ἲࢆ⾜ࡗࡓࠋ(B)Neuro2a ⣽⬊࣐࢘ࢫ Chac1 Ⓨ⌧ࢥࣥࢫࢺࣛࢡࢺࢆࢺࣛࣥ ࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ24 㛫ᚋࠊTg (0.1 PM)ࠊTm (2 Pg/mL)ࠊMG132 (MG) (20 PM) ࡲࡓ ࡣ DMSO (con) ࡛ 12 㛫ฎ⌮ࡋࡓࠋࡑࡋ࡚ࠊ⣽⬊⁐ゎ≀ࢆ Chac1 ࡲࡓࡣ Actin ᢠయࢆ ⏝࠸࡚࢙࢘ࢫࢱࣥࣈࣟࢵࢺἲࡼࡾゎᯒࡋࡓࠋ㻌
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2-4. ⪃ᐹ
HAEC ࢆࢸ࣮࣒ࣟᛶື⬦◳ࡢᕢ୰࡛ቑຍࡍࡿ㓟ࣜࣥ⬡㉁࡛࠶ࡿ ox-PAPC ่࡛⃭ࡋࡓㄏᑟࡉࢀࡿ㑇ఏᏊࡋ࡚ Chac1 ࡀྠᐃࡉࢀ࡚௨㝆ࠊᵝࠎ࡞ࣔࢹ ࣝ࠾࠸࡚ Chac1 mRNA ࡢⓎ⌧ㄏᑟࡀሗ࿌ࡉࢀ࡚ࡁࡓ (43-49ࠊ52)ࠋᡃࠎࡣ Tg ㄏᑟ ᛶࡢ ER ࢫࢺࣞࢫࡀ Neuro2a ⣽⬊࠾࠸࡚ࡶ Chac1 mRNA Ⓨ⌧ࣞ࣋ࣝࢆቑຍࡉࡏ ࡿࡇࢆ DNA ࣞ RT-PCR ἲࢆ⏝࠸࡚♧ࡋࡓࠋࡲࡓࠊChac1 Ⓨ⌧ࡀ㌿࠾ࡼࡧ ⩻ヂᚋࡢ୧᪉࡛ไᚚࡉࢀ࡚࠸ࡿࡇࢆ᫂ࡽࡋࡓࠋNeuro2a ⣽⬊࠾ࡅࡿ Chac1 mRNA ࡢⓎ⌧ࣃࢱ࣮ࣥࡣ GADD153 (60)ࠊGRP78 (61ࠊ62) ࡢࡼ࠺࡞᪤▱ࡢ ER ࢫࢺ ࣞࢫㄏᑟᛶࡢ㑇ఏᏊྠᵝ࡛࠶ࡗࡓ (ᅗ 2-1)ࠋ࣐࢘ࢫ Chac1 㑇ఏᏊࡢࣉ࣮ࣟࣔࢱ࣮ୖ ࡣ CREB/ATFࠊAARE ࡤࢀࡿ 2 ࡘࡢ≉ᚩⓗ࡞㓄ิࡀᏑᅾࡋࠊ࣐࢘ࢫࣄࢺྵࡵ ࡓࡢ⬨᳝ື≀㛫࡛ࡼࡃಖᏑࡉࢀ࡚࠸ࡿ (ᅗ 2-2)ࠋ୍⯡ⓗࠊ3 ࡘࡢ ER ᒁᅾࢫࢺࣞ ࢫࢭࣥࢧ࣮࡛࠶ࡿ PERK (2)ࠊIRE1 (63)ࠊATF6 (64) ࡣࠊER ᶵ⬟ࢆᘬࡁ㉳ࡇࡍᵝࠎ ࡞่⃭ࡼࡗ࡚ྠάᛶࡉࢀࠊୗὶࡢᶆⓗᅉᏊࡢⓎ⌧ࢆㄏᑟࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ ࡿ (65-68)ࠋάᛶࡋࡓ PERK ࡣ eIF2D ࢆࣜࣥ㓟ࡋࠊࡑࢀࡼࡾ ATF4 ࡢ⩻ヂࡀ ୖ᪼ࡍࡿࠋࡉࡽࠊࣜࣥ㓟ࡉࢀࡓ eIF2D ࡣయⓗṤࡢࢱࣥࣃࢡ㉁ࡢ⩻ヂࢆᢚไ ࡍࡿ (69)ࠋER ࢫࢺࣞࢫᛂ⟅ࡼࡗ࡚ቑຍࡋࡓ ATF4 ࡣ AARE 㓄ิࢆㄆ㆑ࡋࠊ GADD153 (60ࠊ70)ࠊTRIB3 (tribbles 3 homolog) (71)ࠊASNS (asparagine synthetase) (72) ࡞ࡢᶆⓗ㑇ఏᏊࡢ㌿ࢆㄏᑟࡍࡿࠋRomanski ࡽࡣࣄࢺ Chac1 ࣉ࣮ࣟࣔࢱ࣮ୖ ࡢ AARE 㓄ิୖὶ࠶ࡿ CREB/ATF 㓄ิࡀ ATF4 ᙉไⓎ⌧ᑐࡋⴭࡋࡃᛂ⟅ࡍࡿ ࡇࢆ HAEC ࡛ࡢࣉ࣮ࣟࣔࢱ࣮άᛶࢆゎᯒࡍࡿࡇࡼࡗ࡚ሗ࿌ࡋࡓ (50)ࠋᮏ◊✲࡛ ࡣࠊ࣐࢘ࢫ-ࣄࢺ㛫࡛ಖᏑࡉࢀ࡚࠸ࡿ࣐࢘ࢫ Chac1 ࣉ࣮ࣟࣔࢱ࣮ୖࡢ CREB/ATF 㓄ิ ࡣᇶ♏㌿άᛶ㔜せ࡛࠶ࡿࡀࠊ୍᪉ࠊࡑࡢ㓄ิࢆḞ࠸ࡓ 210/+112)ࠊኚ␗ᆺ (-262/+112 m1) ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺࣛࢡࢺࡀ Tm ฎ⌮ࡸ ATF4 ᙉไⓎ⌧ᛂ⟅ࡍࡿࡇ ࢆ♧ࡋࡓ (ᅗ 2-3ࠊᅗ 2-5 (B)ࠊ(C))ࠋࡉࡽࠊAARE 㓄ิኚ␗ᆺ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺ ࣛࢡࢺ (-262/+112 m2) ࡣࡑࢀࡽࡢ่⃭ᑐࡋ࡚ࠊCREB/ATF 㓄ิኚ␗ᆺ (-262/+112 m1) ྠᵝࡢᛂ⟅ࢆ♧ࡋࡓࠋࡇࢀࡽḞᦆᆺࠊኚ␗ᆺ Chac1 ࣉ࣮ࣟࣔࢱ࣮࣏࣮ࣞࢱ࣮ࢥ ࣥࢫࢺࣛࢡࢺࡢゎᯒ⤖ᯝࡽࠊCREB/ATFࠊAARE 㓄ิࡣࡑࢀࡒࢀ ER ࢫࢺࣞࢫ่⃭ ᛂ⟅ࡍࡿࡀࠊ┦ⓗാ࡞࠸ࡇࡀ♧၀ࡉࢀࡓࠋ୍᪉࡛ࠊࡇࢀࡽࡢ㓄ิࡣ Chac1 ࣉ࣮ࣟࣔࢱ࣮ࡢᇶ♏㌿άᛶࡢ⥔ᣢ㛵ࡋ࡚ࡣ༠ㄪࡋ࡚ാࡃྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋ࡞ࡐ ࡞ࡽࠊ୧㓄ิኚ␗ᆺ࣏࣮ࣞࢱ࣮ࢥࣥࢫࢺࣛࢡࢺ (-262/+112 m3) ࡣ∦᪉ࡢ㓄ิࡢኚ␗ᆺ
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ࢥࣥࢫࢺࣛࢡࢺ (-262/+112 m1ࠊm2) ẚ㍑ࡋ࡚ⴭࡋࡃࣉ࣮ࣟࣔࢱ࣮άᛶࡀపୗࡋࡓ ࡽ࡛࠶ࡿ (ᅗ 2-5 (B)ࠊ(C))ࠋ
CREB2 ࡶࡤࢀࡿ ATF4 ࡣ CREB/ATF ࣇ࣑࣮ࣜ㌿ᅉᏊࡢ୍ࡘ࡛࠶ࡿ (73)ࠋ CREB ࡣ PKA ࡸ MAPK (mitogen-activated protein kinase) ࢆྵࡴᵝࠎ࡞㓝⣲ࡼࡗ ࡚ࣜࣥ㓟ࡉࢀࠊᵝࠎ࡞㑇ఏᏊⓎ⌧ࢆไᚚࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ (74)ࠋForskolin ࠶ ࡿ࠸ࡣ PMA ฎ⌮ࡼࡿ Chac1 mRNA ࡢⓎ⌧ࣝࢩࣇ࢙࣮ࣛࢮ࣏࣮ࣞࢱ࣮ࢵࢭ ࡢ⤖ᯝࡽࠊPKAࠊPKC ౫Ꮡⓗ࡞ࢩࢢࢼࣝ⤒㊰ࡣ CERB/ATFࠊAARE 㓄ิࢆࡍࡿ Chac1 㑇ఏᏊࡢⓎ⌧ㄏᑟࡣ㛵ࡋ࡚࠸࡞࠸ࡇࡀศࡗࡓ (ᅗ 2-4)ࠋᚑࡗ࡚ࠊ࣐࢘ࢫ Chac1 㑇ఏᏊⓎ⌧ࡣ ER ࢫࢺࣞࢫㄏᑟᛶࡢ่⃭ࡼࡗ࡚ࠊ㑅ᢥⓗಁ㐍ࡉࢀࡿࡇࡀ ᫂ࡽ࡞ࡗࡓࠋ Chac1 mRNA Ⓨ⌧ࡣᵝࠎ࡞⌮Ꮫⓗ≧ἣ࠾࠸࡚ୖ᪼ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࡀ (43-49ࠊ52)ࠊChac1 ࢱࣥࣃࢡ㉁ࡢไᚚᶵᵓࡣ࠶ࡲࡾゎᯒࡉࢀ࡚࠸࡞࠸ࠋMungrue ࡽࡣ GFP (green fluorescent protein) ࢱࢢࡀຍࡉࢀࡓ Chac1 ࡢᙉไⓎ⌧ࡣ HEK293 ⣽ ⬊ࡸ HAEC ࣏ࢺ࣮ࢩࢫࢆᘬࡁ㉳ࡇࡍࡇࢆሗ࿌ࡋ࡚࠸ࡿ (43)ࠋࡲࡓࠊࡑࡢࠊ ࣏ࢺ࣮ࢩࢫάᛶᅉᏊ࡛࠶ࡿ࢝ࢫࣃ࣮ࢮ3ࠊAIF (apoptosis inducing factor) Ⓨ⌧ࡣୖ᪼ ࡋ ࠊ ࣏ ࢺ ࣮ ࢩ ࢫ 㜼 ᐖ ᅉ Ꮚ ࡛ ࠶ ࡿ TNFRSF6B (tumor necrosis factor receptor superfamily member 6B) Ⓨ⌧ࡣῶᑡࡍࡿࡇࢆぢฟࡋ࡚࠸ࡿࠋࡋࡋࠊChac1 ࢱࣥࣃ ࢡ㉁Ⓨ⌧ไᚚࡘ࠸࡚ࡣゎᯒࡉࢀ࡚࠸࡞࠸ࠋணഛᐇ㦂࠾࠸࡚ࠊMG132 ࡸࡢ ER ࢫ ࢺࣞࢫㄏᑟ࡛ Neuro2a ⣽⬊ࢆฎ⌮ࡋࡓࡢෆᅉᛶ Chac1 ࢱࣥࣃࢡ㉁ࡢ᳨ฟࢆヨ ࡳࡓࡀࠊᮏ◊✲ࡢ᮲௳࡛ࡣ≉␗ⓗ࡞ࣂࣥࢻࡣ᳨ฟ࡛ࡁ࡞ࡗࡓࠋࡑࡇ࡛ࠊࢱࢢࡢ࠸࡚ ࠸࡞࠸࣐࢘ࢫ Chac1 ࢆᙉไⓎ⌧ࡋࡓ Neuro2a ⣽⬊࠾࠸࡚ࠊࡑࡢࢱࣥࣃࢡⓎ⌧ࢆ᳨ ウࡋࡓࡇࢁ MG132 ࡛ฎ⌮ࡋࡓࡢࡳ᳨ฟ࡛ࡁࡓ (ᅗ 2-6 (B))ࠋ௨ୖࡢ⤖ᯝࡣࠊ Chac1 ࢱࣥࣃࢡ㉁ࡣࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ࢩࢫࢸ࣒࡛㎿㏿ศゎࡉࢀ࡚࠾ࡾࠊ⣽ ⬊ෆ࡛㠀ᖖᏳᐃ࡛࠶ࡿࡇࢆ♧၀ࡍࡿࡶࡢ࡛࠶ࡿࠋUb ࢆࡋࡓࣉࣟࢸࢯ࣮࣒ ࡼࡿࢱࣥࣃࢡ㉁ศゎࡣ␗ᖖࢱࣥࣃࢡ㉁㝖ཤ࣭࢚ࣥࢻࢧࢺ࣮ࢩࢫ࣭ࢩࢢࢼࣝఏ㐩࣭DNA ಟ࣭㌿ไᚚࢆྵࡴᗈ⠊࡞⣽⬊ෆᶵᵓ㛵ࢃࡗ࡚࠸ࡿ (14-17)ࠋྠᵝࠊER ࢫࢺࣞ ࢫᛂ⟅㛵ࢃࡿከࡃࡢᅉᏊࡢⓎ⌧ࡀࠊࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ࢩࢫࢸ࣒ࡼࡗ࡚㌿ ࠾ࡼࡧ⩻ヂ࡛ࣞ࣋ࣝไᚚࡉࢀ࡚࠸ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋJiang Wek ࡽࡣ࣐࢘ ࢫ⥺⥔ⱆ⣽⬊࠾࠸࡚ࣉࣟࢸࢯ࣮࣒㜼ᐖࡀ ATF4 mRNA Ⓨ⌧ࣞ࣋ࣝࢆኚࡉࡏࡿࡇ ࡞ࡃࠊATF4 ࢱࣥࣃࢡ㉁✚ࢆᑟࡃࡇࢆሗ࿌ࡋ࡚࠸ࡿ (75)ࠋᙼࡽࡣ MG132 ㄏᑟ
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ᛶࡢ ATF4 Ᏻᐃࣜࣥ㓟 eIF2D ࡼࡿ ATF4 ✚ࡢ⤖ᯝࡋ࡚ GADD153 mRNA ࢱࣥࣃࢡ㉁Ⓨ⌧ࣞ࣋ࣝࡀቑຍࡍࡿࡇࡶሗ࿌ࡋ࡚࠸ࡿࠋᮏ◊✲࡛ࡶࠊATF4 ࢱ ࣥࣃࢡ㉁ࡣ MG132 ࡛ฎ⌮ࡋࡓ Neuro2a ⣽⬊࠾࠸࡚ቑຍࡍࡿ࠸࠺⤖ᯝࢆணഛᐇ 㦂ࡽᚓ࡚࠸ࡿࠋࡇࡢ ATF4 ቑຍࡣ TgࠊTm ฎ⌮ࡼࡾࡶ MG132 ฎ⌮ࡼࡗ࡚ⴭࡋࡃ ቑຍࡋࡓࠋࡋࡋࠊMG132 ࡛ฎ⌮ࡋࡓ Neuro2a ⣽⬊࠾ࡅࡿ ATF4 ࢱࣥࣃࢡ㉁ࡢ ✚ࡣ Chac1 mRNA Ⓨ⌧ࣞ࣋ࣝᙳ㡪ࢆ࠼࡞ࡗࡓ (ᅗ 2-6 (A))ࠋࡲࡓࠊࡑࡢࡢ GADD153 mRNA Ⓨ⌧ࡣ⦆ࡸ࡞ୖ᪼ࡔࡗࡓࠋMG132 ࡛ฎ⌮ࡋࡓ Neuro2a ⣽⬊࠾ ࡅࡿ ATF4 ࢱࣥࣃࢡ㉁ࡢቑຍࡀࠊChac1 GADD153 mRNA Ⓨ⌧ẚࡋ࡞ࡗࡓ ᶵᵓࡘ࠸࡚ࡣ࡛᫂࠶ࡿࠋbZIP (basic leucine zipper domain) ࢆᣢࡘ ATF4 ࡢࡼ࠺ ࡞㌿ᅉᏊࡢ࠸ࡃࡘࡣྠᵝࡢᵓ㐀ࢆᣢࡘ㌿ᅉᏊ┦స⏝ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚ ࠸ ࡿ (76) ࠋ ATF4 ࡣ ᶆ ⓗ ࡞ ࡿ 㑇 ఏ Ꮚ ࡢ Ⓨ ⌧ ࢆ ㄏ ᑟ ࡍ ࡿ ࡓ ࡵ C/EBPE (CCAAT/enhancer-binding protein E)ࠊGADD153ࠊNrf2 (NF-E2-like basic leucine zipper transcriptional activator) ┦స⏝ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ (72ࠊ77ࠊ78)ࠋᚑࡗ࡚ࠊ ᵝࠎ࡞⣽⬊✀࠾ࡅࡿࡇࢀࡽᅉᏊࡢࡉࡽ࡞ࡿゎᯒࡀࠊChac1 ࡢ㌿ไᚚࢆ᫂ࡽࡍ ࡿࡢᚲせ࡛࠶ࡿ⪃࠼ࡽࢀࡿࠋ ௨ୖࡼࡾࠊࣝࢩࣇ࢙࣮ࣛࢮ࣏࣮ࣞࢱ࣮ࢵࢭࢆ⏝࠸ࡿࡇ࡛࣐࢘ࢫ Chac1 ࣉࣟࣔ ࣮ࢱ࣮ୖࡢ 2 ࡘࡢ ATF4 ᛂ⟅ᛶࡢ㓄ิࡋ࡚ࡢ CREB/ATFࠊAARE 㓄ิࢆึࡵ࡚ྠ ᐃࡋࡓࠋER ࢫࢺࣞࢫࠊ㓟ࢫࢺࣞࢫࠊ࢘ࣝࢫឤᰁࡼࡗ࡚άᛶࡍࡿከࡃࡢࢩࢢࢼ ࣝࡣ eIF2D ࡢࣜࣥ㓟ࢆࡍࡿ ATF4 ⤒㊰ࡢάᛶࡀሗ࿌ࡉࢀ࡚࠸ࡿ (79-82)ࠋᚑ ࡗ࡚ࠊ␗࡞ࡿ⣽⬊✀࠾ࡅࡿࡉࡽ࡞ࡿ Chac1 ࣉ࣮ࣟࣔࢱ࣮ࡢゎᯒࡀᵝࠎ࡞⣽⬊ᛂ⟅ࡸ ᝈ (⚄⤒ኚᛶᝈࠊࡀࢇࠊ⅖) ࡢ⌮ゎᐤࡍࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿ (33-36ࠊ38ࠊ 83ࠊ84)ࠋᮏ◊✲࡛ࡣࠊChac1 ࢱࣥࣃࢡ㉁ࡣ⣽⬊ෆ࡛Ᏻᐃ࡛࠶ࡾࠊࡑࡢ⣽⬊ෆྵ㔞ࡣ ࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ࢩࢫࢸ࣒ࡼࡗ࡚ཝᐦไᚚࡉࢀ࡚࠸ࡿࡇࡀ♧၀ࡉࢀࡓ (ᅗ 2-6 (B))ࠋ᭱㏆ࠊKumar ࡽࡀ࣏ࢺ࣮ࢩࢫㄏᑟᛶࡢ≉ᚩࢆᣢࡘ Chac1 ࡀࠊࡑࡢ J-glutamyl cyclotransferase άᛶࡼࡗ࡚ GSH ࢆ≉␗ⓗศゎࡍࡿࡇࢆሗ࿌ࡋࡓ (54)ࠋᚑࡗ࡚ࠊChac1 ࣉ࣮ࣟࣔࢱ࣮ࡘ࠸࡚ࡢᮏ◊✲ࡣ ER ࢫࢺࣞࢫࡀ⣽⬊ෆ㓟㑏 ඖ≧ែࡢ␗ᖖࢆᘬࡁ㉳ࡇࡍᶵᵓࢆࡅࡿࡶࡢ⪃࠼ࡽࢀࡿ (85)ࠋᚋࡢࡉࡽ࡞ࡿ Chac1 ࡢゎᯒࡀࠊeIF2D-ATF4 ⤒㊰ࢆࡋࡓࢫࢺࣞࢫᛂ⟅ࡼࡗ࡚ᘬࡁ㉳ࡇࡉࢀࡿ ᵝࠎ࡞ᝈࡢⓎ㐍⾜ࢆ㜵ࡄ᪂ࡓ࡞▱ぢࢆ࠼ࡿ⪃࠼ࡽࢀࡿࠋ
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➨
➨୕❶
Chac1 ࡢ⩻ヂ࠾ࡼࡧ⩻ヂᚋࡢไᚚᶵᵓࡢゎᯒ
3-1. ᢒ㘓 Chac1 ࡣ ER ࢫ ࢺ ࣞ ࢫ ㄏ ᑟ ᛶ ࡢ 㑇 ఏ Ꮚ ࡛ ࠶ ࡾ ࠊ GSH ࢆ ศ ゎ ࡍ ࡿ J-glutamyl cyclotransferase ࡋ࡚ࡢᶵ⬟ࢆᣢࡘࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ (43-49ࠊ54ࠊ55)ࠋᮏ◊✲ ࡛ࡣ Chac1 ࡢ⩻ヂᏳᐃᛶࡘ࠸࡚ゎᯒࡋࠊChac1 mRNA ෆࡢ 5’UTR Ꮡᅾࡍࡿ Kozak ᵝ㓄ิࡀ Chac1 ࡢ⩻ヂᐤࡍࡿࡇࢆぢฟࡋࡓࠋຍ࠼࡚ࠊ5’UTR ࢆྵࡲ࡞ ࠸ Chac1 Ⓨ⌧ࢥࣥࢫࢺࣛࢡࢺࡽࡣࠊChac1 ࢱࣥࣃࢡ㉁ࢥ࣮ࢻ㡿ᇦࡢ 2 ␒┠ࡢ ATG ࢥࢻࣥࡼࡾ⩻ヂࡉࢀࡓపศᏊᆺ Chac1 ('Chac1) ࡀ⏘⏕ࡉࢀࡿࡇࡀศࡗࡓࠋ ➨❶ࡼࡾᡃࠎࡣࠊࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ࢩࢫࢸ࣒ࡀ Chac1 ࢱࣥࣃࢡ㉁ࡢศゎ 㛵ࡋ࡚࠸ࡿࡇࢆ᫂ࡽࡋ࡚࠸ࡿࠋࡋࡋࠊChac1 Ⓨ⌧ࡣ Ub 㑇ఏᏊࢆඹⓎ⌧ ࡉࡏࡿࡇࡼࡗ࡚㢧ⴭୖ᪼ࡋࡓࠋࡉࡽࠊචỿ㝆ἲࢆ⏝࠸࡚ࠊUb ศᏊࡀ┤᥋ Chac1 ⤖ྜࡋ࡚࠸ࡿࡇࠊࣜࢪࣥṧᇶࢆ࡚ࣝࢠࢽࣥ⨨ࡋࡓኚ␗ᆺ Chac1 ࡶ Ub ࡉࢀ࡚࠸ࡿࡇࢆ᫂ࡽࡋࡓࠋ᭱ᚋࠊ㔝⏕ᆺ Chac1 ࡣ⣽⬊ෆ GSH ࣞ࣋ ࣝࢆῶᑡࡉࡏࡿࡀࠊ'Chac1 ࡣῶᑡࡉࡏ࡞࠸ࡇࢆ♧ࡋࡓࠋ௨ୖࡼࡾࠊChac1 ࢱࣥࣃࢡ ㉁Ⓨ⌧ࡣ 5’UTR ෆࡢ Kozak ᵝ㓄ิ Ub ௰ᛶࡢ⤒㊰ࡼࡗ࡚ࠊ⩻ヂ࠾ࡼࡧ⩻ヂᚋ ࡛ࣞ࣋ࣝไᚚࡉࢀ࡚࠸ࡿࡇࢆ᫂ࡽࡋࡓࠋChac1 ࢱࣥࣃࢡ㉁ࡢⓎ⌧ㄪ⠇࠾ࡅࡿ Ub ࡼࡿ᪉ྥᛶࡢไᚚࡣࠊែ⏕⌮Ꮫⓗ࡞≧ἣ࠾ࡅࡿ GSH ᜏᖖᛶࢆ⌮ゎࡍࡿୖ ࡛᪂ࡋ࠸▱ぢ࡞ࡿ⪃࠼ࡽࢀࡿࠋ33
3-2. ᮦᩱ᪉ἲ
3-2-1. ྛ✀⸆ᢠయ
CHX (cycloheximide) ࠊ Baf (bafilomycin A1) ࠊ ALLN (N-Acetyl-L-leucyl-L-leucyl-L- norleucinal) ࡣ Sigma-Aldrich ࡼࡾ㉎ධࡋࡓࠋSuc-LLVY-MCA ࡣ Peptide Institute ࡽ㉎ධࡋࡓࠋCMA (concanamycin A) ࡣ Wako ࡼࡾ㉎ධࡋࡓࠋc-MycࠊHAࠊLC-3 ᑐ ࡍࡿᢠయࡣࡑࢀࡒࢀ Santa Cruz BiotechnologyࠊClontech LaboratoriesࠊMedical & Biological Laboratories ࡽ㉎ධࡋࡓࠋ
3-2-2. ࣉࣛࢫ࣑ࢻࡢస〇
࣐࢘ࢫ Chac1 㑇ఏᏊ (NM_026929) ࡣ PCR ἲࡼࡗ࡚ C57/BL6 ࣐࢘ࢫ⬻⏤᮶ࡢ mRNA ࡽ RT-PCR ࡼࡗ࡚ cDNA ࢆቑᖜࡋࠊChac1 ࢱࣥࣃࢡ㉁ࢥ࣮ࢻ㡿ᇦ (+162/+833) ࢆ pcDNA3.1 ࣋ࢡࢱ࣮ࢡ࣮ࣟࢽࣥࢢࡋࡓ (pcDNA3.1-Chac1)ࠋc-Myc ᢠయࡼࡗ࡚ Chac1 ࢱࣥࣃࢡ㉁ࢆ᳨ฟࡍࡿࡓࡵࠊMyc ࢚ࣆࢺ࣮ࣉࢆࣥࢳࢭࣥࢫ ࣉ࣐࣮ࣛ (5’-TCACAGATCCTCTTCTGAGATGAGTTTTTGTTCGGTCAGTGCCAGA GGC-3’) ࢆ ⏝࠸ ࡚ PCR ἲ ࡼ ࡾ pcDNA3.1-Chac1 ࡢ C ᮎ ➃ ┤ ᥋ ຍ ࡋ ࠊ pcDNA3.1-Chac1-Myc (+162/+830; Chac1-Myc) ࢆస〇ࡋࡓ (ᅗ 3-1 (A))ࠋ5’UTR 㛗 (+1/+830; 5’-Chac1-Myc)ࠊ5’UTR ࡢ୍㒊 (+82/+830; '5’Chac1-Myc)ࠊ⩻ヂ㛤ጞⅬ┤๓ ࡢ Kozak ᵝ㓄ิ (+156/+830; Kozak-Chac1-Myc) ࢆྵࡴ Chac1 ࢥࣥࢫࢺࣛࢡࢺ 2 ␒┠ࡢ࣓ࢳ࢜ࢽࣥࡽ⩻ヂࡉࢀࡓపศᏊᆺ Chac1 ࢥࣥࢫࢺࣛࢡࢺ (+393/+830; 'Chac1-Myc) ࡣ pcDNA3.1-Chac1-Myc ࢆ㗪ᆺࡋ࡚ PCR ἲࡼࡗ࡚స〇ࡋࡓ (ᅗ 3-1 (A))ࠋࡉࡽࠊpcDNA3.1-Chac1-Myc ࢆ PCR ἲࡢ㗪ᆺࡋ࡚࣓ࢳ࢜ࢽࣥṧᇶࢆ ࢯࣟࢩࣥ⨨ࡍࡿࡇ࡛ࠊ3 ࡘࡢኚ␗ᆺࢥࣥࢫࢺࣛࢡࢺ (M1IࠊM78IࠊM187I; ᅗ 3-2 (C)) ࢆస〇ࡋࠊࡲࡓࠊpcDNA3.1-5’-Chac1-Myc ࢆ PCR ἲࡢ㗪ᆺࡋ࡚ࢥ࣮ࢻ㡿ᇦ ࡢ࡚ࡢࣜࢪࣥṧᇶࢆࣝࢠࢽࣥ⨨ࡍࡿࡇ࡛ࠊ5’-Chac1 (K0)-Myc (ᅗ 3-4 (A)) ࢆ స〇ࡋࡓࠋຍ࠼࡚ࠊpcDNA3.1-5’-Chac1-Myc (WTࠊK0)-Myc ࡽ PCR ἲࡼࡗ࡚⤊ Ṇࢥࢻࣥ 5’UTR ࢆ㝖ࡁࠊࡑࡢ PCR ⏘≀ࢆ pcDNA3.1-MycHis ࣋ࢡࢱ࣮ (Life Technologies) ࢡ࣮ࣟࢽࣥࢢࡍࡿࡇࡼࡗ࡚ Chac1 (WTࠊK0)-MycHis ࢆస〇ࡋࡓࠋ Chac1 ࢥ࣮ࢻ㡿ᇦࡢ 2-6 ࣑ࣀ㓟 2-26 ࣑ࣀ㓟ࢆḞ࠸ࡓ 2 ࡘࡢ N ᮎ➃Ḟᦆᆺ ࢥࣥࢫࢺࣛࢡࢺ (Kozak-Chac1 ('5)-MycࠊKozak-Chac1 ('25)-Myc) ࡣ pcDNA3.1-
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Kozak-Chac1-Myc ࢆ㗪ᆺࡋ࡚ PCR ἲࡼࡗ࡚స〇ࡋࡓ (ᅗ 3-5 (B))ࠋN ᮎ➃ Myc ࢱࢢࡢ࠸ࡓ Chac1 ࢥࣥࢫࢺࣛࢡࢺ (Myc-Chac1; ᅗ 3-5 (A)) ࢆస〇ࡍࡿࡓࡵ ࠊpcDNA3.1-Chac1 ࡢ Chac1 ࢥ ࣮ ࢻ 㡿 ᇦ ࢆ pCMV-Myc ࣋ ࢡ ࢱ ࣮ (Clontech Laboratories) ࢧࣈࢡ࣮ࣟࢽࣥࢢࡋ࡚ N ᮎ➃ Myc ࢚ࣆࢺ࣮ࣉࢆຍࡋࠊࡑࡢᚋࠊ pcDNA3.1 ࣋ࢡࢱ࣮ࢧࣈࢡ࣮ࣟࢽࣥࢢࡋࡓࠋMyc ࢱࢢෆࡢࣜࢪࣥࢆࣝࢠࢽࣥ⨨ ࡋࡓኚ␗ᆺ Chac1 (K0) ࢥࣥࢫࢺࣛࢡࢺ (+162/+830; Chac1 (K0)-Myc(KR)His) ࡣ Chac1 (K0)-MycHis ࢆ㗪ᆺࡋ࡚ PCR ἲࡼࡗ࡚స〇ࡋࡓࠋN ➃ HA ࢱࢢࡀຍ ࡉࢀࡓ㔝⏕ᆺ (HA-Ub (WT)) 48ࠊ63 ␒┠ࡢࣜࢪࣥࡢࡳࢆᣢࡘ༢୍ࣜࢪࣥኚ␗ᆺ Ub ࢥࣥࢫࢺࣛࢡࢺ (HA-Ub (K48ࠊK63)) ࡣ Dr. Kah-Leong LIM ࡼࡾ౪ࡉࢀࡓ (86)ࠋ
3-2-3. ⣽⬊ᇵ㣴⸆ฎ⌮
HEK293 ⣽⬊ࡣ 8% ࢘ࢩ⫾ඣ⾑Ύ 1% ࣌ࢽࢩࣜࣥ-ࢫࢺࣞࣉࢺ࣐ࢩࣥࢆྵࢇࡔ Dulbecco’s Modified Eagle’s Medium ࡼࡗ࡚ᇵ㣴ࡋࡓࠋࡇࡢ◊✲࠾ࡅࡿྛࢥࣥࢫࢺ ࣛ ࢡ ࢺࡢ ࢺ ࣛࣥ ࢫࣇ ࢙ࢡ ࢩ ࣙ ࣥ ࡣ ࣓࣮ ࣮࢝ ࡢㄝ ᫂ ᚑ ࠸ ࠊpolyethylenimine Max (Polysciences) ࢆ⏝࠸࡚⾜ࡗࡓ (87)ࠋHEK293 ⣽⬊ࡣ Tg (0.1 PM)ࠊTm (2 Pg/mL)ࠊ MG132 (20 PM)ࠊCHX (10 Pg/mL)ࠊBaf (50 nM)ࠊCMA (50 nM)ࠊALLN (10 PM) ࡚グ ㍕ࡢ㛫ฎ⌮ࡋࡓࠋ 3-2-4. RT-PCR TRIzol ࡚⣽⬊ࢆ⁐ゎᚋࠊࢡ࣒ࣟࣟ࣍ࣝࢆຍ࠼࡚ΰࡋࠊịୖ࡛ 10 ศ㟼⨨ࡋࡓࠋࡑ ࡋ࡚ࠊ15,000 rpmࠊ4Υ ࡛ 10 ศ㐲ᚰᚋࠊୖᒙࢆ୍ᐃ㔞⛣ࡋࠊࢯࣉࣟࣃࣀ࣮ࣝࢆຍ࠼ ࡚ΰࡋࡓࠋịୖ࡛ 15 ศᨺ⨨ᚋࠊ15,000 rpmࠊ4Υ ࡛ 10 ศ㐲ᚰࡋࡓࠋୖΎࢆ㝖ཤࡢ ᚋࠊ70% ࢚ࢱࣀ࣮ࣝࢆຍ࠼ࠊ8,500 rpmࠊ4Υ ࡛ 5 ศ㐲ᚰᚋࠊୖΎࢆ㝖ཤࡋࠊỿẊ≀ ࢆ RNA ࡋ࡚⏝ࡋࡓࠋࡑࡋ࡚ࠊDNase ฎ⌮ࢆ⾜ࡗࡓ RNA ࢆ 72Υ ࡛ 10 ศ ಖ ࡋࡓᚋࠊ RNA (0.5 Pg/10 PL)ࠊDTT (10 mM)ࠊdNTP (0.5 mM)ࠊrandom ninemers (0.05 Pg/10 PL) ࠊ RNaseOUT (2 U/10 PL) ࠊ prime superscript III RNase reverse transcriptase (25 U/10 PL) ࢆΰྜࡋࠊ42Υ ࡛ 60 ศಖ ࡍࡿࡇ࡛ cDNA ࡋࡓࠋ ≉ᐃࡢ㑇ఏᏊⓎ⌧ࡣ௨ୗ♧ࡍࣉ࣐࣮ࣛ࣌ (1 PM)ࠊdNTP (0.1 mM)ࠊrTaq (0.25 U/10 PL) ࢆྵࡴ PCR reaction mixture ࢆ࠺ࡇ࡛ቑᖜࡋࡓࠋእᅉᛶࡢ Chac1 mRNA
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ࢆ᳨ฟࡍࡿࡓࡵ T7 primerࠊChac1 antisense primer ࢆ⏝࠸ࠊG3PDH mRNA ࢆ᳨ฟ ࡍࡿࡓࡵࠊG3PDH sense primerࠊG3PDH antisense primer ࢆ⏝࠸ࡓࠋ
T7 primer; 5’-TAATACGACTCACTATAGGG-3’ࠊ
Chac1 antisense primer; 5’-CTGTGTGGCAATGACCTCTTC-3’ࠊ G3PDH sense primer; 5’-ACCACAGTCCATGCCATCAC-3’ࠊ G3PDH antisense primer; 5’-TCCACCACCCTGTTGCTGTA-3’
ᆺⓗ࡞ᛂ᮲௳ࡣ 96Υ ࡛ 30 ⛊ࠊ58Υ ࡛ 30 ⛊ࠊ72Υ ࡛ 30 ⛊࡛⾜ࡗࡓࠋ⤖ᯝ ࡣྛ㑇ఏᏊ㐺ษ࡞ࢧࢡࣝ (20 ࡽ 34 ࢧࢡࣝ) ࡛ቑᖜࡋࡓࡢࡶࡢࡀ♧ࡋ࡚࠶ࡿࠋ cDNA ቑᖜ⏘≀ࡣ 2.0% ࡢ࣮࢞ࣟࢫࢤࣝࢆ⏝࠸࡚㟁ẼὋືࡼࡗ࡚ศ㞳ࡋࠊ࢚ࢳࢪ ࣒࢘ࣈ࣐ࣟࢻ࡛ྍどࡋࡓࠋእᅉᛶ Chac1 㑇ఏᏊࡢ┦ᑐⓗ࡞ mRNA Ⓨ⌧ࣞ࣋ࣝࡣ Image J software (National Institutes of Health; NIH) ࢆ⏝࠸࡚ᩘ್ࡋࠊChac1-Myc ࡢ ್࡛ᶆ‽ࡋࡓࡶࡢࢆ♧ࡋࡓࠋᐇ㦂ࡣ」ᩘᅇ⾜࠸ࠊ⌧ᛶࢆ☜ㄆࡋࡓࠋ
3-2-5. ࢙࢘ࢫࢱࣥࣈࣟࢵࢺἲ
⣽⬊ࡣ homogenate buffer [20 mM Tris-HCl (pH 8.0)ࠊ137 mM NaClࠊ2 mM EDTAࠊ10% ࢢࣜࢭ࣮ࣟࣝࠊ1% Triton X-100ࠊ1 mM PMSFࠊ10 Pg/mL ࣟ࣌ࣉࢳࣥࠊ10 Pg/mL ࣉࢫࢱࢳࣥA ] ࡛⁐ゎࡋࡓࠋྛ⣽⬊⁐ゎ≀ࢆ Bradford ἲ࡚ࠊࢱࣥࣃࢡ㔞 ᐃᚋࠊྛ ⣽⬊⁐ゎ≀ࡣ SDS Laemmli sample buffer [62.5 mM Tris-HCl (pH 6.8)ࠊ2% SDSࠊ10% ࢢࣜࢭ࣮ࣟࣝ] ΰࡋࡓࠋ➼㔞ࡢ⣽⬊⁐ゎ≀ࢆ 12.5% ࡲࡓࡣ 15% ࣏ࣜࢡࣜࣝ ࣑ࢻ㟁ẼὋືࢤࣝࢆ⏝࠸࡚ศ㞳ࡋࠊpolyvinylidene difluoride membranes ㌿ࡋࡓࠋ ࡑࡋ࡚ࠊChac1 (1:1,000)ࠊc-Myc (1:1,000)ࠊHA (1:1,000)ࠊLC3 (1:3,000)ࠊActin (1:5,000) ᑐࡍࡿᢠయࢆ⏝࠸࡚ࠊECL Detection System ࡲࡓࡣ Western Blotting Substrate Plus (Thermo Fisher Scientific) ࡼࡗ࡚┠ⓗࡢࣂࣥࢻࢆ᳨ฟࡋࡓࠋࣂࣥࢻᙉᗘࡣ Image J software ࡼࡗ࡚ゎᯒࡋࡓࠋᐇ㦂ࡣ」ᩘᅇ⾜࠸ࠊ⌧ᛶࢆ☜ㄆࡋࡓࠋ
3-2-6. චỿ㝆ἲ
ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࢆ⾜ࡗࡓ 60 mm ࢹࢵࢩࣗୖࡢ HEK293 ⣽⬊ࢆᅇࡋࠊlysis buffer [20 mM Tris-HCl (pH8.0)ࠊ150 mMNaClࠊ1% Nonidet P-40ࠊ1 mM EDTAࠊ1 mM PMSFࠊ10 Pg/mL ࣟ࣌ࣉࢳࣥࠊ10 Pg/mL ࣉࢫࢱࢳࣥ A] ࡛⁐ゎࡋࡓࠋ⁐ゎ≀ࡣ 12,000 x g ࡛ 6 ศ㐲ᚰࡋࠊྍ⁐ᛶ⏬ศࢆᅇᚋࠊ2 Pg ࡢ c-Myc ᢠయ Protein G
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Sepharose (GE Healthcare Bioscience) ࢆ⏝࠸࡚චỿ㝆ࢆ⾜ࡗࡓࠋࣞࢪࣥ⤖ྜࡋࡓ ࢱࣥࣃࢡ㉁ࡣ wash buffer [20 mM Tris-HCl (pH 8.0)ࠊ150 mMNaClࠊ0.2% Nonidet P-40ࠊ1 mM EDTAࠊ1 mM PMSF] ࡛ 3 ᅇὙίᚋࠊSDS Laemmli sample buffer ᠱ⃮ ࡋࠊ࢙࢘ࢫࢱࣥࣈࣟࢵࢺἲࡼࡗ࡚ゎᯒࡋࡓࠋᐇ㦂ࡣ」ᩘᅇ⾜࠸ࠊ⌧ᛶࢆ☜ㄆࡋࡓࠋ
3-2-7. GSH 㔞ࡢ ᐃ
⣽⬊ෆࡢ GSH ྵ㔞ࡣ Hisson Hill ࡢ᪉ἲᚑ࠸ࠊ⺯ගἲࡼࡾᐃ㔞ࡋࡓ (88)ࠋ⡆ ₩㏙ࡿࠊᅇࡋࡓ⣽⬊ỿẊ≀ࡣ 5 mM EDTA 25% (w/v) ࣓ࢱࣜࣥ㓟⁐ᾮࢆྵ ࡴ 0.1 M sodium phosphate buffer (pH 8.0) ᠱ⃮ࡋࡓࠋ10,000 x g ࡛ 10 ศ㐲ᚰᚋࠊ ྛୖΎࢆ 5 mM EDTA 0.1% o-phthalaldehyde ࢆྵࡴ 0.1 M sodium phosphate buffer (pH 8.0) 15 ศᛂࡉࡏࡓࠋྛࢧࣥࣉࣝࡢ⺯ගᙉᗘࡣບ㉳Ἴ㛗 350 nm/⺯ග Ἴ㛗 420 nm ࡛ ᐃࡋࡓࠋᐇ㦂ࡣ」ᩘᅇ⾜࠸ࠊ⌧ᛶࢆ☜ㄆࡋࡓࠋ
3-2-8. ࣉࣟࢸࢯ࣮࣒άᛶ ᐃ
グ㍕ࡋࡓ᮲௳ࡢ HEK293 ⣽⬊࠾ࡅࡿ 26S ࣉࣟࢸࢯ࣮࣒άᛶࢆྵࡴ࢟ࣔࢺࣜࣉࢩ ࣥᵝࣉࣟࢸࢯ࣮࣒άᛶࡣ Mark FP ࡽࡢ᪉ἲᚑ࠸ࠊ⺯ගἲࡼࡾᐃ㔞ࡋࡓ (89)ࠋ⡆ ₩㏙ࡿࠊᅇࡋࡓ⣽⬊ỿẊ≀ࡣ lysis buffer [1% (v/v) Triton X-100 ࢆྵࡴ PBS (phosphate buffered saline)] ᠱ⃮ࡋࠊ30 ศịୖ㟼⨨ࡋࡓࠋ16,500 x g ࡛ 15 ศ㐲 ᚰᚋࠊྛୖΎࢆ 20 PM MG132 Ꮡᅾୗࠊ㠀Ꮡᅾୗ࡛ assay solution [30 mM Tris-HCl (pH 7.6)ࠊ2 mM MgClࠊ10 mM NaClࠊ10 mM KClࠊ0.5 mM DTTࠊ1 mM ATPࠊ100 PM Suc-LLVY-MCA] 37Υ ࡛ 60 ศᛂࡉࡏࡓࠋྛࢧࣥࣉࣝࡢ⺯ගᙉᗘࡣບ㉳Ἴ㛗 360 nm/ ⺯ගἼ㛗 450 nm ࡛ ᐃࡋࡓࠋᐇ㦂ࡣ」ᩘᅇ⾜࠸ࠊ⌧ᛶࢆ☜ㄆࡋࡓࠋ
3-2-9. ⤫ィゎᯒ
⤖ᯝࡣ♧ࡋ࡚࠶ࡿᅇᩘࡢᖹᆒ s ᶆ‽೫ᕪ࡛⾲ࡋࡓࠋ⤫ィゎᯒࡣ one way-ANOVA ࡼࡾ್ࢆゎᯒࡋࠊTukey’s ᳨ᐃࢆ⾜ࡗࡓࠋ**p < 0.01 ࡣ⤫ィⓗ᭷ពࡳ࡞ࡍࠋ
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3-3. ⤖ᯝ
3-3-1. ࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ࢩࢫࢸ࣒ࡼࡿ Chac1 Ⓨ⌧ไᚚ
࣐࢘ࢫ Chac1 mRNA ࡣ 5’UTR (+1/+161) ࡑࡢୗὶࡢࢱࣥࣃࢡ㉁ࢥ࣮ࢻ㡿ᇦ (+162/+833)ࠊ3’UTR (+834/+1583) ࡛ᡂࡾ❧ࡗ࡚࠾ࡾࠊ⩻ヂࡉࢀࡿ 223 ࣑ࣀ㓟 (aa) ࡼ ࡾ ᡂ ࡿ ( ᅗ 3-1 (A)) ࠋ ➨ ❶ ࡢ ⤖ ᯝ ࡼ ࡾ ࠊ Chac1 Ⓨ ⌧ ࢥ ࣥ ࢫ ࢺ ࣛ ࢡ ࢺ (+162/+833) ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓ Neuro2a ⣽⬊࠾࠸࡚ Chac1 ࡣࣉࣟࢸ ࢯ࣮࣒㜼ᐖ࡛࠶ࡿ MG132 ฎ⌮ࡼࡗ࡚Ᏻᐃࡍࡿࡇࡀศࡗࡓࠋࡼࡗ࡚ࠊḟ Chac1 mRNA ෆࡢ 5’UTR 㡿ᇦࠊ≉⩻ヂ㛤ጞⅬ┤๓Ꮡᅾࡍࡿ Kozak ᵝ㓄ิ (GGCACC) ╔┠ࡋࡓࠋࡑࡇ࡛ࠊ5’UTR 㛗 (+1/+830; 5’-Chac1-Myc)ࠊ5’UTR ࡢ୍ 㒊 (+82/+830; '5’Chac1-Myc)ࠊ⩻ヂ㛤ጞⅬ┤๓ࡢ Kozak ᵝ㓄ิ (+156/+830; Kozak-Chac1-Myc) ࢆྵࡴ Chac1 ࢥࣥࢫࢺࣛࢡࢺ 5’UTR ࢆྵࡲ࡞࠸ࢥࣥࢫࢺࣛࢡࢺ (+162/+830; Chac1-Myc) ࢆస〇ࡋࡓ (ᅗ 3-1 (A))ࠋࡲࡎጞࡵࠊస〇ࡋࡓࢥࣥࢫࢺࣛࢡ ࢺࡢ୰࡛ Chac1-Myc ࢆ HEK293 ⣽⬊ࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࠊER ࢫࢺࣞࢫㄏᑟ ࡛࠶ࡿ (TgࠊTm) ࣉࣟࢸࢯ࣮࣒㜼ᐖ࡛࠶ࡿ MG132 ࡛ฎ⌮ࡋࡓ (ᅗ 3-1 (B))ࠋ ➨❶ࡢ⤖ᯝྠᵝࠊChac1 ࢱࣥࣃࢡ㉁ࡣ MG132 ฎ⌮ࡼࡗ࡚Ᏻᐃࡋࠊࡢ⸆ ࡛ࡣࢇຠᯝࡀ࡞ࡗࡓࠋෆᅉᛶ Chac1 ࢱࣥࣃࢡ㉁ࡀ MG132 ฎ⌮ࡼࡗ࡚ ᏳᐃࡍࡿࢆゎᯒࡍࡿࡓࡵࠊChac1 ᢠయࢆ⏝࠸࡚ MG132 ฎ⌮ࠊᮍฎ⌮ୗࡢ HEK293 ⣽⬊࠾ࡅࡿⓎ⌧ࢆ᳨ウࡋࡓ (ᅗ 3-1 (C))ࠋ⤖ᯝࡋ࡚ࠊෆᅉᛶ Chac1 Ⓨ⌧ ࡣᮍฎ⌮ୗࡢ⣽⬊࡛ࡣࡈࡃࢃࡎ࡛࠶ࡾࠊMG132 ฎ⌮ࡼࡗ࡚ⴭࡋࡃቑຍࡋࡓࠋᚑࡗ ࡚ࠊ௨㝆ࡢᐇ㦂ࡣྛ Chac1 ࢥࣥࢫࢺࣛࢡࢺࢆ୍㐣ⓗࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡍࡿࡇ ࡼࡾࠊࢱࣥࣃࢡ㉁Ⓨ⌧ไᚚࢆゎᯒࡋࡓࠋḟࠊMG132 ฎ⌮ࡼࡿ Chac1 Ⓨ⌧ୖ᪼ ࡀ⩻ヂᚋࡢࢱࣥࣃࢡ㉁ศゎ㜼ᐖࡼࡿࡶࡢࢆゎᯒࡍࡿࡓࡵࠊࢱࣥࣃࢡ㉁⩻ヂ㜼ᐖ ࡛࠶ࡿ CHX ࢆ⏝࠸ࡓࠋᙉไⓎ⌧ࡋࡓ 5’-Chac1-Myc 㑇ఏᏊࡽࡢ Chac1 ࡣ CHX ฎ ⌮ࡼࡗ࡚㛫౫ᏑⓗศゎࡉࢀࠊCHX MG132 ࡢඹฎ⌮ࡼࡗ࡚ⴭࡋࡃᏳᐃࡋ ࡓ (ᅗ 3-1 (D))ࠋMG132 ࡣ࣮࢜ࢺࣇࢪ࣮ࢆάᛶࡍࡿ࠸࠺ሗ࿌ࡀ࠶ࡿࡢ࡛ (90)ࠊ Chac1 ࡢⓎ⌧ไᚚࡘ࠸࡚ࠊ࣮࢜ࢺࣇࢪ࣮ࡢ㛵ࢃࡾࡘ࠸᳨࡚ウࡋࡓࠋChac1-Myc ࢆᙉไⓎ⌧ࡋࡓ HEK293 ⣽⬊ࢆ MG132ࠊ࣮࢜ࢺࣇࢪ࣮㜼ᐖ (BafࠊCMA)ࠊ࢝ࣝࣃ ࣥI 㜼ᐖ (ALLN) ࡛ฎ⌮ࡋࡓࠋ⤖ᯝࡋ࡚ࠊChac1 ࡣ MG132 ฎ⌮࡛Ᏻᐃࡋࡓ ࡀࠊBafࠊCMAࠊALLN ฎ⌮࡛ࡣ⦆ࡸ࡞Ᏻᐃࡋぢࡽࢀ࡞ࡗࡓ (ᅗ 1 (E))ࠋᅗ
3-38
1 (E) ࠾࠸࡚ࠊ࣮࢜ࢺࣇࢪ࣮࣐࣮࣮࡛࢝࠶ࡿ LC3-II (LC3-I ࣍ࢫࣇࢳࢪ࢚ࣝࢱ ࣀ࣮࣑ࣝࣥࡀ⤖ྜ) ࡣ BafࠊCMA ฎ⌮ࡼࡗ࡚ⴭࡋࡃቑຍࡋࡓࡀ (91)ࠊMG132 ฎ ⌮࡛ࡣࢃࡎ࡞ᙳ㡪ࡋぢࡽࢀ࡞ࡗࡓࠋࡉࡽࠊࣉࣟࢸࢯ࣮࣒ࡼࡿศゎࡢᇶ㉁ ࡋ࡚▱ࡽࢀ࡚࠸ࡿෆᅉᛶࡢ c-Myc ࢱࣥࣃࢡ㉁ࡣ MG132 ࡢฎ⌮࡛ࡢࡳᏳᐃࡋ (92ࠊ 93)ࠊࡢ⸆ฎ⌮࡛ࡣຠᯝࡣ࡞ࡗࡓ (ᅗ 3-1 (E))ࠋࡇࢀࡽࡢ⤖ᯝࡽࠊࣘࣅ࢟ࢳ ࣥ-ࣉࣟࢸࢯ࣮࣒ࢩࢫࢸ࣒ࡼࡗ࡚ Chac1 ࢱࣥࣃࢡ㉁Ⓨ⌧ࡀไᚚࡉࢀ࡚࠸ࡿࡇࡀ ♧ࡉࢀࡓࠋ
39 ᅗ
ᅗ3-1. ࣘࣅ࢟ࢳࣥ-ࣉࣟࢸࢯ࣮࣒ࢩࢫࢸ࣒ࡼࡿ Chac1 Ⓨ⌧ไᚚ
㻌
(A) ᮏ◊✲࡛⏝ࡋࡓ Chac1 Ⓨ⌧ࢥࣥࢫࢺࣛࢡࢺࡢᶍᘧᅗ Chac1 ࢥ࣮ࢻ㡿ᇦෆࡢ࣓ ࢳ࢜ࢽࣥࠊࣜࢪࣥṧᇶࡢ⨨ (NM_026929ࠊNCBI ࢹ࣮ࢱ࣮࣋ࢫࡼࡾྲྀᚓ)ࠋ(B)HEK293 ⣽ ⬊✵࣋ࢡࢱ࣮ (mock) Chac1-Myc ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ24 㛫ᚋࠊTg (0.1 PM)ࠊTm (2 Pg/mL)ࠊMG132 (MG) (20 PM) ࡲࡓࡣ⁐፹࡛࠶ࡿ DMSO (con) ࡛ 12 㛫ฎ ⌮ࡋࡓࠋࡑࡋ࡚ࠊ⣽⬊⁐ゎ≀ࢆ c-Myc ࡲࡓࡣ Actin ᢠయࢆ⏝࠸࡚࢙࢘ࢫࢱࣥࣈࣟࢵࢺἲ ࡼࡾゎᯒࡋࡓࠋ(C)HEK293 ⣽⬊ࢆ MG132 (MG) (20 PM) ࡲࡓࡣ DMSO (con) ࡛ 12 㛫ฎ⌮ࡋࡓࠋࡑࡋ࡚ࠊ⣽⬊⁐ゎ≀ࢆ Chac1 ࡲࡓࡣ Actin ᢠయࢆ⏝࠸࡚࢙࢘ࢫࢱࣥࣈࣟࢵࢺ ἲࡼࡾゎᯒࡋࡓࠋ(D) HEK293 ⣽⬊ 5’-Chac1-Myc ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ24 㛫ᚋࠊCHX (10 Pg/mL) ༢⊂ࡲࡓࡣ CHX MG132 (MG) (20 PM) ࡛ グ㍕ࡢ㛫ฎ⌮ࡋ ࡓࠋࡑࡋ࡚ࠊ⣽⬊⁐ゎ≀ࢆ c-Myc ࡲࡓࡣ Actin ᢠయࢆ⏝࠸࡚࢙࢘ࢫࢱࣥࣈࣟࢵࢺἲࡼࡾ ゎᯒࡋࡓࠋࣂࣥࢻᙉᗘࡣ Image J software ࡼࡗ࡚ᩘ್ࡋࠊActin ࡢ್࡛ᶆ‽ࡋࡓࠋ (E)HEK293 ⣽⬊ Chac1-Myc ࢆࢺࣛࣥࢫࣇ࢙ࢡࢩࣙࣥࡋࡓࠋ24 㛫ᚋࠊBaf (50 nM)ࠊ CMA (50 nM)ࠊALLN (10 PM)ࠊMG132 (MG) (20 PM) ࡲࡓࡣ DMSO (con) ࡛ 12 㛫ฎ⌮ ࡋࡓࠋࡑࡋ࡚ࠊ⣽⬊⁐ゎ≀ࢆ c-MycࠊLC3 ࡲࡓࡣ Actin ᢠయࢆ⏝࠸࡚࢙࢘ࢫࢱࣥࣈࣟࢵࢺ ἲࡼࡾゎᯒࡋࡓࠋ
