་ ඹ ࣜ ࣏ ᵝ ᘧ 㸯
ᶵ 㛵 䝸䝫䝆䝖䝸Ⓩ 㘓 ⏝ ㄽ ᩥ 䛾せ ⣙
ㄽ ᩥ ᥦ ฟ ⪅ Ặ ྡ ᚠ ⎔ ჾ ⭈ ⮚ ෆ ⛉ Ꮫ ㅮ ᗙ Ặ ྡ Ἑ ྜ ῝ 㔛
㸦 ㄽ ᩥ 㢟 ┠ 㸧
Mitochondrial inhibitory factor protein 1 functions as an endogenous inhibitor for coupling factor 6
(Inhibitory factor 1 ࡣ coupling factor 6 ࡢ ෆ ᅉ ᛶ 㜼 ᐖ ≀ ㉁ ࡋ ࡚ ᶵ ⬟ ࡍ ࡿ )
㸦 ෆ ᐜ ࡢ せ ⣙ 㸧
⫼⫼ ᬒ࣭┠ ⓗ㸸ATP ྜ ᡂ 㓝 ⣲ ࡣ ࣑ ࢺ ࢥ ࣥ ࢻ ࣜ ෆ ⭷ ⣽ ⬊ ⭷ ᒁ ᅾ ࡋ ࠊATP ࢆ 㥑 ື ຊ ࡋ
࡚ ィ ᪉ ྥ ᅇ ㌿ ࡍ ࡿ
F
1ศ Ꮚ ࣔ ࣮ ࢱ ࣮ ࣉ ࣟ ࢺ ࣥ ࢆ 㥑 ື ຊ ࡋ ࡚ ィ ᪉ ྥ ᅇ ㌿ ࡍࡿ
F
oศ Ꮚ ࣔ ࣮ ࢱ ࣮ ࡽ ᡂ ࡿ ࠋ ࣑ ࢺ ࢥ ࣥ ࢻ ࣜ ࡛ ࡣ 㓟 ⓗ ࣜ ࣥ 㓟 ࡼ ࡾ ⏘ ⏕ ࡉ ࢀ ࡓ ࣉࣟ ࢺ ࣥ ࡼ ࡾ ࠊ
F
oศ Ꮚ ࣔ ࣮ ࢱ ࣮ ࡢ ᅇ ㌿ ࡀF
1ศ Ꮚ ࣔ ࣮ ࢱ ࣮ ࡢ ᅇ ㌿ ࢆ 㥙 ࡋ ࠊࡑ ࡢ ⤖ ᯝF
1 ศ Ꮚ ࣔ ࣮ ࢱ ࣮ ࡣ ィ ᅇ ㌿ ࡋ ATP ࡀ ⏘ ⏕ ࡉ ࢀ ࡿ ࠋ ୍ ᪉ ࠊ ⣽ ⬊ ⭷ ࡛ ࡣ ⾑ ὶ ഃ ✺ ฟ ࡋ ࡓF
1ศ Ꮚ ࣔ ࣮ ࢱ ࣮ ࡢ ᅇ ㌿ ࡀ ⣽ ⬊ ⭷ ෆ ࡢ
F
oศ Ꮚ ࣔ ࣮ ࢱ ࣮ ࡢ ᅇ ㌿ ࢆ 㥙 ࡋ ࠊ ィ ᅇ ㌿ ࡍ ࡿ ࡇ ࡼ ࡾ ATPศ ゎ ࣉ ࣟ ࢺ ࣥ ࡢ ⣽ ⬊ ෆ ὶ ධ ࡀ ⏕ ࡌ ࡿ ࠋᡃ ࠎ ࡣ ࠊcoupling factor 6 (CF6) ࡀ ࠊ⣽ ⬊ ⭷ ATPྜ ᡂ 㓝 ⣲ ࡢ
F
1ศ Ꮚ ࣔ ࣮ ࢱ ࣮ ⤖ ྜ ࡋ ィ ᅇ ㌿ ࢆ ቑ ᙉ ࡉ ࡏ ࡿ ࡇ ࡼࡾ ⣽ ⬊ ෆ 㓟 ᛶ ࢆ ច ㉳ ࡍ ࡿ ࡇ ࢆ ᫂ ࡽ ࡋ ࡓ(1)ࠋࡑ ࡢ ⤖ ᯝ ࠊෆ ⓶ ᶵ ⬟ 㞀 ᐖ ࠊ⾑ ⟶ ⦰ ࠊ apoptosis ➼ ࢆ ᘬ ࡁ ㉳ ࡇ ࡍ(2-5)ࠋ᭱ ㏆ ࠊ
F
1ศ Ꮚ ࣔ ࣮ ࢱ ࣮ ࡢ ィ ᅇ ㌿ ࡔ ࡅ ࢆ 㜼 ᐖ ࡋ ࠊ ィ ᅇ ㌿ ࡣ ᙳ 㡪 ࡋ ࡞ ࠸ ࣌ ࣉ ࢳ ࢻ ࡀ ྠ ᐃ ࡉ ࢀinhibitory factor protein 1 (
IF1) ྡ ࡉࢀ ࡓ ࠋ ᮏ ◊ ✲ ࡢ ┠ ⓗ ࡣ ࠊ ࡇ ࡢ IF1 ࡀ CF6 ࡢ స ⏝ ࢆ 㜼 ᐖ ࡋ ࠊ ෆ ᅉ ᛶ ࡢ 㜼 ᐖ ≀ ㉁ ࡛ ࠶ ࡿ ྍ
⬟ ᛶ ࢆ ᳨ ウ ࡍ ࡿ ࡇ ࡛ ࠶ ࡿ ࠋ
᪉ ἲ 㸸1. IF1-pcDNA3 ୪ ࡧ IF1-pAcGFP1-C3ࣉ ࣛ ࢫ ࣑ ࢻ ࡢ స 〇 㸸Human mature ୪ ࡧ immature IF1 ࢆ pCMV-SPORT6 (Open Biosystems, Invitrogen™ MHS1010- 73732)ࢆ ࢸ ࣥ ࣉ ࣞ ࣮ ࢺ ࡋ PCR ἲ ࡛ స 〇 ᚋ ࠊpcDNA3 ୪ ࡧ pAcGFP1-C3 ࣋ ࢡ ࢱ ࣮ (Clontech Laboratories, Inc, Mountain View, CA, USA) ᤄ ධ ࡋ ࡓ ࠋ2. IF1 ⣽ ⬊ ᒁ ᅾ 㸸 HEK-293⣽ ⬊ mature୪ ࡧ immature IF1-pAcGFP1-C3ࣉ ࣛ ࢫ ࣑ ࢻ ࢆ transfection ࡋ BZ-X710 Fluorescence microscope ࡛ ほ ᐹ ࡋ ࡓ ࠋ3. IF1 ࡢ ศ Ἢ ᐃ 㸸HEK-293 ⣽ ⬊ mature୪ ࡧ immature IF1-pcDNA3ࣉ ࣛ ࢫ ࣑ ࢻ ࢆ transfectionᚋ ᇵ 㣴 ୖ Ύ ࢆ ᥇ ྲྀ ࡋ ࠊ exosome ⏬ ศ ࢆ ᢳ ฟ ᚋ Western blot ἲ ࡼ ࡾ IF1 ࢆ ᰁ Ⰽ ࡋ ࡓ ࠋ4. ATP ᐃ ἲ 㸸 ᇵ 㣴 ᾮ
୰ ࡢ ATP ⃰ ᗘ ࢆ ATP bioluminescent somatic cell assay kit (Sigma Chemical, St.
Louis, MO, USA)ࢆ ⏝ ࠸ ࡚ ࣝ ࣑ ࣀ ࣓ ࣮ ࢱ ࣮ ࡛ ᐃ ࡋ ࡓ ࠋ5. ⣽ ⬊ ෆ pH ᐃ㸸HEK-293 ⣽
⬊ ⣽ ⬊ ෆ pH dye ࡛ ࠶ ࡿ BCECF (2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein)- AMࢆ loadࡋ ࠊ495 nm/440 nmࡢ Ἴ 㛗 ࡛ ᐃ ࡋ ࡓ ࠋ6. Apoptosis ࡢ ᐃ 㸸HEK-293 ⣽ ⬊
IF1 ࢆ 㐣 Ⓨ ⌧ ࡉ ࡏ CF6 ࡢ apoptosis ཬ ࡰ ࡍ ᙳ 㡪 ࢆ Annexin V-propidium iodide (PI)࢟ ࢵ ࢺ ࢆ ⏝ ࠸ ࡚ flow cytometryἲ ࡛ ᐃ ࡋ ࡓ ࠋ7. Akt ࣜ ࣥ 㓟 ࡢ ᐃ㸸ᢠ Akt ୪ ࡧ
ᢠ ࣜ ࣥ 㓟 Aktᢠ య ࢆ ⏝ ࠸ ࡚ Western blot ἲ ࡛ ᐃ ࡋ ࡓ ࠋ
⤖ ᯝ 㸸1. IF1 ⣽ ⬊ ᒁ ᅾ 㸸Mature IF1 㐣 Ⓨ ⌧ ⣽ ⬊ ࡛ ࡣ ࠊ ⣽ ⬊ ㉁ ࡛ ࡢ ࡳ IF1 ࡢ Ⓨ ⌧ ࡀ ㄆ
ࡵ ࡽ ࢀ ࡓ ࠋ ୍ ᪉ ࠊimmature IF1-GFP 㐣 Ⓨ ⌧ ⣽ ⬊ ࡛ ࡣ 24 㛫 ᚋ ࣑ ࢺ ࢥ ࣥ ࢻ ࣜ ⭷ ࡀ Ⓨ Ⰽ ࡋ ࠊ48-72 㛫 ᚋ ࡣ ⣽ ⬊ ⭷ ࡢ Ⓨ Ⰽ ࡶ ほ ᐹ ࡉ ࢀ ࡓ ࠋ2. IF1 ศ Ἢ 㸸 ᇵ 㣴 ୖ Ύ ࡽ exosome ⏬ ศ ࢆ ᢳ ฟ ࡋ ࠊWestern blot ἲ ࡼ ࡾ IF1 ࢆ ᰁ Ⰽ ࡍ ࡿ ࠊIF1 ࡢ 㐣 Ⓨ ⌧ ࡼ
ࡾ IF1 ࡢ ศ Ἢ ࡀ ⏕ ࡌ ࡿ ࡇ ࢆ ☜ ㄆ ࡋ ࡓ ࠋ3. ATP ⃰ ᗘ㸸HEK-293 ⣽ ⬊ CF6 10-7M ࢆ ῧ ຍ ࡍ ࡿ ᇵ 㣴 ୖ Ύ ࡢ ATP ࡣ ῶ ᑡ ࡋ ࡓ ࡀ ࠊIF1 㐣 Ⓨ ⌧ ⣽ ⬊ ࡛ ࡣ ῶ ᑡ ࡋ ࡞ ࡗ ࡓ ࠋ4. ⣽
⬊ ෆ pH㸸CF6 10-7Mῧ ຍ ┤ ᚋ ࡽ ⣽ ⬊ ෆ pH ࡣ ῶ ᑡ ࡋ ࡓ ࡀ ࠊIF1 㐣 Ⓨ ⌧ ⣽ ⬊ ࡛ ࡣ ῶ ᑡ ࡀ ぢ ࡽ ࢀ ࡞ ࡗ ࡓ ࠋ5. Apoptosis㸸Annexin V-PIἲ ࡛ ⣽ ⬊ apoptosis ࢆ ᐃ ࡍ ࡿ ࠊCF6 10-7M ῧ ຍ ࡼ ࡾ HEK-293 ⣽ ⬊ ࡢ apoptosis ࡣ 4.5s1.2% ࡽ 6.4s1.3% ቑ ຍ (p<0.05)ࡋ ࡓ ࡀ ࠊIF1 㐣 Ⓨ ⌧ ⣽ ⬊ ࡛ ࡣ ቑ ຍ ࡋ ࡞ ࡗ ࡓ ࠋ6. Akt ࣜ ࣥ 㓟 㸸CF6 ࡢ apoptosis ಁ 㐍 స ⏝ ࡣ Akt ࡢ ࣜ ࣥ 㓟 ࡢ ᢚ ไ(52s13%,p<0.05)ࢆ క ࡗ ࡓ ࡀ ࠊIF1 ࡢ 㐣
Ⓨ ⌧ ⣽ ⬊ ࡛ ࡣ CF6ࡢ ῧ ຍ ࡛ Akt ࡢ ࣜ ࣥ 㓟 ࡣ ᢚ ไ ࡉ ࢀ ࡞ ࡗ ࡓ ࠋ
⪃
⪃ ᐹ 㸸CF6 ࡣ ࣉ ࣟ ࢫ ࢱ ࢧ ࢡ ࣜ ࣥ ⏘ ⏕ 㜼 ᐖ ≀ ㉁ ࡋ ࡚ ᡃ ࠎ ࡀ ྠ ᐃ ࡋ ࡓ ࣌ ࣉ ࢳ ࢻ ࡛ ࠶ ࡿ (2)ࠋCF6 ࡣ ୍ 㓟 ❅ ⣲ ࡢ ⏘ ⏕ ࡶ 㜼 ᐖ ࡋ ࠊෆ ⓶ ᶵ ⬟ ࡢ 㞀 ᐖ ࠊ᭦ ࡣ 㣗 ሷ ឤ ཷ ᛶ 㧗 ⾑ ᅽ ࠊ⢾
ᒀ ࢆ ច ㉳ ࡍ ࡿ ࡇ ࡶ ᫂ ࡽ ࡞ ࡗ ࡓ(1,3-5)ࠋᚑ ࡗ ࡚ ࠊCF6 ࡼ ࡿ ࡇ ࢀ ࡽ ࡢ స ⏝ ᣕ ᢠ ࡍ ࡿ ศ Ꮚ ࡢ ྠ ᐃ ࡣ 㔜 せ ⪃ ࠼ ࡽ ࢀ ࡓ ࠋCF6 ࡢ స ⏝ Ⅼ ࡽ ⪃ ࠼ ࡚ ࠊIF1 ࠸ ࠺ ࣌ ࣉ ࢳ ࢻ ࡣ CF6 ᣕ ᢠ ࡍ ࡿ ྍ ⬟ ᛶ ࡀ ᥎ ᐹ ࡉ ࢀ ࠊ ᮏ ◊ ✲ ࡛ ࡑ ࡢ ᳨ ウ ࢆ ⾜ ࡗ ࡓ ࠋ
ᅇ ึ ࡵ ࡚ ࠊCF6 ᣕ ᢠ ⓗ స ⏝ ࡍ ࡿ ෆ ᅉ ᛶ ࡢ ࣌ ࣉ ࢳ ࢻ ࡀ ྠ ᐃ ࡉ ࢀ ࡓ ࠋIF1 ࡣ ࣑ ࢺ ࢥ
ࣥ ࢻ ࣜ ࡛ ࡢ ATP ⏘ ⏕ ࡣ ᙳ 㡪 ࢆ ࠼ ࡎ ࠊCF6 ࡢ ࡳ ࢆ ᢚ ไ ࡍ ࡿ ྍ ⬟ ᛶ ࢆ ᭷ ࡍ ࡿ ࠋ ᮏ ◊
✲ ࡛ ࡣ ࠊ⏕ ⌮ స ⏝ ࡋ ࡚ CF6 ࡼ ࡿ apoptosis స ⏝ ࡢ ࡳ ࡢ ᳨ ウ ࡛ ࠶ ࡗ ࡓ ࡀ ࠊCF6 ࡣ ࡑ ࡢ
✀ ࠎ ࡢ ⏕ ⌮ ά ᛶ ࢆ ᭷ ࡍ ࡿ ࡓ ࡵ ࠊࡑ ࢀ ࡽ ࡢ స ⏝ ᑐ ࡋ ࡚ ࡶ ᣕ ᢠ ⓗ ࠺ ࡢ ᳨ ウ ࡀ ᚲ せ
ᛮ ࢃ ࢀ ࡓ ࠋ
IF1ࡣATP⏘ ⏕ ᝏ ᙳ 㡪 ࢆ ࠼ ࡎ CF6ࡢ స ⏝ ᣕ ᢠ ࡍ ࡿ ྍ ⬟ ᛶ ࡀ ♧ ࡉ ࢀ ࡓ ࡇ ࡽ ࠊ ࡑ ࡢ ⏕ య ෆ ࡛ ࡢ ෆ ᅉ ᛶ ᙺ ࡢ ゎ ᫂ ࡶ ࡉ ࡿ ࡇ ࡞ ࡀ ࡽ ࠊCF6ᣕ ᢠ ⸆ ࡢ ᵓ 㐀 ࢹ ࢨ ࣥ ࡋ ࡶ
᭷ ⏝ ⪃ ࠼ ࡽ ࢀ ࡿ ࠋ ᚋ ࡢ ⸆ ࡀ ᮇ ᚅ ࡉ ࢀ ࡿ ࡇ ࢁ ࡛ ࠶ ࡿ ࠋ
⤖ ㄽ 㸸CF6 ࡣ ATP ࡢ ᾘ ㈝ ࢆ ࡋ ࡚ ⣽ ⬊ ෆ 㓟 ᛶ ࢆ ច ㉳ ࡋ ࠊIF1 ࡣ ࡑ ࢀ ࢆ ᢚ ไ ࡍ ࡿ ࡇ
ࢆ ☜ ㄆ ࡋ ࡓ ࠋࡲ ࡓ ࠊIF1 ࡣ CF6 ࡼ ࡿ apoptosisࡢ ಁ 㐍 ᑐ ࡋ ࡚ ᣕ ᢠ ⓗ స ⏝ ࡋ ࠊࡑ ࡢ ᶵ ᗎ ࡋ ࡚ ࠊCF6 ࡼ ࡿ ࣜ ࣥ 㓟 Akt ࡢ ⬺ ࣜ ࣥ 㓟 ࢆ ᢚ ไ ࡍ ࡿ ࡇ ࡀ ☜ ㄆ ࡉ ࢀ ࡓ ࠋ௨ ୖ
ࡼ ࡾ ࠊIF1 ࡣ CF6 ࡢ ෆ ᅉ ᛶ 㜼 ᐖ ≀ ㉁ ࡋ ࡚ ᶵ ⬟ ࡍ ࡿ ྍ ⬟ ᛶ ࡀ ♧ ၀ ࡉ ࢀ ࡓ ࠋ
ཧ ⪃ ᩥ ⊩
1. Osanai T, Magota K, Tanaka M, Shimada M, Murakami R, Sasaki S, Tomita H, Maeda N, Okumura K. Intracellular signaling for vasoconstrictor coupling factor 6:
novel function of E-subunit of ATP synthase as receptor. Hypertension 2005;46:1140-1146.
2. Osanai T, Kamada T, Fujiwara N, Katoh T, Takahashi K, Kimura M, Satoh K, Magota K, Kodama S, Tanaka T, Okumura K. A novel inhibitory effect on prostacyclin synthesis of coupling factor 6 extracted from the heart of spontaneously hypertensive rats. J Biol Chem 1998;273:31778-31783.
3. Osanai T, Tanaka M, Kamada T, Nakano T, Takahashi K, Okada S, Sirato K, Magota K, Kodama S, Okumura K. Mitochondrial coupling factor 6 as a potent endogenous vasoconstrictor. J Clin Invest 2001;108:1023-1030.
4. Osanai T, Tanaka M, Magota K, Tomita H, Okumura K. Coupling factor 6-induced activation of ecto-F1Fo complex induces insulin resistance, mild glucose intolerance and elevated blood pressure in mice. Diabetologia 2012;55:520-529.
5. Suzuki A, Osanai T, Tanaka M, Tomita H, Magota K, Okumura K. Coupling factor 6 attenuates CXCR4 expression through the HIF-1ǂ and c-Src pathways and promotes endothelial apoptosis and inflammation. Hypertens Res 2014;37:708-715.
