4-3-1
䠊GroEL-AD
䛻䜘䜛ᵝ䚻䛺䝍䞁䝟䜽㉁䜈䛾┦స⏝䛸⥺⥔ᙧᡂᢚไ䃐-Synuclein 䛿䝟䞊䜻䞁䝋䞁䛻㛵䛧䠈A䃑1-42 䛿䜰䝹䝒䝝䜲䝬䞊䛻㛵䛧䛶䛔䜛䚹
GroES
䛿≉ᐃ䛾Ẽ䜈䛾㛵䛿䜏䜙䜜䛺䛔䛜䠈Gdn-HCl 䛾Ꮡᅾୗ䛺䛹䛾ኚᛶ᮲௳ୗ䛻䛚䛔䛶䜰䝭䝻䜲䝗⥺⥔䜢ᙧᡂ䛩䜛䝰䝕䝹⺮ⓑ㉁䛸䛧䛶䛾≉ᚩ䜢ᣢ䛳䛶䛔䜛䚹䝬䜴䝇⚄⤒⣽⬊䛻䛚 䛔䛶䠈GroES 䛾⥺⥔䛻䛚䛔䛶ᙧᡂ䛥䜜䜛䜸䝸䝂䝬䞊ศᏊ䛿䠈⣽⬊ẘᛶ䛜♧䛥䜜䛶䛔䜛䚹
3
䛴䛾䝍䞁䝟䜽㉁(䃐-Synuclein䠈A䃑1-42䠈GroES)䛾จ 㞟䛻䛚䛡䜛GroEL-AD
䛾ᙳ 㡪 䜢Thioflavin-T
⺯ග ᐃ䛻䜘䜚ゎᯒ䛧䛯(ᅗ4-4)䚹3
䛴䛾䝍䞁䝟䜽㉁䛿䜲䞁䜻䝳䝧䞊䝖䜢㛤ጞ䛧 䛶䛛䜙0~6
㛫ᚋ䛻⥺⥔ᙧᡂ䛾㛤ጞ䛜䜏䜙䜜䜛䚹ྛ䝍䞁䝟䜽㉁䛻䛚䛔䛶䠈GroEL-AD 䜢ῧ ຍ䛩䜛䛣䛸䛷⥺⥔ᙧᡂ䛻䛚䛡䜛ᙳ㡪䛜䜏䜙䜜䛯䚹䃐-Synuclein 䛾䝰䝹ẚ1:0.5䠈A䃑1-42
䛾 䝰䝹ẚ1䠖1䠈GroES
䛾䝰䝹ẚ1䠖2
䛷GroEL-AD
䜢ῧຍ䛩䜛䛸䠈ึ䜑䛾⥺⥔᰾ᙧᡂẁ㝵䛻GroEL-AD
䛜ᙳ㡪䛧䠈䛭䛾ᚋ䛾䜰䝭䝻䜲䝗⥺⥔ᙧᡂ䜒ᚎ䚻䛻ῶᑡ䛧䛶䛔䛯䚹䜘䜚㧗䛔ẚ⋡䛷GroEL-AD
䜢ῧຍ䛩䜛䛣䛸䛷䛻⥺⥔ᙧᡂ䛜ᢚไ䛥䜜䠈GroEL-AD 䛻䜘䜛ᙉ䛔㜼ᐖຠᯝ 䛜♧䛥䜜䛯䚹GroEL-AD䛾䜏䛾䝁䞁䝖䝻䞊䝹ᐇ㦂䛷䛿⥺⥔䛾ᙧᡂ䛿䜏䜙䜜䛺䛛䛳䛯䚹ᅗ4-4D
䛾䜘䛖䛻䠈3 䛴䛾䝍䞁䝟䜽㉁䛾䛺䜰䝭䝻䜲䝗⥺⥔䛾ᙧᡂ䜢ᢚไ䛩䜛䛯䜑䛻䠈䃐-Synuclein 䛾䝰䝹ẚ1:3䠈A䃑1-42
䛾䝰䝹ẚ1䠖20䠈GroES
䛾䝰䝹ẚ1䠖4
䛸GroEL-AD
䛾ᚲせ㔞䛜㐪 䛖䛣䛸䛛䜙䠈ྛ䝍䞁䝟䜽㉁䛾䜰䝭䝻䜲䝗⥺⥔䜢GroEL-AD
䛜Ṇ䜑䜛䛾䛻␗䛺䜛ᙳ㡪䜢ཬ䜌䛧䛶 䛔䜛䛸♧䛥䜜䛯䚹107
4-4䠊GroEL-AD䜢క䛳䛯䃐-Synuclein䠈A䃑1-42䠈GroES䛾Thioflavin-T ᐃ (A)GroEL-ADᏑᅾୗཪ䛿㠀Ꮡᅾୗ䛻䛚䛡䜛䃐-Synuclein䛾⥺⥔ᙧᡂ (B)GroEL-ADᏑᅾୗཪ䛿㠀Ꮡᅾୗ䛻䛚䛡䜛A䃑1-42䛾⥺⥔ᙧᡂ (C)GroEL-ADᏑᅾୗཪ䛿㠀Ꮡᅾୗ䛻䛚䛡䜛GroES䛾⥺⥔ᙧᡂ (D)GroEL-AD䛸3䛴䛾ᶆⓗ䝍䞁䝟䜽㉁䛾⥺⥔ᙧᡂᢚไ䛾㛵ಀ
䃐-Synuclein(䖃)䠈A䃑1-42(䖃)䠈GroES(䖃)䜢䛭䜜䛮䜜♧䛩䚹
108
44-3-2
䠊GroEL-AD
䛾Ꮡᅾୗ䛻䛚䛔䛶ᙧᡂ䛥䜜䛯䜰䝭䝻䜲䝗⥺⥔䛾ᙧែほᐹGroEL-AD
䛾Ꮡᅾୗ䛻䛚䛔䛶ᙧᡂ䛥䜜䛯䜰䝭䝻䜲䝗⥺⥔䛾ᙧែ䜢ほᐹ䛩䜛䛯䜑䛻䠈AFM
䛻䜘䜛ほᐹ䜢⾜䛳䛯(ᅗ
4-5)䚹୍␒ᕥഃ䛾ᅗ䛻䛚䛔䛶䛭䜜䛮䜜䛾䝍䞁䝟䜽㉁䛜 GroEL-AD
䛾 㠀Ꮡᅾୗ䛻䛚䛔䛶␗䛺䜛ᙧែ䛾䜰䝭䝻䜲䝗⥺⥔䜢ᙧᡂ䛩䜛䛾䛜☜ㄆ䛷䛝䜛䚹GroEL-AD 䛾䜏 䛷䛿䠈จ㞟䛫䛪䠈䜰䝭䝻䜲䝗⥺⥔䜢ᙧᡂ䛧䛶䛔䛺䛔䚹GroEL-AD
䛾Ꮡᅾୗ䛷䛭䜜䛮䜜䛾䝍䞁䝟 䜽㉁䛿䠈ᙧᡂ䛥䜜䛯䜰䝭䝻䜲䝗⥺⥔䛾ᙧែ䛜᫂☜䛻ኚ䛧䛶䛔䜛䚹䜘䜚㧗䛔ྜ䛷GroEL-AD
䜢ῧຍ䛧䛶䜒䜰䝭䝻䜲䝗⥺⥔䛿䜏䜙䜜䜛䛜䠈⥺⥔䛿▷䛟䛺䜚䠈యⓗ䛺⥺⥔䛾㔞䛿ᴟ䜑䛶ᑡ㔞䜎 䛷ῶᑡ䛧䛶䛔䛯䚹䛣䜜䜙䛾᮲௳䛻䛚䛔䛶䠈⥺⥔䛸ྠᵝ䛻䛔䛟䛴䛛䛾ᑠ䛥䛔䜰䝰䝹䝣䜯䝇䛺จ㞟 䛜☜ㄆ䛷䛝䛯䚹䃐-Synuclein䛾䝰䝹ẚ1:3䠈A䃑1-42
䛾䝰䝹ẚ1䠖20䠈GroES
䛾䝰䝹ẚ1䠖4
䛸㐣㔞䛾GroEL-AD
䛾Ꮡᅾୗ䛻䛚䛔䛶᭱⤊ⓗ䛻ᡂ⇍䛧䛯⥺⥔䛿䜏䜙䜜䛪䠈䛔䛟䛴䛛䛾ᑠ 䛥䛔⌫≧䛾จ㞟䛜௦䜟䜚䛻䜏䜙䜜䜛䚹୍␒ྑ䛻♧䛧䛯ᅗ䛸GroEL-AD only
䛷♧䛧䛯ᅗ䜢ẚ㍑䛩䜛䛣䛸䛷䠈ᶆⓗ䛸䛺䜛䝍䞁䝟䜽㉁䜎䛯䛿㐣㔞䛾
GroEL-AD
䛾䜰䝰䝹䝣䜯䝇䛺จ㞟䛷䛒䜛 䛣䛸䛜⪃䛘䜙䜜䜛䚹௨ୖ䛾⤖ᯝ䜘䜚䠈⥺⥔ᙧᡂᛂ䛻䛚䛡䜛
GroEL-AD
䛾㛵䛿䠈ᶆⓗ䛸䛺䜛䝍䞁䝟䜽㉁䛜ᙧ ᡂ䛩䜛䜰䝭䝻䜲䝗⥺⥔䛾ᙧែ䜢䛝䛟ኚ䛥䛫䜛䛾䛷䛿䛺䛟䠈ྛᶆⓗ 䝍䞁䝟䜽㉁䛜᭱⤊ⓗ䛻ᙧ ᡂ䛩䜛⥺⥔㔞䜢㜼ᐖ䛩䜛ാ䛝䛜䛒䜛䛸⪃䛘䜙䜜䛯䚹㻌
109
ᅗ4-5䠊GroEL-AD䜢క䛳䛯⥺⥔ᙧᡂᚋ䛾AFMほᐹ 䝇䜿䞊䝹䝞䞊䛿䠈1 䃛m䜢♧䛩䚹
110
ḟ䛻䠈3 䛴䛾ᶆⓗ䝍䞁䝟䜽㉁䛜ᙧᡂ䛩䜛⥺⥔䛾ᙧែ䜈䛾
GroEL-AD
䛾ᙳ㡪䜢䜘䜚ヲ⣽䛻 ゎᯒ䛩䜛䛯䜑䛻䠈TEM 䛻䜘䜛ゎᯒ䜢⾜䛳䛯(ᅗ4-6)䚹䛣䛾䛸䛝䝁䞁䝖䝻䞊䝹䛸䛧䛶 GroEL-AD
䛾௦䜟䜚䛻➼䝰䝹⃰ᗘ䛾∵⾑Ύ䜰䝹䝤䝭䞁(BSA)䜢⏝䛧䛯䚹ᅗ4-6
䛻䛚䛔䛶䠈BSA䛿3
䛴 䛾ᶆ ⓗ 䝍䞁䝟䜽㉁䛾⥺ ⥔ ᙧ ᡂ ᛂ 䛻ᙳ 㡪 䛧䛶䛔䜛䛾䛜☜ ㄆ 䛷䛝䜛䚹䛧䛛䛧䠈➼ 䝰䝹⃰ ᗘ 䛾GroEL-AD
䛿3
䛴䛾ᶆⓗ䝍䞁䝟䜽㉁䛾⥺⥔ᙧᡂ䜢䛥䜙䛻㜼ᐖ䛧䛶䛔䜛䛣䛸䛛䜙䠈⥺⥔ᙧᡂ䛻 䛚䛡䜛BSA
䛾ᙳ㡪䛿㠀≉␗ⓗ䛷䛒䜛䛾䛜♧䛥䜜䜛䚹BSA䛾ྛᶆⓗ䝍䞁䝟䜽㉁䜈䛾㠀≉␗ⓗ䛺ᙳ㡪䛿䝍䞁䝟䜽㉁䛷␗䛺䛳䛶䛔䜛䚹BSA Ꮡᅾୗ䛾
A䃑1-42
䛿Thioflavin-T
⺯ග䛜య 䛷䜟䛪䛛䛻ῶᑡ䛧䛶䛔䜛䛾䛜䜏䜙䜜䜛䛜䠈⥺⥔᰾ᙧᡂཪ䛿ᙧᡂ䛥䜜䜛⥺⥔㔞䛻ᙳ㡪䛧䛺䛔䚹 䛭䜜䛻ᑐ䛧䠈BSA Ꮡᅾୗ䛾GroES
䛿⥺⥔᰾ᙧᡂ䛜ⴭ䛧䛟㐜ᘏ䛧䛶䛔䜛䚹BSAᏑᅾୗ䛾䃐-Synuclein
䛿⥺⥔᰾ᙧᡂ䛸⥺⥔ఙ㛗䛾୧᪉䛻ᙳ㡪䛧䛶䛔䜛䚹䛣䛾BSA
䛾ῧຍ䛻䛚䛡䜛␗䛺 䜛ᙳ㡪䛿䠈ྛᶆⓗ䝍䞁䝟䜽㉁䛾⥺⥔ᛂ䜢᥎㐍䛩䜛≉␗ⓗ䛺ศᏊ㛫┦స⏝䛾㐪䛔䛜ᙳ 㡪䛧䛶䛔䜛䚹ྛᶆⓗ䝍䞁䝟䜽㉁䛾␗䛺䜛᮲௳䛷ᙧᡂ䛥䜜䛯⥺⥔䛾ᙧែ䛻䛝䛺㐪䛔䛿䛺䛛䛳 䛯䚹䛧䛛䛧䠈እ䛜2
䛴䛒䜛䚹1 䛴䛿䠈BSA 䛾Ꮡᅾୗ䛷ᙧᡂ䛧䛯A䃑1-42
䛾⥺⥔䛿䠈A䃑1-42
༢⊂䛸GroEL-AD
Ꮡᅾୗ䛾A䃑1-42
䛻䛚䛔䛶ᙧᡂ䛧䛯⥺⥔䜘䜚䜒▷䛔䛣䛸䛷䛒䜛䚹䛭䛧 䛶䠈GroEL-AD䛾Ꮡᅾୗ䛷ᙧᡂ䛧䛯α-Synuclein䛾⥺⥔䛿䠈䛾2
᮲௳䛷ᙧᡂ䛧䛯⥺⥔䜘 䜚䜒ᖜ䛜ⴭ䛧䛟⣽䛟䜏䛘䜛䛣䛸䛷䛒䜛䚹⥺⥔䛾యⓗ䛺ᙧែ䛿ᅗ4-5
䛻䛚䛡䜛AFM
䛷ほᐹ䛥䜜 䛯⤖ᯝ䛸୍⮴䛧䛶䛔䜛䚹111
ᅗ4-6䠊GroEL-AD䜢క䛳䛯⥺⥔ᙧᡂᚋ䛾TEMほᐹ
ᕥ䠖a䠊A䃑1-42䠈b䠊䃐-Synuclein䠈c䠊GroES䛾Thioflavin-T⺯ග ᐃ⤖ᯝ GroEL-AD㠀Ꮡᅾୗ(䖃)䠈GroEL-ADᏑᅾୗ(䖃)䠈BSAᏑᅾୗ(䖃)
ྑ䠖ᕥᅗ䛾30㛫(A䃑1-42䠈GroES) 䛸40㛫(䃐-Synuclein)䛾⥺⥔ᙧᡂᚋ䛾TEM⏬ീ
ᅗୖ䛻♧䛧䛯ᩥᏐ䛾Ⰽ䛿䛭䜜䛮䜜ᕥᅗ䛾䜒䛾䜢ᑐ㇟䛻䛧䛶䛔䜛䚹
112
4-3-3
䠊␗␗䛺䜛㛫䛻䛚䛡䜛GroEL-AD
䜢క䛳䛯䜰䝭䝻䜲䝗⥺⥔ᙧᡂ䜈䛾ᙳ㡪 ḟ䛻䠈ྛᶆⓗ䝍䞁䝟䜽㉁䛾⥺⥔ᙧᡂ䛻䛚䛡䜛␗䛺䜛㛫䛷GroEL-AD
䜢ῧຍ䛧䛯䛸䛝䛾ᙳ 㡪䛻䛴䛔䛶᳨ド䛧䛯(ᅗ4-7)䚹ྛᶆⓗ䝍䞁䝟䜽㉁䛻䛚䛔䛶䠈α-Synuclein
䛷䛿0䠈3䠈8䠈24
㛫䛻䠈A䃑1-42 䛷䛿0䠈0.5䠈1.5䠈8
㛫䛻䠈GroES 䛷䛿0䠈6䠈10䠈24
㛫䛻GroEL-AD
䜢ῧຍ䛧䛯䚹4-3-1
䛷♧䛧䛯䜘䛖䛻⥺⥔ᙧᡂ䜢䛻ᢚไ䛩䜛䜘䛖䛻䛭䜜䛮䜜䃐-Synuclein
䛾 䝰䝹ẚ1䠖3䠈A䃑1-42
䛾䝰䝹ẚ1䠖20䠈GroES
䛾䝰䝹ẚ1䠖4
䛷GroEL-AD
䜢ῧຍ䛧䛯䚹ྛᐇ㦂䛻䛚䛔䛶䠈
GroEL-AD
䜢⥺⥔䛾㏵୰䛷ῧຍ䛩䜛䛣䛸䛷ᙧᡂ䛧䛯⥺⥔䛿䜒䛸䛾≧ែ䛻ᡠ 䜙䛺䛔䛜䠈᭦䛺䜛⥺⥔ఙ㛗䜢㜼ᐖ䛩䜛䛣䛸䛿䛷䛝䛯䚹GroEL-AD 䛿ྛ㛫䛻䛚䛔䛶ῧຍ䛧䛶 䛩䛠 ᙳ 㡪 䜢ཬ 䜌䛧䛶䛔䜛 䛾䛜ぢ 䛶ྲྀ 䜜䜛 䚹ᶆ ⓗ 䝍䞁䝟䜽 ㉁ 䛻 㛵 ಀ 䛺䛟 䠈䛸 䛶䜒᪩ 䛔 㛫 䛻GroEL-AD
䜢ῧຍ䛩䜛䛣䛸䛷⥺⥔ᙧᡂ䜢䛻㜼ᐖ䛷䛝䜛䛾䛜♧䛥䜜䛯䚹䛣䛾⤖ᯝ䛸4-3-2
䛷♧䛧䛯AFM䠈TEM
䛾ᅗ䛸ྜ䜟䛫䛶䠈GroEL-AD 䛿᳨ฟྍ⬟䛺䝍䞁䝟䜽㉁⥺⥔䛾ᵓ㐀䜢 ಟṇ䛩䜛⬟ຊ䛜䛒䜛䛾䛷䛿䛺䛟䠈䜰䞁䝣䜷䞊䝹䝗䛧䛯ྍ⁐ᛶ䛾༢㔞య䝍䞁䝟䜽㉁䛻┤᥋స⏝䛧 䛶䛔䜛䛾䛛䠈⥺⥔䜢ᙧᡂ䛥䛫䜛ᵝ䚻䛺୰㛫యศᏊ✀䛾⃰ᗘ䜢ῶᑡ䛥䛫䛶䛔䜛ാ䛝䛜䛒䜛䛸⪃䛘䜙䜜䛯䚹ከ㔞య䝝䞁䝏䞁䝏䞁䛾ᵓ㐀䛻ᙳ㡪䜢ཬ䜌䛩㐟㞳䝍䞁䝟䜽㉁⃰ᗘ䜢ㄪ⠇䛩䜛㢮ఝ䛧 䛯ศᶵᵓ䛜
HSP70
䛸HSP40
䛷ሗ࿌䛥䜜䛶䛔䜛䚹䛧䛛䛧䠈ᮏ◊✲䛻䛚䛡䜛䠈GroEL-AD Ꮡᅾୗ䛷䛾䃐-Synuclein 䛾ሙྜ䠈ᙧᡂ䛥䜜䜛⥺⥔䛾ᙧែ䛜䜟䛪䛛䛻ኚ䛧䛶䛔䜛䛣䛸䛜♧၀䛥䜜䛯䚹
113
ᅗ4-7䠊␗䛺䜛㛫䛻GroEL-AD䜢ῧຍ䛧䛯䛸䛝䛾⥺⥔ᙧᡂᛂ (a)䃐-Synuclein䠈(b)A䃑1-42䠈(c)GroES
ྛ䝍䞁䝟䜽㉁䛻䛚䛡䜛GroEL-AD䛾ῧຍ㛫䛿௨ୗ䛾㏻䜚䛷䛒䜛䚹 (a)0 㛫(䖃)䠈3㛫(䖃)䠈8㛫(䖃)䠈24㛫(䖃) (b)0 㛫(䖃)䠈0.5 㛫(䖃)䠈1.5㛫(䖃)䠈8㛫(䖃) (c)0 㛫(䖃)䠈6 㛫(䖃)䠈10 㛫(䖃)䠈24 㛫(䖃)
䛻䛚䛔䛶GroEL-AD䜢ῧຍ䛧䛯䚹
(䖃)䛿䠈GroEL-AD㠀Ꮡᅾୗ䛻䛚䛡䜛⥺⥔ᙧᡂ⤖ᯝ䜢♧䛧䛶䛔䜛䚹
114
44-3-4
䠊GroEL-AD
䛸ᶆⓗ䝍䞁䝟䜽㉁䛸䛾┦స⏝ゎᯒ┤᥋ⓗ䛺㉁㔞䛾ゎᯒ䛜⾜䛘䜛
QCM
䜢⏝䛧䛶GroEL-AD
䛸ᶆⓗ䝍䞁䝟䜽㉁㛫䛾┦స⏝䜢䜘䜚ヲ⣽䛻ゎᯒ䛧䛯䚹QCM 䛿䠈䝉䞁䝃䞊㟁ᴟ䛻䝍䞁䝟䜽㉁䜢ᅛᐃ䛧䛶䛚䛝䠈┦స⏝
䛩䜛䝍䞁䝟䜽㉁䜢ῧຍ䛧䛶䛔䛟䛣䛸䛷⏕䛨䜛ᚤ㔞䛺㉁㔞ኚ䜢ືᩘኚ䛸䛧䛶䝸䜰䝹䝍䜲䝮䛻
᳨ฟ䛷䛝䜛䚹䛭䛾䛯䜑䠈䝍䞁䝟䜽㉁㛫䛾┦స⏝䜢㧗ឤᗘ䛻 ᐃ䛷䛝䜛᪉ἲ䛷䛒䜛䚹ᅗ
4-8a
䛻GroEL-AD
䛸3
䛴䛾ᶆⓗ䝍䞁䝟䜽㉁䛾┦స⏝ゎᯒ⤖ᯝ䜢♧䛧䛯䚹ᛂBuffer
䜢ῧຍ 䛧䛯䛸䛝䠈ືᩘ䛿ኚ䛧䛶䛺䛔䛜䠈ྛᶆⓗ 䝍䞁䝟䜽㉁䛾ῧຍ䛩䜛䛣䛸䛻䜘䜚᪩ᛴ䛺ືᩘኚ䛜⏕䛨䛶䛔䜛䚹ືᩘ䛾ῶᑡ㔞䛿ᶆⓗ䝍䞁䝟䜽㉁䛾✀㢮䛻䜘䜚␗䛺䛳䛶䛔䜛䚹GroEL-AD䛸
GroES
ཪ䛿䃐-Synuclein䛾┦స⏝䛿䠈䜋䛸䜣䛹ືᩘ䛜ῶᑡ䛧䛶䛔䛺䛔䚹䛭䜜䛻ᑐ䛧䠈A 䃑1-42 䛿ῧຍ䛩䜛䛸䛩䛠䛻ືᩘ䛜ῶᑡ䛧䛶䛔䜛䚹䛣䛾ኚ䛿䠈ᛂ㔞䛜ከ䛔䛾䛛䠈㠀≉␗ⓗ䛺⤖ྜ䜢ᫎ䛧䛶䛔䜛䛣䛸䛜⪃䛘䜙䜜䜛䚹A䃑1-42 䛻䜘䜛ゎᯒ䛿䠈䛾
2
䛴䛾ᶆⓗ䝍䞁䝟䜽㉁䛸ẚ䜉䠈䛭䛾ᛂ䛿Ᏻᐃ䛷䛒䛳䛯䚹䛣䜜䛿䠈A䃑1-42 䛾ศᏊ䝃䜲䝈䛜ᑠ䛥䛩䛞䜛䛾䛛䠈๓
㏙䛧䛯䜘䛖䛻
A䃑1-42
䛾⥺⥔ᢚไ䛻ከ㔞䛾GroEL-AD
䛜ᚲせ䛺ᛶ㉁䛜ᫎ䛥䜜䠈ṇ☜䛺 ゎᯒ䛜⾜䛘䛺䛔䛣䛸䛛䜙䠈QCM䛻䜘䜛ศᯒ䛻㐺䛧䛶䛔䛺䛔䜘䛖䛻⪃䛘䜙䜜䜛䚹㻌
A䃑1-42
䛸GroEL-AD
䛾┦స⏝ゎᯒ䛸ẚ㍑䛧䠈GroEL-AD䠖GroES䛸GroEL-AD䠖䃐-Synuclein
䛾┦స⏝ゎᯒ䛿䜘䜚ṇ☜䛺 ᐃ䜢⾜䛖䛣䛸䛜ྍ⬟䛷䛒䜛䚹ᅗ4-8b-d
䛻0.4 M Gdn-HCl
Ꮡᅾୗ䛻䛚䛡䜛GroEL-AD
䛸GroES
䛾┦స⏝ゎᯒ⤖ᯝ䜢♧䛧䛯䚹ᅗ4-8b
䛻 䛚䛔䛶䠈GroES䜈䛾⃰ᗘ౫Ꮡⓗ䛺GroEL-AD
䛾⤖ྜ䜢♧䛧䛯䚹䛣䛾⤖ᯝ䛷䛿䠈GroEL-AD 䜢ῧຍ䛧䛯ྛ⃰ᗘ䛻䛚䛡䜛㣬྾╔㔞䂴F䜢⟬ฟ䛧䠈ᶓ㍈䜢GroEL-AD
䛾⃰ᗘ䠈ᶓ㍈䜢㣬྾╔㔞䛸䛧䛶䝥䝻䝑䝖䛩䜛䛣䛸䛷䠈ゎ㞳ᐃᩘ
K d
䛾⟬ฟ䜢⾜䛳䛯䚹ᅗ4-8c
䛻䛚䛔䛶♧䛧䛯䜘䛖 䛻䠈|䂴F|䜢䝷䜲䞁䝣䜱䝑䝔䜱䞁䜾䛩䜛䛣䛸䛷(7.0㼼1.6)㽢10
-6䠄M䠅䛾K d
್䜢ᚓ䜙䜜䜛䚹䜎䛯䠈 ᅗ4-8b
䛷☜ㄆ䛷䛝䜛GroES
䛸ᵝ䚻䛺⃰ᗘ䛻䛚䛡䜛GroEL-AD
䛾྾╔䜹䞊䝤䜢䝣䜱䝑䝔䜱䞁 䜾䛩䜛䛣䛸䛷䠈ᶓ㍈䜢GroEL-AD
䛾⃰ᗘ䠈⦪㍈䜢k
obs 䛸䛧䛶䝥䝻䝑䝖䛩䜛䛣䛸䛷䠈ゎ㞳㏿ᗘk
off䠈⤖ྜ㏿ᗘ
k
on 䜢⟬ฟ䛧䛯䚹ᅗ4-8d
䛻䛚䛔䛶k
obsvs 䠷GroEL-AD䠹䛾䝥䝻䝑䝖䜘䜚⟬ฟ䛧䛯
115
k
off 䛜䝛䜺䝔䜱䝤䛺್䜢♧䛩䛾䛷䠈ᅗ4-8d
䛻䛚䛡䜛 ᐃ䛷䛿䛣䛾ᩘ್䛿0
䛻タᐃ䛧䛯䚹䛣䛾 ᮲௳䛻䛚䛔䛶⟬ฟ䛧䛯k
on 䛿4.1㽢10䈭(M
-1s
-1)䛷䛒䛳䛯䚹䛣䛾᮲௳ୗ䛻䛚䛡䜛䝕䞊䝍ศᯒ䛷
䛿䠈ᑐ䛻䛷䛝䜛䜹䜲䝛䝔䜱䝑䜽⋡ᐃᩘ䛾ᴫ⟬䜢㏻䛧䛯K d
䜢ᴫ⟬䛷䛝䛺䛛䛳䛯䚹䛧䛛䛧䠈ᅗ4-8c
䛷䛿䠈GroES 䛸GroEL-AD
䛾㛫䛻䛚䛔䛶䠈ྍ㏫䛺䜹䜲䝛䝔䜱䝑䜽⋡ᐃᩘ䛻䛚䛡䜛┦స⏝䛸୍⮴䛧䛶䛔䛯䚹
116
ᅗ4-8䠊GroEL-AD䛸䃐-Synuclein䠈A䃑1䉄42䠈GroES㛫䛾AffinixQN䃛䛻䛚䛡䜛┦స⏝ゎᯒ 䠄a䠅GroEL-AD䛸䃐-Synuclein(䇶)䠈GroES䠄䇶䠅䠈A䃑1-42䠄䠉䠅䛸䛾┦స⏝ゎᯒ
䠄b䠅GroEL-AD䛸ኚᛶ᮲௳ୗ䛻䛚䛡䜛GroES䛸䛾┦స⏝ゎᯒ
0 䃛M(䇶)䠈0.495 䃛M(ຑ)䠈1.23 䃛M(ຑ)䠈2.48 䃛M(ຑ)䠈2.97 䃛M(ຑ)䠈3.71 䃛M(ຑ)䠈 4.95 䃛M(ຑ)䠈9.90 䃛M(ຑ) 䛻䛚䛡䜛䝥䝻䝑䝖䜢䛭䜜䛮䜜♧䛧䛶䛔䜛䚹
(c)␗䛺䜛⃰ᗘ䛻䛚䛡䜛GroEL-AD䜢ῧຍ䛧䛯䛸䛝䛾GroES䛸䛾|䂴F|䛾್䛾䝥䝻䝑䝖 (d)␗䛺䜛⃰ᗘ䛻䛚䛡䜛GroEL-AD䛸GroES䛻䛚䛔䛶ほ 䛥䜜䛯kobs 䛾⥺ᙧᅇᖐ⥺
⾲4-1䠊GroES䛸GroEL-AD䛾┦స⏝䛻䛚䛔䛶⟬ฟ䛧䛯䜹䜲䝛䝔䜱䝑䜽䝟䝷䝯䞊䝍䞊 䝉䞁䝃䞊ୖ䛻䛿GroES䜢ᅛᐃ䛧䠈0.4 M Gdn-HClᏑᅾୗ䛷䛾ゎᯒ䜢⾜䛳䛯䚹
Sample k
on(M
-1䞉S-1) k
off(S
-1) K d (M)
GroES 4.1㽢10䈭 0.29 7.0㽢10
-6117
ᅗ
4-9
䛻GroEL-AD
䛸䃐-Synuclein䛸䛾┦స⏝䛾ゎᯒ⤖ᯝ䜢♧䛧䛯䚹䛣䛾⤖ᯝ䛛䜙䛿䠈GroES
䛸GroEL-AD
㛫䛾┦స⏝䛸䛿ẚ䜉䠈␗䛺䛳䛯ᛂ䜢♧䛧䛶䛔䜛䛾䛜☜ㄆ䛷䛝䜛䚹AffinixQN䃛䜢⏝䛔䛯 QCM
ᐃ䛻䜘䜚ほ 䛥䜜䛯⤖ྜ䜹䞊䝤䛿䠈᥎ዡ䛥䜜䛶䛔䜛༢ᣦᩘᛂ䜈㐺ྜ䛧䛺䛛䛳䛯䚹䛥䜙䛺䜛ศᯒ䛷䛿䠈ྛ䃐-Synuclein ⃰ᗘ䛻䛚䛡䜛
GroEL-AD
䜈䛾⤖ྜ䛿䠈␗䛺䜛እぢୖ䛾ᐃᩘ⋡䛻䛚䛡䜛
2
䛴䛾ู䚻䛺⤖ྜᛂ䛷䛒䜛㔜ᣦᩘ㛵ᩘ䛻᭱䜒㐺ྜ䛧䛶䛔䛯䚹┦స⏝䛾ゎᯒ䜘䜚ᖜ䛾ྜィ䜢⏝䛧䠈ᅗ
4-9b
䜘䜚䠈K d䠙(1.23㼼0.31)㽢 10
-6(M)
䜢⟬ฟ䛧䛯䚹እぢୖ䛾㏿䛔ᐃᩘ⋡(k
obs fast)
䛸䜘䜚㐜䛔ᐃᩘ⋡(k
obs slow)
䛾୧᪉䜢䛭䜜 䛮䜜⏝䛔䛶⤖ྜᛂ䛻䛚䛡䜛k
on/k
off䜢⟬ฟ䛧䛯(ᅗ4-9c䠈d)䚹ᅗ 4-8
䛻♧䛩䜘䛖䛻䠈kobs fast䜘䜚䠈
k
on= 1.20㽢10㼾 (M
-1s
-1)䛸 k
off= 0.25 (s
-1)䛾್䜢ᑟ䛝ฟ䛧䛯䚹䛣䜜䜘䜚䠈 K d䠙2.1㽢10
-4(M)䜢⟬ฟ䛧䛯䚹ḟ䛻䠈ᅗ 4-9d
䛷♧䛧䛯䜘䛖䛻䠈kobs slow 䛛䜙k
on= 9.2㽢10㼽 (M
-1s
-1)䛸 k
off䠙0.017 (s-1
)䛾䜹䜲䝛䝔䜱䝑䜽⋡䜢䛭䜜䛮䜜ᑟ䛝ฟ䛧䛯䚹䛣䜜䜘䜚䠈ゎ㞳ᐃᩘ K d = 1.8㽢10
-5(M)䛾ᩘ್䜢⟬ฟ䛧䛯䚹
118
ᅗ4-9䠊QCM䜢⏝䛔䛯GroEL-ADศᏊ䛸䃐-Synuclein䛾┦స⏝ゎᯒ
(a) ␗䛺䜛⃰ᗘ䛻䛚䛔䛶ῧຍ䛧䛯䃐-Synuclein䛸GroEL-AD䛾QCM䛻䜘䜛┦స⏝ゎᯒ 1.72 䃛M(䖃)䠈3.45 䃛M(䖃)䠈6.90 䃛M(䖃)䠈13.8 䃛M(䖃)䜢䛭䜜䛮䜜♧䛧䛶䛔䜛䚹 (b) ␗䛺䜛⃰ᗘ䛻䛚䛔䛶ῧຍ䛧䛯䃐-Syn䛸GroEL-AD䛸䛾|䂴F|್䛾㠀⥺ᙧ䝣䜱䝑䝔䜱䞁䜾
(c) ␗䛺䜛⃰ᗘ䛻䛚䛔䛶ῧຍ䛧䛯䃐-Syn䛾GroEL-AD䛻ᑐ䛩䜛kobsfast䛾⥺ᙧᅇᖐศᯒ (d) ␗䛺䜛⃰ᗘ䛻䛚䛔䛶ῧຍ䛧䛯䃐-Syn䛾GroEL-AD䛻ᑐ䛩䜛kobsslow䛾⥺ᙧᅇᖐศᯒ
⾲4-2䠊䃐-Synuclein䛸GroEL-AD䛾┦స⏝䛻䛚䛔䛶ほ 䛥䜜䛯2䛴䛾䝰䞊䝗䛻䛚䛔䛶㻌 䛭䜜䛮䜜⟬ฟ䛧䛯䜹䜲䝛䝔䜱䝑䜽䝟䝷䝯䞊䝍䞊
䝉䞁䝃䞊ୖ䛻䛿GroEL-AD䜢ᅛᐃ䛧䛶ᛂ䜢⾜䛳䛯䚹
k
obsk
on(M
-1䞉s-1) k
off(s
-1) K d (M)
Fast 1.2㽢10
30.25 2.1㽢10
-4Slow 9.2㽢10㼽 0.017 1.8㽢10
-5119
䛣䜜䜙䛾ศᯒ䛻䛚䛔䛶䠈䃐-Synuclein䛾
GroEL-AD
䜈䛾⤖ྜ䛾㔜せ䛺≉ᚩ䛷䛒䜛ⴭ䛧䛔k
off ㏿ᗘ䛻㛵ಀ䛩䜛⤖ྜᶵᵓ䜢᫂䜙䛛䛻䛧䛯䚹GroEL-AD 䛸GroES
䛸䛾⤖ྜ䛿䠈䠍䛴䛾ྍ㏫䛺⤖ྜ䛷䛒䜛䛾䛻ᑐ䛧䠈ᅗ
4-9
䛷♧䛧䛯䜘䛖䛻䠈䃐-Synuclein䛸GroEL-AD
㛫䛾1
䛴䛾 ༢⣧䛷䛿䛺䛔ከᵝ䛺ᵝᘧ䛻䛚䛡䜛ືⓗ䛺⤖ྜ䛻䜘䜛ᖹ⾮䜢♧䛧䛶䛔䛯䚹GroEL-AD 䛿䠈in
vitro
䛻䛚䛔䛶䠈䜰䝭䝻䜲䝗䜢ᙧᡂ䛩䜛䝍䞁䝟䜽㉁䛾⥺⥔ᙧᡂ䛻䛚䛡䜛ᵝ䚻䛺ᙧែ䜢ㄆ㆑䛧⤖ྜ䛩䜛䛣䛸䛜䛷䛝䜛⬟ຊ䜢ᮏ㉁ⓗ䛻ഛ䛘䛶䛔䜛䚹䛣䜜䛻䜘䜚䠈GroEL-AD 䛿ᵝ䚻䛺䜰䝭䝻䜲䝗≧
⢏Ꮚ䛾ᙧᡂ䜢ᢚไ䛩䜛䛣䛸䛜ྍ⬟䛻䛺䜛䛸⪃䛘䜙䜜䜛䚹