細胞膜媒介麥角固醇與 α-crystallin 的作用

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劉諭庭(Yu-Ting Liu) 查詢紙本館藏

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物理學系

論文名稱

細胞膜媒介麥角固醇與 α-crystallin 的作用
(Interaction between ergosterol and α-crystallin mediated by membranes)

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★ 羊毛脂固醇對 α-crystallin 和 SM 細胞膜間作用的影響

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摘要(中)

白內障是老年人常見的疾病，成因是水晶體混濁讓進入眼睛的光產生散射，導致視力模糊甚至失明，目前置換人工水晶體的手術是主要的治療方法，技術雖已成熟但仍有不可避免的風險，因此發展非手術及預防性的療法及藥物，已成為老年化社會的重要議題。
α-crystallin 是水晶體中主要的水溶性蛋白，主要由 αA 和 αB 組成，其功能為維持水晶體中其他蛋白，如 β 和 γ-crystallin，的正常狀態，防止蛋白質聚集，以維持水晶體的透明度，而 α-crystallin 抗聚集功能的喪失，被認為是白內障的主要原因。
動物實驗的結果表示，羊毛脂固醇可以抑制白內障的形成，甚至可以使混濁的水晶體恢復透明，但其機制仍不清楚；固醇分子一般存在於細胞膜上，與組成細胞膜的脂質分子有很強的交互作用，而有研究顯示，隨著年齡增長與水晶體細胞膜結合的 α-crystallin比例會增加，因此我們提出固醇分子以細胞膜作為媒介恢復 α-crystallin 的模型來解釋固醇分子治療白內障的機制。
本論文中，我們利用基因轉殖、表達與蛋白質純化的技術製作 αA-crystallin 和 αB-crystallin，並使用植物中常見的固醇分子-麥角固醇與 DOPC 和 Di20:1PC 脂質分子構成模型脂膜，研究 α-crystallin、麥角固醇和細胞膜間的交互作用；首先，利用伴護蛋白活性測量研究細胞膜微胞對 α-crystallin 伴護功能的影響，並且使用圓二色光譜技術決定 αcrystallin 與細胞膜微胞結合的比例，最後，製備多片層細胞膜樣品，利用 X 光繞射技術且經過處理後得到脂質雙層膜電子密度結構，研究麥角固醇與 α-crystallin 對脂質雙層膜結構的影響，藉由實驗結果，我們討論麥角固醇、α-crystallin 與細胞膜間的作用。
綜合三種實驗結果，顯示麥角固醇不僅使膜的厚度變薄，也使 α-crystallin 更容易與膜結合進入脂膜間，且麥角固醇並不能恢復因細胞膜所造成的 α-crystallin 抗聚集能力下降，這些結果與膽固醇對 α-crystallin 細胞膜作用的影響不同。

摘要(英)

A cataract, resulting in blurred vision or even blindness, is a common disease for aged people. It is caused by the light scattering in the eye lens. Nowadays, the regular treatment is to replace the cloudy eye lens with artificial ones. Although the surgery is well developed, it is also
accompanied by high risks. Therefore, developing non-surgical as well as preventive therapies and drugs is an urgent issue in an aging society.
Alpha-crystallin(α-crystallin) is the major water-soluble protein in the eye lens and consists of two subunits, αA and αB, their function is to maintain the native state of other proteins, such as β and γ-crystallin, in the eye lens. That is to retain the transparency of the eye lens by
inhibiting protein aggregation. The function loss of α-crystallin is known as a reason for cataracts.
The results of animal experiments indicate that lanosterol can inhibit the formation of cataracts as well as restore the cloudy lens back to the transparent state. Its mechanism is still a puzzle until now. Sterol molecules generally exist in cell membranes and have a strong interaction with lipids. Furthermore, previous studies show α-crystallins bound to membranes increase with age. We propose a model based on sterol-protein interaction mediated by membranes to clarify the mechanism of inhibition of cataracts induced by sterols.
In this study, αA-crystallin and αB-crystallin were produced via gene transfer, expression, and protein purification. A sterol from plants, ergosterol, DOPC and Di20:1PC lipids were used as model membranes for studying the interactions between α-crystallin, ergosterol, and lipid
membranes. First, the effect of membranes on the chaperone activity of αA and αB was checked by the ADH and lysozyme assays. Then, circular dichroism spectroscopy was used to probe the ratio of α-crystallin binding to membranes. Finally, X-ray diffraction was used to determine the
electron density of the lipid bilayers of the lamellar thin film sample. The structural change of the lipid bilayer induced by α-crystallin and ergosterol binding was extracted from X-ray data. This paper will discuss the observed interaction between ergosterol, α-crystallin and membrans
from the experimental results.
Combining the three experimental results, it was shown that ergosterol not only made the thickness of the membrane thinner, but also made it easier for α-crystallin to bind to membranes and enter membranes. Then, ergosterol could not restore the anti-aggregation ability of αcrystallin caused by membranes. These results are different from the effect of cholesterol on membranes.

關鍵字(中)

★ 細胞膜
★ 麥角固醇
★ α-crystallin
★ 伴護活性
★ 圓二色光譜
★ 多片層X光繞射

關鍵字(英)

論文目次

摘要 i
Abstract ii
致謝 iii
目錄 iv
圖目錄 vi
符號說明 ix
第一章序論 1
1-1 前言 1
1-2 水晶體 2
1-3 α水晶體蛋白（α-crystallin） 3
1-4 固醇分子（sterols） 4
1-5 動機 5
1-6 實驗方法 6
1-7 結論 7
第二章材料、樣品製備和實驗方法 8
2-1 材料 8
2-1-1 磷脂質 8
2-1-2 固醇分子 8
2-1-3 蛋白質 8
2-1-4 基因片段 8
2-1-5 菌 8
2-1-6 藥品 8
2-1-7 儀器 9
2-2 樣品製備 10
2-2-1 αA- crystallin、αB- crystallin製備步驟 10
2-2-2 小型單層膜微胞（Small Unilamellar Vesicles, SUVs）製備步驟 13
2-2-3 小型多層膜微胞（Small Multi-lamellar Vesicles, SMVs）製備步驟 14
2-2-4 水合法製作的多片層X光繞射樣品步驟 14
2-3 實驗原理與方法 15
2-3-1 圓二色光譜儀（J-815, JASCO, Japan）測量α-crystallin伴護活性 15
2-3-2 圓二色光譜儀（J-810, JASCO, Japan）偏振光譜測量（CD） 17
2-3-3 多片層X光繞射（Lamellar X-ray Diffraction, LXD） 22
第三章結果與討論 29
3-1 伴護活性（抗聚集能力）的測量 29
3-1-1 蛋白質的效應 29
3-1-2 加入細胞膜微胞的效應 33
3-1-3 麥角固醇的效應 36
3-2 圓二色光譜儀測量蛋白質二級結構結果 37
3-2-1 蛋白質與細胞膜微胞的效應 41
3-2-2 麥角固醇的效應 41
3-3 多片層X光繞射結果 42
3-3-1 蛋白質與細胞膜的效應 44
3-3-2 麥角固醇的效應 46
第四章結論 48
參考資料 49

參考文獻

[1] N. Congdon et al., “Prevalence of cataract and pseudophakia/ aphakia among adults
in the United States,” Arch. Ophthalmol, Vol 122, April 2004, pp. 487–494.
[2] 衛生福利部官網的衛生福利統計專區之性別統計專區中衛生類, 2019, 取自
https://dep.mohw.gov.tw/dos/cp-5339-59467-113.html。
[3] K. L. Moreau, J. A. King, “Protein misfolding and aggregation in cataract disease and
prospects for prevention,” Trends in Molecular Medicine, Vol 18, May 2012, pp. 273-
282.
[4] R. C. Augusteyn, “On the growth and internal structure of the human lens,” R.C.
Augusteyn / Experimental Eye Research, Vol 90, June 2010, pp. 643-654.
[5] B. A. Cobb and J. M. Petrash, “α-crystallin Chaperone-like Activity and Membrane
Binding in Age-Related Cataracts,” Biochemistry, Vol 41, January 2002, pp. 483-490.
[6] Ling Zhao et al., “Lanosterol reverses protein aggregation in cataracts,” Nature, Vol
523, July 2015, pp. 607-611.
[7] R. Michael and A. J. Bron, “The ageing lens and cataract: a model of normal and
pathological ageing,” Philosophical Transactions of the Royal Society B, Vol 366,
April 2011, pp. 1278–1292.
[8] T. Leo, Jr. Chylack “Mechanisms of senile cataract formation,” Ophthalmology, Vol
91, June 1984, pp. 596-602.
[9] S.J. Landry, “Protein interactions and molecular chaperones,” Biochemistry, Vol 601,
September1998.
[10] Masahide Yamamoto et al., “Characterization of the Hydrophobic Region of Heat
Shock Protein 90,” Japanese Biochemical Society, Vol 110 (1), July 1991, pp. 141–
145.
50
[11] D. L. Nelson, M. M. Cox, “Lehninger′s Principles of Biochemistry,” Fourth
Edition, New York: Freeman and Company, 2005.
[12] C. Bagneris et al., “Crystal structures of alpha-crystallin domain dimers of alphaBcrystallin and Hsp20,” J. Mol. Biol., Vol 392, October 2009, pp. 1242-1252.
[13] A. Laganowsky et al., “Crystal structures of truncated alpha and alphaB crystallins
reveal structural mechanisms of polydispersity important for eye lens function,”
Protein Sci., Vol 19, May 2010, pp. 1031-1043.
[14] Ligia Acosta-Sampson, Jonathan King, “Partially folded aggregation intermediates
of human gammaD-, gammaC-, and gammaScrystallin are recognized and bound by
human alphaB-crystallin chaperone,” J. Mol. Biol., Vol 401, August 2010, pp. 134–
152.
[15] K.R. Heys et al., “Presbyopia and heat: changes associated with aging of the human
lens suggest a functional role for the small heat shock protein, alpha-crystallin, in
maintaining lens flexibility,” Aging Cell, Vol 6, December 2007, pp. 807-815.
[16] J. A. Carver et al., “Age-related changes in bovine alpha-crystallin and highmolecular-weight protein,” Exp. Eye Res., Vol 63, December 1996, pp. 639-647.
[17] D. L. Boyle, L. Takemoto, “EM immunolocalization of alpha-crystallins: association
with the plasma membrane from normal and cataractous human lenses.,” Curr. Eye
Res., Vol 15, May 1996, pp. 577-582.
[18] R. J. Cenedella, C. R. Fleschner, “Selective association of crystallins with lens
′native′ membrane during dynamic cataractogenesis,” Curr. Eye Res., Vol 11, August
1992, pp. 801-815.
[19] M. H. Sweeney, R. J. Truscott, “An impediment to glutathione diffusion in older
normal human lenses: a possible precondition for nuclear cataract,” Exp. Eye Res., Vol
67, November 1998, pp.587-595.
[20] B. A. Moffat et al., “Age-related changes in the kinetics of water transport in normal
51
human lenses,” Exp. Eye Res., Vol 69, December 1999, pp. 663-669.
[21] R. J. Truscott, “ Age-related nuclear cataract: a lens transport problem, ”
Ophthalmic Res., Vol 32, September 2000, pp. 185-194.
[22] K. Mitra et al., “Modulation of the bilayer thickness of exocytic pathway membranes
by membrane proteins rather than cholesterol,” Proc. Natl. Acad. Sci. USA, Vol 101,
March 2004, pp. 4083–4088.
[23] O. G. Mouritsen, M. Bloom, “ Mattress model of lipid-protein interactions in
membranes,” Biophys, Vol 46, August 1984, pp. 141–153.
[24] J. A Killian, “Hydrophobic mismatch between proteins and lipids in membranes,
Biochim,” Biophys. Acta., Vol 1376, November 1988, pp. 401–415.
[25] N. L. Gershfeld, “ Equilibrium studies of lecithin-cholesterol interactions I.
Stoichiometry of lecithin-cholesterol complexes in bulk systems,” Biophys J., Vol 22,
June 1978, pp. 469–488.
[26] S. Subramaniam, H. M. McConnell, “Critical mixing in monolayer mixtures of
phospholipid and cholesterol,” J. Phys. Chem., Vol 91, 1987, pp. 1715–1718.
[27] A. Radhakrishnan, H. M. McConnell, “ Cholesterol-phospholipid complexes in
membranes,” J. Am. Chem. Soc., Vol 121, January 1999, pp. 486–487.
[28] Y. K. Levine, M. H. Wilkins, “Structure of oriented lipid bilayers,” Nat. New Biol.,
Vol 230, March 1971, pp. 69–72.
[29] D. L. Worcester, N. P. Franks, “Structural analysis of hydrated egg lecithin and
cholesterol bilayers. II. Neutrol diffraction,” J. Mol. Biol., Vol 100, January 1976, pp.
359–378.
[30] T. J. McIntosh, P. W. Holloway, “Determination of the depth of bromine atoms in
bilayers formed from bromolipid probes,” Biochemistry, Vol 26, March 1987, pp.
1783–1788.
[31] N. P. Franks, “Structural analysis of hydrated egg lecithin and cholesterol bilayers. I.
52
X-ray diffraction,” J. Mol. Biol., Vol 100, January 1976, pp. 345–358.
[32] Michael Rappolt et al., “Structural, dynamic and mechanical properties of POPC at
low cholesterol concentration studied in pressure/temperature space,” Eur. Biophys,
Vol 31, February 2003, pp. 575–585.
[33] J. Gallova´ et al., “Bilayer thickness in unilamellar extruded 1,2-dimyristoleoyl and
1,2-dierucoyl phosphatidylcholine vesicles: SANS contrast variation study of
cholesterol effect,” Colloids Surf. B Biointerfaces, Vol 38, October 2004, pp. 11–14.
[34] J. Pencer et al., “ Bilayer thickness and thermal response of
dimyristoylphosphatidylcholine unilamellar vesicles containing cholesterol, ergosterol
and lanosterol: a small-angle neutron scattering study,” Biochim. Biophys. Acta.,
Vol 1720, December 2005, pp. 84–91.
[35] H. Schaller, “The role of sterols in plant growth and development,” Progress in
Lipid Research, Vol 42, May 2003, pp. 163–175.
[36] P Mahesh Shanmugam et al.,“Effect of lanosterol on human cataract nucleus,”
Indian Journal of Ophthalmology, Vol 63, December 2015, pp. 888-890.
[37] W. R. Nes, M. L. McKean, “Biochemistry of Steroids and other Isopentenoids,”
Baltimore: University Park Press, 1977.
[38] J. Henriksen, “Universal behavior of membranes with sterols,” Biophys, Vol 90,
March 2006, pp. 1639–1649.
[39] P. L. Yeagle, “The Membranes of Cells,” Second edition, U.S.A.: Academic Press,
1993.
[40] J. Czub, M. Baginski, “Comparative molecular dynamics study of lipid membranes
containing cholesterol and ergosterol,” Biophys, Vol 90, April 2006, pp. 2368–2382.
[41] Z. Cournia, G. M. Ullmann, J. C. Smith, “ Differential effects of cholesterol,
ergosterol and lanosterol on a dipalmitoyl phosphatidylcholine membrane: a molecular
dynamics simulation study,“ J. Phys. Chem. B., Vol 111, February 2007, pp. 1786–
53
1801.
[42] R. Semer, E. Gelerinter, “A spin label study of the effects of sterols on egg lecithin
bilayers,” Chem. Phys. Lipids., Vol 23, February 1979, pp. 201–211.
[43] Wei-Chin Hung et al., “Comparative study of the condensing effects of ergosterol
and cholesterol,” Biophysical Journal, Vol 110, May 2016, pp. 2026–2033.
[44] F. Magaraci et al., “ Azasterols as inhibitors of sterol 24-Methyltransferase in
leishmania species and trypanosoma cruzi,” J. Med. Chem., Vol 46, October 2003, pp.
4714-4727.
[45] D. Borchman, “Lipid conformational order and the etiology of cataract and dry eye,”
J. Lipid Res., Vol 62, February 2021.
[46] J. Peschek et al., “ The eye lens chaperone -crystallin forms defined globular
assemblies,” PNAS, Vol 106, August 2009, pp. 13272-13277.
[47] N. Sreerama, R. W. Woody, “ Computation and Analysis of Protein Circular
Dichroism Spectra,” Methods in Enzymology, Vol 383, 2004, pp. 318-351.
[48] Ming-Tao Lee, Yu-Yung Chang, Wei-Chin Hung, “Conformational Changes of αcrystallin Proteins Induced by Heat Stress,” Biophysical Journal, Vol 114, February
2018.
[49] Ming-Tao Lee, “Biophysical characterization of peptide–membrane interactions,”
Advances in Physics: X, Vol 3, June 2018, pp. 144-164.
[50] J. Torbet, M. H. F. Wilkins, “X-ray diffraction studies of lecithin bilayers,” J. Theor.
Biol., Vol 62, October 1976, pp. 447-458.

指導教授

李明道(Ming-Tao Lee)

審核日期

2022-6-15

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