臺灣博碩士論文加值系統

膽酸負責人體腸道中的脂質和維生素吸收。當脂質消化結束後，大部分的膽酸會被轉運蛋白回收到小腸內，接著再經過腸肝循環，最後會再被儲存於肝臟細胞中。Apical Sodium-dependent Bile acid Transporter (ASBT) 是其中一個位在小腸絨毛細胞上協助回收的轉運蛋白，而ASBT是利用鈉離子的濃度梯度將腸道中的牛磺膽酸(taurocholate) 運送到小腸表皮細胞內。最近的研究已經解出ASBT細菌同源蛋白的三種不同晶體結構，第一個結構是ASBTNM，在此結構中發現到有兩個鈉離子和一個taurocholate的inward-open構形。其他的兩個結構是ASBTYf，分別解出inward-open and outward-open的構形，而在這兩個結構裡都沒有發現到任何鈉離子與的存在。雖然這三個不同構形的晶體結構表示ASBT可能是利用不同的構形變化對taurocholate進行運輸，但是造成這種明顯構形變化的關鍵因素和蛋白本身的運輸機制仍然是不清楚的。因此在本研究中，我們開發了一種非侵入式的生物化學技術，PEGylation In Gel Fluorescence (PIGF)，用來檢測不同環境下ASBTNM受質結合口袋的溶劑可接觸性。在我們設計的不同緩衝液環境之下，其PIGF實驗結果跟目前解出的三種ASBT細菌同源蛋白晶體結構是非常一致的。而且PIGF的結果也提供對於ASBTNM產生構形變化的分子機制之洞見。此外，我們使用ASBTNM L44C之DNA作為模板，並在鈉離子結合胺基酸進行alanine單點突變。我們發現N115A突變株顯示出的PIGF實驗結果比其他的L44C突變們有非常明顯的程度差異出現，因此我們推論N115A突變蛋白可能會讓ASBTNM偏好處在outward-open的構形狀態。

Bile acid is responsible for lipid and vitamin absorption in human’s intestine. After lipid digestion, bile acids are recycled at the ileal epithelium by Apical Sodium-dependent Bile acid Transporter (ASBT), and undergo enterohepatic circulation, by which most of bile acids are reabsorbed back to the liver. Recent studies have determined three different crystal structures of ASBT bacterial homologous proteins. The first structure of the ASBT bacterial homologous protein was ASBTNM, showing an inward-open conformation in complex with two sodium ions and one taurocholate. The others were ASBTYf, revealing both inward-open and outward-open conformations, respectively, but no sodium ions and taurocholate were observed. Although the three crystal structures demonstrated that ASBT may adopt different conformations for taurocholate translocation, the critical factor triggering such drastic conformational changes and the mechanism of transport remains unclear. In this study, we developed a non-invasive biochemical approach, PEGylation In Gel Fluorescence (PIGF) to detect the solvent accessibility of ASBTNM substrate-binding crevice in different conditions. The results indicate that the PIGF profiles are in great agreement with crystal structures and the results further provide deep insight into the mechanistic determinants for conformational alternation of ASBT. Besides, we used ASBTNM L44C mutant as the DNA template and replaced the Na+-binding residues individually with alanine. We found that L44C/N115A mutant showed the highest extent of PEGlyation, suggesting N115A substitution may lock ASBTNM in outward-facing conformation.

中文摘要 i
Abstract ii
目錄 iii
圖表目錄 v
1. 研究背景介紹 1
1.1 前言 1
1.2 二級主動轉運蛋白 2
1.3 交替通透機制(Alternating Access Mechanism) 2
1.3.1 兩種不同的交替通透機制模型 2
1.4 ASBT(Apical Sodium-dependent Bile acid Transporter) 3
1.5 研究目的和實驗設計 4
1.6 PIGF (PEGylation In Gel Fluorescence)的實驗設計 5
2. 實驗與方法 6
2.1 Site-Directed Mutagenesis 6
2.2 PEGylation In Gel Fluorescence (PIGF) 7
2.3 蛋白純化與結晶 9
2.3.1 Membrane製備 9
2.3.2 純化 10
2.3.3 結晶 12
2.4 蛋白的Functional assay 12
3. 結果 14
3.1 ASBTNM cysteine less的PEGylation測試 14
3.2 mPEG-maleimide-5K在鉀離子與鈉離子環境下的反應性控制實驗 14
3.3 ASBTNM的各個突變株在4種環境下的PEGylation實驗 15
3.3.1 分析在鉀離子以及鈉離子/taurocholate環境下的PEGylation結果 15
3.3.2 分析在鉀離子以及鉀離子/taurocholate環境下的PEGylation結果 16
3.3.3 分析在鈉離子以及鈉離子/taurocholate環境下的PEGylation結果 16
3.4 四個環境下PEGylation顯著差異的總比較 17
3.5 以L44C當作indicator來對ASBTNM的Sodium binding site進行PEGylation實驗 17
3.6 針對ASBTNM-N115A進行蛋白純化以及結晶條件篩選 18
3.7 ASBTNM突變株的Functional assay 18
4. 討論 20
4.1 PIGF實驗結果的發現 20
4.2 mPEG-maleimide-5K在兩個環境下的反應性比較中的發現 20
4.3 mPEG-maleimide-5K是否結合到ASBTNM上時，是否會影響ASBTNM與taurocholate的結合與運輸 21
4.4 PIGF實驗的其他應用 21
4.5 PIGF實驗的缺點與其他可以彌補的新技術: 電子自旋共振技術（ESR） 22
4.6 DEER技術應用到觀察ASBTNM的構型變化與運輸機制 22
4.7 Functional assay實驗結果的發現 23
4.8 結論 23
5. 參考文獻 51
6. 附錄 54
附錄1: 54
附錄2: 55

1.Jardetzky, O., Simple Allosteric Model for Membrane Pumps. Nature, 1966. 211(5052): p. 969-970.
2.Mitchell, P., A General Theory of Membrane Transport From Studies of Bacteria. Nature, 1957. 180(4577): p. 134-136.
3.DeFelice, L.J., Transporter structure and mechanism. Trends in Neurosciences, 2004. 27(6): p. 352-359.
4.Abramson, J., et al., Structure and Mechanism of the Lactose Permease of Escherichia coli. Science, 2003. 301(5633): p. 610.
5.Dang, S., et al., Structure of a fucose transporter in an outward-open conformation. Nature, 2010. 467(7316): p. 734-738.
6.Quistgaard, E.M., et al., Structural basis for substrate transport in the GLUT-homology family of monosaccharide transporters. Nat Struct Mol Biol, 2013. 20(6): p. 766-768.
7.Wisedchaisri, G., et al., Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE. Nature Communications, 2014. 5: p. 4521.
8.Coincon, M., et al., Crystal structures reveal the molecular basis of ion translocation in sodium/proton antiporters. Nat Struct Mol Biol, 2016. 23(3): p. 248-255.
9.Mirza, O., et al., Structural evidence for induced fit and a mechanism for sugar/H(+) symport in LacY. The EMBO Journal, 2006. 25(6): p. 1177-1183.
10.Guan, L., et al., Structural determination of wild-type lactose permease. Proceedings of the National Academy of Sciences of the United States of America, 2007. 104(39): p. 15294-15298.
11.Hakizimana, P., et al., Interactions between Phosphatidylethanolamine Headgroup and LmrP, a Multidrug Transporter: A CONSERVED MECHANISM FOR PROTON GRADIENT SENSING? Journal of Biological Chemistry, 2008. 283(14): p. 9369-9376.
12.Lee, C., et al., A two-domain elevator mechanism for sodium/proton antiport. Nature, 2013. 501(7468): p. 573-577.
13.Yernool, D., et al., Structure of a glutamate transporter homologue from Pyrococcus horikoshii. Nature, 2004. 431(7010): p. 811-818.
14.Boudker, O., et al., Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter. Nature, 2007. 445(7126): p. 387-393.
15.Reyes, N., C. Ginter, and O. Boudker, Transport mechanism of a bacterial homologue of glutamate transporters. Nature, 2009. 462(7275): p. 880-885.
16.Mulligan, C. and J.A. Mindell, Mechanism of Transport Modulation by an Extracellular Loop in an Archaeal Excitatory Amino Acid Transporter (EAAT) Homolog. The Journal of Biological Chemistry, 2013. 288(49): p. 35266-35276.
17.Rogina, B., et al., Extended Life-Span Conferred by Cotransporter Gene Mutations in Drosophila. Science, 2000. 290(5499): p. 2137.
18.Mancusso, R., et al., Structure and mechanism of a bacterial sodium-dependent dicarboxylate transporter. Nature, 2012. 491(7425): p. 622-626.
19.Vergara-Jaque, A., et al., Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms. Frontiers in Pharmacology, 2015. 6: p. 183.
20.Mulligan, C., et al., The bacterial dicarboxylate transporter, VcINDY, uses a two-domain elevator-type mechanism. Nature structural & molecular biology, 2016. 23(3): p. 256-263.
21.Fredriksson, R., et al., The solute carrier (SLC) complement of the human genome: Phylogenetic classification reveals four major families. FEBS Letters, 2008. 582(27): p. 3811-3816.
22.Hediger, M.A., et al., The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteins. Pflügers Archiv, 2004. 447(5): p. 465-468.
23.Geyer, J., T. Wilke, and E. Petzinger, The solute carrier family SLC10: more than a family of bile acid transporters regarding function and phylogenetic relationships. Naunyn-Schmiedeberg's Archives of Pharmacology, 2006. 372(6): p. 413-431.
24.Pols, T.W.H., et al., The bile acid membrane receptor TGR5 as an emerging target in metabolism and inflammation. Journal of hepatology, 2011. 54(6): p. 1263-1272.
25.Dawson, P.A., Role of the Intestinal Bile Acid Transporters in Bile Acid and Drug Disposition. Handbook of experimental pharmacology, 2011(201): p. 169-203.
26.Braun, A., et al., Inhibition of intestinal absorption of cholesterol by ezetimibe or bile acids by SC-435 alters lipoprotein metabolism and extends the lifespan of SR-BI/apoE double knockout mice. Atherosclerosis, 2008. 198(1): p. 77-84.
27.Lewis, M.C., L.E. Brieaddy, and C. Root, Effects of 2164U90 on ileal bile acid absorption and serum cholesterol in rats and mice. Journal of Lipid Research, 1995. 36(5): p. 1098-1105.
28.Bhat, B.G., et al., Inhibition of ileal bile acid transport and reduced atherosclerosis in apoE−/− mice by SC-435. Journal of Lipid Research, 2003. 44(9): p. 1614-1621.
29.Lazaridis, K.N., et al., Alternative splicing of the rat sodium/bile acid transporter changes its cellular localization and transport properties. Proceedings of the National Academy of Sciences of the United States of America, 2000. 97(20): p. 11092-11097.
30.Dawson, P.A., et al., Targeted Deletion of the Ileal Bile Acid Transporter Eliminates Enterohepatic Cycling of Bile Acids in Mice. Journal of Biological Chemistry, 2003. 278(36): p. 33920-33927.
31.Hu, N.J., et al., Crystal structure of a bacterial homologue of the bile acid sodium symporter ASBT. Nature, 2011. 478(7369): p. 408-11.
32.Zhou, X., et al., Structural basis of the alternating-access mechanism in a bile acid transporter. Nature, 2014. 505(7484): p. 569-73.
33.Bird, L.E., et al., Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli. Journal of Visualized Experiments : JoVE, 2015(95): p. 52357.
34.Drew, D., et al., GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nature protocols, 2008. 3(5): p. 784-798.
35.Drew, D., et al., Optimization of membrane protein overexpression and purification using GFP fusions. Nat Meth, 2006. 3(4): p. 303-313.
36.Weinman, S.A., M.W. Carruth, and P.A. Dawson, Bile Acid Uptake via the Human Apical Sodium-Bile Acid Cotransporter Is Electrogenic. Journal of Biological Chemistry, 1998. 273(52): p. 34691-34695.
37.Claxton, D.P., et al., Ion/substrate-dependent conformational dynamics of a bacterial homolog of neurotransmitter:sodium symporters. Nat Struct Mol Biol, 2010. 17(7): p. 822-829.
38.McHaourab, H.S., P.R. Steed, and K. Kazmier, Toward the Fourth Dimension of Membrane Protein Structure: Insight into Dynamics from Spin-labeling EPR Spectroscopy. Structure (London, England : 1993), 2011. 19(11): p. 1549-1561.
39.Yang, C., et al., Revealing Structural Changes of Prion Protein during Conversion from α-Helical Monomer to β-Oligomers by Means of ESR and Nanochannel Encapsulation. ACS Chemical Biology, 2015. 10(2): p. 493-501.