利用單分子螢光共振能量轉移光譜觀察鈉離子依賴性膽酸轉運蛋白(ASBT)的動態變化

Conformational Dynamics of the Apical Sodium-dependent Bile acid Transporter (ASBT) Studied by Single-Molecule Fluorescence Resonance Energy Transfer Spectroscopy

古芝綺(Ku Chih-Chi)

指導教授 ： 李以仁

國立臺灣師範大學/理學院/化學系/碩士(2021年)

https://doi.org/10.6345/NTNU202101169

摘要

鈉離子依賴性膽酸轉運蛋白(Apical sodium-dependent bile acid transporter; ASBT)利用膜內外的鈉離子濃度梯度驅動運輸膽酸(bile acid)，被認為是腸肝循環(enterohepatic circulation)中膽酸回收的第一步。抑制ASBT功能會減少膽酸的回收，導致膽酸的合成增加和降低膽固醇，可用於治療高膽固醇血症(hypercholesterolemia)。目前已有三種ASBT晶體結構被解出，分別為含有兩個鈉離子與一個受質結合的向內開口型(inward-facing)，與沒有任何離子與受質的結合時，向內開口型及向外開口型(outward-facing)。然而，這些靜態結構不足以提供一個完整的運輸機制，我們利用單分子螢光共振能量轉移光譜觀察蛋白脂質體與微胞中ASBT的構型動力學。我們在實驗中觀察到可互相轉換的三種構型，其狀態轉變之動力學受溶液中的離子與膽酸濃度影響，可以提供膜蛋白更詳細的運輸機制。

關鍵字

鈉離子依賴性膽酸轉運蛋白（ASBT） ； 腸肝循環 ； 高膽固醇血症 ； 單分子螢光共振能量轉移

並列摘要

Apical sodium-dependent bile acid transporter (ASBT) utilizes sodium gradient to transport bile acid and is considered the first step for bile acid reabsorption in the enterohepatic circulation. Inhibiting the ASBT function reduces the reabsorption of bile acid synthesized from cholesterol. Inhibition of ASBT promotes the synthesis of bile acid and reduction of cholesterol for the treatment of hypercholesterolemia. Three crystal structures of ASBT have been reported: Inward-open structure binding with bile acid taurocholate and two sodium cations was found in ASBT homolog from Neisseria meningitides (ASBTNM). Apo structures with inward- and outward-open structures were resolved in ASBT homolog from Yersinia frederiksenii (ASBTYF). However, these static structures are insufficient to provide a comprehensive transportation mechanism, which is expected to be highly dynamic. We utilize single-molecule fluorescence resonance energy transfer (smFRET) microscopy to observe the direct conformation dynamics in the micelle and proteoliposome. Our result revealed that there are transitions between three major conformations. Cation- (K+ and Na+), as well as the cargo- (taurocholate in Na+), dependent transition kinetics was observed. Thus, the direct observation of conformational change can provide more detailed information on the transport mechanism.

並列關鍵字

Apical sodium-dependent bile acid transporter(ASBT) ； enterohepatic circulation ； hypercholesterolemia ； single-molecule fluorescence resonance energy transfer(smFRET)

參考文獻

[1] Wallin, E.; von Heijne, G., Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Sci. 1998, 7 (4), 1029-38.

[2] Stillwell, W., Chapter 19 - Membrane Transport. In An Introduction to Biological Membranes (Second Edition), Stillwell, W., Ed. Elsevier: 2016; pp 423-451.

[3] Perland, E.; Lekholm, E.; Eriksson, M. M.; Bagchi, S.; Arapi, V.; Fredriksson, R., The Putative SLC Transporters Mfsd5 and Mfsd11 Are Abundantly Expressed in the Mouse Brain and Have a Potential Role in Energy Homeostasis. PLoS One. 2016, 11 (6), e0156912.

[4] Lazaridis, K. N.; Pham, L.; Tietz, P.; Marinelli, R. A.; deGroen, P. C.; Levine, S.; Dawson, P. A.; LaRusso, N. F., Rat cholangiocytes absorb bile acids at their apical domain via the ileal sodium-dependent bile acid transporter. J Clin Invest. 1997, 100 (11), 2714-21.

[5] Hofmann, A. F.; Hagey, L. R., Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics. Cell Mol Life Sci. 2008, 65 (16), 2461-83.