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首页> 外文学位 >Understanding structure-function relationships and protein stability of the human apical sodium-dependent bile acid transporter (ASBT, SLC10A2).
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Understanding structure-function relationships and protein stability of the human apical sodium-dependent bile acid transporter (ASBT, SLC10A2).

机译：了解人心钠依赖性胆汁酸转运蛋白（ASBT，SLC10A2）的结构-功能关系和蛋白质稳定性。

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摘要

The apical sodium-dependent bile acid transporter (ASBT, SLC10A2) is renowned as the major bile acid transporter in the intestine. It utilizes cellular sodium gradient to actively concentrate bile acids in the enterocytes, thereby playing a key role in the enterohepatic circulation of bile acids (EHC). ASBT is a promising target for prodrug approaches aiming at improving drug bioavailability, and for drugs to treat hypercholesterolaemia, since cholesterol metabolism is induced upon bile acid depletion. Moreover, its contributing role in drug-drug interactions is rapidly emerging. Topologically, the human (hASBT) is a glycoprotein that spans the membrane bilayer seven times, oriented with an extracellular N-terminus and a cytoplasmic C-terminus (Nexo/Ccyt). Despite its physiological and pharmacological relevance, ASBT remains to be fully characterized at the molecular level. Here, we summarize advances made by our group and others in the quest to understand ASBT's structure-function relationships and its complex mechanism of bile acid transport. We also report our novel findings regarding protein regions relevant for function and protein stability in the hASBT. Our observations indicate that residues located at the transmembrane 1 (TM1) play a pivotal role in hASBT function, and that Gly50, placed at the interface of TM1 with the intracellular loop 1 (IL1), is critical for hASBT stability. Expanding our studies to IL1, we identified a cluster of amino acids comprising Cys51 – Lys57, which are likely involved in hASBT protein stability, whereas residues downstream Lys57 appear to be relevant for transport. We have demonstrated that successful mapping of regions implicated in hASBT's transport cycle can be achieved with a combination of site-directed mutagenesis, bile acid uptake and kinetics, sodium-activation assays and the substituted-cysteine accessibility method. Moreover, inhibition of the proteasome with MG132, and of prolyl-peptidyl isomerases with cyclosporine A and FK506, are valuable approaches to reveal the contribution of specific amino acids to hASBT stability. Finally, we integrate our data to propose an overall schematic of hASBT transport, which will contribute to a better understanding on ASBT physiology and, potentially, on other proteins in the SLC10 family.
机译：根尖钠依赖性胆汁酸转运蛋白（ASBT，SLC10A2）是肠道中主要的胆汁酸转运蛋白。它利用细胞钠梯度使胆汁酸活跃地集中在肠细胞中，从而在胆汁酸（EHC）的肠肝循环中起关键作用。 ASBT是旨在改善药物生物利用度的前药方法和治疗高胆固醇血症药物的有希望的靶标，因为胆汁酸耗尽会诱导胆固醇代谢。而且，它在药物相互作用中的作用正在迅速显现。拓扑学上，人（hASBT）是一种糖蛋白，跨膜双层达七次，以细胞外N端和胞质C端（Nexo / Ccyt）定向。尽管具有生理和药理学意义，ASBT仍需在分子水平上得到充分表征。在这里，我们总结了我们小组和其他人在了解ASBT的结构-功能关系及其胆汁酸转运的复杂机制方面所取得的进展。我们还报告了有关与hASBT功能和蛋白质稳定性有关的蛋白质区域的新发现。我们的观察结果表明，位于跨膜1（TM1）上的残基在hASBT功能中起关键作用，而位于TM1与细胞内环1（IL1）界面的Gly50对hASBT的稳定性至关重要。将我们的研究扩展到IL1，我们发现了一个包含Cys51 – Lys57的氨基酸簇，这些氨基酸可能与hASBT蛋白的稳定性有关，而Lys57下游的残基似乎与运输有关。我们已经证明，通过定点诱变，胆汁酸摄取和动力学，钠活化测定法和取代的半胱氨酸可及性方法的组合，可以成功地绘制涉及hASBT转运周期的区域图。此外，用MG132抑制蛋白酶体以及用环孢霉素A和FK506抑制脯氨酰-肽基异构酶是揭示特定氨基酸对hASBT稳定性的贡献的有价值的方法。最后，我们整合我们的数据以提出hASBT转运的总体示意图，这将有助于更好地了解ASBT生理学，并可能进一步了解SLC10家族中的其他蛋白质。

著录项

  • 作者

    Claro da Silva, Tatiana.;

  • 作者单位

    University of Maryland, Baltimore.;

  • 授予单位 University of Maryland, Baltimore.;
  • 学科 Health Sciences Pharmacy.
  • 学位 Ph.D.
  • 年度 2011
  • 页码 134 p.
  • 总页数 134
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 地球物理学;
  • 关键词

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