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首页> 外文期刊>Biochimica et biophysica acta. Biomembranes >Human bile acid transporter ASBT (SLC10A2) forms functional non-covalent homodimers and higher order oligomers
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Human bile acid transporter ASBT (SLC10A2) forms functional non-covalent homodimers and higher order oligomers

机译：人胆酸转运蛋白ASBT（SLC10A2）形成功能性非共价偶联双层和高阶低聚物

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The human apical sodium-dependent bile acid transporter, hASBT/SLC10A2, plays a central role in cholesterol homeostasis via the efficient reabsorption of bile acids from the distal ileum. hASBT has been shown to self associate in higher order complexes, but while the functional role of endogenous cysteines has been reported, their implication in the oligomerization of hASBT remains unresolved. Here, we determined the self-association architecture of hASBT by site-directed mutagenesis combined with biochemical, immunological and functional approaches. We generated a cysteine-less form of hASBT by creating point mutations at all 13 endogenous cysteines in a stepwise manner. Although Cysless hASBT had significantly reduced function correlated with lowered surface expression, it featured an extra glycosylation site that facilitated its differentiation from wt-hASBT on immunoblots. Decreased protein expression was associated with instability and subsequent proteasome-dependent degradation of Cysless hASBT protein. Chemical cross-linking of wild-type and Cysless species revealed that hASBT exists as an active dimer and/or higher order oligomer with apparently no requirement for endogenous cysteine residues. This was further corroborated by co-immunoprecipitation of differentially tagged (HA-, Flag-) wild-type and Cysless hASBT. Finally, Cysless hASBT exhibited a dominant-negative effect when co expressed with wild-type hASBT which validated heterodimerization/oligomerization at the functional level. Combined, our data conclusively demonstrate the functional existence of hASBT dimers and higher order oligomers irrespective of cysteine-mediated covalent bonds, thereby providing greater understanding of its topological assembly at the membrane surface.
机译：人的顶端依赖性胆汁酸转运蛋白HASBT / SLC10A2在胆固醇稳态中通过远离回肠的有效重吸收的胆汁酸来起到核心作用。哈斯特已被证明是在高阶复合物中自我助理，但虽然报告了内源性半胱氨酸的功能作用，但它们对HARBT的寡聚化的含义仍未解决。在这里，我们确定哈斯特的自我关联体系结构通过地点诱变与生化，免疫和功能方法相结合。我们通过以逐步的方式产生所有13个内源性半胱氨酸的点突变来产生半胱氨酸的哈斯特形式。尽管无菌HABT具有显着降低的功能与降低表面表达相关的功能，但它具有额外的糖基化位点，便于其在免疫印迹上的WT-HABT分化。蛋白质表达减少与不稳定性和随后的蛋白酶体依赖性降解的无菌HABT蛋白有关。野生型和无菌栽物种的化学交联显示HARBT作为活性二聚体和/或高阶低聚物，显然不要求内源性半胱氨酸残基。这通过差异标记（HA-，FLAG-）野生型和无菌HABBT的共同免疫沉淀进一步证实。最后，当Cy型HASBT表达时，无菌HABT表达了野生型HASBT的野生型HATBT，该伴有在功能水平处验证了异二聚化/寡聚化。结合，我们的数据得出了无论半胱氨酸介导的共价键如何，无论半胱氨酸介导的共价键如何，如何证明HARBT二聚体和更高阶低聚物的功能存在。

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