Cholesterol-induced conformational changes in the sterol-sensing domain of the Scap protein suggest feedback mechanism to control cholesterol synthesis
Cholesterol-induced conformational changes in the sterol-sensing domain of the Scap protein suggest feedback mechanism to control cholesterol synthesis
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DOI：
10.1074/jbc.m117.783894
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发表时间：
2017-05-26
影响因子：
4.8
通讯作者：
Radhakrishnan, Arun
中科院分区：
文献类型：
--
作者：
Gao, Yansong;Zhou, Yulian;Radhakrishnan, Arun
SCAP是内质网(ER)膜的一种多聚体蛋白，它将固醇调节元件结合蛋白运输到高尔基复合体进行蛋白水解性激活。胆固醇在内质网膜上的积累阻止了SCAP的转运，并减少了胆固醇的合成。以前，我们提供了证据表明，当胆固醇与SCAP的环路1结合时，胆固醇抑制就开始了，SCAP环路投射到内质网管腔。在细胞内，这种结合导致环1与环7分离，环7是另一个管腔SCAP环。然而，当我们将胆固醇添加到环1和环7的分离的络合物中时，我们一直无法证明这种解离。因此，我们推测这种解离需要分隔环1和环7的中间多面体序列的构象变化。在这里，我们使用密封的膜小泡中的蛋白酶保护实验来证明这种变化。在没有胆固醇、胰酶或蛋白酶K的情况下，胞质环4被切割，产生一个受保护的片段，我们用抗环1的单抗观察到这个片段。当胆固醇加入到这些膜上时，环4的切割被取消。由于环4是分隔环1和环7的所谓的类固醇敏感结构域的一部分，这些结果支持胆固醇与环1结合改变了类固醇敏感结构域的构象的假设。他们还提出，这种构象变化有助于将胆固醇信号从环1传递到环7，从而允许环分离并促进胆固醇合成的反馈抑制。这些见解提出了一种新的结构模型，用于胆固醇介导的SCAP活性调节。
Scap is a polytopic protein of endoplasmic reticulum (ER) membranes that transports sterol regulatory element-binding proteins to the Golgi complex for proteolytic activation. Cholesterol accumulation in ER membranes prevents Scap transport and decreases cholesterol synthesis. Previously, we provided evidence that cholesterol inhibition is initiated when cholesterol binds to loop 1 of Scap, which projects into the ER lumen. Within cells, this binding causes loop 1 to dissociate from loop 7, another luminal Scap loop. However, we have been unable to demonstrate this dissociation when we added cholesterol to isolated complexes of loops 1 and 7. We therefore speculated that the dissociation requires a conformational change in the intervening polytopic sequence separating loops 1 and 7. Here we demonstrate such a change using a protease protection assay in sealed membrane vesicles. In the absence of cholesterol, trypsin or proteinase K cleaved cytosolic loop 4, generating a protected fragment that we visualized with a monoclonal antibody against loop 1. When cholesterol was added to these membranes, cleavage in loop 4 was abolished. Because loop 4 is part of the so-called sterol-sensing domain separating loops 1 and 7, these results support the hypothesis that cholesterol binding to loop 1 alters the conformation of the sterol-sensing domain. They also suggest that this conformational change helps transmit the cholesterol signal from loop 1 to loop 7, thereby allowing separation of the loops and facilitating the feedback inhibition of cholesterol synthesis. These insights suggest a new structural model for cholesterol-mediated regulation of Scap activity.