Identification of TRAMs as sphingolipid-binding proteins using a photoactivatable and clickable short-chain ceramide analog.
Identification of TRAMs as sphingolipid-binding proteins using a photoactivatable and clickable short-chain ceramide analog.
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DOI：
10.1016/j.jbc.2021.101415
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发表时间：
2021-12
期刊：
影响因子：
--
通讯作者：
Ye J
中科院分区：
文献类型：
--
作者：
Deng Y;You L;Lu Y;Han S;Wang J;Vicas N;Chen C;Ye J
神经酰胺是一种脂质分子，部分通过反转某些跨膜蛋白的拓扑结构来调节多种生理和病理反应。这种拓扑倒置是通过调节的选择性易位（RAT）实现的，它在翻译过程中逆转了膜蛋白在内质网上易位的方向。然而，由于研究蛋白质-神经酰胺相互作用的技术挑战，尚不清楚细胞中如何感知神经酰胺水平以触发RAT。在这里，我们报道了pac-C7-Cer的合成，pac-C7-Cer是一种可光激活和可点击的短链神经酰胺类似物，可以用作研究蛋白质-神经酰胺相互作用的探针。我们证明了易位链相关膜蛋白2 (TRAM2)，一个已知控制跨膜4 L6亚家族成员20的RAT的蛋白质，和TRAM2的同源物TRAM1，与pac-C7-Cer衍生的分子相互作用。这种相互作用被天然存在的长链神经酰胺分子所取代。我们发现神经酰胺及其类似物与TRAM2的结合与它们诱导跨膜4l6亚家族成员20的RAT的能力相关。除了探测神经酰胺- tram相互作用外，我们提供的证据表明pac-C7-cer可用于蛋白质组范围内的神经酰胺结合蛋白鉴定。我们的研究通过鉴定TRAMs作为潜在的神经酰胺结合蛋白提供了RAT的机制见解，并建立了pac-C7-Cer作为未来研究神经酰胺-蛋白质相互作用的有价值的工具。
Ceramide is a lipid molecule that regulates diverse physiological and pathological reactions in part through inverting the topology of certain transmembrane proteins. This topological inversion is achieved through regulated alternative translocation (RAT), which reverses the direction by which membrane proteins are translocated across the endoplasmic reticulum during translation. However, owing to technical challenges in studying protein–ceramide interaction, it remains unclear how ceramide levels are sensed in cells to trigger RAT. Here, we report the synthesis of pac-C7-Cer, a photoactivatable and clickable short-chain ceramide analog that can be used as a probe to study protein–ceramide interactions. We demonstrate that translocating chain-associated membrane protein 2 (TRAM2), a protein known to control RAT of transmembrane 4 L6 subfamily member 20, and TRAM1, a homolog of TRAM2, interacted with molecules derived from pac-C7-Cer. This interaction was competed by naturally existing long-chain ceramide molecules. We showed that binding of ceramide and its analogs to TRAM2 correlated with their ability to induce RAT of transmembrane 4 L6 subfamily member 20. In addition to probing ceramide–TRAM interactions, we provide evidence that pac-C7-cer could be used for proteome-wide identification of ceramide-binding proteins. Our study provides mechanistic insights into RAT by identifying TRAMs as potential ceramide-binding proteins and establishes pac-C7-Cer as a valuable tool for future study of ceramide–protein interactions.
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影响因子：
7.7
作者：
Denard B;Lee C;Ye J
通讯作者：
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影响因子：
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作者：
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DOI：
10.1083/jcb.113.6.1267
发表时间：
1991-06
期刊：
The Journal of cell biology
影响因子：
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作者：
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通讯作者：
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影响因子：
4.8
作者：
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通讯作者：
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影响因子：
64.5
作者：
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通讯作者：
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