Bioconjugation Strategies for Revealing the Roles of Lipids in Living Cells.
Bioconjugation Strategies for Revealing the Roles of Lipids in Living Cells.
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DOI：
10.1021/acs.accounts.2c00511
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发表时间：
2022-11-01
影响因子：
18.3
通讯作者：
Knittel, Caroline H.
中科院分区：
文献类型：
--
作者：
Devaraj, Neal K.;Knittel, Caroline H.
活细胞的结构边界由许多成膜脂质组成。脂质不仅对细胞区室化至关重要，而且还参与细胞信号传导和能量储存。脂质水平异常与严重的人类疾病有关，例如癌症、多发性硬化症、神经退行性疾病以及溶酶体贮积症。鉴于其生物学意义，人们对研究脂质及其对细胞的影响产生了极大的兴趣。然而，限制因素包括脂质的低溶解度、其结构复杂性以及使用遗传技术直接操纵脂质结构的挑战。目前研究脂质的方法主要依赖于脂质组学，它使用质谱法分析脂质提取物的成分。尽管这些努力已经成功地对细胞中的大量脂质进行了编目和分析，但关于它们的确切功能作用和亚细胞分布的许多方面仍然是个谜。在本报告中，我们概述了我们的实验室如何开发和应用不同的生物共轭策略来研究脂质和脂质修饰在细胞中的作用。受到我们正在进行的开发脂质生物共轭策略以生成人造细胞膜的工作的启发，我们开发了一种在活细胞中合成神经酰胺的方法，使用水杨醛酯，该酯很容易与鞘氨醇以无痕神经酰胺连接的形式发生反应。我们的研究不仅证实了关于神经酰胺与细胞死亡关联的现有知识，而且还提供了关于神经酰胺在细胞凋亡中的结构与功能关系的有趣的新发现。我们最初的努力促使我们研究使用活细胞成像检测内源性鞘脂的探针。我们描述了一种荧光探针的开发，该探针可与活细胞中的鞘氨醇发生化学选择性反应，从而能够检测人类疾病中这种生物标志物内源水平升高的情况。基于我们对脂质荧光标记的兴趣，我们还探索了使用生物正交反应来标记化学合成的脂质探针。我们讨论了光笼二氢四嗪脂质的开发，其中生物正交反应的启动可以由可见光触发，从而允许通过时空控制对膜进行活细胞修饰。最后，蛋白质经常被脂质进行翻译后修饰，这对蛋白质的亚细胞定位和功能具有重要影响。用小分子探针控制脂质修饰有助于揭示脂质翻译后修饰的功能，并可能激发新的治疗策略。我们描述了我们之前对合成膜形成的研究如何启发我们开发一种两亲性半胱氨酸衍生物，该衍生物可以使活细胞中膜结合的 S-酰化蛋白去棕榈酰化。最终，我们将这种两亲物介导的去棕榈酰化（AMD）应用于研究健康和患病细胞中癌症相关棕榈酰化蛋白的棕榈酰化。
The structural boundaries of living cells are composed of numerous membrane-forming lipids. Lipids not only are crucial for the cellular compartmentalization but also are involved in cell signaling as well as energy storage. Abnormal lipid levels have been linked to severe human diseases such as cancer, multiple sclerosis, neurodegenerative diseases, as well as lysosomal storage disorders. Given their biological significance, there is immense interest in studying lipids and their effect on cells. However, limiting factors include the low solubility of lipids, their structural complexity, and the challenge of using genetic techniques to directly manipulate lipid structure. Current methods to study lipids rely mostly on lipidomics, which analyzes the composition of lipid extracts using mass spectrometry. Although, these efforts have successfully catalogued and profiled a great number of lipids in cells, many aspects about their exact functional role and subcellular distribution remain enigmatic. In this Account, we outline how our laboratory developed and applied different bioconjugation strategies to study the role of lipids and lipid modifications in cells. Inspired by our ongoing work on developing lipid bioconjugation strategies to generate artificial cell membranes, we developed a ceramide synthesis method in live cells using a salicylaldehyde ester that readily reacts with sphingosine in form of a traceless ceramide ligation. Our study not only confirmed existing knowledge about the association of ceramides with cell death, but also gave interesting new findings about the structure—function relationship of ceramides in apoptosis. Our initial efforts led us to investigate probes that detect endogenous sphingolipids using live cell imaging. We describe the development of a fluorogenic probe that reacts chemoselectively with sphingosine in living cells, enabling the detection of elevated endogenous levels of this biomarker in human disease. Building on our interest in the fluorescence labeling of lipids, we have also explored the use ofbioorthogonal reactions to label chemically synthesized lipid probes. We discuss the development of photocaged dihydrotetrazine lipids, where the initiation of the bioorthogonal reaction can be triggered by visible light, allowing for live cell modification of membranes with spatiotemporal control. Finally, proteins are often post-translationally modified by lipids, which have important effects on protein subcellular localization and function. Controlling lipid modifications with small molecule probes could help reveal the function of lipid post-translational modifications and could potentially inspire novel therapeutic strategies. We describe how our previous studies on synthetic membrane formation inspired us to develop an amphiphilic cysteine derivative that depalmitoylates membrane-bound S-acylated proteins in live cells. Ultimately, we applied this amphiphile mediated depalmitoylation (AMD) in studies investigating the palmitoylation of cancer relevant palmitoylated proteins in healthy and diseased cells.
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