Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Center for Motor Neuron Biology and Diseases, Department of Neurology. Columbia University Irving Medical Center. New York, NY 10032, USA; Department of Pathology & Cell Biology. Columbia University Irving Medical Center. New York, NY 10032, USA.
Bioorg Chem. 2024 Jun;147:107365. doi: 10.1016/j.bioorg.2024.107365. Epub 2024 Apr 16.
Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally identify and monitor levels of all prenylated proteins in a cell simultaneously, our laboratory and others have developed chemical proteomic approaches that rely on the metabolic incorporation of isoprenoid analogues bearing bio-orthogonal functionality followed by enrichment and subsequent quantitative proteomic analysis. Here, several improvements in the synthesis of the alkyne-containing isoprenoid analogue C15AlkOPP are reported to improve synthetic efficiency. Next, metabolic labeling with C15AlkOPP was optimized to obtain useful levels of metabolic incorporation of the probe in several types of primary cells. Those conditions were then used to study the prenylomes of motor neurons (ES-MNs), astrocytes (ES-As), and their embryonic stem cell progenitors (ESCs), which allowed for the identification of 54 prenylated proteins from ESCs, 50 from ES-MNs, and 84 from ES-As, representing all types of prenylation. Bioinformatic analysis revealed specific enriched pathways, including nervous system development, chemokine signaling, Rho GTPase signaling, and adhesion. Hierarchical clustering showed that most enriched pathways in all three cell types are related to GTPase activity and vesicular transport. In contrast, STRING analysis showed significant interactions in two populations that appear to be cell type dependent. The data provided herein demonstrates that robust incorporation of C15AlkOPP can be obtained in ES-MNs and related primary cells purified via magnetic-activated cell sorting allowing the identification and quantification of numerous prenylated proteins. These results suggest that metabolic labeling with C15AlkOPP should be an effective approach for investigating the role of prenylated proteins in primary cells in both normal cells and disease pathologies, including ALS.
蛋白质的 prenylation 是一类广泛的翻译后修饰的一个例子，其中蛋白质被共价连接到各种疏水性部分。为了全局地识别和监测细胞中所有 prenylated 蛋白的水平，我们的实验室和其他实验室已经开发了化学蛋白质组学方法，这些方法依赖于异戊烯类似物的代谢掺入，这些类似物带有生物正交功能，然后进行富集和随后的定量蛋白质组学分析。在这里，报道了几种改进炔烃含异戊烯类似物 C15AlkOPP 的合成方法，以提高合成效率。接下来，优化了 C15AlkOPP 的代谢标记，以获得探针在几种类型的原代细胞中有用的代谢掺入水平。然后，使用这些条件来研究运动神经元 (ES-MNs)、星形胶质细胞 (ES-As) 和它们的胚胎干细胞前体 (ESCs) 的 prenylomes，这使得能够从 ESCs 中鉴定出 54 种 prenylated 蛋白，从 ES-MNs 中鉴定出 50 种，从 ES-As 中鉴定出 84 种，代表所有类型的 prenylation。生物信息学分析显示了特定的富集途径，包括神经系统发育、趋化因子信号、Rho GTPase 信号和黏附。层次聚类显示，所有三种细胞类型中大多数富集的途径都与 GTPase 活性和囊泡运输有关。相比之下，STRING 分析显示在两个似乎依赖于细胞类型的群体中存在显著的相互作用。本文提供的数据表明，通过磁性激活细胞分选纯化的 ES-MNs 和相关原代细胞中可以获得 C15AlkOPP 的稳健掺入，从而鉴定和定量大量 prenylated 蛋白。这些结果表明，C15AlkOPP 的代谢标记应该是研究 prenylated 蛋白在正常细胞和疾病病理中的原代细胞中的作用的有效方法，包括 ALS。
