Dissecting intracellular metabolite trafficking using chemoproteomics

摘要 信号代谢产物控制各种细胞过程，包括细胞周期、分化和适应 环境刺激。细胞内信号代谢产物的运输对维持细胞 动态平衡和整合代谢和转录反应。代谢物运输缺陷和 分布可能导致多种疾病，包括癌症、免疫学、炎症性和新陈代谢。 精神错乱。亚细胞区隔作用允许相同的分子参与不同的生物 流程。信号代谢物通常作为特定蛋白质或传感器配体的第二信使。 核受体(NR)，配体激活的转录因子，感知环境信号并驱动 细胞反应。由于其固有的反应性，NR配体的细胞内水平以及它们的 亚细胞定位，受到严格控制，可能会根据营养状态和 病理生理条件。尽管我们了解它们的功能，但我们对核能如何 由于缺乏针对受体配体的特定工具，受体配体在细胞器之间的传递仍然有限 机械装置。我们建议将化学蛋白质组学、代谢组学和细胞分析相结合，以开发新的 化学工具，询问蛋白质相互作用的NR配体，并确定他们的细胞内伴侣。 利用这些技术，我们打算揭示细胞内运输的分子和功能基础 信号代谢物，并确定在其合成位置结合NR配体的专用蛋白伴侣 或者进入细胞，将它们运输到细胞核，并将它们传递给NRS。我们的初步调查结果令人振奋 工作是发现了PGRMC2作为一种细胞内的血红素伴侣，从线粒体运输血红素 并调节血红素反应转录因子的转录活性，如REV- ERB和BACH1.我们将利用从这项初步工作中获得的经验，将我们的研究扩展到 鉴定已知的NR配体的其他运输机制，如激活PPAR的脂肪酸，a 配体激活的转录因子家族，调节新陈代谢和全身能量平衡。这个 这项提议的第二个主要目标是开发空间和时间分辨的蛋白质代谢物图谱，我们 预计将超越细胞内贩运机制的确定，并对 通过提供一种强有力的策略来研究代谢物-蛋白质串扰。最后，这个项目独一无二地结合了 我们在转录调控、新陈代谢和化学生物学方面的多学科专业知识 探索细胞生物学中一项令人兴奋的新发现。

ABSTRACT Signaling metabolites control various cellular processes, including cell cycle, differentiation, and adaptations to environmental stimuli. Intracellular trafficking of signaling metabolites is crucial for maintaining cellular homeostasis and integrate metabolic and transcriptional responses. Defects in metabolite transport and distribution may lead to multiple diseases, including cancer, immunological, inflammatory, and metabolic disorders. Subcellular compartmentalization allows the same molecules to partake in distinct biological processes. Signaling metabolites generally act as second messengers for specific proteins or ligands for sensors and nuclear receptors (NR), ligand-activated transcription factors that sense environmental signals and drive cellular response. Because of their intrinsic reactivity, the intracellular levels of NR ligands, along with their subcellular localization, are tightly controlled and may oscillate greatly depending on nutritional states and pathophysiological conditions. Despite our understanding of their functions, our knowledge of how nuclear receptor ligands travel across organelles remains limited due to the lack of specific tools to target such mechanisms. We propose to integrate chemoproteomics, metabolomics, and cellular assays, to develop novel chemical tools to interrogate the protein interactomes of NR ligands and identify their intracellular chaperones. Leveraging these technologies, we intend to reveal the molecular and functional basis of intracellular trafficking of signaling metabolites and identify dedicated protein chaperones that bind NR ligands at their site of synthesis or entry into the cell, transport them to the nucleus, and deliver them to NRs. A driving finding of our preliminary work was the discovery of PGRMC2 as an intracellular heme chaperone that transports heme from mitochondria to the nucleus and regulates the transcriptional activity of heme-responsive transcription factors such as Rev- Erb and BACH1. We will use the experience acquired from this initial work to extend our studies to the identification of other transport mechanisms for known NR ligands, such as fatty acids, that activate PPARs, a family of ligand-activated transcription factors that regulate metabolism and systemic energy homeostasis. The second major goal of this proposal is to develop spatial- and time-resolved protein-metabolite maps, which we expect to go beyond the identification of intracellular trafficking mechanisms and have a broader impact on the field by providing a powerful strategy to study metabolite-protein crosstalk. Lastly, this project uniquely combines our multidisciplinary expertise in transcriptional regulation, metabolism, and chemical biology to lead the exploration of a new exciting findings in cell biology.