Dissecting intracellular metabolite trafficking using chemoproteomics

摘要 信号代谢物控制各种细胞过程，包括细胞周期、分化和适应 环境刺激。信号代谢物的细胞内运输对于维持细胞 稳态并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、炎症和代谢疾病 失调。亚细胞区室化允许相同的分子参与不同的生物活动 流程。信号代谢物通常充当特定蛋白质或传感器配体的第二信使 和核受体（NR），配体激活的转录因子，可感知环境信号并驱动 细胞反应。由于其固有的反应性，NR 配体的细胞内水平及其 亚细胞定位受到严格控制，并且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，但我们对核如何运作的了解 由于缺乏针对此类受体配体的特定工具，受体配体跨细胞器的传播仍然受到限制 机制。我们建议整合化学蛋白质组学、代谢组学和细胞检测，开发新的 化学工具来询问 NR 配体的蛋白质相互作用组并鉴定其细胞内伴侣。 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 信号代谢物并鉴定在其合成位点结合 NR 配体的专用蛋白伴侣 或进入细胞，将它们转运至细胞核，并将其递送至NR。我们初步的一个重要发现 工作是发现 PGRMC2 作为细胞内血红素伴侣，从线粒体运输血红素 进入细胞核并调节血红素反应性转录因子（例如 Rev-）的转录活性 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 识别已知 NR 配体的其他转运机制，例如激活 PPAR 的脂肪酸， 调节新陈代谢和全身能量稳态的配体激活转录因子家族。的 该提案的第二个主要目标是开发空间和时间分辨的蛋白质代谢图谱，我们将其 期望超越细胞内贩运机制的识别，并对细胞内运输产生更广泛的影响 通过提供强大的策略来研究代谢物-蛋白质串扰。最后，该项目独特地结合了 我们在转录调控、代谢和化学生物学方面的多学科专业知识引领 探索细胞生物学中令人兴奋的新发现。

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.