Precise and In Vivo-Compatible Spatial Proteomics via Bioluminescence-Triggered Photocatalytic Proximity Labeling

IF 10.4 1区 化学 Q1 CHEMISTRY, MULTIDISCIPLINARY
Xuege Sun, , , Yanling Zhang, , , Wenjie Lu, , , Hongyang Guo, , , Guodong He, , , Siyuan Luo, , , Haodong Guo, , , Zijuan Zhang, , , Wenjing Wang, , , Ling Chu, , , Xiangyu Liu, , and , Wei Qin*, 
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引用次数: 0

Abstract

Protein function is closely tied to its localization and interactions, which can be mapped using proximity labeling (PL). Traditional PL methods, such as peroxidases and biotin ligases, suffer from toxicity or high background. While visible-light-triggered photocatalytic labeling offers great potential, it is limited by light-induced background and restricted in vivo applications. Here we present BRET-ID, an in vivo-compatible PL technology for precise mapping of membraneless organelles and transient protein–protein interactions with subminute temporal resolution. BRET-ID combines a genetically encoded photocatalyst and NanoLuc luciferase, locally generating blue light to activate the photocatalyst via bioluminescence resonance energy transfer (BRET). This activation produces singlet oxygen, which oxidizes nearby proteins for analysis with a streamlined chemoproteomic workflow. BRET-ID enables precise mapping of ER membrane proteins, exhibiting high spatial specificity. Leveraging its high temporal resolution, BRET-ID provides 1 min snapshots of dynamic GPCR interactions during ligand-induced endocytosis. Additionally, BRET-ID identifies G3BP1-interacting proteins in arsenite-stressed cells and tumor xenografts, uncovering novel stress granule components, including the mTORC2 subunit RICTOR. BRET-ID serves as a powerful genetically encoded tool for studying protein localization and molecular interactions in living organisms.

A genetically encoded BRET-activated proximity labeling tool enables high-resolution mapping of protein localizations and interactions in live cells and in vivo.

通过生物发光触发光催化接近标记精确和体内兼容的空间蛋白质组学
蛋白质的功能与其定位和相互作用密切相关,这可以使用接近标记(PL)来绘制。传统的PL方法,如过氧化物酶和生物素连接酶,存在毒性或高本底的问题。虽然可见光触发的光催化标记具有巨大的潜力,但受光诱导背景和体内应用的限制。在这里,我们提出BRET-ID,一种体内兼容的PL技术,用于精确绘制无膜细胞器和瞬态蛋白质-蛋白质相互作用的亚分钟时间分辨率。BRET- id结合了基因编码的光催化剂和NanoLuc荧光素酶,局部产生蓝光,通过生物发光共振能量转移(BRET)激活光催化剂。这种激活产生单线态氧,氧化附近的蛋白质,用流线型的化学蛋白质组学工作流程进行分析。BRET-ID能够精确定位内质网膜蛋白,具有很高的空间特异性。BRET-ID利用其高时间分辨率,在配体诱导的内吞作用过程中提供1分钟动态GPCR相互作用的快照。此外,BRET-ID鉴定了亚砷酸盐应激细胞和肿瘤异种移植物中g3bp1相互作用蛋白,揭示了新的应激颗粒成分,包括mTORC2亚基RICTOR。BRET-ID是研究生物体中蛋白质定位和分子相互作用的强大遗传编码工具。基因编码bret激活的接近标记工具可以实现活细胞和体内蛋白质定位和相互作用的高分辨率制图。
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来源期刊
ACS Central Science
ACS Central Science Chemical Engineering-General Chemical Engineering
CiteScore
25.50
自引率
0.50%
发文量
194
审稿时长
10 weeks
期刊介绍: ACS Central Science publishes significant primary reports on research in chemistry and allied fields where chemical approaches are pivotal. As the first fully open-access journal by the American Chemical Society, it covers compelling and important contributions to the broad chemistry and scientific community. "Central science," a term popularized nearly 40 years ago, emphasizes chemistry's central role in connecting physical and life sciences, and fundamental sciences with applied disciplines like medicine and engineering. The journal focuses on exceptional quality articles, addressing advances in fundamental chemistry and interdisciplinary research.
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