An engineered trafficking biosensor reveals a role for DNAJC13 in DOR downregulation

IF 12.9 1区生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY

Nature chemical biology Pub Date : 2024-09-02 DOI:10.1038/s41589-024-01705-2

Brandon Novy, Aleksandra Dagunts, Tatum Weishaar, Emily E. Holland, Hayden Adoff, Emily Hutchinson, Monica De Maria, Martin Kampmann, Nikoleta G. Tsvetanova, Braden T. Lobingier

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Abstract

Trafficking of G protein-coupled receptors (GPCRs) through the endosomal–lysosomal pathway is critical to homeostatic regulation of GPCRs following activation with agonist. Identifying the genes involved in GPCR trafficking is challenging due to the complexity of sorting operations and the large number of cellular proteins involved in the process. Here, we developed a high-sensitivity biosensor for GPCR expression and agonist-induced trafficking to the lysosome by leveraging the ability of the engineered peroxidase APEX2 to activate the fluorogenic substrate Amplex UltraRed (AUR). We used the GPCR–APEX2/AUR assay to perform a genome-wide CRISPR interference screen focused on identifying genes regulating expression and trafficking of the δ-opioid receptor (DOR). We identified 492 genes consisting of both known and new regulators of DOR function. We demonstrate that one new regulator, DNAJC13, controls trafficking of multiple GPCRs, including DOR, through the endosomal–lysosomal pathway by regulating the composition of the endosomal proteome and endosomal homeostasis.

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工程化贩运生物传感器揭示了DNAJC13在DOR下调中的作用

G 蛋白偶联受体（GPCR）通过内泌体-溶酶体途径的转运对于 GPCR 在激动剂激活后的平衡调节至关重要。由于分拣操作的复杂性以及参与该过程的细胞蛋白数量庞大，因此鉴定参与 GPCR 转运的基因具有挑战性。在这里，我们利用工程过氧化物酶 APEX2 激活荧光底物 Amplex UltraRed (AUR) 的能力，开发了一种高灵敏度生物传感器，用于检测 GPCR 的表达和激动剂诱导的向溶酶体的迁移。我们利用 GPCR-APEX2/AUR 试验进行了全基因组 CRISPR 干扰筛选，重点是鉴定调控δ-阿片受体（DOR）表达和转运的基因。我们鉴定了 492 个基因，其中既有已知的 DOR 功能调控因子，也有新的调控因子。我们证明，一个新的调控因子 DNAJC13 通过调控内泌体蛋白组的组成和内泌体稳态，控制包括 DOR 在内的多种 GPCR 通过内泌体-溶酶体途径的转运。

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来源期刊

Nature chemical biology 生物-生化与分子生物学

CiteScore

23.90

自引率

1.40%

发文量

238

审稿时长

12 months

期刊介绍： Nature Chemical Biology stands as an esteemed international monthly journal, offering a prominent platform for the chemical biology community to showcase top-tier original research and commentary. Operating at the crossroads of chemistry, biology, and related disciplines, chemical biology utilizes scientific ideas and approaches to comprehend and manipulate biological systems with molecular precision. The journal embraces contributions from the growing community of chemical biologists, encompassing insights from chemists applying principles and tools to biological inquiries and biologists striving to comprehend and control molecular-level biological processes. We prioritize studies unveiling significant conceptual or practical advancements in areas where chemistry and biology intersect, emphasizing basic research, especially those reporting novel chemical or biological tools and offering profound molecular-level insights into underlying biological mechanisms. Nature Chemical Biology also welcomes manuscripts describing applied molecular studies at the chemistry-biology interface due to the broad utility of chemical biology approaches in manipulating or engineering biological systems. Irrespective of scientific focus, we actively seek submissions that creatively blend chemistry and biology, particularly those providing substantial conceptual or methodological breakthroughs with the potential to open innovative research avenues. The journal maintains a robust and impartial review process, emphasizing thorough chemical and biological characterization.