AID2 与 BromoTag 的结合拓展了基于降解子的蛋白质敲除技术的用途。

IF 6.5 1区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY EMBO Reports Pub Date : 2024-09-01 Epub Date: 2024-08-23 DOI:10.1038/s44319-024-00224-4
Yuki Hatoyama, Moutushi Islam, Adam G Bond, Ken-Ichiro Hayashi, Alessio Ciulli, Masato T Kanemaki
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引用次数: 0

摘要

急性蛋白质敲除是剖析动态细胞过程中蛋白质功能的有力方法。我们以前曾报道过一种改进的辅助素诱导的降解子系统 AID2,但最近注意到它诱导降解一些重要复制因子(如 ORC1 和 CDC6)的能力不足以诱导致死。在这里,我们提出了组合降解子技术,以控制两种蛋白或加强目标耗竭。为此,我们首先将基于 PROTAC 的脱胶子 dTAG 和 BromoTag 与 AID2 进行了比较,揭示了它们的主要特征,然后演示了用 AID2 和 BromoTag 分别控制凝聚素和凝集素的过程。我们利用 AID2 和 BromoTag 开发了一种双脱氧核苷酸系统,以加强目标耗竭和加速耗竭动力学,并证明 ORC1 和 CDC6 对 MCM 负载至关重要。最后,我们证明了双脱殖子系统对 ORC1 和 CDC6 的共同耗竭完全抑制了 DNA 复制,细胞以单染色体进入有丝分裂,表明 DNA 复制与细胞周期控制脱钩。我们的组合脱粒技术将扩大功能分析的应用范围。
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Combination of AID2 and BromoTag expands the utility of degron-based protein knockdowns.

Acute protein knockdown is a powerful approach to dissecting protein function in dynamic cellular processes. We previously reported an improved auxin-inducible degron system, AID2, but recently noted that its ability to induce degradation of some essential replication factors, such as ORC1 and CDC6, was not enough to induce lethality. Here, we present combinational degron technologies to control two proteins or enhance target depletion. For this purpose, we initially compare PROTAC-based degrons, dTAG and BromoTag, with AID2 to reveal their key features and then demonstrate control of cohesin and condensin with AID2 and BromoTag, respectively. We develop a double-degron system with AID2 and BromoTag to enhance target depletion and accelerate depletion kinetics and demonstrate that both ORC1 and CDC6 are pivotal for MCM loading. Finally, we show that co-depletion of ORC1 and CDC6 by the double-degron system completely suppresses DNA replication, and the cells enter mitosis with single-chromatid chromosomes, indicating that DNA replication is uncoupled from cell cycle control. Our combinational degron technologies will expand the application scope for functional analyses.

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来源期刊
EMBO Reports
EMBO Reports 生物-生化与分子生物学
CiteScore
11.20
自引率
1.30%
发文量
267
审稿时长
1 months
期刊介绍: EMBO Reports is a scientific journal that specializes in publishing research articles in the fields of molecular biology, cell biology, and developmental biology. The journal is known for its commitment to publishing high-quality, impactful research that provides novel physiological and functional insights. These insights are expected to be supported by robust evidence, with independent lines of inquiry validating the findings. The journal's scope includes both long and short-format papers, catering to different types of research contributions. It values studies that: Communicate major findings: Articles that report significant discoveries or advancements in the understanding of biological processes at the molecular, cellular, and developmental levels. Confirm important findings: Research that validates or supports existing knowledge in the field, reinforcing the reliability of previous studies. Refute prominent claims: Studies that challenge or disprove widely accepted ideas or hypotheses in the biosciences, contributing to the correction and evolution of scientific understanding. Present null data: Papers that report negative results or findings that do not support a particular hypothesis, which are crucial for the scientific process as they help to refine or redirect research efforts. EMBO Reports is dedicated to maintaining high standards of scientific rigor and integrity, ensuring that the research it publishes contributes meaningfully to the advancement of knowledge in the life sciences. By covering a broad spectrum of topics and encouraging the publication of both positive and negative results, the journal plays a vital role in promoting a comprehensive and balanced view of scientific inquiry. 
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