在N-degron路径的工程N-degron的生成和表征使用ubiquitin-reference技术
Hye Yeon Kim1, Tae Hyun Bae2, Su Jin Lee1
1Department of Biomedical Sciences, College of Medicine, Seoul National University, Seoul, Republic of Korea; Cellular Degradation Biology Center, College of Medicine, Seoul National University, Seoul, Republic of Korea.
Methods in enzymology
|September 24, 2025
概括
N-降解途径通过N-终端氨基酸控制蛋白质降解. 本研究详细介绍了乌比基融合技术和乌比基参考技术的协议,以研究N-降解子及其与N-识别素的相互作用.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 细胞生物学 细胞生物学
背景情况:
- 该N-degron途径通过破坏N-终端氨基酸 (N-degrons) 的稳定性来调节蛋白质和细胞成分降解.
- N-degrons与N-recognins相互作用,将基质连接到无素-蛋白酶体系统 (UPS) 或自-溶酶体系统 (ALS).
- 产生N-degron基板是具有挑战性的,因为它依赖于特定的第二位置残留物来启动 metionin 裂变.
研究的目的:
- 描述用于设计和利用Ubiquitin融合技术 (UFT) 和Ubiquitin参考技术 (URT) 结构的协议.
- 为了能够描述基质降解动力学.
- 为了验证假定的N-degrons并评估它们与N-recognins的相互作用.
主要方法:
- 乌比奎融合技术 (UFT) 创建了嵌合结构,其中乌比奎与基质 (X基质) 融合,允许在乌比奎被二基酶 (DUBs) 分裂后暴露各种N端氨基酸.
- 乌比奎丁参考技术 (URT) 通过测量从参考结构 (M-参考-Ub-X基板) 中的裂变产品的比率来量化X基板衰变.
- 提供了用于设计URT结构的协议,以研究基质降解和N-recognin相互作用.
主要成果:
- URT允许精确测量工程N-degron基板的半衰期.
- 在Arg/N-degron路径中,URT在识别和表征N-识别素方面发挥了重要作用.
- 描述的协议有助于研究各种N-降解子及其功能作用.
结论:
- UFT 和 URT 提供了可靠的方法来研究 N-degron 途径.
- 这些技术对于理解蛋白质降解机制和识别新型N-degrons和N-recognins至关重要.
- 这里详细介绍的协议将促进细胞降解过程和相关途径的研究.
相关概念视频
01:13The Proteasome
1.6K
Eukaryotic cells can degrade proteins through several pathways. One of the most important among these is the ubiquitin-proteasome pathway. It helps the cell eliminate the misfolded, damaged, or unwarranted cytoplasmic proteins in a highly specific manner.
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. This involves participation of a series of enzymes including— E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. This involves participation of a series of enzymes including— E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...
1.6K
02:18The Proteasome
10.1K
Eukaryotic cells can degrade proteins through several pathways. One of the most important amongst these is the ubiquitin-proteasome pathway. It helps the cell eliminate the misfolded, damaged, or unwarranted cytoplasmic proteins in a highly specific manner.
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. A series of enzymes carry out the ubiquitination of the target proteins - E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. A series of enzymes carry out the ubiquitination of the target proteins - E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...
10.1K
02:58Regulated Protein Degradation
8.7K
It is vital to regulate the activity of enzymatic as well as non-enzymatic proteins inside the cell. This can be achieved either through creating a balance between their rate of synthesis and degradation or regulating the intrinsic activity of the protein. Both these regulation mechanisms play an essential role in the normal functioning of cells.
Protein degradation plays two important roles in the cells. It helps to protect cells from misfolded or damaged proteins before they lead to a...
Protein degradation plays two important roles in the cells. It helps to protect cells from misfolded or damaged proteins before they lead to a...
8.7K
01:32Export of Misfolded Proteins out of the ER
5.0K
After folding, the ER assesses the quality of secretory and membrane proteins. The correctly folded proteins are cleared by the calnexin cycle for transport to their final destination, while misfolded proteins are held back in the ER lumen. The ER chaperones attempt to unfold and refold the misfolded proteins but sometimes fail to achieve the correct native conformation. Such terminally misfolded proteins are then exported to the cytosol by ER-associated degradation or ERAD pathway for...
5.0K