Tsang Tiffany, Huerta Fidel, Liu Yingpeng, Che Jianwei, Metivier Rebecca J, Ferrao Silas, Donovan Katherine A, Jones Lyn H, Zerfas Breanna L, Nowak Radosław P
Center for Protein Degradation, Dana-Farber Cancer Institute, Boston Massachusetts 02215, United States.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston Massachusetts 02115, United States.
ACS Chem Biol. 2023 Apr 21;18(4):933-941. doi: 10.1021/acschembio.3c00084. Epub 2023 Apr 5.
The ability to rapidly and selectively modulate cellular protein levels using small molecules is essential for studying complex biological systems. Degradation tags, such as dTAG, allow for selective protein removal with a specific degrader molecule, but their utility is limited by the large tag size (>12 kDa) and the low efficiency of fusion product gene knock-in. Here, we describe the development of a short 24 amino acid peptide tag that enables cell-based quantification and covalent functionalization of proteins to which it is fused. The minimalistic peptide, termed HiBiT-SpyTag, incorporates the HiBiT peptide for protein level quantification and SpyTag, which forms a spontaneous isopeptide bond in the presence of the SpyCatcher protein. Transient expression of dTAG-SpyCatcher efficiently labels HiBiT-SpyTag-modified BRD4 or IRE1α in cells, and subsequent treatment with the dTAG13 degrader results in efficient protein removal without the need for full dTAG knock-in. We also demonstrate the utility of HiBiT-SpyTag for validating the degradation of the endoplasmic reticulum (ER) stress sensor IRE1α, which led to the development of the first PROTAC degrader of the protein. Our modular HiBiT-SpyTag system represents a valuable tool for the efficient development of degraders and for studying other proximity-induced pharmacology.
利用小分子快速、选择性地调节细胞蛋白质水平的能力对于研究复杂生物系统至关重要。降解标签，如dTAG，可通过特定的降解分子实现蛋白质的选择性去除，但其效用受到标签尺寸大（>12 kDa）和融合产物基因敲入效率低的限制。在此，我们描述了一种由24个氨基酸组成的短肽标签的开发，该标签能够对与其融合的蛋白质进行基于细胞的定量和共价功能化。这种简约的肽称为HiBiT-SpyTag，它包含用于蛋白质水平定量的HiBiT肽和SpyTag，SpyTag在SpyCatcher蛋白存在的情况下会形成自发的异肽键。dTAG-SpyCatcher的瞬时表达可有效标记细胞中HiBiT-SpyTag修饰的BRD4或IRE1α，随后用dTAG13降解剂处理可有效去除蛋白质，而无需完全敲入dTAG。我们还展示了HiBiT-SpyTag在验证内质网（ER）应激传感器IRE1α降解方面的效用，这促成了该蛋白首个PROTAC降解剂的开发。我们的模块化HiBiT-SpyTag系统是开发降解剂以及研究其他邻近诱导药理学的宝贵工具。
