HiBiT-SpyTag: A Minimal Tag for Covalent Protein Capture and Degrader Development

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 siz...

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Vydáno v:	ACS chemical biology Ročník 18; číslo 4; s. 933
Hlavní autoři:	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
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	United States 21.04.2023
Témata:	Chromatography, Affinity - methods Endoribonucleases Molecular Probes - chemistry Molecular Probes - metabolism Nuclear Proteins Peptides - chemistry Protein Serine-Threonine Kinases Proteolysis Proteolysis Targeting Chimera - chemistry Proteolysis Targeting Chimera - metabolism Transcription Factors
ISSN:	1554-8937, 1554-8937
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Shrnutí:	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.
Bibliografie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1554-8937 1554-8937
DOI:	10.1021/acschembio.3c00084