Paudel Yagya Prasad, Valiente Pedro A, Kim Jisun, Kim Philip M
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON M5S 3E1, Canada.
Department of Computer Science, University of Toronto, Toronto, ON M5S 3E1, Canada.
ACS Omega. 2024 Nov 22;9(49):48471-48479. doi: 10.1021/acsomega.4c07071. eCollection 2024 Dec 10.
Here, we describe an innovative and efficient method for screening peptide activators of G-protein-coupled receptors (GPCRs) utilizing a protein-protein interaction (PPI) approach. We designed a library of 92,918 peptides fused with transmembrane domains of glycosylphosphatidylinositol-anchored proteins (GPI-APs). We employed a pooled lentiviral system to promote the expression of these proteins at the cellular membrane and evaluate their ability to activate GPCRs. We then used fluorescence-activated cell sorting (FACS) to screen the GPI-AP-peptide library and identify novel peptide activators of the glucagon-like peptide-1 receptor (GLP-1R). We discovered one peptide PepA3 derived from the Frizzled-like (FZ) domain of human Carboxypeptidase Z (CPZ), a regulated secreted metallocarboxypeptidase. Notably, PepA3 and its two related variants, PepA and PepA2, activated the GLP-1R receptor with less potency but comparable efficacy to that of GLP-1. We then hypothesized that all of these peptides will bind differently to the GLP-1R than the normal ligand. Our technology could identify novel GPCR-activating peptides for structure-function or drug discovery research.
在此，我们描述了一种利用蛋白质-蛋白质相互作用（PPI）方法筛选G蛋白偶联受体（GPCR）肽激活剂的创新且高效的方法。我们设计了一个包含92,918个与糖基磷脂酰肌醇锚定蛋白（GPI-AP）跨膜结构域融合的肽库。我们采用了一种汇集慢病毒系统来促进这些蛋白在细胞膜上的表达，并评估它们激活GPCR的能力。然后，我们使用荧光激活细胞分选（FACS）来筛选GPI-AP-肽库，并鉴定胰高血糖素样肽-1受体（GLP-1R）的新型肽激活剂。我们发现了一种源自人羧肽酶Z（CPZ，一种受调控的分泌型金属羧肽酶）的卷曲样（FZ）结构域的肽PepA3。值得注意的是，PepA3及其两个相关变体PepA和PepA2激活GLP-1R受体的效力较低，但与GLP-1的效力相当。然后我们推测，所有这些肽与GLP-1R的结合方式将与正常配体不同。我们的技术可以为结构-功能或药物发现研究鉴定新型GPCR激活肽。
