The study of gut-microbe and host interaction has drawn significant interest in recent years, since gut microbiota play an important role in human health and disease, especially in cardiovascular disease (CVD). Employing untargeted metabolomics as a discovery platform, our laboratory has identified a key gut-microbe derived metabolite phenylacetylglutamine (PAG), which is pro-thrombotic, induces platelet hypersensitivity and significantly contributes to CVD. My goal is to identify the receptor(s), PAG mediates cellular events through leading to CVD progression. My preliminary studies using a label-free whole-cell assay technology dynamic mass redistribution (DMR), suggest that PAG undergoes saturable and specific binding to cells, suggesting receptor-ligand interaction. Secondary messenger assays along with DMR studies confirm that PAG functions through G-protein coupled receptors (GPCRs). Interestingly, loss of function studies in megakaryoblast cells suggest that PAG is likely to mediate cellular response through adrenergic receptors (ADRs), which potentially regulates heart function and circulatory system. We propose to characterize of PAG responsive ADRs present on human platelets (alpha 2A, alpha 2B and & beta 2) by performing gain of function and radioligand binding studies in HEK293 cell lines. Further, we plan to identify and characterize other possible ADRs (alpha 1A, alpha 1B, alpha 1D, alpha 2C, beta 1 and beta 3) triggered by PAG performing gain and loss of function studies in different cell lines. To test the hypothesis that PAG may function through GPCRs other than ADRs in other tissues, we propose to screen a wide range of human nonolfactory GPCRs through DMR using silencer select GPCR-siRNA library. As a complementary approach, we plan to perform the beta-arrestin2 recruitment assay named PRESTO-Tango to validate all the positive GPCR hits including the ADRs in HTLA cells.The study of receptors plays a very crucial role in biomedical research today and fascinated the scientific world for the last few decades. Interestingly, GPCRs are the targets for drugs accounting for more than half of all prescribed drugs available in the world. Our key observation of PAG interaction with GPCRs, specifically with ADRs opens a great possibility to target the receptors with small molecule drugs to combat PAG-mediated CVD progression.
|Program type||Postdoctoral Fellowship|
|Effective start/end date||01/01/2020 → 12/31/2021|