Chemoproteomics of microbiota metabolites reveals small-molecule agonists for orphan receptor GPRC5A

Abstract

The microbiota generates diverse metabolites that can engage multiple pathways to modulate host physiology and disease, but their protein targets and mechanism(s) of action have not been fully elucidated. To address this challenge, we focused on indole-3-acetic acid (IAA), a prominent microbiota metabolite, and developed IAA-based chemical reporters for proteomic studies. We discovered that IAA interacts with many proteins in host cells, including small-molecule transporters, receptors and metabolic enzymes. Notably, our functional studies revealed that IAA binds to orphan G protein-coupled receptors such as GPRC5A, but only aromatic monoamines were capable of inducing GPRC5A signaling. Functional profiling of microbiota uncovered specific bacterial species and enzymes that generate GPRC5A agonists. Finally, biochemical characterization of GPRC5A activation identified more potent synthetic agonists as well as key amino acid residues involved in ligand binding. These studies highlight the utility of chemoproteomics to dissect protein targets and mechanisms of action for microbiota metabolites.

Chemoproteomic Analysis of Microbiota Metabolite–Protein Targets and Mechanisms

“…Nonetheless, the identification of novel metabolite protein interactions may guide the exploration of structurally related metabolites that may yield more active agonists or antagonists of newly identified protein targets. 108 Given the increasingly frequent discoveries of microbiota and their metabolites in host physiology and disease, we hope this Perspective on chemoproteomics inspires the development and application of new chemical reporters to elucidate the mechanisms and functions of microbiota metabolites in health and disease.…”

“…To explore additional indole metabolite protein targets, our laboratory developed metabolic and photoaffinity reporters derived from indole acetic acid (IAA) (Figure 5A,B) and employed chemoproteomic profiling of IAA-interacting proteins in intestinal epithelial cells. 108 Interestingly, we identified many unpredicted IAA-interacting proteins, including small molecule transporters, metabolic enzymes, innate immune-associated effectors, and orphan GPCRs. Notably, our functional studies showed that IAA can interact with GPRC5A, an orphan GPCR tumor suppressor, but did not affect its activity.…”

“…However, our additional experiments revealed that aromatic amino-derived monoamines such as tryptamine and phenethylamine as well as their microbiota-generating species can activate GPRC5A-mediated β-arrestin signaling, which provides the first agonists of this tumor-and immunityassociated GPCR. 108 The microbiota metabolism of aromatic amino acids into monoamines is important for modulating host physiology. For example, Ruminococcus gnavus generates tryptamine, 109 while Morganella morganii has been shown to produce phenylethylamine.…”

“…In this regard, aAA-based reporters could facilitate the identification of additional protein targets and cellular pathways in epithelial and immune cells. To explore additional indole metabolite protein targets, our laboratory developed metabolic and photoaffinity reporters derived from indole acetic acid (IAA) (Figure A,B) and employed chemoproteomic profiling of IAA-interacting proteins in intestinal epithelial cells . Interestingly, we identified many unpredicted IAA-interacting proteins, including small molecule transporters, metabolic enzymes, innate immune-associated effectors, and orphan GPCRs.…”

“…(A) Indole acetic acid metabolic reporter (alk-IAA) 108. (B) Indole acetic acid photoaffinity reporter (x-alk-IAA) 108. (C) 5-Hydroxytryptamine metabolic reporter (5-PT) 134.…”