Isom Lab Research Milestones
In our proton-related research program, we use a variety of human and yeast cell models, synthetic biology, molecular biophysics and pharmacology, and data science to illuminate the molecular and cellular basis of proton signaling and sensing. In the Fall of 2023, we will add structural biology to our growing capabilities, a new opportunity made possible by our Molecular Electron Microscopy Center at the Frost Institute for Chemistry and Molecular Sciences.
Putting the pH into pHarmacology
Molecular pharmacology and molecular biophysics teamed with synthetic biology:
Many factors, including coincident pH signals, regulate the interaction of endogenous ligands and drugs with their targets. Until our work (1-3), there were no cell-based platforms for studying how coincident pH signals regulate protein and receptor function(s) in processes such as cell signaling. By engineering innovative synthetic biology solutions—humanizing a pH-tolerant yeast model, developing high throughput CRISPR editing capabilities, and designing pooled experiments deconvoluted by cell sorting—we have created a yeast-based platform for human G protein-coupled receptors (GPCRs) that enables the study and discovery of endogenous GPCR ligands and drugs at any physiologic pH. Thus far, we have used our platform to study 100+ human GPCRs to discover new GPCR ligands (3,4). Our long-term goal is to port all 900+ human GPCRs to this system.
Proton gating of GPCR signaling
Molecular pharmacology and molecular biophysics teamed with synthetic biology:
Coincident pH signals are critical to the (patho)physiology of cells, tissues, and organs. However, our understanding of the molecular and cellular basis of proton sensing and regulation is limited. For example, once activated, many cell-surface receptors are internalized by endocytosis, and their activity is thought to be regulated by progressive endosomal acidification. For GPCRs, this coincident pH signaling could inhibit, enhance, or have no effect on GPCR activity, degradation, and recycling back to the cell membrane. We have begun to address this question at scale using our yeast-based platform (3). Our initial study of 28 human GPCRs has systematically shown that proton gating is a common feature of GPCR signaling (1). Building on this advance, we are pursuing the study and discovery of pH-intelligent GPCR ligands, metabolites, and drugs that selectively regulate GPCR function at specific pH values.
Illuminating mechanisms of GPCR proton sensing
Molecular biophysics and synthetic biology teamed with data science:
Three proton-sensing GPCRs were discovered over two decades ago: GPR4, GPR65, and GPR68. Until our recent work (5), these receptors were thought to be activated by extracellular histidine residues sensing changes in extracellular pH. Combining data science, molecular biophysics, and synthetic biology, we showed that proton sensing is instead regulated by a triad of conserved acidic residues buried deep in the receptor core (5). Mechanistically, this triad controls receptor activation by functioning as a H+ and Na+ coincidence detector. As such, this work is the first to demonstrate the phenomenon of molecular coincidence detection in response to coincident pH signals. We are now using this insight to illuminate the (patho)physiologic behavior of these receptors to develop therapeutic inhibitors and modulators.
GPCRs lack concensus cholesterol-binding motifs
Data science:
By virtue of their residence in membrane bilayers, the structure and function of transmembrane proteins and receptors are often regulated by specific lipid and sterol species, such as cholesterol. Early structures of GPCRs contained bound cholesterol, creating the perception that they contain cholesterol-binding motifs. We discovered this perception was inaccurate by exhaustively characterizing GPCR-cholesterol binding sites in the Protein Data Bank. Our computations show that the locations of cholesterol binding sites in GPCRs are recurrent and highly predictable even though they lack consensus, 3-dimensional amino acid motifs (6).
References
1 -- Kapolka, N. J., Rowe, J. B., Taghon, G. J., Morgan, W. M., O'Shea, C. R. & Isom, D. G. Proton-gated coincidence detection is a common feature of GPCR signaling. Proc Natl Acad Sci U S A 118 (2021). https://doi.org:10.1073/pnas.2100171118
2 -- Rowe, J. B., Taghon, G. J., Kapolka, N. J., Morgan, W. M. & Isom, D. G. CRISPR-addressable yeast strains with applications in human G protein-coupled receptor profiling and synthetic biology. J Biol Chem 295, 8262-8271 (2020). https://doi.org:10.1074/jbc.RA120.013066
3 -- Kapolka, N. J. et al. DCyFIR: a high-throughput CRISPR platform for multiplexed G protein-coupled receptor profiling and ligand discovery. Proc Natl Acad Sci U S A 117, 13117-13126 (2020). https://doi.org:10.1073/pnas.2000430117
4 -- Kapolka, N. J. & Isom, D. G. HCAR3: an underexplored metabolite sensor. Nat Rev Drug Discov 19, 745 (2020). https://doi.org:10.1038/d41573-020-00173-2
5 -- Rowe, J. B., Kapolka, N. J., Taghon, G. J., Morgan, W. M. & Isom, D. G. The evolution and mechanism of GPCR proton sensing. J Biol Chem 296, 100167 (2021). https://doi.org:10.1074/jbc.RA120.016352
6 -- Taghon, G. J., Rowe, J. B., Kapolka, N. J. & Isom, D. G. Predictable cholesterol binding sites in GPCRs lack consensus motifs. Structure 29, 499-506 e493 (2021). https://doi.org:10.1016/j.str.2021.01.004