Biology subjects

Isom, D. G.

Publications and source records attributed to Isom, D. G..

12 recordsLinked to original sources

bioRxiv2026

Modulating proton sensing by the adenosine receptor A2

For years, it was believed that G-protein-coupled receptors (GPCRs) activated or modulated by changes in physiological pH were mechanistically regulated by pH-sensitive histidine residues on the receptors exterior. More recent studies have shown that traditional acid-sensing GPCRs (GPR4, GPR65, GPR68) contain buried acidic amino acids in their 7-transmembrane regions and can be activated by pH-dependent mechanisms. The focus of our research has been the adenosine A2A receptor (A2AR) and its ability to activate at low pH levels without having the typical acidic triad structure found in previously studied acid-sensing GPCRs. We used a combination of bioinformatics and a humanized yeast-based platform, DCyFIR, to identify potential structures involved in the proton-sensing mechanism of A2AR. We also validated some of our A2AR variant yeast phenotypes using mammalian cells. Our data suggest that certain mutations eliminate the pH sensitivity of the A2AR while preserving agonist-induced signaling. Additionally, we demonstrated that one of our mutations (N284D) eliminates pH sensing but increases agonist potency, thereby providing a structural explanation for how pH-sensing and sodium binding occur that differs from previously established models. Overall, these data indicate that G-protein-coupled receptors can sense pH in several ways and demonstrate the potential of a novel, pH-insensitive A2AR in acid-related contexts.

molecular biology↗

bioRxiv2026

Proton-gating of opioid receptors and isoform functionality

The three opioid receptors, mu (mu-OR), delta (delta-OR), and kappa (kappa-OR), are key G-protein-coupled receptors (GPCRs) involved in mediating pain relief. Yet, there has been a lack of understanding regarding how changes in the extracellular pH associated with inflammation affect their signal transduction pathways. This study demonstrates that all opioid receptors act as proton-gated coincidence detectors. A humanized yeast-based platform called DCyFIR, which isolates the pH environment outside the cell from potential confounding factors inside the cell, was employed to examine the activation of opioid receptors over a range of physiologically relevant to pathologically low pH environments. As such, the results show that agonist efficacy decreases with decreasing pH. This indicates that the receptor itself contains one or more protonatable sidechains that act as an intrinsic "pH sensor". To determine the structural basis of this pH-sensing mechanism, structure-based pHinder calculations were used to identify potential pH sensing residues. Subsequent variant profiling identified the protonation of one conserved aspartic acid residue, D149 (also known as D3.32), located in a region near the binding site of opioid ligands, which, when exposed to acidic pH conditions, resulted in the disruption of the salt bridge necessary for effective docking of opioid ligands with the receptor. Consequently, the receptor lost all ability to be activated by agonists. Further characterization using a variety of functional assays on more than twenty different mu-OR alternative splicing variants indicated that all isoforms that possessed a continuous 7-transmembrane domain retained this proton-sensing capability. These studies collectively provide insight into the structural mechanisms underlying pH-dependent opioid receptor activation and lay the foundation for the design of new generations of selective analgesics that activate opioid receptors only in acidic inflammatory tissue.

molecular biology↗

bioRxiv2026

Next-generation genetically encoded biosensors for spatiotemporal intracellular pH monitoring, mapping, and profiling

Bioluminescence resonance energy transfer (BRET) systems are widely used for live-cell spectroscopy and biosensor engineering, yet the intrinsic pH sensitivity of commonly used BRET components has not been systematically examined. Here, we show that major BRET luciferase donors, fluorescent acceptors, and donor-acceptor assay pairs exhibit pronounced pH-dependent spectroscopic behavior across physiologically relevant conditions, identifying environmental pH responsiveness as a fundamental property of widely used BRET systems and a potential source of previously underappreciated assay artifacts. Leveraging these principles, we engineered ORION (ratiOmetRIc prOton seNsor), a genetically encoded ratiometric BRET pH sensor based on the NanoLuc-mVenus fusion. ORION exhibited strong brightness, an approximately 9-fold dynamic range, and robust responsiveness across a substantially broader pH range than that of existing genetically encoded sensors. Compared to pHluorin2, ORION maintained substantially improved quantitative performance at acidic pH values below 6.0. To demonstrate its utility in a biological application, we applied ORION across diverse cancer cell models and identified heterogeneous acid imprinting states, suggesting that tumor cells can retain persistent physiological memory of adaptation to acidic microenvironments even after prolonged ex vivo culture. Together, these findings establish pH responsiveness as a fundamental property of BRET systems and position ORION as a best-in-class platform for interrogating and quantifying pH regulation of biology in living systems.

biochemistry↗

bioRxiv2026

Systematic mapping of chimera-permissive sites by CRISPR-guided PAM scanning

Engineered protein chimeras enable new biological functions but remain difficult to design due to context-dependent constraints on insertion tolerance and the need to preserve host protein function. Here, we report CRISPR-guided protospacer adjacent motif (PAM) scanning in yeast to map chimera-permissive sites in living cells. We apply this approach to peptide and reporter insertions. In the first application, we generated 91 insertion chimeras encoding a defined protease cleavage sequence across six components of a model G protein-coupled receptor (GPCR) signaling pathway. Sixty-three percent of sites retain signaling, identifying positions that preserve host function and reveal broad, position-dependent tolerance. Coupling insertional scanning with cognate proteases enables site-resolved mapping of in-cell accessibility, distinguishing protected and exposed regions and defining EX1- and EX2-like regimes. These chimeras are responsive to proteolysis and pharmacological inhibition, enabling reversible control of protein activity. In a second application, we scanned 32 positions in yeast Ste2 and human A2A and MTNR1A receptors to engineer bi-functional chimeras that retain native function while incorporating reporter activity. Together, these results establish PAM scanning as a scalable, protein-agnostic framework for mapping insertion tolerance, interrogating protein accessibility in vivo, and enabling scalable ground-truth benchmarking of predictive chimera engineering.

synthetic biology↗

bioRxiv2026

Rapid CRISPR-Cas9 Genome Editing in S. cerevisiae

This protocol enables rapid CRISPR-Cas9 genome editing in Saccharomyces cerevisiae by replacing restriction/ligation guide cloning with PCR-based protospacer installation and seamless plasmid recircularization. It describes in silico HDR donor and SgRNA design, install guide sequences into cas9 plasmid by PCR and seamless assembly, plasmid cloning and sequence verification in E. coli, and LiAc/PEG co-transformation of yeast with Cas9-sgRNA plasmid plus HDR donor. The workflow selects yeast colonies on G418 and confirms edits by PCR and sequencing.

cell biology↗

bioRxiv2025

PIONEER: A Periplasmic Display Platform for Synthetic Biology-Based Screening of Genetically Encoded Protein Regulators

Periplasmic display in yeast is a promising but underdeveloped method for screening and studying genetically encoded biomolecules. We present PIONEER, a modular platform that localizes peptides, proteins, and nanobodies to the membrane-proximal periplasmic space of S. cerevisiae, enabling direct interrogation of membrane protein function. By optimizing secretion signals and display scaffolds, PIONEER ensures stable ligand retention and robust autocrine signaling. The ability to assess peptide and protein ligand activity through signaling, rather than binding alone, marks a key advance toward function-first screening. Applied to human G protein-coupled receptors (GPCRs), the system enables detection of surface expression, ligand activity profiling, and classification of nanobody regulators, including antagonists and conformational stabilizers. It also distinguishes intrabodies based on their functional effects and chaperone activity. Although demonstrated with GPCRs, PIONEER is adaptable to a wide range of membrane and soluble protein targets. As AI-driven design expands the space of candidate ligands and binders, this platform offers a scalable method for linking predicted sequences and their structures to biological function.

synthetic biology↗

bioRxiv2025

Non-redundant roles for paralogous proteins in the yeast glucose-sensing pathway

Paralogs engage in biological processes through both redundant and non-redundant functions. In the yeast Saccharomyces cerevisiae, approximately one-fifth of the genome consists of paralogs, with their encoded proteins involved in multiple pathways. However, the unique contributions of individual paralogs have remained poorly defined. Here, we undertook a systematic examination of eight paralog pairs in the glucose-sensing pathways, deleting each component and measuring the resulting changes in gene expression. To that end we established a new transcription reporter system to monitor the response to glucose as well as to non-preferred sugars in single cells. Focusing on the PKA catalytic subunits, comprised of the paralogs Tpk1 and Tpk3 as well as the isomorphic kinase Tpk2, we employed mass spectrometry to identify their contribution to cellular metabolism, used a GFP-based sensor to follow changes in cytosolic pH, and used BioID to identify unique and shared binding partners. Our data reveal that paralogs in the glucose-sensing pathway contribute in multiple and unique ways to signal transduction, and establish potential mechanisms driving the preservation of these and other duplicated genes throughout long periods of evolution.

genetics↗

bioRxiv2023

Compartment-Specific Activation of the Proton-Sensor GPR65 is Uncoupled from Receptor Trafficking

The canonical view of G protein-coupled receptor (GPCR) function is that receptor trafficking is tightly coupled to signaling. GPCRs remain on the plasma membrane (PM) at the cell surface until they are activated, after which they are desensitized and internalized into endosomal compartments. This canonical view presents an interesting context for proton-sensing GPCRs because they are more likely to be activated in acidic endosomal compartments than at the PM. Here we show that the trafficking of the prototypical proton-sensor GPR65 is fully uncoupled from signaling, unlike that of other known mammalian GPCRs. GPR65 internalized and localized to early and late endosomes, from where they signal at steady state, irrespective of extracellular pH. Acidic extracellular environments stimulated receptor signaling at the PM in a dose-dependent manner, although endosomal GPR65 was still required for a full signaling response. Receptor mutants that were incapable of activating cAMP trafficked normally, internalized, and localized to endosomal compartments. Our results show that GPR65 is constitutively active in endosomes, and suggest a model where changes in extracellular pH reprograms the spatial pattern of receptor signaling and biases the location of signaling to the cell surface.

pharmacology and toxicology↗

bioRxiv2021

Proton-gated coincidence detection is a common feature of GPCR signaling

The evolutionary expansion of G protein-coupled receptors (GPCRs) has produced a rich diversity of transmembrane sensors for many physical and chemical signals. In humans alone, over 800 GPCRs detect stimuli such as light, hormones, and metabolites to guide cellular decision making primarily using intracellular G protein signaling networks. This diversity is further enriched by GPCRs that function as molecular logic gates capable of discerning multiple inputs to transduce cues encoded in complex, context-dependent signals. Here, we show that many GPCRs are switch-like Boolean-gated coincidence detectors that link proton (H+) binding to GPCR signaling. Using a panel of 28 receptors, covering 280 individual GPCR-G coupling combinations, we show that H+ gating both positively and negatively modulates and controls GPCR signaling. Notably, these observations extend to all modes of GPCR pharmacology including ligand efficacy, potency, and cooperativity. Additionally, we show that GPCR antagonism and constitutive activity are regulated by H+ gating and report the discovery of a new acid sensor, the adenosine A2a receptor (ADORA2A), which can be activated solely by acidic pH. Together, these findings establish a new paradigm for GPCR biology and pharmacology in acidified microenvironments such as endosomes, synapses, tumors, and ischemic vasculature.

pharmacology and toxicology↗

bioRxiv2020

One Year of SARS-CoV-2: How Much Has the Virus Changed?

SARS-CoV-2 coronavirus has caused a world-wide crisis with profound effects on both healthcare and the economy. In order to combat the COVID-19 pandemic, research groups have shared viral genome sequence data through the GISAID initiative. We collected and computationally profiled [~]223,000 full SARS-CoV-2 proteome sequences from GISAID over one year for emergent nonsynonymous mutations. Our analysis shows that SARS-CoV-2 proteins are mutating at substantially different rates, with most viral proteins exhibiting little mutational variability. As anticipated, our calculations capture previously reported mutations occurred in the first period of the pandemic, such as D614G (Spike), P323L (NSP12), and R203K/G204R (Nucleocapsid), but also identify recent mutations like A222V and L18F (Spike) and A220V (Nucleocapsid). Our comprehensive temporal and geographical analyses show two periods with different mutations in the SARS-CoV-2 proteome: December 2019 to June 2020 and July to November 2020. Some mutation rates differ also by geography; the main mutations in the second period occurred in Europe. Furthermore, our structure-based molecular analysis provides an exhaustive assessment of mutations in the context of 3D protein structure. Emerging sequence-to-structure data is beginning to reveal the site-specific mutational tolerance of SARS-CoV2 proteins as the virus continues to spread around the globe.

bioinformatics↗

bioRxiv2020

An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity

Isocitrate dehydrogenase 1 (IDH1) catalyzes the reversible NADP+-dependent conversion of isocitrate to -ketoglutarate (-KG) to provide critical cytosolic substrates and drive NADPH-dependent reactions like lipid biosynthesis and glutathione regeneration. In biochemical studies, the forward reaction is studied at neutral pH, while the reverse reaction is typically characterized in more acidic buffers. This led us to question whether IDH1 catalysis is pH-regulated, which would have functional implications under conditions that alter cellular pH, like apoptosis, hypoxia, cancer, and neurodegenerative diseases. Here, we show evidence of catalytic regulation of IDH1 by pH, identifying a trend of increasing kcat values for -KG production upon increasing pH in the buffers we tested. To understand the molecular determinants of IDH1 pH sensitivity, we used the pHinder algorithm to identify buried ionizable residues predicted to have shifted pKa values. Such residues can serve as pH sensors, with changes in protonation states leading to conformational changes that regulate catalysis. We identified an acidic residue buried at the IDH1 dimer interface, D273, with a predicted pKa value upshifted into the physiological range. D273 point mutations had decreased catalytic efficiency and, importantly, loss of pH-regulated catalysis. Based on these findings, we conclude that IDH1 activity is regulated, at least in part, by pH. We show this regulation is mediated by at least one buried acidic residue [~]12 [A] from the IDH1 active site. By establishing mechanisms of regulation of this well-conserved enzyme, we highlight catalytic features that may be susceptible to pH changes caused by cell stress and disease.

biochemistry↗

bioRxiv2020

DCyFIR: a high-throughput CRISPR platform for multiplexed G protein-coupled receptor profiling and ligand discovery

More than 800 G protein-coupled receptors (GPCRs) comprise the largest class of membrane receptors in humans. While there is ample biological understanding and many approved drugs for prototypic GPCRs, most GPCRs still lack well-defined biological ligands and drugs. Here, we report our efforts to tap the potential of understudied GPCRs by developing yeast-based technologies for high-throughput CRISPR engineering and GPCR ligand discovery. We refer to these technologies collectively as Dynamic Cyan induction by Functional Integrated Receptors: DCyFIR. A major advantage of DCyFIR is that GPCRs and other assay components are CRISPR-integrated directly into the yeast genome, making it possible to decode ligand specificity by profiling mixtures of GPCR-barcoded yeast strains in a single tube. To demonstrate the capabilities of DCyFIR, we engineered a yeast strain library of 30 human GPCRs and their 300 possible GPCR-G coupling combinations. Profiling of these 300 strains, using parallel (DCyFIRscreen) and multiplex (DCyFIRplex) DCyFIR modes, recapitulated known GPCR agonism with 100% accuracy, and identified unexpected interactions for the receptors ADRA2B, HCAR3, MTNR1A, S1PR1, and S1PR2. To demonstrate DCyFIR scalability, we profiled a library of 320 human metabolites and observed new GPCR-ligand interactions with amino acid, lipid, sugar, and steroid metabolites. Remarkably, many of these findings pertained to understudied "pharmacologically dark" receptors GPR4, GPR65, GPR68, and HCAR3. For example, we found that kynurenic acid activated HCAR3 with a nearly 20-fold lower EC50 than GPR35, its known receptor. Taken together, these findings demonstrate the power of DCyFIR for identifying novel ligand interactions with prototypic and understudied GPCRs. Significance StatementG protein-coupled receptors (GPCRs) are the largest class of membrane receptors in humans. As such, GPCR signaling is central to human biology and medicine. While more than 30% of approved drugs target roughly 150 GPCRs, most receptors lack well-defined endogenous ligands and are currently not druggable. To address this challenge, we created a GPCR screening platform for ligand and drug discovery. This innovative technology enables the cost-effective profiling of ligands and drug compounds against mixtures of hundreds of GPCR-barcoded cell strains in a single experiment. Because a ligand or drug is tested against a collection of receptors all at once, our novel method accelerates the process of identifying potential GPCR ligands and drugs.

pharmacology and toxicology↗