Biology subjects

Lee, K. D.

Publications and source records attributed to Lee, K. D..

6 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↗

bioRxiv2020

Poor Quality Vβ Recombination Signal Sequences Enforce TCRβ Allelic Exclusion by Limiting the Frequency of Vβ Recombination

Monoallelic expression (allelic exclusion) of T and B lymphocyte antigen receptor genes is achieved by the assembly of a functional gene through V(D)J recombination on one allele and subsequent feedback inhibition of recombination on the other allele. There has been no validated mechanism for how only one allele of any antigen receptor locus assembles a functional gene prior to feedback inhibition. Here, we demonstrate that replacement of a single V{beta} recombination signal sequence (RSS) with a better RSS increases V{beta} rearrangement, reveals Tcrb alleles compete for utilization in the {beta} T cell receptor (TCR) repertoire, and elevates the fraction of {beta} T cells expressing TCR{beta} protein from both alleles. The data indicate that poor qualities of V{beta} RSSs for recombination with D{beta} and J{beta} RSSs enforces allelic exclusion by stochastically limiting the incidence of functional V{beta} rearrangements on both alleles before feedback inhibition terminates V{beta} recombination.

immunology↗

bioRxiv2019

Assemblage of Focal Species Recognizers - AFSR: A technique for decreasing false positive rates of acoustic automatic identification in a multiple species context

Passive acoustic monitoring (PAM) coupled with automated species identification is a promising tool for species monitoring and conservation worldwide. However, high false positive rates are still an important limitation and a crucial factor for acceptance of these techniques in wildlife surveys. Here we present the Assemblage of Focal Species Recognizers - AFSR, a novel approach for decreasing false positives and increasing models precision in multispecies contexts. AFSR focusses on decreasing false positives by excluding unreliable sound file segments that are prone to misidentification. We used MatlabHTK, a hidden Markov models interface for bioacoustics analyses, for illustrating AFSR technique by comparing two approaches, 1) a multispecies recognizer where all species are identified simultaneously, and 2) an assemblage of focal species recognizers (AFSR), where several recognizers that each prioritise a single focal species are then summarised into a single output, according to a set of rules designed to exclude unreliable segments. Both approaches (the multispecies recognizer and AFSR) used the same sound files training dataset, but different processing workflow. We applied these recognisers to PAM recordings from a remote island colony with five seabird species and compared their outputs with manual species identifications. False positive rates and precision improved for all the five species when using AFSR, achieving remarkable 0% false positives and 100% precision for three of five seabird species, and < 6% false positive rates, and >90% precision for the other two species. AFSR output was also used to generate daily calling activity patterns for each species. Instead of attempting to withdraw useful information from every fragment in a sound recording, AFSR prioritises more trustworthy information from sections with better quality data. AFSR can be applied to automated species identification from multispecies PAM recordings worldwide.

ecology↗