Biology subjects

Dohlman, H. G.

Publications and source records attributed to Dohlman, H. G..

10 recordsLinked to original sources

bioRxiv2026

A mechanistic classification of GNAO1 mutations links Gβ γ sequestration to hyperkinetic disease and treatment response

Go is the most prevalent G protein subunit in the brain and is critical for signal transduction through G protein-coupled receptors. Mutations in the gene encoding Go (GNAO1) cause a rare neurodevelopmental disorder with involuntary movements (NEDIM) and, in some patients, developmental and epileptic encephalopathy (DEE). Here, we integrate human genetic analysis with cellular assays to define mechanistic classes of GNAO1 mutations and in vivo approaches to evaluate therapeutic strategies. We grouped variants into two functional categories: those predicted to sequester G{beta}{gamma} and those with reduced Go expression and therefore increased G{beta}{gamma} signaling. Analysis of patient data revealed that mutations predicted to impair G{beta}{gamma} release are significantly associated with NEDIM. Using the GNAO1R209H/+ mouse as a model of NEDIM, we evaluated two small molecules: caffeine, a nonselective adenosine receptor antagonist, and gallein, a selective modulator of G{beta}{gamma}-effector interactions. Both compounds significantly reduced hyperlocomotion in the open field test. Whole-cell patch-clamp electrophysiology revealed sex- and cell type-specific hyperexcitability in spiny projection neurons within the dorsolateral striatum of GNAO1R209H/+ mice. Together, these findings implicate dysfunction of dorsolateral striatal pathways in GNAO1-related disorder and suggest that mechanistic understanding of disease variants can guide targeted therapeutic strategies.

pharmacology and toxicology↗

bioRxiv2026

Disrupted RGS coupling underlies a clinically distinct subset of GNAO1-related disorder

Heterotrimeric G proteins are transducers for a wide variety of neurotransmitter receptors. Mutations in the G protein o subunit are implicated in an early-onset neurological condition, GNAO1-related disorder, that manifests as movement disorders, developmental delays, and/or epileptic seizures. In a screen of disease-linked mutations in Go, we identified a subset that had no obvious biochemical defects, warranting further investigation. By structural modeling, protein-protein binding experiments in vitro and in cells, as well as measures of downstream ion channel electrophysiology, we identified a subset that impairs interactions with regulator of G protein signaling (RGS) proteins. RGS proteins accelerate G protein GTPase activity and are therefore critical for proper neurotransmitter signaling. Further, synthesis of current clinical data revealed that patients harboring these variants have comparatively mild symptoms. Our study demonstrates the importance of RGS regulation in G protein function, cell signaling, and human neurobiology.

biochemistry↗

bioRxiv2026

Conserved Residues in the Gα interface show subtype specificity in Gβγ coupling

Although the structural basis of selective G-protein coupling to G protein coupled receptors (GPCRs) is well characterized, the mechanisms underlying selective interactions between distinct G subtypes (Gs, Gi, Gq, and G12/13) and G{beta}{gamma} remain poorly understood. While conserved residues in G subtypes are often assumed to have similar functions, they may instead modulate coupling selectivity by altering the frequency and stability of contacts at the G:G{beta}{gamma} interface. Using molecular dynamics (MD) simulations combined with the interpretable machine learning method, Bayesian Network Model (BNM), and protein-protein proximity (BRET) assays, we show that conserved residues in the two closely related Gi/o and Gq/11 subfamilies contribute differentially to G{beta}{gamma} coupling. These conserved residue "hotspots" on Gi and Gq produced divergent functional effects on G{beta}{gamma} coupling, indicating that conservation does not ensure functional equivalence. These findings suggest that local microenvironment and paralog-specific allosteric coupling shape how conserved interface residues contribute to protein-protein coupling. The framework provides a systematic approach for dissecting subtype-specific mechanisms, with implications for drug design and for annotating the functional relevance of disease-associated variants. The computational methods used here are broadly applicable to other homologous protein families.

molecular 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↗

bioRxiv2021

Multi-omics analysis of multiple glucose-sensing receptor systems in yeast

The yeast Saccharomyces cerevisiae has long been used to produce alcohol from glucose and other sugars. While much is known about glucose metabolism, relatively little is known about the receptors and signaling pathways that indicate glucose availability. Here we compare the two glucose receptor systems in S. cerevisiae. The first is a heterodimer of transporter-like proteins (transceptors), while the second is a seven-transmembrane receptor coupled to a large G protein (Gpa2) and two small G proteins (Ras1 and Ras2). Through comprehensive measurements of glucose-dependent transcription and metabolism, we demonstrate that the two receptor systems have distinct roles in glucose signaling: the G protein-coupled receptor directs carbohydrate and energy metabolism, while the transceptors regulate ancillary processes such as ribosome, amino acids, cofactor and vitamin metabolism. The large G protein transmits the signal from its cognate receptor, while the small G protein Ras2 (but not Ras1) integrates responses from both receptor pathways. Collectively, our analysis reveals the molecular basis for glucose detection and the earliest events of glucose-dependent signal transduction in yeast.

genomics↗

bioRxiv2021

Multi-omics analysis of glucose-mediated signaling by a moonlighting Gβ protein Asc1/RACK1

G proteins were originally discovered through efforts to understand the effects of hormones, such as glucagon and epinephrine, on glucose metabolism. On the other hand, many cellular metabolites, including glucose, serve as ligands for G protein-coupled receptors. Here we investigate the consequences of glucose-mediated receptor signaling, and in particular the role of a G subunit Gpa2 and a non-canonical G{beta} subunit, known as Asc1 in yeast and RACK1 in animals. Asc1/RACK1 is of particular interest because it has multiple, seemingly unrelated, functions in the cell. The existence of such "moonlighting" operations has complicated the determination of phenotype from genotype. Through a comparative analysis of individual gene deletion mutants, and by integrating transcriptomics and metabolomics measurements, we have determined the relative contributions of the G and G{beta} protein subunits to glucose-initiated processes in yeast. We determined that Gpa2 is primarily involved in regulating sugar metabolism while Asc1 is primarily involved in amino acid metabolism. Both proteins are involved in regulating purine metabolism. Of the two subunits, Gpa2 regulates a greater number of gene transcripts and was particularly important in determining the amplitude of response to glucose addition. We conclude that the two G protein subunits regulate distinct but complementary processes downstream of the glucose-sensing receptor, as well as processes that lead ultimately to changes in cell growth and metabolism. AUTHOR SUMMARYDespite the societal importance of glucose fermentation in yeast, the mechanisms by which these cells detect and respond to glucose have remained obscure. Glucose detection requires a cell surface receptor coupled to a G protein that is comprised of two subunits, rather than the more typical heterotrimer: an subunit Gpa2 and the {beta} subunit Asc1 (or RACK1 in humans). Asc1/RACK1 also serves as a subunit of the ribosome, where it regulates the synthesis of proteins involved in glucose fermentation. This manuscript uses global metabolomics and transcriptomics to demonstrate the distinct roles of each G protein subunit in transmitting the glucose signal. Whereas Gpa2 is primarily involved in the metabolism of sugars, Asc1/RACK1 contributes to production of amino acids necessary for protein synthesis and cell division. These findings reveal the initial steps of glucose signaling and several unique and complementary functions of the G protein subunits. More broadly, the integrated approach used here is likely to guide efforts to determine the topology of complex G protein and metabolic signaling networks in humans.

genomics↗

bioRxiv2020

A Universal Allosteric Mechanism for G Protein Activation

G proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote GTP binding and the dissociation of G from the G{beta}{gamma} subunits. Structural studies have revealed the molecular basis for subunit association with receptors, RGS proteins and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We used cell signaling assays, MD simulations, biochemistry and structural analysis to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This "G-R-E motif" secures GTP and, through an allosteric link, discharges the G{beta}{gamma} dimer. Replacement of network residues prevents subunit dissociation, regardless of agonist or GTP binding. These findings reveal the molecular basis for the final committed step of G protein activation. HIGHLIGHTSO_LIReceptors promote GTP-GDP exchange and dissociation of G protein and {beta}{gamma} subunits C_LIO_LIWe find an allosteric network linking the {gamma} phosphate of GTP with release of G{beta}{gamma} C_LIO_LIThe network consists of a conserved Gly-Arg-Glu "activation triad" C_LIO_LITriad mutations prevent subunit dissociation, regardless of agonist or GTP binding C_LIO_LITriad mutations are responsible for human endocrine and neurological disorders C_LI

biochemistry↗

bioRxiv2020

Potassium starvation induces autophagy in yeast

ABSTRACTAutophagy is a conserved process that recycles cellular contents to promote survival. Although nitrogen starvation is the canonical inducer of autophagy, recent studies have revealed several other nutrients important to this process. In this study, we used a quantitative, high-throughput assay to identify potassium starvation as a new and potent inducer of autophagy. We found that potassium-dependent autophagy requires the core pathway kinases Atg1, Atg5, Vps34, as well as other components of Phosphatidylinositol 3-kinase Complex I. Transmission electron microscopy revealed abundant autophagosome formation in response to both stimuli. RNA sequencing indicated distinct transcriptional responses – nitrogen affects transport of ions such as copper while potassium targets the organization of other cellular components. Thus, nitrogen and potassium share the ability to influence metabolic supply and demand but do so in different ways. Both inputs promote catabolism through bulk autophagy, but inhibit cellular anabolism through distinct mechanisms.Competing Interest StatementThe authors have declared no competing interest.ABBREVIATIONSPIPhosphatidylinositolATGAutophagy-related geneSCDSynthetic Complete medium with DextroseYNBYeast Nitrogen BaseTEMTransmission Electron MicroscopyMAPKMitogen-Activated Protein KinaseDEGDifferentially Expressed Gene.View Full Text

biochemistry↗

bioRxiv2020

A predictive model of gene expression reveals the role of regulatory motifs in the mating response of yeast

ABSTRACTCells use signaling pathways to receive and process information about their environment. These systems are nonlinear, relying on feedback and feedforward regulation to respond appropriately to changing environmental conditions. Mathematical models developed to describe signaling pathways often fail to show predictive power, because the models are not trained on data that probe the diverse time scales on which feedforward and feedback regulation operate. We addressed this limitation using microfluidics to expose cells to a broad range of dynamic environmental conditions. In particular, we focus on the well-characterized mating response pathway of S. cerevisiae (yeast). This pathway is activated by mating pheromone and initiates the transcriptional changes required for mating. Although much is known about the molecular components of the mating response pathway, less is known about how these components function as a dynamical system. Our experimental data revealed that pheromone-induced transcription persists following removal of pheromone and that long-term adaptation of the transcriptional response occurs when pheromone exposure is sustained. We developed a model of the regulatory network that captured both persistence and long-term adaptation of the mating response. We fit this model to experimental data using an evolutionary algorithm and used the parameterized model to predict scenarios for which it was not trained, including different temporal stimulus profiles and genetic perturbations to pathway components. Our model allowed us to establish the role of four regulatory motifs in coordinating pathway response to persistent and dynamic stimulation.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗

bioRxiv2020

Model-driven experimental design identifies counter-acting feedback regulation in the osmotic stress response of yeast

Cells rely on mitogen-activated protein kinases (MAPKs) to survive environmental stress. In yeast, activation of the MAPK Hog1 is known to mediate the response to high osmotic conditions. Recent studies of Hog1 revealed that its temporal activity is subject to both negative and positive feedback regulation, yet the mechanisms of feedback remain unclear. By designing mathematical models of increasing complexity for the Hog1 MAPK cascade, we identified pathway circuitry sufficient to capture Hog1 dynamics observed in vivo. We used these models to optimize experimental designs for distinguishing potential feedback loops. Performing experiments based on these models revealed mutual inhibition between Hog1 and its phosphatases as the likely positive feedback mechanism underlying switch-like, dose-dependent MAPK activation. Importantly, our findings reveal a new signaling function for MAPK phosphatases. More broadly, they demonstrate the value using mathematical models to infer targets of feedback regulation in signaling pathways.

systems biology↗