Biology subjects

Prosser, R. S.

Publications and source records attributed to Prosser, R. S..

5 recordsLinked to original sources

bioRxiv2026

Local Confinement within Plasma Membrane Nanodomains Drives Constitutive Activity of GPCRs

Many G protein coupled receptors (GPCRs) exhibit constitutive (basal) activity, where they can signal in the absence of ligand binding through spontaneous conformational transitions that facilitate G protein coupling and downstream signaling. This intrinsic baseline activity is critical for cellular homeostasis and can be modulated by the receptors conformational ensemble, membrane organization, and interactions with intracellular effectors. In this study, we use live-cell signaling assays, fluorescence cross-correlation spectroscopy (FCCS), and single-particle tracking (SPT) to investigate how membrane organization influences the basal activity of two class A GPCRs: the M1 muscarinic receptor (M1R) and the adenosine A2A receptor (A2AR). In live-cell signalling assays, M1R showed minimal agonist-independent Ca{superscript 2} responses, while A2AR exhibited significant basal cAMP production that was eliminated by an inverse agonist. FCCS showed that, without ligand, only a small portion of M1R co-diffuses with its cognate G11 protein, whereas a much larger fraction of A2AR co-diffuses with the GS protein. SPT revealed that A2AR, but not M1R, is enriched in slowly diffusing, confined states with spatial scales around 150-200 nm and sensitivity to cholesterol- and raft-modulating agents, consistent with localization in lipid-raft nanodomains. Dual-color tracking and diffusion mapping demonstrated that a significant portion of A2AR and GS share confinement domains under basal conditions, while M1R and G11 only show such co-confinement in the active state. These findings support a model where the co-confinement of GPCRs and G proteins within plasma membrane nanodomains--rather than stable pre-coupled RG complexes-- determines the level of constitutive GPCR activity.

biophysics↗

bioRxiv2021

Allosteric modulation of the adenosine A2A receptor by cholesterol

Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A2A receptor (A2AR) in reconstituted phospholipid bilayers. GTP hydrolysis assays show that cholesterol is a weak positive allosteric modulator of A2AR, as seen through enhanced basal signaling and a small decrease in agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy suggests that this enhancement arises from an increase in the receptors active state populations and stronger G protein coupling. 19F NMR of fluorinated cholesterol analogs reveals transient and non-specific interactions with A2AR, indicating a lack of high-affinity binding sites or direct allosteric modulation. This is confirmed by computational analysis which suggests that cholesterol contacts confer a weak and possibly negative allosteric effect. The combined results suggest that the observed cholesterol allostery in A2AR is likely a result of indirect membrane effects through cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.

biophysics↗

bioRxiv2020

Acute oral toxicity and risks of exposure to the neonicotinoid thiamethoxam, and other classes of systemic insecticide, for the Common Eastern Bumblebee (Bombus impatiens)

The Common Eastern Bumblebee (Bombus impatiens) is native to North America with an expanding range across Eastern Canada and the USA. This species is commercially produced primarily for greenhouse crop pollination and is a common and abundant component of the wild bumblebee fauna in agricultural, suburban and urban landscapes. However, there is a dearth of pesticide toxicity information about North American bumblebees. The present study determines the acute oral toxicity (48-hour LD50) of cyantraniliprole (>0.54 g/bee), flupyradifurone (>1.7 g/bee), sulfoxaflor (0.0194 g/bee), and thiamethoxam (0.0012 g/bee). Compared with published honey bee (Apis mellifera) LD50 values, the present study shows that thiamethoxam and sulfoxaflor are 4.2x and 7.5x more acutely toxic to B. impatiens, whereas flupyradifurone is more acutely toxic to A. mellifera. The current rule of thumb for toxicity extrapolation beyond the honey bee as a model species, termed 10x safety factor, may be sufficient for bumblebee acute oral toxicity. A comparison of three risk assessment equations suggested that the Standard Risk Approach (SRA) and Fixed Dose Risk Approach (FDRA) provide more nuanced levels of risk evaluation compared to the European Plant Protection Organization (EPPO) Hazard Quotient (HQ), primarily because SRA and FDRA take into account real world variability in pollen and nectar pesticide residues and the chances that bees are exposed to them.

pharmacology and toxicology↗

bioRxiv2018

Risks of exposure to systemic insecticides in agricultural soil in Ontario, Canada for the hoary squash bee (Peponapis pruinosa) and other ground-nesting bee species

Insecticide exposure is an environmental factor of concern relating to pollinator health. Recent increases in use of systemic insecticides, particularly neonicotinoids, have led to considerable research into the potential impacts for bees of exposure to these insecticide residues via the nectar and pollen of treated crops or contaminated wild flowers. However, although the majority of bee species are ground-nesting, the risk of exposure to insecticides in soil has not yet been evaluated. Here we use the hoary squash bee (Peponapis pruinosa) as a model system to provide the first evaluation of the risk of exposure to insecticide residues in soil to ground-nesting solitary bee species. The evaluation assessed agrochemical residues from the nectar, pollen and soil from Cucurbita-crops (e.g. squash and pumpkin) grown in southern Ontario in 2016, and soil insecticide residue data collected from field crops by the Ontario government. Systemic insecticide residues were rarely detected in the nectar and pollen of Cucurbita-crops in 2016, and hazard assessment deemed these potential routes of exposure to be non-hazardous for honey bee lethal dose endpoints. In contrast, quantifiable pesticide residues were frequently detected in soil suggesting this route of exposure to be hazardous for honey bee lethal dose endpoints, leading to further assessment using probabilistic risk assessments. Concentrations of clothianidin, imidacloprid, thiamethoxam (neonicotinoid insecticides) and chlorantraniliprole (an anthranilic diamide insecticide) in soil samples were plotted to produce an environmental exposure distribution for each insecticide. A honey bee LC50 and a solitary bee LC50 endpoint were converted to exposure endpoints using the amount of soil excavated by hoary squash bees during nest construction, for both acute and chronic exposure scenarios. The probability of exceedance of each exposure endpoint was calculated and compared to an acceptable risk threshold (i.e. 5% exceedance). In the acute exposure scenario, risk to hoary squash bees was below acceptable threshold levels for all residues evaluated using the honey bee LC50, but exceeded the threshold for clothianidin and imidacloprid using the solitary bee LC50. In the chronic exposure scenario, risk from exposure to clothianidin and imidacloprid exceeded the threshold for both the honey bee and solitary bee LC50s, and for chlorantraniliprole risk only exceeded the threshold for the solitary bee LC50. Using the hoary squash bee as a model, the wider implications for other ground-nesting bees that pollinate crops was explored using insecticide residues from soil samples taken from Ontario field crops. Probabilistic risk assessments suggest that risk to ground-nesting bees is high from clothianidin residues in soils, even when exposure is acute, and substantial for both thiamethoxam and imidacloprid under chronic exposure scenarios. These results demonstrate the urgent need to consider direct exposure to pesticides in soil for ground-nesting bees as part of risk assessments and provides a potential model for evaluating risk from this type of exposure.

ecology↗

bioRxiv2017

Notes On Synthesis Of Perdeutero-5-13C,5,5,5-Trifluoroisoleucine VI

The 13CF3 group is a promising label for heteronuclear (19F,13C) NMR studies of proteins. Desirable locations for this NMR spin label include the branched chain amino acid methyl groups. It is known that replacement of CH3 by CF3 at such locations preserves protein structure and function and enhances stability. 13CF3 may be introduced at the a position of isoleucine and incorporated biosynthetically in highly deuterated proteins. This paper reports our work in synthesis and purification of 5,5,5-trifluoroisoleucine, its perdeutero and 5-13C versions and of 2-13C-trifluoroacetate and its utility as a precursor for introduction of the 13CF3 group into proteins.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC=\"FIGDIR/small/140681_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (36K):\norg.highwire.dtl.DTLVardef@1531c5eorg.highwire.dtl.DTLVardef@16b8e29org.highwire.dtl.DTLVardef@423d0aorg.highwire.dtl.DTLVardef@b5951a_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗