11 research outputs found
Multi-Drug Resistance Transporters and a Mechanism-Based Strategy for Assessing Risks of Pesticide Combinations to Honey Bees
Annual losses of honey bee colonies remain high and pesticide exposure is one possible cause. Dangerous combinations of pesticides, plant-produced compounds and antibiotics added to hives may cause or contribute to losses, but it is very difficult to test the many combinations of those compounds that bees encounter. We propose a mechanism-based strategy for simplifying the assessment of combinations of compounds, focusing here on compounds that interact with xenobiotic handling ABC transporters. We evaluate the use of ivermectin as a model substrate for these transporters. Compounds that increase sensitivity of bees to ivermectin may be inhibiting key transporters. We show that several compounds commonly encountered by honey bees (fumagillin, Pristine, quercetin) significantly increased honey bee mortality due to ivermectin and significantly reduced the LC50 of ivermectin suggesting that they may interfere with transporter function. These inhibitors also significantly increased honey bees sensitivity to the neonicotinoid insecticide acetamiprid. This mechanism-based strategy may dramatically reduce the number of tests needed to assess the possibility of adverse combinations among pesticides. We also demonstrate an in vivo transporter assay that provides physical evidence of transporter inhibition by tracking the dynamics of a fluorescent substrate of these transporters (Rhodamine B) in bee tissues. Significantly more Rhodamine B remains in the head and hemolymph of bees pretreated with higher concentrations of the transporter inhibitor verapamil. Mechanism-based strategies for simplifying the assessment of adverse chemical interactions such as described here could improve our ability to identify those combinations that pose significantly greater risk to bees and perhaps improve the risk assessment protocols for honey bees and similar sensitive species
Targeting spike glycans to inhibit SARS-CoV2 viral entry
SARS-CoV-2 spike harbors glycans which function as ligands for lectins. Therefore, it should be possible to exploit lectins to target SARS-CoV-2 and inhibit cellular entry by binding glycans on the spike protein. Burkholderia oklahomensis agglutinin (BOA) is an antiviral lectin that interacts with viral glycoproteins via N-linked high mannose glycans. Here, we show that BOA binds to the spike protein and is a potent inhibitor of SARS-CoV-2 viral entry at nanomolar concentrations. Using a variety of biophysical approaches, we demonstrate that the interaction is avidity driven and that BOA cross-links the spike protein into soluble aggregates. Furthermore, using virus neutralization assays, we demonstrate that BOA effectively inhibits all tested variants of concern as well as SARS-CoV 2003, establishing that multivalent glycan-targeting molecules have the potential to act as pan-coronavirus inhibitors
Cosolute and Crowding Effects on a Side-By-Side Protein Dimer
The effects of small
(∼10<sup>2</sup> Da) and larger (>10<sup>3</sup> Da) cosolutes
on the equilibrium stability of monomeric globular
proteins are broadly understood, excluding volume stabilizes proteins
and chemical interactions are stabilizing when repulsive, but destabilizing
when attractive. Proteins, however, rarely work alone. Here, we investigate
the effects of small and large cosolutes on the equilibrium stability
of the simplest defined protein–protein interactions, the side-by-side
homodimer formed by the A34F variant of the 56-residue B1 domain of
protein G. We used <sup>19</sup>F nuclear magnetic resonance spectroscopy
to quantify the effects of urea, trimethylamine oxide, Ficoll, and
more physiologically relevant cosolutes on the dimer dissociation
constant. The data reveal the same stabilizing and destabilizing influences
from chemical interactions as observed in studies of protein stability.
Results with more physiologically relevant molecules such as bovine
serum albumin, lysozyme, and reconstituted <i>Escherichia coli</i> cytosol reflect the importance of chemical interactions between
these cosolutes and the test protein. Our study serves as a stepping-stone
to a more complete understanding of crowding effects on protein–protein
interactions
Surface Charge Modulates Protein–Protein Interactions in Physiologically Relevant Environments
Protein–protein
interactions are fundamental to biology
yet are rarely studied under physiologically relevant conditions where
the concentration of macromolecules can exceed 300 g/L. These high
concentrations cause cosolute–complex contacts that are absent
in dilute buffer. Understanding such interactions is important because
they organize the cellular interior. We used <sup>19</sup>F nuclear
magnetic resonance, the dimer-forming A34F variant of the model protein
GB1, and the cosolutes bovine serum albumin (BSA) and lysozyme to
assess the effects of repulsive and attractive charge–charge
dimer–cosolute interactions on dimer stability. The interactions
were also manipulated via charge-change variants and by changing the
pH. Charge–charge repulsions between BSA and GB1 stabilize
the dimer, and the effects of lysozyme indicate a role for attractive
interactions. The data show that chemical interactions can regulate
the strength of protein–protein interactions under physiologically
relevant crowded conditions and suggest a mechanism for tuning the
equilibrium thermodynamics of protein–protein interactions
in cells
Multi-Drug Resistance Transporters and a Mechanism-Based Strategy for Assessing Risks of Pesticide Combinations to Honey Bees.
Annual losses of honey bee colonies remain high and pesticide exposure is one possible cause. Dangerous combinations of pesticides, plant-produced compounds and antibiotics added to hives may cause or contribute to losses, but it is very difficult to test the many combinations of those compounds that bees encounter. We propose a mechanism-based strategy for simplifying the assessment of combinations of compounds, focusing here on compounds that interact with xenobiotic handling ABC transporters. We evaluate the use of ivermectin as a model substrate for these transporters. Compounds that increase sensitivity of bees to ivermectin may be inhibiting key transporters. We show that several compounds commonly encountered by honey bees (fumagillin, Pristine, quercetin) significantly increased honey bee mortality due to ivermectin and significantly reduced the LC50 of ivermectin suggesting that they may interfere with transporter function. These inhibitors also significantly increased honey bees sensitivity to the neonicotinoid insecticide acetamiprid. This mechanism-based strategy may dramatically reduce the number of tests needed to assess the possibility of adverse combinations among pesticides. We also demonstrate an in vivo transporter assay that provides physical evidence of transporter inhibition by tracking the dynamics of a fluorescent substrate of these transporters (Rhodamine B) in bee tissues. Significantly more Rhodamine B remains in the head and hemolymph of bees pretreated with higher concentrations of the transporter inhibitor verapamil. Mechanism-based strategies for simplifying the assessment of adverse chemical interactions such as described here could improve our ability to identify those combinations that pose significantly greater risk to bees and perhaps improve the risk assessment protocols for honey bees and similar sensitive species
Increased sensitivity of honey bees to ivermectin (1ug/ml) following oral exposure to standard and tested inhibitors (1mM) and LC50 of ivermectin following treatment of bees with 1 mM inhibitor solution.
<p>All inhibitor + ivermectin treatments had significantly higher mean mortality than the ivermectin treatment (p < 0.0001).</p
Mean 24 h mortality (±SE) of honey bees fed 1 μg/ml ivermectin treated with low concentrations of inhibitors.
<p>Bees fed Pristine (Prist), quercetin (Querc), fumagillin (Fumag) and verapamil (Verap) in sucrose syrup. All inhibitor/doses shown had significantly greater mortality than the ivermectin-only control (P < 0.002).</p
Mortality of honey bees (24 h ±SE) pretreated with a series of concentrations of candidate inhibitors.
<p>Bees were fed verapamil, quercetin, fumagillin or Pristine before and during oral exposure to 1 μg/ml ivermectin.</p
Transporter function assays.
<p>Bees treated with a series of dosages of the MDR transporter-inhibitor verapamil before and after ingestion of the fluorescent substrate Rhodamine B solution, and assayed at two times (24 and 48 h post ingestion) show greater fluorescence at increased verapamil concentrations in both tissues.</p
Thermodynamic consequences of Tyr to Trp mutations in the cation-Ï€-mediated binding of trimethyllysine by the HP1 chromodomain.
Evolution has converged on cation-Ï€ interactions for recognition of quaternary alkyl ammonium groups such as trimethyllysine (Kme3). While computational modelling indicates that Trp provides the strongest cation-Ï€ interaction of the native aromatic amino acids, there is limited corroborative data from measurements within proteins. Herein we investigate a Tyr to Trp mutation in the binding pocket of the HP1 chromodomain, a reader protein that recognizes Kme3. Binding studies demonstrate that the Trp-mediated cation-Ï€ interaction is about -5 kcal mol-1 stronger, and the Y24W crystal structure shows that the mutation is not perturbing. Quantum mechanical calculations indicate that greater enthalpic binding is predominantly due to increased cation-Ï€ interactions. NMR studies indicate that differences in the unbound state of the Y24W mutation lead to enthalpy-entropy compensation. These results provide direct experimental quantification of Trp versus Tyr in a cation-Ï€ interaction and afford insight into the conservation of aromatic cage residues in Kme3 reader domains