Superposition of an AC field improves the discrimination between peptides in nanopore analysis

Jakova, Elisabet; Lee, Jeremy S.

doi:10.1039/c4an02180k

Cited by 3 publications

(12 citation statements)

References 41 publications

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“…At 500 MHz and 1 GHz, the translocation peak shifts to −84 pA and the bumping peak shifts to −24 pA. These blockade current values are similar to those observed for AS in the absence of drug , suggesting that the AC field may be capable of causing the drug to be stripped off the AS.…”

Section: Resultssupporting

confidence: 71%

“…At 50 MHz the magnitude of the bumping peak increases to 72%. As discussed previously, the AC field is expected to cause the complex to oscillate so that for much of the time it is in an unfavorable orientation to enter the lumen of the pore . At higher frequencies, there are small changes in the proportion of the events in each peak but there are large changes in the blockade currents.…”

Section: Resultsmentioning

confidence: 93%

“…It was found that as the frequency increased the proportion of bumping events increased as expected for a molecule with a very large dipole moment . Seven drugs were chosen for analysis based on their known binding sites.…”

Section: Resultsmentioning

confidence: 99%

“…The standard nanopore analysis has been described in detail previously . The AC voltage was applied via Agilent E4420B signal generator (TRS‐RenTelco, Dollard‐des‐Ormeaux, QC, Canada).…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we have described a further modification to nanopore analysis in which an AC field is superimposed on the electrophoretic DC voltage . This allows discrimination between peptides based on their dipole moment .…”

Section: Introductionmentioning

confidence: 99%

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Behavior of α‐synuclein–drug complexes during nanopore analysis with a superimposed AC field

Jakova

Lee

2016

Electrophoresis

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Seven α-synuclein-drug complexes have been studied by nanopore analysis in which an AC field of 200 mV from 10 MHz to 1 GHz has been superimposed on the standard electrophoretic DC voltage of 100 mV. α-Synuclein has a large dipole moment and in the absence of drug the AC field causes the molecule to oscillate at the entrance to the pore and reduces its ability to translocate through the pore. Thus more bumping events are observed in the current blockade histograms. The binding of drugs to α-synuclein has a large effect on the event profiles depending on the region of α-synuclein to which the drugs bind. Caffeine and (-)-nicotine bind both the N- and C-termini causing the protein to adopt a loop conformation that allows translocation even in the AC field. Metformin, which binds only to the C-terminus also facilitates translocation. For these drugs there is good evidence that the AC field is causing the complex to dissociate as it enters the pore that has not been observed previously. In contrast, complexes with (+)-amphetamine that has an N-terminal binding site and cocaine that binds to the central region of the protein, show only small changes in the event profiles in an AC field.

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Section: Resultssupporting

confidence: 71%

Section: Resultsmentioning

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Behavior of α‐synuclein–drug complexes during nanopore analysis with a superimposed AC field

Jakova

Lee

2016

Electrophoresis

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Adenosine A1 receptor ligands bind to α-synuclein: implications for α-synuclein misfolding and α-synucleinopathy in Parkinson’s disease

Jakova

Moutaoufik

Lee

et al. 2022

Transl Neurodegener

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Background Accumulating α-synuclein (α-syn) aggregates in neurons and glial cells are the staples of many synucleinopathy disorders, such as Parkinson’s disease (PD). Since brain adenosine becomes greatly elevated in ageing brains and chronic adenosine A1 receptor (A1R) stimulation leads to neurodegeneration, we determined whether adenosine or A1R receptor ligands mimic the action of known compounds that promote α-syn aggregation (e.g., the amphetamine analogue 2-aminoindan) or inhibit α-syn aggregation (e.g., Rasagiline metabolite 1-aminoindan). In the present study, we determined whether adenosine, A1R receptor agonist N6-Cyclopentyladenosine (CPA) and antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) could directly interact with α-syn to modulate α-syn aggregation and neurodegeneration of dopaminergic neurons in the substantia nigra (SN). Methods Nanopore analysis and molecular docking were used to test the binding properties of CPA and DPCPX with α-syn in vitro. Sprague–Dawley rats were administered with 7-day intraperitoneal injections of the A1R ligands and 1- and 2-aminoindan, and levels of α-syn aggregation and neurodegeneration were examined in the SN pars compacta and hippocampal regions using confocal imaging and Western blotting. Results Using nanopore analysis, we showed that the A1R agonists (CPA and adenosine) interacted with the N-terminus of α-syn, similar to 2-aminoindan, which is expected to promote a “knot” conformation and α-syn misfolding. In contrast, the A1R antagonist DPCPX interacted with the N- and C-termini of α-syn, similar to 1-aminoindan, which is expected to promote a “loop” conformation that prevents α-syn misfolding. Molecular docking studies revealed that adenosine, CPA and 2-aminoindan interacted with the hydrophobic core of α-syn N-terminus, whereas DPCPX and 1-aminoindan showed direct binding to the N- and C-terminal hydrophobic pockets. Confocal imaging and Western blot analyses revealed that chronic treatments with CPA alone or in combination with 2-aminoindan increased α-syn expression/aggregation and neurodegeneration in both SN pars compacta and hippocampus. In contrast, DPCPX and 1-aminoindan attenuated the CPA-induced α-syn expression/aggregation and neurodegeneration in SN and hippocampus. Conclusions The results indicate that A1R agonists and drugs promoting a “knot” conformation of α-syn can cause α-synucleinopathy and increase neuronal degeneration, whereas A1R antagonists and drugs promoting a “loop” conformation of α-syn can be harnessed for possible neuroprotective therapies to decrease α-synucleinopathy in PD.

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Adenosine A1 Receptor Ligands Bind to α-Synuclein: Implications for α-Synuclein Misfolding and α-Synucleinopathy in Parkinson’s Disease

Jakova¹,

Moutaoufik²,

Lee³

et al. 2020

Preprint

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Background:Accumulating α-synuclein (α-Syn) aggregates in neurons and glial cells are the staples of many synucleinopathy disorders, such as Parkinson’s disease. Since brain adenosine becomes greatly elevated in ageing brains and chronic adenosine A1 receptor (A1R) stimulation leads to neurodegeneration, we determined whether adenosine or A1R receptor ligands mimic the action of known compounds that promote α-Syn aggregation (e.g., the amphetamine analogue 2-aminoindan) or inhibit α-Syn aggregation (e.g., Rasagiline metabolite 1-aminoindan). In the present study, we determined whether adenosine, the A1R receptor agonist N6-Cyclopentyladenosine (CPA) and antagonist 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) directly interact with α-Syn to modulate α-Syn aggregation and neurodegeneration of dopaminergic neurons in the substantia nigra (SN). Methods:Nanopore analysis and molecular dynamics simulation were used to test the binding properties of CPA and DPCPX with α-Syn in vitro. Sprague-Dawley rats were administered 7-day intraperitoneal injections of the A1R ligands and 1- and 2-aminoindan, and levels of α-Syn aggregation and neurodegeneration were examined in the substantia nigra pars compacta region. Results:Using nanopore analysis, we showed that the A1R agonists (CPA and adenosine) interacted with the N-terminus of α-Syn, similar to 2-aminoindan, which is expected to promote a “knot” conformation and α-Syn misfolding. In contrast, the A1R antagonist DPCPX interacted with the N- and C-termini of α-Syn, similar to 1-aminoindan, which is expected to promote a “loop” conformation which prevents α-Syn misfolding. Molecular docking studies revealed that adenosine, CPA and 2-aminoindan interacted with the hydrophobic core of α-Syn N-terminus, whereas DPCPX and 1-aminoindan showed direct binding to the N- and C-terminal hydrophobic pockets. Histological and confocal imaging studies revealed that chronic treatments with CPA alone or in combination with 2-aminoindan increased α-Syn aggregation and neurodegeneration in SN pars compacta. In contrast, DPCPX and 1-aminoindan attenuated CPA-induced neurodegeneration but did not significantly reduce α-Syn aggregation.Conclusions:The results indicate A1R agonists and drugs promoting a “knot” conformation of α-Syn can cause α-synucleinopathy and increase neuronal degeneration, whereas A1R antagonists and drugs promoting a “loop” conformation of α-Syn can be harnessed for possible neuroprotective therapies to decrease α-synucleinopathy in Parkinson’s disease.

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Superposition of an AC field improves the discrimination between peptides in nanopore analysis

Cited by 3 publications

References 41 publications

Behavior of α‐synuclein–drug complexes during nanopore analysis with a superimposed AC field

Behavior of α‐synuclein–drug complexes during nanopore analysis with a superimposed AC field

Adenosine A1 receptor ligands bind to α-synuclein: implications for α-synuclein misfolding and α-synucleinopathy in Parkinson’s disease

Adenosine A1 Receptor Ligands Bind to α-Synuclein: Implications for α-Synuclein Misfolding and α-Synucleinopathy in Parkinson’s Disease

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