scholarly journals Label-Free Assay of Protein Kinase A Activity and Inhibition Using a Peptide-Based Electrochemical Sensor

Biomedicines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 423
Author(s):  
Hyunju Cho ◽  
Chang-Seuk Lee ◽  
Tae Hyun Kim
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Self Assembly ◽  
Dynamic Range ◽  
Electrochemical Impedance ◽  
Protein Kinase Activity ◽  
Label Free ◽  
Transfer Resistance ◽  
Substrate Peptide ◽  
Kinase A

We propose a simple label-free electrochemical biosensor for monitoring protein kinase activity and inhibition using a peptide-modified electrode. The biosensor employs cys-kemptide (CLRRASLG) as a substrate peptide which was immobilized on the surface of a gold electrode via the self-assembly of the thiol terminals in cysteine (C) residues. The interaction between protein kinase A (PKA) and adenosine 5′-triphosphate (ATP) on the cys-kemptide immobilized electrode can cause the transfer of ATP terminal phosphates to the peptide substrates at serine (S) residues, which alters the surface charge of the electrode, thus enabling monitoring of the PKA activity via measuring the interfacial electron transfer resistance with electrochemical impedance spectroscopy. The proposed sensor showed reliable, sensitive, and selective detection of PKA activity with a wide dynamic range of 0.1–100 U/mL and a detection limit of 56 mU/mL. The sensor also exhibited high selectivity, rendering it possible to screen PKA inhibitors. Moreover, the sensor can be employed to evaluate the activity and inhibition of PKA in real samples.

Charge-Balanced Metal Fluoride Complexes for Protein Kinase A with Adenosine Diphosphate and Substrate Peptide SP20

2012 ◽  
Vol 124 (49) ◽  
pp. 12408-12411 ◽  
Author(s):  
Yi Jin ◽  
Matthew J. Cliff ◽  
Nicola J. Baxter ◽  
Hugh R. W. Dannatt ◽  
Andrea M. Hounslow ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Adenosine Diphosphate ◽  
Metal Fluoride ◽  
Substrate Peptide ◽  
Fluoride Complexes ◽  
Kinase A

scholarly journals Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter

2019 ◽  
Author(s):  
Allen K. Kim ◽  
Helen D. Wu ◽  
Takanari Inoue
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Electrostatic Interactions ◽  
Rational Design ◽  
Dynamic Range ◽  
Single Cell Analysis ◽  
Resonance Energy ◽  
Sequence Motif ◽  
Recognition Mechanism ◽  
Kinase A

AbstractProtein Kinase A (PKA) exists as a tetrameric holoenzyme which activates with increase of cAMP and plays an important role in many physiological processes including cardiac physiology, neuronal development, and adipocyte function. Although this kinase has been the subject of numerous biosensor designs, a single-fluorophore reporter that performs comparably to Förster resonance energy transfer (FRET) has not yet been reported. Here, we have used basic observations of electrostatic interactions in PKA substrate recognition mechanism and nucleus localization sequence motif to design a phosphorylation switch that shuttles between the cytosol and the nucleus, a strategy that should be generalizable to all basophilic kinases. The resulting reporter yielded comparable kinetics and dynamic range to the PKA FRET reporter, AKAR3EV. We also performed basic characterization and demonstrated its potential use in monitoring multiple signaling molecules inside cells using basic fluorescence microscopy. Due to the single-fluorophore nature of this reporter, we envision that this could find broad applications in studies involving single cell analysis of PKA activity.

scholarly journals Rapid Determination of Ochratoxin A in Grape and Its Commodities Based on a Label-Free Impedimetric Aptasensor Constructed by Layer-by-Layer Self-Assembly

Toxins ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 71 ◽  
Author(s):  
Mina Nan ◽  
Yang Bi ◽  
Huali Xue ◽  
Sulin Xue ◽  
Haitao Long ◽  
...  
Keyword(s):  
Ochratoxin A ◽  
Self Assembly ◽  
Electrochemical Impedance ◽  
Rapid Determination ◽  
Grape Juice ◽  
High Sensitivity ◽  
Layer By Layer ◽  
Label Free ◽  
Transfer Resistance

A simple and sensitive label-free impedimetric aptasensor for rapid determination of ochratoxin A (OTA) has been developed, which was based on the combination between thiolated aptamer and gold nanoparticles by layer-by-layer self-assembly. Because of the interaction between aptamer and OTA, the relative normalized electron-transfer resistance (ΔRct) values obtained by electrochemical impedance spectroscopy (EIS) was proportional to the concentration of OTA and showed a good linear relationship from 0.1 to 10.0 ng/mL, with a lower detection limit (0.030 ng/mL) than one-step thiolated DNA aptasensor. The established method was successfully applied to detect and analyze OTA in table wine and grape juice, and the recovery was 90.56%–104.21% when PVP effective removed of phenolic substances. The label-free impedimetric aptasensor was used for rapid detection and quantitation of OTA in the inoculated grapes with the Aspergillus Nigri (H1), and the production of OTA (62.4 μg/kg, 20 μg/kg) far exceeded the maximum levels of 2 μg/kg after inoculation for three days. The developed method exhibited a good specificity, high sensitivity, time-efficient, and it could be applied to detect the OTA concentration in grape and its commodities.

Ultrasensitive amplification-free detection of protein kinase based on catalyzed assembly and enumeration of gold nanoparticles

2019 ◽  
Vol 55 (17) ◽  
pp. 2505-2508 ◽  
Author(s):  
Tongtong Tian ◽  
Yuanyuan Yao ◽  
Beibei Yang ◽  
Kun Zhang ◽  
Baohong Liu
Keyword(s):  
Gold Nanoparticles ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Single Particle ◽  
Label Free ◽  
Kinase A

A novel single-particle enumeration method for label-free, amplification-free and ultrasensitive probing of protein kinase A activity based on catalyzed assembly.

scholarly journals A Subunit of Protein Kinase A Regulates Growth and Differentiation in the Fungus Mucor circinelloides

2009 ◽  
Vol 8 (7) ◽  
pp. 933-944 ◽  
Author(s):  
J. Ocampo ◽  
L. Fernandez Nuñez ◽  
F. Silva ◽  
E. Pereyra ◽  
S. Moreno ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Developmental Stages ◽  
Mrna Level ◽  
Mucor Circinelloides ◽  
Binding Activity ◽  
Regulatory Subunit ◽  
Tube Length ◽  
Protein Kinase Activity ◽  
Kinase A

ABSTRACT The cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling pathway plays a role in regulating development, growth, and virulence in a number of fungi. To determine whether PKA plays a similar function in zygomycete fungi, a mutant of Mucor circinelloides was generated that lacks pkaR1, one of the regulatory subunits of PKA. The mutant showed a reduction in growth and alterations in germination rates, cell volume, germ tube length, and asexual sporulation. The lack of pkaR1 gene resulted in a highly decreased, but not null, cAMP binding activity and in a protein kinase activity that was still dependent on cAMP, although with a higher −/+ cAMP activity ratio, suggesting the existence of other cAMP binding activities. Consequently, three proteins analogous to pkaR1 were predicted from the recently sequenced genome of M. circinelloides and were named pkaR2, pkaR3, and pkaR4. Two of the proteins, pkaR2 and pkaR3, with cAMP binding activity were isolated from the wild-type strain and identified by mass spectrometry. The expression of all genes was detected at the mRNA level by semiquantitative reverse transcription-PCR, and they showed a differential expression at different developmental stages. This is the first time that a fungus is reported to have more than one gene encoding the regulatory subunit of PKA.

scholarly journals An Electrochemical Immunosensor Based on a Self-Assembled Monolayer Modified Electrode for Label-Free Detection of α-Synuclein

Sensors ◽  
2020 ◽  
Vol 20 (3) ◽  
pp. 617 ◽  
Author(s):  
Chuang-Ye Ge ◽  
Md. Mahbubur Rahman ◽  
Wei Zhang ◽  
Nasrin Siraj Lopa ◽  
Lei Jin ◽  
...  
Keyword(s):  
Dynamic Range ◽  
Electrochemical Impedance ◽  
Charge Transfer Resistance ◽  
Label Free ◽  
Serum Samples ◽  
Self Assembled Monolayer ◽  
Electrochemical Impedance Spectra ◽  
Transfer Resistance ◽  
Label Free Detection ◽  
Self Assembled

This research demonstrated the development of a simple, cost-effective, and label-free immunosensor for the detection of α-synuclein (α-Syn) based on a cystamine (CYS) self-assembled monolayer (SAM) decorated fluorine-doped tin oxide (FTO) electrode. CYS-SAM was formed onto the FTO electrode by the adsorption of CYS molecules through the head sulfur groups. The free amine (–NH2) groups at the tail of the CYS-SAM enabled the immobilization of anti-α-Syn-antibody, which concurrently allowed the formation of immunocomplex by covalent bonding with α-Syn-antigen. The variation of the concentrations of the attached α-Syn at the immunosensor probe induced the alternation of the current and the charge transfer resistance (Rct) for the redox response of [Fe(CN)6]3−/4−, which displayed a linear dynamic range from 10 to 1000 ng/mL with a low detection limit (S/N = 3) of ca. 3.62 and 1.13 ng/mL in differential pulse voltammetry (DPV) and electrochemical impedance spectra (EIS) measurements, respectively. The immunosensor displayed good reproducibility, anti-interference ability, and good recoveries of α-Syn detection in diluted human serum samples. The proposed immunosensor is a promising platform to detect α-Syn for the early diagnose of Parkinson’s disease, which can be extended for the determination of other biologically important biomarkers.

scholarly journals FRET and LRET Biosensors for Cell-based Imaging and Screening of Rac1 Activation

2021 ◽  
Author(s):  
Ha Pham ◽  
Mona Hoseini Soflaee ◽  
Andrei V Karginov ◽  
Lawrence Miller
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Hek293t Cell ◽  
Resonance Energy ◽  
Terbium Complex ◽  
A Cell ◽  
Fret Biosensors ◽  
Kinase A

Rac1 is a key regulator of several cell signaling pathways and dysregulated Rac1 activation has been implicated in cancer. Genetically encoded Forster resonance energy transfer (FRET) biosensors with enhanced dynamic range enabled live cell fluorescence imaging of Rac1 activity and a cell lysate-based assay of Rac1 inhibition in 96-well plates. We prepared HEK293T cell lines that stably expressed polypeptides with a general domain sequence (N- to C-terminus) of 1) FRET acceptor; 2) Rac/Cdc42 binding domain of human p21 protein kinase A (residues 68-150); 3) a linker domain; 4) FRET donor; and 5) full-length Rac1. Activated Rac1 binds to the protein kinase A domain, bringing donors and acceptors close together to increase FRET. We evaluated the effects on FRET signal dynamic range of alpha helical linkers comprised of alternating repeats of roughly four glutamate and four arginine or lysine residues. So-called ER/K linkers had limited effects on conventional FRET biosensors that incorporated the fluorescent protein (FP) pairs mCerulean/YPet, or mTFP1(cp227)/mVenus(cp229). Fluorometric measurements of cells that co-expresssed biosensors with positive (TIAM1) or negative (RhoGDI) Rac1 regulators revealed significant dynamic range enhancement in only one FP construct (mCerulean/YPet with 20 nm ER/K linker) relative to an analogous structure that incorporated an unstructured linker. We transfected this construct into a cell line that stably expressed a rapamycin-inducible c-Src analog (RapR-Src) and observed activation of Rac1 at protruding edges following rapamycin stimulation. In cells that expressed lanthanide-based FRET (LRET biosensors) that incorporated a luminescent terbium complex donor and GFP fluorescent acceptor, time-gated luminescence (TGL) measurements showed substantial gains in dynamic range that increased with linker length (up to 1200%). We robustly detected small molecule Rac1 inhibition following lysis of LRET biosensor-expressing cells grown directly in 96-well plates. The results herein highlight the potential of FRET and LRET biosensors with ER/K linkers for cell-based imaging and screening of protein activities.

Charge-Balanced Metal Fluoride Complexes for Protein Kinase A with Adenosine Diphosphate and Substrate Peptide SP20

2012 ◽  
Vol 51 (49) ◽  
pp. 12242-12245 ◽  
Author(s):  
Yi Jin ◽  
Matthew J. Cliff ◽  
Nicola J. Baxter ◽  
Hugh R. W. Dannatt ◽  
Andrea M. Hounslow ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Adenosine Diphosphate ◽  
Metal Fluoride ◽  
Substrate Peptide ◽  
Fluoride Complexes ◽  
Kinase A
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