Localized surface plasmon resonance aptasensor for selective detection of SARS-CoV-2 S1 protein.

In this work, we designed and developed a method to detect S1 spike protein of SARS-CoV-2. The portable Localized Surface Plasmon Resonance instrument equipped with a two-channel system was combined with the biotin-streptavidin platform on a nanogold surface to immobilize biotinylated aptamers. The proposed assay does not utilize antibodies or enzyme-based reagents, further simplifying the detection method. Using aptamer-protein bioaffinity interactions, the aptasensor selectively and specifically detected in real-time S1 spike protein, rather than S2 spike protein, RBD spike protein, or bovine serum albumin. The dynamic range and limit of detection of the aptasensor was determined to be 1 nM-100 nM and 0.26 nM, respectively. Notably, aptasensor detected preferentially S1 protein of SARS-CoV-2 compared to SARS-CoV and detected S1 protein with >95% recovery in artificial saliva, and serum albumin, excellent repeatability and shelf-life stability. The method may provide a low-cost, rapid, and real-time detection and monitoring of viruses in the general public.

Dual roles of tau R peptides on Cu(II)/(I)-mediated reactive oxygen species formation.

Metal dyshomeostasis plays a critical role in the reactive oxygen species (ROS) formation and protein misfolding and aggregation; hence, contributing to neurodegeneration. Tau protein plays a key role in normal cellular function by maintaining microtubule formation in brain. The role of metal ions on tau protein biochemistry has not been systematically evaluated, but earlier reports indicated that metal ions modulate the complex biochemistry of this protein and its peptides. Herein, we evaluated interactions of biologically-relevant Cu(II) ions with the four repeat peptides of tau protein (R1 through R4) and their role on the formation of ROS, Cu(II) to Cu(I) reduction, and ultimately, peptide aggregation. The role of R peptides on ROS formation was characterized in the absence and presence of biological reducing agent, ascorbate by using UV-Vis and fluorescence spectroscopy. In the presence of the reducing agent, all Cu(II)-peptide complexes reduced hydroxyl radical (OH·), while only Cu(II)-R3 complex depleted the hydrogen peroxide (H2O2). In the absence of a reducing agent, only Cu(II)-R2 and Cu(II)-R3 complexes, which contain Cys and His residues, produced OH· and H2O2. Only R2 and R3 peptides, but not R1 and R4, reduced Cu(II) to Cu(I). The aggregation propensities of R peptides were modulated by Cu(II) and ascorbate, and were imaged by transmission electron microscopy. All metallo-peptides were characterized predominantly as singly charged mononuclear complexes by mass spectrometry. The data indicate that Cu(II)-peptide complexes may act as pro-oxidants or antioxidants and exhibit unique aggregation propensities under specific environmental conditions, with implications in the biological setting.

Aggregation of gelsolin wild-type and G167K/R, N184K, and D187N/Y mutant peptides and inhibition.

Gelsolin, an actin-binding protein, is localized intra- and extracellularly in the bloodstream and throughout the body. Gelsolin amyloidosis is a disease characterized by several point mutations that lead to cleavage and fibrillization of gelsolin. The D187 mutation to N or Y leads to aggregation of peptide fragments with shortest aggregating peptide identified as 182SFNNGDCFILD192. Recently, G167 has also been identified as relevant gelsolin mutation, which leads to gelsolin deposits in kidneys, but its aggregation is much less understood. Hence, we systematically investigated in vitro the aggregation propensities of the following gelsolin peptides: 167GRRVV171 (1), 161RLFQVKG167 (2), 184NNGDCFILDL193 (3), 188CFILDL193 (4), 187DCFILDL193 (5), and their respective mutants (G167K, G167R, N184K, D187Y, D187N), by using spectroscopic methods [fluorescence Proteostat, Thioflavin T (ThT), turbidity assay, and Dynamic Light Scattering (DLS)], and Transmission Electron Microscopy (TEM). The (non) mutant peptides containing CFILDL sequence aggregated into fibrillar networks, while G167R mutation promoted aggregation compared to the wild-type sequence. In the presence of inhibitors, Methylene Blue (MB) and epigallocatechin gallate (EGCG), the gelsolin peptide (3-5) aggregation was reduced with the IC50 values in the 2-13 µM range. We discovered that inhibitors have dual functionality, as aggregation inhibitors and disaggregation promoters, potentially allowing for the prevention and reversal of gelsolin amyloidosis. Such therapeutic strategies may improve outcomes related to other amyloidogenic diseases of the heart, brain, and eye.

Structural evaluations of tau protein conformation: methodologies and approaches.

Protein-misfolding diseases are based on a common principle of aggregation initiated by intra- and inter-molecular contacts. The structural and conformational changes induced by biochemical transformations such as post-translational modifications (PTMs), often lead to protein unfolding and misfolding. Thus, these order-to-disorder or disorder-to-order transitions may regulate cellular function. Tau, a neuronal protein, regulates microtubule (MT) structure and overall cellular integrity. However, misfolded tau modified by PTMs results in MT destabilization, toxic tau aggregate formation, and ultimately cell death, leading to neurodegeneration. Currently, the lack of structural information surrounding tau severely limits understanding of neurodegeneration. This minireview focuses on the current methodologies and approaches aimed at probing tau conformation and the role of conformation in various aspects of tau biochemistry. The recent applications of nuclear magnetic resonance, mass spectrometry, Förster resonance electron transfer, and molecular dynamics simulations toward structural analysis of conformational landscapes of tau will be described. The strategies developed for structural evaluation of tau may significantly improve our understanding of misfolding diseases.

Selective Electrochemical versus Chemical Oxidation of Bulky Phenol.

The electrochemical oxidation of selected tert-butylated phenols 2,6-di-tert-butyl-4-methylphenol (1), 2,6-di-tert-butylphenol (2), 2,4,6-tri-tert-butylphenol (3), 2-tert-butylphenol (4), and 4-tert-butylphenol (5) was studied in an aprotic environment using cyclic voltammetry, square-wave voltammetry, and UV-vis spectroscopy. All compounds exhibited irreversible oxidation of the corresponding phenol or phenolate ion. Compound 2 was selectively electrochemically oxidized, while other phenol analogues underwent mostly chemical oxidation. The electrochemical oxidation of 2 produced a highly absorbing product, 3,5,3',5'-tetra-tert-butyl-4,4'-diphenoquinone, which was characterized by X-ray crystal diffraction. The electrochemical oxidation was monitored as a function of electrochemical parameters and concentration. Experimental and theoretical data indicated that the steric hindrance, phenoxyl radical stability, and hydrogen bonding influenced the outcome of the electrochemical oxidation. The absence of the substituent at the para position and the presence of the bulky substituents at ortho positions were structural and electrostatic requirements for the selective electrochemical oxidation.

Evaluation of ferritin and transferrin binding to tau protein.

Tau protein is a neurodegeneration biomarker. Due to the high concentration of metal ions in the brain, the metallation of tau proteins and their catalytic role in reactive oxygen formation have been identified as a major biochemical pathway of neurodegeneration. High levels of iron ions have been detected in Alzheimer's disease brains. One of biological sources of iron ions are iron-rich proteins, such as transferrin or ferritin. However, the interactions between tau and these metallo-proteins have not been fully characterized. Here, the interactions between the longest form of full-length tau protein (tau441) with iron-rich proteins were detected using electrochemical impedance spectroscopy. Tau441 was immobilized on Au surface, via N-terminal (N-tau-Au film) or Cys-residues (Cys-tau-Au film), and the charge-transfer resistance, Rct, was monitored prior and post ferritin or transferrin binding. Significant increase in Rct was observed post transferrin binding above 50µgmL-1, but not ferritin regardless of concentration with N-tau-Au film. Additionally, the electrochemical trend was linear with respect to transferrin concentration. Electrochemical data indicated low binding by ferritin to N-tau-Au or Cys-tau-Au films. The interaction of apotransferrin or apoferritin with tau films was also evaluated. Electrochemical data may be pointing to the differences in protein binding modes by transferrin compared to ferritin as well as to importance of metal ions in protein-protein interactions.

Electrochemical detection of anti-tau antibodies binding to tau protein and inhibition of GSK-3ß-catalyzed phosphorylation.

Tau protein hyperphosphorylation triggers tau aggregation and its toxicity, leading to neuronal death and cell-to-cell toxicity. Hence, inhibition of protein kinases is a viable tool toward reduction of tau toxicity. By targeting various epitopes of Tau441 protein immobilized on Au surface, the protein kinase inhibition by anti-tau antibodies was measured by surface electrochemistry. The electrochemical impedance spectroscopy was used to measure the charge transfer resistance (Rct) of nonphosphorylated tau-Au film (nTau-Au) and compared with the phosphorylated tau-Au film (pTau-Au). The pTau-Au films were characterized by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS), which indicated high phosphorus content. The Rct factor was used as the measure of inhibition efficacies by anti-tau antibodies (D8, A10, P262, and Tau46) in addition to antibody formulation intravenous immunoglobulin (IVIG). The Rct factor for pTau-Au in the absence of antibodies was 0.25 ± 0.08, indicating a dramatic decrease in Rct on phosphorylation. The Rct factors for Tau46 and A10 were 0.57 ± 0.22 and 0.65 ± 0.26, respectively, indicating phosphorylation inhibition. All antibodies exhibited similar binding to nTau-Au. The proposed electrochemical assay may be used for detection of other posttranslational modifications.

Electrochemical Characterization of Protein Adsorption onto YNGRT-Au and VLGXE-Au Surfaces.

The adsorption of the proteins CD13, mucin and bovine serum albumin on VLGXE-Au and YNGRT-Au interfaces was monitored by electrochemical impedance spectroscopy in the presence of [Fe(CN)6](3-/4-). The hydrophobicity of the Au surface was tailored using specific peptides, blocking agents and diluents. The combination of blocking agents (ethanolamine or n-butylamine) and diluents (hexanethiol or 2-mercaptoethanol) was used to prepare various peptide-modified Au surfaces. Protein adsorption onto the peptide-Au surfaces modified with the combination of n-butylamine and hexanethiol produced a dramatic decrease in the charge transfer resistance, Rct, for all three proteins. In contrast, polar peptide-surfaces induced a minimal change in Rct for all three proteins. Furthermore, an increase in Rct was observed with CD13 (an aminopeptidase overexpressed in certain cancers) in comparison to the other proteins when the VLGXE-Au surface was modified with n-butylamine as a blocking agent. The electrochemical data indicated that protein adsorption may be modulated by tailoring the peptide sequence on Au surfaces and that blocking agents and diluents play a key role in promoting or preventing protein adsorption. The peptide-Au platform may also be used for targeting cancer biomarkers with designer peptides.