The multifunctional dopamine D2/D3 receptor agonists also possess inhibitory activity against the full-length tau441 protein aggregation.

Neurodegeneration leads to variety of diseases which are linked to aberrant protein or peptide aggregation, as a one possible mechanism. Hence, small drug molecules targeting aggregation are of interest. Tau protein aggregation is one of the biomarkers of neurodegenerative diseases and is a viable drug target. Toward multifunctional inhibitors, we aim to incorporate structural elements in a potential drug in order to preserve dopamine agonist activity, which elevates disease symptoms associated with motor skills, and promote inhibitory activity against aggregation of the full-length tau (2N4R, tau441) protein. In our design, we introduced various moieties (catechol, non-catechol, biphenyl, piperazine, and thiazole) to determine which functional group leads to the greatest aggregation inhibition of tau. In vitro, tau aggregation was induced by heparin and monitored by using fluorescence aggregation assay, transmission electron microscopy and 4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (Bis-ANS) fluorescence spectroscopy. The catechol containing compounds, D-519 and D-520, prevented aggregation of tau. By contrast, non-catechol and thiazole containing compounds (D-264 and D-636) were poor inhibitors. The Bis-ANS studies revealed that the potent inhibitors bound solvent-exposed hydrophobic sites. Based on the density functional theory calculations on inhibitors tested, the compounds characterized with the high polarity and polarizability were more effective aggregation inhibitors. These findings could lead to the development of small multifunctional drug inhibitors for the treatment of tau-associated neurodegeneration.

A dip-and-read optical aptasensor for detection of tau protein.

Neurodegeneration currently remains without a differential diagnosis or cure. Tau protein is one of the biomarkers of neurodegenerative diseases commonly known as tauopathies. Tau protein plays an integral role in stabilizing microtubules and cell structure; however, due to post-translational modifications, tau protein undergoes self-assembly into cytotoxic structures and is co-localized intra- and extracellularly. Hence, tau protein is a viable biomarker associated with protein pathogenesis and neurodegeneration. The novel optical biosensor for tau441 protein is based on the aptamer recognition probe and the biolayer interferometry (BLI) method for detection. The current biotin-aptasensor in combination with the streptavidin surface provides real-time monitoring of tau441 protein in the nanomolar range, with the limit of detection at 6.7 nM in vitro. The tau441 detection is achieved with high selectivity over other neurodegeneration biomarkers which include amyloid-ß and α-synuclein. The aptasensor also allows for tau441 protein detection in a complex matrix such as fetal bovine serum, indicating its utility in other biological fluids for diagnostic applications. The optical method is simple, rapid and highly selective for point-of-care application which is critical for achieving the early and differential diagnosis of neurodegenerative diseases and identifying their treatments. Graphical abstract.

A Bioorganometallic Approach to Study Histidine Kinase Autophosphorylations.

Auto-phosphorylation of bacterial histidine kinases PhoR, PhoQ, and EnvZ has been investigated using adenosine-5'-[γ-ferrocene] triphosphate (Fc-ATP) as a cosubstrate for the first time. The study has been carried out in solution and on surface. Results from biochemical multiplex assay and surface electrochemical/optical methods are consistent, which successfully demonstrates that Fc-ATP is an efficient cosubstrate for histidine kinase auto-phosphorylations. The study also has discovered that the concentration of Fc-ATP influences the autophosphorylation efficiency. This developed methodology will provide a powerful tool in studying such biological processes towards further understanding of the involved mechanism.

Electron transfer in peptides.

In this review, we discuss the factors that influence electron transfer in peptides. We summarize experimental results from solution and surface studies and highlight the ongoing debate on the mechanistic aspects of this fundamental reaction. Here, we provide a balanced approach that remains unbiased and does not favor one mechanistic view over another. Support for a putative hopping mechanism in which an electron transfers in a stepwise manner is contrasted with experimental results that support electron tunneling or even some form of ballistic transfer or a pathway transfer for an electron between donor and acceptor sites. In some cases, experimental evidence suggests that a change in the electron transfer mechanism occurs as a result of donor-acceptor separation. However, this common understanding of the switch between tunneling and hopping as a function of chain length is not sufficient for explaining electron transfer in peptides. Apart from chain length, several other factors such as the extent of the secondary structure, backbone conformation, dipole orientation, the presence of special amino acids, hydrogen bonding, and the dynamic properties of a peptide also influence the rate and mode of electron transfer in peptides. Electron transfer plays a key role in physical, chemical and biological systems, so its control is a fundamental task in bioelectrochemical systems, the design of peptide based sensors and molecular junctions. Therefore, this topic is at the heart of a number of biological and technological processes and thus remains of vital interest.

Effects of tau domain-specific antibodies and intravenous immunoglobulin on tau aggregation and aggregate degradation.

Tau pathology, including neurofibrillary tangles, develops in Alzheimer's disease (AD). The aggregation and hyperphosphorylation of tau are potential therapeutic targets for AD. Administration of anti-tau antibodies reduces tau pathology in transgenic "tauopathy" mice; however, the optimal tau epitopes and conformations to target are unclear. Also unknown is whether intravenous immunoglobulin (IVIG) products, currently being evaluated in AD trials, exert effects on pathological tau. This study examined the effects of anti-tau antibodies targeting different tau epitopes and the IVIG Gammagard on tau aggregation and preformed tau aggregates. Tau aggregation was assessed by transmission electron microscopy and fluorescence spectroscopy, and the binding affinity of the anti-tau antibodies for tau was evaluated by enzyme-linked immunosorbent assays. Antibodies used were anti-tau 1-150 ("D-8"), anti-tau 259-266 ("Paired-262"), anti-tau 341-360 ("A-10"), and anti-tau 404-441 ("Tau-46"), which bind to tau's N-terminus, microtubule binding domain (MBD) repeat sequences R1 and R4, and the C-terminus, respectively. The antibodies Paired-262 and A-10, but not D-8 and Tau-46, reduced tau fibrillization and degraded preformed tau aggregates, whereas the IVIG reduced tau aggregation but did not alter preformed aggregates. The binding affinities of the antibodies for the epitope for which they were specific did not appear to be related to their effects on tau aggregation. These results confirm that antibody binding to tau's MBD repeat sequences may inhibit tau aggregation and indicate that such antibodies may also degrade preformed tau aggregates. In the presence of anti-tau antibodies, the resulting tau morphologies were antigen-dependent. The results also suggested the possibility of different pathways regulating antibody-mediated inhibition of tau aggregation and antibody-mediated degradation of preformed tau aggregates.

Clickable 5'-γ-ferrocenyl adenosine triphosphate bioconjugates in kinase-catalyzed phosphorylations.

Clickable co-substrate: A tri-functional 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) derivative containing a clickable site was synthesized. This compound is an effective co-substrate in kinase-catalyzed phosphorylation reactions, which can be detected by both electrochemical and immunoassay detection methods. The clickable reaction site makes direct modification possible, which greatly expands its application.

A protein-based electrochemical biosensor for detection of tau protein, a neurodegenerative disease biomarker.

A protein-based electrochemical biosensor was developed for detection of tau protein aimed towards electrochemically sensing misfolding proteins. The electrochemical assay monitors tau-tau binding and misfolding during the early stage of tau oligomerization. Electrochemical impedance spectroscopy was used to detect the binding event between solution tau protein and immobilized tau protein (tau-Au), acting as a recognition element. The charge transfer resistance (Rct) of tau-Au was 2.9 ± 0.6 kΩ. Subsequent tau binding to tau-Au decreased the Rct to 0.3 ± 0.1 kΩ (90 ± 3% decrease) upon formation of a tau-tau-Au interface. A linear relationship between the Rct and the solution tau concentration was observed from 0.2 to 1.0 µM. The Rct decrease was attributed to an enhanced charge permeability of the tau-tau-Au surface to a redox probe [Fe(CN)6](3-/4-). The electrochemical and surface characterization data suggested conformational and electrostatic changes induced by tau-tau binding. The protein-based electrochemical platform was highly selective for tau protein over bovine serum albumin and allowed for a rapid sample analysis. The protein-based interface was selective for a non-phosphorylated tau441 isoform over the paired-helical filaments of tau, which were composed of phosphorylated and truncated tau isoforms. The electrochemical approach may find application in screening of the early onset of neurodegeneration and aggregation inhibitors.

Surface plasmon resonance imaging of amyloid-ß aggregation kinetics in the presence of epigallocatechin gallate and metals.

A number of human protein misfolding disorders, including Alzheimer's disease (AD), are closely related to the accumulation of ß-sheet-rich amyloid fibrils or aggregates. Neuronal toxicity in AD has been linked to the interactions of amyloid-ß (Aß) with metals, especially Zn(2+), Cu(2+), and Fe(3+), which leads to the production of reactive oxygen species. Nucleation-dependent Aß aggregation, or "seeding", is thought to propagate fibril formation. In this surface plasmon resonance imaging (SPRi) study, we have shown that the fibril seeds formed with the incubation of Aß in the presence of metals are better at promoting monomer elongation compared to Aß alone or in the presence of a well-described polyphenol, (-)-epigallocatechin-3-gallate (EGCG). This is a novel attempt to simultaneously monitor the effects of multiple modulators on fibril elongation using a single chip. EGCG was shown in transmission electron microscopy (TEM) and thioflavin T (ThT) studies to promote the formation of off-pathway, highly stable unstructured oligomers, supporting the SPRi results. These findings suggest that SPRi provides a promising platform as a screening tool for small molecules that can affect the aggregation pathways in neurodegenerative diseases.

Probing copper/tau protein interactions electrochemically.

Alzheimer's disease (AD) is a neurodegenerative disorder that is characterized by peptide and protein misfolding and aggregation, in part due to the presence of excess metal ions such as copper(II) [Cu(II)]. Recently, the brain levels of Cu(II) complexes in vivo were linked to the oxidative stress in neurodegenerative disorders, including AD. Amyloid ß-peptide (Aß), found outside neuronal cells, has been investigated extensively in connection with Cu(II) ion toxicity; however, the effects of metallation on tau are less known. Normal tau protein binds and stabilizes the microtubules in neurons, but in diseased cells tau hyperphosphorylation and aggregation are evident and compromise tau function. There is increasing evidence that the Cu(II) ion may play an important role in tau biochemistry. Here, we present an electrochemical study of the interactions between full-length tau-410 and Cu(II) ions. The coordination of Cu(II) ions to tau immobilized on gold surfaces induces an electrochemical signal at approximately 140±5mV versus Ag/AgCl due to the Cu(II)/Cu(I) redox couple. Redox potentials and current intensities of Cu(II)-containing nonphosphorylated tau (nTau) and phosphorylated tau (pTau) films were determined at different pH conditions. Greater Cu(II) uptake by pTau over nTau films was observed at low pH. Competitive zinc(II) [Zn(II)] ion binding studies revealed significant Cu(II) ion displacement in pTau films. X-ray photoelectron spectroscopy analysis indicated the presence of Cu 2p and Zn 2p binding energies in protein samples, further supporting metal ion coordination to protein films. The surface-based electrochemical technique requires a minimal protein amount (a few microliters) and allows monitoring the bound Cu(II) ions and the redox activities of the resulting metalloprotein films.

Role of Triggers on the Structural and Functional Facets of TAR DNA-binding Protein 43.

Nuclear TAR DNA-binding protein 43 (TDP-43) mitigates cellular function, but the dynamic nucleus-cytoplasm shuttling of TDP-43 is disrupted in diseases, such as Amyotrophic Lateral Sclerosis (ALS). The polymorphic nature of the TDP-43 structures in vitro and in vivo is a result of environmental factors leading to the protein pathogenesis. Once the triggers which mitigate TDP-43 biochemistry are identified, new therapies can be developed. This review aims to illustrate recent discoveries in the diversity of TDP-43 structures (amyloidogenic and non-amyloidogenic) and highlight the triggers which result in their formation.

Profiling of adenine-derived signaling molecules, cytokinins, in myotubes reveals fluctuations in response to lipopolysaccharide-induced cell stress.

Cytokinins (CTKs) are a diverse collection of evolutionarily conserved adenine-derived signaling molecules classically studied as phytohormones; however, their roles and production have been less studied in mammalian systems. Skeletal muscles are sensitive to cellular cues such as inflammation and in response, alter their secretome to regulate the muscle stem cell and myofiber niche. Using cultured C2C12 muscle cells, we profiled CTK levels to understand (1) whether CTKs are part of the muscle secretome and (2) whether CTKs are responsive to cellular stress. To induce cellular stress, C2C12 myotubes were treated with lipopolysaccharides (LPS) for 24 h and then media and cell fractions were collected for ultra high-performance liquid chromatography tandem mass spectrometry with electrospray ionization (UHPLC-(ESI+)-HRMS/MS) for metabolomics and CTK profiling. Across LPS-treated and control cells, 11 CTKs were detected in the extracellular space while 6 were detected intracellularly. We found that muscle cells are enriched in isopentenyladenine (iP) species (from free base, riboside to nucleotide forms), and that extracellular levels are increased after LPS treatment. Our study establishes that muscle cells express various forms of CTKs, and that CTK levels are responsive to LPS-induced cell stress, suggesting a role for CTKs in intra- and extracellular signaling of mammalian cells.

Versatile strategy for biochemical, electrochemical and immunoarray detection of protein phosphorylations.

Protein kinases catalyze the phosphorylation of cellular proteins involved in the regulation of many cellular processes and have emerged as promising targets for the treatment of several diseases. Conventional assays to monitor protein kinase activity are limited because they typically rely on transfer of radioactive phosphate or phospho-specific antibodies that recognize specific substrates or sequence motifs. To overcome the limitations of conventional assays, we have developed a versatile approach based on transfer of ferrocene-phosphate that can be readily monitored using electrochemical detection or detection with antiferrocene antibodies in an immunoarray format. This assay is readily adapted to multiplex arrays and can be employed for monitoring kinase activity in complex mixtures and for kinase inhibitor profiling.

Hierarchical organization of ferrocene-peptides.

Hierarchical self-assembly of disubstituted ferrocene (Fc)-peptide conjugates that possess Gly-Val-Phe and Gly-Val-Phe-Phe peptide substituents leads to the formation of nano- and micro-sized assemblies. Hydrogen-bonding and hydrophobic interactions provide directionality to the assembly patterns. The self-assembling behavior of these compounds was studied in solution by using (1)H NMR and circular dichroism (CD) spectroscopies. In the solid state, attenuated total reflectance (ATR) FTIR spectroscopy, single-crystal X-ray diffraction (XRD), powder X-ray diffraction (PXRD), and scanning electron microscopy (SEM) methods were used. Spontaneous self-assembly of Fc-peptides through intra- and intermolecular hydrogen-bonding interactions induces supramolecular assemblies, which further associate and give rise to fibers, large fibrous crystals, and twisted ropes. In the case of Fc[CO-Gly-Val-Phe-OMe](2) (1), molecules initially interact to form pleated sheets that undergo association into long fibers that form bundles and rectangular crystalline cuboids. Molecular offsets and defects, such as screw dislocations and solvent effects that occur during crystal growth, induce the formation of helical arrangements, ultimately leading to large twisted ropes. By contrast, the Fc-tetrapeptide conjugate Fc[CO-Gly-Val-Phe-Phe-OMe](2) (2) forms a network of nanofibers at the supramolecular level, presumably due to the additional hydrogen-bonding and hydrophobic interactions that stem from the additional Phe residues.

Electrochemical screening of the indole/quinolone derivatives as potential protein kinase CK2 inhibitors.

An electrochemical method based on the bioorganometallic Fc-ATP cosubstrate for kinase-catalyzed phosphorylation reactions was used for monitoring casein kinase 2 (CK2) phosphorylations in the absence and presence of five indole/quinolone-based potential inhibitors. Fc-phosphorylation of immobilized peptide RRRDDDSDDD on Au surfaces resulted in a current density at approximately 460 ± 10 mV. An electrochemical redox signal was significantly decreased in the presence of inhibitors. In addition, the electrochemical signal was concentration dependent with respect to the potential inhibitors 1 to 5, which proved to be viable CK2 drug targets with estimated IC50 values in the nanomolar range.

Electrochemical "signal-on" reporter for amyloid aggregates.

The synthesis and characterization of four new Ferrocene (Fc) bioconjugates, bearing a podant (Lys)-Leu-Val-Phe-Phe motif, namely the hydrophobic sequence of amyloid-ß-peptides (Aß), is reported. The Fc-peptide conjugates are characterized by a reversible redox activity and the ability to undergo hydrophobic and hydrogen bonding interactions. Biomolecular interactions between Fc-bioconjugates with Aß(12-28) fragments were studied by circular dichroism (CD), transmission electron microscopy (TEM), and electrochemistry. All four Fc-peptides interacted favourable with Aß(12-28) and prevented fibril formation, the extent of which depended on the length of the peptide and the nature of the C-terminal group. The aggregates obtained for the Aß(12-28)/Fc-peptide mixtures range from short fibrils to spherical aggregates. We demonstrated that in solution the peptide sequence and peptide charge affect the biomolecular interactions. Fc-peptide interactions with immobilized Aß(12-28)-Cys films on Au surfaces were detected by measuring the current response of the Fc redox process. The formal redox potential, E(0), at ~440 (10) mV and i(pc)/i(pa) at 0.9 were observed characteristic for the monosubstituted Fc-derivative undergoing a one-electron redox process. On the surface, methyl ester-protected Fc-peptides (1 and 3) interacted only weakly with Aß(12-28)-Cys films, giving rise to minimal redox activity. In contrast, charged Fc-peptides (2 and 4) gave a significant electrochemical readout following the interaction with Aß(12-28)-Cys films. Interestingly, the Fc-peptide charge dictates the surface-assisted interactions, while hydrophobic and ionic effects contribute to the overall solution behaviour of the Fc-bioconjugates with Aß(12-28).

Electrochemical investigations into Tau protein phosphorylations.

Hyperphosphorylation of Tau, a protein that stabilizes microtubules, leads to the breakdown of the microtubular structure and ultimately to the formation of neurofibrillar tangles within neurons. Here, we report monitoring of Tau phosphorylations electrochemically, using Tau protein films chemically linked to gold surfaces and 5'-γ-ferrocenyl (Fc) adenosine triphosphate (Fc-ATP) as a co-substrate. Fc-phosphorylation reactions of Tau are explored using the three protein kinases, glycogen synthase kinase (GSK-3ß), sarcoma (Src)-related kinase, and protein kinase A (PKA), which catalyze Fc-phosphorylation of different residues and regions within Tau. The kinetic parameters of the biochemical process (K(M) and V(max)) were determined.

Electrochemical investigations of Tau protein phosphorylations and interactions with Pin1.

Phosphorylation of Tau by the protein kinase GSK-3ß was monitored by electrochemical impedance spectroscopy of immobilized Tau on gold surfaces. As a result of Tau phosphorylation, the film resistance decreases significantly due to conformational changes and reorganization of the immobilized phosphorylated Tau (pTau) protein, which in turn enables the interactions of pTau with the peptidyl-prolyl cis/trans isomerase, Pin1. Interactions are specific to phospho-Ser (pSer) and phospho-Thr (pThr) residues of pTau. Impedance changes occurred as a function of pTau-Pin1 interactions and are related to the amount of Pin1 bound, which resulted in an increase of the charge-transfer resistance, R(CT). Our results clearly indicate that the isomerase Pin1 interacts favorably with pSer/pThr-Pro residues in Tau, but does not bind non-phosphorylated Tau or phospho-Tyr residues in Tau films. Our study demonstrates the utility of electrochemical impedance studies to probe protein modifications and biomolecular interactions.

Selective detection of nitrotyrosine using dual-fluorescent carbon dots.

The post-translational modification of amino acid plays a critical role in normal and diseased biological states. Specifically, nitrotyrosine (nTyr) has been linked to diseases, including neurodegeneration, among others. Hence, alternative methods are required for detection and differentiation of nTyr from other structurally similar analogues, such as Tyrosine (Tyr) or phosphotyrosine (pTyr). Herein, the selective detection of nTyr, over other congeners, was achieved by using dual-fluorescent carbon dots (CDs) in buffered solution, artificial saliva, bovine serum albumin and diluted equine serum. The nTyr induced fluorescence quenching of the blue and red emissions of CDs, in the 20-105 µM linear range, and with the limit of detection (LOD) at 34 µM, which was well below the physiological concentration required for detection. The sensor was functional at biological pH values, with optimal quenching efficiency at basic pH. The sensor was highly selective for nTyr even in the presence of common biological interferences (metal cations, organic anions, amino acids, nucleosides and other biologicals). The mechanism of quenching (a combination of static and dynamic) was ascribed to the nonradiative energy transfer, due to electronic overlap between nTyr absorbance and CDs fluorescence emission, and electron transfer from excited CDs state to nTyr as an electron acceptor. The dual-fluorescent CDs represent viable sensors for key biological modifications, and their selectivity and sensitivity may be further improved through tailored chemical synthesis of CDs, such as tunable surface chemistry to promote selective recognition of analyte of interest.

Use of 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates having poly(ethylene glycol) spacers in kinase-catalyzed phosphorylations.

The 5'-γ-ferrocenyl adenosine triphosphate (Fc-ATP) bioconjugates (3 and 4), containing the poly(ethylene glycol) spacers, were synthesized and compared to a hydrophobic analogue as co-substrates for the following protein kinases: sarcoma related kinase (Src), cyclin-dependent kinase (CDK), casein kinase II (CK2α), and protein kinase A (PKA). Electrochemical kinase assays indicate that the hydrophobic Fc-ATP analogue was an optimal co-substrate for which K(M) values were determined to be in the 30-200 µM range, depending on the particular protein kinase. The luminescence kinase assay demonstrated the kinase utility for all Fc-ATP conjugates, which is in line with the electrochemical data. Moreover, Fc-ATP bioconjugates exhibit competitive behavior with respect to ATP. Relatively poor performance of the polar Fc-ATP bioconjugates as co-substrates for protein kinases was presumably due to the additional H-bonding and electrostatic interactions of the poly(ethylene glycol) linkers of Fc-ATP with the kinase catalytic site and the target peptides. Phosphorylation of the full-length protein, His-tagged pro-caspase-3, was demonstrated through Fc-phosphoamide transfer to the Ser residues of the surface-bound protein by electrochemical means. These results suggest that electrochemical detection of the peptide and protein Fc-phosphorylation via tailored Fc-ATP co-substrates may be useful for probing protein-protein interactions.

Probing the role of the linker in ferrocene-ATP conjugates: monitoring protein kinase catalyzed phosphorylations electrochemically.

The synthesis and electrochemical properties of ferrocene conjugates are presented for the purpose of investigating adenosine 5'-[γ-ferrocenoylalkyl] triphosphate (1 a-4 a, ferrocene (Fc)-ATP) as co-substrates for phosphorylation reactions. Compounds 1 a-4 a were synthesized, purified by HPLC, and characterized by NMR spectroscopy and mass spectrometry. In solution, all Fc-ATP bioconjugates exhibit a reversible one-electron redox process with a half-wave potential (E(1/2)) in the 390-430 mV range, peak separations (ΔE(p)) in the 40-70 mV range, and the peak current ratio (i(pa)/i(pc)) near unity. The peptide-modified surface Glu-Gly-Ile-Tyr-Asp-Val-Pro was used to study the sarcoma-related protein (Src) kinase activity by employing the Fc-ATP bioconjugates as co-substrates. Subsequent kinase-catalyzed transfer of the γ-Fc-phosphate group to the tyrosine residues of the surface-bound peptides was characterized by a formal potential (E°) ≈390 mV (vs. Ag/AgCl). The Fc-coverage, estimated by time-of-flight secondary-ion mass spectrometry (TOF-SIMS) and cyclic voltammetry (CV), suggested validity of Fc-ATP conjugates as kinase co-substrates. Depending on the length of the alkyl spacer of the Fc-ATP conjugate, different current densities were obtained, pointing to a direct correlation between the two. Molecular modeling revealed that the structural constraint imposed by the short alkyl spacer (1 a) causes a steric congestion and negatively affects the outcome of phosphorylation reaction. An optimal analytical response was obtained with the Fc-ATP conjugates with linker lengths longer than six CH(2) groups.