Oligomerization and aggregation of NAP-22 with several metal ions.

Metal ions participate in various biochemical processes such as electron transport chain, gene transcription, and enzymatic reactions. Furthermore, the aggregation promoting effect of several metal ions on neuronal proteins such as prion, tau, Aß peptide, and α-synuclein, has been reported. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the detergent-resistant membrane microdomain fraction of the neuronal cell membrane. Previously, we showed oligomer formation of NAP-22 in the presence of several phospholipids and fatty acids. In this study, we found the aggregation of NAP-22 by FeCl2, FeCl3, and AlCl3 using native-PAGE. Oligomer or aggregate formation of NAP-22 by ZnCl2 or CuSO4 was shown with SDS-PAGE after cross-linking with glutaraldehyde. Morphological analysis with electron microscopy revealed the formation of large aggregates composed of small annular oligomers in the presence of FeCl3, AlCl3, or CuSO4. In case of FeCl2 or ZnCl2, instead of large aggregates, scattered annular and globular oligomers were observed. Interestingly, metal ion induced aggregation of NAP-22 was inhibited by several coenzymes such as NADP+, NADPH, or thiamine pyrophosphate. Since NAP-22 is highly expressed in the presynaptic region of the synapse, this result suggests the participation of metal ions not only on the protein and membrane dynamics at the presynaptic region, but also on the metabolic regulation though the interaction with coenzymes.

An Ultra-low Detection Limit Fe3+ Optical Fiber Fluorescent Sensor Based on a Anti-B18H22 Derivative with Aggregation-induced Emission Enhancement.

A fluorescent Fe3+ probe ((C10H7NO2)2B18H20, M1) by introducing two isoquinoline-1-carboxylic acid group into the 6,9-position of anti-B18H22 was designed and synthesized. The structure of M1 was investigated by 1H NMR, MS, FT-IR and theoretical calculation, and its optical properties were characterized with UV-Vis and PL. M1 showed aggregation induced emission enhancement (AIEE) properties in THF/H2O solution, and exhibited an excellent selectivity toward Fe3+ in THF/H2O (v/v, ƒw = 95%) solution with a detection limit of 1.93 × 10-5 M. The interaction mechanism of probe for detecting Fe3+ is attributed to the involvement of intramolecular charge transfer (ICT) process. Furthermore, a optical fiber fluorescent Fe3+ sensor based on M1 sensing film was developed, the detection limit of the optical fiber Fe3+ fluorescent sensor could be improved to13.8 pM, the ultra-low detection limit is superior to most reported fluorescent probes (or sensors) towards Fe3+. This method has the advantages of high sensitivity, anti-interference and easy to operate, and has great potential in the field of the analysis of environmental and biological samples.

Photoactivation of Solid-State Fluorescence through Controllable Intermolecular [2+2] Photodimerization.

Intermolecular [2+2] photodimerization provides a distinctive approach to construct photoresponsive fluorescent materials in a manner of switching on solid-state fluorescence. Herein, we report efficient photoactivation of bright solid-state fluorescence based on controllable intermolecular [2+2] photodimerization reaction of benzo[b]thiophene 1,1-dioxide (BTO) derivatives, which provides a simple and effective way to construct smart photoresponsive solid-state fluorescent materials. Rational choice of substituents in BTO molecular skeleton enables them to efficiently undergo photodimerization through regulating molecular stacking in crystal, and also leads to photoactivation of solid-state fluorescence due to the generation of brightly fluorescent photodimers. This intermolecular photodimerization reaction also offers an effective method to synthesize photostable AIEgens with purely through-space conjugation.

Design, synthesis, and evaluation of 2,2'-bipyridyl derivatives as bifunctional agents against Alzheimer's disease.

Alzheimer's disease (AD) is a complex multifactorial neurodegenerative disease. Metal ion dyshomeostasis and Aß aggregation have been proposed to contribute to AD progression. Metal ions can bind to Aß and promote Aß aggregation, and ultimately lead to neuronal death. Bifunctional (metal chelation and Aß interaction) compounds are showing promise against AD. In this work, eleven new 3,3'-diamino-2,2'-bipyridine derivatives 4a-4k were synthesized, and evaluated as bifunctional agents for AD treatment. In vitro Aß aggregation inhibition assay confirmed that most of the synthesized compounds exhibited significant self-induced Aß1-42 aggregation inhibition. Among them, compound 4d displayed the best inhibitory potency of self-induced Aß1-42 aggregation with IC50 value of 9.4 µM, and it could selectively chelate with Cu2+ and exhibited 66.2% inhibition of Cu2+-induced Aß1-42 aggregation. Meanwhile, compound 4d showed strong neuroprotective activity against Aß1-42 and Cu2+-treated Aß1-42 induced cell damage. Moreover, compound 4d in high dose significantly reversed Aß-induced memory impairment in mice.

Photoinduced Fluorescence Switching in Molecular Aggregates by Topological [2+2] Cycloaddition.

The optical modulation of fluorescence characteristics in molecular aggregates, which mainly involves diverse molecular stacking and the consequent intermolecular interactions, remains a significant challenge for potential applications in optical anticounterfeiting, data storage, and imaging. Here we successfully realize in situ fluorescence switching in molecular aggregates of thianaphthene-dioxide derivatives by topological [2+2] photocycloaddition, which represents a promising way to regulate the molecular stacking and alter photophysical processes. Notably, 2-(3,5-bis-trifluoromethylphenyl)benzo[b]thiophene-dioxide (BTO-TF) in both crystal and powder forms exhibits a unique switching from an initial nonfluorescent state to a highly fluorescent state (ΦPL =0.46) upon UV irradiation, because of the destruction of the [2+2] cycloaddition process by volume expansion of the photodimer. Furthermore, we demonstrate such a [2+2] photocycloaddition can occur when 2-(4-carboxypheny)benzo[b]thiophene-dioxide (BTO-OH) is doped within selective polymer matrixes, and can be utilized for the visualization of macrophase separation in polymer blends.

A novel AIEE active anti-B18H22derivative-based Cu2+and Fe3+fluorescence off-on-off sensor.

A novel fluorescence sensor for successive detection of Cu2+and Fe3+based on anti-B18H22derivative which possesses 5-hydroxyisoquinoline as an ionophore was synthesized via a one-pot and its structure and photophysical properties were characterized by NMR, HRMS, FTIR, UV-vis, PL and theoretical calculation. The fluorophore displays two emission peaks at 460 nm and 670 nm in THF solution coming from the emission of the locally excited state and intramolecular charge transfer fluorescence, respectively. The complex exhibited obvious aggregation-induced emission enhancement (AIEE) characteristics in THF/H2O solution by increasing the aqueous concentration from 70% to 95%. The AIEE molecules showed a high selectivity towards Cu2+over other metal ions by forming a 2:1 metal-to-ligand complex in THF/H2O (fw = 20%) solution, the fluorescence intensity increased as a linear function of the Cu2+concentration at 460 nm due to the inhibition of PET effect. The fluorescent emission was quenched linearly by the addition of Fe3+, which provides a method for successive determination of Cu2+and Fe3+based on 'off-on-off' fluorescence of the fluorescent. The detection limit of Cu2+and Fe3+was 5.7 × 10-6M and 7.2 × 10-5M respectively. Morever, a rapid identification of Cu2+in the aqueous solution by naked eyes can be realized. In addition, the molecules were pH-sensitive, the fluorescence quenching can be observed in strongly alkaline environment. The method has been applied to the determination of copper ions in water samples with satisfactory results.

Pseudopeptide Amyloid Aggregation Inhibitors: In Silico, Single Molecule and Cell Viability Studies.

Neurodegeneration in Alzheimer's disease (AD) is defined by pathology featuring amyloid-ß (Aß) deposition in the brain. Aß monomers themselves are generally considered to be nontoxic, but misfold into ß-sheets and aggregate to form neurotoxic oligomers. One suggested strategy to treat AD is to prevent the formation of toxic oligomers. The SG inhibitors are a class of pseudopeptides designed and optimized using molecular dynamics (MD) simulations for affinity to Aß and experimentally validated for their ability to inhibit amyloid-amyloid binding using single molecule force spectroscopy (SMFS). In this work, we provide a review of our previous MD and SMFS studies of these inhibitors and present new cell viability studies that demonstrate their neuroprotective effects against Aß(1-42) oligomers using mouse hippocampal-derived HT22 cells. Two of the tested SG inhibitors, predicted to bind Aß in anti-parallel orientation, demonstrated neuroprotection against Aß(1-42). A third inhibitor, predicted to bind parallel to Aß, was not neuroprotective. Myristoylation of SG inhibitors, intended to enhance delivery across the blood-brain barrier (BBB), resulted in cytotoxicity. This is the first use of HT22 cells for the study of peptide aggregation inhibitors. Overall, this work will inform the future development of peptide aggregation inhibitors against Aß toxicity.

1,2,4-trihydroxynaphthalene-2-O-ß-D-glucopyranoside: A new powerful antioxidant and inhibitor of Aß42 aggregation isolated from the leaves of Lawsonia inermis.

Mounting evidence indicates free radicals as toxic species causing damage to human cells leading to the pathogenesis of many diseases such as neurodegenerative disease. Plant derived antioxidants are considered as promising strategy to prevent free radical toxicity. In this study, the crude extract (CE), 50%MeOH, Petroleum Ether (PE) and Ethyl acetate (EA) fractions of Lawsonia inermis leaves were investigated for their antioxidant activity and their ability to counteract amyloid-ß42 (Aß42) aggregation. Elution of the most bioactive fraction (EA) on silica gel column chromatography led to six sub-fractions. The most active sub-fraction (1) was further resolved on silica gel column chromatography. A new compound with powerful antioxidant and anti-Aß42 aggregation properties was purified and characterised by spectroscopic methods as 1,2,4-trihydroxynaphthalene-2-O-ß-D-glucopyranoside (THNG). This finding suggests that the antioxidant and anti-Aß42 aggregation activities of L. inermis leaves are strongly correlated to this compound.

Molecular Dynamics Simulations on the Bioactive Molecule of hIAPP22-29 (NFGAILSS) and Rational Drug Design.

This chapter includes information about the structure in equilibrium of the bioactive molecule hIAPP22-29 (NFGAILSS). The experimental structure was derived using X-ray and its 2D NOESY NMR experiments in d 6-DMSO and d-HFIP solvents. This molecule contains eight of the ten amino acids of the 20-29 region of the human islet amyloid polypeptide (hIAPP) often referred as the "amyloidogenic core." Amyloid deposits are well-known to cause as many as 20 pathological neurodegenerative disorders such as Alzheimer, Parkinson, Huntington, and Creutzfeldt-Jakob. The experimental structure was relaxed using molecular dynamics (MD) in simulation boxes consisting in DMSO and HFIP; the latter not provided by the applied software. The calculations were performed in GPUs and supercomputers, and some basic scripting is described for reference. The simulations confirmed the inter- and intramolecular forces that led to an "amyloidogenic core" observed from NOE experiments. The results showed that in DMSO and HFIP environment, Phe is not in spatial proximity with Leu or Ile, and this is consistent with an amyloidogenic core. However, in an amphipathic environment such as the model lipid bilayers, this communication is possible and may influence peptide amyloidogenic properties. The knowledge gained through this study may contribute to the rational drug design of novel peptides or organic molecules acting by modifying preventing amyloidogenic properties of the hIAPP peptide.

Similar interaction chromatography of proteins: A cross interaction chromatographic approach to estimate the osmotic second virial coefficient.

Self-interaction chromatography (SIC) has established itself as an important experimental technique for the measurement of the second osmotic virial coefficients B22. B22 data are critical for understanding a range of protein solution phenomena, particularly aggregation and crystallisation. A key limitation to the more extensive use of SIC is the need to develop a method for immobilising each specific protein of interest onto a chromatographic support. This requirement is both a time and protein consuming constraint, which means that SIC cannot be used as a high throughput method for screening a wide range of proteins and their variants. Here an experimental framework is presented for estimating B22 values using Similar Interaction Chromatography (SimIC). This work uses experimental B23 and B32 data for lysozyme, lactoferrin, catalase and concanavalin A to reliably estimate B22 using arithmetic mean field approximations and is demonstrated to give good agreement with SIC measurements of B22 for the same proteins. SimIC could form the basis of a rapid protein variant screening methods to assess the developability of protein therapeutic candidates for industrial and academic researchers with respect to aggregation behaviour by eluting target proteins through a series of well-characterised protein immobilized reference columns.

Aggregation of Polyglutamine-expanded Ataxin 7 Protein Specifically Sequesters Ubiquitin-specific Protease 22 and Deteriorates Its Deubiquitinating Function in the Spt-Ada-Gcn5-Acetyltransferase (SAGA) Complex.

Human ataxin 7 (Atx7) is a component of the deubiquitination module (DUBm) in the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex for transcriptional regulation, and expansion of its polyglutamine (polyQ) tract leads to spinocerebellar ataxia type 7. However, how polyQ expansion of Atx7 affects DUBm function remains elusive. We investigated the effects of polyQ-expanded Atx7 on ubiquitin-specific protease (USP22), an interacting partner of Atx7 functioning in deubiquitination of histone H2B. The results showed that the inclusions or aggregates formed by polyQ-expanded Atx7 specifically sequester USP22 through their interactions mediated by the N-terminal zinc finger domain of Atx7. The mutation of the zinc finger domain in Atx7 that disrupts its interaction with USP22 dramatically abolishes sequestration of USP22. Moreover, polyQ expansion of Atx7 decreases the deubiquitinating activity of USP22 and, consequently, increases the level of monoubiquitinated H2B. Therefore, we propose that polyQ-expanded Atx7 forms insoluble aggregates that sequester USP22 into a catalytically inactive state, and then the impaired DUBm loses the function to deubiquitinate monoubiquitinated histone H2B or H2A. This may result in dysfunction of the SAGA complex and transcriptional dysregulation in spinocerebellar ataxia type 7 disease.

Mutation in transforming growth factor beta induced protein associated with granular corneal dystrophy type 1 reduces the proteolytic susceptibility through local structural stabilization.

Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/ßig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3' containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions.