Artificial Molecular Chaperone Research Articles

Bifunctional cysteine gold nanoclusters for β-amyloid fibril inhibition and fluorescence imaging: a distinctive approach to manage Alzheimer's disease.

Alzheimer's disease (AD) is a progressive complex neurodegenerative disorder affecting millions of individuals worldwide. Currently, there is no effective treatment for AD. AD is characterized by the deposition of amyloid plaques/fibrils. One major strategy for managing this disease is by slowing the progression of AD using different drugs which could potentially limit free-radical formation, oxidative stress and lipid peroxidation and promote the survival of neurons exposed to β-amyloid. Inhibition of amyloid fibrillization and clearance of amyloid plaques/fibrils are essential for the prevention and treatment of AD. The thiophilic interaction between the side chain of an aromatic residue in a polypeptide and a sulphur atom of the compound can effectively inhibit amyloid fibril formation. In this work, we have synthesized cysteine-capped gold nanoclusters (Cy-AuNCs) which exhibit inherent red emission and can disintegrate amyloid fibrils through the aforementioned thiophilic interactions. Herein, we also used molecular docking to study the thiophilic interactions between the sulphur atom of Cy-AuNCs and the aromatic rings of the protein. Finally, the gold cluster was functionalized with a brain targeting molecule, Levodopa (AuCs-LD), to specifically target the brain and to facilitate passage through the blood brain barrier (BBB). Both Cy-AuNCs and AuCs-LD showed good biocompatibility and the inherent fluorescence properties of nanoclusters enabled real time imaging. The efficacy of the nanoclusters to disintegrate amyloid fibrils and their ability to cross the BBB were demonstrated both in vitro and in vivo in the BBB model and the AD animal model respectively. Our results imply that nanoparticle-based artificial molecular chaperones may offer a promising therapeutic approach for AD.

Active Regulation of Supramolecular Chirality through Integration of CdSe/CdS Nanorods for Strong and Tunable Circular Polarized Luminescence.

Building the cooperativity in artificial self-assembling systems will synergistically reshape their properties and expand their application spectrum. Here, we show how the cooperativity between achiral CdSe/CdS nanorods (NRs) and chiral perylene diimide (PDI)-based molecules is built upon their coassembly. We demonstrate that chirality transfer from chiral molecular assemblies to CdSe/CdS NRs is enabled by the encapsulation of NRs into PDI suprascrolls through chain-chain van der Waals interactions, which in turn gives rise to markedly enhanced circularly polarized luminescence of the nanocomposites. Additionally, the circularly polarized emissive bands of the nanocomposites could be finely tuned by engineering the emissive bands of NRs. More importantly, these nanocomposites are able to invert their chirality when NRs are self-assembled into chiral superlattices under a larger amount of NRs. Detailed mechanistic studies unveil that these jammed NRs assemblies hamper the folding of two-dimensional (2D) chiral nanosheets, which consequently suppress the interlayer excitonic coupling and implement the nanocomposites with inverted chirality. Our finding exemplifies a way to invert the chirality switching from folding to unfolding of 2D supramolecular nanosheets, akin to the chirality inversion in the helix-to-superhelix transition in conventional one-dimensional (1D) supramolecular systems. We also show that the strong van der Waals interactions from aliphatic chains are crucial in achieving such chirality transfer and inversion. Overall, we demonstrate that these achiral CdS/CdSe NRs could serve as artificial molecular chaperones to aid the unfolding of supramolecular nanosheets with controlled chirality.

Artificial Molecular Chaperone Systems for Proteins, Nucleic Acids, and Synthetic Molecules.

Molecular chaperones play critical roles in biological functions. They are closely involved in the maintenance of cell homeostasis, proper folding of proteins and nucleic acids, and inhibition of irreversible aggregation in denatured proteins. In addition to protein production, molecular chaperone function is widely recognized as important for peptide and protein drug delivery systems. Therefore, much effort has been made in recent decades to develop chaperone-mimetic molecules that have similar structures and biological functions to natural chaperones. These artificial molecular chaperone systems have been demonstrated to facilitate proper protein and nucleic acid folding, in addition to the formation of higher-order structures of synthetic molecules. Furthermore, the functions of these artificial systems show promising clinical applications in drug delivery and biomolecule detection. This topical review focuses on recent advances in the design, construction, characterization, and potential applications of different artificial molecular systems with distinct functional roles, such as the folding of water-soluble and membrane proteins, nucleic acids, and the self-assembly of synthetic molecules. Strategies used in the construction of some artificial molecule chaperone systems for proteins (such as pairs of amphiphilic molecules or self-assembled nanogels) and their applications as biomaterials are described. Specific examples from each design strategy are also highlighted to demonstrate the mechanisms, challenges, and limitations of the different artificial molecular systems. By highlighting the many new developments that have expanded the applications of the artificial chaperones beyond protein folding, this review aims to stimulate further studies on their design and applications.

β-Cyclodextrin-Modified Magnetic Nanoparticles Immobilized on Sepharose Surface Provide an Effective Matrix for Protein Refolding.

In this article, we propose an impressive and facile strategy to improve protein refolding using solid phase artificial molecular chaperones consisting of the surface-functionalized magnetic nanoparticles. Specifically, monotosyl-β-cyclodextrin connected to the surface of 3-aminopropyltriethoxysilane (APES)-modified magnetic nanoparticles is immobilized on the sepharose surface to promote interaction with exposed hydrophobic surfaces of partially folded (intermediates) and unfolded states of proteins. Their efficiencies were investigated by circular dichroism spectroscopy and photoluminescence spectroscopy of the protein. Although the mechanism of this method is based on principles of hydrophobic chromatography, this system is not only purging the native protein from inactive inclusion bodies but also improving the protein refolding process. We chose β-cyclodextrin (β-CD) considering multiple reports in the literature about its efficiency in protein refolding and its biocompatibility. To increase the surface area/volume ratio of the sepharose surface by nanoparticles, more β-CD molecules are connected to the sepharose surface to make a better interaction with proteins. We suppose that proteins are isolated in the nanospace created by bound cyclodextrins on the resin surface so intermolecular interactions are reduced. The architecture of nanoparticles was characterized by Fourier transform infrared spectra, X-ray diffraction, scanning electron microscopy images, energy dispersive X-ray spectroscopy, nuclear magnetic resonance (1H NMR and 13C NMR), and dynamic light scattering.

Inhibition of Amyloid Fibril Growth and Dissolution of Amyloid Fibrils by Curcumin–Gold Nanoparticles

Inhibition of amyloid fibrillation and clearance of amyloid fibrils/plaques are essential for the prevention and treatment of various neurodegenerative disorders involving protein aggregation. Herein, we report curcumin-functionalized gold nanoparticles (Au-curcumin) of hydrodynamic diameter 10-25 nm, which serve to inhibit amyloid fibrillation and disintegrate/dissolve amyloid fibrils. In nanoparticle form, curcumin is water-soluble and can efficiently interact with amyloid protein/peptide, offering enhanced performance in inhibiting amyloid fibrillation and dissolving amyloid fibrils. Our results imply that nanoparticle-based artificial molecular chaperones may offer a promising therapeutic approach to combat neurodegenerative disease.

Amphiphilic nanogel of enzymatically synthesized glycogen as an artificial molecular chaperone for effective protein refolding

Enzymatically synthesized glycogen (ESG) bearing hydrophobic groups were prepared to construct a new artificial molecular chaperone system. The radius of the amphiphilic ESG nanogel was approximately 15 nm. The nanogels prevented irreversible aggregation of carbonic anhydrase. Cyclodextrin induced the release of carbonic anhydrase in its active form via a one-step mechanism without dissociation of the nanogels.

Artificial Chaperone Polysaccharide Nanogels for Protein Delivery: A Thermodynamic Study of Protein-Nanogel Interactions Using Fluorescence Correlation Spectroscopy

Molecular chaperones selectively trap heat-denatured proteins or their intermediates, primarily by hydrophobic interactions, to prevent irreversible aggregation resulting from macromolecular host (molecular chaperone)-guest (protein) interactions. The molecular chaperone function is an important concept that is expected to lead to breakthroughs in drug delivery systems, especially for protein or peptide delivery in regenerative medicine, such as bone regeneration. We have reported that polysaccharide nanogels act as artificial molecular chaperones. To further clarify the molecular chaperone function of nanogels as protein carriers, the elucidation of nanogel-protein interactions are especially important. Here, we investigated the interaction of a protein with a polysaccharide nanogel using fluorescence correlation spectroscopy at variable temperatures, using fluorescence-labeled bovine serum albumin (BSA) as a model protein. In particular, thermodynamic parameters of the heat-induced complexation of protein with CHP nanogels were evaluated using the van't Hoff plot. The plot shows that the CHP nanogels strongly complexed with heat-denatured BSA. The increased hydrophobicity of the denatured, unfolded protein may prefer complexation with amphiphilic hydrogel nanoparticles over complexation with the completely folded native protein. Thermodynamic parameters suggest that the complexation is entropically driven, rather than enthalpically, under the conditions studied.

Cyclodextrin-responsive nanogel as an artificial chaperone for horseradish peroxidase

The thermal stabilization and refolding of horseradish peroxidase (HRP) upon heating were investigated using an artificial molecular chaperone consisting of cholesterol-bearing pullulan (CHP) nanogels. The CHP nanogels inhibited the aggregation of HRP under heating by complexation with the denatured HRP. The enzyme activity of HRP complexed with CHP nanogels was not detected. However, the enzyme activity recovered up to 80% of native HRP after the addition of cyclodextrin (CD) to the complex. The dissociation of CHP nanogels was induced by the formation of an inclusion complex of cholesterol groups of CHP with CD. The enzyme activity of HRP was only significantly recovered by the addition of β-CD or its derivatives. Natural molecular chaperones, such as GroEL/ES, trap, fold, and release the nonnative proteins by changing the hydrophobicity of the specific sites of the molecular chaperone that interact with the nonnative protein. The functional mechanism of the nanogel chaperon system is similar to that of natural molecular chaperones. The nanogel chaperone system is a useful tool to aid the refolding and thermal stabilization of unstable proteins for post-genome research, and in medical and biological applications.

Refolding of denatured/reduced lysozyme at high concentrations by artificial molecular chaperone‐ion exchange chromatography

Development of high efficiency and low cost protein refolding methods is a highlighted research focus in biotechnology. Artificial molecular chaperone (AMC) and protein folding liquid chromatography (PFLC) are two attractive refolding methods developed in recent years. In the present work, AMC and one branch of PFLC, ion exchange chromatography (IEC), are integrated to form a new refolding method, artificial molecular chaperone-ion exchange chromatography (AMC-IEC). This new method is applied to the refolding of a widely used model protein, urea-denatured/dithiothreitol-reduced lysozyme. Many factors influencing the refolding of lysozyme, such as urea concentration, beta-cyclodextrin concentration, molar ratio of detergent to protein, mobile phase flow rate, and type of detergent, were investigated, respectively, to optimize the conditions for lysozyme refolding by AMC-IEC. Compared with normal IEC refolding method, the activity recoveries of lysozyme obtained by AMC-IEC were much higher in the investigated range of initial protein concentrations. Moreover, the activity recoveries obtained by using this newly developed refolding method were still quite high for denatured/reduced lysozyme at high initial concentrations. When the initial protein concentration was 200 mg mL(-1), the activity recovery was over 60%. In addition, the lifetime of the chromatographic column during AMC-IEC was much longer than that during protein refolding by normal IEC. Therefore, AMC-IEC is a high efficient and low cost protein refolding method.

A novel protein refolding method integrating ion exchange chromatography with artificial molecular chaperone

Artificial molecular chaperone (AMC) and ion exchange chromatography (IEC) were integrated, thus a new refolding method, artificial molecular chaperone–ion exchange chromatography (AMC–IEC) was developed. Compared with AMC and IEC, the activity recovery of lysozyme obtained by AMC–IEC was much higher in the investigated range of initial protein concentrations, and the results show that AMC–IEC is very efficient for protein refolding at high concentrations. When the initial concentration of lysozyme is 180 mg/mL, its activity recovery obtained by AMC–IEC is still as high as 76.6%, while the activity recoveries obtained by AMC and IEC are 45.6% and 42.4%, respectively.

Thermoresponsive Controlled Association of Protein with a Dynamic Nanogel of Hydrophobized Polysaccharide and Cyclodextrin: Heat Shock Protein-Like Activity of Artificial Molecular Chaperone

Dynamic CHP-CD nanogels, which consisted of a self-assembly of cholesteryl-group-bearing pullulan (CHP) and beta-cyclodextrin (CD), were characterized by SEC and SEC-MALS methods. The nanogels prevented the thermal aggregation of carbonic anhydrase B (CAB) by selective trapping of the heat-denatured protein. After the complex between the CHP-CD nanogels and CAB was cooled, the enzyme activity of CAB spontaneously recovered upon release from the complex. The dynamic nanogels self-regulated an association of heat denatured protein and dissociation of native protein depending on the concentration of CD. The thermal stability of CAB was improved by thermoresponsive controlled association between the proteins and the artificial molecular chaperone.

Photoresponsive nanogels formed by the self-assembly of spiropyrane-bearing pullulan that act as artificial molecular chaperones.

Novel photoresponsive nanogels were prepared by the self-assembly of spiropyrane-bearing pullulan (SpP). The solution properties of the nanogels could be controlled by photostimulation via isomerization between hydrophobic spiropyrane and hydrophilic merocyanine. The molecular chaperone-like activity of the nanogels in protein refolding was investigated. The activity of citrate synthase significantly increased when the amphiphilicity of SpP nanogels was switched by photostimulation.

Protein refolding assisted by self-assembled nanogels as novel artificial molecular chaperone

Molecular chaperone-like activity for protein refolding was investigated using nanogels of self-assembly of cholesterol-bearing pullulan. Nanogels effectively prevented protein aggregation (i.e. carbonic anhydrase and citrate synthase) during protein refolding from GdmCl denaturation. Enzyme activity recovered in high yields upon dissociation of the gel structure in which the proteins were trapped, by the addition of cyclodextrins. The nanogels assisted protein refolding in a manner similar to the mechanism of molecular chaperones, namely by catching and releasing proteins. The nanogels acted as a host for the trapping of refolded intermediate proteins. Cyclodextrin is an effector molecule that controls the binding ability of these host nanogels to proteins. The present nanogel system was also effective at the renaturation of inclusion body of a recombinant protein of the serine protease family.