The conformation of silk fibroin (SF) frozen with polyethylene glycol (PEG) at a molecular weight from 2 kDa to 20 kDa and a mass ratio of PEG:SF from 1:5 to 10:1 was studied by spectral and microscopic methods. It is found that the conformation transition of SF from random coil to β-sheet could be induced by the stress resulting from PEG crystallization at −20 °C, and greatly depended on the cooling rate, PEG:SF mass ratio and PEG molecular weight. These findings provide a new method for the preparation of desired SF nanofibers.

A novel protein tyrosine phosphatase 1B (PTP1B) inhibitor, FYGL, extracted from Ganoderma lucidum, was first reported to have an efficient hypoglycemic effect and high safety in vivo in our previous study. However, the underlying mechanism of the anti-diabetes activity was still unclear. The dominant effective component of FYGL was demonstrated to be a neutral hyperbranched proteoglycan macromolecule. Therefore, it is necessary to first elucidate how the biomacromolecule works at the cellular level, in order to understand its possible functional mechanism in vivo. Herein, we focused on demonstrating the cell internalization and endocytosis mechanism of FYGL in HepG2 cells. A series of cellular uptake pathways was explored, including clathrin-mediated endocytosis, caveolae/lipid-mediated endocytosis, and macropinocytosis. Confocal laser scanning microscopy and flow cytometry were used to demonstrate the absorption of FYGL by HepG2 cells. Furthermore, pharmacologically selective inhibitors and Western blot methods were used to demonstrate that macropinocytosis mediated by c-Src/PI3K cascades was the preferred route for the uptake of FYGL in cells. Our study provided the basis of uptake of FYGL for an efficient hypoglycemic effect. The functional mechanism of the signaling pathway in vivo will be reported in the future.

α-Synuclein (α-Syn) aggregates are the major component of Lewy bodies (LB), which is a pathological hallmark in the brain tissue of Parkinson's disease (PD) patients. It has been reported that (−)-epigallocatechin-3-gallate (EGCG) is biologically able to penetrate the blood–brain barrier and inhibit the fibrillation of amyloid proteins. This study aimed to provide insight into the possible mechanism of EGCG as a potential candidate agent for the prevention and treatment of PD on the basis of the interaction between α-Syn and EGCG. In the present study, the effects of EGCG on the fibrillation and disaggregation of α-Syn were investigated by thioflavin T (ThT) fluorescence spectroscopy, circular dichroism spectroscopy (CD), nuclear magnetic resonance (NMR) spectroscopy, atomic force microscopy (AFM) and transmission electron microscopy (TEM) on a molecular level. In addition, on the cellular level, we investigated the protective effects of EGCG on α-Syn-induced cell death in the transduced PC12 cells which overexpressed α-Syn, using the techniques of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) assay, western blot and confocal laser scanning microscopy. It was found that EGCG not only significantly inhibited the conformational transition of α-Syn from random coil to β-sheet conformers through binding to Ile, Phe and Tyr amino residues, but also disaggregated the amyloid fibrils of α-Syn in a dose-dependent manner, through binding to Leu, His, Phe and Tyr amino residues. It is also demonstrated that EGCG can protect PC12 cells against α-Syn-induced damage by inhibiting the overexpression and fibrillation of α-Syn in the cells.

In order to overcome the drawbacks of silk fibroin (SF)-based materials, SF has been blended with some polymers. Before using the blend material, understanding of the structures and phase behaviors of the blend is thought to be essential. In this study, solid-state 13C CP-MAS NMR and Raman imaging techniques were used to study the structures and phase behaviors of blends of SF with polyethylene glycol (PEG) at a molecular weight that varied from 2 to 20 kDa and a blend ratio of SF/PEG from 95/5 to 70/30 (w/w%) at the molecular and microscopic levels. It is found that the conformational transition of SF to the β-sheet increased as the PEG content increased, while the amount of the formed β-sheet conformers was decreased as the PEG molecular weight increased for a given content. It is also observed that SF was incompatible with PEG to some extent. The phase separation into “sea” and “island” domains took place in the SF/PEG blend films. SF was dominantly present in the “sea” domain, while PEG in the “island” domains. The conformation of SF in the interface between SF and PEG was changed to the β-sheet, while that in the protein-rich domain remained in the random coil and/or helix conformation. These observations suggest that the specifically expected materials, for example, the silk-based microspheres or scaffold materials can be manufactured by controlling the molecular weight and content of PEG in the blend system.

Abnormal denaturation and aggregation of human amylin or islet amyloid polypeptide (IAPP) into amyloid fibrils has been implicated in the pathogenesis of type 2 diabetic mellitus. Trehalose, a super-hydrophilic molecule, has been shown to prevent denaturation of biomolecules when they are under environmental stress. In this work, we sought to investigate the effects of trehalose on the fibrillation and aggregation of human IAPP (hIAPP) by using circular dichroism spectrum, thioflavin-T fluorescence spectrum, dynamic light scattering, transmission electronic microscopy, atomic force microscopy and quartz crystal microbalance. We demonstrated that (1) the conformation of hIAPP changed from α-helix to β-sheet, followed by fibrillation and aggregation, (2) a low dose of trehalose (under 100 mM) inhibited or delayed the conformation transition of hIAPP and (3) a high dose (more than 500 mM) induced the conformation transition, and promoted the fibrillation and aggregation of hIAPP. These findings are in agreement with the hypothesis of the water replacement and volume exclusion effect on the proteins. The lower concentration of trehalose could replace the water molecules surrounding the hIAPP, and interact with proteins through hydrogen bonding, leading to a reduction in the protein interaction itself, and therefore inhibiting or delaying the protein fibrillation and aggregation. In contrast, the higher concentration of trehalose might interact with itself to form macromolecular clusters, acting as a crowding agent, leading to the hIAPP molecules being excluded by the trehalose clusters and interacting between each other, and therefore promoting the hIAPP fibrillation and aggregation.

•A noval proteoglycan, named FYGL-n, was extracted from G. lucidum.•FYGL-n was an efficient PTP1B inhibitor with IC50 7.8 ± 0.2 μg/mL.•The polysaccharide moiety of FYGL-n was composed of Ara, Gal, Rha and Glc.•The molecular weight of the FYGL-n was 72.9 kDa.•The complete sequence of FYGL-n was established indicating a hyperbranched biopolymer.Presently, an efficient protein tyrosine phosphatase 1B (PTP1B) inhibitor, named FYGL-n, was isolated from Ganoderma Lucidum and characterized for its structure and bioactivity. Structure and chain conformation of FYGL-n based on both chemical and spectroscopic analysis showed that FYGL-n was a hyperbranched heteropolysaccharide bonded with protein via both serine and threonine residues by O-type glycoside, and showed a sphere observed by AFM. Specifically, monosaccharide composition indicated that FYGL-n consisted of d-arabinose, d-galactose, l-rhamnose and d-glucose in a mole ratio of 0.08:0.21:0.24:0.47, with a molecular mass of 72.9 kDa. The analysis of amino acids in FYGL-n indicated that there were 16 common amino acids, among which aspartic acid, glycine, serine, alanine, glutamic acid and threonine were the dominant components. Also it was demonstrated that FYGL-n could inhibit the PTP1B activity on a competitive mechanism in vitro.

Misfolding or β-sheet nano-fibrillation of specific proteins is considered to be an underlying pathogenic mechanism of neurodegenerative diseases. It was found previously that Al(III) can affect the β-sheet nano-fibrillation and deposit neurodegenerative disease related proteins, and that curcumin can interact with metal ions and those proteins. In this work, silk fibroin (SF) was used as a model fibrillation protein for the investigation of the influence of Al(III) and curcumin on SF conformation transition. The effects of Al(III) and curcumin on SF were investigated using circular dichroism, thioflavin T fluorescence, 1-anilino-8-naphthalene sulfonate fluorescence, turbidity assays, atomic force microscope and Fourier transform infrared. This research demonstrated that Al(III) can bind with specific amino acid residues of SF, and then accelerate the formation of intermediates and also the formation of nanofibrils. The concentration of Al(III) is an important factor that influences the folding speed of SF. Furthermore, curcumin cannot only restrain the conformation transition of silk fibroin, but can also reverse the conformation transition of SF and Al(III)-induced SF from insoluble β-sheet to soluble random coil. Curcumin may prevent the neurodegenerative related proteins from nano-fibrillating and aggregating.

Ganoderma lucidum (G. lucidum) is a mushroom which has been used for health promotion for a long time in China. In the present work a neutral hetero-polysaccharide, named FYGL-1, was isolated from FYGL which was reported previously capable of antihyperglycemia in vivo for further detailed chemical structure investigation. The results of monosaccharide composition and GPC analysis indicated that FYGL-1 consisted of galactose, rhamnose and glucose in mole ratio of 1.00:1.15:3.22 with a molecular weight of 78 kDa. The detailed structure of FYGL-1 was characterized by periodate oxidation, Smith degradation, methylation analysis, along with FT-IR, GC, GC–MS, 1D 1H and 13C NMR and 2D NMR (HSQC, COSY, NOESY and TOCSY). Based on the analysis of the results, the structure of the repeating unit of FYGL-1 was established as:Figure optionsHighlights► A novel polysaccharide was extracted from Ganoderma lucidum. ► The polysaccharide was composed of Gal, Rha and Glc. ► The molecular weight of the polysaccharide was 78 kDa. ► The structure contained 1,2-β-l-Rhap, 1,3,6-α-d-Galp, 1,2,6-α-d-Glcp and 1-α-d-Glcp.

Misfolding or amyloid β-sheet aggregation of specific proteins is considered to be an underlying pathogenic mechanism of neurodegenerative diseases, such as Alzheimer's disease (AD). Some metallic ions, such as Zn(II), have been reported to promote the conformational transition of neurodegenerative disease-related proteins, whereas (-)-epigallocatechine gallate (EGCG) displays an inhibitory effect on the aggregation of those proteins. Few studies have investigated the secondary structure changes of those pathological proteins affected by Zn(II), EGCG and their co-existence. In the present work we used silk fibroin (SF), which has a similar conformational transition mechanism to neurodegenerative diseases-related proteins, as a model protein to reveal the influence of Zn(II) and EGCG on the SF conformation and the interference of EGCG on the Zn(II)-induced SF conformational transition. The results indicate that low concentrations of Zn(II) ions promotes the aggregation of SF, while EGCG significantly inhibits the aggregation. Importantly, EGCG can inhibit and even dissociate the Zn(II)-induced β-sheet aggregation of SF in a dose dependent manner. These findings provide a significant theoretical basis for understanding the effect of Zn(II) and EGCG on the conformational transition of proteins involved in neurodegenerative diseases and developing agents like EGCG as potential treatments.

Neurodegenerative diseases involve the disorder of protein folding in which normally soluble proteins are deposited as abnormally insoluble fibrils that disrupt tissue structure and cause disease. Aluminum ion, Al(III), is known to induce formation of the insoluble β-sheet-rich fibrils, while curcumin is considered capable of binding both metal ions and neurodegenerative disease-related proteins. In the present work, silk fibroin (SF) which have essential and sufficient amino acid sequences for the protein fibrillation on nucleation-dependent conformation transition mechanism similar to that for the fibrillation of neurodegenerative proteins, was used as a model protein to reveal the influence of curcumin on Al(III)-induced conformation transition of proteins. It was demonstrated that Al(III) could induce the conformation of SF into β-sheet, while curcumin could inhibit the event. In particular, Al(III)–curcumin complexes formed at [Al(III)]/[curcumin] ratios from 1:1 to 1:2 could reverse the conformation of SF from β-sheet into random coil. These findings would be of revelatory significance for developing an efficient approach inhibiting the fibrillation of neurodegenerative proteins.

Inhibition of protein tyrosine phosphatase 1B (PTP1B) activity has been considered to be a promising therapy approach to treat type 2 diabetes. In this work, a novel PTP1B activity inhibitor, named FYGL (Fudan–Yueyang–G. lucidum), was screened from the fruiting bodies of Ganoderma lucidum and showed an efficient PTP1B inhibitory potency with IC50 = 5.12 ± 0.05 μg/mL. FYGL is a water-soluble macromolecular proteoglycan with a protein to polysaccharide ratio of 17:77 and a viscosity-average molecular weight (Mη) of 2.6 × 105. The type 2 diabetic mice treated orally by FYGL showed an obvious decrease in plasma glucose level compared with the diabetic controls without drug treatment, comparable with that of diabetic mice treated with metformin, a clinical drug. The toxicity of FYGL is very low. The results indicate that FYGL may serve as a drug candidate or a health-care food for diabetic therapy or protection.

Curcumin has been recognized as a potential natural drug to treat the Alzheimer’s disease (AD) by chelating baleful metal ions, scavenging radicals and preventing the amyloid β (Aβ) peptides from the aggregation. In this paper, Al(III)–curcumin complexes with Al(III) were synthesized and characterized by liquid-state 1H, 13C and 27Al nuclear magnetic resonance (NMR), mass spectroscopy (MS), ultraviolet spectroscopy (UV) and generalized 2D UV–UV correlation spectroscopy. In addition, the density functional theory (DFT)-based UV and chemical shift calculations were also performed to view insight into the structures and properties of curcumin and its complexes. It was revealed that curcumin could interact strongly with Al(III) ion, and form three types of complexes under different molar ratios of [Al(III)]/[curcumin], which would restrain the interaction of Al(III) with the Aβ peptide, reducing the toxicity effect of Al(III) on the peptide.Highlights► Curcumin can form three types of complexes with Al(III). ► The interaction of Al(III) with the Aβ peptide would be limited by curcumin. ► Curcumin may weaken the Aβ toxicity induced by metal ions.

Curcumin and its complexes with Zn2+ and Cu2+ ions were synthesized and characterized by elemental analysis, mass spectroscopy, IR spectroscopy, UV spectroscopy, solution 1H and solid-state 13C NMR spectroscopy, EPR spectroscopy. In addition, the density functional theory (DFT)-based UV and 13C chemical shift calculations were also performed to view insight into those compound structures and properties. The results show that curcumin easily chelate the metal ions, such as Zn2+ and Cu2+, and the Cu(II)-curcumin complex has an ability to scavenge free-radicals. We demonstrated the differences between Zn(II)-curcumin and Cu(II)-curcumin complexes in structure and properties, enhancing the comprehensions about the curcumin roles in the Alzhermer’s disease treatment.

Cell affinity is one of the important issues required for developing tissue engineering materials. Although the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) has been attractive for its controllable mechanical properties recent years, its cell affinity is still necessary to be improved for the requirements. For this purpose, the regenerated silk fibroin (SF) was coated on the PHBHHx films and its porous scaffolds. The mechanical test showed that SF-modified PHBHHx (SF/PHBHHx) film has a maximum tensile strength of 11.5 ± 0.5 MPa and elongation at break of 175 ± 5%. ATR-FTIR spectroscopy demonstrated that SF firmly attached on the scaffold by the hydrogen bonding interaction between SF and PHBHHx even flushed for 21 days in the phosphate-buffer saline (PBS) solution (pH = 7.4). In order to characterize the cell affinity of the SF-modified material, endothelial-like cell line ECV304 cells were seeded on the SF/PHBHHx films and its porous scaffolds. The histochemical analyses of cells stained by the hematoxylin and eosin (HE) as well as cell nuclei stained by the 4′,6-diamindine-2′-phenylindole (DAPI) demonstrated that cell attached and reached nearly 100% confluence on the SF/PHBHHx films when cultured for 4 days, which was much faster than that on the pure PHBHHx film. Moreover, the assay of cell activity by the 3-(4, 5-dimethyl thiazol -2-yl)-2, 5-diphenyl terazolium bromide (MTT) showed quantitatively that the number of cells on the SF/PHBHHx porous scaffolds was significant more than that on the unmodified ones after 4, 8, and 14 days culture, respectively. Scanning electron microscopy (SEM) revealed the similar results. Therefore, the SF-modified PHBHHx material is maybe a potential material applicable in the cardiovascular tissue engineering.

In the present work, we investigated Na+ ion effect on the silk fibroin (SF) conformation. Samples are Na+-involved regenerated silk fibroin films. 13C CP-MAS NMR demonstrates that as added [Na+] increases, partial silk fibroin conformation transit from helix-form to β-form at certain Na+ ion concentration which is much higher than that in Bombyx mori silkworm gland. The generalized two-dimensional NMR–NMR correlation analysis reveals that silk fibroin undergoes several intermediate states during its conformation transition process as [Na+] increase. The appearance order of the intermediates is followed as: helix and/or random coil → helix-like → β-sheet-like → β-sheet, which is the same as that produced by pH decrease from 6.8 to 4.8 in the resultant regenerated silk fibroin films. The binding sites of Na+ to silk fibroin might involve the carbonyl oxygen atom of certain amino acids sequence which could promote the formation of β-sheet conformation. Since the Na+O bond is weak, the ability of Na+ inducing the secondary structure transition is weaker than those of Ca2+, Cu2+ and even K+. It is maybe a reason why the sodium content is much lower than potassium in the silkworm gland.

In order to overcome the drawbacks of silk fibroin (SF)-based materials, SF has been blended with some polymers. Before using the blend material, understanding of the structures and phase behaviors of the blend is thought to be essential. In this study, solid-state 13C CP-MAS NMR and Raman imaging techniques were used to study the structures and phase behaviors of blends of SF with polyethylene glycol (PEG) at a molecular weight that varied from 2 to 20 kDa and a blend ratio of SF/PEG from 95/5 to 70/30 (w/w%) at the molecular and microscopic levels. It is found that the conformational transition of SF to the β-sheet increased as the PEG content increased, while the amount of the formed β-sheet conformers was decreased as the PEG molecular weight increased for a given content. It is also observed that SF was incompatible with PEG to some extent. The phase separation into “sea” and “island” domains took place in the SF/PEG blend films. SF was dominantly present in the “sea” domain, while PEG in the “island” domains. The conformation of SF in the interface between SF and PEG was changed to the β-sheet, while that in the protein-rich domain remained in the random coil and/or helix conformation. These observations suggest that the specifically expected materials, for example, the silk-based microspheres or scaffold materials can be manufactured by controlling the molecular weight and content of PEG in the blend system.