Tomonori Waku

The Mechanism of Fibril Formation of a Non-inhibitory Serpin Ovalbumin Revealed by the Identification of Amyloidogenic Core Regions

Ovalbumin (OVA), a non-inhibitory member of the serpin superfamily, forms fibrillar aggregates upon heat-induced denaturation. Recent studies suggested that OVA fibrils are generated by a mechanism similar to that of amyloid fibril formation, which is distinct from polymerization mechanisms proposed for other serpins. In this study, we provide new insights into the mechanism of OVA fibril formation through identification of amyloidogenic core regions using synthetic peptide fragments, site-directed mutagenesis, and limited proteolysis. OVA possesses a single disulfide bond between Cys73 and Cys120 in the N-terminal helical region of the protein. Heat treatment of disulfide-reduced OVA resulted in the formation of long straight fibrils that are distinct from the semiflexible fibrils formed from OVA with an intact disulfide. Computer predictions suggest that helix B (hB) of the N-terminal region, strand 3A, and strands 4–5B are highly β-aggregation-prone regions. These predictions were confirmed by the fact that synthetic peptides corresponding to these regions formed amyloid fibrils. Site-directed mutagenesis of OVA indicated that V41A substitution in hB interfered with the formation of fibrils. Co-incubation of a soluble peptide fragment of hB with the disulfide-intact full-length OVA consistently promoted formation of long straight fibrils. In addition, the N-terminal helical region of the heat-induced fibril of OVA was protected from limited proteolysis. These results indicate that the heat-induced fibril formation of OVA occurs by a mechanism involving transformation of the N-terminal helical region of the protein to β-strands, thereby forming sequential intermolecular linkages.

Preparation of heat-induced artificial collagen gels based on collagen-mimetic dendrimers

Collagen, a major component of the extracellular matrix, contains Gly-Pro-Hyp repeats that form a hydrogel. In this study, artificial collagen-mimetic materials were designed. Synthetic dendritic macromolecules were fully modified with (Pro-Hyp-Gly)n and named collagen-mimetic dendrimers. A collagen-like triple helical structure was observed by circular dichroism spectrometry, with an efficiency that depended on the peptide length. A (Pro-Hyp-Gly)10-modified dendrimer exhibited the most efficient triple helix formation. Thermal stability was enhanced by clustering at the surface of the dendrimer. The (Pro-Hyp-Gly)10-modified dendrimer was assembled by heating and the assembly was affected by temperature, time and concentration. Hydrogels based on the (Pro-Hyp-Gly)10-modified dendrimer, but not on the peptide itself, were successfully prepared by heating. The sol–gel transition behavior was similar to natural collagen but not gelatin, which is thermally denatured collagen. Dynamic rheological analysis showed that the sol–gel transition temperature and the strength depended on the concentration. Thus, the collagen-mimetic dendrimer incorporating (Pro-Hyp-Gly)10 is an injectable and controllable artificial collagen gel.

Temperature-dependent higher order structures of the (Pro-Pro-Gly)10-modified dendrimer†

Collagen is the most abundant protein in mammals and is widely used as a biomaterial for tissue engineering and drug delivery. We previously reported that dendrimers and linear polymers, modified with collagen model peptides (Pro-Pro-Gly)₅, form a collagen-like triple-helical structure; however, its triple helicity needs improvement. In this study, a collagen-mimic dendrimer modified with the longer collagen model peptides, (Pro-Pro-Gly)₁₀, was synthesized and named PPG10-den. Circular dichroism analysis shows that the efficiency of the triple helix formation in PPG10-den was much improved over the original. The X-ray diffraction analysis suggests that the higher order structure was similar to the collagen triple helix. The thermal stability of the triple helix in PPG10-den was higher than in the PPG10 peptide itself and our previous collagen-mimic polymers using (Pro-Pro-Gly)₅. Interestingly, PPG10-den also assembled at low temperatures. Self-assembled structures with spherical and rod-like shapes were observed by transmission electron microscopy. Furthermore, a hydrogel of PPG10-den was successfully prepared which exhibited the sol-gel transition around 45°C. Therefore, the collagen-mimic dendrimer is a potential temperature-dependent biomaterial.

Ovalbumin Delivery by Guanidine-Terminated Dendrimers Bearing an Amyloid-Promoting Peptide via Nanoparticle Formulation

Development of protein delivery systems is important for biomedical applications such as immunotherapy. Ovalbumin (OVA) is a major component of egg whites, and is a possible cause of egg allergy. In this study, OVA was used as a model protein to develop a delivery system using guanidine-terminated dendrimers (Gdn-den) bearing an amyloid-promoting peptide derived from the helix B (hB) region of OVA (hB-Gdn-den). OVA nanoparticles (NPs) were prepared by heat treatment of OVA/hB-Gdn-den mixtures. The NP size and the surface charge were controlled by adjusting the ratio of hB-Gdn-den to OVA. The NPs were around 200 nm in diameter and stably dispersed, and their encapsulation efficiency for OVA was more than 80%. Although OVA NPs were also prepared using Gdn-den, the NPs aggregated readily. Complexation with hB-Gdn-den induced conformational changes in the OVA, and the hB peptide promoted digestion of OVA. These suggest that the hB peptide of the Gdn-den works as a possible anchor to OVA. The positively charged OVA NPs effectively associated with RAW264 cells. Thus, the amyloid-promoting Gdn-den, when mixed with OVA at a suitable molar ratio to form NPs, could act as a carrier for delivery of antigen proteins to immune cells.

Ice Growth Inhibition in Antifreeze Polypeptide Solution by Short-Time Solution Preheating

The objective of this study is to enhance the inhibition of ice growth in the aqueous solution of a polypeptide, which is inspired by winter flounder antifreeze protein. We carried out measurements on unidirectional freezing of the polypeptide solution. The thickness of the solution was 0.02 mm, and the concentration of polypeptide was varied from 0 to 2 mg/mL. We captured successive microscopic images of ice/solution interfaces, and measured the interface velocity from the locations of tips of the pectinate interface in the images. We also simultaneously measured the temperature by using a small thermocouple. The ice/solution interface temperature was defined by the temperature at the tips. It was found that the interface temperature was decreased with an increasing concentration of polypeptide. To try varying the activity of the polypeptide, we preheated the polypeptide solution and cooled it before carrying out the measurements. Preheating for 1–5 hours was found to cause a further decrease in the interface temperature. Furthermore, wider regions of solution and ice with inclined interfaces in the pectinate interface structure were observed, compared with the case where the solution was not preheated. Thus, the ice growth inhibition was enhanced by this preheating. To investigate the reason for this enhancement, we measured the conformation and aggregates of polypeptide in the solution. We also measured the local concentration of polypeptide. It was found that the polypeptide aggregates became larger as a result of preheating, although the polypeptide conformation was unchanged. These large aggregates caused both adsorption to the interface and the wide regions of supercooled solution in the pectinate interface structure.

Self-Assembled Structure of Peptide Nanospheres Induces High Stability against Hydrolysis and Sterilization

The hydrolysis properties of peptide nanospheres consisting of a high-density poly(ethylene glycol) (PEG) brush layer and a poly(L-phenylalanine) (PPhe) core were evaluated in relation to their self-assembled nanostructure. To clarify the effect of the self-assembled structure of the nanospheres on their hydrolysis properties, deformed nanospheres possessing exactly the same components were used as a comparison sample. The desorption of the PEG chains on the peptide nanospheres occurred weakly in alkaline solution at 80° C for 7 days, whereas the deformed nanospheres showed drastic desorption of 70% of the PEG chains within 3 days, possibly due to a low density of PEG chains on their surfaces. The content ratio of the β-sheet conformation in the core increased with increasing hydrolysis time, suggesting that the α-helix and random coil conformations were more easily hydrolyzed. Furthermore, the peptide nanospheres retained all their properties, even after autoclaving and ethanol sterilization. These results demonstrated that the self-assembled spherical morphology induced extreme stability and sterilizable peptide nanospheres can be useful as drug-delivery system.

PE859, A Novel Curcumin Derivative, Inhibits Amyloid-β and Tau Aggregation, and Ameliorates Cognitive Dysfunction in Senescence-Accelerated Mouse Prone 8

Aggregation of amyloid-β (Aβ) and tau plays a crucial role in the onset and progression of Alzheimers disease (AD). Therefore, the inhibition of Aβ and tau aggregation may represent a potential therapeutic target for AD. Herein, we designed and synthesized both Aβ and tau dual aggregation inhibitors based on the structure of curcumin and developed the novel curcumin derivative PE859. In this study, we investigated the inhibitory activity of PE859 on Aβ aggregationin vitro and the therapeutic effects of PE859 on cognitive dysfunction via dual inhibition of Aβ and tau aggregation in vivo. PE859 inhibited Aβ aggregation in vitro and protected cultured cells from Aβ-induced cytotoxicity. Furthermore, PE859 ameliorated cognitive dysfunction and reduced the amount of aggregated Aβ and tau in brains of senescence-accelerated mouse prone 8 (SAMP8). These results warrant consideration of PE859 as a candidate drug for AD.

Effects of preheating on ice growth in antifreeze polypeptides solutions in a narrow space

We conducted measurements on the unidirectional freezing of aqueous solutions of polypeptide or of winter flounder antifreeze protein. The polypeptide was based on a part of the antifreeze protein. We measured temperatures in the solutions and ice with a small thermocouple, and defined the interface temperature as the temperature at the tip of the serrated or pectinate interface. It was found that the interface temperature of these solutions was lower than that of pure water. To vary the activity of these solutes, we preheated the solutions and cooled them before conducting the measurements. We found that preheating for several hours caused further decreases in the interface temperature and a decrease in the interface velocity. In addition, the inclined interfaces became wider as a result of the preheating. Thus, the supercooled states in the solutions were enhanced by the preheating. To investigate the reasons for these changes, we measured the aggregates of the solutes in the solutions. These aggregates became larger as a result of preheating. It can therefore be concluded that these large aggregates attenuated the ice growth by their interaction with the ice surfaces.

Suppression of droplets freezing on glass surfaces on which antifreeze polypeptides are adhered by a silane coupling agent

The development of ice-phobic, glass-substrate surfaces is important for many reasons such as poor visibility through the ice-covered windshields of vehicles. The present authors have developed new glass surfaces coated with a silane coupling agent and polypeptides whose amino-acid sequence is identical to a partial sequence of winter flounder antifreeze protein. We have conducted experiments on the freezing of sessile water droplets on the glass surfaces, and measured the droplet temperature, contact angle, contact area and surface roughness. The results show that the supercooling temperature decreased noticeably in the case where a higher concentration solution of polypeptide was used for the coating. The adhesion strength of frozen droplets was lowest in the same case. In addition, we observed many nanoscale humps on the coated surface, which were formed by polypeptide aggregates in the solution. We argue that the combination of the hydrophilic humps and the hydrophobic base surfaces causes water molecules adjacent to the surfaces to have a variety of orientations in that plane, even after the ice layer started to grow. This then induces a misfit of water-molecule spacing in the ice layers and consequent formation of fragile polycrystalline structure. This explains the lower values of ice adhesion strength and supercooling enhancement in the cases of the polypeptide-coated glass plates.

Fusion of polymeric material-binding peptide to cell-adhesion artificial proteins enhances their biological function

Orientation-controlled protein immobilization on a solid substrate surface is important for the development of biomedical materials such as scaffolds used in tissue engineering. In this study, the authors demonstrated that the introduction of material-binding peptides (MBPs) in Arg-Gly-Asp (RGD)-fused artificial proteins called blocking peptide fragment (BPF), which are fragments (residues 419-607) of the molecular chaperone DnaK, enhances the oriented adsorption of proteins on the polymer surface and improves their cell adhesion capability. The authors used isotactic poly(methyl methacrylate) (it-PMMA) binding peptides (c02 peptide; ELWRPTR) as a model system. A quartz crystal microbalance study showed that the fusion of c02 peptide with BPF-RGD proteins slightly enhanced adsorption on it-PMMA surfaces. On the other hand, atomic force microscopic images of it-PMMA surfaces adsorbed with c02-BPF-RGD proteins showed a dotlike pattern, with the sizes of the dots comparable to those of BPF protein dimers, indicating that the immobilization of c02-BPF-RGD partially occurred in an oriented manner via specific interaction between the c02 peptide and it-PMMA. This is in sharp contrast to the random adsorption of BPF-RGD and BPF. These results were supported by results of the enzyme-linked immunosorbent assay using an antihistidine tag antibody. In addition, c02-BPF-RGD adsorbed on it-PMMA showed better cell attachment and spreading ability than BPF-RGD and BPF. This methodology can be applied to other MBP systems and cell-binding motifs. Thus, BPF-based artificial cell adhesion proteins fused with MBPs might be useful as surface modifiers of polymer materials for improving their cell adhesion ability.