A Comparative Functional Analysis of Pea Protein and Grass Carp Protein Mixture via Blending and Co-Precipitation

Currently, the application of protein mixture derived from plants and animals is of great interest to the food industry. However, the synergistic effects of isolated protein blends (BL) are not well established. Herein, the development of a more effective method (co-precipitation) for the production of protein mixtures from pea and grass carp is reported. Pea protein isolate (PPI), grass carp protein isolate (CPI), and pea–carp protein co-precipitates (Co) were prepared via isoelectric solubilization/precipitation using peas and grass carp as raw materials. Meanwhile, the BL was obtained by blending PPI with CPI. In addition, the subunit composition and functional properties of Co and BL were investigated. The results show that the ratios of vicilin to legumin α + β and the soluble aggregates of Co were 2.82- and 1.69-fold higher than that of BL. The surface hydrophobicity of Co was less than that of BL, PPI, and CPI (p < 0.05). The solubility of Co was greater than that of BL, PPI, and CPI (p < 0.05), and the foaming activity was higher than that of BL and CPI (p < 0.05) but slightly lower than that of PPI. In addition, based on the emulsifying activity index, particle size, microstructure, and viscosity, Co had better emulsifying properties than BL, PPI, and CPI. The study not only confirmed that co-precipitation was more effective than blending for the preparation of mixed protein using PPI and CPI but also provided a standard of reference for obtaining a mixture of plant and animal proteins.

Characterization of divalent cation interactions with AASTY nanodiscs

Amphiphilic copolymers show great promise in extracting membrane proteins directly from lipid bilayers into 'native nanodiscs'. However, many such copolymers are polyanionic and sensitive to divalent cations, limiting their applicability towards Ca2+ or Mg2+ dependent proteins. Here, we characterize the Ca2+ and Mg2+ sensitivity of poly(acrylic acid-co-styrene) (AASTY) copolymers using analytical UV and fluorescent size exclusion chromatography, enabling us to separate signals from nanodiscs, copolymers, and soluble aggregates. Determination of free Ca2+ ion concentrations in the presence of copolymer shows that divalent cation tolerance is dependent on not only specific characteristics of a copolymer, but also on its concentration. We see that high ionic strength protects against aggregation facilitated by divalent cations, which is prominent in nanodiscs isolated from excess free copolymer through dialysis. Overall, we conclude that the behavior of amphiphilic copolymers in the presence of divalent cations is more complex than precipitation beyond a specific cation concentration.

Hyperphosphorylated tau self-assembles into amorphous aggregates eliciting TLR4-dependent inflammatory responses

Soluble aggregates of the microtubule-associated protein tau have been challenging to assemble and characterize, despite their important role in the development of tauopathies. We found that sequential hyperphosphorylation by PKA in conjugation with either GSK3-β or SAPK4 enabled recombinant wild-type (WT) tau of isoform 0N4R to spontaneously polymerize into small amorphous aggregates in vitro. We employed tandem mass spectrometry to determine the phosphorylation sites and the degree of phosphorylation, and super-resolution microscopy and electron microscopy to characterize the morphology of aggregates formed. Functionally, in comparison with the unmodified aggregates, which require heparin induction to assemble, these self-assembled hyperphosphorylated tau aggregates more efficiently disrupt membrane bilayers and induce Toll-like receptor 4 (TLR4)-dependent inflammatory responses. Together, our results demonstrate that tau hyperphosphorylation is potentially damaging to cells, providing a mechanistic model of how hyperphosphorylation of tau aggregates drives neuroinflammation in tauopathies.

Improving the Solubility and Digestibility of Potato Protein with an Online Ultrasound-Assisted PH Shifting Treatment at Medium Temperature

Ultrasonic (US) treatment was combined with pH shifting (pHS) and mild thermal (40 °C) (T40) treatment (US/T40/pHS) to improve the solubility of potato protein. The effects of the ultrasonication frequency, ultrasonication time, and incorporation sequence on the solubility of potato protein were investigated. The results showed that online US/T40/pHS treatment resulted in higher solubility of potato protein and enhanced free amino group release during in vitro digestion. The solubility of potato protein treated with online US/T40/pHS at a mono-frequency of 40 kHz for 15 min increased by 1.73 times compared with the control (p < 0.05). The digestibility rate increased by 16.0% and 30.8% during gastric and intestinal digestion, respectively, compared with the control (p < 0.05). It was demonstrated that online US/T40/pHS treatment significantly changed the secondary and tertiary structures of potato protein according to the results of circular dichroism and internal fluorescence. SDS-PAGE, particle size, and atomic force microscopy (AFM) showed that structural changes led to the formation of large soluble aggregates. The results suggested that the improvement in the solubility and digestibility of potato protein treated with online US/T40/pHS may be due to the formation of large soluble aggregates, which are more hydrophilic and sensitive to digestive enzymes.

Trodusquemine displaces protein misfolded oligomers from cell membranes and abrogates their cytotoxicity through a generic mechanism

The onset and progression of numerous protein misfolding diseases are associated with the presence of oligomers formed during the aberrant aggregation of several different proteins, including amyloid-β (Aβ) in Alzheimer’s disease and α-synuclein (αS) in Parkinson’s disease. These small, soluble aggregates are currently major targets for drug discovery. In this study, we show that trodusquemine, a naturally-occurring aminosterol, markedly reduces the cytotoxicity of αS, Aβ and HypF-N oligomers to human neuroblastoma cells by displacing the oligomers from cell membranes in the absence of any substantial morphological and structural changes to the oligomers. These results indicate that the reduced toxicity results from a mechanism that is common to oligomers from different proteins, shed light on the origin of the toxicity of the most deleterious species associated with protein aggregation and suggest that aminosterols have the therapeutically-relevant potential to protect cells from the oligomer-induced cytotoxicity associated with numerous protein misfolding diseases.