Heat-induced Protein Aggregation Research Articles

Aggregation and Size Attributes Analysis of Unadsorbed and Adjuvant-adsorbed Antigens using a Multispectral Imaging Flow Cytometer Platform

Various vaccine quality attributes should be monitored to ensure consistency, potency, purity, and safety of vaccine products prior to lot release. Vaccine particle size and protein antigen aggregation are two important considerations for particle-adsorbed vaccines. In this study, we evaluated the use of imaging flow cytometry as a potential all-in-one platform to measure adjuvant particle size and to detect protein aggregates through a combination of brightfield microscopy, side scatter detection, and fluorescence microscopy. An aluminum phosphate adjuvant was analyzed for size using the brightfield function, and the size measurement was compared against laser diffraction. Heat-induced protein aggregates of either unadsorbed antigens or aluminum phosphate adjuvant-adsorbed antigens were stained with the fluorescent ProteoStat aggregation dye, followed by detection and analysis using a combination of the brightfield and fluorescence microscopy functions. The change in aggregation of unadsorbed antigens was confirmed using dynamic light scattering. These results demonstrate the versatility of the imaging flow cytometry platform for the evaluation of multiple vaccine quality characteristics.

Rehydration Properties of Whey Protein Isolate Powders Containing Nanoparticulated Proteins

The rehydration properties of original whey protein isolate (WPIC) powder and spray-dried WPI prepared from either unheated (WPIUH) or nanoparticulated WPI solutions were investigated. Nanoparticulation of whey proteins was achieved by subjecting reconstituted WPIC solutions (10% protein, w/w, pH 7.0) to heat treatment at 90 °C for 30 s with no added calcium (WPIH) or with 2.5 mM added calcium (WPIHCa). Powder surface nanostructure and elemental composition were investigated using atomic force microscopy and X-ray photoelectron spectroscopy, followed by dynamic visualisation of wetting and dissolution characteristics using environmental scanning electron microscopy. The surface of powder particles for both WPIUH and WPIC samples generally appeared smooth, while WPIH and WPIHCa displayed micro-wrinkles with more significant deposition of nitrogen and calcium elements. WPIH and WPIHCa exhibited lower wettability and solubility performance than WPIUH and WPIC during microscopic observation. This study demonstrated that heat-induced aggregation of whey proteins, in the presence or absence of added calcium, before drying increases aggregate size, alters the powder surface properties, consequently impairing their wetting characteristics. This study also developed a fundamental understanding of WPI powder obtained from nanoparticulated whey proteins, which could be applied for the development of functional whey-based ingredients in food formulations, such as nanospacers to modulate protein–protein interactions in dairy concentrates.

Heat-induced denaturation and aggregation of protein in quinoa (Chenopodium quinoa Willd.) seeds and whole meal

Physical barriers hinder about 20–25% of the protein from being extracted from whole meal. Heat-induced denaturation and aggregation of protein in quinoa seeds and in whole meal was investigated. Maximally 37% of the protein in seeds covalently aggregate when boiling for 15 min. Although embryonic cell walls surrounding protein bodies remain intact during boiling of seeds, protein aggregation is not hindered. 11S Globulin monomers first dissociate into their acidic and basic subunits which further assemble into large (> 500 kDa) mainly disulfide-linked aggregates. 2S Albumins are not involved in covalent aggregation but partially leach during seed boiling. The presence of disrupted food matrix constituents in whole meal delays denaturation and causes less aggregation of protein in whole meal than in seeds. Globulins still dissociate into their subunits but less and mainly small covalent aggregates (ca. 100–500 kDa) are formed. These novel insights allow developing new quinoa-based food products.

Effect of Heat Treatment on the Property, Structure, and Aggregation of Skim Milk Proteins.

To study the mechanism of heat-induced protein aggregates, skim milk was heated at 55, 65, 75, 85, and 95°C for 30 s. Then, the sulfhydryl content, surface hydrophobicity, and secondary structure of heat-treated skim milk were studied. Treating skim milk at different temperatures induced a decrease in sulfhydryl content (75.9% at 95°C) and an increase in surface hydrophobicity (44% at 95°C) with a disrupted secondary structure containing random coil, β-sheet, and β-turn of skim milk proteins. The change in these properties facilitated aggregate formation through disulfide bonds and hydrophobicity interaction. Microstructural observation also showed a higher degree of aggregation when skim milk was heated at 85 and 95°C. The result of two-dimensional polyacrylamide gel electrophoresis demonstrated that the aggregates consisted of a high proportion of κ-casein, β-lactoglobulin, and other whey proteins.

The interfacial covalent bonding of whey protein hydrolysate and pectin under high temperature sterilization: Effect on emulsion stability

In this study, the effect of high-pressure steam sterilization (121 °C for 15 min) on whey protein hydrolysate-pectin solutions and emulsions was studied. The interaction and emulsification characteristics of pectin and whey protein concentrate (WPC) were evaluated from the solution system to the emulsion system. Enzymatic hydrolysis of WPC (WPH, 2 % and 8 % degree of hydrolysis) increased the covalent binding with pectin, which reduced the heat-induced aggregation of protein and improved emulsification. The thermodynamic incompatibility between WPC and pectin was not conducive to the covalent bonding under high temperature sterilization and produced serious aggregates, which also made a rapid increase in particle size (up to ∼3 μm), compared to WPH-pectin emulsion (∼ 400 nm). In addition, if emulsion was stirred during the sterilization, the creaming and protein aggregation could be avoided. By comparing low methoxy pectin (LMP) and high methoxy pectin (HMP), it was found that the whey protein-HMP complex had better emulsification stability, and the steric stabilization played a more important role in emulsion stability than the electrostatic repulsion. The changes of whey protein and pectin at the oil-water interface of the emulsion during the sterilization process may provide a reference for the sterilized bioactive ingredient delivery system.

Stabilization of hempseed protein dispersion: Influence of sonication, hydrocolloids, and sodium hexametaphosphate

Nowadays, there is a growing demand for consumption of protein-based healthy drinks. However, the thermal process usually leads to heat-induced aggregation of proteins, consequently phase separation. Hence, the aim of the present study was to enhance the thermal stability of hemp protein concentrate (HPC) dispersion using physical processes, hydrocolloids, and sodium hexametaphosphate (SHMP). The rheological and sensory properties of the dispersions as well as the electrical charge and size of the particles were evaluated. The lowest and highest solubility of HPC belonged to pH 3.0 and 7.0, respectively. For stabilization at these pHs, the nonadsorbing hydrocolloid (guar) was required much more than adsorbing ones (xanthan, gum tragacanth, and high methoxyl pectin). In order to produce a heat-stable HPC dispersion, the protein concentration needs to be ≤1.5 wt.%. Furthermore, both the sonication and homogenization processes improved the stability against sterilization. Sonication led to lower particle size than homogenization (562 vs. 1,018 nm). To increase protein concentration (up to 3.0 wt.%), SHMP (0.01–0.04 wt.%) was essentially required. It was also found that the guar induced the highest apparent viscosity and the lowest sensory acceptance, while the most preferred formulae were the one with HPC (1.5 wt.%), sugar (10.0 wt.%), and xanthan (0.4 wt.%). Practical applications The findings of the present study highlighted the importance of hemp protein concentrate (HPC) thermal instability and various approaches to its prevention or improvement which could be potentially beneficial for the commercialization of heat-resistant HPC-based healthy beverage with high protein content for elders, athletes, and diet-watchers.

The Putative RNA-Binding Protein Dri1 Promotes the Loading of Kinesin-14/Klp2 to the Mitotic Spindle and Is Sequestered into Heat-Induced Protein Aggregates in Fission Yeast.

Cells form a bipolar spindle during mitosis to ensure accurate chromosome segregation. Proper spindle architecture is established by a set of kinesin motors and microtubule-associated proteins. In most eukaryotes, kinesin-5 motors are essential for this process, and genetic or chemical inhibition of their activity leads to the emergence of monopolar spindles and cell death. However, these deficiencies can be rescued by simultaneous inactivation of kinesin-14 motors, as they counteract kinesin-5. We conducted detailed genetic analyses in fission yeast to understand the mechanisms driving spindle assembly in the absence of kinesin-5. Here, we show that deletion of the dri1 gene, which encodes a putative RNA-binding protein, can rescue temperature sensitivity caused by cut7-22, a fission yeast kinesin-5 mutant. Interestingly, kinesin-14/Klp2 levels on the spindles in the cut7 mutants were significantly reduced by the dri1 deletion, although the total levels of Klp2 and the stability of spindle microtubules remained unaffected. Moreover, RNA-binding motifs of Dri1 are essential for its cytoplasmic localization and function. We have also found that a portion of Dri1 is spatially and functionally sequestered by chaperone-based protein aggregates upon mild heat stress and limits cell division at high temperatures. We propose that Dri1 might be involved in post-transcriptional regulation through its RNA-binding ability to promote the loading of Klp2 on the spindle microtubules.

Modulating the in vitro gastric digestion of heat-induced beta-lactoglobulin aggregates: Incorporation with polysaccharide

Three heat-induced protein aggregates, beta-lactoglobulin fibrils (BLGF), nanoparticles (BLGN), and worm-like aggregates (BLGW) were chosen to probe the effect of disulfide bond and surface hydrophobicity on their gastric digestion behavior. Furthermore, the effect of polysaccharide (dextran sulfate sodium, DSS) on the digestion behavior of the protein aggregates was investigated. Results showed that disulfide bond had a mild restraint on the digestion extent (maximum up to 4.65%), especially when its content was below 1 mol/mol, while the surface hydrophobicity had a stronger influence (up to 8.96%), and there is definitive positive linear relationship between the surface hydrophobicity and the digestion extent. When incorporated with DSS, both the disulfide bond content and surface hydrophobicity of the aggregates decreased, consequently, and the digestion was impeded, confirming the stronger effect from the surface hydrophobicity. The digestion extent of the heat-induced protein aggregates could be modulated linearly by incorporation of polysaccharide.

Reduced Adhesive Force Leading to Enhanced Thermal Stability of Soy Protein Particles by Combined Preheating and Ultrasonic Treatment.

Developing liquid systems with high protein contents is drawing intensive attention; however, this is challenged by heat-induced aggregation and gelation of proteins. Herein, we described a facile but robust approach of combined preheating and ultrasonic treatment (CPUT) to fabricate soy protein particles (SPPs) with enhanced heat stability. Results showed that these heat-stable particles, upon reheating at 1% (w/v), showed antiaggregation property evidenced from no obvious changes of the particle size distributions of suspensions. Besides, no gelation was found in the reheated test for SPPs suspended even at a concentration of 10% (w/v). In contrast, the control formed sol-gel after heating. The rearrangements of soy protein molecules by CPUT led to the formation of SPPs with reduced surface energy, which was primarily responsible for their heat stability. These findings highlighted that the CPUT could prepare thermally stable soy proteins, providing insights into the application of soy proteins in protein-enriched beverages.

Application of Glyceric Acid to Bio-related Functional Materials and Improvement of Microbial Production.

Glyceric acid (GA) is an oxidative product of glycerol, and its d-isomer is obtained as a phytochemical from tobacco leaves and fruits of some plants. However, the production and applications of GA have not yet been fully investigated. In this review, recent developments in the microbial production of GA and its application to bio-related materials are summarized. The sodium salt of diacylated GA showed superior surface tension-lowering activity and antitrypsin activity. GA and its glucosyl derivative had positive effects on the viability and collagen production of skin cells in vitro, respectively. Glucosyl derivatives of GA showed protective effects against heat-induced protein aggregation. In addition, the microbial production of GA using raw glycerol as the starting material was investigated. The effect of methanol, a major impurity in raw glycerol, on GA production was investigated, and mutant strains to tolerate methanol in the culture were constructed. Enantioselective production of GA using newly isolated microbial strains has also been developed.

Trehalose Inhibits the Heat-Induced Formation of the Amyloid-Like Structure of Soluble Proteins Isolated from Human Cataract Lens.

The age-dependent loss of solubility and aggregation of crystallins constitute the pathological hallmarks of cataract. Several biochemical and biophysical factors are responsible for the reduction of crystallins' solubility and formation of irreversible protein aggregates, which display amyloid-like characteristics. The present study reports the heat-induced aggregation of soluble proteins isolated from human cataract lenses and the formation of amyloid-like structures. Exposure of protein at 55 °C for 4 h resulted in extensive (≈ 60%) protein aggregation. The heat-induced protein aggregates displayed substantial (≈ 20nm) redshift in the wavelength of maximum absorption (λmax) of Congo red (CR) and increase in Thioflavin T (ThT) fluorescence emission intensity, indicating the presence of amyloid-like structures in the heat-induced protein aggregates. Subsequently, the addition of trehalose resulted in substantial inhibition of heat-induced aggregation and the formation of amyloid-like structure. The ability of trehalose to inhibit the heat-induced aggregation was found to be linearly dependent upon its concentrationused. The optimum effect was observed in the presence of 30-40% (w/v) trehalose where the aggregated was found to be reduced from 60 to 30%. The present study demonstrated the ability to trehalose to inhibit the protein aggregation and interfere with the formation of amyloid-like structures.

Preheat-induced soy protein particles with tunable heat stability

Recently, there is a growing interest in developing protein-enriched beverages with improved nutritional and functional properties. However, this is challenged by heat-induced aggregation and gelation of edible proteins, which limits their practical applications in high protein systems. In this study, soy protein particles (SPPs) with tunable heat stability were prepared by simply preheating soy proteins suspensions (pH 6.4 and 1% (w/v) concentration) at different temperatures and times. Results showed that heat-stabled SPPs were successfully obtained at high preheating temperatures with prolonged time. The SPPs structures were found to be highly unfolded, denatured, and compact. In addition, these particles exhibited lower viscosities and higher flow behavior index without gelation, whereas those prepared at lower preheating temperatures were found to readily gel after reheating. These results provide useful insights on how heat stable SPPs can be prepared, which extends their further application in protein-enriched beverages and relevant products.

Bulk and interfacial properties of milk fat emulsions stabilized by whey protein isolate and whey protein aggregates

Whey protein is widely used in the food industry as an emulsion stabilizer because of its outstanding emulsifying ability. Recent studies have shown that heat-induced whey protein aggregates may also have potential to stabilize emulsions. The interfacial behavior of whey protein and whey protein aggregates adsorbed at the milk fat-water interface has not been well investigated, especially not in the nonlinear regime, which is highly relevant for the preparation of products such as recombined dairy cream.In this study, the interfacial properties of milk fat-water interfaces stabilized by whey protein isolate (WPI) and whey protein aggregates (WPA) at different bulk concentrations (0.1 wt% - 4.0 wt%) were studied by Large Amplitude Oscillatory Dilatation (LAOD). Lissajous plots were used to analyse the nonlinear response of the interfaces as a function of strain amplitude and frequency. The elastic modulus was quantified based on the tangent modulus at zero instantaneous strain in expansion and in compression. Bulk stability of creams stabilized with the mentioned proteins was studied by determining creaming rate, droplet size distribution, ζ-potential and viscosity of the continuous phase.At low concentrations (<2.0 wt%), WPI-stabilized cream had smaller oil droplets than WPA-stabilized cream, indicating that at these concentrations WPI had better emulsifying ability. For concentrations higher than 2.0 wt%, WPA was a better emulsifier in terms of creaming stability because of the higher viscosity of the continuous phase of the emulsions. Both WPI and WPA could prevent coalescence equally well if the concentration was higher than 0.5 wt%. LAOD measurements showed that at a protein concentration of 0.1 wt%, there was little difference between WPI- and WPA-stabilized interfaces. At 4.0 wt%, WPI showed abrupt intra-cycle yielding followed by a predominantly viscous behavior at large expansion. The WPA interfacial layer had a larger maximum linear strain, and showed a more gradual softening in expansion and mild strain hardening in compression. We hypothesize that WPI formed denser and more brittle (quasi-) 2d structures at the interface, while the interfaces formed by WPA might have a thicker and more stretchable 3d structure. The WPA-stabilized emulsion was less resistant to coalescence upon drastic stirring, which can be explained with its different large deformation behavior, and is relevant for applications where the cream is subjected to large deformations (whipping or stirring).

Structural characterization of heat-induced protein aggregates in model infant milk formulas

Heat treatments induce structural modifications of bovine milk proteins. In this study, we aimed to investigate how these structural modifications are affected by the whey protein profile of infant milk formulas (IMFs) and heat treatment parameters. Three model IMFs (1.3% of proteins; casein:whey protein ratio 40:60) differing by the whey protein profile (1:3.37 α-lactalbumin (α-LA):β-lactoglobulin (β-LG), 1:3.26:1.80 α-LA:β-LG:lactoferrin (LF) or 1:0.13:0.40 α-LA:β-LG:LF), were heated at 67.5 °C or 80 °C to reach an akin whey protein denaturation extent of 65%. Protein structures were analyzed by asymmetrical flow field-flow fractionation coupled with multiangle light scattering and differential refractometer, transmission electron microscopy and electrophoresis. The unheated IMFs were used as reference. The results showed that LF addition in IMFs induced partial casein micelle disintegration before heating. In the absence of added LF, the heat-denatured whey proteins either formed soluble whey protein aggregates or casein micelle-bound whey protein aggregates. The latter were favored at the expense of soluble aggregates in the heated IMFs with the LF content increase and the concomitant β-LG content decrease. Consequently, the casein micelle structure was strongly dependent on the β-LG and LF amounts in IMFs and on the heating temperature. In the IMFs containing β-LG and LF and at temperature greater than the β-LG denaturation temperature, the casein micelles exhibited filamentous appendages on its surface after heating. Below the denaturation temperature of β-LG and in IMFs containing trace β-LG amount, the heated casein micelles were perfectly spherical with a smooth surface.

Influence of desialylation of caseinomacropeptide on the denaturation and aggregation of whey proteins

The effect of the addition of caseinomacropeptide (CMP) or desialylated CMP on the heat-induced denaturation and aggregation of whey proteins was investigated in the pH range 3 to 7 after heating at 80°C for 30 min. The rate and temperature of denaturation, the extent of aggregation, and the changes in secondary structure of the whey proteins heated in presence of CMP or desialylated CMP were measured. The sialic acid bound to CMP favored the denaturation and aggregation of whey proteins when the whey proteins were oppositely charged to CMP at pH 4. A transition occurred at pH 6, below which the removal of sialic acid enhanced the stabilizing properties of CMP against the denaturation and aggregation of the whey proteins. At pH >6, the interactions between desialylated CMP and the whey proteins led to more extensive denaturation and aggregation. Sialic acid bound to CMP influenced the denaturation and aggregation behavior of whey proteins in a pH-dependent manner, and this should be considered in future studies on the heat stability of such systems containing CMP.

Amyloid aggregates of the deubiquitinase OTUB1 are neurotoxic, suggesting that they contribute to the development of Parkinson's disease

Parkinson's disease (PD) is a multifactorial malady and the second most common neurodegenerative disorder, characterized by loss of dopaminergic neurons in the midbrain. A hallmark of PD pathology is the formation of intracellular protein inclusions, termed Lewy bodies (LBs). Recent MS studies have shown that OTU deubiquitinase ubiquitin aldehyde-binding 1 (OTUB1), a deubiquitinating enzyme of the OTU family, is enriched together with α-synuclein in LBs from individuals with PD and is also present in amyloid plaques associated with Alzheimer's disease. In the present study, using mammalian cell cultures and a PD mouse model, along with CD spectroscopy, atomic force microscopy, immunofluorescence-based imaging, and various biochemical assays, we demonstrate that after heat-induced protein aggregation, OTUB1 reacts strongly with both anti-A11 and anti-osteocalcin antibodies, detecting oligomeric, prefibrillar structures or fibrillar species of amyloidogenic proteins, respectively. Further, recombinant OTUB1 exhibited high thioflavin-T and Congo red binding and increased β-sheet formation upon heat induction. The oligomeric OTUB1 aggregates were highly cytotoxic, characteristic of many amyloid proteins. OTUB1 formed inclusions in neuronal cells and co-localized with thioflavin S and with α-synuclein during rotenone-induced stress. It also co-localized with the disease-associated variant pS129-α-synuclein in rotenone-exposed mouse brains. Interestingly, OTUB1 aggregates were also associated with severe cytoskeleton damage, rapid internalization inside the neuronal cells, and mitochondrial damage, all of which contribute to neurotoxicity. In conclusion, the results of our study indicate that OTUB1 may contribute to LB pathology through its amyloidogenic properties.

Enhancing the thermal stability of soy proteins by preheat treatment at lower protein concentration

The heat-induced aggregation of edible proteins has been regarded as one of the critical challenges for their application in protein-enriched beverages. Therefore, the formulation of thermal stable proteins to improve the stability of these beverages upon heating is highly desired. In this study, soy proteins (SPs) with enhanced heat stability were obtained by low-concentration-preheating (LCPH). Results from reheating of the above samples showed that pretreatment of SPs at low concentrations (≤1.0%, w/v) increased their resistance against aggregation. Additionally, when the suspensions of the particles were reheated at 10% (w/v) protein concentration, no gelation was found for samples prepared by LCPH, indicating collapsed protein–protein interactions, whereas gelled suspensions were obtained for native SPs and samples prepared by preheating at higher protein concentrations (≥2.0%, w/v). Furthermore, suspensions of particles prepared at lower protein concentration showed lower viscosities and higher flow behavior index values before and after reheat treatment. These findings highlighted that LCPH would provide fundamental information on the application of SPs in high protein beverages.

Mycobacterial transcript cleavage factor Gre, exhibits chaperone-like activity

Gre factors reactivate stalled elongation complexes by enhancing the intrinsic transcript cleavage activity of RNA polymerase. Previous work by us has shown that unlike in Escherichia coli (E.coli), Mycobacterium tuberculosis Gre factor is essential for its survival. Apart from their role in transcription regulation Gre factors have been implicated in stress response. A recent study has shown the role of E.coli GreA as a cellular chaperone, which inhibits aggregation of substrate proteins under heat stress condition. Moreover it was shown that GreA enables E.coli to survive heat shock and oxidative stress. In the current work, we have characterized the moonlighting chaperone activity and its plausible mechanism in Mycobacterium smegmatis Gre (MsGre) factor. We show here that MsGre prevents heat-induced aggregation of the substrate protein and also protects enzymatic activity. Interestingly Gre factor exists as a dimer in solution and does not undergo heat induced oligomerization. From the 8-anilino-1-naphthalene sulfonate (ANS) binding studies MsGre was shown to expose hydrophobic surface upon heat stress that would allow binding to unfolded or partially folded substrate protein. From Circular Dichroism (CD) studies, we also show that MsGre has a stable secondary structure under thermal stress. We propose that the presence of C-terminal FKBP-like fold in MsGre factor that might contribute to its chaperone-like function.

Rat Glioma Cell-Based Functional Characterization of Anti-Stress and Protein Deaggregation Activities in the Marine Carotenoids, Astaxanthin and Fucoxanthin.

Stress, protein aggregation, and loss of functional properties of cells have been shown to contribute to several deleterious pathologies including cancer and neurodegeneration. The incidence of these pathologies has also been shown to increase with age and are often presented as evidence to the cumulative effect of stress and protein aggregation. Prevention or delay of onset of these diseases may prove to be unprecedentedly beneficial. In this study, we explored the anti-stress and differentiation-inducing potential of two marine bioactive carotenoids (astaxanthin and fucoxanthin) using rat glioma cells as a model. We found that the low (nontoxic) doses of both protected cells against UV-induced DNA damage, heavy metal, and heat-induced protein misfolding and aggregation of proteins. Their long-term treatment in glioma cells caused the induction of physiological differentiation into astrocytes. These phenotypes were supported by upregulation of proteins that regulate cell proliferation, DNA damage repair mechanism, and glial differentiation, suggesting their potential for prevention and treatment of stress, protein aggregation, and age-related pathologies.

Heat-induced aggregation and gelation of proteins from edible honey bee brood (Apis mellifera) as a function of temperature and pH

Edible insects offer a huge potential as a new source of proteins for food and feed and as a promising functional agent. In this regard, our study aimed to evaluate the effect of pH and temperature on aggregation and gelation of honey bee brood (Apis mellifera). Aggregation of soluble proteins was dependent on temperature and pH value and the highest coagulation was reached at 85 °C at pH 5 and 7 (73.7 and 68.4%, respectively). Changes in protein surface hydrophobicity, net charge and amount of available and buried sulfhydryl groups demonstrated the contribution of either covalent or non-covalent intermolecular interactions resulted in aggregation at various pH. The least gelation concentration of raw powder from honey bee brood varied from 5 to 11% on raw powder basis at pH 7 and 3, respectively. Moreover, the effect of pH on rheological and textural properties, as well as gel microstructure from honey bee brood is discussed. Therefore, freeze dried raw powder of honey bee brood has rich nutritional value and demonstrated gelation concentration comparable to conventional protein sources that reveal its potential as an ingredient for insect-based gel product.