Heat-induced Aggregation Research Articles

Influence of L-homoarginine as an analogue of L-arginine on the heat-induced aggregation of proteins.

The influence of l-homoarginine on the heat-induced aggregation of three model proteins, i.e. porcine, mink, and human growth hormones was investigated by circular dichroism spectroscopy. It was found that the effect of l-homoarginine as an analogue of arginine depends on the concentration of the additive as well as the protein itself. l-Homoarginine increased the onset temperature of heat-induced aggregation of both porcine and mink growth hormones. However, the formation of human growth hormone aggregates was increased at low concentrations of l-homoarginine. Only at higher concentrations of the additive was the onset temperature of human growth hormone aggregation found to increase. Additional experiments of human growth hormone melting in the presence of histidine, lysine, and sodium chloride were performed. The effect of lysine was similar as in the presence of l-homoarginine. It follows that in protein formulations low concentrations of amino acids should be used with some precaution. At low concentration of additive, depending on the charge of both protein and amino acid used, the promotion of aggregation of unfolding intermediates may occur.

Foaming Characteristics of Commercial Soy Protein Isolate as Influenced by Heat-Induced Aggregation

The foaming properties of commercial soy protein isolate subjected to different temperatures (20–90°C) were assessed. The results revealed that the solubility and surface hydrophobicity of a 5% (w/v) commercial soy protein isolate suspension increased with increasing temperature, which increased foaming capacity and reduced foaming stability. Commercial soy protein isolate supernatant (i.e., soluble fraction) had higher foaming capacity at low temperatures (20–50°C). A high content of commercial soy protein isolate soluble fraction increased foaming capacity but decreased foaming stability. The SDS-PAGE patterns and molecular weight distribution of commercial soy protein isolate revealed that there were soluble, large molecular weight aggregates (>400 kDa) formed mainly from A and B-11S polypeptides of commercial soy protein isolate via disulfide bonds. Additionally, some aggregates also dissociated into small polypeptides and subunits after heat treatment. Commercial soy protein isolate precipitate (i.e., insoluble fraction) had a high content of proline and cysteine, which probably contributed to the foaming stability of commercial soy protein isolate.

Impact of environment conditions on physicochemical characteristics of ovalbumin heat-induced nanoparticles and on their ability to bind PUFAs

In this work, OVA heat-induced aggregates were obtained by controlled heat treatment varying temperature (60, 70 and 80 °C), heating time (3, 5 and10 min), aqueous medium pH (5.5, 6.0 and 6.5) and protein concentration (5, 10 and 14.2 g/L). Particle size distribution and surface characteristics derived from fluorescence spectroscopy (both intrinsic and extrinsic) were determined. Evaluation of these physicochemical properties allowed knowing experimental conditions under which nanometric OVA aggregates (OVAn), with suitable surface hydrophobicity, could be produced. OVAn ability to bind polyunsaturated fatty acid (PUFA) was carried out by turbidity measurement. In these experiments, linoleic acid (LA) was used as a PUFA model. In general, OVA aggregate sizes increased with temperature, time and protein concentration, furthermore at higher pH value, OVA aggregate sizes were lower. OVA aggregates surface hydrophobicity increased with rising heating temperature and the pH value indicating, on one hand, larger protein unfolding and on the other hand lower aggregation via hydrophobic interaction at higher pH value, respectively. However, surface hydrophobicity decreased with concentration suggesting greater aggregation. OVAn obtained at 70 °C, 10 g/L, pH 6–6.5 and 70 °C, 5 g/L, pH 6 were assayed by LA binding ability. This property was 1.4–2.0 folds greater than for native OVA. Information derived from this work could be of practical interest for the development of innovative PUFAs carrier systems.

Effect of Ca2+ and Mg2+ ions on oligomeric state and chaperone-like activity of αB-crystallin in crowded media

The main function of small heat shock proteins acting as suppressors of aggregation of non-native proteins is greatly influenced by crowded environment in the cell and the presence of divalent metal ions. The goal of the present work was to study the effects of Ca2+ and Mg2+ ions on the quaternary structure and anti-aggregation activity of αB-crystallin under crowding conditions. We showed that Ca2+ and Mg2+ ions induced formation of suboligomeric forms of αB-crystallin. This effect was retained in the presence of crowder (polyethylene glycol), although to a lesser degree. The chaperone-like activity of αB-crystallin was analyzed using heat-induced aggregation of myosin subfragment 1 (S1) at 40°C. In the absence of crowding agents chaperone-like activity of αB-crystallin exhibited low sensitivity to the presence of Ca2+ and Mg2+ ions. The addition of the crowding agents (polyethylene glycol 20000, Ficoll 70) dramatically increased S1 aggregation rates and significantly depressed anti-aggregation activity of αB-crystallin. Low concentrations of Ca2+ (0.1mM) and Mg2+ (10mM) partially restored the chaperone-like activity of αB-crystallin in the presence of crowders. This effect was observed at relatively low values of [αB-crystallin]/[S1] molar ratio, however, at [αB-crystallin]/[S1]>0.2 the stimulating effect of Ca2+ became less pronounced. These findings might indicate that under crowded cell conditions different factors, including divalent cations, can effectively modulate chaperone-like activity of protein chaperones, which otherwise cannot properly cope with crowding-provoked accelerated rates of substrates aggregation.

Enhancement of ultrafiltration-performance and improvement of hygienic quality during the production of whey concentrates

Ultrafiltration (UF) is used for the concentration of whey proteins from whey. Membrane fouling during processing results in a decrease in flux, while the predominant molecular fouling reaction and fouling species are still largely unknown. Heat-induced whey protein aggregates are inevitably generated in industrial cheese-making processes, and were found to accelerate membrane fouling during whey concentration. By means of upstream microfiltration prior to UF, these aggregates can be selectively removed. By this means, UF flux could be increased up to twofold for the UF of sweet whey and threefold for acid whey. For filtration of sweet whey, significant calcium retention was observed. Therefore, membrane fouling for both whey types seem follow different molecular mechanisms. Besides the removal of whey protein aggregates, the retention of microorganisms and a log-reduction of up to log 3.5 was achieved by pre-filtration. Shelf-life of whey could be increased to two weeks without pasteurisation.

Functional adaptations of the bacterial chaperone trigger factor to extreme environmental temperatures.

Trigger factor (TF) is the first molecular chaperone interacting cotranslationally with virtually all nascent polypeptides synthesized by the ribosome in bacteria. Thermal adaptation of chaperone function was investigated in TFs from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli and the hyperthermophile Thermotoga maritima. This series covers nearly all temperatures encountered by bacteria. Although structurally homologous, these TFs display strikingly distinct properties that are related to the bacterial environmental temperature. The hyperthermophilic TF strongly binds model proteins during their folding and protects them from heat-induced misfolding and aggregation. It decreases the folding rate and counteracts the fast folding rate imposed by high temperature. It also functions as a carrier of partially folded proteins for delivery to downstream chaperones ensuring final maturation. By contrast, the psychrophilic TF displays weak chaperone activities, showing that these functions are less important in cold conditions because protein folding, misfolding and aggregation are slowed down at low temperature. It efficiently catalyses prolyl isomerization at low temperature as a result of its increased cellular concentration rather than from an improved activity. Some chaperone properties of the mesophilic TF possibly reflect its function as a cold shock protein in E. coli.

Effect of heating whey proteins in the presence of milk fat globule membrane extract or phospholipids from buttermilk

The objective of this study was to determine the contribution of phospholipids from buttermilk as a nucleus in the heat-induced aggregation of whey proteins. Solutions of whey proteins (5%, w/v) were adjusted to pH 4.6 or 6.8 and then heated at 65 or 80 °C for 25 min with or without 1% (w/v) of milk fat globule membrane (MFGM) extract or phospholipid powder. The aggregation mechanisms were characterised using analysis with Ellman's reagent, one-dimensional gel electrophoresis, thin-layer chromatography, and three-dimensional confocal laser-scanning microscopy. Three-dimensional images showed protein/phospholipid interactions in the presence of MFGM extract or phospholipids, and thin-layer chromatography plates showed no trace of free phospholipids after 20 min at pH 4.6. Overall, the results demonstrate that phospholipids from buttermilk were involved in the formation of protein aggregates through the MFGM fragments at a low temperature, whereas phospholipids could interact directly with the proteins at a higher temperature (80 °C).

Enhanced heat stability of high protein emulsion systems provided by microparticulated whey proteins

There is a desire to incorporate increasingly high concentrations of whey proteins into nutritional beverages to improve their nutritional content and amino acid profile. However, typical thermal treatments for sterility or extended shelf life cause undesirable aggregation/gelation of the whey proteins. To address this instability issue at high protein concentration, there is a need to develop new protein ingredient technologies that can provide resistance to heat-induced aggregation. In this study, the heat stability characteristics of model food emulsions containing standard or microparticulated whey protein concentrate (WPC80) were compared. Oil-in-water emulsions [10% (wt/wt) sunflower oil] containing increasing concentrations of protein (from 2 to 12 wt%) were prepared at pH 7.0. The effects of retorting at 120 °C for 10 min on particle size distribution, rheological properties, susceptibility to heat-induced coagulation, microstructure and surface protein concentration of the standard and microparticulated WPCs were compared. Also, various levels of NaCl were added to examine the heat stability of micro-aggregated WPC in the presence of additional salts.Microparticulated WPC emulsions showed significantly enhanced heat stability compared with standard WPC emulsions. Emulsions with up to 11 wt% protein and no visible aggregation or gelation after retorting were produced using microparticulated WPC. For standard WPC emulsions under the same heating conditions, large aggregates were formed and there was a change in flow behaviour to non-Newtonian at 3 wt% protein. With this specific technology, high protein whey-based nutritional beverages can be produced using conventional thermal treatments.

Tauroursodeoxycholic acid prevents stress induced aggregation of proteins in vitro and promotes PERK activation in HepG2 cells

Tauroursodeoxycholic acid (TUDCA) a bile salt and chemical chaperone reduces stress-induced aggregation of proteins; activates PERK [PKR (RNA-dependent protein kinase)-like ER (endoplasmic reticulum) kinase] or EIF2AK3, one of the hall marks of ER stress induced unfolded protein response (UPR) in human hepatoblastoma HepG2 cells; prevents heat and dithiothreitol (DTT) induced aggregation of BSA (bovine serum albumin), and reduces ANS (1-anilino-naphthalene-8-sulfonate) bound BSA fluorescence in vitro. TUDCA inactivates heat treated, but not the native EcoR1 enzyme, and reduces heat-induced aggregation and activity of COX-1 (cyclooxygenase enzyme-1) in vitro. These findings suggest that TUDCA binds to the hydrophobic regions of proteins and prevents their subsequent aggregation. This may stabilize unfolded proteins that can mount UPR or facilitate their degradation through cellular degradation pathways.

Heat-induced aggregation of thylakoid membranes affect their interfacial properties.

Many of our most popular lipid containing foods are in emulsion form. These foods are often highly palatable with high caloric density, that subsequently increases the risk of overconsumption and possibly lead to obesity. Regulating the lipid bioavailability of high-fat foods is one approach to prevent overconsumption. Thylakoids, the chloroplast membrane, creates a barrier around lipid droplets, which prolong lipolysis and increase satiety as demonstrated both in animal and human studies. However, a reduced lipase inhibiting capacity has been reported after heat treatment but the mechanism has not yet been fully established. The aim of this study was to investigate thylakoids' emulsifying properties post heat-treatment and possible links to alterations in lipase inhibiting capacity and chlorophyll degradation. Heat-treatment of thylakoids at either 60 °C, 75 °C or 90 °C for time interval ranging from 15 s to 4 min reduced ability to stabilise emulsions, having increased lipid droplets sizes, reduced emulsification capacity, and elevated surface load as consequence. Emulsifying properties were also found to display a linear relationship to both chlorophyll and lipase inhibiting capacity. The correlations support the hypothesis that heat-treatment induce chlorophyll degradation which promote aggregation within proteins inside the thylakoid membrane known to play a decisive role in interfacial processes. Therefore, heat-treatment of thylakoids affects both chlorophyll content, lipase inhibiting capacity and ability to stabilise the oil-water interface. Since the thylakoid's appetite reducing properties are a surface-related phenomenon, the results are useful to optimize the effect of thylakoids as an appetite reducing agent.

Effect of polyol sugars on the stabilization of monoclonal antibodies

We investigate the impact of sugars and polyols on the heat-induced aggregation of a model monoclonal antibody whose monomer depletion is rate-limited by protein unfolding. We follow the kinetics of monomer consumption by size exclusion chromatography, and we interpret the results in the frame of two mechanistic schemes describing the enhanced protein stability in the presence of polyols. It is found that the stabilization effect increases with increasing polyol concentration with a comparable trend for all of the tested polyols. However, the stabilization effect at a given polyol concentration is polyol specific. In particular, the stabilization effect increases as a function of polyol size until a plateau is reached above a critical polyol size corresponding to six carbon atoms. Our results show that the stabilization by polyols does not depend solely on the volume fraction filled by the polyol molecules, but is also affected by the polyol chemistry.

Effect of globular pea proteins fractionation on their heat-induced aggregation and acid cold-set gelation

This report focuses on cold-set gelation of pea (Pisum sativum L.) proteins and related globulin fractions, namely Vicilin 7S and Legumin 11S. Protein thermal denaturation and aggregation were investigated using differential scanning calorimetry (DSC) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and sulfhydryl (S−)/disulfide (S–S) bond assessment. While the denaturation temperature (Td) of pea proteins increased from about 69 to 77 °C with increasing legumin content, the disulfide-linked acidic (α) and basic (β) legumin subunits (56–58 kDa) denatured and aggregated in a temperature range of 75–85 °C. Dissociation of legumin oligomers and their rearrangements via hydrophobic interactions and sulfhydryl/disulfide bonds exchange reactions would occur concomitantly during the heat-treatment, giving rise mainly to high-molecular weight aggregates of random structure. However thermal denaturation and aggregation of vicilin molecules would involve solely non-covalent interactions. Then glucono-δ-lactone (GDL) acid-induced gelation of protein thermal aggregates was evaluated by means of G′ and G″ moduli. The preheated mixed pea globulins and vicilin-enriched samples gave rise to increased final moduli values of the acid gels, while legumin-enriched samples displayed low gelling properties. Improving functional properties of pea proteins would help to promote their application in plant-based gelled products such as tofu, alternatively to their soy counterparts.

Spatial quality control bypasses cell-based limitations on proteostasis to promote prion curing.

The proteostasis network has evolved to support protein folding under normal conditions and to expand this capacity in response to proteotoxic stresses. Nevertheless, many pathogenic states are associated with protein misfolding, revealing in vivo limitations on quality control mechanisms. One contributor to these limitations is the physical characteristics of misfolded proteins, as exemplified by amyloids, which are largely resistant to clearance. However, other limitations imposed by the cellular environment are poorly understood. To identify cell-based restrictions on proteostasis capacity, we determined the mechanism by which thermal stress cures the [PSI(+)]/Sup35 prion. Remarkably, Sup35 amyloid is disassembled at elevated temperatures by the molecular chaperone Hsp104. This process requires Hsp104 engagement with heat-induced non-prion aggregates in late cell-cycle stage cells, which promotes its asymmetric retention and thereby effective activity. Thus, cell division imposes a potent limitation on proteostasis capacity that can be bypassed by the spatial engagement of a quality control factor.

Thermally-induced structural changes in an armadillo repeat protein suggest a novel thermosensor mechanism in a molecular chaperone

Molecular chaperones are commonly identified by their ability to suppress heat-induced protein aggregation. The muscle-specific molecular chaperone UNC-45B is known to be involved in myosin folding and is trafficked to the sarcomeres A-band during thermal stress. Here, we identify temperature-dependent structural changes in the UCS chaperone domain of UNC-45B that occur within a physiologically relevant heat-shock range. We show that distinct changes to the armadillo repeat protein topology result in exposure of hydrophobic patches, and increased flexibility of the molecule. These rearrangements suggest the existence of a novel thermosensor within the chaperone domain of UNC-45B. We propose that these changes may function to suppress aggregation under stress by allowing binding to a wide variety of aggregation prone loops on its client.

Heat-induced formation of myosin oligomer-soluble filament complex in high-salt solution

Heat-induced aggregation of myosin into an elastic gel plays an important role in the water-holding capacity and texture of meat products. Here, we investigated thermal aggregation of porcine myosin in high-salt solution over a wide temperature range by dynamic light scattering experiments. The myosin samples were readily dissolved in 1.0M NaCl at 25°C followed by dilution into various salt concentrations. The diluted solutions consistently contained both myosin monomers and soluble filaments. The filament size decreased with increasing salt concentration and temperature. High temperatures above Tm led to at least partial dissociation of soluble filaments and thermal unfolding, resulting in the formation of soluble oligomers and binding to the persistently present soluble filaments. Such a complex formation between the oligomers and filaments has never been observed. Our results provide new insight into the heat-induced myosin gelation in high-salt solution.

A highly charged region in the middle domain of plant endoplasmic reticulum (ER)-localized heat-shock protein 90 is required for resistance to tunicamycin or high calcium-induced ER stresses.

Heat-shock protein 90 (HSP90) is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes under both physiological and stress conditions. In Arabidopsis, there are seven HSP90 isoforms (HSP90.1-HSP90.7) that are localized in the cytoplasm/nucleus, mitochondrion, chloroplast, and endoplasmic reticulum (ER) where protein folding actively takes place. In this study, we analysed the sequence of ER-localized Arabidopsis HSP90.7 and the other ER GRP94 proteins from plants and animals, and identified a short, charged region that is specifically present in the middle domain of plant-derived GRP94 proteins. To understand the role of this charged region, we analysed transgenic plants that expressed a mutant protein, HSP90.7(Δ22), which had this charged region deleted. We showed that seedlings expressing HSP90.7(Δ22) had significantly enhanced sensitivity to ER stress induced by tunicamycin or a high concentration of calcium, although its general chaperone activity in preventing the model protein from heat-induced aggregation was not significantly affected. We also analysed the ATP-binding and hydrolysis activity of both wild-type and mutant HSP90.7 proteins, and found that they had slightly different ATP-binding affinities. Finally, using a yeast two-hybrid screen, we identified a small set of HSP90.7 interactors and showed that the charged region is not required for the candidate client interaction, although it may affect their binding affinity, thus providing potential targets for further investigation of HSP90.7 functions.

Conformational stability, reversibility and heat-induced aggregation of α-1-acid glycoprotein.

To investigate the relationship between conformational stability, reversibility of denaturation and aggregation of protein, we determined the conformation, melting temperature (Tm), and reversibility of heat-induced denaturation of α-1-acid glycoprotein (AGP) in aqueous solutions at various pH values using circular dichroism (CD) and differential scanning microcalorimetry. To quantitate and characterize heat-induced AGP aggregation under the same pH conditions, solutions of AGP were incubated at 50°C and then analysed by size exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis, CD and SEC in the presence of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid. The conformational stability of AGP was reduced at lower pH, whereas the reversibility of protein denaturation was reduced at higher pH. AGP formed some large non-covalent aggregates during incubation at lower pH, whereas incubation at higher pH tended to cause the formation of dimer species without the formation of large aggregates. These results indicated that lower conformational stability was related to the formation of non-covalent large aggregates, whereas reduced reversibility was related to dimer formation. Thus, evaluating both conformational stability and reversibility is necessary for developing optimal formulations and to predict the kinds of aggregates that will be induced during protein storage.

Essential genetic interactors of SIR2 required for spatial sequestration and asymmetrical inheritance of protein aggregates.

Sir2 is a central regulator of yeast aging and its deficiency increases daughter cell inheritance of stress- and aging-induced misfolded proteins deposited in aggregates and inclusion bodies. Here, by quantifying traits predicted to affect aggregate inheritance in a passive manner, we found that a passive diffusion model cannot explain Sir2-dependent failures in mother-biased segregation of either the small aggregates formed by the misfolded Huntingtin, Htt103Q, disease protein or heat-induced Hsp104-associated aggregates. Instead, we found that the genetic interaction network of SIR2 comprises specific essential genes required for mother-biased segregation including those encoding components of the actin cytoskeleton, the actin-associated myosin V motor protein Myo2, and the actin organization protein calmodulin, Cmd1. Co-staining with Hsp104-GFP demonstrated that misfolded Htt103Q is sequestered into small aggregates, akin to stress foci formed upon heat stress, that fail to coalesce into inclusion bodies. Importantly, these Htt103Q foci, as well as the ATPase-defective Hsp104Y662A-associated structures previously shown to be stable stress foci, co-localized with Cmd1 and Myo2-enriched structures and super-resolution 3-D microscopy demonstrated that they are associated with actin cables. Moreover, we found that Hsp42 is required for formation of heat-induced Hsp104Y662A foci but not Htt103Q foci suggesting that the routes employed for foci formation are not identical. In addition to genes involved in actin-dependent processes, SIR2-interactors required for asymmetrical inheritance of Htt103Q and heat-induced aggregates encode essential sec genes involved in ER-to-Golgi trafficking/ER homeostasis.

The chaperone-like activity of α-synuclein attenuates aggregation of its alternatively spliced isoform, 112-synuclein in vitro: plausible cross-talk between isoforms in protein aggregation.

Abnormal oligomerization and aggregation of α-synuclein (α-syn/WT-syn) has been shown to be a precipitating factor in the pathophysiology of Parkinson's disease (PD). Earlier observations on the induced-alternative splicing of α-syn by Parkinsonism mimetics as well as identification of region specific abnormalities in the transcript levels of 112-synclein (112-syn) in diseased subjects underscores the role of 112-syn in the pathophysiology of PD. In the present study, we sought to identify the aggregation potential of 112-syn in the presence or absence of WT-syn to predict its plausible role in protein aggregation events. Results demonstrate that unlike WT-syn, lack of 28 aa in the C-terminus results in the loss of chaperone-like activity with a concomitant gain in vulnerability to heat-induced aggregation and time-dependent fibrillation. The effects were dose and time-dependent and a significant aggregation of 112-syn was evident at as low as 45°C following 10 min of incubation. The heat-induced aggregates were found to be ill-defined structures and weakly positive towards Thioflavin-T (ThT) staining as compared to clearly distinguishable ThT positive extended fibrils resulting upon 24 h of incubation at 37°C. Further, the chaperone-like activity of WT-syn significantly attenuated heat-induced aggregation of 112-syn in a dose and time-dependent manner. On contrary, WT-syn synergistically enhanced fibrillation of 112-syn. Overall, the present findings highlight a plausible cross-talk between isoforms of α-syn and the relative abundance of these isoforms may dictate the nature and fate of protein aggregates.

Effect of whey concentration on protein recovery in fresh ovine ricotta cheese

Ricotta cheese, particularly the ovine type, is a typical Italian dairy product obtained by heat-coagulation of the proteins in whey. The aim of this work was to investigate the influence of whey protein concentration, obtained by ultrafiltration, on yield of fresh ovine ricotta cheese. Ricotta cheeses were obtained by thermocoagulation of mixtures with protein content of 1.56, 3.10, 4.16, and 7.09g/100g from the mixing of skim whey and ultrafiltered skim whey. A fat-to-protein ratio of 1.1 (wt/wt) was obtained for all mixtures by adding fresh cream. The initial mixtures, as well as the final ricotta cheeses, were analyzed for their composition and by SDS-PAGE. Protein bands were quantified by QuantityOne software (Bio-Rad, Hercules, CA) and identified by liquid chromatography-tandem mass spectrometry. Significant differences in the composition of the ricotta cheese were observed depending on protein concentration. Particularly, ricotta cheese resulting from the mixture containing 7.09g/100g of protein presented higher moisture (72.88±1.50g/100g) and protein (10.18±0.45g/100g) contents than that prepared from the mixture with 1.56g/100g of protein (69.52±1.75 and 6.70±0.85g/100g, respectively), and fat content was lower in this sample (12.20±1.60g/100g) compared with the other treatments, with mean values between 15.72 and 20.50g/100g. Each protein fraction presented a different behavior during thermocoagulation. In particular, the recovery of β-lactoglobulin and α-lactalbumin in the cheese increased as their content increased in the mixtures. It was concluded that concentrating ovine rennet whey improved the extent of heat-induced protein aggregation during the thermal coagulation process. This resulted in a better recovery of each protein fraction in the product, and in a consequent increase of ricotta cheese yield.