This research paper addresses the hypothesis that powdered milk may contain amyloid fibrils. Amyloids are fibrillar aggregates of proteins. Up to this time, research on the presence of amyloids in food products are scarce. To check the hypothesis we performed thioflavin T fluorescence assay, X-ray powder diffraction, atomic force microscopy and fluorescence microscopy imaging. Our preliminary results show that commercially available milks contain fibrils that have features characteristic to amyloids. The obtained results can be interpreted in two opposite ways. The presence of amyloids could be considered as a hazard due to the fact that food products may induce amyloid related diseases. On the other hand, the presence of amyloids in traditionally consumed foodstuffs could serve as proof that fibrils of food proteins do not pose a threat for consumers.

Although the consequences of the crowded cell environments may affect protein folding, function and misfolding reactions, these processes are often studied in dilute solutions in vitro. We here used biophysical experiments to investigate the amyloid fibril formation process of the fish protein apo-β-parvalbumin in solvent conditions that mimic steric and solvation aspects of the in vivo milieu. Apo-β-parvalbumin is a folded protein that readily adopts an amyloid state via a nucleation–elongation mechanism. Aggregation experiments in the presence of macromolecular crowding agents (probing excluded volume, entropic effects) as well as small molecule osmolytes (probing solvation, enthalpic effects) revealed that both types of agents accelerate overall amyloid formation, but the elongation step was faster with macromolecular crowding agents but slower in the presence of osmolytes. The observations can be explained by the steric effects of excluded volume favoring assembled states and that amyloid nucleation does not involve monomer unfolding. In contrast, the solvation effects due to osmolyte presence promote nucleation but not elongation. Therefore, the amyloid-competent nuclei must be compact with less osmolytes excluded from the surface than either the folded monomers or amyloid fibers. We conclude that, in contrast to other amyloidogenic folded proteins, amyloid formation of apo-β-parvalbumin is accelerated by crowded cell-like conditions due to a nucleation process that does not involve large-scale protein unfolding.

α-Synuclein (α-syn) is an intrinsically disordered protein with a highly asymmetric charge distribution, whose aggregation is linked to Parkinson’s disease. The effect of ionic strength was investigated at mildly acidic pH (5.5) in the presence of catalytic surfaces in the form of α-syn seeds or anionic lipid vesicles using thioflavin T fluorescence measurements. Similar trends were observed with both surfaces: increasing ionic strength reduced the rate of α-syn aggregation although the surfaces as well as α-syn have a net negative charge at pH 5.5. This anomalous salt dependence implies that short-range attractive electrostatic interactions are critical for secondary nucleation as well as heterogeneous primary nucleation. Such interactions were confirmed in Monte Carlo simulations of α-syn monomers interacting with surface-grafted C-terminal tails, and found to be weakened in the presence of salt. Thus, nucleation of α-syn aggregation depends critically on an attractive electrostatic component that is screened by salt to the extent that it outweighs the screening of the long-range repulsion between negatively charged monomers and negative surfaces. Interactions between the positively charged N-termini of α-syn monomers on the one hand, and the negatively C-termini of α-syn on fibrils or vesicles surfaces on the other hand, are thus critical for nucleation.

The bulk of the major core protein VP7 in African horse sickness virus (AHSV) self-assembles into flat, hexagonal crystalline particles in a process appearing unrelated to viral replication. Why this unique characteristic of AHSV VP7 is genetically conserved, and whether VP7 aggregation and particle formation have an effect on cellular biology or the viral life cycle, is unknown. Here we investigated how different small peptide and enhanced green fluorescent protein (eGFP) insertions into the VP7 top domain affected VP7 localization, aggregation, and particle formation. This was done using a dual laser scanning confocal and transmission electron microscopy approach in conjunction with analyses of the solubility, aggregation, and fluorescence profiles of the proteins. VP7 top domain modifications did not prevent trimerization, or intracellular trafficking, to one or two discrete sites in the cell. However, modifications that resulted in a misfolded and insoluble VP7-eGFP component blocked trafficking, and precluded protein accumulation at a single cellular site, perhaps by interfering with normal trimer–trimer interactions. Furthermore, the modifications disrupted the stable layering of the trimers into characteristic AHSV VP7 crystalline particles. It was concluded that VP7 trafficking is driven by a balance between VP7 solubility, trimer forming ability, and trimer–trimer interactions.

Aberrant aggregation of improperly folded proteins is the hallmark of several human neurodegenerative disorders, including Huntington's Disease (HD) with autosomal-dominant inheritance. In HD, expansion of the CAG-repeat-encoded polyglutamine (polyQ) stretch beyond ~40 glutamines in huntingtin (Htt) and its N-terminal fragments leads to the formation of large (up to several μm) globular neuronal inclusion bodies (IBs) over time. We report direct observations of aggregating Htt exon 1 in living and fixed cells at enhanced spatial resolution by stimulated emission depletion (STED) microscopy and single-molecule super-resolution optical imaging. Fibrils of Htt exon 1 arise abundantly across the cytosolic compartment and also in neuritic processes only after nucleation and aggregation into a fairly advanced stage of growth of the prominent IB have taken place. Structural characterizations of fibrils by STED show a distinct length cutoff at ~1·5 µm and reveal subsequent coalescence (bundling/piling). Cytosolic fibrils are observed even at late stages in the process, side-by-side with the mature IB. Htt sequestration into the IB, which in neurons has been argued to be a cell-protective phenomenon, thus appears to saturate and over-power the cellular degradation systems and leaves cells vulnerable to further aggregation producing much smaller, potentially toxic, conformational protein species of which the fibrils may be comprised. We further found that exogenous delivery of the apical domain of the chaperonin subunit CCT1 to the cells via the cell medium reduced the aggregation propensity of mutant Htt exon 1 in general, and strongly reduced the occurrence of such late-stage fibrils in particular.

Caprine and bovine milks have a similar overall gross composition, but vary considerably in the ratios of their casein components. These differences in colloidal casein micelles could affect directly or indirectly the heat stability of caprine and bovine milks at their natural pH. In the present work, the differences in colloidal stability of caprine and bovine milk have been studied by analysing the effect of heat treatment and skimming on precipitation of proteins. Raw and heated milk samples (70 °C/5 min, 80°C/5 min and 90°C/5 min) were centrifuged at 600, 2000, and 4500 g. The amount of precipitate formed after skimming was measured and the protein composition of both precipitates and supernatants analysed using the SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and densitometry. In caprine milk, the heat treatment prior to skimming had a statistically significant effect on protein precipitation. Centrifugal force had a statistically significant effect on amount of precipitate for both milks, but the amount was 2 to 4 times higher for caprine milk. When defatting the milk for electrophoresis, a centrifugal force of 600 g appeared to be the most appropriate, in order to avoid protein loss and a possible error in the interpretation of results. Results of this study could also serve as the basis for further investigations on adjusting the skimming conditions for caprine milk in industrial dairy processing environment.

The effects of a bovine whey peptide product enriched in proline (wPRP) on the solubility of milk proteins were tested under ambient conditions or following heat treatment at 75 and 100 °C, for 1 and 15 min, followed by post-incubation storage at either ambient temperature or 4 °C for up to 7 d. wPRP promoted solubilisation of milk proteins in a concentration-dependent manner without heat treatment and also after heat treatment at 75 and 100 °C, and the effect was enhanced after storage under either ambient or refrigerated storage conditions. Interactions of wPRP and milk proteins were monitored by particle size analysis and tryptic digestion and specifically linked with solubilisation of αS1 casein (αS1-Cn), which supported observed changes in milk protein solubility. The results suggested that wPRP preferably prevented or reversed physical versus covalent protein aggregation, with the relaxation of hydrophobic interactions at 4 °C providing an additive effect. This application of wPRP represents a novel approach to stabilisation of dairy proteins following thermal processing with industrial usefulness yet to be explored.

Numerous human diseases are associated with conformational change and aggregation of proteins, including Alzheimer's, Parkinson's, prion diseases (such as mad cow disease), familial amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease), Huntington's, and type II (mature onset) diabetes. In many cases, it has been demonstrated that conformational change and aggregation can occur outside living cells and complex biochemical networks. Hence, approaches from materials and physical science have enhanced our understanding of the role of protein aggregation in these diseases at the molecular and nanoscale levels. In this article, we will review what is known about these protein structures from the perspective of materials science, focusing on the details of emergent oligomeric and nanotube-like structures, their interactions with model lipid bilayers, how the structures relate to observed biological phenomena, and how protein aggregation and amyloid formation can be employed for the good in biology and materials science.

In this review, we discuss examples that show how glial-cell pathology is increasingly recognized in several neurodegenerative diseases. We also discuss the more provocative idea that some of the disorders that are currently considered to be neurodegenerative diseases might, in fact, be due to primary abnormalities in glia. Although the mechanism of glial pathology (i.e. modulating glutamate excitotoxicity) might be better established for amyotrophic lateral sclerosis (ALS), a role for neuronal–glial interactions in the pathogenesis of most neurodegenerative diseases is plausible. This burgeoning area of neuroscience will receive much attention in the future and it is expected that further understanding of basic neuronal–glial interactions will have a significant impact on the understanding of the fundamental nature of human neurodegenerative disorders.

One of the proposed roles of the GroEL-GroES cavity is to provide an “infinite dilution” folding chamber where protein substrate can fold avoiding deleterious off-pathway aggregation. Support for this hypothesis has been strengthened by a number of studies that demonstrated a mandatory GroES requirement under nonpermissive solution conditions, i.e., the conditions where proteins cannot spontaneously fold. We have found that the refolding of glutamine synthetase (GS) does not follow this pattern. In the presence of natural osmolytes trimethylamine N-oxide (TMAO) or potassium glutamate, refolding GS monomers readily aggregate into very large inactive complexes and fail to reactivate even at low protein concentration. Surprisingly, under these “nonpermissive” folding conditions, GS can reactivate with GroEL and ATP alone and does not require the encapsulation by GroES. In contrast, the chaperonin dependent reactivation of GS under another nonpermissive condition of low Mg2+ (<2 mM MgCl2) shows an absolute requirement of GroES. High-performance liquid chromatography gel filtration analysis and irreversible misfolding kinetics show that a major species of the GS folding intermediates, generated under these “low Mg2+” conditions exist as long-lived metastable monomers that can be reactivated after a significantly delayed addition of the GroEL. Our results indicate that the GroES requirement for refolding of GS is not simply dictated by the aggregation propensity of this protein substrate. Our data also suggest that the GroEL-GroES encapsulated environment is not required under all nonpermissive folding conditions.

The denaturant-induced (un)folding of apoflavodoxin from Azotobacter vinelandii has been followed at the residue level by NMR spectroscopy. NH groups of 21 residues of the protein could be followed in a series of 1H–15N heteronuclear single-quantum coherence spectra recorded at increasing concentrations of guanidinium hydrochloride despite the formation of protein aggregate. These NH groups are distributed throughout the whole apoflavodoxin structure. The midpoints of unfolding determined by NMR coincide with the one obtained by fluorescence emission spectroscopy. Both techniques give rise to unfolding curves with transition zones at significantly lower denaturant concentrations than the one obtained by circular dichroism spectroscopy. The NMR (un)folding data support a mechanism for apoflavodoxin folding in which a relatively stable intermediate is involved. Native apoflavodoxin is shown to cooperatively unfold to a molten globule-like state with extremely broadened NMR resonances. This initial unfolding step is slow on the NMR chemical shift timescale. The subsequent unfolding of the molten globule is faster on the NMR chemical shift timescale and the limited appearance of 1H–15N HSQC cross peaks of unfolded apoflavodoxin in the denaturant range studied indicates that it is noncooperative.

The most common form of systemic amyloidosis originates from antibody light chains. The large number of amino acid variations that distinguish amyloidogenic from nonamyloidogenic light chain proteins has impeded our understanding of the structural basis of light-chain fibril formation. Moreover, even among the subset of human light chains that are amyloidogenic, many primary structure differences are found. We compared the thermodynamic stabilities of two recombinant κ4 light-chain variable domains (VLs) derived from amyloidogenic light chains with a VL from a benign light chain. The amyloidogenic VLs were significantly less stable than the benign VL. Furthermore, only the amyloidogenic VLs formed fibrils under native conditions in an in vitro fibril formation assay. We used site-directed mutagenesis to examine the consequences of individual amino acid substitutions found in the amyloidogenic VLs on stability and fibril formation capability. Both stabilizing and destabilizing mutations were found; however, only destabilizing mutations induced fibril formation in vitro. We found that fibril formation by the benign VL could be induced by low concentrations of a denaturant. This indicates that there are no structural or sequence-specific features of the benign VL that are incompatible with fibril formation, other than its greater stability. These studies demonstrate that the VL β-domain structure is vulnerable to destabilizing mutations at a number of sites, including complementarity determining regions (CDRs), and that loss of variable domain stability is a major driving force in fibril formation.