Fig 4 - uploaded by P. Cooke
Content may be subject to copyright.
Three-dimensional models for -casein. Polypeptide backbone structure for the working molecular model of monomeric -casein (left); Pro is indicated by P (Kumosinski et al., 1993). The C chain trace of the hydrophobic dimer and tetramer of reduced carboxymethylated -casein (right); this structure may simulate the building block of the fibrillar structures.
Source publication
The caseins of milk form a unique calcium–phosphate transport complex that provides these necessary nutrients to the neonate. The colloidal stability of these particles is primarily the result of κ-casein. As purified from milk, this protein occurs as spherical particles with a weight average molecular weight of 1.18 million. The protein exhibits a...
Citations
... Caseins can also act as non-ATP-dependent, holdase-type molecular chaperones to limit globular protein unfolding and aggregation under stress conditions (Bhattacharyya and Das, 1999;Morgan et al., 2005). All four of the cow caseins can form amyloid fibrils (Bahraminejad et al., 2022;Farrell et al., 2003;Thorn et al., 2005;Treweek et al., 2011) but because of their action as molecular chaperones a mixture of two or more different caseins can instead form an amorphous oligomeric structure. Indeed, it has been suggested that the native casein micelle, invariably a mixture of twofive caseins, is the means by which the mammary tissue remains free of amyloid structures even though milk contains mM concentrations of amyloidogenic caseins (Bahraminejad et al., 2022), ...
... Caseins form a supramolecular structure termed a 'casein micelle' to transport calcium and phosphate to the neonate (Holt et al., 2013), and κ-CN is widely recognised as the protein which creates the outer layer of the micelle and provides steric and electrostatic repulsion between micelles to prevent aggregation (De Kruif, 1998;Horne, 2003;Walstra, 1999;Waugh, 1958). κ-CN is 169 amino acids in length, containing one tryptophan residue and belongs to the intrinsically disordered protein family that lacks an ordered tertiary structure (Farrell et al., 2004;Farrell et al., 2003). There are 14 κ-CN genetic variants that have been identified so far and the amino acid sequence for the κ-CN variants reported herein are described in Hewa Nadugala et al. (2022). ...
... No incubation or lag phase in formation was visible, confirming the findings of others for κ-CN (Ecroyd et al., 2008) and other intrinsically disordered proteins. Moreover, protein concentration dependent differences in initial fluorescence were noted for both UG and 2G κ-CN B, in agreement with Ecroyd et al. (2008), Farrell et al. (2003 and Thorn et al. (2005). These concentration dependent differences in thioflavin T fluorescence were observed to continue over time for both UG and 2G κ-CN B, however the rate of increase reduced and almost plateaued for 2G κ-CN B across all protein concentrations from around the 360 min point onwards, indicating that a rate limiting step for fibril formation was reached based on substrate availability as shown by Ecroyd et al. (2008). ...
... The TEM micrographs show that both UG and 2G κ-CN B are present as curly and rugged surface linear aggregates, which aligns with Chun et al. (2012) who observed thicker fibrils with a rugged surface using unreduced forms of κ-CN, while Ecroyd et al. (2008), Ecroyd et al. (2010), Leonil et al. (2008), and Pan and Zhong (2015) observed substantially longer fibrils with a smooth surface using reduced forms of κ-CN. Although disulphide bond reduction does not significantly affect the secondary and tertiary structure of κ-CN (Farrell et al., 2003), it is believed that the ability of κ-CN to form oligomers, and the subsequent propensity for amyloid fibril formation, is influenced by changes in the number of disulphide bonds links of cysteine 11 and cysteine 88 (Hewa Nadugala et al., 2022;Rasmussen et al., 1992). This indicates that β-sheet assembly might be different with the presence or absence of disulphide bonds during fibril formation. ...
In order to explore the functions of glycosylation of κ-Casein (κ-CN) in bovine milk, unglycosylated (UG) and twice glycosylated (2G) forms of κ-CN B were purified by selective precipitation followed by anion exchange chromatography from κ-CN BB milk and tested for their amyloid fibril formation and morphology, oligomerisation states and protein structure. The diameter of self-assembled κ-CN B aggregates of both glyco-form were shown for the first time to be in the same 26.0–28.7 nm range for a 1 mg mL⁻¹ solution. The presence of two bound glycans in the protein structure of 2G κ-CN B led to a greater increase in the maximum amyloid fibril formation rate with increasing protein concentration and a difference in both length (82.0 ± 29.9 vs 50.3 ± 13.7 nm) and width (8.6 ± 2.1 vs 13.9 ± 2.5 nm) for fibril morphology compared to UG κ-CN B. The present results suggest that amyloid fibril formation proceeds at a slow but steady rate via the self-assembly of dissociated, monomeric κ-CN B proteins at concentrations of 0.22–0.44 mg mL⁻¹. However amyloid fibril formation proceeds more rapidly via the assembly of either aggregated κ-CN present in a micelle-like form or dissociated monomeric κ-CN, packed into reorganised formational structures above the critical micellar concentration to form fibrils of differing width. The degree of glycosylation has no effect on the polarity of the adjacent environment, nor non-covalent and disulphide interactions between protein molecules when in the native form. Yet glycosylation can influence protein folding patterns of κ-CN B leading to a reduced tryptophan intrinsic fluorescence intensity for 2G compared to UG κ-CN B. These results demonstrate that glycosylation plays an important role in the modulation of aggregation states of κ-CN and contributes to a better understanding of the role of glycosylation in the formation of amyloid fibrils from intrinsically disordered proteins.
... Amyloid fibril formation by peptides and proteins is associated with a wide range of human disorders including Alzheimer's and Parkinson's diseases [15,16]. In vitro, solutions of purified cow jcasein or a S2 -casein readily form amyloid fibrils under physiological conditions [17][18][19], whereas the other two purified caseins require harsher conditions to do so [20]. Given the intrinsic tendency of caseins to form amyloid fibrils, it is remarkable that the action of caseins as molecular chaperones prevents fibril formation from occurring within the casein micelles [21,22]. ...
... (B) Schematic representation of the weak interactions between intrinsically disordered proteins. Left to right: Caseins contain many steric zipper SLiMs, that is, amyloid fibril-forming hexapeptide segments, consistent with the ability of individual caseins to form stable amyloid fibrils [6,[17][18][19][20]. Low-complexity aromatic-rich kinked segments (LARKS) adopt transient cross b-sheet structures (the characteristic structural feature of amyloid fibrils) that can interact with each other [43,44]. ...
Casein micelles are extracellular polydisperse assemblies of unstructured casein proteins. Caseins are the major component of milk. Within casein micelles, casein molecules are stabilised by binding to calcium phosphate nanoclusters and, by acting as molecular chaperones, through multivalent interactions. In light of such interactions, we discuss whether casein micelles can be considered as extracellular condensates formed by liquid‐liquid phase separation. We analyse the sequence, structure and interactions of caseins in comparison to proteins forming intracellular condensates. Furthermore, we review the similarities between caseins and small heat‐shock proteins whose chaperone activity is linked to phase separation of proteins. By bringing these observations together, we describe a regulatory mechanism for protein condensates, as exemplified by casein micelles. Casein proteins are the major components of milk, where they form large, polydisperse assemblies known as casein micelles. Within these extracellular casein micelles, caseins are stabilised by binding to calcium phosphate nanoclusters and through multivalent interactions. Here, we provide evidence that these interactions are similar to those associated with liquid‐liquid phase separation in intracellular biomolecular condensates such as membraneless organelles.
... Specifically, micelle structures have been used for controlled release of hydrophobic bioactive components (such as vitamins) and/or hydrophilic molecules that can be embedded in these nano-supramolecular structures 28 . Caseins distinctive structural properties of IDPs and the capability to form amyloid fibrils make also them an interesting model system in relation to the analysis of potentially pathological proteinmembrane interactions [29][30][31] . The ability of -casein in binding membranes seems to be related to the antibacterial activity of short peptides isolated within the sequence of this protein 32,33 . ...
Background:
Environmental conditions regulate the association/aggregation states of proteins and their action in cellular compartments. Analysing protein behaviour in presence of lipid membranes is fundamental for the comprehension of many functional and dysfunctional processes. Here, we present an experimental study on the interaction between model membranes and α-casein. α-casein is the major component of milk proteins and it is recognised to play a key role in performing biological functions. The conformational properties of this protein and its capability to form supramolecular structures, like micelles or irreversible aggregates, are key effectors in functional and pathological effects.
Methods:
By means of quantitative fluorescence imaging and complementary spectroscopic methods, we were able to characterise α-casein association state and the course of events induced by pH changes, which regulate the interaction of this molecule with membranes.
Results:
The study of these complex dynamic events revealed that the initial conformation of the protein critically regulates the fate of α-casein, size and structure of the newly formed aggregates and their effect on membrane structures. Disassembly of micelles due to modification in electrostatic interactions results in increased membrane structure rigidity which accompanies the formation of protein lipid flower-like co-aggregates with protein molecules localised in the external part.
General significance:
These results may contribute to the comprehension of how the initial state of a protein establishes the course of events that occur upon changes in the molecular environment. These events which may occur in cells may be essential to functional, pathological or therapeutical properties specifically associated to casein proteins.
... In general, IDPs have an open and solvent-exposed conformation due to a predominantly hydrophilic sequence (Bowman et al., 2020;Schneider, Jensen, & Blackledge, 2019;van der Lee et al., 2014). More recently, other key similarities between caseins and many IDPs have been identified, including a tendency to form amyloid fibrils (Farrell, Cooke, Wickham, Piotrowski, & Hoagland, 2003;Thorn, Ecroyd, Sunde, Poon, & Carver, 2008;Thorn et al., 2005), a preponderance of the helical but thermally unstable poly-L-proline type II (PP-II) secondary structure (Greenfield, 2006;Syme et al., 2002;van der Lee et al., 2014) and an ability to act as molecular chaperones to prevent or slow amorphous aggregation or amyloid fibril formation by unfolding globular proteins or destabilised peptides or IDPs (Bhattacharyya & Das, 1999;Morgan, Treweek, Lindner, Price, & Carver, 2005). The PP-II structure is characteristic of IDPs but almost absent in globular proteins and hence the interpretation of, for example, circular dichroism spectra of IDPs, using globular protein models, overestimates the proportions of non-PP-II regular secondary structures (Barron, Blanch, & Hecht, 2002;Greenfield, 2006). ...
... The dynamics of exchange are of crucial importance in the role of caseins in suppressing the highly sequence-specific formation of amyloid fibrils by kand/or a S2 -caseins and the aggregation of unfolding whey proteins. Under in vitro physiological conditions, kand a S2 -casein form amyloid fibrils in the absence of the other caseins, with k-casein having the greater proclivity to do so (Farrell et al., 2003;Thorn et al., 2005Thorn et al., , 2008. For k-casein, dissociation from its oligomeric state to form the monomer is the ratedetermining step in fibril formation Ecroyd, Thorn, Liu, & Carver, 2010). ...
Compatible theories describing the size distribution of milk casein micelles, the partition of milk salts and the formation of calcium phosphate nanocluster complexes are combined to provide the first quantitative model of the cow casein micelle. Complexes of caseins with calcium phosphate nanoclusters associate with each other and the remaining (free) caseins to form a stable, polydisperse distribution of micelle sizes. The micelles in the distribution have a highly hydrated coat-core structure in which the calcium phosphate nanoclusters, and caseins bound directly to them, occur only in the core whereas the free caseins are found in both the coat and core. The theory is used to describe the structure, substructure, surface potential, size distribution, and the salt and protein composition of casein micelles in a standard cow milk. Multivalent casein-casein interactions through short, linear, amino acid sequence motifs determine many aspects of casein micelle formation and stability.
... Fibril formation is uncommon in liquid milk, although some evidence suggests the possible existence of very short (7-10 nm) fibril-like structures in bovine casein micelles (Lencki, 2007). Amyloid fibril formation has been documented for κ-CN (Farrell et al., 2003;Thorn et al., 2005;Leonil et al., 2008), for α S2 -CN (Thorn et al., 2008), and to some extent for isolated β-CN (Pan and Zhong, 2015). The latter, however, formed fibrils only under nonphysiological conditions at pH 2 and 90°C over 30 h. ...
... Based on the significant increase of κ-CN in the pellet with concurrent significant decrease in the supernatant (and the opposite development for β-CN in the supernatant), it appears that, out of the 3 fibrillogenic candidates (α S2 -CN, β-CN, κ-CN; Farrell et al., 2003;Thorn et al., 2005;Thorn et al., 2008), κ-CN is the most likely constituent of the fibrils seen in this study. The diverging but complimentary trends in the distribution of κ-CN and β-CN in the pellet and supernatant, respectively, mirror the fibrillar and amorphous structure formation within the casein matrix and indicate a functional relationship where fibril formation is Vollmer et al.: CASEIN FIBRILS IN A PROCESSED CHEESE MODEL no longer suppressed by the chaperone activity of β-CN on its natural target, κ-CN. ...
The effects of varying the concentration of pentasodium triphosphate (PP) emulsifying salt [0, 0.6, 1.2, 1.5, and 1.8%, plus 0.9% of a mixture of citric acid (CA) and disodium phosphate (DSP) to adjust cheese pH to 5.85] on rheological, textural, physicochemical, and microstructural properties were studied in a processed cheese model system containing ~20% micellar casein concentrate, ~20% sunflower oil, and ~59% water. Special emphasis was placed on the unique casein fibrils recently described in a comparable processed cheese model system. Our results show that during processing (90°C, 17.37 rpm over 270 min) the apparent viscosity increased more and faster for formulations containing higher concentrations of PP, in analogy to the so-called creaming reaction, a general thickening of the molten cheese mass with prolonged processing. We found that 1.2% PP (plus 0.9% CA-DSP) appeared to be the threshold for the creaming reaction to take place. With increasing PP concentrations, cheese hardness increased in a sigmoidal fashion, and insoluble (protein-bound) calcium concentration decreased exponentially. Light micrographs of samples taken at the end of processing indicated initially large and dense casein aggregates within the matrix that disappeared with higher levels of PP, in parallel with the development of a finer emulsion. With transmission electron microscopy analysis on the same samples, the highly complex restructuring of the casein matrix was evident; casein fibrils had formed de novo at the periphery of the loosening casein aggregates. With higher levels of PP, amorphous areas were observed in place of the dense casein aggregates that appeared progressively void of protein, whereas fibril concentration increased throughout the rest of the matrix. Fibrils progressively attached to the surface of fat globules, thereby emulsifying them. Reverse-phase HPLC analysis of insoluble and soluble fractions indicated κ-casein to be the most likely constituent of the newly formed fibrils. The results of this study suggest that PP induced a concentration-dependent dissociation of caseins (through increased calcium chelation) and further led to their spatial separation. In essence, their chaperone activity was hindered, which resulted in amorphous aggregation on the one hand and fibril formation on the other.
... Fibril formation has been demonstrated for native and reduced forms of κ-CN (Farrell et al., 2003;Thorn et al., 2005Thorn et al., , 2008Lee et al., 2019), α S2 -CN (Thorn et al., 2008), and, to some degree, β-CN, although not under physiological conditions (Pan and Zhong, 2015). Fibril formation could not be induced for α S1 -CN (Thorn et al., 2008). ...
The “creaming reaction,” a general thickening of the molten cheese mass during the manufacture of processed cheese, which is often seen to occur in a stepwise fashion, affects the viscosity and texture of the finished product. Thus, this phenomenon is of critical importance for the processed cheese industry, yet mechanisms underlying the structure formation in this surprisingly complex and dynamic food system are only poorly understood. Using a model system consisting of micellar casein concentrate, vegetable oil, water, and a mixture of melting salts, we followed the characteristic viscosity profile with its primary and secondary increase over time. A rheometer equipped with a custom-made cup geometry was used, which served as a mini-reaction vessel to simulate the conditions during the manufacture of processed cheese. The mixture was subjected to constant heat (90°C) and stirring (7.93 rpm), comparable to processed cheese cooking, for up to 410 min. At specific time points, samples were taken, and the micro- and ultrastructure was investigated with light and transmission electron microscopy. Results from our extensive study uncovered the following key steps: (1) a decrease in fat globule size with concomitant increase in the number of fat globules, which were also more evenly distributed; (2) a progressive separation of the casein matrix into fibrillogenic and nonfibrillogenic fractions; (3) formation of fibrils and their higher-order structuring followed by their partial degradation; and (4) increasing interactions of the fibrils with the fat globule surface leading to a higher degree of emulsification. Of these different observations, results indicate that after the caseins dissociated under the influence of the melting salts, protein–protein interactions were the primary driver of the structure formation and thus contributed to the initial viscosity increase. Fat globules were involved in the structure formation at later time points. Therefore, fat–protein interactions in addition to continued protein–protein interactions were assumed to contribute to the secondary viscosity increase. An updated processed cheese creaming model is presented. The use of the term “texturization” instead of “creaming” is proposed.
... This is the smallest of the caseins and is responsible for the steric stability of the casein micelles [36]. In native environments, κ-casein forms multimeric colloidal systems through several types of interactions, such as disulfide bonds, electrostatic, hydrogen bonds, and hydrophobic interactions [37][38][39][40]. Researchers observed that κ-casein, both in its native and reduced-carboxymethylated form, is able to form amyloid fibrils in vitro if incubated at 37 • C [41][42][43][44]. ...
... In Figure 4a, the κ-casein spectra at the beginning of the aggregation process, which starts with thermal incubation at 37 • C (t 0 ), and after about 24 h (t end ), corresponding to the plateau in protein fibrillogenesis kinetics ( Figure 1), are reported. We found that, similar to what was reported by Farrell et al. [40], the protein during amyloid formation underwent a conformational transition with two spectral changes, one at 200 nm, and the other, more marked, at around 220 nm. As shown by electron microscopy experiments, these spectral changes are associated, by Farrell et al., with the formation of structures with fibrillar morphology. ...
... A fresh stock solution of κ-casein in 50 mM phosphate buffer pH 7.4 was continuously stirred for 24 h and filtered through 0.22 µm filters before using. As verified by dynamic light scattering, this procedure assures sample homogeneity but not reduction to monomeric species, which cannot be obtained due to the presence, in the κ-casein sample, of stable self-associating oligomeric micelle-like species [40]. The protein concentration was determined by recording the absorbance spectrum after filtering the sample by using an extinction coefficient at 280 nm of 0.95 mg −1 ·mL·cm −1 [39] and considering the purity of the sample (70%) provided by Sigma Aldrich. ...
Waste valorization represents one of the main social challenges when promoting a circular economy and environmental sustainability. Here, we evaluated the effect of the polyphenols extracted from apple peels, normally disposed of as waste, on the amyloid aggregation process of κ-casein from bovine milk, a well-used amyloidogenic model system. The effect of the apple peel extract on protein aggregation was examined using a thioflavin T fluorescence assay, Congo red binding assay, circular dichroism, light scattering, and atomic force microscopy. We found that the phenolic extract from the peel of apples of the cultivar “Fuji”, cultivated in Sicily (Caltavuturo, Italy), inhibited κ-casein fibril formation in a dose-dependent way. In particular, we found that the extract significantly reduced the protein aggregation rate and inhibited the secondary structure reorganization that accompanies κ-casein amyloid formation. Protein-aggregated species resulting from the incubation of κ-casein in the presence of polyphenols under amyloid aggregation conditions were reduced in number and different in morphology.
... They are analogous to the P-rich domain-linking disordered sequences in multi-domain globular proteins which have a low tendency to interact with other sequences [25]. Caseins are also typical of many other IDPs in having a tendency to form amyloid fibrils; bovine κ-and α S2 -caseins do so under physiological conditions [26][27][28]. Caseins can act as molecular chaperones [29][30][31][32] to limit amorphous and fibrillar aggregation of destabilised proteins. Indeed, within the amorphous casein micelle, the caseins act on each other as chaperones to limit or prevent amyloid formation, even at the high casein concentrations in the ducts and cisterns of the mammary gland [33]. ...
Bovine milk αS2-casein, an intrinsically disordered protein, readily forms amyloid fibrils in vitro and is implicated in the formation of amyloid fibril deposits in mammary tissue. Its two cysteine residues participate in the formation of either intra- or intermolecular disulphide bonds, generating monomer and dimer species. X-ray solution scattering measurements indicated that both forms of the protein adopt large, spherical oligomers at 20 °C. Upon incubation at 37 °C, the disulphide-linked dimer showed a significantly greater propensity to form amyloid fibrils than its monomeric counterpart. Thioflavin T fluorescence, circular dichroism and infrared spectra were consistent with one or both of the dimer isomers (in a parallel or antiparallel arrangement) being predisposed toward an ordered, amyloid-like structure. Limited proteolysis experiments indicated that at least part of the A⁸¹ − K¹¹³ region is incorporated into the fibril core, implying that this region, which is predicted by several algorithms to be amyloidogenic, initiates fibril formation of αS2-casein. The partial conservation of the cysteine motif and the frequent occurrence of disulphide-linked dimers in mammalian milks despite the associated risk of mammary amyloidosis, suggest that the dimeric conformation of αS2-casein is a functional, yet amyloidogenic, structure.
... Work done in the present century has only confirmed this conclusion by showing that caseins are largely hydrophilic compared to most globular proteins, although they are more hydrophobic than most other IDPs [28]. Thus, like many other intrinsically disordered proteins, caseins can form amyloid fibrils [29][30][31][32], act as molecular chaperones [33][34][35][36][37] and form homotypic or heterotypic disordered and highly hydrated amorphous oligomers and larger aggregates [38,39], sometimes called fuzzy complexes [40][41][42]. Intrinsically disordered proteins are more hydrophilic than globular proteins and normally form multivalent interactions in which the hydrophobic effect plays a minor part compared to polar interactions [42][43][44][45][46]. ...
