Structure of biomimetic casein micelles: Critical tests of the hydrophobic colloid and multivalent-binding models using recombinant deuterated and phosphorylated ß-casein.

Milk contains high concentrations of amyloidogenic casein proteins and is supersaturated with respect to crystalline calcium phosphates such as apatite. Nevertheless, the mammary gland normally remains unmineralized and free of amyloid. Unlike κ-casein, ß- and αS-caseins are highly effective mineral chaperones that prevent ectopic and pathological calcification of the mammary gland. Milk invariably contains a mixture of two to five different caseins that act on each other as molecular chaperones. Instead of forming amyloid fibrils, several thousand caseins and hundreds of nanoclusters of amorphous calcium phosphate combine to form fuzzy complexes called casein micelles. To understand the biological functions of the casein micelle its structure needs to be understood better than at present. The location in micelles of the highly amyloidogenic κ-casein is disputed. In traditional hydrophobic colloid models, it, alone, forms a stabilizing surface coat that also determines the average size of the micelles. In the recent multivalent-binding model, κ-casein is present throughout the micelle, in intimate contact with the other caseins. To discriminate between these models, a range of biomimetic micelles was prepared using a fixed concentration of the mineral chaperone ß-casein and nanoclusters of calcium phosphate, with variable concentrations of κ-casein. A biomimetic micelle was also prepared using a highly deuterated and in vivo phosphorylated recombinant ß-casein with calcium phosphate and unlabelled κ-casein. Neutron and X-ray scattering experiments revealed that κ-casein is distributed throughout the micelle, in quantitative agreement with the multivalent-binding model but contrary to the hydrophobic colloid models.

When case reporting becomes untenable: Can sewer networks tell us where COVID-19 transmission occurs?

Monitoring SARS-CoV-2 in wastewater is a valuable approach to track COVID-19 transmission. Designing wastewater surveillance (WWS) with representative sampling sites and quantifiable results requires knowledge of the sewerage system and virus fate and transport. We developed a multi-level WWS system to track COVID-19 in Atlanta using an adaptive nested sampling strategy. From March 2021 to April 2022, 868 wastewater samples were collected from influent lines to wastewater treatment facilities and upstream community manholes. Variations in SARS-CoV-2 concentrations in influent line samples preceded similar variations in numbers of reported COVID-19 cases in the corresponding catchment areas. Community sites under nested sampling represented mutually-exclusive catchment areas. Community sites with high SARS-CoV-2 detection rates in wastewater covered high COVID-19 incidence areas, and adaptive sampling enabled identification and tracing of COVID-19 hotspots. This study demonstrates how a well-designed WWS provides actionable information including early warning of surges in cases and identification of disease hotspots.

Amyloid fibril formation by αS1- and ß-casein implies that fibril formation is a general property of casein proteins.

Caseins are a diverse family of intrinsically disordered proteins present in the milks of all mammals. A property common to two cow paralogues, αS2- and κ-casein, is their propensity in vitro to form amyloid fibrils, the highly ordered protein aggregates associated with many age-related, including neurological, diseases. In this study, we explored whether amyloid fibril-forming propensity is a general feature of casein proteins by examining the other cow caseins (αS1 and ß) as well as ß-caseins from camel and goat. Small-angle X-ray scattering measurements indicated that cow αS1- and ß-casein formed large spherical aggregates at neutral pH and 20°C. Upon incubation at 65°C, αS1- and ß-casein underwent conversion to amyloid fibrils over the course of ten days, as shown by thioflavin T binding, transmission electron microscopy, and X-ray fibre diffraction. At the lower temperature of 37°C where fibril formation was more limited, camel ß-casein exhibited a greater fibril-forming propensity than its cow or goat orthologues. Limited proteolysis of cow and camel ß-casein fibrils and analysis by mass spectrometry indicated a common amyloidogenic sequence in the proline, glutamine-rich, C-terminal region of ß-casein. These findings highlight the persistence of amyloidogenic sequences within caseins, which likely contribute to their functional, heterotypic self-assembly; in all mammalian milks, at least two caseins coalesce to form casein micelles, implying that caseins diversified partly to avoid dysfunctional amyloid fibril formation.

Are casein micelles extracellular condensates formed by liquid-liquid phase separation?

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 the 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 with 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.

A quantitative calcium phosphate nanocluster model of the casein micelle: the average size, size distribution and surface properties.

Caseins (αS1, αS2, ß and κ) are the main protein fraction of bovine milk. Together with nanoclusters of amorphous calcium phosphate (CaP) and divalent cations, they combine to form a polydisperse distribution of particles called casein micelles. A casein micelle model is proposed which is consistent with the way in which intrinsically disordered proteins interact through predominantly polar, short, linear, motifs. Using the model, an expression is derived for the size distribution of casein micelles formed when caseins bind to the CaP nanoclusters and the complexes further associate with each other and the remaining mixture of free caseins. The result is a refined coat-core model in which the core is formed mainly by the nanocluster complexes and the coat is formed exclusively by the free caseins. Example calculations of the size distribution and surface composition of an average bovine milk are compared with experiment. The average size, size distribution and surface composition of the micelles is shown to depend on the affinity of the nanocluster complexes for each other in competition with their affinity for free caseins, and on the concentrations of free caseins, calcium ions and other salts in the continuous phase.

Native disulphide-linked dimers facilitate amyloid fibril formation by bovine milk αS2-casein.

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 the region from Ala81 to Lys113 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.

Salt partition, ion equilibria, and the structure, composition, and solubility of micellar calcium phosphate in bovine milk with added calcium salts.

Increasing dietary calcium has been suggested to have a range of health benefits, such as reducing the risk of osteoporosis and hypertension. However, producing calcium-fortified products is challenging due to the destabilizing effect caused by added calcium. We provide new data on the effect of adding either calcium gluconate or calcium lactate at up to 50 mM on the partition of salts and the structure and solubility of micellar calcium phosphate (MCP). The empirical chemical formula of the MCP in milk with added calcium was Ca(HPO4)0.6(PO4)0.267, similar to that previously reported for the MCP in native bovine casein micelles. Ion equilibria calculations showed that the solubility of the MCP was decreased as measured by an increase in negative logarithm of the solubility constant (pKS) from 6.8 to 7.3 ± 0.1 and 7.5 ± 0.1 for milk with added calcium gluconate and calcium lactate, respectively. No substantial change in the amorphous structure of the MCP was observed by either X-ray powder diffraction or infrared spectroscopy of dried casein micelles as a result of added calcium. The conclusion is that the added calcium caused an increase in the concentration of the MCP and decreased its solubility without changing its amorphous structure or chemical composition.

Sequence characteristics responsible for protein-protein interactions in the intrinsically disordered regions of caseins, amelogenins, and small heat-shock proteins.

Milk caseins and dental amelogenins are intrinsically disordered proteins (IDPs) that associate with themselves and others. Paradoxically, they are also described as hydrophobic proteins, which is difficult to reconcile with a solvent-exposed conformation. We attempt to resolve this paradox. We show that caseins and amelogenins are not hydrophobic proteins but they are more hydrophobic than most IDPs. Remarkably, uncharged residues from different regions of these mature proteins have a nearly constant average hydropathy but these regions exhibit different charged residue frequencies. A novel sequence analysis method was developed to identify hydrophobic and order-promoting regions that would favor conformational collapse. We found that such regions were uncommon; most hydrophobic and order-promoting residues were adjacent to hydrophilic or disorder-promoting residues. A further reason why caseins and amelogenins do not collapse is their high proportion of disorder-promoting proline residues. We conclude that in these proteins the hydrophobic effect is not large enough to cause conformational collapse but it can contribute, along with polar interactions, to protein-protein interactions. This behaviour is similar to the interaction of the disordered N-terminal region of small heat-shock proteins with either themselves during oligomer formation or other, unfolding, proteins during chaperone action.

Functional and dysfunctional folding, association and aggregation of caseins.

Caseins are a group of closely related intrinsically disordered proteins (IDPs), best known for their occurrence in milk as stable, polydisperse, roughly spherical, amorphous particles, typically containing thousands of protein chains and hundreds of nanoclusters of calcium phosphate. The particles are called casein micelles though their structure bears no resemblance to detergent micelles. Caseins have an open and flexible conformation with a preponderance of poly-l-proline II secondary structure and hence cannot be described as hydrophobic proteins. Individually, and in combination, they associate through polar and non-polar interactions to form polydisperse fuzzy complexes (including the native casein micelle) while retaining their hydrated and flexible conformation to a large degree. Like many other IDPs, caseins are prone to form cytotoxic amyloid fibrils. However, they are also highly effective molecular chaperones so that a mixture of different caseins can form fuzzy complexes that are often self-limiting in size and, within which, amyloid fibril formation is suppressed. The remarkable ability of caseins to sequester nanoclusters of calcium phosphate in stable complexes is due to their flexible conformation and multiply-phosphorylated short sequences. These features combine to form a dense peptide shell around the calcium phosphate making the core-shell complex thermodynamically stable, even at high calcium and phosphate concentrations. Thus, the casein micelle provides a readily digested, high calcium food for the neonate. It also preserves the functional properties of caseins as IDPs and protects the mammary gland against amyloid formation and pathological calcification, dysfunctional processes that would reduce the future reproductive success of the mother.

A quantitative model of the bovine casein micelle: ion equilibria and calcium phosphate sequestration by individual caseins in bovine milk.

The white appearance of skim milk is due to strong light scattering by colloidal particles called casein micelles. Bovine casein micelles comprise expressed proteins from four casein genes together with significant fractions of the total calcium, inorganic phosphate, magnesium and citrate ions in the milk. Thus, the milk salts are partitioned between the casein micelles, where they are mostly in the form of nanoclusters of an amorphous calcium phosphate sequestered by caseins through their phosphorylated residues, with the remainder in the continuous phase. Previously, a salt partition calculation was made assuming that the nanoclusters are sequestered only by short, highly phosphorylated casein sequences, sometimes called phosphate centres. Three of the four caseins have a proportion of their phosphorylated residues in either one or two phosphate centres and these were proposed to react with the nanoclusters equally and independently. An improved model of the partition of caseins and salts in milk is described in which all the phosphorylated residues in competent caseins act together to bind to and sequester the nanoclusters. The new model has been applied to results from a recent study of variation in salt and casein composition in the milk of individual cows. Compared to the previous model, it provides better agreement with experiment of the partition of caseins between free and bound states and equally good results for the partition of milk salts. In addition, new calculations are presented for the charge on individual caseins in their bound and free states.

Proteostasis and the Regulation of Intra- and Extracellular Protein Aggregation by ATP-Independent Molecular Chaperones: Lens α-Crystallins and Milk Caseins.

Molecular chaperone proteins perform a diversity of roles inside and outside the cell. One of the most important is the stabilization of misfolding proteins to prevent their aggregation, a process that is potentially detrimental to cell viability. Diseases such as Alzheimer's, Parkinson's, and cataract are characterized by the accumulation of protein aggregates. In vivo, many proteins are metastable and therefore under mild destabilizing conditions have an inherent tendency to misfold, aggregate, and hence lose functionality. As a result, protein levels are tightly regulated inside and outside the cell. Protein homeostasis, or proteostasis, describes the network of biological pathways that ensures the proteome remains folded and functional. Proteostasis is a major factor in maintaining cell, tissue, and organismal viability. We have extensively investigated the structure and function of intra- and extracellular molecular chaperones that operate in an ATP-independent manner to stabilize proteins and prevent their misfolding and subsequent aggregation into amorphous particles or highly ordered amyloid fibrils. These types of chaperones are therefore crucial in maintaining proteostasis under normal and stress (e.g., elevated temperature) conditions. Despite their lack of sequence similarity, they exhibit many common features, i.e., extensive structural disorder, dynamism, malleability, heterogeneity, oligomerization, and similar mechanisms of chaperone action. In this Account, we concentrate on the chaperone roles of α-crystallins and caseins, the predominant proteins in the eye lens and milk, respectively. Intracellularly, the principal ATP-independent chaperones are the small heat-shock proteins (sHsps). In vivo, sHsps are the first line of defense in preventing intracellular protein aggregation. The lens proteins αA- and αB-crystallin are sHsps. They play a crucial role in maintaining solubility of the crystallins (including themselves) with age and hence in lens proteostasis and, ultimately, lens transparency. As there is little metabolic activity and no protein turnover in the lens, crystallins are very long lived proteins. Lens proteostasis is therefore very different to that in normal, metabolically active cells. Crystallins undergo extensive post-translational modification (PTM), including deamidation, racemization, phosphorylation, and truncation, which can alter their stability. Despite this, the lens remains transparent for tens of years, implying that lens proteostasis is intimately integrated with crystallin PTMs. Many PTMs do not significantly alter crystallin stability, solubility, and functionality, which thereby facilitates lens transparency. In the long term, however, extensive accumulation of crystallin PTMs leads to large-scale crystallin aggregation, lens opacification, and cataract formation. Extracellularly, various ATP-independent molecular chaperones exist that exhibit sHsp-like structural and functional features. For example, caseins, the major milk proteins, exhibit chaperone ability by inhibiting the amorphous and amyloid fibrillar aggregation of a diversity of destabilized proteins. Caseins maintain proteostasis within milk by preventing deleterious casein amyloid fibril formation via incorporation of thousands of individual caseins into an amorphous structure known as the casein micelle. Hundreds of nanoclusters of calcium phosphate are sequestered within each casein micelle through interactions with short, highly phosphorylated casein sequences. This results in a stable biofluid that contains a high concentration of potentially amyloidogenic caseins and concentrations of calcium and phosphate that can be far in excess of the solubility of calcium phosphate. Casein micelle formation therefore performs vital roles in neonatal nutrition and calcium homeostasis in the mammary gland.

Effect of Phosphorylation on a Human-like Osteopontin Peptide.

The last decade established that the dynamic properties of the phosphoproteome are central to function and its modulation. The temporal dimension of phosphorylation effects remains nonetheless poorly understood, particularly for intrinsically disordered proteins. Osteopontin, selected for this study due to its key role in biomineralization, is expressed in many species and tissues to play a range of distinct roles. A notable property of highly phosphorylated isoforms of osteopontin is their ability to sequester nanoclusters of calcium phosphate to form a core-shell structure, in a fluid that is supersaturated but stable. In Biology, this process enables soft and hard tissues to coexist in the same organism with relative ease. Here, we extend our understanding of the effect of phosphorylation on a disordered protein, the recombinant human-like osteopontin rOPN. The solution structures of the phosphorylated and unphosphorylated rOPN were investigated by small-angle x-ray scattering and no significant changes were detected on the radius of gyration or maximum interatomic distance. The picosecond-to-nanosecond dynamics of the hydrated powders of the two rOPN forms were further compared by elastic and quasi-elastic incoherent neutron scattering. Phosphorylation was found to block some nanosecond side-chain motions while increasing the flexibility of other side chains on the faster timescale. Phosphorylation can thus selectively change the dynamic behavior of even a highly disordered protein such as osteopontin. Through such an effect on rOPN, phosphorylation can direct allosteric mechanisms, interactions with substrates, cofactors and, in this case, amorphous or crystalline biominerals.

Structural studies of hydrated samples of amorphous calcium phosphate and phosphoprotein nanoclusters.

There are abundant examples of nanoclusters and inorganic microcrystals in biology. Their study under physiologically relevant conditions remains challenging due to their heterogeneity, instability, and the requirements of sample preparation. Advantages of using neutron diffraction and contrast matching to characterize biomaterials are highlighted in this article. We have applied these and complementary techniques to search for nanocrystals within clusters of calcium phosphate sequestered by bovine phosphopeptides, derived from osteopontin or casein. The neutron diffraction patterns show broad features that could be consistent with hexagonal hydroxyapatite crystallites smaller than 18.9 Å. Such nanocrystallites are, however, undetected by the complementary X-ray and FTIR data, collected on the same samples. The absence of a distinct diffraction pattern from the nanoclusters supports the generally accepted amorphous calcium phosphate structure of the mineral core.

A review of the biology of calcium phosphate sequestration with special reference to milk.

In milk, a stable fluid is formed in which sequestered nanoclusters of calcium phosphate are substructures in casein micelles. As a result, calcium and phosphate concentrations in milk can be far in excess of their solubility. Variations of calcium, phosphate and casein concentrations in milks, both within and among species, are mainly due to the formation of the nanocluster complexes. Caseins evolved from tooth and bone proteins well before the evolution of lactation. It has therefore been suggested that the role of caseins in milk is an adaptation of an antecedent function in the control of some aspect of biomineralisation. There is new evidence that nanocluster-type complexes are also present in blood serum and, by implication, in many other closely related biofluids. Because such fluids are stable but nevertheless supersaturated with respect to the bone and tooth mineral hydroxyapatite, they allow soft and mineralised tissues to co-exist in the same organism with relative ease. An appreciable concentration of nanocluster complexes exists in fresh saliva. Such saliva may stabilise tooth mineral and help to repair demineralised lesions. In the extracellular matrix of bone, nanocluster complexes may be involved in directing the amorphous calcium phosphate to intrafibrillar spaces in collagen where they can mature into oriented apatite crystals. Thus, evidence is accumulating that calcium phosphate sequestration by phosphopeptides to form equilibrium complexes, first observed in milk, is more generally important in the control of physiological calcification.

Mineralisation of soft and hard tissues and the stability of biofluids.

Evidence is provided from studies on natural and artificial biofluids that the sequestration of amorphous calcium phosphate by peptides or proteins to form nanocluster complexes is of general importance in the control of physiological calcification. A naturally occurring mixture of osteopontin peptides was shown, by light and neutron scattering, to form calcium phosphate nanoclusters with a core-shell structure. In blood serum and stimulated saliva, an invariant calcium phosphate ion activity product was found which corresponds closely in form and magnitude to the ion activity product observed in solutions of these osteopontin nanoclusters. This suggests that types of nanocluster complexes are present in these biofluids as well as in milk. Precipitation of amorphous calcium phosphate from artificial blood serum, urine and saliva was determined as a function of pH and the concentration of osteopontin or casein phosphopeptides. The position of the boundary between stability and precipitation was found to agree quantitatively with the theory of nanocluster formation. Artificial biofluids were prepared that closely matched their natural counterparts in calcium and phosphate concentrations, pH, saturation, ionic strength and osmolality. Such fluids, stabilised by a low concentration of sequestering phosphopeptides, were found to be highly stable and may have a number of beneficial applications in medicine.

Multifunctional role of osteopontin in directing intrafibrillar mineralization of collagen and activation of osteoclasts.

Mineralized collagen composites are of interest because they have the potential to provide a bone-like scaffold that stimulates the natural processes of resorption and remodeling. Working towards this goal, our group has previously shown that the nanostructure of bone can be reproduced using a polymer-induced liquid-precursor (PILP) process, which enables intrafibrillar mineralization of collagen with hydroxyapatite to be achieved. This prior work used polyaspartic acid (pASP), a simple mimic for acidic non-collagenous proteins, to generate nanodroplets/nanoparticles of an amorphous mineral precursor which can infiltrate the interstices of type-I collagen fibrils. In this study we show that osteopontin (OPN) can similarly serve as a process-directing agent for the intrafibrillar mineralization of collagen, even though OPN is generally considered a mineralization inhibitor. We also found that inclusion of OPN in the mineralization process promotes the interaction of mouse marrow-derived osteoclasts with PILP-remineralized bone that was previously demineralized, as measured by actin ring formation. While osteoclast activation occurred when pASP was used as the process-directing agent, using OPN resulted in a dramatic effect on osteoclast activation, presumably because of the inherent arginine-glycine-aspartate acid ligands of OPN. By capitalizing on the multifunctionality of OPN, these studies may lead the way to producing biomimetic bone substitutes with the capability of tailorable bioresorption rates.

Unfolded phosphopolypeptides enable soft and hard tissues to coexist in the same organism with relative ease.

Unfolded phosphopolypeptides that contain one or more multiply phosphorylated short sequences can sequester amorphous calcium phosphate to form stable complexes of constant average size and chemical composition. A biofluid containing such complexes is supersaturated with respect to the bone and tooth mineral, hydroxyapatite but is undersaturated with respect to the amorphous precursor phase. Thus, soft tissues permeated by the biofluid should not experience ectopic calcification and hard tissues should remain mineralised. Sequestration by caseins allows high concentrations of calcium and phosphate to be attained in milk while osteopontin, fetuin and other phosphopolypeptides may act in a similar way in blood, other biofluids, soft and hard tissues.

Aggregation behavior of bovine κ- and ß-casein studied with small angle neutron scattering, light scattering, and cryogenic transmission electron microscopy.

In the native bovine casein micelle the calcium sensitive caseins (α(S1)-, α(S2)- and ß-casein) sequester amorphous calcium phosphate in nanometer-sized clusters, whereas the calcium-insensitive κ-casein limits the growth of the micelle. In this paper, we further investigate the self-association of κ- and ß-casein, which are two of the key proteins that control the substructure of the milk casein micelle, using neutron and light scattering techniques and cryogenic transmission electron microscopy. Results demonstrate that κ-casein can, apart from the known self-assembly, form amyloid-like fibrils already at temperatures of 25 °C when subject to agitation. This extended aggregation behavior of κ-casein is inhibited by ß-casein, as reported by others. These findings have implications for the structure and stability of casein micelles. The neutron scattering data was used to gain information on the self-assembly structure of κ-casein. ß-Casein shows similar self-association behavior as κ-casein, but unlike κ-casein, the self-association exhibits temperature dependence within the studied temperatures (6 and 25 °C). Here, we will discuss our extended study of the known self-assembly of casein in the context of the fibrillation of κ-casein.

An E. coli over-expression system for multiply-phosphorylated proteins and its use in a study of calcium phosphate sequestration by novel recombinant phosphopeptides.

Phosphoproteins and phosphopeptides were expressed by E. coli to give yields of 30-200mg of purified protein per litre with an average degree of phosphorylation at multiple sites of 61-83%. The method employed two compatible cohabiting plasmids having low and high copy number, expressing a protein kinase and, more abundantly, the substrate (poly)peptide, respectively. It was used to phosphorylate recombinant beta-casein or osteopontin at multiple casein kinase-2 sites. Two constructs were designed to produce shorter peptides containing one or more clusters of phosphorylation sites resembling the phosphate centres of caseins. In the first, a 53-residue 6-His tagged phosphopeptide was expressed at a 5-fold higher molar yield. The second had multiple tandem repeats of a tryptic phosphopeptide sequence to give a similar increase in efficiency. Each recombinant phosphopeptide was purified (30-100mg) and small-angle X-ray scattering measurements showed that they, like certain casein and osteopontin phosphopeptides, sequester amorphous calcium phosphate to form calcium phosphate nanoclusters. In principle, the method can provide novel phosphopeptides for the control of biocalcification or be adapted for use with other kinases and cognate proteins or peptides to study the effect of specific phosphorylations on protein structure. Moreover, the insertion of a phosphate centre sequence, possibly with a linker peptide, may allow thermodynamically stable, biocompatible nanoparticles to be made from virtually any sequence.