Caseins are a unique and diverse group of proteins present in bovine milk. While their function is presumed to be primarily nutritional, caseins have a remarkable ability to stabilize proteins, i.e., to inhibit protein aggregation and precipitation, that is comparable to molecular chaperones of the small heat-shock protein (sHsp) family. Additionally, sHsps have been shown to inhibit the formation of amyloid fibrils. This study investigated (i) the fibril-forming propensities of casein proteins and their mixture, sodium caseinate, and (ii) the ability of caseins to prevent in vitro fibril formation by kappa-casein. Transmission electron microscopy (TEM) and X-ray fiber diffraction data demonstrated that kappa-casein readily forms amyloid fibrils at 37 degrees C particularly following reduction of its disulfide bonds. The time-dependent increase in thioflavin T fluorescence observed for reduced and nonreduced kappa-casein at 37 degrees C was suppressed by stoichiometric amounts of alphaS- and beta-casein and by the hydrophobic dye 8-anilino-1-naphthalene sulfonate; the inhibition of kappa-casein fibril formation under these conditions was verified by TEM. Our findings suggest that alphaS- and beta-casein are potent inhibitors of kappa-casein fibril formation and may prevent large-scale fibril formation in vivo. Casein proteins may therefore play a preventative role in the development of corpora amylacea, a disorder associated with the accumulation of amyloid deposits in mammary tissue.

No abstract

Small heat-shock proteins (sHsps) and clusterin are molecular chaperones that share many functional similarities despite their lack of significant sequence similarity. These functional similarities, and some differences, are discussed. sHsps are ubiquitous intracellular proteins whereas clusterin is generally found extracellularly. Both chaperones potently prevent the amorphous aggregation and precipitation of target proteins under stress conditions such as elevated temperature, reduction and oxidation. In doing so, they act on the slow off-folding protein pathway. The conformational dynamism and aggregated state of both proteins may be crucial for their chaperone function. Subunit exchange is likely to be important in regulating chaperone action; the dissociated form of the protein is probably the chaperone-active species rather than the aggregated state. They both exert their chaperone action without the need for hydrolysis of ATP and have little ability to refold target proteins. Increased expression of sHsp and clusterin accompanies a range of diseases, e.g. Alzheimer's, Creutzfeldt-Jakob and Parkinson's diseases, that arise from protein misfolding and deposition of highly structured protein aggregates known as amyloid fibrils. The interaction of sHsps and clusterin with fibril-forming species is discussed along with their ability to prevent fibril formation, probably via utilization of their chaperone ability. Disciplines Life Sciences | Physical Sciences and Mathematics | Social and Behavioral Sciences Publication DetailsThis article was originally published as Carver, JA, Rekas, A, Thorn, DC and Wilson, MR, Small heat-shock proteins and clusterin: intra-and extracellular molecular chaperones with a common mechanism of action and function, IUBMB Life, 55, 2003, 661-668 AbstractSmall heat-shock proteins (sHsps) and clusterin are molecular chaperones that share many functional similarities despite their lack of significant sequence similarity. These functional similarities, and some differences, are discussed. sHsps are ubiquitous intracellular proteins whereas clusterin is generally found extracellularly. Both chaperones potently prevent the amorphous aggregation and precipitation of target proteins under stress conditions such as elevated temperature, reduction and oxidation. In doing so, they act on the slow off-folding protein pathway. The conformational dynamism and aggregated state of both proteins may be crucial for their chaperone function. Subunit exchange is likely to be important in regulating chaperone action; the dissociated form of the protein is probably the chaperone-active species rather than the aggregated state. They both exert their chaperone action without the need for hydrolysis of ATP and have little ability to refold target proteins. Increased expression of sHsp and clusterin accompanies a range of diseases, e.g. Alzheimer's, Creutzfeldt-Jakob and Parkinson's diseases, that arise from protein misfolding and deposition of highly structured protein aggregates known as amyloid f...

Yellow emission variants of green fluorescent protein (GFP) have been found useful in a variety of applications in biological systems due to their red-shifted emission spectrum and sensitivity to environmental parameters, such as pH and ionic strength. However, slow maturation properties and new requirements for more intense fluorescence necessitated further mutagenesis studies of these proteins. Venus, a new variant with improved maturation and brightness, as well as reduced environmental dependence, was recently developed by introducing five mutations into the well characterized variant, enhanced yellow fluorescent protein (EYFP). In this paper, we present the crystal structure of Venus at 2.2 Å resolution, which enabled us to correlate its novel features with these mutation points. The rearrangement of several side chains near the chromophore, initiated by the F46L mutation, was found to improve maturation at 37°C by removing steric and energetic constraints, which may hinder folding of the polypeptide chain, and by accelerating the oxidation of the C␣-C␤ bond of Tyr 66 during chromophore formation. M153T, V163A, and S175G were also found to improve the rate of maturation by creating regions of greater flexibility. F64L induced large conformational changes in the molecule, leading to the removal of halide sensitivity by preventing ion access to the binding site. Green fluorescent protein (GFP),1 originally isolated from the jellyfish Aequorea victoria, has been the subject of continued interest since its gene was first cloned in 1992 (1). The high stability of mature GFP over various environment conditions, the spontaneous autocatalytical generation of the fluorophore of GFP, and the possibility of spectral manipulation by mutagenesis, make GFP and its related proteins attractive tools for numerous biological applications (2).Maturation of GFP proceeds in three major steps, beginning as the 238-residue single GFP polypeptide folds into its nearly native conformation. Residues 65-67 of the folded protein then undergo several chemical reactions necessary for chromophore formation, including cyclization and dehydration (3). The final rate-limiting step in the maturation process involves the oxidation of the C␣-C␤ bond of Tyr 66 by aerial oxygen. This maturation process takes ϳ3 h at room temperature and its efficiency further decreases at 37°C, hindering the use of GFP in some biological applications. To improve maturation and performance at 37°C, mutations such as F99S, M153T, and V163A have been introduced, creating valuable GFP variants for a wider range of applications (4, 5).Thorough understanding of the relationship between the protein sequence and its physico-chemical properties requires three-dimensional structure determination of GFP at atomic resolution. Crystal structures of both the monomeric and dimeric forms of GFP have been solved previously (6, 7). The wild-type GFP (wtGFP) chromophore, which exists in equilibrium between two ground states, produces two maxima in the light absorption spectrum. Th...

Inclusions of aggregated alpha-synuclein (alpha-syn) in dopaminergic neurons are a characteristic histological marker of Parkinson's disease (PD). In vitro, alpha-syn in the presence of dopamine (DA) at physiological pH forms SDS-resistant non-amyloidogenic oligomers. We used a combination of biophysical techniques, including sedimentation velocity analysis, small angle X-ray scattering (SAXS) and circular dichroism spectroscopy to study the characteristics of alpha-syn oligomers formed in the presence of DA. Our SAXS data show that the trimers formed by the action of DA on alpha-syn consist of overlapping worm-like monomers, with no end-to-end associations. This lack of structure contrasts with the well-established, extensive beta-sheet structure of the amyloid fibril form of the protein and its pre-fibrillar oligomers. We propose on the basis of these and earlier data that oxidation of the four methionine residues at the C- and N-terminal ends of alpha-syn molecules prevents their end-to-end association and stabilises oligomers formed by cross linking with DA-quinone/DA-melanin, which are formed as a result of the redox process, thus inhibiting formation of the beta-sheet structure found in other pre-fibrillar forms of alpha-syn.

No abstract

The vertebrate lens is composed of a very high concentration of proteins, the main group of which is the crystallins, the higher-order structural arrangement of which enables the refraction of light to ensure proper vision. The principal lens protein is a-crystallin which, in addition to its structural role, also functions as a molecular chaperone to interact and complex with the b-crystallins and c-crystallins to prevent their aggregation and precipitation [1]. The crystallins are very stable proteins, and the lack of protein turnover in all but the outer (epithelial) layer of the lens means that they have to be very long lived. With age, however, many a-Crystallin is the principal lens protein which, in addition to its structural role, also acts as a molecular chaperone, to prevent aggregation and precipitation of other lens proteins. One of its two subunits, aB-crystallin, is also expressed in many nonlenticular tissues, and a natural missense mutation, R120G, has been associated with cataract and desmin-related myopathy, a disorder of skeletal muscles [Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D & Fardeau M (1998) Nat Genet 20, 92-95]. In the present study, realtime 1 H-NMR spectroscopy showed that the ability of R120G aB-crystallin to stabilize the partially folded, molten globule state of a-lactalbumin was significantly reduced in comparison with wild-type aB-crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced a-lactalbumin, but showed limited chaperone activity for other target proteins. Using NMR spectroscopy, gel electrophoresis, and MS, we observed that, unlike the wild-type protein, R120G aB-crystallin is intrinsically unstable in solution, with unfolding of the protein over time leading to aggregation and progressive truncation from the C-terminus. Light scattering, MS, and size-exclusion chromatography data indicated that R120G aB-crystallin exists as a larger oligomer than wild-type aB-crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of aB-crystallin causes partial unfolding, increased exposure of hydrophobic regions, and enhances its susceptibility to proteolysis, thus reducing its solubility and promoting its aggregation and complexation with other proteins. These characteristics may explain the involvement of R120G aB-crystallin with human disease states.

The molecular chaperone, alpha-crystallin, has the ability to prevent the fibrillar aggregation of proteins implicated in human diseases, for example, amyloid beta peptide and alpha-synuclein. In this study, we examine, in detail, two aspects of alpha-crystallin's fibril-suppressing ability: (a) its temperature dependence, and (b) the nature of the aggregating species with which it interacts. First, the efficiency of alpha-crystallin to suppress fibril formation in kappa-casein and alpha-synuclein increases with temperature, despite their rate of fibrillation also increasing in the absence of alpha-crystallin. This is consistent with an increased chaperone ability of alpha-crystallin at higher temperatures to protect target proteins from amorphous aggregation [GB Reddy, KP Das, JM Petrash & WK Surewicz (2000) J Biol Chem275, 4565-4570]. Second, dual polarization interferometry was used to monitor real-time alpha-synuclein aggregation in the presence and absence of alphaB-crystallin. In contrast to more common methods for monitoring the time-dependent formation of amyloid fibrils (e.g. the binding of dyes like thioflavin T), dual polarization interferometry data did not reveal any initial lag phase, generally attributed to the formation of prefibrillar aggregates. It was shown that alphaB-crystallin interrupted alpha-synuclein aggregation at its earliest stages, most likely by binding to partially folded monomers and thereby preventing their aggregation into fibrillar structures.