John Carver

SUPRAMOLECULAR ORDER WITHIN THE LENS - H-1-NMR SPECTROSCOPIC EVIDENCE FOR SPECIFIC CRYSTALLIN-CRYSTALLIN INTERACTIONS

Publisher: Elsevier BV

Date: 11-1994

DOI: 10.1006/EXER.1994.1146

Abstract: alpha-, beta- and gamma-crystallins from bovine lens contain flexible terminal extensions which are readily observed by NMR spectroscopy. By monitoring these resonances, NMR spectroscopy therefore offers a means of examining specific protein-protein interactions in crystallin mixtures. In this paper, a 1H NMR spectroscopic study of bovine lens nuclear and cortical homogenates and various crystallin mixtures is presented. In both homogenates, resonances from the flexible C-terminal extensions of alpha-crystallin and the N-terminal extension of beta B2-crystallin are readily observed suggesting that these regions are not involved in crystallin-crystallin interactions. In the cortical homogenate, resonances from the short N-terminal extension of gamma S-crystallin are also present. The cortical homogenate gives rise to more intense resonances than the nuclear homogenate, suggesting that the cortical region has many more mobile crystallin regions. In both homogenates, the C-terminal extension of beta B2-crystallin and the very short C-terminal extension of gamma B-crystallin are not observed. Thus, the C-terminal regions of these proteins are involved in interactions with other crystallins. Similar effects are observed upon mixing of the in idual crystallins, e.g. the C-terminal extension of gamma B-crystallin is absent in spectra of mixtures of total gamma-crystallin and high-molecular-weight beta-crystallin aggregates (beta H). Overall, the results are consistent with a short-range order for the crystallins within the lens.

Small heat-shock proteins and clusterin: Intra- and extracellular molecular chaperones with a common mechanism of action and function?

Publisher: Wiley

Date: 12-2003

DOI: 10.1080/15216540310001640498

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 sHsps and clusterin accompanies a range of diseases that arise from protein misfolding and deposition of highly structured protein aggregates known as amyloid fibrils, e.g., Alzheimer's, Creutzfeldt-Jakob and Parkinson's diseases. The interaction of sHsps and clusterin with fibril-forming species is discussed along with their ability to prevent fibril formation.

The dissociated form of κ-casein is the precursor to its amyloid fibril formation

Publisher: Portland Press Ltd.

Date: 28-06-2010

DOI: 10.1042/BJ20091949

Abstract: Bovine milk κ-casein forms a self-associating oligomeric micelle-like species, in equilibrium with dissociated forms. In its native form, intra- and inter-molecular disulfide bonds lead to the formation of multimeric species ranging from monomers to decamers. When incubated under conditions of physiological pH and temperature, both reduced and non-reduced κ-casein form highly structured β-sheet amyloid fibrils. We investigated whether the precursor to κ-casein fibril formation is a dissociated state of the protein or its oligomeric micelle-like form. We show that reduced κ-casein is capable of forming fibrils well below its critical micelle concentration, i.e. at concentrations where only dissociated forms of the protein are present. Moreover, by regulating the degree of disulfide linkages, we were able to investigate how oligomerization of κ-casein influences its propensity for fibril formation under conditions of physiological pH and temperature. Thus, using fractions containing different proportions of multimeric species, we demonstrate that the propensity of the disulfide-linked multimers to form fibrils is inversely related to their size, with monomeric κ-casein being the most aggregation prone. We conclude that dissociated forms of κ-casein are the amyloidogenic precursors to fibril formation rather than oligomeric micelle-like species. The results highlight the role of oligomerization and natural binding partners in preventing amyloid fibril formation by disease-related proteins in vivo.

Model for amorphous aggregation processes

Publisher: American Physical Society (APS)

Date: 09-11-2009

DOI: 10.1103/PHYSREVE.80.051907

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

Publisher: Elsevier BV

Date: 11-2022

DOI: 10.1016/J.BBAPAP.2022.140854

Abstract: Caseins are a erse family of intrinsically disordered proteins present in the milks of all mammals. A property common to two cow paralogues, α S2 - 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 (α S1 and β) as well as β-caseins from camel and goat. Small-angle X-ray scattering measurements indicated that cow α S1 - and β-casein formed large spherical aggregates at neutral pH and 20°C. Upon incubation at 65°C, α S1 - and β-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 β-casein exhibited a greater fibril-forming propensity than its cow or goat orthologues. Limited proteolysis of cow and camel β-casein fibrils and analysis by mass spectrometry indicated a common amyloidogenic sequence in the proline, glutamine-rich, C-terminal region of β-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 ersified partly to avoid dysfunctional amyloid fibril formation.

Non-oxidative modification of lens crystallins by kynurenine: a novel post-translational protein modification with possible relevance to ageing and cataract

Publisher: Elsevier BV

Date: 02-2000

DOI: 10.1016/S0167-4838(99)00234-4

Abstract: In humans, the crystallin proteins of the ocular lens become yellow-coloured and fluorescent with ageing. With the development of senile nuclear cataract, the crystallins become brown and additional fluorophores are formed. The mechanism underlying crystallin colouration is not known but may involve interaction with kynurenine-derived UV filter compounds. We have recently identified a sulphur-linked glutathionyl-3-hydroxykynurenine glucoside adduct in the lens and speculated that kynurenine may also form adducts with GSH and possibly with nucleophilic amino acids of the crystallins (e.g. Cys). Here we show that kynurenine modifies calf lens crystallins non-oxidatively to yield coloured (365 nm absorbing), fluorescent (Ex 380 nm/Em 450-490 nm) protein adducts. Carboxymethylation and succinylation of crystallins inhibited kynurenine-mediated modification by approx. 90%, suggesting that Cys, Lys and possibly His residues may be involved. This was confirmed by showing that kynurenine formed adducts with GSH as well as with poly-His and poly-Lys. NMR studies revealed that the novel poly-Lys-kynurenine covalent linkage was via the epsilon-amino group of the Lys side chain and the betaC of the kynurenine side chain. Analysis of tryptic peptides of kynurenine-modified crystallins revealed that all of the coloured peptides contained either His, Cys or an internal Lys residue. We propose a novel mechanism of kynurenine-mediated crystallin modification which does not require UV light or oxidative conditions as catalysts. Rather, we suggest that the side chain of kynurenine-derived lens UV filters becomes deaminated to yield an alpha,beta-unsaturated carbonyl which is highly susceptible to attack by nucleophilic amino acid residues of the crystallins. The inability of the lens fibre cells to metabolise their constituent proteins results in the accumulation of coloured/fluorescent crystallins with age.

Protein nanostructures in food - Should we be worried?

Publisher: Elsevier BV

Date: 05-2014

DOI: 10.1016/J.TIFS.2014.02.003

The elusive role of the N-terminal extension of beta A3- and beta A1-crystallin

Publisher: Oxford University Press (OUP)

Date: 1996

DOI: 10.1093/PROTEIN/9.11.1021

Abstract: beta-Crystallins are structural lens proteins with a conserved two-domain structure and variable N- and C-terminal extensions. These extensions are assumed to be involved in quaternary interactions within the beta-crystallin oligomers or with other lens proteins. Therefore, the production of beta A3- and beta A1-crystallin from the single beta A3/A1 mRNA by dual translation initiation is of interest. These crystallins are identical, except that beta A1 has a much shorter N-terminal extension that beta A3. This rare mechanism has been conserved for over 250 million years during the evolution of the beta A3/A1 gene, suggesting that the generation of different N-terminal extensions confers a selective advantage. We therefore compared the stability and association behaviour of recombinant beta A3- and beta A1-crystallin. Both proteins are equally stable in urea- and pH-induced denaturation experiments. Gel filtration and analytical ultracentrifugation established that beta A3 and beta A1 both form homodimers. In the water-soluble proteins of bovine lens, beta A3 and beta A1 are present in the same molecular weight fractions, indicating that they oligomerize equally with other beta-crystallins. 1H-NMR spectroscopy showed that residues Met1 to Asn22 of the N-terminal extension of beta A3 have great flexibility and are solvent exposed, excluding them from protein interactions in the homodimer. These results indicate that the different N-terminal extensions of beta A3 and beta A1 do not affect their homo- or heteromeric interactions.

Proteostasis and the regulation of intra- and extracellular protein aggregation by ATP-independent molecular chaperones: Lens alpha-crystallins and milk caseins

Publisher: American Chemical Society (ACS)

Date: 14-02-2018

DOI: 10.1021/ACS.ACCOUNTS.7B00250

Abstract: Molecular chaperone proteins perform a ersity 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 ex le, caseins, the major milk proteins, exhibit chaperone ability by inhibiting the amorphous and amyloid fibrillar aggregation of a ersity of destabilized proteins. Caseins maintain proteostasis within milk by preventing deleterious casein amyloid fibril formation via incorporation of thousands of in idual 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.

The interaction of αB-crystallin with mature α-synuclein amyloid fibrils inhibits their elongation

Publisher: Elsevier BV

Date: 03-2010

DOI: 10.1016/J.BPJ.2009.10.056

Recognizing and analyzing variability in amyloid formation kinetics: Simulation and statistical methods

Publisher: Elsevier BV

Date: 2012

DOI: 10.1016/J.AB.2016.07.013

Abstract: We examine the phenomenon of variability in the kinetics of amyloid formation and detail methods for its simulation, identification and analysis. Simulated data, reflecting intrinsic variability, were produced using rate constants, randomly s led from a pre-defined distribution, as parameters in an irreversible nucleation-growth kinetic model. Simulated kinetic traces were reduced in complexity through description in terms of three characteristic parameters. Practical methods for assessing convergence of the reduced parameter distributions were introduced and a bootstrap procedure was applied to determine convergence for different levels of intrinsic variation. Statistical methods for assessing the significance of shifts in parameter distributions, relating to either change in parameter mean or distribution shape, were tested. Robust methods for analyzing and interpreting kinetic data possessing significant intrinsic variance will allow greater scrutiny of the effects of anti-amyloid compounds in drug trials.

Coaggregation of κ-Casein and β-Lactoglobulin Produces Morphologically Distinct Amyloid Fibrils

Publisher: Wiley

Date: 02-2017

DOI: 10.1002/SMLL.201603591

Abstract: The unfolding, misfolding, and aggregation of proteins lead to a variety of structural species. One form is the amyloid fibril, a highly aligned, stable, nanofibrillar structure composed of β-sheets running perpendicular to the fibril axis. β-Lactoglobulin (β-Lg) and κ-casein (κ-CN) are two milk proteins that not only in idually form amyloid fibrillar aggregates, but can also coaggregate under environmental stress conditions such as elevated temperature. The aggregation between β-Lg and κ-CN is proposed to proceed via disulfide bond formation leading to amorphous aggregates, although the exact mechanism is not known. Herein, using a range of biophysical techniques, it is shown that β-Lg and κ-CN coaggregate to form morphologically distinct co-amyloid fibrillar structures, a phenomenon previously limited to protein isoforms from different species or different peptide sequences from an in idual protein. A new mechanism of aggregation is proposed whereby β-Lg and κ-CN not only form disulfide-linked aggregates, but also amyloid fibrillar coaggregates. The coaggregation of two structurally unrelated proteins into cofibrils suggests that the mechanism can be a generic feature of protein aggregation as long as the prerequisites for sequence similarity are met.

A NEW UV-FILTER COMPOUND IN HUMAN LENSES

Publisher: Wiley

Date: 11-07-1994

DOI: 10.1016/0014-5793(94)00601-6

Abstract: A new UV-filter compound, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside, has been identified in human lenses. The structure suggests that it is derived biosynthetically from tryptophan. Quantification studies on the new compound show that it is the second-most abundant UV-filter compound in the lens with an absorption and fluorescence spectrum similar to that of 3-hydroxykynurenine glucoside.

The effect of small molecules in modulating the chaperone activity of αB-crystallin against ordered and disordered protein aggregation

Publisher: Wiley

Date: 23-01-2008

DOI: 10.1111/J.1742-4658.2008.06257.X

Abstract: Protein aggregation can proceed via disordered or ordered mechanisms, with the latter being associated with amyloid fibril formation, which has been linked to a number of debilitating conditions including Alzheimer's, Parkinson's and Creutzfeldt-Jakob diseases. Small heat-shock proteins (sHsps), such as alphaB-crystallin, act as chaperones to prevent protein aggregation and are thought to play a key role in the prevention of protein-misfolding diseases. In this study, we have explored the potential for small molecules such as arginine and guanidine to affect the chaperone activity of alphaB-crystallin against disordered (amorphous) and ordered (amyloid fibril) forms of protein aggregation. The effect of these additives is highly dependent upon the target protein undergoing aggregation. Importantly, our results show that the chaperone action of alphaB-crystallin against aggregation of the disease-related amyloid fibril forming protein alpha-synucleinA53T is enhanced in the presence of arginine and similar positively charged compounds (such as lysine and guanidine). Thus, our results suggest that target protein identity plays a critical role in governing the effect of small molecules on the chaperone action of sHsps. Significantly, small molecules that regulate the activity of sHsps may provide a mechanism to protect cells from the toxic protein aggregation that is associated with some protein-misfolding diseases.

Carboxymethylated-κ-casein: A convenient tool for the identification of polyphenolic inhibitors of amyloid fibril formation

Publisher: Elsevier BV

Date: 2010

DOI: 10.1016/J.BMC.2009.10.063

Abstract: Reduced and carboxymethylated-kappa-casein (RCM-kappa-CN) is a milk-derived amyloidogenic protein that readily undergoes nucleation-dependent aggregation and amyloid fibril formation via a similar pathway to disease-specific amyloidogenic peptides like amyloid beta (Abeta), which is associated with Alzheimer's disease. In this study, a series of flavonoids, many known to be inhibitors of Abeta fibril formation, were screened for their ability to inhibit RCM-kappa-CN fibrilisation, and the results were compared with literature data on Abeta inhibition. Flavonoids that had a high degree of hydroxylation and molecular planarity gave good inhibition of RCM-kappa-CN fibril formation. IC(50) values were between 10- and 200-fold higher with RCM-kappa-CN than literature results for Abeta fibril inhibition, however, with few exceptions, they showed a similar trend in potency. The convenience and reproducibility of the RCM-kappa-CN assay make it an economic alternative first screen for Abeta inhibitory activity, especially for use with large compound libraries.

Dissociation from the oligomeric state is the rate-limiting step in fibril formation by κ-casein

Publisher: Elsevier BV

Date: 04-2008

DOI: 10.1074/JBC.M709928200

Role of salt bridges in the dimer interface of 14-3-3ζ in dimer dynamics, N-terminal α-helical order, and molecular chaperone activity

Publisher: Elsevier BV

Date: 2018

DOI: 10.1074/JBC.M117.801019

Measurement of amyloid formation by turbidity assay - seeing through the cloud

Publisher: Springer Science and Business Media LLC

Date: 23-11-2016

DOI: 10.1007/S12551-016-0233-7

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

Publisher: Wiley

Date: 08-07-2019

DOI: 10.1002/BIP.23319

Abstract: 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.

Site-directed mutations in the C-terminal extension of human αB-crystallin affect chaperone function and block amyloid fibril formation

Publisher: Public Library of Science (PLoS)

Date: 17-10-2007

DOI: 10.1371/JOURNAL.PONE.0001046

STRUCTURE-FUNCTION STUDIES ON BOMBESIN AND RELATED PEPTIDES - BIOLOGICAL EFFECTS ON SWISS 3T3 CELLS AND TWO-DIMENSIONAL H-1-NMR ANALYSIS

Publisher: Wiley

Date: 12-1988

DOI: 10.1111/J.1749-6632.1988.TB23927.X

NMR IDENTIFICATION OF A PARTIAL HELICAL CONFORMATION FOR BOMBESIN IN SOLUTION

Publisher: Wiley

Date: 02-1990

DOI: 10.1111/J.1432-1033.1990.TB15348.X

Abstract: The conformation of bombesin in trifluoroethanol/water mixtures has been studied using 1H-NMR spectroscopy. By a combination of two-dimensional 1H-NMR techniques and measurement of vicinal NH-alpha-CH spin-spin coupling constants, the secondary structure of the molecule has been determined. Bombesin adopts a helical structure in the region from Asn-6 to Met-14 with the remaining N-terminal portion existing as a more extended structure. The structure is very similar to that proposed from Fourier-transform infrared spectroscopic measurements for bombesin inserted into lipid bilayers [D. Erne & R. Schwyzer (1987) Biochemistry 26, 6316-6319]. The absence of a hydrogen bond between the sidechains of Trp-8 and His-12 is discussed in terms of the ionization state of His-12. Stabilisation of the helix results when His-12 is in the ionized state.

Maculatin 1.1, an anti-microbial peptide from the Australian tree frog, Litoria genimaculata. Solution structure and biological activity

Publisher: Wiley

Date: 04-2000

DOI: 10.1046/J.1432-1327.2000.01089.X

Abstract: The dorsal glands of Australian tree frogs from the Litoria species contain a ersity of antibiotic peptides that forms part of the defence system of the animal. Here, the antibiotic activity and structure of maculatin 1.1, a 21 amino acid peptide from Litoria genimaculata, are compared. The activity data on maculatin 1.1 and a series of its analogues imply that the mechanism of action of maculatin 1.1 involves binding to, and subsequent lysis of, the bacterial cell membrane. The structure of maculatin 1.1 was determined using NMR spectroscopy in a trifluoroethanol/water mixture and when incorporated into dodecylphosphocholine micelles. Under both conditions, the peptide adopts a very similar conformation, i.e. a helical structure with a central kink in the vicinity of Pro15. The kink allows the peptide to adopt a well-defined hipathic conformation along its entire length. The similar structures determined under both solvent conditions imply that structures of membrane-interacting peptides in trifluoroethanol/water mixtures are representative of those adopted in a membrane environment, e.g. when incorporated into micelles. The synthetic Ala15 analogue of maculatin 1.1 has markedly reduced activity and its NMR-derived structure is a well-defined helix, which lacks the central kink and flexibility of the parent molecule. It is concluded that the kink is important for full biological activity of the peptide, probably because it allows maximum hipathicity of the peptide to facilitate interaction with the membrane. The structure of maculatin 1.1 is compared with a related peptide, caerin 1.1 [Wong, H., Bowie, J.H. and Carver, J.A. (1997) Eur. J. Biochem. 247, 545-557], which has an additional central proline residue and enhanced central flexibility compared with maculatin 1.1. The role of central flexibility within antibiotic peptides in their interaction with bacterial membranes is discussed.

Crystallins, cataract, and dynamic lens proteostasis. A commentary on P.W.N. Schmid, N.C.H. Lim, C. Peters, K.C. Back, B. Bourgeois, F. Pirolt, B. Richter, J. Peschek, O. Puk, O.V. Amarie, C. Dalke, M. Haslbeck, S. Weinkauf, T. Madl, J. Graw, and J. Buchner (2021) Imbalances in the eye lens proteome are linked to cataract formation, Nat. Struct. Mol. Biol. 28, 143–151. doi: 10.1038/s41594-020-00543-9

Publisher: Elsevier BV

Date: 07-2021

DOI: 10.1016/J.EXER.2021.108619

Clusterin is an ATP-independent chaperone with very broad substrate specificity that stabilizes stressed proteins in a folding-competent state

Publisher: American Chemical Society (ACS)

Date: 12-2000

DOI: 10.1021/BI002189X

Abstract: We recently reported that the ubiquitous, secreted protein clusterin has chaperone activity in vitro [Humphreys et al. (1999) J. Biol. Chem. 274, 6875-6881]. In this study, we demonstrate that clusterin (i) inhibits stress-induced precipitation of a very broad range of structurally ergent protein substrates, (ii) binds irreversibly via an ATP-independent mechanism to stressed proteins to form solubilized high molecular weight complexes, (iii) lacks detectable ATPase activity, (iv) when acting alone, does not effect refolding of stressed proteins in vitro, and (v) stabilizes stressed proteins in a state competent for refolding by heat shock protein 70 (HSP70). Furthermore, we show that, at physiological levels, clusterin inhibits stress-induced precipitation of proteins in undiluted human serum. Clusterin represents the first identified secreted mammalian chaperone. However, reports from others suggest that, at least under stress conditions, clusterin may be retained within cells to exert a protective effect. Regardless of the topological site(s) of action, the demonstration that clusterin can stabilize stressed proteins in a refolding-competent state suggests that, during stresses, the action of clusterin may inhibit rapid and irreversible protein precipitation and produce a reservoir of inactive but stabilized molecules from which other refolding chaperones can subsequently salvage functional proteins.

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

Publisher: Elsevier BV

Date: 03-2021

DOI: 10.1016/J.BPC.2020.106530

IDENTIFICATION BY H-1-NMR SPECTROSCOPY OF FLEXIBLE C-TERMINAL EXTENSIONS IN BOVINE LENS ALPHA-CRYSTALLIN

Publisher: Wiley

Date: 19-10-1992

DOI: 10.1016/0014-5793(92)81386-Z

Abstract: Two-dimensional 1H NMR spectroscopy of bovine eye lens alpha-crystallin and its isolated alpha A and alpha B subunits reveals that these aggregates have short and very flexible C-terminal extensions of eight (alpha A) and ten (alpha B) amino acids which adopt little preferred conformation in solution. Total alpha-crystallin forms a tighter aggregate than the isolated alpha A and alpha B subunit aggregates. Our results are consistent with a micelle model for alpha-crystallin quaternary structure. The presence of terminal extensions is a general feature of those crystallins, alpha and beta, which form aggregates.

The chaperone action of bovine milk alpha(S1)- and alpha(S2)-caseins and their associated form alpha(S)-casein

Publisher: Elsevier BV

Date: 06-2011

DOI: 10.1016/J.ABB.2011.03.012

Abstract: α(S)-Casein, the major milk protein, comprises α(S1)- and α(S2)-casein and acts as a molecular chaperone, stabilizing an array of stressed target proteins against precipitation. Here, we report that α(S)-casein acts in a similar manner to the unrelated small heat-shock proteins (sHsps) and clusterin in that it does not preserve the activity of stressed target enzymes. However, in contrast to sHsps and clusterin, α-casein does not bind target proteins in a state that facilitates refolding by Hsp70. α(S)-Casein was also separated into α- and α-casein, and the chaperone abilities of each of these proteins were assessed with amorphously aggregating and fibril-forming target proteins. Under reduction stress, all α-casein species exhibited similar chaperone ability, whereas under heat stress, α-casein was a poorer chaperone. Conversely, α(S2)-casein was less effective at preventing fibril formation by modified κ-casein, whereas α- and α(S1)-casein were comparably potent inhibitors. In the presence of added salt and heat stress, α(S1)-, α- and α(S)-casein were all significantly less effective. We conclude that α(S1)- and α-casein stabilise each other to facilitate optimal chaperone activity of α(S)-casein. This work highlights the interdependency of casein proteins for their structural stability.

H-1 NUCLEAR-MAGNETIC-RESONANCE STUDIES OF AN INTEGRAL MEMBRANE-PROTEIN - SUBUNIT-C OF THE F1F0 ATP SYNTHASE

Publisher: Elsevier BV

Date: 02-0011

DOI: 10.1016/0022-2836(87)90357-3

Abstract: The membrane-traversing subunit c parallel from the F0 part of the ATP synthase molecule has been studied in chloroform/methanol by high-resolution 1H n.m.r. Various one-dimensional and two-dimensional techniques have been used for assignment purposes, some NOE connectivities were established and some 3JHN alpha coupling constants were measured from spin--echo experiments. The effects of varying pH, solvent composition, lanthanide concentration and temperature have been investigated. Evidence is presented that the molecule has extensive alpha-helical segments, and the hairpin structure suggested by other groups is supported by our n.m.r. data. Only one ionizable group, assigned to the C-terminal carboxyl, is observed to titrate in the pH range 2 to 10 so the conserved residue, Asp61, which binds dicyclohexylcarbodiimide, presumably has (at least in this solvent system) an abnormally high pK value.

A H-1-NMR SPECTROSCOPIC COMPARISON OF GAMMA(S)-CRYSTALLIN AND GAMMA(B)-CRYSTALLIN

Publisher: Elsevier BV

Date: 08-1994

DOI: 10.1006/EXER.1994.1099

Abstract: Two-dimensional 1H NMR spectroscopic studies are presented on bovine gamma S- and gamma B-crystallin. In gamma S-crystallin, the four N-terminal residues have great flexibility compared with the rest of the molecule and assume a random coil conformation. NMR spectroscopy and electrospray mass spectrometry show that the N-terminal residue is acetylated. Thus, gamma S-crystallin is similar to the acidic beta-crystallins in having a flexible N-terminal extension and an N-terminus that is blocked with an acetyl group but no C-terminal extension. In addition to the short N-terminal extension in gamma S-crystallin, other unassigned resonances are also observed in the NMR spectra. In gamma B-crystallin, however, cross-peaks in the NH to alpha-CH region of the spectrum are essentially restricted to the last three residues of the C-terminal domain. The NMR data imply that gamma S-crystallin has a more flexible structure than gamma B-crystallin. Sedimentation equilibrium studies on gamma S-crystallin are consistent with this proposal. Resonances from the N-terminal extension of gamma S-crystallin are not affected by the presence of alpha-crystallin implying that this region is not involved in interactions between the two molecules. It is concluded that gamma S-crystallin shares structural properties which are intermediate between the beta- and gamma-crystallins.

The solution structure of frenatin 3, a neuronal nitric oxide synthase inhibitor from the giant tree frog, Litoria infrafrenata

Publisher: Wiley

Date: 11-2003

DOI: 10.1002/BIP.10524

Abstract: The peptide frenatin 3 is a major component of the skin secretion of the Australian giant tree frog, Litoria infrafrenata. Frenatin 3 is 22 amino acids in length, and shows neither antimicrobial nor anticancer activity. It inhibits the production of nitric oxide by the enzyme neuronal nitric oxide synthase at a micromolar concentration by binding to its regulatory protein, Ca2+ calmodulin, a protein known to recognize and bind hipathic alpha-helices. The solution structure of frenatin 3 has been investigated using NMR spectroscopy and restrained molecular dynamics calculations. In trifluoroethanol/water mixtures, the peptide forms an hipathic alpha-helix over residues 1-14 while the C-terminal eight residues are more flexible and less structured. The flexible region may be responsible for the lack of antimicrobial activity. In water, frenatin 3 exhibits some alpha-helical character in its N-terminal region.

The small heat-shock chaperone protein, alpha-crystallin, does not recognise stable molten globule states of cytosolic proteins

Publisher: Elsevier BV

Date: 08-2000

DOI: 10.1016/S0167-4838(00)00109-6

Abstract: The small heat-shock protein (sHsp), alpha-crystallin, acts as a molecular chaperone by interacting with destabilized 'substrate' proteins to prevent their precipitation from solution under conditions of stress. alpha-Crystallin and all sHsps are intracellular proteins. Similarly to other chaperones, the 'substrate' protein is in an intermediately folded, partly structured molten globule state when it interacts and complexes with alpha-crystallin. In this study, stable molten globule states of the cytosolic proteins, gamma-crystallin and myoglobin, have been prepared. Within the lens, gamma-crystallin naturally interacts with alpha-crystallin and myoglobin and alpha-crystallin are present together in muscle tissue. The molten globule states of gamma-crystallin and myoglobin were prepared by reacting gamma-crystallin with glucose 6-phosphate and by removing the haem group of myoglobin. Following spectroscopic characterisation of these modified proteins, their interaction with alpha-crystallin was examined by a variety of spectroscopic and protein chemical techniques. In both cases, there was no interaction with alpha-crystallin that led to complexation. It is concluded that alpha-crystallin does not recognise stable molten globule states of cytosolic 'substrate' proteins and only interacts with molten globule states of proteins that are on the irreversible pathway towards an aggregated and precipitated form.

H-1-NMR SPECTROSCOPY REVEALS THAT MOUSE HSP25 HAS A FLEXIBLE C-TERMINAL EXTENSION OF 18 AMINO-ACIDS

Publisher: Wiley

Date: 07-08-1995

DOI: 10.1016/0014-5793(95)00770-A

Abstract: The small heat-shock proteins (Hsps) exist as large aggregates and function by interacting and stabilising non-native proteins in a chaperone-like manner. Two-dimensional 1H NMR spectroscopy of mouse Hsp25 reveals that the last 18 amino acids have great flexibility with motion that is essentially independent of the domain core of the protein. The lens protein, alpha-crystallin, is homologous to Hsp25 and its two subunits also have flexible C-terminal extensions. The flexible region in Hsp25 encompasses exactly that expected from sequence comparison with alpha-crystallin implying that both proteins have similar structures and that the C-terminal extensions could be of functional importance for both proteins.

Real-time monitoring of amyloid growth in a rigid gel matrix

Publisher: Elsevier BV

Date: 10-2016

DOI: 10.1016/J.AB.2016.07.024

Abstract: We demonstrate the real-time monitoring of the growth of amyloid-protein aggregates in a semi-rigid gel environment constructed from a 5% w/v gelatin solution. The kinetics of amyloid fibril growth from reduced and carboxy-methylated κ-casein occurring in the gel medium was contrasted against that obtained in a regular solution assay. Aggregation kinetics were recorded using Thioflavin T fluorescence. Transmission electron microscopy was used to confirm the aggregates' existence and morphology. The current demonstration of controlled amyloid growth in a gel environment represents the first step towards development of an experimental model for investigating the role of spatial and medium factors in the kinetics of aggregation-based proteopathies.

Amyloid aggregation and membrane activity of the antimicrobial peptide uperin 3.5

Publisher: Wiley

Date: 23-02-2018

DOI: 10.1002/PEP2.24052

STRUCTURAL CHARACTERIZATION OF PIPERIDINE ALKALOIDS FROM PANDANUS-AMARYLLIFOLIUS BY INVERSE-DETECTED 2D NMR TECHNIQUES

Publisher: Elsevier BV

Date: 11-1993

DOI: 10.1016/S0031-9422(00)90735-0

Mildly acidic pH activates the extracellular molecular chaperone clusterin

Publisher: Elsevier BV

Date: 10-2002

DOI: 10.1074/JBC.M204855200

Identification of glutathionyl-3-hydroxykynurenine glucoside as a novel fluorophore associated with aging of the human lens

Publisher: Elsevier BV

Date: 07-1999

DOI: 10.1074/JBC.274.30.20847

HIGH-RESOLUTION H-1-NMR STUDY OF THE SOLUTION STRUCTURE OF ALAMETHICIN

Publisher: American Chemical Society (ACS)

Date: 02-1987

DOI: 10.1021/BI00378A010

Abstract: A 1H NMR study of the peptide alamethicin, which forms voltage-gated ion channels in membranes, is described. The molecule was studied in methanol as a function of temperature and pH. A complete assignment of the spectra is given, including several stereospecific assignments. Alamethicin was found to have a structure substantially similar to the crystal although, in solution, the C-terminal dipeptide adopts a somewhat extended conformation. The overall conformation was insensitive to the ionization of the side chain of the only ionizable group, Glu-18.

Host-defence peptides of Australian anurans: structure, mechanism of action and evolutionary significance

Publisher: Elsevier BV

Date: 06-2004

DOI: 10.1016/J.PEPTIDES.2004.03.006

H-1-NMR SPECTROSCOPY OF BETA-B2-CRYSTALLIN FROM BOVINE EYE LENS - CONFORMATION OF THE N-TERMINAL AND C-TERMINAL EXTENSIONS

Publisher: Wiley

Date: 04-1993

DOI: 10.1111/J.1432-1033.1993.TB17764.X

Abstract: 1H-NMR spectroscopic studies of a 46-kDa homodimer, beta B2-crystallin, from bovine eye lens are presented. beta B2-crystallin has terminal extensions extending from globular N- and C-terminal domains that are well resolved in the NMR spectra, whereas, in the main, resonances from the bulk of the protein are not observed. Using two-dimensional NMR methods on beta B2-crystallin, its synthesised terminal extensions and a proteolysed s le of beta B2-crystallin with a portion of its C-terminus removed, it was possible to assign resonances to most of the amino acids in the terminal extensions. One-dimensional experiments at various pH values provided H-2 chemical shifts for the three terminal extension histidines from which their pKa values were measured. It is concluded that the terminal extensions appear to be of little ordered conformation, are accessible to solvent and flex freely from the main body of the protein. The results of the NMR spectroscopic studies of beta B2-crystallin are in excellent agreement with those for the X-ray crystal structure [Bax, B., Lapatto, R., Nalini, V., Driessen, H., Lindley, P. F., Mahadevan, D., Blundell, T. L. & Slingsby, C. (1990) Nature 347, 776-780]. No change in the spectrum of beta B2-crystallin was observed in the presence of calcium, suggesting that the termini are not involved in calcium binding.

Hemin as a generic and potent protein misfolding inhibitor

Publisher: Elsevier BV

Date: 11-2014

DOI: 10.1016/J.BBRC.2014.10.062

Abstract: Protein misfolding causes serious biological malfunction, resulting in diseases including Alzheimer's disease, Parkinson's disease and cataract. Molecules which inhibit protein misfolding are a promising avenue to explore as therapeutics for the treatment of these diseases. In the present study, thioflavin T fluorescence and transmission electron microscopy experiments demonstrated that hemin prevents amyloid fibril formation of kappa-casein, amyloid beta peptide and α-synuclein by blocking β-sheet structure assembly which is essential in fibril aggregation. Further, inhibition of fibril formation by hemin significantly reduces the cytotoxicity caused by fibrillar amyloid beta peptide in vitro. Interestingly, hemin degrades partially formed amyloid fibrils and prevents further aggregation to mature fibrils. Light scattering assay results revealed that hemin also prevents protein amorphous aggregation of alcohol dehydrogenase, catalase and γs-crystallin. In summary, hemin is a potent agent which generically stabilises proteins against aggregation, and has potential as a key molecule for the development of therapeutics for protein misfolding diseases.

Selective NMR Experiments on Macromolecules: Implementation and Analysis of QUIET-NOESY

Publisher: Elsevier BV

Date: 06-1998

DOI: 10.1006/JMRE.1998.1430

Abstract: The QUIET-NOESY experiment (Zwahlen et al., J. Am. Chem Soc. 116, 362-368, 1994) is applied to measure the mobility of the flexible extensions in the large aggregate (800 kDa) of a small heat-shock protein. The proper choices of the experimental protocol and parameters are discussed in order to employ a simplified data analysis procedure. Further experimental verification of the proposed strategy is also presented using the cyclic peptide gramicidin S as a model compound. Under suitable conditions, the determinations based on the analysis of QUIET-NOESY data are affected to a negligible extent by the approximations that are introduced by the proposed approach. Copyright 1998 Academic Press.

The Amyloid Fibril-Forming β-Sheet Regions of Amyloid β and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ

Publisher: MDPI AG

Date: 11-10-2021

DOI: 10.3390/MOLECULES26206120

Abstract: 14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid β (Aβ) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer’s and Parkinson’s diseases, respectively, a process that is intimately linked to the diseases’ progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aβ (Aβ40) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of 15N-labeled Aβ40 and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aβ40 (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt β-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased in iduals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aβ40 and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.

The Quaternary Organization and Dynamics of the Molecular Chaperone HSP26 Are Thermally Regulated

Publisher: Elsevier BV

Date: 09-2010

DOI: 10.1016/J.CHEMBIOL.2010.06.016

A spectroscopic study of glycated bovine alpha-crystallin: investigation of flexibility of the C-terminal extension, chaperone activity and evidence for diglycation

Publisher: Elsevier BV

Date: 12-1997

DOI: 10.1016/S0167-4838(97)00145-3

Abstract: The effect of glycating the C-terminal extensions of alpha-crystallin on their flexibility was investigated. In the course of the study the reaction sites were identified and double glycation of single lysine residues was found. Alpha-crystallin was incubated until approximately one mole of the sugar had reacted per subunit of the crystallin. The reaction sites were investigated by mass spectrometry and H NMR spectroscopy, and were found to be principally in the short and flexible C-terminal extensions. The chaperone ability of alpha-crystallin was unaffected by this limited glycation. There was little effect on the flexibility of the C-terminal extensions. This result supports the view that the flexibility of the C-terminal extensions of alpha-crystallin is important for chaperone activity. As alpha-crystallin consists of a mixture of unmodified and phosphorylated subunits, a detailed investigation was undertaken of the reaction of galactose with peptides comprising the C-terminal extensions of alphaA- and alphaB-crystallin. The alphaA peptide was incubated with galactose until 0.79 mole of sugar was bound per mole of peptide and the alphaB peptide reacted until 2.2 moles of galactose had been incorporated. The purified glycated peptides were examined by NMR and mass spectrometry to identify glycation site(s), and the effect of glycation on the conformation of the peptides. For both peptides, it was found that extensive glycation of the constituent lysine residues occurred. The addition of two galactose molecules to some lysine residues of the peptides was also noted. This diglycation was confirmed in control experiments with N-acetyl-lysine.

Functional and dysfunctional folding, association and aggregation of caseins

Publisher: Elsevier

Date: 2020

DOI: 10.1016/BS.APCSB.2019.09.002

Abstract: 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. In idually, 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.

[2,3] SIGMATROPIC REARRANGEMENT OF 1-VINYLIC TETRAHYDROISOQUINOLINE N-YLIDES AND N-OXIDES

Publisher: Elsevier BV

Date: 05-1993

DOI: 10.1016/S0040-4039(00)73696-3

Investigating the importance of the flexible hinge in caerin 1.1: Solution structures and activity of two synthetically modified caerin peptides

Publisher: American Chemical Society (ACS)

Date: 03-01-2004

DOI: 10.1021/BI035760B

Abstract: Caerin 1.1 is a potent broad-spectrum antibacterial peptide isolated from a number of Australian frogs of the Litoria genus. In membrane-like media, this peptide adopts two alpha-helices, separated by a flexible hinge region bounded by Pro15 and Pro19. Previous studies have suggested that the hinge region is important for effective orientation of the two helices within the bacterial cell membrane, resulting in lysis via the carpet mechanism. To evaluate the importance of the two Pro residues, they were replaced with either Ala or Gly. The antibacterial activity of these two peptides was tested, and their three-dimensional structures were determined using two-dimensional NMR spectroscopy and restrained molecular dynamics calculations. The resulting structures indicate that the central hinge angle decreases significantly upon replacement of the Pro residues with Gly and to a further extent with Ala. This trend was mirrored by a corresponding decrease in antibiotic activity, further exemplifying the necessity of the hinge in caerin 1.1 and related peptides. In a broader context, the use of Pro, Gly, and Ala variants of caerin 1.1 has enabled the relationship between conformational flexibility and activity to be directly investigated in a systematic manner.

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

Publisher: Wiley

Date: 18-07-2022

DOI: 10.1002/1873-3468.14449

Abstract: 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 HIGH-RESOLUTION H-1-NMR STUDY OF THE SOLUTION STRUCTURE OF HUMAN EPIDERMAL GROWTH-FACTOR

Publisher: Wiley

Date: 09-1986

DOI: 10.1016/0014-5793(86)80869-9

Abstract: 500 MHz 1H NMR studies of human epidermal growth factor are described. The backbone resonances of the 1-48 derivative of hEGF have been assigned using two-dimensional techniques. Analysis of the type and magnitude of the observed sequential nuclear Overhauser effects and the NH-alpha CH spin-spin coupling constants allowed prediction of the secondary structure. Aspects of the tertiary structure are also identified. A pair of antiparallel beta-sheets involving residues 18-23 and 28-34 is a dominant feature of the solution structure.

Oxidation products of 3-hydroxykynurenine bind to lens proteins: Relevance for nuclear cataract

Publisher: Elsevier BV

Date: 05-1997

DOI: 10.1006/EXER.1996.0258

Abstract: 3-Hydroxykynurenine (3OHKyn), present as a human lens UV filter, has also been implicated as a carcinogen and neurotoxin. It has been suggested that oxidation of 3OHKyn is involved in each of these effects. In the presence of oxygen, 3OHKyn has been found to react with bovine crystallins, to give brown-coloured products (Stutchbury and Truscott, 1993). In this study the roles of UV-light, pH, glutathione and oxygen were examined, with the objective of determining how these factors may affect the binding of 3OHKyn to crystallins under the conditions found within the lens itself. The presence of oxygen was found to be an important parameter for determining the extent to which 3OHKyn reacts with protein, and when it was totally excluded, little modification was observed. UV-light was not required for activation, but was found to augment the extent of modification and cross-linking, while an elevated pH, which is known to accelerate the rate of 3OHKyn oxidation, did not markedly increase the extent of reaction with the crystallins. 3OHKyn binding was accompanied by crystallin aggregation, pigmentation, and development of non-tryptophan fluorescence, all of which have been associated with cataract formation. The inclusion of glutathione, a ubiquitous antioxidant, in reaction mixtures resulted in a delayed onset of crystallin modification. This effect was apparent at concentrations of glutathione greater than 1 mM. When glutathione levels fell below 1 mM, crystallins became modified by 3OHKyn. Since lens glutathione concentrations decrease with age, and are known to be lower in the lens nucleus than the cortex, this region appears particularly vulnerable to modification by this UV filter. Thus, whilst the other human lens UV filters, kynurenine (Kyn) and 3-hydroxykynurenine glucoside (3HKG), appear to require activation by UV-light in order to react with proteins, 3OHKyn can modify crystallins in the absence of light, under conditions of low oxygen tension, and in the presence of glutathione concentrations found in the nucleus of an aged lens. Its reactivity is increased in the presence of both light and oxygen. The contributions of these parameters to the reactivity of 3OHKyn are discussed, with respect to the aetiology of senile nuclear cataract.

The effect of dextran on subunit exchange of the molecular chaperone alpha A-crystallin

Publisher: Elsevier BV

Date: 2007

DOI: 10.1016/J.BBAPAP.2006.10.002

Abstract: Alpha-crystallin, a member of small heat shock protein (sHsp) family, is comprised of alphaA and alphaB subunits and acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation. The alphaA-crystallin homopolymer consists of 30-40 subunits that are undergoing dynamic exchange. In vivo, alpha-crystallin elicits its chaperone action in a crowded cellular environment (e.g. in the lens). In vitro, inert molecular crowding agents (e.g. dextran) are often used to mimic crowded conditions. In this study, it was found that alpha-crystallin and alphaA-crystallin are poorer chaperones in the presence of dextran. Using fluorescence resonance energy transfer, it is shown that the alphaA-crystallin subunit exchange rate strongly increases with temperature. Binding of reduced ovotransferrin to alphaA-crystallin markedly decreases the rate of subunit exchange, as does the presence of dextran. In addition, in the presence of dextran the effect of reduced ovotransferrin on decreasing the rate of subunit exchange of alphaA-crystallin is greater than in the absence of dextran. Under the conditions of molecular crowding, the alphaA-crystallin subunit exchange rate is not temperature-dependent. In the absence of dextran, the exchange rate of alphaA-crystallin subunits correlates with its chaperone efficiency, i.e. the chaperone ability of alphaA-crystallin increases with temperature. However in the presence of dextran, the temperature dependence of the chaperone ability of alphaA-crystallin is eliminated.

Functional amyloid protection in the eye lens: Retention of α-crystallin molecular chaperone activity after modification into amyloid fibrils

Publisher: MDPI AG

Date: 12-09-2017

DOI: 10.3390/BIOM7030067

The chaperone activity of a-synuclein: Utilizing deletion mutants to map its interaction with target proteins

Publisher: Wiley

Date: 10-02-2012

DOI: 10.1002/PROT.24028

Abstract: α-Synuclein is the principal component of the Lewy body deposits that are characteristic of Parkinson's disease. In vivo, and under physiological conditions in vitro, α-synuclein aggregates to form amyloid fibrils, a process that is likely to be associated with the development of Parkinson's disease. α-Synuclein also possesses chaperone activity to prevent the precipitation of amorphously aggregating target proteins, as demonstrated in vitro. α-Synuclein is an intrinsically disordered (i.e., unstructured) protein of 140 amino acids in length, and therefore studies on its fragments can be correlated directly to the functional role of these regions in the intact protein. In this study, the fragment containing residues 61-140 [α-syn(61-140)] was observed to be highly amyloidogenic and was as effective a chaperone in vitro as the full-length protein, while the N- and C-terminal fragments α-syn(1-60) and α-syn(96-140) had no intrinsic chaperone activity. Interestingly, full-length fibrillar α-synuclein had greater chaperone activity than nonfibrillar α-synuclein. It is concluded that the amyloidogenic NAC region (residues 61-95) contains the chaperone-binding site which is optimized for target protein binding as a result of its β-sheet formation and/or ordered aggregation by α-synuclein. On the other hand, the first 60 residues of α-synuclein modulate the protein's chaperone-active site, while at the same time protecting α-synuclein from fibrillation. On its own, however, this fragment [α-syn(1-60)] had a tendency to aggregate amorphously. As a result of this study, the functional roles of the various regions of α-synuclein in its chaperone activity have been delineated.

Facile detection of organometallic derivatives of peptides using electrospray mass spectrometry

Publisher: Informa UK Limited

Date: 04-1995

DOI: 10.1080/00958979508024323

Monitoring the prevention of amyloid fibril formation by alpha-crystallin

Publisher: Wiley

Date: 14-11-2007

DOI: 10.1111/J.1742-4658.2007.06144.X

Abstract: The molecular chaperone, alpha-crystallin, has the ability to prevent the fibrillar aggregation of proteins implicated in human diseases, for ex le, 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.

The effect of milk constituents and crowding agents on amyloid fibril formation by κ-casein

Publisher: American Chemical Society (ACS)

Date: 04-02-2016

DOI: 10.1021/ACS.JAFC.5B04977

Abstract: When not incorporated into the casein micelle, κ-casein, a major milk protein, rapidly forms amyloid fibrils at physiological pH and temperature. In this study, the effects of milk components (calcium, lactose, lipids, and heparan sulfate) and crowding agents on reduced and carboxymethylated (RCM) κ-casein fibril formation was investigated using far-UV circular dichroism spectroscopy, thioflavin T binding assays, and transmission electron microscopy. Longer-chain phosphatidylcholine lipids, which form the lining of milk ducts and milk fat globules, enhanced RCM κ-casein fibril formation irrespective of whether the lipids were in a monomeric or micellar state, whereas shorter-chain phospholipids and triglycerides had little effect. Heparan sulfate, a component of the milk fat globule membrane and catalyst of amyloid deposition in extracellular tissue, had little effect on the kinetics of RCM κ-casein fibril formation. Major nutritional components such as calcium and lactose also had no significant effect. Macromolecular crowding enhances protein-protein interactions, but in contrast to other fibril-forming species, the extent of RCM κ-casein fibril formation was reduced by the presence of a variety of crowding agents. These data are consistent with a mechanism of κ-casein fibril formation in which the rate-determining step is dissociation from the oligomer to give the highly amyloidogenic monomer. We conclude that the interaction of κ-casein with membrane-associated phospholipids along its secretory pathway may contribute to the development of amyloid deposits in mammary tissue. However, the formation of spherical oligomers such as casein micelles is favored over amyloid fibrils in the crowded environment of milk, within which the occurrence of amyloid fibrils is low.

Evidence That Clusterin Has Discrete Chaperone and Ligand Binding Sites

Publisher: American Chemical Society (ACS)

Date: 08-12-2001

DOI: 10.1021/BI0157666

Abstract: Clusterin is the first identified extracellular mammalian chaperone and binds to a wide variety of partly unfolded, stressed proteins.Clusterin also binds to many different unstressed ligands including the cell surface receptor low-density lipoprotein receptor-related protein-2 (LRP-2). It is unknown whether clusterin binds to all of these many ligands via one or more binding sites. Furthermore, the region(s) of clusterin involved in these many binding interactions remain(s) to be identified. As part of an investigation of these issues, we expressed recombinant human clusterin in the yeast Pichia pastoris. The resultant protein had variable proteolytic truncations of the C-terminal region of the alpha-chain and the N-terminal region of the beta-chain. We compared the chaperone and ligand binding activities of this recombinant product with those of clusterin purified from human serum. We also tested whether the binding of clusterin to ligands could be inhibited by competitive binding with other clusterin ligands or by anti-clusterin monoclonal antibodies. Collectively, our results indicate that (i) clusterin has three independent classes of binding sites for LRP-2, stressed proteins, and unstressed ligands, respectively, and (ii) the binding sites for LRP-2 and stressed proteins are likely to be in parts of the molecule other than the C-terminal region of the alpha-chain or the N-terminal region of the beta-chain. It has been suggested that, in vivo, clusterin binds to toxic molecules in the extracellular environment and carries these to cells expressing LRP-2 for uptake and degradation. This hypothesis is supported by our demonstration that clusterin has discrete binding sites for LRP-2 and other (potentially toxic) molecules.

SELECTIVE LABELING OF PEPTIDES USING (DIENYL) IRON TRICARBONYL CATIONS

Publisher: Royal Society of Chemistry (RSC)

Date: 1993

DOI: 10.1039/C39930000928

Enhanced molecular chaperone activity of the small heat-shock protein αB-crystallin following covalent immobilization onto a solid-phase support

Publisher: Wiley

Date: 10-01-2011

DOI: 10.1002/BIP.21584

Abstract: The well-characterized small heat-shock protein, alphaB-crystallin, acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation and precipitation. Structural perturbation (e.g., partial unfolding) enhances the in vitro chaperone activity of alphaB-crystallin. Proteins often undergo structural perturbations at the surface of a synthetic material, which may alter their biological activity. This study investigated the activity of alphaB-crystallin when covalently bound to a support surface alphaB-crystallin was immobilized onto a range of solid material surfaces, and its characteristics and chaperone activity were assessed. Immobilization was achieved via a plasma-deposited thin polymeric interlayer containing aldehyde surface groups and reductive amination, leading to the covalent binding of alphaB-crystallin lysine residues to the surface aldehyde groups via Schiff-base linkages. Immobilized alphaB-crystallin was characterized by X-ray photoelectron spectroscopy, atomic force microscopy, and quartz crystal microgravimetry, which showed that 300 ng cm(-2) (dry mass) of oligomeric alphaB-crystallin was bound to the surface. Immobilized alphaB-crystallin exhibited a significant enhancement (up to 5000-fold, when compared with the equivalent activity of alphaB-crystallin in solution) of its chaperone activity against various proteins undergoing both amorphous and amyloid fibril forms of aggregation. The enhanced molecular chaperone activity of immobilized alphaB-crystallin has potential applications in preventing protein misfolding, including against amyloid disease processes, such as dialysis-related amyloidosis, and for biodiagnostic detection of misfolded proteins.

Unraveling the mysteries of protein folding and misfolding

Publisher: Wiley

Date: 12-2008

DOI: 10.1002/IUB.117

Abstract: This mini-review focuses on the processes and consequences of protein folding and misfolding. The latter process often leads to protein aggregation and precipitation with the aggregates adopting either highly ordered (amyloid fibril) or disordered (amorphous) forms. In particular, the amyloid fibril is discussed because this form has gained considerable notoriety due to its close links to a variety of debilitating diseases including Alzheimer's, Parkinson's, Huntington's, and Creutzfeldt-Jakob diseases, and type-II diabetes. In each of these diseases a different protein forms fibrils, yet the fibrils formed have a very similar structure. The mechanism by which fibrils form, fibril structure, and the cytotoxicity associated with fibril formation are discussed. The generic nature of amyloid fibril structure suggests that a common target may be accessible to treat amyloid fibril-associated diseases. As such, the ability of some molecules, for ex le, the small heat-shock family of molecular chaperone proteins, to inhibit fibril formation is of interest due to their therapeutic potential.

Casein structures in the context of unfolded proteins

Publisher: Elsevier BV

Date: 07-2015

DOI: 10.1016/J.IDAIRYJ.2014.07.008

Amyloid-β Oligomers are Sequestered by both Intracellular and Extracellular Chaperones

Publisher: American Chemical Society (ACS)

Date: 08-11-2012

DOI: 10.1021/BI301277K

Application of the Double-Mutant Cycle Strategy to Protein Aggregation Reveals Transient Interactions in Amyloid-β Oligomers

Publisher: American Chemical Society (ACS)

Date: 08-11-2021

DOI: 10.1021/ACS.JPCB.1C05829

Abstract: Transient oligomeric intermediates in the peptide or protein aggregation pathway are suspected to be the key toxic species in many amyloid diseases, but deciphering their molecular nature has remained a challenge. Here we show that the strategy of "double-mutant cycles", used effectively in probing protein-folding intermediates, can reveal transient interactions during protein aggregation. It does so by comparing the changes in thermodynamic parameters between the wild type, and single and double mutants. We demonstrate the strategy by probing the possible transient salt bridge partner of lysine 28 (K28) in the oligomeric states of amyloid β-40 (Aβ

SEVI, the semen enhancer of HIV infection along with fragments from its central region, form amyloid fibrils that are toxic to neuronal cells

Publisher: Elsevier BV

Date: 09-2014

DOI: 10.1016/J.BBAPAP.2014.06.006

Abstract: Semen-derived enhancer of viral infection (SEVI) is the term given to the amyloid fibrils formed by a 39-amino acid fragment (PAP248-286) of prostatic acidic phosphatase (PAP) found in human semen. SEVI enhances human immunodeficiency virus (HIV) infectivity by four to five orders of magnitude (Münch et al., 2007). Here, we show by various biophysical techniques including Thioflavin T fluorescence, circular dichroism spectroscopy and transmission electron microscopy that fragments encompassing the central region of SEVI, i.e. PAP248-271 and PAP257-267, form fibrils of similar morphology to SEVI. Our results show that the central region, residues PAP267-271, is crucially important in promoting SEVI fibril formation. Furthermore, SEVI and fibrillar forms of these peptide fragments are toxic to neuronal pheochromocytoma 12 cells but not to epithelial colon carcinoma cells. These findings imply that although SEVI assists in the attachment of HIV-1 to immune cells, it may not facilitate HIV entry by damaging the epithelial cell layer that presents a barrier to the HIV.

THE CONFORMATION OF BOMBESIN IN SOLUTION AS DETERMINED BY TWO-DIMENSIONAL H-1-NMR TECHNIQUES

Publisher: Wiley

Date: 10-1987

DOI: 10.1111/J.1432-1033.1987.TB13404.X

Abstract: The 1H nuclear magnetic resonance spectrum of the tetradecapeptide, bombesin, has been assigned in (2H6)dimethyl sulphoxide solution and aqueous solution using two-dimensional techniques. The chemical shifts in both solvents indicate that the molecule has little secondary structure and adopts a random coil conformation. A comparison is made between the spectra of various smaller bombesin fragments and the intact polypeptide.

Monitoring the Interaction between β2-Microglobulin and the Molecular Chaperone αB-crystallin by NMR and Mass Spectrometry

Publisher: Elsevier BV

Date: 06-2013

DOI: 10.1074/JBC.M112.448639

Structural alterations of alpha-crystallin during its chaperone action

Publisher: Wiley

Date: 15-11-1998

DOI: 10.1046/J.1432-1327.1998.2580170.X

Abstract: The small heat-shock protein, alpha-crystallin, has chaperone ability whereby it stabilises proteins under stress conditions. In this study, alterations in the structure of alpha-crystallin during its interaction with a variety of substrate proteins (insulin, alpha-lactalbumin, ovotransferrin and serum albumin) under stress conditions have been examined using visible absorption, 31P-NMR and 1H-NMR and fluorescence spectroscopy. The fluorescence and 31P-NMR data imply that during the chaperone action of alpha-crystallin under reducing conditions, there is a slight increase in hydrophilicity of its N-terminal region and an alteration in flexibility of its C-terminal region, but overall, alpha-crystallin does not undergo a gross structural change. The fluorescence data suggest that substrate proteins interact with alpha-crystallin in a molten globule or intermediately folded state. The same conclusion is made from 1H-NMR spectroscopic monitoring of the interaction of alpha-crystallin with substrate proteins, e.g. the insulin B chain. The stoichiometry of interaction between alpha-crystallin and the various substrate proteins reveals that steric factors are important in determining the efficiency of interaction between the two proteins, i.e. on a molar subunit basis, alpha-crystallin is a more efficient chaperone protein with smaller substrate proteins. Comparison is also made between the high-molecular-mass (HMM) complexes formed between alpha-crystallin and ovotransferrin when reduced and heat stressed. Under heating conditions, fluorescence spectroscopy indicates that the HMM complex has a greater exposure of hydrophobicity to solution than that formed by reduction. Furthermore, in interacting with heated ovotransferrin, the C-terminal extension of the alphaB-crystallin subunit preferentially loses its flexibility suggesting that it is involved in stabilising bound ovotransferrin. By contrast, this extension is only partially reduced in flexibility in the HMM complex formed after reduction of ovotransferrin. The functional role of the C-terminal extensions in the chaperone action and the overall quaternary structure of alpha-crystallin is discussed.

Gallic acid interacts with α-synuclein to prevent the structural collapse necessary for its aggregation

Publisher: Elsevier BV

Date: 09-2014

DOI: 10.1016/J.BBAPAP.2014.04.013

Abstract: The accumulation of protein aggregates containing amyloid fibrils, with α-synuclein being the main component, is a pathological hallmark of Parkinson's disease (PD). Molecules which prevent the formation of amyloid fibrils or disassociate the toxic aggregates are touted as promising strategies to prevent or treat PD. In the present study, in vitro Thioflavin T fluorescence assays and transmission electron microscopy imaging results showed that gallic acid (GA) potently inhibits the formation of amyloid fibrils by α-synuclein. Ion mobility-mass spectrometry demonstrated that GA stabilises the extended, native structure of α-synuclein, whilst NMR spectroscopy revealed that GA interacts with α-synuclein transiently.

The functional roles of the unstructured N- and C-terminal regions in αB-crystallin and other mammalian small heat-shock proteins

Publisher: Springer Science and Business Media LLC

Date: 08-04-2017

DOI: 10.1007/S12192-017-0789-6

A multi-pathway perspective on protein aggregation: Implications for control of the rate and extent of amyloid formation

Publisher: Wiley

Date: 31-01-2015

DOI: 10.1016/J.FEBSLET.2015.01.032

The Structure and Stability of the Disulfide-Linked γS-Crystallin Dimer Provide Insight into Oxidation Products Associated with Lens Cataract Formation

Publisher: Elsevier BV

Date: 02-2019

DOI: 10.1016/J.JMB.2018.12.005

Abstract: The reducing environment in the eye lens diminishes with age, leading to significant oxidative stress. Oxidation of lens crystallin proteins is the major contributor to their destabilization and deleterious aggregation that scatters visible light, obscures vision, and ultimately leads to cataract. However, the molecular basis for oxidation-induced aggregation is unknown. Using X-ray crystallography and small-angle X-ray scattering, we describe the structure of a disulfide-linked dimer of human γS-crystallin that was obtained via oxidation of C24. The γS-crystallin dimer is stable at glutathione concentrations comparable to those in aged and cataractous lenses. Moreover, dimerization of γS-crystallin significantly increases the protein's propensity to form large insoluble aggregates owing to non-cooperative domain unfolding, as is observed in crystallin variants associated with early-onset cataract. These findings provide insight into how oxidative modification of crystallins contributes to cataract and imply that early-onset and age-related forms of the disease share comparable development pathways.

The molecular chaperone β-casein prevents amorphous and fibrillar aggregation of α-lactalbumin by stabilisation of dynamic disorder

Publisher: Portland Press Ltd.

Date: 11-02-2020

DOI: 10.1042/BCJ20190638

Abstract: Deficits in protein homeostasis (proteostasis) are typified by the partial unfolding or misfolding of native proteins leading to amorphous or fibrillar aggregation, events that have been closely associated with diseases including Alzheimer's and Parkinson's diseases. Molecular chaperones are intimately involved in maintaining proteostasis, and their mechanisms of action are in part dependent on the morphology of aggregation-prone proteins. This study utilised native ion mobility–mass spectrometry to provide molecular insights into the conformational properties and dynamics of a model protein, α-lactalbumin (α-LA), which aggregates in an amorphous or amyloid fibrillar manner controlled by appropriate selection of experimental conditions. The molecular chaperone β-casein (β-CN) is effective at inhibiting amorphous and fibrillar aggregation of α-LA at sub-stoichiometric ratios, with greater efficiency against fibril formation. Analytical size-exclusion chromatography demonstrates the interaction between β-CN and amorphously aggregating α-LA is stable, forming a soluble high molecular weight complex, whilst with fibril-forming α-LA the interaction is transient. Moreover, ion mobility–mass spectrometry (IM-MS) coupled with collision-induced unfolding (CIU) revealed that α-LA monomers undergo distinct conformational transitions during the initial stages of amorphous (order to disorder) and fibrillar (disorder to order) aggregation. The structural heterogeneity of monomeric α-LA during fibrillation is reduced in the presence of β-CN along with an enhancement in stability, which provides a potential means for preventing fibril formation. Together, this study demonstrates how IM-MS and CIU can investigate the unfolding of proteins as well as examine transient and dynamic protein–chaperone interactions, and thereby provides detailed insight into the mechanism of chaperone action and proteostasis mechanisms.

The solution structure and activity of caerin 1.1, an antimicrobial peptide from the Australian green tree frog, Litoria splendida

Publisher: Wiley

Date: 15-07-1997

DOI: 10.1111/J.1432-1033.1997.00545.X

Abstract: Caerin 1.1 is one of the major antimicrobial peptides isolated from the skin of the Australian green tree frog, Litoria splendida. Two-dimensional 1H-1H and 1H-13C NMR spectroscopy in trifluoroethanol/H2O (50:50, by vol.) have been used to assign the 1H and 13C-NMR spectra of this 25-amino-acid peptide. From an examination of these data, and using distance geometry and molecular dynamics calculations, the solution conformation of caerin 1.1 has been determined. The peptide adopts two well-defined helices from Leu2 to Lys11 and from Val17 to His24 separated by a region of less-defined helicity and greater flexibility. Overall, the peptide has a distinct hipathic charge distribution. The solution structure of caerin 1.1 is compared with activity data against a variety of micro-organisms for the parent peptide and some naturally occurring and synthetic variants of caerin 1.1. The structural and activity data are consistent with caerin 1.1 interacting with membranes in a similar manner to other antimicrobial peptides, i.e. via a carpet-like mechanism whereby the in idual peptides aggregate in a helical manner and orient themselves parallel to the membrane in a sheet-like arrangement [Shai, Y. (1995) Trends Biochem. Sci. 20, 460-464].

Gallic acid is the major component of grape seed extract that inhibits amyloid fibril formation

Publisher: Elsevier BV

Date: 12-2013

DOI: 10.1016/J.BMCL.2013.09.071

Abstract: Many protein misfolding diseases, for ex le, Alzheimer's, Parkinson's and Huntington's, are characterised by the accumulation of protein aggregates in an amyloid fibrillar form. Natural products which inhibit fibril formation are a promising avenue to explore as therapeutics for the treatment of these diseases. In this study we have shown, using in vitro thioflavin T assays and transmission electron microscopy, that grape seed extract inhibits fibril formation of kappa-casein (κ-CN), a milk protein which forms amyloid fibrils spontaneously under physiological conditions. Among the components of grape seed extract, gallic acid was the most active component at inhibiting κ-CN fibril formation, by stabilizing κ-CN to prevent its aggregation. Concomitantly, gallic acid significantly reduced the toxicity of κ-CN to pheochromocytoma12 cells. Furthermore, gallic acid effectively inhibited fibril formation by the amyloid-beta peptide, the putative causative agent in Alzheimer's disease. It is concluded that the gallate moiety has the fibril-inhibitory activity.

The multifaceted nature of αB-crystallin

Publisher: Springer Science and Business Media LLC

Date: 07-05-2020

DOI: 10.1007/S12192-020-01098-W

An Enantiopure Hydrogen‐Bonded Octameric Tube: Self‐Sorting and Guest‐Induced Rearrangement

Publisher: Wiley

Date: 05-11-2015

DOI: 10.1002/ANIE.201508362

Abstract: The assembly of a discrete hydrogen-bonded molecular tube from eight small identical monomers is reported. Tube assembly was accomplished by means of selective heterodimerization between isocytosine and ureidopyrimidinone hydrogen-bonding motifs embedded in an enantiopure bicyclic building block, leading to the selective formation of an octameric supramolecular tube. Upon introduction of a fullerene guest molecule, the octameric tube rearranges into a tetrameric inclusion complex and the hydrogen-bonding mode is switched. The dynamic behavior of the system is further explored in solvent- and guest-responsive self-sorting experiments.

Amyloid fibril formation by lens crystallin proteins and its implications for cataract formation.

Publisher: Elsevier BV

Date: 2004

DOI: 10.1074/JBC.M308203200

β-Synuclein: An Enigmatic Protein with Diverse Functionality

Publisher: MDPI AG

Date: 16-01-2022

DOI: 10.3390/BIOM12010142

Abstract: α-Synuclein (αS) is a small, unstructured, presynaptic protein expressed in the brain. Its aggregated form is a major component of Lewy bodies, the large proteinaceous deposits in Parkinson’s disease. The closely related protein, β-Synuclein (βS), is co-expressed with αS. In vitro, βS acts as a molecular chaperone to inhibit αS aggregation. As a result of this assignation, βS has been largely understudied in comparison to αS. However, recent reports suggest that βS promotes neurotoxicity, implying that βS is involved in other cellular pathways with functions independent of αS. Here, we review the current literature pertaining to human βS in order to understand better the role of βS in homeostasis and pathology. Firstly, the structure of βS is discussed. Secondly, the ability of βS to (i) act as a molecular chaperone (ii) regulate synaptic function, lipid binding, and the nigrostriatal dopaminergic system (iii) mediate apoptosis (iv) participate in protein degradation pathways (v) modulate intracellular metal levels and (vi) promote cellular toxicity and protein aggregation is explored. Thirdly, the P123H and V70M mutations of βS, which are associated with dementia with Lewy bodies, are discussed. Finally, the importance of post-translational modifications on the structure and function of βS is reviewed. Overall, it is concluded that βS has both synergistic and antagonistic interactions with αS, but it may also possess important cellular functions independent of αS.

Clusterin has chaperone-like activity similar to that of small heat shock proteins

Publisher: Elsevier BV

Date: 03-1999

DOI: 10.1074/JBC.274.11.6875

A POSSIBLE CHAPERONE-LIKE QUATERNARY STRUCTURE FOR ALPHA-CRYSTALLIN

Publisher: Elsevier BV

Date: 08-1994

DOI: 10.1006/EXER.1994.1101

Methionine Oxidation Enhances kappa-Casein Amyloid Fibril Formation

Publisher: American Chemical Society (ACS)

Date: 11-04-2012

DOI: 10.1021/JF205168T

Abstract: The effects of protein oxidation, for ex le of methionine residues, are linked to many diseases, including those of protein misfolding, such as Alzheimer's disease. Protein misfolding diseases are characterized by the accumulation of insoluble proteinaceous aggregates comprised mainly of amyloid fibrils. Amyloid-containing bodies known as corpora amylacea (CA) are also found in mammary secretory tissue, where their presence slows milk flow. The major milk protein κ-casein readily forms amyloid fibrils under physiological conditions. Milk exists in an extracellular oxidizing environment. Accordingly, the two methionine residues in κ-casein (Met(95) and Met(106)) were selectively oxidized and the effects on the fibril-forming propensity, cellular toxicity, chaperone ability, and structure of κ-casein were determined. Oxidation resulted in an increase in the rate of fibril formation and a greater level of cellular toxicity. β-Casein, which inhibits κ-casein fibril formation in vitro, was less effective at suppressing fibril formation of oxidized κ-casein. The ability of κ-casein to prevent the amorphous aggregation of target proteins was slightly enhanced upon methionine oxidation, which may arise from the protein's greater exposed surface hydrophobicity. No significant changes to κ-casein's intrinsically disordered structure occurred upon oxidation. The enhanced rate of fibril formation of oxidized κ-casein, coupled with the reduced chaperone ability of β-casein to prevent this aggregation, may affect casein-casein interaction within the casein micelle and thereby promote κ-casein aggregation and contribute to the formation of CA.

Cumulative deamidations of the major lens protein γS ‐crystallin increase its aggregation during unfolding and oxidation

Publisher: Wiley

Date: 28-08-2020

DOI: 10.1002/PRO.3915

Ion Mobility Mass Spectrometry Studies of the Inhibition of Alpha Synuclein Amyloid Fibril Formation by ( - )-Epigallocatechin-3-Gallate

Publisher: CSIRO Publishing

Date: 2011

DOI: 10.1071/CH10334

Abstract: Ion mobility-mass spectrometry (IM-MS) is emerging as an important biophysical technique for the structural analysis of proteins and their assemblies, in particular for structurally heterogeneous systems such as those on the protein misfolding and aggregation pathway. Using IM-MS we have monitored amyloid fibril formation of A53T α-synuclein, a mutant synuclein protein associated with Parkinson’s disease, and identified that a conformational change towards a more compact structure occurs during the initial stages of aggregation. Binding of A53T α-synuclein to a flavenoid based amyloid fibril inhibitor, (–)-epigallocatechin-3-gallate, has been observed with a 1:1 stoichiometry. By analysis of ion collision cross-sections, we show epigallocatechin gallate binding prevents protein conformational change, and in turn decreases the formation of fibrillar aggregates.

ASSIGNMENT OF H-1-NMR RESONANCES OF HISTIDINE AND OTHER AROMATIC RESIDUES IN MET-MYOGLOBIN, CYANO-MYOGLOBIN, OXY-MYOGLOBIN, AND (CARBON MONOXY)MYOGLOBIN

Publisher: American Chemical Society (ACS)

Date: 09-10-1984

DOI: 10.1021/BI00316A012

Abstract: The resolved 1H NMR resonances of the aromatic region in the 270-MHz NMR spectrum of sperm whale, horse, and pig metmyoglobin (metMb) have been assigned, including the observable H-2 and H-4 histidine resonances, the tryptophan H-2 resonances, and upfield-shifted resonances from one tyrosine residue. The use of different Mb species, carboxymethylation, and matching of pK values allows the assignment of the H-4 resonances, which agree in only three cases out of seven with scalar-correlated two-dimensional NMR spectroscopy assignments by others. The conversion to hydroxymyoglobin at high pH involves rearrangements throughout the molecule and is observed by many assigned residues. In sperm whale ferric cyanomyoglobin, nine H-2 and eight H-4 histidine resonances have been assigned, including the His-97 H-2 resonance and tyrosine resonances from residues 103 and 146. The hyperfine-shifted resonances from heme and near-heme protons observe a shift with a pK = 5.3 +/- 0.3 (probably due to deprotonation of His-97, pK = 5.6) and another shift at pK = 10.8 +/- 0.3. The spectrum of high-spin ferrous sperm whale deoxymyoglobin is very similar to that of metMb, which allows the assignment of seven surface histidine H-2 and H-4 resonances and also resonances from the two tryptophan residues and one tyrosine. In diamagnetic sperm whale (carbon monoxy)myoglobin (COMb), 10 His H-2 and 11 His H-4 resonances are observed, and 8 H-2 and 9 H-4 resonances are assigned, including His-64 H-4, the distal histidine. This important resonance is not observed in sperm whale oxymyoglobin, which in general shows very similar titration curves to COMb. Histidine-36 shows unusual titration behavior in the paramagnetic derivatives but normal behavior in the diamagnetic derivatives, which is discussed in the accompanying paper [Bradbury, J. H., & Carver, J. A. (1984) Biochemistry (following paper in this issue)].

LOSS OF THE C-TERMINAL SERINE RESIDUE FROM BOVINE BETA-B-2-CRYSTALLIN

Publisher: Elsevier BV

Date: 05-1995

DOI: 10.1016/S0014-4835(05)80061-2

Abstract: Electrospray mass spectrometric (ES-MS) examination of bovine beta-crystallins showed a significant component corresponding in mass to beta B2-crystallin less one serine residue. Tryptic digestion, followed by isolation and characterisation of the C-terminal peptide, demonstrated that this new species has arisen by the loss of the C-terminal serine residue. This phenomenon appears to be age-related since no truncation was detected in beta B2-crystallin from foetal lenses and the proportion of the truncated form, as judged by ES-MS, was lower in beta-crystallin isolated from calf lenses than that from the lenses of 3-year-old animals. This process therefore is similar to a recently reported loss of the C-terminal serine from alpha A-crystallin, which we have confirmed using ES-MS. Loss of a C-terminal serine from both crystallins may indicate the presence of carboxypeptidase-A-like activity in bovine lenses. ES-MS data provided no evidence for a significant degree of phosphorylation of beta B2-crystallin.

Dynamism in Molecular Chaperones

Publisher: Elsevier BV

Date: 10-2011

DOI: 10.1016/J.JMB.2011.08.052

Small heat-shock proteins prevent α-synuclein aggregation via transient interactions and their efficacy is affected by the rate of aggregation

Publisher: Elsevier BV

Date: 10-2016

DOI: 10.1074/JBC.M116.739250

A quantitative NMR spectroscopic examination of the flexibility of the C-terminal extensions of the molecular chaperones, αA- and αB-crystallin

Publisher: Elsevier BV

Date: 11-2010

DOI: 10.1016/J.EXER.2010.08.015

Abstract: The principal lens proteins αA- and αB-crystallin are members of the small heat-shock protein (sHsp) family of molecular chaperone proteins. Via their chaperone action, αA- and αB-crystallin play an important role in maintaining lens transparency by preventing crystallin protein aggregation and precipitation. αB-crystallin is found extensively extralenticularly where it is stress inducible and acts as a chaperone to facilitate general protein stabilization. The structure of either αA- or αB-crystallin is not known nor is the mechanism of their chaperone action. Our earlier (1)H NMR spectroscopic studies determined that mammalian sHsps have a highly dynamic, polar and unstructured region at their extreme C-terminus (summarized in Carver (1999) Prog. Ret. Eye Res. 18, 431). This C-terminal extension acts as a solubilizing agent for the relatively hydrophobic protein and the complex it makes with its target proteins during chaperone action. In this study, αA- and αB-crystallin were (15)N-labelled and their (1)H-(15)N through-bond correlation, heteronuclear single-quantum coherence (HSQC) NMR spectra were assigned via standard methods. (1)H-(15)N spin-lattice (T(1)) and spin-spin (T(2)) relaxation times were measured for αA- and αB-crystallin in the absence and presence of a bound target protein, reduced α-lactalbumin. (1)H-(15)N Nuclear Overhauser Effect (NOE) values provide an accurate measure, on a residue-by-residue basis, of the backbone flexibility of polypeptides. From measurement of these NOE values, it was determined that the flexibility of the extension in αA- and αB-crystallin increased markedly at the extreme C-terminus. By contrast, upon chaperone interaction of αA-crystallin with reduced α-lactalbumin, flexibility was maintained in the extension but was distributed evenly across all residues in the extension. Two mutants of αB-crystallin in its C-terminal region: (i) I159A and I161A and (ii) K175L, have altered chaperone ability (Treweek et al. (2007) PLoS One 2, e1046). Comparison of (1)H-(15)N NOE values for these mutants with wild type αB-crystallin revealed alteration in flexibility of the extension, particularly at the extremity of K175L αB-crystallin, which may affect chaperone ability.

AN INVESTIGATION INTO THE STABILITY OF ALPHA-CRYSTALLIN BY NMR-SPECTROSCOPY, EVIDENCE FOR A 2-DOMAIN STRUCTURE

Publisher: Elsevier BV

Date: 06-1993

DOI: 10.1016/0167-4838(93)90107-3

Abstract: The stability of bovine lens alpha-crystallin with respect to temperature, pH and urea has been investigated by 1H and 31P-NMR spectroscopy. The 1H and 31P-NMR spectra of alpha-crystallin show little change with temperature up to 75 degrees C, indicating that alpha-crystallin has great thermal stability and does not undergo any major change in structure with temperature. 1H spectral studies of alpha-crystallin and its isolated alpha A and alpha B subunits reveal a marked difference in the stability of these species. It is found that, at pH 2.5, alpha A-crystallin adopts a native conformation whereas alpha B-crystallin is denatured. On the other hand, the two subunits when part of the total alpha-crystallin aggregate adopt a native conformation at pH 2.5, but in the presence of 0.1 M glycine the alpha B subunits become denatured. Thus, alpha A-crystallin and total alpha-crystallin are more stable species than alpha B-crystallin and, in total alpha-crystallin, there is an interaction between the compact domains of the alpha A and alpha B subunits that leads to enhanced stability. Finally, changes in the 1H and 31P-NMR spectra of alpha A-crystallin and alpha B-crystallin in the presence of varying concentrations of urea are consistent with a two-domain model for alpha-crystallin subunits with the C-terminal domain being less stable and unfolding first in the presence of urea.

Amyloid fibril formation, structure and domain swapping of acyl‐coenzyme A thioesterase‐7

Publisher: Wiley

Date: 27-04-2023

DOI: 10.1111/FEBS.16795

Abstract: Acyl‐coenzyme A thioesterase (Acot) enzymes are involved in a broad range of essential intracellular roles including cell signalling, lipid metabolism, inflammation and the opening of ion channels. Dysregulation in lipid metabolism has been linked to neuroinflammatory and neurological disorders such as Alzheimer's and Parkinson's diseases. Structurally, Acot enzymes adopt a circularised trimeric arrangement with each monomer containing an N‐ and a C‐terminal hotdog domain. Acot7 spontaneously forms amyloid fibrils in vitro under physiological conditions. The resultant amyloid fibrillar structures were characterised by dye‐binding fluorescence assays, far‐UV circular dichroism spectroscopy, transmission electron microscopy and X‐ray fibre diffraction. Acot7 has an unusual mechanism of aggregation with no lag phase. The initial phase (~ 18 h) of aggregation involves conformational rearrangement within the oligomers to form species of enhanced β‐sheet character. The subsequent loss of α‐helical structure is accompanied by large‐scale amyloid fibril formation. The crystal structure of Acot7 revealed an unexpected arrangement of the two domains within the circularised trimeric structure, which is the basis for a proposed mechanism of amyloid fibril formation involving domain swapping during the initial phase of aggregation. Acot7 formed fibrils in the presence of its substrate arachidonoyl‐CoA and its inhibitors and maintained its enzyme activity during fibril assembly. It is proposed that the Acot7 fibrillar form acts as functional amyloid.

Terminal regions confer plasticity to the tetrameric assembly of human HspB2 and HspB3

Publisher: Elsevier BV

Date: 09-2018

DOI: 10.1016/J.JMB.2018.06.047

Amyloid fibril formation by bovine milk αs2-casein occurs under physiological conditions yet is prevented by its natural counterpart, αs1-casein

Publisher: American Chemical Society (ACS)

Date: 27-02-2008

DOI: 10.1021/BI701278C

Abstract: The calcified proteinaceous deposits, or corpora amylacea, of bovine mammary tissue often comprise a network of amyloid fibrils, the origins of which have not been fully elucidated. Here, we demonstrate by transmission electron microscopy, dye binding assays, and X-ray fiber diffraction that bovine milk alpha s2-casein, a protein synthesized and secreted by mammary epithelial cells, readily forms fibrils in vitro. As a component of whole alpha s-casein, alpha s2-casein was separated from alpha s1-casein under nonreducing conditions via cation-exchange chromatography. Upon incubation at neutral pH and 37 degrees C, the spherical particles typical of alpha s2-casein rapidly converted to twisted, ribbon-like fibrils approximately 12 nm in diameter, which occasionally formed loop structures. Despite their irregular morphology, these fibrils possessed a beta-sheet core structure and the ability to bind amyloidophilic dyes such as thioflavin T. Fibril formation was optimal at pH 6.5-6.7 and was promoted by higher incubation temperatures. Interestingly, the protein appeared to be less prone to fibril formation upon disulfide bond reduction with dithiothreitol. Thus, alpha s2-casein is particularly susceptible to fibril formation under physiological conditions. However, our findings indicate that alpha s2-casein fibril formation is potently inhibited by its natural counterpart, alpha s1-casein, while is only partially inhibited by beta-casein. These findings highlight the inherent propensity of casein proteins to form amyloid fibrils and the importance of casein-casein interactions in preventing such fibril formation in vivo.

Age-related changes in bovine alpha-crystallin and high-molecular-weight protein

Publisher: Elsevier BV

Date: 12-1996

DOI: 10.1006/EXER.1996.0158

Abstract: The high-molecular-weight (HMW) protein from the lens is composed mostly of alpha-crystallin in a highly aggregated state. Bovine HMW protein was carefully separated from alpha-crystallin by size-exclusion chromatography. alpha-Crystallin has chaperone-like ability whereby it stabilizes other proteins under conditions of stress (e.g. heat). Comparison of bovine HMW protein and alpha-crystallin shows that the HMW protein has a markedly reduced chaperone ability compared to alpha-crystallin. However, in contrast to the results of other workers, we observe no alteration with age in the ability of alpha-crystallin to act as a chaperone. Using electrospray ionisation mass spectrometry, changes in the phosphorylation of the alpha-crystallin subunits with age have been quantified. Phosphorylation of alpha-crystallin occurs early in life but does not alter in proportion after about three years of age. In addition, phosphorylation of the A subunit of alpha-crystallin has little effect on its chaperone ability. As is found in the artificially prepared HMW complex of alpha- and gamma-crystallin, NMR spectroscopy shows that in the naturally occurring HMW protein, the short C-terminal extension of the alpha B subunit has lost its flexibility whereas the alpha A subunit extension is still flexible. Post-translational modifications therefore seem to have little effect on the chaperone action of alpha-crystallin, but alterations in the quaternary structure of alpha-crystallin via incorporation into the HMW aggregate, lead to major changes in the chaperone ability of the protein. The results are consistent with the notion that one of the contributing factors to cataract formation in the lens is the depletion of alpha-crystallin with age as it is converted into the HMW protein.

ON THE INTERACTION OF ALPHA-CRYSTALLIN WITH UNFOLDED PROTEINS

Publisher: Elsevier BV

Date: 10-1995

DOI: 10.1016/0167-4838(95)00146-L

Abstract: alpha-Crystallin, a major protein component of the lens, has chaperone-like properties whereby it prevents destabilised proteins from precipitating out of solution. It does so by forming a soluble high-molecular-weight (HMW) complex. A spectroscopic investigation of the HMW complex formed between a variety of unfolded proteins and bovine alpha-crystallin is presented in this paper. As monitored by fluorescence spectroscopy, a large amount of the hydrophobic probe, 8-anilino-1-naphthalene sulfonate (ANS) binds to the HMW complex implying that the complexed proteins (alcohol dehydrogenase (ADH), gamma-crystallin and rhodanese) are bound in an unfolded, possibly molten-globule state. The interaction between the anionic surfactant, sodium dodecyl sulfate (SDS) and ADH at high temperatures gives rise to a similar large increase in ANS fluorescence to that for the complex between alpha-crystallin and ADH. SDS, like alpha-crystallin, therefore complexes to proteins in their unfolded state leaving a large hydrophobic surface exposed to solvent. Unlike other chaperones (e.g., GroEL, DnaK and SecB), alpha-crystallin does not interact with unfolded, hydrophobic but stable proteins (e.g., reduced and carboxymethylated alpha-lactalbumin and alpha-casein). It is concluded that alpha-crystallin will only complex with proteins that are about to precipitate out of solution, i.e., ones that are severely compromised. 1H-NMR spectroscopy of the HMW complex formed between alpha-crystallin and gamma-crystallin indicates that the short C-terminal extension of alpha B-crystallin, but not that of alpha A-crystallin, has lost its flexibility in the complex implying that the former is involved in interactions with the unfolded gamma-crystallin molecule, possibly electrostatically via its two C-terminal lysine residues.

Secondary structure determination of N-15-labelled human Long-[Arg-3]-insulin-like growth factor 1 by multidimensional NMR spectroscopy

Publisher: Wiley

Date: 22-12-1997

DOI: 10.1016/S0014-5793(97)01496-8

Abstract: Insulin-like growth factors (IGFs) are a group of proteins that promote cell growth and differentiation. Long-[Arg-3]-IGF-I (Francis et al. (1992) J. Mol. Endocrinol. 8, 213-223), a potent analogue of IGF-I, which has a Glu-3 to Arg-3 substitution and a hydrophobic, thirteen amino acid N-terminal extension, has been studied by 1H,15N NMR spectroscopy. All the backbone 1H and 15N assignments and most of the 1H sidechain assignments have been completed. The secondary structure elements were identified by determining the sequential and medium range NOEs from sensitivity-enhanced 15N-NOESY-HSQC and sensitivity-enhanced 15N-HSQC-NOESY-HSQC spectra. The IGF-I domain of Long-[Arg-3]-IGF-I was found to have an almost identical structure to IGF-I. The N-terminal seven amino acid residues of the extension have very few medium range or long range NOEs but the next five amino acids form a turn-like structure that is spatially close to the beginning of helix 1 in the IGF-I domain. Hydrogen-deuterium exchange experiments show that all the slowly exchanging backbone amide protons in the IGF-I domain are either in the helical or the extended structural elements. Many of the amide protons in the N-terminal extension are also protected from the solvent although the residues in this part of the extension do not have any identifiable secondary structure. The results are interpreted in terms of the increased biological potency of Long-[Arg-3]-IGF-I and the decreased binding to insulin-like growth factor binding proteins.

NMR-STUDIES OF THE NA+, MG2+ AND CA2+ COMPLEXES OF CYCLOSPORINE-A

Publisher: Royal Society of Chemistry (RSC)

Date: 1992

DOI: 10.1039/C39920001682

THE STRUCTURE OF MELITTIN - A H-1-NMR STUDY IN METHANOL

Publisher: Wiley

Date: 04-1988

DOI: 10.1111/J.1432-1033.1988.TB13977.X

Abstract: The conformation of the 26-residue polypeptide melittin has been studied using 1H-NMR spectroscopy in methanolic solution. The 1H-NMR spectrum of melittin has been assigned using two-dimensional NMR techniques and the secondary structure has been calculated from nuclear Overhauser enhancement data using distance geometry and restrained molecular dynamics analyses. The structure is found to be mainly helical, and similar to that found in crystals from diffraction data: residues 2-11 and 13-26 form regular alpha-helices joined by a 'hinge' between residues 11-12. The structure in this hinge region is shown to be significantly different from that in the crystal structure, leading to a smaller angle between the two helices. The possible significance of the proline residues in this and similar membrane-spanning peptides is discussed.

Interaction of the Molecular Chaperone αB-Crystallin with α-Synuclein: Effects on Amyloid Fibril Formation and Chaperone Activity

Publisher: Elsevier BV

Date: 07-2004

DOI: 10.1016/J.JMB.2004.05.054

METABOLIC EFFECTS OF INTERLEUKIN-3 ON 32D CL23 CELLS ANALYZED BY NMR

Publisher: Wiley

Date: 11-1987

DOI: 10.1002/JCP.1041330220

Abstract: 31P NMR of living 32D cl23 cells and 1H NMR of cell extracts were used to study the metabolic effects of interleukin 3 (IL3). When IL3 was removed from 32D cl23 for 9-10 hours 31P spectra showed a decrease in sugar phosphate, gamma ATP/ADP, alpha ATP/ADP/NAD, and beta ATP resonances which declined progressively over a time period of up to 16 hours. By comparison, ATP measurements using the luciferin/luciferase method resulted in the decline of ATP levels from 12 hours in the absence of IL3. At this time, viability of the cells was unaffected. For 1H NMR experiments cells were grown in the presence and absence of IL3 for 4 and 24 hours, after which acid cell extracts were prepared. These spectra revealed a four-fold decrease in lactate 4 hours post-IL3 removal. Alanine levels were unchanged but glycine was elevated 1.5-fold whilst various other amino acids were elevated slightly. After 24 hours without IL3, only 22% of cells were viable which was reflected in a general decline of most resonance intensities. These findings suggest that IL3 exerts its effect primarily on glucose metabolism and has a delayed secondary effect on maintenance of ATP levels in the cell. We have demonstrated the applicability of high resolution 1H and 31P NMR to the study of cellular metabolism in hemopoietic cells.

Crystallin proteins and amyloid fibrils

Publisher: Springer Science and Business Media LLC

Date: 22-09-2009

DOI: 10.1007/S00018-008-8327-4

Abstract: Improper protein folding (misfolding) can lead to the formation of disordered (amorphous) or ordered (amyloid fibril) aggregates. The major lens protein, alpha-crystallin, is a member of the small heat-shock protein (sHsp) family of intracellular molecular chaperone proteins that prevent protein aggregation. Whilst the chaperone activity of sHsps against amorphously aggregating proteins has been well studied, its action against fibril-forming proteins has received less attention despite the presence of sHsps in deposits found in fibril-associated diseases (e.g. Alzheimer's and Parkinson's). In this review, the literature on the interaction of alphaB-crystallin and other sHsps with fibril-forming proteins is summarized. In particular, the ability of sHsps to prevent fibril formation, their mechanisms of action and the possible in vivo consequences of such associations are discussed. Finally, the fibril-forming propensity of the crystallin proteins and its implications for cataract formation are described along with the potential use of fibrillar crystallin proteins as bionanomaterials.

Preventing α-synuclein aggregation: The role of the small heat-shock molecular chaperone proteins

Publisher: Elsevier BV

Date: 09-2014

DOI: 10.1016/J.BBADIS.2014.06.024

Abstract: Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. The failure of proteostasis can result in the accumulation of non-native proteins leading to their aggregation and deposition in cells and in tissues. The amyloid fibrillar aggregation of the protein α-synuclein into Lewy bodies and Lewy neuritis is associated with neurodegenerative diseases classified as α-synucleinopathies, which include Parkinson's disease and dementia with Lewy bodies. The small heat-shock proteins (sHsps) are molecular chaperones that are one of the cell's first lines of defence against protein aggregation. They act to stabilise partially folded protein intermediates, in an ATP-independent manner, to maintain cellular proteostasis under stress conditions. Thus, the sHsps appear ideally suited to protect against α-synuclein aggregation, yet these fail to do so in the context of the α-synucleinopathies. This review discusses how sHsps interact with α-synuclein to prevent its aggregation and, in doing so, highlights the multi-faceted nature of the mechanisms used by sHsps to prevent the fibrillar aggregation of proteins. It also examines what factors may contribute to α-synuclein escaping the sHsp chaperones in the context of the α-synucleinopathies.

H-1-NMR ASSIGNMENTS OF ANONAINE AND XYLOPINE DERIVATIVES FROM TALAUMA-GITINGENSIS

Publisher: American Chemical Society (ACS)

Date: 11-1990

DOI: 10.1021/NP50072A043

The eye lens protein alpha A-crystallin of the blind mole rat Spalax ehrenbergi: Effects of altered functional constraints

Publisher: Elsevier BV

Date: 02-2002

DOI: 10.1006/EXER.2001.1124

Small heat-shock proteins: Important players in regulating cellular proteostasis

Publisher: Springer Science and Business Media LLC

Date: 29-10-2015

DOI: 10.1007/S00018-014-1754-5

Abstract: Small heat-shock proteins (sHsps) are a erse family of intra-cellular molecular chaperone proteins that play a critical role in mitigating and preventing protein aggregation under stress conditions such as elevated temperature, oxidation and infection. In doing so, they assist in the maintenance of protein homeostasis (proteostasis) thereby avoiding the deleterious effects that result from loss of protein function and/or protein aggregation. The chaperone properties of sHsps are therefore employed extensively in many tissues to prevent the development of diseases associated with protein aggregation. Significant progress has been made of late in understanding the structure and chaperone mechanism of sHsps. In this review, we discuss some of these advances, with a focus on mammalian sHsp hetero-oligomerisation, the mechanism by which sHsps act as molecular chaperones to prevent both amorphous and fibrillar protein aggregation, and the role of post-translational modifications in sHsp chaperone function, particularly in the context of disease.

Small heat-shock proteins interact with a flanking domain to suppress polyglutamine aggregation

Publisher: Proceedings of the National Academy of Sciences

Date: 19-05-2010

DOI: 10.1073/PNAS.0914773107

Abstract: Small heat-shock proteins (sHsps) are molecular chaperones that play an important protective role against cellular protein misfolding by interacting with partially unfolded proteins on their off-folding pathway, preventing their aggregation. Polyglutamine (polyQ) repeat expansion leads to the formation of fibrillar protein aggregates and neuronal cell death in nine diseases, including Huntington disease and the spinocerebellar ataxias (SCAs). There is evidence that sHsps have a role in suppression of polyQ-induced neurodegeneration for ex le, the sHsp alphaB-crystallin (αB-c) has been identified as a suppressor of SCA3 toxicity in a Drosophila model. However, the molecular mechanism for this suppression is unknown. In this study we tested the ability of αB-c to suppress the aggregation of a polyQ protein. We found that αB-c does not inhibit the formation of SDS-insoluble polyQ fibrils. We further tested the effect of αB-c on the aggregation of ataxin-3, a polyQ protein that aggregates via a two-stage aggregation mechanism. The first stage involves association of the N-terminal Josephin domain followed by polyQ-mediated interactions and the formation of SDS-resistant mature fibrils. Our data show that αB-c potently inhibits the first stage of ataxin-3 aggregation however, the second polyQ-dependent stage can still proceed. By using NMR spectroscopy, we have determined that αB-c interacts with an extensive region on the surface of the Josephin domain. These data provide an ex le of a domain/region flanking an amyloidogenic sequence that has a critical role in modulating aggregation of a polypeptide and plays a role in the interaction with molecular chaperones to prevent this aggregation.

A Spectroscopic Marker for Structural Transitions Associated with Amyloid-β Aggregation

Publisher: American Chemical Society (ACS)

Date: 24-04-2020

DOI: 10.1021/ACS.BIOCHEM.0C00113

The solution structures and activity of caerin 1.1 and caerin 1.4 in aqueous trifluoroethanol and dodecylphosphocholine micelles

Publisher: Wiley

Date: 21-04-2003

DOI: 10.1002/BIP.10324

Abstract: The caerin 1 peptides are among the most powerful of the broad-spectrum antibiotic hibian peptides. Caerin 1.1 has previously been shown to form an hipathic helix-bend-helix structure in aqueous trifluoroethanol (H. Wong, J. H. Bowie, and J. A. Carver European Journal Biochemistry, 1997, Vol. 247, pp. 545-557) and structure-activity relationship studies indicate that both helices are required for activity, as well as flexibility in the bend region connecting the two. The structure of caerin 1.1 in dodecylphosphocholine micelles was investigated and shown to be very similar to that determined in aqueous trifluoroethanol. Caerin 1.4, which is identical to caerin 1.1, but with serine residues replacing Val5 and Gly7, is less active than caerin 1.1 against most bacterial species but has improved activity against Escherichia coli and Micrococcus luteus. The solution NMR structure of caerin 1.4 was determined in both aqueous trifluoroethanol and dodecylphosphocholine micelles, and was shown to be similar to caerin 1.1. It was concluded that differences in the hydrophobicity and hydrophilic angle of the first helix are probably responsible for the different spectra of antibacterial activity. The similarity of the structures calculated in aqueous trifluoroethanol and dodecylphosphocholine micelles suggests that, for caerin 1.1 and 1.4, these solvent systems are equally as good at representing a membrane environment.

The Extracellular Molecular Chaperone Clusterin Inhibits Amyloid Fibril Formation and Suppresses Cytotoxicity Associated with Semen-Derived Enhancer of Virus Infection (SEVI)

Publisher: MDPI AG

Date: 17-10-2022

DOI: 10.3390/CELLS11203259

Abstract: Clusterin is a glycoprotein present at high concentrations in many extracellular fluids, including semen. Its increased expression accompanies disorders associated with extracellular amyloid fibril accumulation such as Alzheimer’s disease. Clusterin is an extracellular molecular chaperone which prevents the misfolding and amorphous and amyloid fibrillar aggregation of a wide variety of unfolding proteins. In semen, amyloid fibrils formed from a 39-amino acid fragment of prostatic acid phosphatase, termed Semen-derived Enhancer of Virus Infection (SEVI), potentiate HIV infectivity. In this study, clusterin potently inhibited the in vitro formation of SEVI fibrils, along with dissociating them. Furthermore, clusterin reduced the toxicity of SEVI to pheochromocytoma-12 cells. In semen, clusterin may play an important role in preventing SEVI amyloid fibril formation, in dissociating SEVI fibrils and in mitigating their enhancement of HIV infection.

The extracellular chaperone clusterin prevents primary and secondary nucleation of an amyloidogenic variant of β2-microglobulin

Publisher: CSIRO Publishing

Date: 11-07-2023

DOI: 10.1071/CH23082

Monitoring Early-Stage Protein Aggregation by an Aggregation-Induced Emission Fluorogen

Publisher: American Chemical Society (ACS)

Date: 23-08-2017

DOI: 10.1021/ACS.ANALCHEM.7B02090

Abstract: Highly ordered protein aggregates, termed amyloid fibrils, are associated with a broad range of diseases, many of which are neurodegenerative, for ex le, Alzheimer's and Parkinson's. The transition from soluble, functional protein into insoluble amyloid fibril occurs via a complex process involving the initial generation of highly dynamic early stage aggregates or prefibrillar species. Amyloid probes, for ex le, thioflavin T and Congo red, have been used for decades as the gold standard for detecting amyloid fibrils in solution and tissue sections. However, these well-established dyes do not detect the presence of prefibrillar species formed during the early stages of protein aggregation. Prefibillar species have been proposed to play a key role in the cytotoxicity of amyloid fibrils and the pathogenesis of neurodegenerative diseases. Herein, we report a novel fluorescent dye (bis(triphenylphosphonium) tetraphenylethene (TPE-TPP)) with aggregation-induced emission characteristics for monitoring the aggregation process of amyloid fibrils. An increase in TPE-TPP fluorescence intensity is observed only with ordered protein aggregation, such as amyloid fibril formation, and not with stable molten globules states or amorphously aggregating species. Importantly, TPE-TPP can detect the presence of prefibrillar species formed early during fibril formation. TPE-TPP exhibits a distinctive spectral shift in the presence of prefibrillar species, indicating a unique structural feature of these intermediates. Using fluorescence polarization, which reflects the mobility of the emitting entity, the specific oligomeric pathways undertaken by various proteins during fibrillation could be discerned. Furthermore, we demonstrate the broad applicability of TPE-TPP to monitor amyloid fibril aggregation, including under erse conditions such as at acidic pH and elevated temperature, or in the presence of amyloid inhibitors.

αB-Crystallin inhibits the cell toxicity associated with amyloid fibril formation by κ-casein and the amyloid-β peptide

Publisher: Springer Science and Business Media LLC

Date: 15-07-2010

DOI: 10.1007/S12192-010-0212-Z

CONFORMATIONAL DIFFERENCES BETWEEN VARIOUS MYOGLOBIN LIGATED STATES AS MONITORED BY H-1-NMR SPECTROSCOPY

Publisher: American Chemical Society (ACS)

Date: 09-10-1984

DOI: 10.1021/BI00316A013

Abstract: In paramagnetic metmyoglobin, cyanomyoglobin (CNMb), and deoxymyoglobin, His-36 has a high pK (approximately 8), and the NMR titration behavior of the H-2 resonance is perturbed, due to the presence at low pH of a hydrogen bond with Glu-38, which is broken at high pH. The His-36 H-4 resonance shows no shift with pK approximately 8 because of two opposing chemical shift effects but monitors the titration of nearby Glu-36 (pK = 5.6). In diamagnetic derivatives [(carbon monoxy)myoglobin (COMb) and oxymyoglobin (oxyMb)], the titration behavior of His-36 H-2 and H-4 resonances is normalized (pK approximately 6.8). The very slight alkaline Bohr effect in sperm whale myoglobin (Mb) is interpreted in terms of the pK change of His-36 from deoxyMb to oxyMb and compensating pK changes in the opposite direction of other unspecified groups. In sperm whale COMb at 40 degrees C, the distal histidine (His-64) and His-97 have pK values of 5.0 and 5.9. The meso proton resonances remote from these groups do not show a titration shift, but the nearby gamma-meso proton (pK = 5.3) responds to titration of both histidines, and the upfield Val-68 methyl at -2.3 ppm (pK = 4.7) witnesses the titration of nearby His-64. At 20 degrees C, the latter resonance is reduced in size, and a second resonance occurs at -2.8 ppm, which is insensitive to pH and, hence, more remote from His-64. Both resonances arise from two conformations of Val-68 in slow equilibrium. In oxyMb at 20 degrees C, only the latter resonance is observed, presumably because of the steric restrictions imposed by the hydrogen bond between ligand and His-64 in oxyMb, which is not present in COMb. In oxyMb the pK of His-97 (5.6) is similar to that of the meso proton resonances (5.5) and to the pK of other pH-dependent processes, including the very small acid Bohr effect. It is likely that these processes are controlled by the titration of His-97.(ABSTRACT TRUNCATED AT 250 WORDS)

The Effect of Oxidized Dopamine on the Structure and Molecular Chaperone Function of the Small Heat-Shock Proteins, αB-Crystallin and Hsp27

Publisher: MDPI AG

Date: 02-04-2021

DOI: 10.3390/IJMS22073700

Abstract: Oxidation of the neurotransmitter, dopamine (DA), is a pathological hallmark of Parkinson’s disease (PD). Oxidized DA forms adducts with proteins which can alter their functionality. αB-crystallin and Hsp27 are intracellular, small heat-shock molecular chaperone proteins (sHsps) which form the first line of defense to prevent protein aggregation under conditions of cellular stress. In vitro, the effects of oxidized DA on the structure and function of αB-crystallin and Hsp27 were investigated. Oxidized DA promoted the cross-linking of αB-crystallin and Hsp27 to form well-defined dimer, trimer, tetramer, etc., species, as monitored by SDS-PAGE. Lysine residues were involved in the cross-links. The secondary structure of the sHsps was not altered significantly upon cross-linking with oxidized DA but their oligomeric size was increased. When modified with a molar equivalent of DA, sHsp chaperone functionality was largely retained in preventing both amorphous and amyloid fibrillar aggregation, including fibril formation of mutant (A53T) α-synuclein, a protein whose aggregation is associated with autosomal PD. In the main, higher levels of sHsp modification with DA led to a reduction in chaperone effectiveness. In vivo, DA is sequestered into acidic vesicles to prevent its oxidation and, intracellularly, oxidation is minimized by mM levels of the antioxidant, glutathione. In vitro, acidic pH and glutathione prevented the formation of oxidized DA-induced cross-linking of the sHsps. Oxidized DA-modified αB-crystallin and Hsp27 were not cytotoxic. In a cellular context, retention of significant chaperone functionality by mildly oxidized DA-modified sHsps would contribute to proteostasis by preventing protein aggregation (particularly of α-synuclein) that is associated with PD.

nNOS inhibition, antimicrobial and anticancer activity of the amphibian skin peptide, citropin 1.1 and synthetic modifications. The solution structure of a modified citropin 1.1

Publisher: Wiley

Date: 03-2003

DOI: 10.1046/J.1432-1033.2003.03462.X

Abstract: A large number of bioactive peptides have been isolated from hibian skin secretions. These peptides have a variety of actions including antibiotic and anticancer activities and the inhibition of neuronal nitric oxide synthase. We have investigated the structure-activity relationship of citropin 1.1, a broad-spectrum antibiotic and anticancer agent that also causes inhibition of neuronal nitric oxide synthase, by making a number of synthetically modified analogues. Citropin 1.1 has been shown previously to form an hipathic alpha-helix in aqueous trifluoroethanol. The results of the structure-activity studies indicate the terminal residues are important for bacterial activity and increasing the overall positive charge, while maintaining an hipathic distribution of residues, increases activity against Gram-negative organisms. Anticancer activity generally mirrors antibiotic activity suggesting a common mechanism of action. The N-terminal residues are important for inhibition of neuronal nitric oxide synthase, as is an overall positive charge greater than three. The structure of one of the more active synthetic modifications (A4K14-citropin 1.1) was determined in aqueous trifluoroethanol, showing that this peptide also forms an hipathic alpha-helix.

Caerin 4.1, an antibiotic peptide from the Australian tree frog, Litoria caerulea. The NMR-derived solution structure

Publisher: CSIRO Publishing

Date: 2000

DOI: 10.1071/CH99108

Abstract: Caerin 4.1 (GLWQK5IKSAA10GDLAS15GIEVG20IKS-NH2) is an antibiotic peptide isolated from the Australian tree frog Litoria caerulea. Unlike caerin 1.1, the major peptide isolated from this species, caerin 4.1 has a narrow spectrum of antibiotic activity, e.g. it shows selective activity against Pasteurella haemolytica and Escherichia coli. Caerin 4.1 consists of 23 amino acid residues and is comparable in size with other wide-spectrum antibiotic peptides isolated from Australian hibians, e.g. caerin 1.1 and maculatin 1.1. An n.m.r. study in trifluoroethanol/water indicates that caerin 4.1 forms an hipathic α-helix with distinct hydrophilic and hydrophobic zones. Two regions of well defined helicity (from Gln4 to Ala10 and from Ile17 to Ile21) are separated by a central helical region of greater conformational variability. The enhanced disorder in this region arises from the presence of two central glycine residues at positions 11 and 16. However, the degree of disorder and hence flexibility is much less than in caerin 1.1 where central proline residues are present instead. This reduced central flexibility may account for the narrow spectrum of biological activity of caerin 4.1, i.e. because biological membranes of the various bacteria have different composition and topology, their optimal interaction with the relatively rigid caerin 4.1 peptide is not possible.

Host defence peptides from the skin glands of the Australian Blue Mountains tree-frog Litoria citropa . Solution structure of the antibacterial peptide citropin 1.1

Publisher: Wiley

Date: 10-1999

DOI: 10.1046/J.1432-1327.1999.00750.X

Abstract: Nineteen citropin peptides are present in the secretion from the granular dorsal glands of the Blue Mountains tree-frog Litoria citropa 15 of these peptides are also present in the secretion from the submental gland. Two major peptides, citropin 1.1 (GLFDVIKKVASVIGGL-NH2), citropin 1.2 (GLFDIIKKVASVVGGL-NH2) and a minor peptide, citropin 1.3 (GLFDIIKKVASVIGGL-NH2) are wide-spectrum antibacterial peptides. The hibian has an endoprotease which deactivates these membrane-active peptides by removing residues from the N-terminal end: loss of three residues gives the most abundant degradation products. The solution structure of the basic peptide citropin 1.1 has been determined by NMR spectroscopy [in a solvent mixture of trifluoroethanol/water (1 : 1)] to be an hipathic alpha-helix with well-defined hydrophobic and hydrophilic regions. The additional four peptides produced by the dorsal glands are structurally related to the antibacterial citropin 1 peptides but contain three more residues at their C-terminus [e.g. citropin 1.1.3 (GLFDVIKKVASVIGLASP-OH)]. These peptides show minimal antibacterial activity their role in the hibian skin is not known.

Structural differences between bovine A¹ and A² β-casein alter micelle self-assembly and influence molecular chaperone activity

Publisher: American Dairy Science Association

Date: 04-2015

DOI: 10.3168/JDS.2014-8800

Abstract: Within each milk protein there are many in idual protein variants and marked alterations to milk functionality can occur depending on the genetic variants of each protein present. Bovine A(1) and A(2) β-casein (β-CN) are 2 variants that contribute to differences in the gelation performance of milk. The A(1) and A(2) β-CN variants differ by a single AA, the substitution of histidine for proline at position 67. β-Casein not only participates in formation of the casein micelle but also forms an oligomeric micelle itself and functions as a molecular chaperone to prevent the aggregation of a wide range of proteins, including the other caseins. Micelle assembly of A(1) and A(2) β-CN was investigated using dynamic light scattering and small-angle X-ray scattering, whereas protein functionality was assessed using fluorescence techniques and molecular chaperone assays. The A(2) β-CN variant formed smaller micelles than A(1) β-CN, with the monomer-micelle equilibrium of A(2) β-CN being shifted toward the monomer. This shift most likely arose from structural differences between the 2 β-CN variants associated with the adoption of greater polyproline-II helix in A(2) β-CN and most likely led to enhanced chaperone activity of A(2) β-CN compared with A(1) β-CN. The difference in micelle assembly, and hence chaperone activity, may provide explain differences in the functionality of homozygous A(1) and A(2) milk. The results of this study highlight that substitution of even a single AA can significantly alter the properties of an intrinsically unstructured protein such as β-CN and, in this case, may have an effect on the functionality of milk.

Small heat shock proteins: multifaceted proteins with important implications for life

Publisher: Springer Science and Business Media LLC

Date: 13-02-2019

DOI: 10.1007/S12192-019-00979-Z

NMR spectroscopy of 14-3-3ζ reveals a flexible C-terminal extension: differentiation of the chaperone and phosphoserine-binding activities of 14-3-3ζ

Publisher: Portland Press Ltd.

Date: 13-07-2011

DOI: 10.1042/BJ20102178

Abstract: Intracellular 14-3-3 proteins bind to many proteins, via a specific phosphoserine motif, regulating erse cellular tasks including cell signalling and disease progression. The 14-3-3ζ isoform is a molecular chaperone, preventing the stress-induced aggregation of target proteins in a manner comparable with that of the unrelated sHsps (small heat-shock proteins). 1H-NMR spectroscopy revealed the presence of a flexible and unstructured C-terminal extension, 12 amino acids in length, which protrudes from the domain core of 14-3-3ζ and is similar in structure and length to the C-terminal extension of mammalian sHsps. The extension stabilizes 14-3-3ζ, but has no direct role in chaperone action. Lys49 is an important functional residue within the ligand-binding groove of 14-3-3ζ with K49E 14-3-3ζ exhibiting markedly reduced binding to phosphorylated and non-phosphorylated ligands. The R18 peptide binds to the binding groove of 14-3-3ζ with high affinity and also reduces the interaction of 14-3-3ζ ligands. However, neither the K49E mutation nor the presence of the R18 peptide affected the chaperone activity of 14-3-3ζ, implying that the C-terminal extension and binding groove of 14-3-3ζ do not mediate interaction with target proteins during chaperone action. Other region(s) in 14-3-3ζ are most likely to be involved, i.e. the protein's chaperone and phosphoserine-binding activities are functionally and structurally separated.

HISTIDINE H-2 NMR RESONANCES OF SPERM WHALE OXY-MYOGLOBIN, CARBONYL-MYOGLOBIN, AND MET-MYOGLOBIN

Publisher: Royal Society of Chemistry (RSC)

Date: 1981

DOI: 10.1039/C39810000208

The antibiotic and anticancer active aurein peptides from the Australian Bell Frogs Litoria aurea and Litoria raniformis - The solution structure of aurein 1.2

Publisher: Wiley

Date: 09-2000

DOI: 10.1046/J.1432-1327.2000.01536.X

Abstract: Seventeen aurein peptides are present in the secretion from the granular dorsal glands of the Green and Golden Bell Frog Litoria aurea, and 16 from the corresponding secretion of the related Southern Bell Frog L. raniformis. Ten of these peptides are common to both species. Thirteen of the aurein peptides show wide-spectrum antibiotic and anticancer activity. These peptides are named in three groups (aureins 1-3) according to their sequences. Amongst the more active peptides are aurein 1.2 (GLFDIIKKIAESF-NH2), aurein 2.2 (GLFDIVKKVVGALGSL-NH2) and aurein 3.1 (GLFDIVKKIAGHIAGSI-NH2). Both L. aurea and L. raniformis have endoproteases that deactivate the major membrane-active aurein peptides by removing residues from both the N- and C-termini of the peptides. The most abundant degradation products have two residues missing from the N-terminal end of the peptide. The solution structure of the basic peptide, aurein 1.2, has been determined by NMR spectroscopy to be an hipathic alpha-helix with well-defined hydrophilic and hydrophobic regions. Certain of the aurein peptides (e.g. aureins 1.2 and 3.1) show anticancer activity in the NCI test regime, with LC50 values in the 10-5-10-4 M range. The aurein 1 peptides have only 13 amino-acid residues: these are the smallest antibiotic and anticancer active peptides yet reported from an anuran. The longer aurein 4 and 5 peptides, e.g. aurein 4.1 (GLIQTIKEKLKELAGGLVTGIQS-OH) and aurein 5. 1 (GLLDIVTGLLGNLIVDVLKPKTPAS-OH) show neither antibacterial nor anticancer activity.

Mimicking phosphorylation of αB-crystallin affects its chaperone activity

Publisher: Portland Press Ltd.

Date: 11-12-2007

DOI: 10.1042/BJ20060981

Abstract: αB-crystallin is a member of the sHsp (small heat-shock protein) family that prevents misfolded target proteins from aggregating and precipitating. Phosphorylation at three serine residues (Ser19, Ser45 and Ser59) is a major post-translational modification that occurs to αB-crystallin. In the present study, we produced recombi-nant proteins designed to mimic phosphorylation of αB-crystallin by incorporating a negative charge at these sites. We employed these mimics to undertake a mechanistic and structural invest-igation of the effect of phosphorylation on the chaperone activity of αB-crystallin to protect against two types of protein misfolding, i.e. amorphous aggregation and amyloid fibril assembly. We show that mimicking phosphorylation of αB-crystallin results in more efficient chaperone activity against both heat-induced and reduc-tion-induced amorphous aggregation of target proteins. Mimick-ing phosphorylation increased the chaperone activity of αB-crystallin against one amyloid-forming target protein (κ-casein), but decreased it against another (ccβ-Trp peptide). We observed that both target protein identity and solution (buffer) conditions are critical factors in determining the relative chaperone ability of wild-type and phosphorylated αB-crystallins. The present study provides evidence for the regulation of the chaperone activity of αB-crystallin by phosphorylation and indicates that this may play an important role in alleviating the pathogenic effects associated with protein conformational diseases.

Avoiding the oligomeric state: αB-crystallin inhibits fragmentation and induces dissociation of apolipoprotein C-II amyloid fibrils

Publisher: Wiley

Date: 16-11-2013

DOI: 10.1096/FJ.12-220657

Abstract: The in vivo aggregation of proteins into amyloid fibrils suggests that cellular mechanisms that normally prevent or reverse this aggregation have failed. The small heat-shock molecular chaperone protein αB-crystallin (αB-c) inhibits amyloid formation and colocalizes with amyloid plaques however, the physiological reason for this localization remains unexplored. Here, using apolipoprotein C-II (apoC-II) as a model fibril-forming system, we show that αB-c binds directly to mature amyloid fibrils (Kd 5.4 ± 0.5 μM). In doing so, αB-c stabilized the fibrils from dilution-induced fragmentation, halted elongation of partially formed fibrils, and promoted the dissociation of mature fibrils into soluble monomers. Moreover, in the absence of dilution, the association of αB-c with apoC-II fibrils induced a 14-fold increase in average aggregate size, resulting in large fibrillar tangles reminiscent of protein inclusions. We propose that the binding of αB-c to fibrils prevents fragmentation and mediates the lateral association of fibrils into large inclusions. We further postulate that transient interactions of apoC-II with αB-c induce a fibril-incompetent monomeric apoC-II form, preventing oligomerization and promoting fibril dissociation. This work reveals previously unrecognized mechanisms of αB-c chaperone action in amyloid assembly and fibril dynamics, and provides a rationale for the in vivo colocalization of small heat-shock proteins with amyloid deposits.-Binger, K. J., Ecroyd, H., Yang, S., Carver, J. A., Howlett, G. J., Griffin, M. D. W. Avoiding the oligomeric state: αB-crystallin inhibits fragmentation and induces dissociation of apolipoprotein C-II amyloid fibrils.

The Aggregation of αB-Crystallin under Crowding Conditions Is Prevented by αA-Crystallin: Implications for α-Crystallin Stability and Lens Transparency

Publisher: Elsevier BV

Date: 09-2020

DOI: 10.1016/J.JMB.2020.08.011

Solution structure and backbone dynamics of long-[Arg(3)]insulin-like growth factor-I

Publisher: Elsevier BV

Date: 04-2000

DOI: 10.1074/JBC.275.14.10009

The solution structure of uperin 3.6, an antibiotic peptide from the granular dorsal glands of the Australian toadlet, Uperoleia mjobergii

Publisher: Wiley

Date: 08-1999

DOI: 10.1034/J.1399-3011.1999.00095.X

Abstract: Uperin 3.6 (GVIDA5AKKVV10NVLKN15LF-NH2) is a wide-spectrum antibiotic peptide isolated from the Australian toadlet, Uperoleia mjobergii. With only 17 amino acid residues, it is smaller than most other wide-spectrum antibiotic peptides isolated from hibians. In 50% (by vol.) trifluoroethanol, an NMR study and structure calculations indicate that uperin 3.6 adopts a well-defined hipathic alpha-helix with distinct hydrophilic and hydrophobic faces. Examination of the activities of synthetic modifications of uperin 3.6 reveal that the three lysine residues are essential for antibiotic activity.

Letter to the Editor: A response to Horne and Lucey (2017)

Publisher: American Dairy Science Association

Date: 07-2017

DOI: 10.3168/JDS.2017-13053

Structural investigation of the hedamycin : d(ACCGGT)(2) complex by NMR and restrained molecular dynamics

Publisher: Elsevier BV

Date: 02-2002

DOI: 10.1006/BBRC.2002.6369

R120G αB-crystallin promotes the unfolding of reduced α-lactalbumin and is inherently unstable

Publisher: Wiley

Date: 13-01-2005

DOI: 10.1111/J.1742-4658.2004.04507.X

Abstract: alpha-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, alphaB-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 Genet20, 92-95]. In the present study, real-time 1H-NMR spectroscopy showed that the ability of R120G alphaB-crystallin to stabilize the partially folded, molten globule state of alpha-lactalbumin was significantly reduced in comparison with wild-type alphaB-crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced alpha-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 alphaB-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 alphaB-crystallin exists as a larger oligomer than wild-type alphaB-crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of alphaB-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 alphaB-crystallin with human disease states.

A novel protein distance matrix based on the minimum arc-length between two amino-acid residues on the surface of a globular protein

Publisher: Elsevier BV

Date: 06-2014

DOI: 10.1016/J.BPC.2014.01.005

Abstract: We present a novel protein distance matrix based on the minimum line of arc between two points on the surface of a protein. Two methods for calculating this distance matrix are developed and contrasted. The first method, which we have called TOPOL, is an approximate rule based algorithm consisting of successive rounds of vector addition. The second method is adapted from the graph theoretic approach of Dijkstra. Both procedures are demonstrated using cytochrome c, a 12,500 Da protein, as a test case. In respect to computational speed and accuracy the TOPOL procedure compares favorably against the more complex method based on shortest path enumeration over a surface manifold grid. Some potential uses of the algorithmic approaches and calculated surface protein distance measurement are discussed.

Quantitative multivalent binding model of the structure, size distribution and composition of the casein micelles of cow milk

Publisher: Elsevier BV

Date: 03-2022

DOI: 10.1016/J.IDAIRYJ.2021.105292

Polypeptide modification and cross-linking by oxidized 3-hydroxykynurenine

Publisher: American Chemical Society (ACS)

Date: 29-11-2000

DOI: 10.1021/BI001230T

Abstract: 3-Hydroxykynurenine (3OHKyn) is present in the mammalian lens as a UV filter and is formed from kynurenine in the tryptophan metabolic pathway. 3OHKyn is a readily autoxidized o-aminophenol which binds to proteins in vitro. The lens, particularly its central region, the nucleus, becomes increasingly oxidized with age. Under such conditions, the oxidation products of 3OHKyn may bind to lens proteins and contribute to nuclear cataract formation. The purpose of this study was to determine the structures of in vitro reaction products of 3OHKyn with model peptides as a general model for 3OHKyn modification of proteins. 3OHKyn was incubated with the dipeptide glycylglycine (GG) and the tetrapeptide tuftsin (sequence TKPR) under oxidizing conditions, and the reaction products were characterized by a variety of spectroscopic techniques. The major 3OHKyn-GG reaction product involves formation of a benzimidazole moiety between the GG N-terminus and the oxidized amino and/or phenol groups of 3OHKyn. In contrast, tuftsin, which has an N-terminal threonine, forms predominantly a cross-linked dimer with oxidized 3OHKyn. This product is analogous in structure to the dimeric reaction product, quinilinobenzoxamine, formed between oxidized 3OHKyn and glycyllysine [Aquilina, J. A., et al. (1999) Biochemistry 38, 11455-11464], which contains a benzoxazole moiety. The identification of a tuftsin dimer suggests that 3OHKyn can react with any peptide having a free alpha-amino group, via a general side chain elimination mechanism. The identification of both benzimidazole and benzoxazole adducts in peptides with a free N-terminus suggests that peptide amino groups can react initially at either the aromatic amino or hydroxyl group of oxidized 3OHKyn. The proportion of each adduct may change, however, depending on the amino acid sequence at the N-terminus.

Glutamic acid residues in the C-terminal extension of small heat shock protein 25 are critical for structural and functional integrity

Publisher: Wiley

Date: 23-10-2008

DOI: 10.1111/J.1742-4658.2008.06719.X

Abstract: Small heat shock proteins (sHsps) are intracellular molecular chaperones that prevent the aggregation and precipitation of partially folded and destabilized proteins. sHsps comprise an evolutionarily conserved region of 80-100 amino acids, denoted the alpha-crystallin domain, which is flanked by regions of variable sequence and length: the N-terminal domain and the C-terminal extension. Although the two domains are known to be involved in the organization of the quaternary structure of sHsps and interaction with their target proteins, the role of the C-terminal extension is enigmatic. Despite the lack of sequence similarity, the C-terminal extension of mammalian sHsps is typically a short, polar segment which is unstructured and highly flexible and protrudes from the oligomeric structure. Both the polarity and flexibility of the C-terminal extension are important for the maintenance of sHsp solubility and for complexation with its target protein. In this study, mutants of murine Hsp25 were prepared in which the glutamic acid residues in the C-terminal extension at positions 190, 199 and 204 were each replaced with alanine. The mutants were found to be structurally altered and functionally impaired. Although there were no significant differences in the environment of tryptophan residues in the N-terminal domain or in the overall secondary structure, an increase in exposed hydrophobicity was observed for the mutants compared with wild-type Hsp25. The average molecular masses of the E199A and E204A mutants were comparable with that of the wild-type protein, whereas the E190A mutant was marginally smaller. All mutants displayed markedly reduced thermostability and chaperone activity compared with the wild-type. It is concluded that each of the glutamic acid residues in the C-terminal extension is important for Hsp25 to act as an effective molecular chaperone.

Dephosphorylation of αS- and β-caseins and its effect on chaperone activity: A structural and functional investigation

Publisher: American Chemical Society (ACS)

Date: 15-06-2009

DOI: 10.1021/JF9008372

Abstract: Milk casein proteins can act as molecular chaperones: under conditions of stress, such as elevated temperature, molecular chaperones stabilize proteins from unfolding, aggregating, and precipitating. In this study, alpha(s)- and beta-caseins were dephosphorylated using alkaline phosphatase. A structural and functional investigation was undertaken to determine the effect of dephosphorylation on the chaperone activity of alpha(s)- and beta-caseins against two types of protein misfolding, i.e., amorphous aggregation and amyloid fibril assembly. The dephosphorylation of alpha(s)- and beta-caseins resulted in a decrease in the chaperone efficiency against both heat- and reduction-induced amorphously aggregating target proteins. In contrast, dephosphorylation had no effect on the chaperone activity of alpha(s)- and beta-caseins against the amyloid-forming target protein kappa-casein. Circular dichroism and fluorescence spectroscopic data indicated that the loss of negative charge associated with dephosphorylation led to an increase in ordered structure of alpha(s)- and beta-caseins. It is concluded that the flexible, dynamic, and relatively unstructured and hiphatic nature of alpha(s)- and beta-caseins is important in their chaperone action.

Elucidation of a novel polypeptide cross-link involving 3-hydroxykynurenine

Publisher: American Chemical Society (ACS)

Date: 08-1999

DOI: 10.1021/BI990458H

ALPHA-CRYSTALLIN - MOLECULAR CHAPERONE AND PROTEIN SURFACTANT

Publisher: Elsevier BV

Date: 02-1994

DOI: 10.1016/0167-4838(94)90009-4

Abstract: Bovine lens alpha-crystallin has recently been shown to function as a molecular chaperone by stabilizing proteins against heat denaturation (Horwitz, J. (1992) Proc. Natl. Acad. Sci. USA, 89, 10449-10453). An investigation, using a variety of physico-chemical methods, is presented into the mechanism of stabilization. alpha-Crystallin exhibits properties of a surfactant. Firstly, a plot of conductivity of alpha-crystallin versus concentration shows a distinct inflection in its profile, i.e., a critical micelle concentration (cmc), over a concentration range from 0.15 to 0.17 mM. Gel chromatographic and 1H-NMR spectroscopic studies spanning the cmc indicate no change in the aggregated state of alpha-crystallin implying that a change in conformation of the aggregate occurs at the cmc. Secondly, spectrophotometric studies of the rate of heat-induced aggregation and precipitation of alcohol dehydrogenase (ADH), beta L- and gamma-crystallin in the presence of alpha-crystallin and a variety of synthetic surfactants show that stabilization against precipitation results from hydrophobic interactions with alpha-crystallin and monomeric anionic surfactants. Per mole of subunit or monomer, alpha-crystallin is the most efficient at stabilization. alpha-Crystallin, however, does not preserve the activity of ADH after heating. After heat inactivation, gel permeation HPLC indicates that ADH and alpha-crystallin form a high molecular weight aggregate. Similar results are obtained following incubation of beta L- and gamma-crystallin with alpha-crystallin. 1H-NMR spectroscopy of mixtures of alpha- and beta L-crystallin, in their native states, reveals that the C-terminus of beta B2-crystallin is involved in interaction with alpha-crystallin. In the case of gamma- and alpha-crystallin mixtures, a specific interaction occurs between alpha-crystallin and the C-terminal region of gamma B-crystallin, an area which is known from the crystal structure to be relatively hydrophobic and to be involved in intermolecular interactions. The short, flexible C-terminal extensions of alpha-crystallin are not involved in specific interactions with these proteins. It is concluded that alpha-crystallin interacts with native proteins in a weak manner. Once a protein has become denatured, however, the soluble complex with alpha-crystallin cannot be readily dissociated. In the aging lens this finding may have relevance to the formation of high molecular weight crystallin aggregates.

The mammalian small heat-shock protein Hsp20 forms dimers and is a poor chaperone

Publisher: Wiley

Date: 15-12-1998

DOI: 10.1046/J.1432-1327.1998.2581014.X

Abstract: Hsp20 is one of the newly described members of the mammalian small heat-shock protein (sHsp) family. It occurs most abundantly in skeletal muscle and heart. We isolated clones for Hsp20 from a rat heart cDNA library, and expressed the protein in Escherichia coli to characterize this little known sHsp. Recombinant Hsp20 displayed similar far-ultraviolet circular dichroism spectra as the most closely related sHsp, alpha B-crystallin, but was less heat stable, denaturing upon heating to 50 degrees C. While other mammalian recombinant sHsps form large multimeric complexes, Hsp20 occurs in two complex sizes, 43-kDa dimers and 470-kDa multimers. The ratio between the two forms depends on protein concentration. Moreover, Hsp20 has a much lower chaperone-like activity than alpha B-crystallin, as indicated by its relatively poor capacity to diminish the reduction-induced aggregation of insulin B chains. Hsp20 is considerably shorter at the C-terminus and less polar than other sHsps, but 1H-NMR spectroscopy reveals that the last 10 residues are flexible, as in the other sHsps. Our findings suggest that Hsp20 is a special member of the sHsp family in being less heat stable and tending to form dimers. These properties, together with the shorter and less polar C-terminal extension, may contribute to the less effective chaperone-like activity.

The molecular chaperone, alpha-crystallin, inhibits amyloid formation by apolipoprotein C-II

Publisher: Elsevier BV

Date: 09-2001

DOI: 10.1074/JBC.M105285200

The interaction of the molecular chaperone, alpha-crystallin, with molten globule states of bovine alpha-lactalbumin

Publisher: Elsevier BV

Date: 10-1997

DOI: 10.1074/JBC.272.44.27722

A TWO-DIMENSIONAL H-1-NMR STUDY OF THE SOLUTION CONFORMATION OF GASTRIN RELEASING PEPTIDE

Publisher: Elsevier BV

Date: 1988

DOI: 10.1016/0006-291X(88)90429-9

Abstract: An almost complete assignment of the 1H NMR spectrum of gastrin releasing peptide in dimethyl sulphoxide solution and aqueous solution has been carried out using two dimensional NMR techniques. The chemical shifts in both solvents have been compared with the corresponding values in random coil polypeptides and it is concluded that gastrin releasing peptide adopts little short or long range order under either solvation conditions.

THE USE OF [(C6H7)FE(CO)3]+ AS A RECOVERABLE PROTECTING GROUP IN PEPTIDE-SYNTHESIS

Publisher: Elsevier BV

Date: 07-1993

DOI: 10.1016/0022-328X(93)83256-U

IDENTIFICATION OF 3-HYDROXYKYNURENINE AS THE LENS PIGMENT IN THE GOURAMI TRICHOGASTER-TRICHOPTERUS

Publisher: Elsevier BV

Date: 06-1992

DOI: 10.1016/0014-4835(92)90167-Q

Abstract: The rapid appearance of many new chemical substances which possess some antineoplastic effects has created a complex problem for the practicing physician. These agents which have shown promise in man and lower animals are grouped according to their modes of action. Each substance is discussed thoroughly with regard to its structure, activity, and influence upon the neoplasms of man. Key references are cited, and the practical value of each chemical agent is defined. The proper methods of administration of the compounds recommended for use are carefully described. In addition a section on agents whose therapeutic value has been disproven is also included.

Amyloid fibril formation by bovine milk κ-casein and its inhibition by the molecular chaperones α3- and β-casein

Publisher: American Chemical Society (ACS)

Date: 12-2005

DOI: 10.1021/BI051352R

Abstract: Caseins are a unique and erse 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.

Darwinian transformation of a 'scarcely nutritious fluid' into milk

Publisher: Wiley

Date: 23-04-2012

DOI: 10.1111/J.1420-9101.2012.02509.X

Abstract: In an early challenge to an aspect of Darwin's theory of natural selection, Jackson Mivart contended that milk could not have evolved 'from a scarcely nutritious fluid from an accidentally hypertrophied cutaneous gland'. The evolutionary change from a gland secretion to milk involves an increase in calcium and protein concentrations by up to 100- and 1000-fold, respectively. Even so, the challenge, we suggest, is not just a problem of scale. An increase in the concentrations of calcium and phosphate brings an increased risk of calcification of the secretory gland because calcium phosphate is highly insoluble. In addition, two of the four constituent milk casein proteins (κ and α(S2)) aggregate to produce toxic amyloid fibrils. It is proposed that both problems were solved through the cosecretion of ancestral β- and κ-caseins to form a stable amorphous aggregate of both proteins with sequestered amorphous calcium phosphate, that is, a primordial casein micelle. Evolutionarily, a gradual increase in the concentration of casein micelles could therefore produce progressively more nutritious fluids for the neonate without endangering the reproductive potential of the mother.

Primary structure of trypsin inhibitors from Sicyos australis

Publisher: Elsevier BV

Date: 03-1996

DOI: 10.1016/0031-9422(95)00734-2

Abstract: Three trypsin inhibitors from Sicyos australis, have been isolated, purified and sequenced. Following protein extraction with ammonium sulphate, the mixture of inhibitors was separated from other proteins by trypsin-affinity chromatography. Subsequent purification of the in idual inhibitors was accomplished by reversed-phase HPLC. The primary structures of each inhibitor were elucidated by a combination of protein sequencing and electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS-MS) on both the untreated and the reduced and S-carboxymethylated inhibitors. All three inhibitors show extensive sequence similarity with inhibitors from cultivated Cucurbitaceae species, although there are a number of novel residues present. One of the inhibitors has a blocked N-terminus (pyroglutamic acid) and the use of MS-MS was crucial to the elucidation of its primary structure. ESI-MS was further used to characterize the non-covalent complex between one of the inhibitors and trypsin.

Binding of the molecular chaperone αb-crystallin to Aβ amyloid fibrils inhibits fibril elongation

Publisher: Elsevier BV

Date: 10-2011

DOI: 10.1016/J.BPJ.2011.07.056

A radish seed antifungal peptide with a high amyloid fibril-forming propensity

Publisher: Elsevier BV

Date: 08-2013

DOI: 10.1016/J.BBAPAP.2013.04.030

Abstract: The amyloid fibril-forming ability of two closely related antifungal and antimicrobial peptides derived from plant defensin proteins has been investigated. As assessed by sequence analysis, thioflavin T binding, transmission electron microscopy, atomic force microscopy and X-ray fiber diffraction, a 19 amino acid fragment from the C-terminal region of Raphanus sativus antifungal protein, known as RsAFP-19, is highly amyloidogenic. Further, its fibrillar morphology can be altered by externally controlled conditions. Freezing and thawing led to amyloid fibril formation which was accompanied by loss of RsAFP-19 antifungal activity. A second, closely related antifungal peptide displayed no fibril-forming capacity. It is concluded that while fibril formation is not associated with the antifungal properties of these peptides, the peptide RsAFP-19 is of potential use as a controllable, highly amyloidogenic small peptide for investigating the structure of amyloid fibrils and their mechanism of formation.

The Amyloid Fibril-Forming Properties of the Amphibian Antimicrobial Peptide Uperin 3.5

Publisher: Wiley

Date: 16-12-2015

DOI: 10.1002/CBIC.201500518

Abstract: The hibian skin is a vast resource for bioactive peptides, which form the basis of the animals' innate immune system. Key components of the secretions of the cutaneous glands are antimicrobial peptides (AMPs), which exert their cytotoxic effects often as a result of membrane disruption. It is becoming increasingly evident that there is a link between the mechanism of action of AMPs and amyloidogenic peptides and proteins. In this work, we demonstrate that the broad-spectrum hibian AMP uperin 3.5, which has a random-coil structure in solution but adopts an α-helical structure in membrane-like environments, forms amyloid fibrils rapidly in solution at neutral pH. These fibrils are cytotoxic to model neuronal cells in a similar fashion to those formed by the proteins implicated in neurodegenerative diseases. The addition of small quantities of 2,2,2-trifluoroethanol accelerates fibril formation by uperin 3.5, and is correlated with a structural stabilisation induced by this co-solvent. Uperin 3.5 fibril formation and the associated cellular toxicity are inhibited by the polyphenol (-)-epigallocatechin-3-gallate (EGCG). Furthermore, EGCG rapidly dissociates fully formed uperin 3.5 fibrils. Ion mobility-mass spectrometry reveals that uperin 3.5 adopts various oligomeric states in solution. Combined, these observations imply that the mechanism of membrane permeability by uperin 3.5 is related to its fibril-forming properties.

Intracellular protein unfolding and aggregation: The role of small heat-shock chaperone proteins

Publisher: CSIRO Publishing

Date: 2003

DOI: 10.1071/CH03031

Abstract: Molecular chaperones are a erse group of proteins that interact with partially folded protein states to stabilize and prevent their mutual (illicit) association. Proteins require involvement with molecular chaperones throughout their lifespan: from their synthesis and folding through intracellular transport, membrane translocation, and to their ultimate degradation. Small heat-shock proteins (sHsps) are a ubiquitous family of molecular chaperones that are found in all organisms. Unlike many of the well-characterized chaperones, for ex le from the Hsp60 and Hsp70 families, sHsps are not involved in regulating protein folding. Instead, under conditions of cellular stress, such as elevated temperatures, they interact and stabilize partially folded target proteins to prevent their aggregation and precipitation. Because of this ability, their expression is elevated in many protein diseases that are characterized by protein aggregation and precipitation, including Alzheimer's, Creutzfeldt–Jakob, and Parkinson's diseases. The principal lens protein, α-crystallin, is a sHsp. Its chaperone ability is important in preventing lens protein precipitation and hence in maintaining lens transparency. This review summarizes the salient structural features of sHsps that enable them to act as highly efficient chaperones to prevent protein precipitation under stress conditions. The mechanism of chaperone action and the state of the target protein when interacting with sHsps are also discussed. Finally, diseases in which sHsp expression is elevated are discussed including the potential roles of sHsps and their therapeutic uses in the treatment of these diseases.

Invited review: Caseins and the casein micelle: Their biological functions, structures, and behavior in foods¹

Publisher: American Dairy Science Association

Date: 10-2013

DOI: 10.3168/JDS.2013-6831

Abstract: A typical casein micelle contains thousands of casein molecules, most of which form thermodynamically stable complexes with nanoclusters of amorphous calcium phosphate. Like many other unfolded proteins, caseins have an actual or potential tendency to assemble into toxic amyloid fibrils, particularly at the high concentrations found in milk. Fibrils do not form in milk because an alternative aggregation pathway is followed that results in formation of the casein micelle. As a result of forming micelles, nutritious milk can be secreted and stored without causing either pathological calcification or amyloidosis of the mother's mammary tissue. The ability to sequester nanoclusters of amorphous calcium phosphate in a stable complex is not unique to caseins. It has been demonstrated using a number of noncasein secreted phosphoproteins and may be of general physiological importance in preventing calcification of other biofluids and soft tissues. Thus, competent noncasein phosphoproteins have similar patterns of phosphorylation and the same type of flexible, unfolded conformation as caseins. The ability to suppress amyloid fibril formation by forming an alternative amorphous aggregate is also not unique to caseins and underlies the action of molecular chaperones such as the small heat-shock proteins. The open structure of the protein matrix of casein micelles is fragile and easily perturbed by changes in its environment. Perturbations can cause the polypeptide chains to segregate into regions of greater and lesser density. As a result, the reliable determination of the native structure of casein micelles continues to be extremely challenging. The biological functions of caseins, such as their chaperone activity, are determined by their composition and flexible conformation and by how the casein polypeptide chains interact with each other. These same properties determine how caseins behave in the manufacture of many dairy products and how they can be used as functional ingredients in other foods.

The Interaction of the Molecular Chaperone α-Crystallin with Unfolding α-Lactalbumin: A Structural and Kinetic Spectroscopic Study

Publisher: Elsevier BV

Date: 05-2002

DOI: 10.1016/S0022-2836(02)00144-4

Probing the structure and interactions of crystallin proteins by NMR spectroscopy

Publisher: Elsevier BV

Date: 1999

DOI: 10.1016/S1350-9462(98)00027-5

Abstract: The lens is composed primarily of proteins, the crystallins, at high concentration whose structure and interactions are responsible for lens transparency. As there is no protein turnover in the majority of the lens, crystallin proteins have to be very stable and long-lived proteins. There are three types of crystallin proteins: alpha, beta and gamma, and they all are composed of a variety of subunits. In addition, extensive post-translational modification is undergone by many of the subunits. Determining the structural features and the preferential interactions and associations undergone by the crystallin proteins in the lens is a large and complex experimental undertaking. Some progress has been made in this area by X-ray crystallographic determination of structures for representative ex les of the beta- and gamma-crystallins [Slingsby, C., Norledge, B., Simpson, A., Bateman, O. A., Wright, G., Driessen H. P. C., Lindley, P. F., Moss, D. S. and Bax, B. (1997) X-ray diffraction and structure of crystallins. Prog. Ret. Eye Res. 16, 3-29]. In this article, a summary is given of nuclear magnetic resonance (NMR) methods to determine information about these aspects of crystallin proteins. It is shown that despite their relatively large size, all crystallins give rise to well-resolved NMR spectra which arise from flexible terminal extensions that extend from the domain core of the proteins. By examining NMR spectra of mixtures of different crystallin subunits, it is possible to determine the role of these extensions in crystallin-crystallin interactions. For ex le, the flexible C-terminal extensions in the two alpha-crystallin subunits are not involved in interacting with the other crystallins but are crucially important in the chaperone action of alpha-crystallin. In this action, alpha-crystallin stabilises other proteins under conditions of stress, e.g. heat. In the lens, this ability probably has important consequences in preventing the precipitation of crystallin proteins with age and thereby contributing to cataract formation. The C-terminal extensions in alpha-crystallin act as solubilising agents for the protein and the high-molecular-weight complex that forms upon chaperone action with a precipitating "substrate" protein. Similar behaviour is observed for a variety of small heat-shock proteins, to which alpha-crystallin is related. NMR studies are also consistent with a two-domain structure for alpha-crystallin. No crystal structure is available for crystallin. Using the NMR data, a model for the quaternary structure of alpha-crystallin is proposed which comprises an annular arrangement for the subunits with a large central cavity.

The growing world of small heat shock proteins: from structure to functions

Publisher: Springer Science and Business Media LLC

Date: 31-03-2017

DOI: 10.1007/S12192-017-0787-8

RNA–LIM: A novel procedure for analyzing protein/single-stranded RNA propensity data with concomitant estimation of interface structure

Publisher: Elsevier BV

Date: 03-2015

DOI: 10.1016/J.AB.2014.11.004

Abstract: RNA-LIM is a procedure that can analyze various pseudo-potentials describing the affinity between single-stranded RNA (ssRNA) ribonucleotides and surface amino acids to produce a coarse-grained estimate of the structure of the ssRNA at the protein interface. The search algorithm works by evolving an ssRNA chain, of known sequence, as a series of walks between fixed sites on a protein surface. Optimal routes are found by application of a set of minimal "limiting" restraints derived jointly from (i) selective s ling of the ribonucleotide amino acid affinity pseudo-potential data, (ii) limited surface path exploration by prior determination of surface arc lengths, and (iii) RNA structural specification obtained from a statistical potential gathered from a library of experimentally determined ssRNA structures. We describe the general approach using a NAST (Nucleic Acid Simulation Tool)-like approximation of the ssRNA chain and a generalized pseudo-potential reflecting the location of nucleic acid binding residues. Minimum and maximum performance indicators of the methodology are established using both synthetic data, for which the pseudo-potential defining nucleic acid binding affinity is systematically degraded, and a representative real case, where the RNA binding sites are predicted by the lified antisense RNA (aaRNA) method. Some potential uses and extensions of the routine are discussed. RNA-LIM analysis programs along with detailed instructions for their use are available on request from the authors.

Characterisation of Amyloid Fibril Formation by Small Heat-shock Chaperone Proteins Human αA-, αB- and R120G αB-Crystallins

Publisher: Elsevier BV

Date: 09-2007

DOI: 10.1016/J.JMB.2007.06.060

Abstract: AlphaB-Crystallin is a ubiquitous small heat-shock protein (sHsp) renowned for its chaperone ability to prevent target protein aggregation. It is stress-inducible and its up-regulation is associated with a number of disorders, including those linked to the deposition of misfolded proteins, such as Alzheimer's and Parkinson's diseases. We have characterised the formation of amyloid fibrils by human alphaB-crystallin in detail, and also that of alphaA-crystallin and the disease-related mutant R120G alphaB-crystallin. We find that the last 12 amino acid residues of the C-terminal region of alphaB-crystallin are predicted from their physico-chemical properties to have a very low propensity to aggregate. (1)H NMR spectroscopy reveals that this hydrophilic C-terminal region is flexible both in its solution state and in amyloid fibrils, where it protrudes from the fibrillar core. We demonstrate, in addition, that the equilibrium between different protofilament assemblies can be manipulated and controlled in vitro to select for particular alphaB-crystallin amyloid morphologies. Overall, this study suggests that there could be a fine balance in vivo between the native functional sHsp state and the formation of amyloid fibrils.

Single Molecule Characterization of the Interactions between Amyloid-β Peptides and the Membranes of Hippocampal Cells

Publisher: American Chemical Society (ACS)

Date: 22-01-2013

DOI: 10.1021/JA3103567

The Kinetics of Amyloid Fibrillar Aggregation of Uperin 3.5 Is Directed by the Peptide’s Secondary Structure

Publisher: American Chemical Society (ACS)

Date: 06-08-2019

DOI: 10.1021/ACS.BIOCHEM.9B00536

Abstract: Many peptides aggregate into insoluble β-sheet rich amyloid fibrils. Some of these aggregation processes are linked to age-related diseases, such as Alzheimer's disease and type 2 diabetes. Here, we show that the secondary structure of the peptide uperin 3.5 directs the kinetics and mechanism of amyloid fibrillar aggregation. Uperin 3.5 variants were investigated using thioflavin T fluorescence assays, circular dichroism spectroscopy, and structure prediction methods. Our results suggest that those peptide variants with a strong propensity to form an α-helical secondary structure under physiological conditions are more likely to aggregate into amyloid fibrils than peptides in an unstructured or "random coil" conformation. This conclusion is in good agreement with the hypothesis that an α-helical transition state is required for peptide aggregation into amyloid fibrils. Specifically, uperin 3.5 variants in which charged amino acids were replaced by alanine were richer in α-helical content, leading to enhanced aggregation compared to that of wild type uperin 3.5. However, the addition of 2,2,2-trifluoroethanol as a major co-solute or membrane-mimicking phospholipid environments locked uperin 3.5 to the α-helical conformation preventing amyloid aggregation. Strategies for stabilizing peptides into their α-helical conformation could provide therapeutic approaches for overcoming peptide aggregation-related diseases. The impact of the physiological environment on peptide secondary structure could explain aggregation processes in a cellular environment.

Mouse Hsp25, a small heat shock protein - The role of its C-terminal extension in oligomerization and chaperone action

Publisher: Wiley

Date: 04-2000

DOI: 10.1046/J.1432-1327.2000.01188.X

Abstract: Under conditions of cellular stress, small heat shock proteins (sHsps), e.g. Hsp25, stabilize unfolding proteins and prevent their precipitation from solution. 1H NMR spectroscopy has shown that mammalian sHsps possess short, polar and highly flexible C-terminal extensions. A mutant of mouse Hsp25 without this extension has been constructed. CD spectroscopy reveals some differences in secondary and tertiary structure between this mutant and the wild-type protein but analytical ultracentrifugation and electron microscopy show that the proteins have very similar oligomeric masses and quaternary structures. The mutant shows chaperone ability comparable to that of wild-type Hsp25 in a thermal aggregation assay using citrate synthase, but does not stabilize alpha-lactalbumin against precipitation following reduction with dithiothreitol. The accessible hydrophobic surface of the mutant protein is less than that of the wild-type protein and the mutant is also less stable at elevated temperature. 1H NMR spectroscopy reveals that deletion of the C-terminal extension of Hsp25 leads to induction of extra C-terminal flexibility in the molecule. Monitoring complex formation between Hsp25 and dithiothreitol-reduced alpha-lactalbumin by 1H NMR spectroscopy indicates that the C-terminal extension of Hsp25 retains its flexibility during this interaction. Overall, these data suggest that a highly flexible C-terminal extension in mammalian sHsps is required for full chaperone activity.

H-1-NMR SPECTROSCOPY OF BOVINE LENS BETA-CRYSTALLIN - THE ROLE OF THE BETA-B2-CRYSTALLIN C-TERMINAL EXTENSION IN AGGREGATION

Publisher: Wiley

Date: 04-1993

DOI: 10.1111/J.1432-1033.1993.TB17765.X

Abstract: 1H-NMR spectroscopic studies of bovine eye lens beta-crystallin aggregates (dimer, trimer and octomer) are presented. The NMR spectra for all three beta-crystallin aggregates are dominated by resonances from the beta B2 subunit, particularly from the N- and C-terminal extensions of this subunit. Resonances from other beta subunits, which all have terminal extensions, are, in general, absent from spectra of the beta-crystallin aggregates. Therefore, the beta B2 subunit and, in particular its terminal extensions, has enhanced flexibility compared to the other beta-crystallin subunits. Furthermore, resonances arising from the C-terminal extension of beta B2-crystallin are not present in the spectrum of the octomer, which is consistent with the C-terminal extension binding in this aggregate and hence being involved in large aggregate formation. A possible interaction between the C-terminal extension of beta B2 and the hydrophobic beta B1 subunit, which is only found in the octomer, is discussed. At higher temperatures (45 degrees C) in the octomer, partial exposure of the C-terminal extension of beta B2 occurs indicating that the octomer may be starting to break up into smaller aggregates.

Casein proteins as molecular chaperones

Publisher: American Chemical Society (ACS)

Date: 12-03-2005

DOI: 10.1021/JF048329H

Abstract: Under conditions of stress, such as elevated temperature, molecular chaperones stabilize proteins from unfolding, aggregating, and precipitating. We have investigated the chaperone activity of the major milk proteins alpha(S)-, beta-, and kappa-casein with reduced insulin and the milk whey proteins, alpha-lactalbumin and beta-lactoglobulin, and compared it with that of the mammalian small heat shock protein (sHsp), alpha-crystallin, and clusterin. alpha(S)-Casein exhibited different chaperone behavior under reduction and heat stresses, i.e., chaperone activity increased with increasing temperature (as observed with alpha-crystallin), but under reduction stress, its chaperone activity increased at lower temperatures. beta- and kappa-casein had comparable chaperone ability with each other but were less effective than alpha(S)-casein. Under molecular crowding conditions, precipitation of stressed protein was accelerated, and alpha(S)-casein was a poorer chaperone. Furthermore, at slightly alkaline pH values, alpha(S)-casein was a less effective chaperone than at neutral pH. Detailed fluorescence, size exclusion chromatography, and real-time NMR studies studies indicated that the casein proteins underwent conformational changes and stabilized the partially unfolded whey proteins prior to formation of high molecular weight soluble complexes. These results are consistent with casein proteins acting as molecular chaperones in a manner similar to sHsps and clusterin.

NMR spectroscopy of alpha-crystallin. Insights into the structure, interactions and chaperone action of small heat-shock proteins

Publisher: Elsevier BV

Date: 05-1998

DOI: 10.1016/S0141-8130(98)00017-8

Abstract: The subunit molecular mass of alpha-crystallin, like many small heat-shock proteins (sHsps), is around 20 kDa although the protein exists as a large aggregate of average mass around 800 kDa. Despite this large size, a well-resolved 1H NMR spectrum is observed for alpha-crystallin which arises from short, polar, highly-flexible and solvent-exposed C-terminal extensions in each of the subunits, alpha A- and alpha B-crystallin. These extensions are not involved in interactions with other proteins (e.g. beta- and gamma-crystallins) under non-chaperone conditions. As determined by NMR studies on mutants of alpha A-crystallin with alterations in its C-terminal extension, the extensions have an important role in acting as solubilising agents for the relatively-hydrophobic alpha-crystallin molecule and the high-molecular-weight (HMW) complex that forms during the chaperone action. The related sHsp, Hsp25, also exhibits a flexible C-terminal extension. Under chaperone conditions, and in the HMW complex isolated from old lenses, the C-terminal extension of the alpha A-crystallin subunit maintains its flexibility whereas the alpha B-crystallin subunit loses, at least partially, its flexibility, implying that it is involved in interaction with the 'substrate' protein. The conformation of 'substrate' proteins when they interact with alpha-crystallin has been probed by 1H NMR spectroscopy and it is concluded that alpha-crystallin interacts with 'substrate' proteins that are in a disordered molten globule state, but only when this state is on its way to large-scale aggregation and precipitation. By monitoring the 1H and 31P NMR spectra of alpha-crystallin in the presence of increasing concentrations of urea, it is proposed that alpha-crystallin adopts a two-domain structure with the larger C-terminal domain unfolding first in the presence of denaturant. All these data have been combined into a model for the quaternary structure of alpha-crystallin. The model has two layers each of approximately 40 subunits arranged in an annulus or toroid. A large central cavity is present whose entrance is ringed by the flexible C-terminal extensions. A large hydrophobic region in the aggregate is exposed to solution and is available for interaction with 'substrate' proteins during the chaperone action.

A structural and functional study of Gln147 deamidation in alpha A-crystallin, a site of modification in human cataract

Publisher: Elsevier BV

Date: 08-2017

DOI: 10.1016/J.EXER.2017.05.005

Abstract: Deamidation of Glu147 in human αA-crystallin is common in aged cataractous lenses (Hains and Truscott, Invest. Ophthalmol. Vis. Sci. 2010, 51, 3107). Accordingly, this modification may have a causative effect in cataract. αA-crystallin is a small heat-shock molecular chaperone protein that prevents aggregation of proteins and is the principal defence against crystallin unfolding and aggregation in the ageing lens. Deamidated Q147E αA-crystallin was structurally characterised using a variety of spectroscopic and biophysical methods, including NMR, circular dichroism and fluorescence spectroscopy and dynamic light scattering. The effect of Glu147 deamidation on αA-crystallin in vitro chaperone ability was determined for a variety of aggregating proteins. Compared to the wild type protein, Q147E αA-crystallin generally exhibited slightly reduced chaperone ability and a small loss of overall structure in its central α-crystallin domain while also showing significantly enhanced thermal stability and a tendency to form slightly larger oligomers. As αA-crystallin is the major lens protein, even a small loss of function could combine with other sources of age-related damage to the crystallins to contribute to lens opacification.

Protein aggregate turbidity: Simulation of turbidity profiles for mixed aggregation reactions

Publisher: Elsevier BV

Date: 04-2016

DOI: 10.1016/J.AB.2015.11.021

Abstract: Due to their colloidal nature, all protein aggregates scatter light in the visible wavelength region when formed in aqueous solution. This phenomenon makes solution turbidity, a quantity proportional to the relative loss in forward intensity of scattered light, a convenient method for monitoring protein aggregation in biochemical assays. Although turbidity is often taken to be a linear descriptor of the progress of aggregation reactions, this assumption is usually made without performing the necessary checks to provide it with a firm underlying basis. In this article, we outline utilitarian methods for simulating the turbidity generated by homogeneous and mixed-protein aggregation reactions containing fibrous, amorphous, and crystalline structures. The approach is based on a combination of Rayleigh-Gans-Debye theory and approximate forms of the Mie scattering equations.

Formation of beta A3/beta B2-crystallin mixed complexes: involvement of N- and C-terminal extensions

Publisher: Elsevier BV

Date: 07-1999

DOI: 10.1016/S0167-4838(99)00123-5

Abstract: The sequence extensions of the beta-crystallin subunits have been suggested to play an important role in the oligomerization of these eye lens proteins. This, in turn, may contribute to maintaining lens transparency and proper light refraction. In homo-dimers of the betaA3- and betaB2-crystallin subunits, these extensions have been shown by (1)H-NMR spectroscopy to be solvent-exposed and highly flexible. In this study, we show that betaA3- and betaB2-crystallins spontaneously form mixed betaA3/betaB2-crystallin complexes, which, from analytical ultracentrifugation experiments, are dimeric at low concentrations (<1 mg ml(-1)) and tetrameric at higher protein concentrations. (1)H-NMR spectroscopy reveals that in the betaA3/betaB2-crystallin tetramer, the N-terminal extensions of betaA3-crystallin remain water-exposed and flexible, whereas both N- and C-terminal extensions of betaB2-crystallin lose their flexibility. We conclude that both extensions of betaB2-crystallin are involved in protein-protein interactions in the betaA3/betaB2-crystallin hetero-tetramer. The extensions may stabilize and perhaps promote the formation of this mixed complex.

Clusterin is an extracellular chaperone that specifically interacts with slowly aggregating proteins on their off-folding pathway

Publisher: Wiley

Date: 02-2002

DOI: 10.1016/S0014-5793(02)02326-8

Abstract: Clusterin is an extracellular mammalian chaperone protein which inhibits stress-induced precipitation of many different proteins. The conformational state(s) of proteins that interact with clusterin and the stage(s) along the folding and off-folding (precipitation-bound) pathways where this interaction occurs were previously unknown. We investigated this by examining the interactions of clusterin with different structural forms of alpha-lactalbumin, gamma-crystallin and lysozyme. When assessed by ELISA and native gel electrophoresis, clusterin did not bind to various stable, intermediately folded states of alpha-lactalbumin nor to the native form of this protein, but did bind to and inhibit the slow precipitation of reduced alpha-lactalbumin. Reduction-induced changes in the conformation of alpha-lactalbumin, in the absence and presence of clusterin, were monitored by real-time (1)H NMR spectroscopy. In the absence of clusterin, an intermediately folded form of alpha-lactalbumin, with some secondary structure but lacking tertiary structure, aggregated and precipitated. In the presence of clusterin, this form of alpha-lactalbumin was stabilised in a non-aggregated state, possibly via transient interactions with clusterin prior to complexation. Additional experiments demonstrated that clusterin potently inhibited the slow precipitation, but did not inhibit the rapid precipitation, of lysozyme and gamma-crystallin induced by different stresses. These results suggest that clusterin interacts with and stabilises slowly aggregating proteins but is unable to stabilise rapidly aggregating proteins. Collectively, our results suggest that during its chaperone action, clusterin preferentially recognises partly folded protein intermediates that are slowly aggregating whilst venturing along their irreversible off-folding pathway towards a precipitated protein.

The selective inhibition of serpin aggregation by the molecular chaperone, alpha-crystallin, indicates a nucleation-dependent specificity

Publisher: Elsevier BV

Date: 12-2003

DOI: 10.1074/JBC.M308376200

Australian National University

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Australian National University

Location: Australia

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University Of Wollongong

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University Of Oxford

Location: United Kingdom of Great Britain and Northern Ireland

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University Of Adelaide

Location: Australia

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