John A. Carver

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

Host-defence peptides secreted from the skin glands of Australian frogs and toads, are, with a few notable exceptions, different from those produced by anurans elsewhere. This review summarizes the current knowledge of the following classes of peptide isolated and characterized from Australian anurans: neuropeptides (including smooth muscle active peptides, and peptides that inhibit the production of nitric oxide from neuronal nitric oxide synthase), antimicrobial and anticancer active peptides, antifungal peptides and antimalarial peptides. Other topics covered include sex pheromones of anurans, and the application of peptide profiling to (i). recognize particular populations of anurans of the same species and to differentiate between species, and (ii). investigate evolutionary aspects of peptide formation.

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

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., Alzheimers, Creutzfeldt‐Jakob and Parkinsons diseases. The interaction of sHsps and clusterin with fibril‐forming species is discussed along with their ability to prevent fibril formation. IUBMB Life, 55: 661‐668, 2003

Identification by 1H NMR spectroscopy of flexible C-terminal extensions in bovine lens α-crystallin

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

Immobilization of the C-terminal extension of bovine alpha A-crystallin reduces chaperone-like activity

α-Crystallins occur as multimeric complexes, which are able to suppress precipitation of unfolding proteins. Although the mechanism of this chaperone-like activity is unknown, the affinity of α-crystallin for aggregation-prone proteins is probably based on hydrophobic interactions. α-Crystallins expose a considerable hydrophobic surface to solution, but nevertheless they are very stable and highly soluble. An explanation for this paradox may be that α-crystallin subunits have a polar and unstructured C-terminal extension that functions as a sort of solubilizer. In this paper we have described five αA-crystallins in which charged and hydrophobic residues were inserted in the C-terminal extension. Introduction of lysine, arginine, and aspartate does not substantially influence chaperone-like activity. In contrast, introduction of a hydrophobic tryptophan greatly diminishes functional activity. CD experiments indicate that this mutant has a normal secondary structure and fluorescence measurements show that the inserted tryptophan is located in a polar environment. However, NMR spectroscopy clearly demonstrates that the presence of the tryptophan residue dramatically reduces the flexibility of the C-terminal extension. Furthermore, the introduction of this tryptophan results in a considerably decreased thermostability of the protein. We conclude that changing the polarity of the C-terminal extension of αA-crystallin by insertion of a highly hydrophobic residue can seriously disturb structural and functional integrity.

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

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 α‐lactalbumin, γ‐crystallin and lysozyme. When assessed by ELISA and native gel electrophoresis, clusterin did not bind to various stable, intermediately folded states of α‐lactalbumin nor to the native form of this protein, but did bind to and inhibit the slow precipitation of reduced α‐lactalbumin. Reduction‐induced changes in the conformation of α‐lactalbumin, in the absence and presence of clusterin, were monitored by real‐time 1H NMR spectroscopy. In the absence of clusterin, an intermediately folded form of α‐lactalbumin, with some secondary structure but lacking tertiary structure, aggregated and precipitated. In the presence of clusterin, this form of α‐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 γ‐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.

On the interaction of α-crystallin with unfolded proteins

α-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 α-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), γ-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 α-crystallin and ADH. SDS, like α-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), α-crystallin does not interact with unfolded, hydrophobic but stable proteins (e.g., reduced and carboxymethylated α-lactalbumin and α-casein). It is concluded that α-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 α-crystallin and γ-crystallin indicates that the short C-terminal extension of αB-crystallin, but not that of αA-crystallin, has lost its flexibility in the complex implying that the former is involved in interactions with the unfolded γ-crystallin molecule, possibly electrostatically via its two C-terminal lysine residues.

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

The unfolding of the apo and holo forms of bovine α-lactalbumin (α-LA) upon reduction by dithiothreitol (DTT) in the presence of the small heat-shock protein α-crystallin, a molecular chaperone, has been monitored by visible and UV absorption spectroscopy, mass spectrometry and 1H NMR spectroscopy. From these data, a description and a time-course of the events that result from the unfolding of both forms of the protein, and the state of the protein that interacts with α-crystallin, have been obtained. α-LA contains four disulphide bonds and binds a calcium ion. In apo α-LA, the disulphide bonds are reduced completely over a period of ∼1500 seconds. Fully reduced α-LA adopts a partly folded, molten globule conformation that aggregates and, ultimately, precipitates. In the presence of an equivalent mass of α-crystallin, this precipitation can be prevented via complexation with the chaperone. α-Crystallin does not interfere with the kinetics of the reduction of disulphide bonds in apo α-LA but does stabilise the molten globule state. In holo α-LA, the disulphide bonds are less accessible to DTT, because of the stabilisation of the protein by the bound calcium ion, and reduction occurs much more slowly. A two-disulphide intermediate aggregates and precipitates rapidly. Its precipitation can be prevented only in the presence of a 12-fold mass excess of α-crystallin. It is concluded that kinetic factors are important in determining the efficiency of the chaperone action of α-crystallin. It interacts efficiently with slowly aggregating, highly disordered intermediate (molten globule) states of α-LA. Real-time NMR spectroscopy shows that the kinetics of the refolding of apo α-LA following dilution from denaturant are not affected by the presence of α-crystallin. Thus, α-crystallin is not a chaperone that is involved in protein folding per se. Rather, its role is to stabilise compromised, partly folded, molten globule states of proteins that are destined for precipitation.

The Interaction of the Molecular Chaperone, α-Crystallin, with Molten Globule States of Bovine α-Lactalbumin

Small heat shock proteins function in a chaperone-like manner to prevent the precipitation of proteins under conditions of stress (e.g. heat). α-Crystallin, the major mammalian lens protein, is a small heat shock protein. The mechanism of chaperone action of these proteins is poorly understood. In this paper, the conformational state of a protein when it forms a high molecular weight complex with α-crystallin is investigated by examining, using NMR spectroscopy and size exclusion high performance liquid chromatography, the interaction of α-crystallin with α-lactalbumin and its various intermediately folded (molten globule) states. The complex is formed following reduction of α-lactalbumin by dithiothreitol in the presence of α-crystallin, and this interaction has been monitored in real time by 1H NMR spectroscopy. It is concluded that α-crystallin interacts with a disordered molten globule state of α-lactalbumin while it is on an irreversible pathway toward aggregation and precipitation. α-Crystallin does not interact, however, with molten globule states of α-lactalbumin that are stable in solution, e.g. the reduced and carboxyamidated species. It is proposed that α-crystallin distinguishes between the various molten globule states of α-lactalbumin on the basis of the lifetimes of these states, i.e. the protein must be in a disordered molten globule state for a significant length of time and on the pathway to aggregation and precipitation for interaction to occur.

α-Crystallin : molecular chaperone and protein surfactant

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.

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

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.