Teresa M. Treweek

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.

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

Small heat-shock proteins (sHsps) are a diverse 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.

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

α‐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, αB‐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 αB‐crystallin to stabilize the partially folded, molten globule state of α‐lactalbumin was significantly reduced in comparison with wild‐type αB‐crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced α‐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 αB‐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 αB‐crystallin exists as a larger oligomer than wild‐type αB‐crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of αB‐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 αB‐crystallin with human disease states.

Intracellular Protein Unfolding and Aggregation: The Role of Small Heat-Shock Chaperone Proteins

Molecular chaperones are a diverse 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 example 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 Alzheimers, Creutzfeldt–Jakob, and Parkinsons 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.

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

Background Alzheimers, Parkinsons and Creutzfeldt-Jakob disease are associated with inappropriate protein deposition and ordered amyloid fibril assembly. Molecular chaperones, including αB-crystallin, play a role in the prevention of protein deposition. Methodology/Principal Findings A series of site-directed mutants of the human molecular chaperone, αB-crystallin, were constructed which focused on the flexible C-terminal extension of the protein. We investigated the structural role of this region as well as its role in the chaperone function of αB-crystallin under different types of protein aggregation, i.e. disordered amorphous aggregation and ordered amyloid fibril assembly. It was found that mutation of lysine and glutamic acid residues in the C-terminal extension of αB-crystallin resulted in proteins that had improved chaperone activity against amyloid fibril forming target proteins compared to the wild-type protein. Conclusions/Significance Together, our results highlight the important role of the C-terminal region of αB-crystallin in regulating its secondary, tertiary and quaternary structure and conferring thermostability to the protein. The capacity to genetically modify αB-crystallin for improved ability to block amyloid fibril formation provides a platform for the future use of such engineered molecules in treatment of diseases caused by amyloid fibril formation.

The chaperone action of bovine milk αS1- and αS2-caseins and their associated form αS-casein.

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.

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

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 α‐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.

Glutamic Acid Residues in the C-Terminal Extension of Hsp25 are Critical for Structural and Functional Integrity

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 α‐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.

Alpha-casein as a molecular chaperone

The caseins are a heterogeneous group of dairy proteins constituting 80% of the protein content of bovine milk. The operational definition of casein is that proportion of total milk protein which precipitates on acidification of milk to a pH value of 4.6 [1]. The remaining dairy proteins, known collectively as whey proteins, do not precipitate. Caseins are synthesised in the mammary gland and are found nowhere else among the plant and animal kingdoms [2]. The casein family of proteins comprises , and -caseins, all with little sequence homology [3]. As their primary function is nutritional, binding large amounts of calcium, zinc and other biologically important metals, amino acid substitutions or deletions have little impact on function. The caseins also lack well-defined structure and as a result their amino acid sequence is less critical to function than in many ‘classic’ globular proteins. As a result, the caseins are one of the most evolutionarily divergent protein families characterised in mammals [2]. Alpha-casein, also known as αS-casein, is in fact two distinct gene products, S1and S2-casein, with the ‘S’ denoting a sensitivity to calcium. Of all the caseins, S1and -casein are predominant in bovine milk, representing 37 and 35% of whole casein respectively, whereas αS2and κ-casein make up 10 and 12% of whole casein, respectively [2].