Stephen Poon

The extracellular chaperone clusterin influences amyloid formation and toxicity by interacting with prefibrillar structures

Clusterin is an extracellular chaperone present in all disease‐associated extracellular amyloid deposits, but its roles in amyloid formation and protein deposition in vivo are poorly understood. The current study initially aimed to characterize the effects of clusterin on amyloid formation in vitro by a panel of eight protein substrates. Two of the substrates (Alzheimers beta peptide and a PI3‐SH3 domain) were then used in further experiments to examine the effects of clusterin on amyloid cytotoxicity and to probe the mechanism of clusterin action. We show that clusterin exerts potent effects on amyloid formation, the nature and extent of which vary greatly with the clusterin: substrate ratio, and provide evidence that these effects are exerted via interactions with prefibrillar species that share common structural features. Proamyloido‐genic effects of clusterin appear to be restricted to conditions in which the substrate protein is present at a very large molar excess;under these same conditions, clusterin coincorporates with substrate protein into insoluble aggregates. However, when clusterin is present at much higher but still substoichiometric levels (e.g., a molar ratio of clusterin:substrate = 1:10), it potently inhibits amyloid formation and provides substantial cytoprotection. These findings suggest that clus‐terin is an important element in the control of extracellular protein misfolding.—Yerbury, J. J., Poon, S., Meehan, S., Thompson, B., Kumita, J. R., Dobson, C. M., Wilson, M. R. The extracellular chaperone clusterin influences amyloid formation and toxicity by interacting with prefibrillar structures. FASEB J. 21, 2312–2322 (2007)

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.

Potential roles of abundant extracellular chaperones in the control of amyloid formation and toxicity

The in vivo formation of fibrillar proteinaceous deposits called amyloid is associated with more than 40 serious human diseases, collectively referred to as protein deposition diseases. In many cases the amyloid deposits are extracellular and are found associated with newly identified abundant extracellular chaperones (ECs). Evidence is presented suggesting an important regulatory role for ECs in amyloid formation and disposal in the body. A model is presented which proposes that, under normal conditions, ECs stabilize extracellular misfolded proteins by binding to them, and then guide them to specific cell receptors for uptake and subsequent degradation. Thus ECs and their receptors may be critical parts of a quality control system to protect the body against dangerously hydrophobic proteins/peptides. However, it also appears possible that in the presence of a high molar excess of misfolded protein, such as might occur during disease, the limited amounts of ECs available may actually exacerbate pathology. Further advances in understanding of the mechanisms that control extracellular protein folding are likely to identify new strategies for effective disease therapies.

Mildly acidic pH activates the extracellular molecular chaperone clusterin.

Many features of the chaperone action of clusterin are similar to those of the intracellular small heat shock proteins (sHSPs) that, like clusterin, exist in solution as heterogeneous aggregates. Increased temperature induces dissociation of some sHSP aggregates and an enhanced chaperone action, suggesting that a dissociated form is the active chaperone species. We recently reported that clusterin aggregates dissociate at mildly acidic pH. To further explore the similarities between clusterin and the sHSPs, we tested the effects of temperature and pH on the structure of clusterin and its chaperone action. Our results demonstrate that increased temperature does not induce dissociation of clusterin aggregates, or other major structural changes, and has little effect on its chaperone action. However, we show that the chaperone action of clusterin is enhanced at mildly acidic pH. Clusterin is the first chaperone shown to be activated by reduced pH. This unique mode of activation appears to result from an increase in regions of solvent-exposed hydrophobicity, which is independent of any major changes in secondary or tertiary structure. We propose a model in which low pH-induced dissociation of clusterin aggregates increases the abundance of the heterodimeric chaperone-active species, which has greater hydrophobicity exposed to solution.

Amyloid fibril formation by bovine milk alpha(s2)-casein occurs under physiological conditions yet is prevented by its natural counterpart, alpha(s1)-casein

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.

Amyloidogenicity and Aggregate Cytotoxicity of Human Glucagon-Like Peptide-1 (hGLP-1)

The potential of human glucagon-like peptide-1 (hGLP-1) as a therapeutic agent is limited by its high aggregation propensity. We show that hGLP-1 forms amyloid-like structures that are preceded by cytotoxic aggregates, suggesting that aggregation of biopharmaceuticals could present a cytotoxic risk to patients besides the reported increased risk in immunogenicity.

Extracellular chaperones and amyloids

The pathology of more than 40 human degenerative diseases is associated with fibrillar proteinaceous deposits called amyloid. Collectively referred to as protein deposition diseases, many of these affect the brain and the central nervous system. In many cases the amyloid deposits are extracellular and are found associated with newly identified abundant extracellular chaperones (ECs). Evidence is discussed which suggests an important regulatory role for ECs in amyloid formation and disposal in vivo. This is emerging as an exciting field. A model is presented in which it is proposed that, under normal conditions, ECs stabilize extracellular misfolded proteins by binding to them, and then guide them to specific receptors for uptake and subsequent degradation. In this scenario, EC receptors are a critical part of a quality control system which protects the brain against dangerously hydrophobic proteins/peptides. However, it also appears possible that in the presence of a high molar excess of misfolded protein, such as might occur during disease, the limited amounts of ECs available may actually exacerabate pathology. Further advances in understanding of the mechanisms that control extracellular protein folding are likely to identify new strategies for effective disease therapies

Polyelectrolyte Complex Materials Consisting of Antibacterial and Cell‐Supporting Layers

The characterization of a polyelectrolyte complex material comprised of two biopolymers, a chitosan upper layer and a gellan gum under layer, is reported. It is shown that the upper layer of chitosan with incorporated levofloxacin displays an antibacterial activity, while the under layer of a gellan gum/TiO(2) composite supports the growth of fibroblastic cells.