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See detailA non-natural variant of human lysozyme (I59T) mimics the in vitro behaviour of the I56T variant that is responsible for a form of familial amyloidosis.
Hagan, Christine L; Johnson, Russell J K; Dhulesia, Anne et al

in Protein Engineering, Design & Selection (2010), 23(7), 499-506

We report here the detailed characterisation of a non-naturally occurring variant of human lysozyme, I59T, which possesses a destabilising point mutation at the interface of the alpha- and beta-domains ... [more ▼]

We report here the detailed characterisation of a non-naturally occurring variant of human lysozyme, I59T, which possesses a destabilising point mutation at the interface of the alpha- and beta-domains. Although more stable in its native structure than the naturally occurring variants that give rise to a familial form of systemic amyloidosis, I59T possesses many attributes that are similar to these disease-associated species. In particular, under physiologically relevant conditions, I59T populates transiently an intermediate in which a region of the structure unfolds cooperatively; this loss of global cooperativity has been suggested to be a critical feature underlying the amyloidogenic nature of the disease-associated lysozyme variants. In the present study, we have utilised this variant to provide direct evidence for the generic nature of the conformational transition that precedes the ready formation of the fibrils responsible for lysozyme-associated amyloid disease. This non-natural variant can be expressed at higher levels than the natural amyloidogenic variants, enabling, for example, singly isotopically labelled protein to be generated much more easily for detailed structural studies by multidimensional NMR spectroscopy. Moreover, we demonstrate that the I59T variant can readily form fibrils in vitro, similar in nature to those of the amyloidogenic I56T variant, under significantly milder conditions than are needed for the wild-type protein. [less ▲]

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See detailThe extracellular chaperone clusterin potently inhibits human lysozyme amyloid formation by interacting with prefibrillar species
Kumita, Janet R.; Poon, Stephen; Caddy, Gemma L. et al

in Journal of Molecular Biology (2007), 369

We have studied the effects of the extracellular molecular chaperone, clusterin, on the in vitro aggregation of mutational variants of human lysozyme, including one associated with familial amyloid ... [more ▼]

We have studied the effects of the extracellular molecular chaperone, clusterin, on the in vitro aggregation of mutational variants of human lysozyme, including one associated with familial amyloid disease. The aggregation of the amyloidogenic variant I56T is inhibited significantly at clusterin to lysozyme ratios as low as 1:80 (i.e. one clusterin molecule per 80 lysozyme molecules). Experiments indicate that under the conditions where inhibition of aggregation occurs, clusterin does not bind detectably to the native or fibrillar states of lysozyme, or to the monomeric transient intermediate known to be a key species in the aggregation reaction. Rather, it seems to interact with oligomeric species that are present at low concentrations during the lag (nucleation) phase of the aggregation reaction. This behavior suggests that clusterin, and perhaps other extracellular chaperones, could have a key role in curtailing the potentially pathogenic effects of the misfolding and aggregation of proteins that, like lysozyme, are secreted into the extracellular environment. [less ▲]

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See detailReduced global copperativity is a common feature underlying the amyloidogenicity of pathogenic lysozyme mutations
Dumoulin, Mireille ULg; Canet, Denis; Last, Alexander M. et al

in Journal of Molecular Biology (2005), 346(3), 773-788

One of the 20 or so human amyloid diseases is associated with the deposition in vital organs of full-length mutational variants of the antibacterial protein lysozyme. Here, we report experimental data ... [more ▼]

One of the 20 or so human amyloid diseases is associated with the deposition in vital organs of full-length mutational variants of the antibacterial protein lysozyme. Here, we report experimental data that permit a detailed comparison to be made of the behaviour of two of these amyloidogenic variants, I56T and D67H, under identical conditions. Hydrogen/deuterium exchange experiments monitored by NMR and mass spectrometry reveal that, despite their different locations and the different effects of the two mutations on the structure of the native state of lysozyme, both mutations cause a cooperative destabilisation of a remarkably similar segment of the structure, comprising in both cases the beta-domain and the adjacent C-helix. As a result, both variant proteins populate transiently a closely similar, partially unstructured intermediate in which the beta-domain and the adjacent C-helix are substantially and simultaneously unfolded, whereas the three remaining a-helices that form the core of the a-domain still have their native-like structure. We show, in addition, that the binding of a camel antibody fragment, cAb-HuL6, which was raised against wild-type lysozyme, restores to both variant proteins the stability and cooperativity characteristic of the wild-type protein; as a consequence, it inhibits the formation of amyloid fibrils by both variants. These results indicate that the reduction in global cooperativity, an associated ability to populate transiently a specific, partly unfolded intermediate state under physiologically relevant conditions, is a common feature underlying the behaviour of these two pathogenic mutations. The formation of intermolecular interactions between lysozyme molecules that are in this partially unfolded state is therefore likely to be the fundamental trigger of the aggregation process that ultimately leads to the formation and deposition in tissue of amyloid fibrils. (C) 2004 Elsevier Ltd. All rights reserved. [less ▲]

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See detailRationalising Lysozyme Amyloidosis: Insights from the Structure and Solution Dynamics of T70N Lysozyme
Johnson, Russell J.K.; Christodoulou, John; Dumoulin, Mireille ULg et al

in Journal of Molecular Biology (2005), 352

T70N human lysozyme is the only known naturally occurring destabilised lysozyme variant that has not been detected in amyloid deposits in human patients. Its study and a comparison of its properties with ... [more ▼]

T70N human lysozyme is the only known naturally occurring destabilised lysozyme variant that has not been detected in amyloid deposits in human patients. Its study and a comparison of its properties with those of the amyloidogenic variants of lysozyme is therefore important for understanding the determinants of amyloid disease. We report here the X-ray crystal structure and the solution dynamics of T70N lysozyme, as monitored by hydrogen/deuterium exchange and NMR relaxation experiments. The X-ray crystal structure shows that a substantial structural rearrangement results from the amino acid substitution, involving residues 45–51 and 68–75 in particular, and gives rise to a concomitant separation of these two loops of up to 6.5 Å. A marked decrease in the magnitudes of the generalised order parameter (S2) values of the amide nitrogen atom, for residues 70–74, shows that the T70N substitution increases the flexibility of the peptide backbone around the site of mutation. Hydrogen/deuterium exchange protection factors measured by NMR spectroscopy were calculated for the T70N variant and the wild-type protein. The protection factors for many of backbone amide groups in the β-domain of the T70N variant are decreased relative to those in the wild-type protein, whereas those in the α-domain display wild-type-like values. In pulse-labelled hydrogen/deuterium exchange experiments monitored by mass spectrometry, transient but locally cooperative unfolding of the β-domain of the T70N variant and the wild-type protein was observed, but at higher temperatures than for the amyloidogenic variants I56T and D67H. These findings reveal that such partial unfolding is an intrinsic property of the human lysozyme structure, and suggest that the readiness with which it occurs is a critical feature determining whether or not amyloid deposition occurs in vivo. [less ▲]

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