Disease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster

in FASEB Journal (2012)

Camelid single-domain antibody fragments as structural probes to study the mechanism of human lysozyme fibrils formation

Poster (2010)

Six variants of human lysozyme (single-point mutations I56T, F57I, W64R, D67H and double mutations F57I/T70N, W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidosis. These ... [more ▼]

Six variants of human lysozyme (single-point mutations I56T, F57I, W64R, D67H and double mutations F57I/T70N, W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidosis. These proteins form extracellular amyloid fibrils that deposit in a wide range of tissues and organs such as liver, spleen and kidneys where they cause damages [1]. It was shown that the D67H and I56T mutations cause a loss in stability and more particularly a loss of global cooperativity of protein [1]. Consequently, under physiologically relevant conditions, these variants can transiently populate a partially unfolded state in which the beta-domain and the C-helix are cooperatively unfolded while the rest of the protein remains native like [1]. The formation of intermolecular interactions between the regions that are unfolded in this intermediate state is likely to be a fundamental trigger of the aggregation process that ultimately leads to the formation and deposition of fibrils in tissues. The binding of three variable domain of camelid antibodies – also named nanobodies - (cAb-HuL 6 [2], cAb-HuL 5 and cAb-HuL 22 [3]) raised against the wild type human lysozyme inhibit in vitro the formation of amyloid fibrils by the lysozyme variants. These three nanobodies bind on different regions of lysozyme and act as amyloid fibrils inhibitor through different mechanisms. On one hand, cAb-HuL 6 and cAb-HuL 22 stabilize the native state of the lysozyme variants thus restoring the global cooperativity characteristic of the wild-type protein. On the other, cAb-HuL 5 probably acts by binding soluble prefibrillar aggregates. In the present work, sixteen other nanobodies specific of human lysozyme have been generated. Competition experiments have shown that they bind to five non overlapping epitopes. The effects of the binding of these nanobodies on the stability of the D67H variant of human lysozyme and on its aggregation into amyloid fibrils will be discussed. References [1] Dumoulin et al, (2006) Acc. Chem. Res, 39, 603-610. [2] Dumoulin et al, (2003) Nature, 424, 783-788. [3] Chan et al. (2008) Biochemistry, 47,11041-11054. [less ▲]

Nanobodies as structural probes to investigate the mechanism of fibril formation by the amyloidogenic variants of human lysozyme.

Poster (2010)

Six variants of human lysozyme (single-point mutations I56T, F57I, W64R, D67H and double mutations F57I/T70N, W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidose. These ... [more ▼]

Six variants of human lysozyme (single-point mutations I56T, F57I, W64R, D67H and double mutations F57I/T70N, W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidose. These proteins form extracellular amyloid fibrils that deposit in a wide range of tissues and organs such as liver, spleen and kidneys where they cause damages [1]. It was shown that the D67H and I56T mutations cause a loss in stability and more particularly a loss of global cooperativity of protein [1]. Consequently, under physiologically relevant conditions, these variants can transiently populate a partially unfolded state in which the beta-domain and the C-helix are cooperatively unfolded while the rest of the protein remains native like [1]. The formation of intermolecular interactions between the regions that are unfolded in this intermediate state is likely to be a fundamental trigger of the aggregation process that ultimately leads to the formation and deposition of fibrils in tissues. The binding of three variable domain of camelid antibodies – also named nanobodies - (cAb-HuL 6 [2], cAb-HuL 5 and cAb-HuL 22 [3]) raised against the wild type human lysozyme inhibit in vitro the formation of amyloid fibrils by the lysozyme variants. These three nanobodies bind on different regions of lysozyme and act as Amyloid fibrils inhibitor through different mechanisms. On one hand, cAb-HuL 6 and cAb-HuL 22 stabilize the native state of the lysozyme variants thus restoring the global cooperativity characteristic of the wild-type protein. On the other, cAb-HuL 5 probably acts by binding soluble prefibrillar aggregates. In the present work, sixteen other nanobodies specific of human lysozyme have been generated. Competition experiments have shown that they bind to five non overlapping epitopes. The effects of the binding of these nanobodies on the stability of the D67H variant of human lysozyme and on its aggregation into amyloid fibrils will be discussed. [less ▲]

Production of four amyloidogenic variants of human lysozyme as inclusion bodies in Escherichia coli

Poster (2009)

Six variants of human lysozyme (I56T, F57I, W64R, D67H, F57I/T70N and W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidosis. This disease involved an extra cellular deposition ... [more ▼]

Six variants of human lysozyme (I56T, F57I, W64R, D67H, F57I/T70N and W112R/T70N) are associated with a hereditary non-neuropathic systemic amyloidosis. This disease involved an extra cellular deposition of amyloid fibrils made of lysozyme variants in a wide range of organs such as liver, spleen and kidneys [1]. The characterisation at the molecular level of two variants, I56T and D67H, has shown that these mutations reduce the stability and more particularly the global cooperativity of the protein. Consequently, under physiologically relevant conditions, these variants can transiently populate a partially unfolded state in which the beta-domain and the C-helix are cooperatively unfolded while the rest of the protein remains native like [1]. The formation of intermolecular interactions between the regions that are unfolded in this intermediate state is likely to be a fundamental trigger of the aggregation process that ultimately leads to the formation and deposition of fibrils in tissues. In order to study the effects of the other amyloidogenic mutations on the properties of lysozyme and thus to get more insight in the mechanism of amyloid formation, it is necessary to produce them in large quantities. The D67H, I56T and F57I variants are currently produced in Aspergillus niger; the expression in this organism is, however, time consuming and the yield is very low. The attempts to use alternative systems such as Pichia pastoris [2], Saccharomyces cerevisiae, and Arabidopsis thaliana have not been conclusive so far. In this work, we have produced the four single-point lysozyme variants as inclusion bodies in Escherichia coli and explored the possibility to refold them. [1] Dumoulin & al., (2006) Acc. Chem. Res., 39, 603 - 610 [2] Kumita & al., (2006) FEBS J., 273, 711-720 [less ▲]

Optimization of the Production of the Amyloidogenic Variants of Human Lysozyme

Poster (2008, February 16)