References of "Dumont, Janice"
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See detailNanobodies as structural probes to investigate the mechanism of fibril formation by the amyloidogenic variants of human lysozyme
Dumont, Janice ULg; pardon, Els; Aumont-Nicaise, Magali et al

Poster (2012, June)

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

Six variants of human lysozyme (single-point mutatants I56T, F57I, W64R, D67H and double mutants 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. We have also shown that the binding of three variable domain of camelid antibodies (VHHs) - raised against the wild type human lysozyme inhibit in vitro the formation of amyloid fibrils by the lysozyme variants. These three VHHs bind on different regions of lysozyme and act as amyloid fibril inhibitor through different mechanisms [2, 3, and unpublished results]. In the present work, sixteen new VHHs specific of human lysozyme have been generated. Competition experiments have shown that they bind to five non-overlapping epitopes. We have demonstrated that five of these VHHs are able to bind lysozyme in conditions used for amyloid fibril formation, and interestingly two of them recognize two epitopes that are different from those of the three VHHs previously characterized [2, 3, and unpublished results]. The effects of these new VHHs on the properties of lysozyme variants such as stability, cooperativity and aggregation will be discussed. [1] Dumoulin, M., J.R. Kumita, and C.M. Dobson, Normal and aberrant biological self-assembly: Insights from studies of human lysozyme and its amyloidogenic variants. Acc Chem Res, 2006, 39(9), 603-610. [2] Dumoulin, M., et al., A camelid antibody fragment inhibits the formation of amyloid fibrils by human lysozyme. Nature, 2003, 424, 783-788. [3] Chan, P.H., et al., Engineering a camelid antibody fragment that binds to the active site of human lysozyme and inhibits its conversion into amyloid fibrils. Biochemistry, 2008, 47, 11041-11054. [less ▲]

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See detailNanobodies as structural probes to investigate the mechanism of fibril formation by the amyloidogenic variants of human lysozyme
Dumont, Janice ULg; Pardon, Els; Aumont-Nicaise, Magalie et al

Poster (2011)

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. We have also shown that the binding of three variable domain of camelid antibodies or (VHHs) - raised against the wild type human lysozyme inhibit in vitro the formation of amyloid fibrils by the lysozyme variants. These three VHHs bind on different regions of lysozyme and act as amyloid fibrils inhibitor through different mechanisms [2, 3, and unpublished results]. In the present work, sixteen new VHHs specific of human lysozyme have been generated. Competition experiments have shown that they bind to five non overlapping epitopes. We have demonstrated that five of these new VHHs are able to bind lysozyme in conditions used for amyloid fibril formation, and interestingly two of them recognize two epitopes that are different from those of the three VHHs previously characterized [2, 3, and unpublished results]. The effects of these new VHHs on the properties of lysozyme variants such as activity, stability, cooperativity and aggregation will be discussed. [less ▲]

Detailed reference viewed: 35 (1 ULg)
See detailCamelid single-domain antibody fragments as structural probes to study the mechanism of human lysozyme fibrils formation
Dumont, Janice ULg; Pardon, Els; Menzer, Linda ULg et al

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 ▲]

Detailed reference viewed: 23 (2 ULg)
See detailNanobodies as structural probes to investigate the mechanism of fibril formation by the amyloidogenic variants of human lysozyme.
Dumont, Janice ULg; Menzer, Linda ULg; Pardon, Els et al

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 ▲]

Detailed reference viewed: 39 (1 ULg)
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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 detailProduction of four amyloidogenic variants of human lysozyme as inclusion bodies in Escherichia coli
Dumont, Janice ULg; Menzer, Linda ULg; Scarafone, Natacha ULg et al

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 ▲]

Detailed reference viewed: 28 (4 ULg)