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See detailCreation of model proteins to investigate the mechanism of aggregation of expanded-polyglutamine proteins. Insertion of polyglutamine tracts into the ß-lactamase BlaP
Scarafone, Natacha ULg

Doctoral thesis (2012)

Polyglutamine (polyQ) diseases are characterized by the formation of intranuclear amyloid-like aggregates by proteins containing an expansion of a polyQ tract above a threshold length. These insoluble ... [more ▼]

Polyglutamine (polyQ) diseases are characterized by the formation of intranuclear amyloid-like aggregates by proteins containing an expansion of a polyQ tract above a threshold length. These insoluble aggregates and/or some of their soluble precursors are thought to play a role in the pathogenesis of the diseases. The only known common point between the causative proteins is the expanded polyQ tract, suggesting that their aggregation critically depends on the expansion of the polyQ tract above a threshold length. Several studies have however shown that the non-polyQ regions can also influence the aggregation behavior of polyQ proteins. In this work, polyQ chimeras were created by inserting different polyQ lengths in two positions (197-198 and 216-217) of the β-lactamase BlaP from Bacillus licheniformis 749/C. The structural and thermodynamic properties of the polyQ chimeras as well as their aggregating properties under native and denaturing conditions were investigated using a range of biophysical techniques including fluorescence, circular dichroism, absorbance, x-ray fiber diffraction and transmission electron microscopy. We have first created and characterized chimeras with 23, 30, 55 and 79Q inserted in position 197-198 (chimeras 197-198). None of these insertions modifies the structure of BlaP; they do, however, significantly destabilize the enzyme by 7.6-8.8 kJ/mol independently of the polyQ length. Similarly to the proteins associated with diseases, there is a threshold number of glutamines above which the BlaP chimeras aggregate into amyloid-like fibrils. It is comprised between 55 and 79Q and between 30 and 55Q under native and denaturing conditions, respectively. This threshold value therefore depends on the structural integrity of BlaP and thus on the steric and/or conformational constraints applied by BlaP to the polyQ tract. We have then compared the properties of these chimeras with those of the chimeras containing polyQ of the same lengths in position 216-217 (chimeras 216-217). The tertiary structure of BlaP is slightly perturbed in the chimeras 216-217 and these chimeras are less stable than the chimeras 197-198. However, the urea-induced equilibrium unfolding experiments suggest that chimeras 216-217 populate a series of discrete intermediate states between the native and unfolded states or that their unfolded state significantly differs from that of chimeras 197-198. Finally, the aggregating properties of the polyQ chimeras 216-217 differ from those of chimeras 197-198. First, the threshold number of glutamines above which chimeras 216-217 readily form fibrils under native conditions (between 30 and 55Q) is lower than that observed for chimeras 197-198 (between 55 and 79Q) suggesting that the steric and/or conformational constraints imposed on the polyQ tract are lower when it is inserted in position 216-217. Secondly, the results obtained under both native and denaturing conditions indicate that the BlaP moiety could assist fibril formation by chimeras 216-217 while it has an inhibiting effect and no effect on fibril formation by the chimeras 197-198 with 55 and 79 glutamines, respectively. Taken together, these results show that the aggregating properties of BlaP chimeras result from a very complex interplay between the propensity of the polyQ tract to mediate fibril formation and the modulating effect of the BlaP moiety. BlaP chimeras present therefore valuable models to investigate in details how the properties of the host protein influence the aggregation behavior of expanded polyQ proteins. [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 ▲]

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See detailThe Bacillus licheniformis BlaP beta-lactamase as a model protein scaffold to study the insertion of protein fragments.
Vandevenne, Marylène ULg; Filée, Patrice ULg; Scarafone, Natacha ULg et al

in Protein Science : A Publication of the Protein Society (2007), 16(10), 2260-71

Using genetic engineering technologies, the chitin-binding domain (ChBD) of the human macrophage chitotriosidase has been inserted into the host protein BlaP, a class A beta-lactamase produced by Bacillus ... [more ▼]

Using genetic engineering technologies, the chitin-binding domain (ChBD) of the human macrophage chitotriosidase has been inserted into the host protein BlaP, a class A beta-lactamase produced by Bacillus licheniformis. The product of this construction behaved as a soluble chimeric protein that conserves both the capacity to bind chitin and to hydrolyze beta-lactam moiety. Here we describe the biochemical and biophysical properties of this protein (BlaPChBD). This work contributes to a better understanding of the reciprocal structural and functional effects of the insertion on the host protein scaffold and the heterologous structured protein fragments. The use of BlaP as a protein carrier represents an efficient approach to the functional study of heterologous protein fragments. [less ▲]

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