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See detailNanobodies as tools to investigate the mechanism of aggregation of chimeric proteins made by the insertion of polyglutamine stretches into the beta-lactamase BlaP
Pain, Coralie ULg

Doctoral thesis (2014)

Among the neurodegenerative amyloidoses, ten disorders, referred to as polyglutamine (polyQ) diseases and including Huntington's disease and several spinocerebellar ataxias, are associated with ten ... [more ▼]

Among the neurodegenerative amyloidoses, ten disorders, referred to as polyglutamine (polyQ) diseases and including Huntington's disease and several spinocerebellar ataxias, are associated with ten proteins within which a polyQ tract is expanded above a threshold of typically 35-45 glutamine residues. Such expanded polyQ tracts lead to the aggregation of the host protein into amyloid fibrils that accumulate in the nucleus of some populations of neurons; these aggregates or some of their precursors are thought to contribute to neuronal death. So far, no preventive or curative treatment exists for these devastating pathologies. While the expansion of the polyQ tract above the threshold is the determinant factor for aggregation, recent studies suggest that non-polyQ regions of these proteins can play a significant role, either preventative or facilitative, in the aggregation process. The general principles governing the complex interplay between the role of the expanded polyQ tract and the role of the non-polyQ regions in the aggregation process are not well understood yet. In order to develop therapeutic strategies, it is important to better understand this complex interplay. To contribute to this aim, we have engineered chimeric proteins via the insertion of polyQ repeats of various lengths (23, 30, 55 and 79Q) into two sites (197 and 216) of the BlaP beta-lactamase from Bacillus licheniformis 749/C. The properties of these chimeric proteins recapitulate the characteristic features of the disease-associated polyQ proteins, i.e. (i) there is a minimum number of inserted glutamines (threshold) required to trigger the aggregation of the chimeras into amyloid fibrils, and (ii) above the threshold, the longer the polyQ tract, the faster the aggregation. Interestingly, for the same polyQ length, the chimeras with insertions in position 216 have an increased propensity to form amyloid fibrils compared to their counterparts with insertions in position 197. These findings highlight the strong influence of the overall protein context on aggregation triggered by expanded polyQ tracts. This thesis addresses the use of the variable domains of camelid heavy-chain antibodies, referred to as nanobodies or VHHs, as structural and mechanistic probes to better understand the different aggregating properties of the two sets of BlaP-polyQ chimeras (197 and 216). We have also performed limited proteolysis experiments and transglutaminase-mediated reactions on the monomeric form of the BlaP-polyQ chimeras to further investigate the effects of the polyQ insertions on the structure and dynamics of the BlaP moiety, as well as the structure of the polyQ tract itself. From the blood of a llama immunised with BlaP197(Gln)55, we isolated more than 60 VHHs specific to the BlaP-polyQ chimeras. Twenty eight of them were produced, purified and characterised. These VHHs were found to be all specific to the BlaP moiety and could be classified into four different groups recognising distinct epitopes on the surface of BlaP. One representative VHH of each group (i.e. cAb-A3S, cAb-H7S, cAb-F11N and cAb-G10S) was selected as probe to investigate the mechanism of aggregation of the BlaP-polyQ chimeras. The epitope of three of them was determined by X-ray diffraction and/or by NMR spectroscopy. Although they recognise distinct epitopes and exhibit different affinities for BlaP, the binding of the four VHHs significantly slows down the aggregation of all the BlaP-polyQ chimeras investigated (i.e. BlaP197(Gln)55, BlaP197(Gln)79 and BlaP216(Gln)79). The extent of inhibition depends however on the chimera and on the experimental conditions. We show that the inhibition of the aggregation of BlaP197(Gln)55 and BlaP197(Gln)79 upon binding of the four VHHs is correlated with the stabilisation of their native state. In the case of BlaP216(Gln)79, the extent of inhibition could not be only correlated to the stabilisation of its native state; the location of the epitope of the VHH is instead also determinant. This observation demonstrates that the lower thermodynamic stability of BlaP216(Gln)79 is not the unique factor responsible for its increased aggregation propensity. It also further highlights the complexity of the aggregation mechanism of polyQ proteins and the strong influence of the non-polyQ regions on the amyloid fibril formation triggered by the expanded polyQ tract. All together our results suggest that antibodies or antibody fragments raised against the non-polyQ regions of polyQ proteins associated with diseases could constitute a relevant therapeutic strategy. They also further demonstrate the power of nanobodies as probes to get a deeper knowledge of the underlying mechanisms of amyloid fibril formation. The preliminary limited proteolysis and transglutamination experiments obtained suggest that the polyQ tracts are all flexible, except that of 23 glutamines inserted in position 197 of BlaP, which seems to be more rigid than the others. The results obtained confirm that, globally, the structure of BlaP is not significantly modified by the insertions while the 216 chimeras seem more dynamic than the 197 chimeras. [less ▲]

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See detailSwitching to the Dark Side: Repositioning of Polyglutamine Repeat Promotes Amyloid Fibril Formation by the Model Protein, β-Lactamase BlaP
Thorn, David ULg; Pain, Coralie ULg; Scarafone, Natacha et al

Conference (2013, November 20)

Background: The expansion of polyglutamine (polyQ) repeats is associated with an increased propensity of the protein to aggregate into amyloid fibrils. There are ten human proteins presently known within ... [more ▼]

Background: The expansion of polyglutamine (polyQ) repeats is associated with an increased propensity of the protein to aggregate into amyloid fibrils. There are ten human proteins presently known within which polyQ expansion above a threshold length, e.g. 35-50 residues, leads to ten distinct neurodegenerative disorders [1], the most well-known being Huntington’s disease. While repeat length, aggregation, and disease are well correlated, recent studies suggest the non-polyQ regions of these proteins can also play a significant role, both preventative and facilitative, in the aggregation process. With the aim of exploring this role in more detail, we have engineered chimeric proteins via the insertion of polyQ repeats of various length (23, 30, 55, 79 Q) into two sites of antibiotic resistance enzyme BlaP β-lactamase from Bacillus licheniformis 749/C [2]. Questions addressed: How does polyQ repeat position affect the structure, stability and aggregation of polyQ proteins? Methods: Aggregation kinetics determined by monitoring the decrease in soluble protein fraction over time. Aggregate morphology examined by transmission electron microscopy. Protein stability derived from thermal or chemical unfolding transitions monitored by far-UV CD and intrinsic fluorescence. Results and discussion: PolyQ insertion at either of the two positions led to a decrease in thermodynamic stability that was largely independent of polyQ length. Chimeras with polyQ insertions at position 216 were destabilised to a much greater extent than those with insertions at position 197. The reduced stability of the 216 chimeras was associated with an increased aggregation propensity: (i) the minimum polyQ length leading to aggregation was lower, and (ii) the aggregation rate was significantly higher than that observed by 197 chimeras with equivalent polyQ lengths. Remarkably, the two sites of polyQ insertion are indeed very similar, both residing within flexible loop regions between stable α-helices. Moreover, the 216 chimeras exhibited a higher aggregation propensity than their 197 counterparts even under denaturing conditions, suggesting the disparity between the two chimeras cannot be accounted for by structural differences alone. These findings highlight the strong and complex influence of the overall protein context on polyQ-mediated aggregation. The molecular basis for the observed changes in stability and aggregation propensity is the subject of on-going work. [less ▲]

Detailed reference viewed: 31 (8 ULg)
See detailGeneration of camelid single-domain antibody fragments raised against proteins containing polyglutamine expansions
Pain, Coralie ULg; Scarafone, Natacha; Jaspar, Aurélie et al

Poster (2010, October 14)

Nine progressive neurodegenerative diseases are associated with the expansion of a polyglutamine (polyQ) tract above a threshold size (~ 35-45 residues) into nine different proteins [1]. These proteins ... [more ▼]

Nine progressive neurodegenerative diseases are associated with the expansion of a polyglutamine (polyQ) tract above a threshold size (~ 35-45 residues) into nine different proteins [1]. These proteins with expanded polyQ repeats have been found to form intranuclear amyloid-like aggregates, and the formation of these aggregates could play an important role in the pathogenesis [2-4]. The polyQ expansion is the only common feature among the proteins involved, suggesting it may be responsible for the aggregation phenomenon. Understanding the molecular mechanism by which the polyQ expansions promote aggregation is therefore crucial for the development of therapeutic strategies. The nine proteins associated with polyQ diseases are difficult to express recombinantly due to their big size and/or their insoluble character. In order to get further insights into the mechanism by which polyQ tracts promote aggregation, we have therefore decided to insert polyQ sequences into a well studied protein, the b-lactamase BlaP from B. licheniformis [5-6]. We have created chimeras containing 23, 30, 55, and 79 glutamines and we have investigated the effects of the insertions on the activity, the structure, the stability of BlaP as well as on its aggregating properties. Preliminary results indicate that BlaP is a good framework to study the molecular mechanism of aggregation associated with expanded polyglutamine tracts. On another hand, our previous work on the amyloidogenic variants of human lysozyme has shown that camelid single domain antibody fragments are very powerful structural probes to understand, at the molecular level, the mechanism of amyloid fibril formation [7]. Moreover, a recent study has suggested that expanded polyQ strectches adopt multiple conformations in solution that can be readily distinguished by monoclonal antibodies [8]. Altogether these results have encouraged us to generate VHHs against our different chimeras and we present here our preliminary results. References [1] Orr and Zoghbi (2007) Annu Rev Neurosci 30, 575-621. [2] DiFiglia et al. (1997) Science 277, 1990-1993. [3] Paulson HL (2000) Brain Pathol 10, 293-299. [4] Sanchez I. et al. (2003) Nature 421, 373-379. [5] Scarafone N. (2008) Mémoire de DEA en Sciences. Université de Liège. [6] Pain C. (2009) Mémoire de Master en Biochimie. Université de Liège. [7] Dumoulin et al. (2003) Nature 424, 783-788. [8] Legleiter J. et al. (2009) J Biol Chem 284, 21647-21648. [less ▲]

Detailed reference viewed: 15 (2 ULg)