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Biophysical study of the aggregation of the androgen receptor protein in spinal bulbar muscular atrophy
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[eng] Spinal bulbar muscular atrophy (SBMA) is a member of the polyglutamine (polyQ) expansion diseases family; the most famous of which is Huntington disease (HD). SBMA is caused by the expansion of the coding region for the polyQ tract in the exon 1 of androgen receptor (AR), which represents the N-terminal intrinsically disordered transactivation domain (NTD). AR is the nuclear receptor sensible to testosterone and aggregates of this polyQ-expanded protein are observed in the motor neurons of SBMA patients. The aggregation mechanism of polyQ proteins depends both on the length of the tract and on the chemical properties of the regions flanking it, that can increase or decrease the rate of aggregation depending on their secondary structure. In order to study the structure of the polyQ tract in AR and the mechanism by which this protein forms aggregates, we developed recombinant proteins designed over the N-terminal fragment of cleavage of a caspase (Caspase 3) associated to the onset of the toxicity in SBMA. We also developed a set of biophysical tools for rendering these aggregation-prone proteins monomeric and to monitor their evolution from the monomer level to the fibril. These methodologically challenging endeavor allowed us to study the secondary structure of this intrinsically disordered protein as a monomer and then to monitor what regions are important in its oligomerization and aggregation. Bulk biophysical experiments and NMR indicated that the polyQ tract of AR is in α-helical conformation, unlike other polyQ tracts described in literature, and we demonstrated that this conformation is caused by the nucleating effect of an N-terminal flanking sequence of four Leu residues (54LLLL58). We also showed that the helical conformation of this tract prevents the polyQ to acquire the ß-sheet conformation and to progress as a fibril, as a deletion mutant of the 54LLLL58 motif aggregates and forms fibrils faster than the wild type. By measuring the aggregation rates of three different AR recombinant proteins with progressively higher polyQ length (4Q, 25Q and 51Q) emerged that the polyQ is not the only region responsible for oligomerization and we identified by NMR that a second region, N- terminal and far apart from the polyQ is responsible of the early oligomerization. By analysis of the chemical shifts in different NMR experiments we obtained that this region (23FQNLF27) however not entirely helical, is prone to interact and acquire secondary structure. Furthermore, this sequence is known to bind to the ligand binding domain (LBD) of AR in an interaction critical for its dimerization and subsequent translocation into the nucleus, which is called N/C interaction. The crystal structure of this complex shows 23FQNLF27 in α-helical conformation when bound to LBD. We then investigated what amino-acids were important in the interaction stabilizing the intereaction of 23FQNLF27. By mutational analysis and measurements of aggregation rates we demonstrated that the helicity of this region is important for the aggregation and mutations that increase the helicity also an increase the aggregation propensity of the protein. We also identified that the residues responsible for the contact are the Gln in position 2, 28 and 36 which form a ‘spine’ of polar residues in register along the α-helix. This polar side of the helix is not the one in contact with LBD during the N/C interaction and it is possible that the two events occur in parallel. In the complex, we characterized the early oligomerization of AR in the aggregation process associated to SBMA with the perspective to provide valuable information for the development of drugs for this diseases that has currently no treatment.
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CHIESA, Giulio. Biophysical study of the aggregation of the androgen receptor protein in spinal bulbar muscular atrophy. [consulta: 15 de desembre de 2025]. [Disponible a: https://hdl.handle.net/2445/106882]