Formation and Structure of Wild Type Huntingtin Exon-1 Fibrils

Abstract

The fact that the heritable neurodegenerative disorder Huntington’s Disease (HD) is autosomal dominant means that there is one wild type and one mutant allele in most HD patients. The CAG repeat expansion in the exon 1 of the protein huntingtin (HTTex1) that causes the disease, leads to the formation of HTT fibrils in vitro and vivo. An important question for understanding the molecular mechanism of HD is which role wild type HTT plays for the formation, propagation, and structure of these HTT fibrils. Here we report that fibrils of mutant HTTex1 are able to seed the aggregation of wild type HTTex1 into amyloid fibrils which in turn can seed the fibril formation of mutant HTTex1. Solid-state NMR and EPR data showed that wild type HTTex1 fibrils closely resemble the structure of mutant fibrils, with small differences indicating a less extended fibril core. These data suggest that wild type fibrils can faithfully perpetuate the structure of mutant fibrils in HD. However, wild type HTTex1 monomers have a much higher equilibrium solubility compared to mutant HTTex1 and only a small fraction incorporates into fibrils.

Huntingtin fibrils with different toxicity, structure, and seeding potential can be reversibly interconverted

“…The N17 was found to be ordered and in agreement with α-helical structure determined by solid-state NMR (Sivanandam et al, 2011). Different circular dichroism (CD) and infra-red (IR) spectral features suggested structural differences between the fibril types of different toxicity (Nekooki-Machida et al, 2009), but in contrast to the structural information available for the fibrils grown at 4°C and room temperature, (Bugg et al, 2012;Hoop et al, 2016;Isas et al, 2017Isas et al, , 2015Lin et al, 2017;Schneider et al, 2011;Sivanandam et al, 2011), little is known about the less toxic fibrils grown at 37°C. Knowing the structural features that modulate toxicity could be important for structure-based therapeutic strategies.…”

“…HTTex1(Q46) was recombinantly expressed as a thioredoxin fusion (Trx-HTTex1) using a pET32a vector in BL21 (DE3) E.coli. The expression and purification of uniformly 15 N-13 C labeled HTTex1(Q46) for NMR measurements and non-isotope labeled Cys mutants for EPR measurements was performed using His60 resin (Clontech) and a subsequent HiTrap Q XL anion exchange chromatography column (GE Healthcare) on an AKTA FPLC system (Amersham Pharmacia Biotech) as previously described (Bugg et al, 2012;Isas et al, 2017).…”

“…HTTex1 seeds were prepared as described previously. (Isas et al, 2017) In short, seed formation was initiated by removing the Trx fusion tag using 1 unit EKMax (Invitrogen) per 280 μg of HTTex1. The mixture was kept at 4°C for several days and sonicated.…”

Insights into protein misfolding and aggregation enabled by solid-state NMR spectroscopy

“…Like other ssNMR-studied amyloids discussed above, the HttEx1 fibrils also have a highly ordered amyloid core that is decorated with exposed non-amyloid flanking regions (with α- and PPII-helical as well as random coil regions; Fig. 5c–e) [87, 91–93]. Delineation of the fibrils’ domain architecture was enabled in large part by ssNMR measurements of the domains’ relative dynamics probed by ssNMR measurements of relaxation rates and dipolar order parameters (Fig.…”

“…In the case of HD it is the huntingtin protein that is mutated, with its polyglutamine domain present within the first exon of the protein. The last five years have seen a number of ssNMR studies of aggregated polyglutamine, both in isolation and in context of the huntingtin exon 1 (HttEx1) [20, 87, 89–93]. HttEx1 is seen as an essential disease-relevant huntingtin N-terminal fragment, as it is observed in patients and can cause HD-like disease in model animals.…”