Among neurodegenerative diseases causing serious medical and social problems, in particular in association with an aging population, Huntington´s disease (HD) is classified as a representative one of nine polyglutamine (polyQ) tract disease families. The polyQ diseases cause dominantly inherited neurodegenerative disorders that typically manifests itself during midlife with motor, psychiatric and cognitive symptoms leading to death in 15-20 years after onset (Fig. 1). They evolve from an expansion of the CAG repeat tract in single responsible genes.

HD results from polyQ expansion at the N-terminal in the huntingtin protein (3,144 amino acid protein). The longer the repeat is, the earlier the onset of disease. A metastudy showed that the prevalence of HD in Europe, North America and Australia was 5.7 per 100 000¹.

Genetic studies showed that huntingtin knock-out cause embryonic lethality implicating the important role of huntingtin during development². Despite that huntingtin was identified to be responsible for HD two decades ago, little is known about its biological function nor the molecular mechanism of the pathogenesis. Even if the polyQ expansion in huntingtin occurs close to the N-terminus it is imperative to investigate the function of huntingtin in full-length context as the expansion is likely to modulate activities of the full-length protein. Huntingtin has several HEAT motifs without any known functional domain. Therefore, huntingtin was predicted to adopt an extended shape as shown in other HEAT repeat proteins.

For the present structural studies, allelic series of huntingtin with various polyQ lengths were successfully expressed and purified. We have obtained the overall structure of full-length huntingtin having 23 (Q23) and 78 (Q78) glutamines at medium-resolution by negatively-stained single-particle electron microscopy (EM) showing the protein adopts a spherical shape³ (Fig. 2). CD spectra were consistent with a predominant α-helical secondary structure. The position of the polyQ region could be mapped onto the structure following antibody-bound amino-terminal FLAG-tags of the Q23- and Q78-huntingtins. Furthermore, cross-linking mass spectrometry analysis revealed a modulated network of intramolecular contacts that together with the EM data suggests that huntingtin is composed of five structural domains. Thus, in a large size protein like huntingtin, HEAT repeat domains can be folded back to form a closed helical solenoid with functional sites in the internal cavity. The difference in structures of the EM maps of Q23 and Q78 huntingtin and between cross-linking patterns of the two species suggest modulation of the overall structure with increasing polyQ lengths.

1. Pringsheim, T. et al. (2012) Mov Disord. 27, 1083-91.
2. Nasir, J. et al. (1995) Cell 81, 811–823.
3. Vijayvargia, R. et al. (2016) eLife, Mar 22;5. pii: e11184. doi: 10.7554/eLife.11184.

Figures:

Fig. 1 Magnetic resonance image scans of a brain from an individual with Huntington's disease (HD) (left) and a normal brain (right). In the HD brain, many brain cells have died and fluid (black areas) has filled in those areas. In the normal brain, those same areas are occupied by brain cells. Adapted from: _x000D_http://ucsdnews.ucsd.edu/pressrelease/uc_san_diego_health_system_named_center_of_excellence_for_huntingtons_disea_x000D_

Fig. 2 Cross-eyed stereo-representation of the 3D maps of huntingtin with Q78 (upper) and Q23 (lower) polyglutamine extensions at the amino-terminals. The protein with a molecular mass of 348 kDa measures approximately 130 x 100 Å. Full-length FLAG-tag huntingtins were applied to ultracentrifugation at 74,329 xg for 16 hr with a 5-20% sucrose gradient in presence of a 0-0.2% glutaraldehyde gradient. Monomeric protein fractions were negatively-stained with 2% (w/v) uranyl acetate for 2 min on 400 mesh carbon grids.

« Back to The 16th European Microscopy Congress 2016

EMC Abstracts - https://emc-proceedings.com/abstract/three-dimensional-maps-of-full-length-huntingtin-and-linkage-to-development-of-huntingtons-disease/