The role of the polyglutamine and N-terminal domains in regulating the aggregation and structural properties of Huntingtin Exon 1

Huntington Disease (HD) is caused by a CAG repeat expansion in the huntingtin gene leading to the formation of mutant Huntingtin protein (Htt) with an expanded polyglutamine (polyQ) domain (>36Q). Generation of short N-terminal Htt fragments by proteolysis plays a key role in HD pathogenesis and Huntingtin exon1 (Httex1) is one of the most used models to study Htt aggregation in vitro. We developed a new expression and purification strategy to produce tag-free Httex1 and Httex1 fragments with different polyQ-lengths from E.coli for biophysical studies and for optimizing protein semisynthesis in our laboratory. Having these proteins in hand we sought to explore the effect of the polyQ and Nt17 domain on Httex1 aggregation and structure, given their essential role in mediating intra- and intermolecular interactions in Htt. We found that polyQ-length inversely correlates with fibril length and that expanded polyQ tracts increase the β-sheet content modulating the roughness and mechanical properties of Httex1 aggregates. This suggests that polyQ-dependent structural differences exist in aggregates formed by wildtype and mutant Httex1. Moreover, using infrared nanospectroscopy we were able to show that Httex1 aggregates are characterized by β-turn structures, whereas fibrils formed by Nt17-truncated Httex1 (∆Nt17-Httex1) contain antiparallel β-sheets, indicating that the NT17 domain influences secondary structure within the polyQ domain. The Nt17 effect on Httex1 structure translates into morphological differences between fibrils formed by Httex1 or ∆Nt17-Httex1 as discerned by high-resolution imaging techniques. The presence of the Nt17 domain induces the formation of smooth and regular amyloid-like fibrils, while in absence of the Nt17 domain irregular and broad fibrils are formed due to strong lateral interaction between fibril filaments. Introduction of a helix-breaking proline into the Nt17 domain (M8P-Httex1) revealed that the Nt17 effect on the aggregate morphology is independent of its conformation. Interestingly, we were able to show that the Nt17 domain is not tightly bound within the fibril core of Httex1 fibrils and able to interact with membranes by performing trypsin digests of Httex1 fibrils and comparing the internalization of Httex1, ∆Nt17-Httex1 and M8P-Httex1 fibrils into primary striatal neurons. The Nt17 domain harbors many post-translational modifications, e.g. phosphorylation, SUMOylation, ubiquitination and acetylation, which modulate Htt aggregation and toxicity. Therefore, we aimed at elucidating the effect of SUMOylation on the aggregation and structural properties of Httex1. After production of site-specifically SUMO1-modified Httex1 by semisynthesis, we observed that SUMO1-modification at K6 or K9 increased the compaction and stability of Httex1 inhibiting aggregation. The developed purification strategy enabled an efficient generation of the native Httex1 sequence. Using unmodified and modified (∆Nt17, M8P, SUMO1) Httex1 variants with varying polyQ lengths we were able to provide novel insights into how the polyQ and Nt17 domains regulate aggregate formation and influence the final aggregate structure. Our results contribute to a better understanding of the molecular determinants of Htt aggregate structure and could facilitate the development of inhibitors or biomarkers of Htt aggregation in vivo.