Huntingtin

Huntingtin (Htt) is the protein coded for in humans by the HTT gene, also known as the IT15 ("interesting transcript 15") gene. Mutated HTT is the cause of Huntington's disease (HD), and has been investigated for this role and also for its involvement in long-term memory storage. It is variable in its structure, as the many polymorphisms of the gene can lead to variable numbers of glutamine residues present in the protein. In its wild-type (normal) form, the polymorphic locus contains 6-35 glutamine residues. However, in individuals affected by Huntington's disease (an autosomal dominant genetic disorder), the polymorphic locus contains more than 36 glutamine residues (highest reported repeat length is about 250). Its commonly used name is derived from this disease; previously, the IT15 label was commonly used. The mass of huntingtin protein is dependent largely on the number of glutamine residues it has; the predicted mass is around 350 kDa. Normal huntingtin is generally acce

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

Sophie Vieweg

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 Î²-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 Î²-turn structures, whereas fibrils formed by Nt17-truncated Httex1 (âNt17-Httex1) contain antiparallel Î²-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 âNt17-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, âNt17-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 (âNt17, 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.

EPFL2017

A New Chemoenzymatic Semisynthetic Approach Provides Insight into the Role of Phosphorylation beyond Exon1 of Huntingtin and Reveals N-Terminal Fragment Length-Dependent Distinct Mechanisms of Aggregation

Rajasekhar Kolla, Hilal Lashuel, Gopinath Pushparathinam, Andreas Reif, Jonathan Jean-Pierre Ricci, Iman Rostami

Huntington’s disease is a neurodegenerative dis- order caused by the expansion of a polyglutamine repeat (>36Q) in the N-terminal domain of the huntingtin protein (Htt), which renders the protein or fragments thereof more prone to aggregate and form inclusions. Although several Htt N-terminal fragments of different lengths have been identified within Htt inclusions, most studies on the mechanisms, sequence, and structural determinants of Htt aggregation have focused on the Httexon1 (Httex1). Herein, we investigated the aggregation properties of mutant N- terminal Htt fragments of various lengths (Htt171, Htt140, and Htt104) in comparison to mutant Httex1 (mHttex1). We also present a new chemoenzymatic semisynthetic strategy that enables site-specific phosphorylation of Htt beyond Httex1. These advances yielded insights into how post-translational modifications (PTMs) and structured domains beyond Httex1 influence aggregation mechanisms, kinetics, and fibril morphology of longer N-terminal Htt fragments. We demonstrate that phosphorylation at T107 significantly slows the aggregation of mHtt171, whereas phosphorylation at T107 and S116 accelerates the aggregation, underscoring the importance of crosstalk between different PTMs. The mHtt171 proteins aggregate via a different mechanism and form oligomers and fibrillar aggregates with morphological properties that are distinct from that of mHttex1. These observations suggest that different N-terminal fragments could have distinct aggregation mechanisms and that a single polyQ-targeting antiaggregation strategy may not effectively inhibit the aggregation of all N-terminal Htt fragments. Finally, our results underscore the need for further studies to investigate the aggregation mechanisms of Htt fragments and how the various fragments interact with each other and influence Htt toxicity and disease progression.

2021

Rajasekhar Kolla, Hilal Lashuel, Gopinath Pushparathinam, Andreas Reif, Jonathan Jean-Pierre Ricci, Iman Rostami

2021

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

Sophie Vieweg

EPFL2017

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

A New Chemoenzymatic Semisynthetic Approach Provides Insight into the Role of Phosphorylation beyond Exon1 of Huntingtin and Reveals N-Terminal Fragment Length-Dependent Distinct Mechanisms of Aggregation

Elucidating the role of N-terminal phosphorylation in regulating the structure and the aggregation propensities of Huntingtin exon 1 using a semisynthetic strategy

A New Chemoenzymatic Semisynthetic Approach Provides Insight into the Role of Phosphorylation beyond Exon1 of Huntingtin and Reveals N-Terminal Fragment Length-Dependent Distinct Mechanisms of Aggregation

Elucidating the role of N-terminal phosphorylation in regulating the structure and the aggregation propensities of Huntingtin exon 1 using a semisynthetic strategy

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