Mutant Huntingtin N-terminal Fragments of Specific Size Mediate Aggregation and Toxicity in Neuronal Cells

Abstract

Huntingtin proteolysis is implicated in Huntington diseasepathogenesis, yet, the nature of huntingtin toxic fragments remains unclear. Huntingtin undergoes proteolysis by calpains and caspases within an N-terminal region between amino acids 460 and 600. We have focused on proteolytic steps producing shorter N-terminal fragments, which we term cp-1 and cp-2 (distinct from previously described cp-A/cp-B). We used HEK293 cells to express the first 511 residues of huntingtin and further define the cp-1 and cp-2 cleavage sites. Based on epitope mapping with huntingtin-specific antibodies, we found that cp-1 cleavage occurs between residues 81 and 129 of huntingtin. Affinity and size exclusion chromatography were used to further purify huntingtin cleavage products and enrich for the cp-1/ cp-2 fragments. Using mass spectrometry, we found that the cp-2 fragment is generated by cleavage of huntingtin at position Arg 167 . This site was confirmed by deletion analysis and specific detection with a custom-generated cp-2 site neo-epitope antibody. Furthermore, alterations of this cleavage site resulted in a decrease in toxicity and an increase in aggregation of huntingtin in neuronal cells. These data suggest that cleavage of huntingtin at residue Arg 167 may mediate mutant huntingtin toxicity in Huntington disease.

Translation of HTT mRNA with expanded CAG repeats is regulated by the MID1–PP2A protein complex

“…The expanded polyQ stretch leads to an increased cleavage of mutant HTT by caspases and calpains, and results in an accumulation of toxic N-terminal HTT-fragments, which form aggregates in HD brains [41][42][43][44][45] . These fragments get degraded via the ubiquitin-proteasome pathway and by autophagy.…”

Huntingtin protein interactions altered by polyglutamine expansion as determined by quantitative proteomic analysis

“…The N-terminal tag was chosen, because the N586 fragment of Htt is likely to undergo further N-terminal proteolysis. 29,30 Short N-terminal fragments of mutant Htt are believed to mediate its toxicity, possibly through aberrant protein interactions. By using a C-terminal tag, such interactions may be overlooked.…”

Aggregation landscapes of Huntingtin exon 1 protein fragments and the critical repeat length for the onset of Huntington’s disease

“…Using the associative memory, water-mediated interactions, structure and energy model (AWSEM), previous simulations by our group have successfully explained how the change of critical nucleus size arises from the differences in the propensity of monomeric polyQ repeats of different lengths to form β-hairpins: the longer repeats fold into hairpins intramolecularly before they aggregate (9). The aggregation of the diseasecausing peptide is, however, further complicated by the presence of flanking amino acid sequences in fragments encoded by HTT exon 1. Experiments indicate that the addition of NT17 at the N terminus of polyQ enormously accelerates the aggregation, probably by encouraging the formation of prefibrillar oligomers (10)(11)(12)(13)(14)(15)(16), whereas the addition of the proline-rich region at the C terminus decreases the rate of aggregation apparently without changing fundamentally the mechanism (10,16,17). Structural characterization of the aggregates (13,14,(18)(19)(20) has shown that, even when there are flanking sequences, polyQ remains the fiber core and adopts a β-hairpin conformation.…”