Lysine residues in the N-terminal huntingtin amphipathicα-helix play a key role in peptide aggregation

Abstract

Huntington's disease is a genetic neurodegenerative disorder caused by an expansion in a polyglutamine domain near the N-terminus of the huntingtin (htt) protein that results in the formation of protein aggregates. Here, htt aggregate structure has been examined using hydrogen-deuterium exchange techniques coupled with tandem mass spectrometry. The focus of the study is on the 17-residue N-terminal flanking region of the peptide that has been shown to alter htt aggregation kinetics and morphology. A top-down sequencing strategy employing electron transfer dissociation is utilized to determine the location of accessible and protected hydrogens. In these experiments, peptides aggregate in a deuterium-rich solvent at neutral pH and are subsequently subjected to deuterium-hydrogen back-exchange followed by rapid quenching, disaggregation, and tandem mass spectrometry analysis. Electrospray ionization of the peptide solution produces the [M + 5H](5+) to [M + 10H](10+) charge states and reveals the presence of multiple peptide sequences differing by single glutamine residues. The [M + 7H](7+) to [M + 9](9+) charge states corresponding to the full peptide are used in the electron transfer dissociation analyses. Evidence for protected residues is observed in the 17-residue N-terminal tract and specifically points to lysine residues as potentially playing a significant role in htt aggregation.

Rapid synthesis of cyclic oligomeric depsipeptides with positional, stereochemical, and macrocycle size distribution control

“…These salt-free conditions led to N-H bassianolide but in only 11% yield. In their synthesis of N-H bassianolide, Suzuki and coworkers attributed the low yield (2.8%) of their macrolactamization (PCl 5 and Et 3 N) to 3 10 -helical intramolecular hydrogen bonding (57,58) in their amino acid octadepsipeptide precursor (59). As a substrate for Mitsunobu esterification, 20 is a more hindered alcohol than 13.…”

Huntingtin N-Terminal Monomeric and Multimeric Structures Destabilized by Covalent Modification of Heteroatomic Residues

“…In Structure 1, residue Lys6 is in relative proximity to Ser16. Interestingly, Lys6 is potentially involved in intermolecular interaction from an early aggregate state (52). In solution, such proximity could lead to charge-dipole interactions between the ε-amino group on Lys6 and the primary alcohol group on Ser16.…”

“…Previous solution docking simulations have alluded to a stabilizing Lys6-Ser16 interaction in antiparallel Nt17 helices (10). Additionally, solution-based deuterium exchange measurements revealed an increased role of Lys6 in Nt17 multimer structure (52). Finally, S13D and S16D mutations in Nt17 prevented aggregation of huntingtin exon 1 synthetic models via the Nt17-mediated pathway (14).…”

“…Previous work highlights the importance of Lys6 and its protection from an aggregated state in a huntingtin exon 1 model, indicating that Lys6 is involved in a stabilizing intra- or intermolecular interaction (52). In previous studies, which utilized top-down deuterium exchange techniques, it was not possible to determine the exact origin of monomeric peptide.…”

“…DEPC targets alcohol and amino groups, such as those found in His, Thr, Lys, and Ser. The Lys6 and Lys15 residues in Nt17 are, at least partially, solvent-inaccessible from an early aggregate state (52), which suggests the presence of an intermolecular side chain interaction. Modification at one of these residues could destabilize oligomerization.…”