The 17-residue-long N terminus in huntingtin controls stepwise aggregation in solution and on membranes via different mechanisms

Abstract

Aggregation of huntingtin protein arising from expanded polyglutamine (polyQ) sequences in the exon-1 region of mutant huntingtin plays a central role in the pathogenesis of Huntington's disease. The huntingtin aggregation pathways are of therapeutic and diagnostic interest, but obtaining critical information from the physiologically relevant htt exon-1 (Httex1) protein has been challenging. Using biophysical techniques and an expression and purification protocol that generates clean, monomeric Httex1, we identified and mapped three distinct aggregation pathways: 1) unseeded in solution; 2) seeded in solution; and 3) membrane-mediated. In solution, aggregation proceeded in a highly stepwise manner, in which the individual domains (N terminus containing 17 amino acids (N17), polyQ, and proline-rich domain (PRD)) become ordered at very different rates. The aggregation was initiated by an early oligomer requiring a pathogenic, expanded Gln length and N17 α-helix formation. In the second phase, β-sheet forms in the polyQ. The slowest step is the final structural maturation of the PRD. This stepwise mechanism could be bypassed by seeding, which potently accelerated aggregation and was a prerequisite for prion-like spreading Remarkably, membranes could catalyze aggregation even more potently than seeds, in a process that caused significant membrane damage. The N17 governed membrane-mediated aggregation by anchoring Httex1 to the membrane, enhancing local concentration and promoting collision via two-dimensional diffusion. Considering its central roles in solution and in membrane-mediated aggregation, the N17 represents an attractive target for inhibiting multiple pathways. Our approach should help evaluate such inhibitors and identify diagnostic markers for the misfolded forms identified here.

Huntingtin fibrils with different toxicity, structure, and seeding potential can be reversibly interconverted

“…HTTex1 consists of three separate domains of which the N17 and the polyQ are typically considered to promote aggregation Pandey et al, 2018), whereas the PRD is sometimes considered to inhibit aggregation (Crick et al, 2013;Darnell et al, 2007).…”

Insights into the molecular mechanism behind solubilization of amyloidogenic polyglutamine‐containing peptides

“…However, recent investigations suggest that it is the oligomeric prefibrillar species on the path of aggregation that renders high cytotoxicity to cells . Growing evidences further showed the ability of amyloid peptides to self‐assemble into α‐helical‐rich oligomers wherein the nucleation of amyloid‐like aggregate structures is observed on binding to the lipid membrane mimetics . Formation of such intermediate structures was found to contribute to the membrane permeation/disruption and membrane‐mediated cell death .…”

“…Growing evidences further showed the ability of amyloid peptides to self‐assemble into α‐helical‐rich oligomers wherein the nucleation of amyloid‐like aggregate structures is observed on binding to the lipid membrane mimetics . Formation of such intermediate structures was found to contribute to the membrane permeation/disruption and membrane‐mediated cell death . Notably, the formation of α‐helical‐rich oligomeric intermediates was observed even in the absence of membranes during the aggregation of huntingtin N‐terminal sequences and α‐Synuclein …”

“…Formation of such intermediate structures was found to contribute to the membrane permeation/disruption and membrane‐mediated cell death . Notably, the formation of α‐helical‐rich oligomeric intermediates was observed even in the absence of membranes during the aggregation of huntingtin N‐terminal sequences and α‐Synuclein …”

N-terminal phosphorylation of Huntingtin: A molecular switch for regulating Htt aggregation, helical conformation, internalization and nuclear targeting

“…The Nt17 has been shown to adopt α-helical structure upon membrane binding (52) and act as an anchor for Httex1 to membranes and mediate its aggregation on membranes (53). To determine the effect of phosphorylation on Nt17 membrane binding properties, we assessed the effect of the phosphorylation at S13 and/or S16 and their corresponding phosphomimetic variants on the binding of the Nt17 peptide to phosphatidylglycerol (POPG) as a membrane model.…”