Title
The 17-residue-long N terminus in huntingtin controls step-wise aggregation in solution and on membranes via different mechanisms
Author
Nitin K. Pandey, J. Mario Isas, Anoop Rawat, Rachel V. Lee, Jennifer Langen, Priyatama Pandey, Ralf Langen
Year
2017
Journal
The Journal of Biological Chemistry
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 step-wise 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 Q 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 step-wise mechanism could be bypassed by seeding, which potently accelerated aggregation and was a prerequisite for prion-like spreading in vivo. 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.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Aggregation, Biochemistry