Role of Hydrophobicity at the N-Terminal Region of Aβ42 in Secondary Nucleation

Abstract

The self-assembly of the amyloid β 42 (Aβ42) peptide is linked to Alzheimer's disease, and oligomeric intermediates are linked to neuronal cell death during the pathology of the disease. These oligomers are produced prolifically during secondary nucleation, by which the aggregation of monomers is catalyzed on fibril surfaces. Significant progress has been made in understanding the aggregation mechanism of Aβ42; still, a detailed molecular-level understanding of secondary nucleation is lacking. Here, we explore the role of four hydrophobic residues on the unstructured N-terminal region of Aβ42 in secondary nucleation. We create eight mutants with single substitutions at one of the four positions�Ala2, Phe4, Tyr10, and Val12�to decrease the hydrophobicity at respective positions (A2T, A2S, F4A, F4S, Y10A, Y10S, V12A, and V12S) and one mutant (Y10F) to remove the polar nature of Tyr10. Kinetic analyses of aggregation data reveal that the hydrophobicity at the Nterminal region of Aβ42, especially at positions 10 and 12, affects the rate of fibril mass generated via secondary nucleation. Cryoelectron micrographs reveal that most of the mutants with lower hydrophobicity form fibrils that are markedly longer than WT Aβ42, in line with the reduced secondary nucleation rates for these peptides. The dominance of secondary nucleation, however, is still retained in the aggregation mechanism of these mutants because the rate of primary nucleation is even more reduced. This highlights that secondary nucleation is a general phenomenon that is not dependent on any one particular feature of the peptide and is rather robust to sequence perturbations.

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“…Despite amyloid proteins being self-assembly units that catalyze their aggregation, the complexity of Aβ aggregation makes it challenging to target with antibodies and molecular receptors, thus redirecting the peptide away from the fibrillation process and necessitating alternative treatment strategies. Recent efforts have focused on creating mutants of pathological Aβ structures to inhibit kinetically amyloid aggregation [95]. However, introducing new antigens may lead to undesirable off-target effects.…”

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“…Developing antibodies for Aβ40 or Aβ42 depends on the specificity of these forms, with Aβ42 being more hydrophobic and primarily responsible for aggregates [ 96 ]—specific and compelling reduction of these aggregates if an antibody is generated to target Aβ42 specifically. However, the protein translated is not the same as Aβ42, leading to adverse effects when the same approach is applied.…”

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“…In recent cryo-EM hIAPP fibril structures, − a significant amount of the hIAPP sequence (residues 1–12) is not resolved in the electron density maps, presumably because of extensive polymorphism. Alternatively, the N-terminus of hIAPP is considered to be dynamic and flexible. , The relevance of the semirigid solvent-facing residues outside the fibril core has been demonstrated for different amyloid-forming proteins. − These regions have been shown to play key parts in the interaction of amyloid fibrils with other cellular components such as RNA molecules, cell membrane binding/disruption, and molecular chaperones . Furthermore, terminal domains of amyloidogenic proteins have been reported to modulate the aggregation mechanism and toxicity. ,− hIAPP fibril assembly is largely controlled by the disulfide bond involving Cys2 and Cys7 at the N-terminus .…”