Charge neutralization of lysine via carbamylation reveals hidden aggregation hot?spots in tau protein flanking regions

Abstract

Tau protein is found abundantly in neurofibrillary tangles in Alzheimer�s disease. The longest human tau isoform (2N4R) has 44 lysine residues. Several lysine-based post-translational modifications (PTMs) such as glycation, acetylation, ubiquitination, and sumoylation have been implicated not only in Alzheimer's disease but also in other tauopathies. Carbamylation is one such lysine neutralizing age-related non-enzymatic PTM which can modulate the aggregation propensity of tau. In the present work, we have studied the aggregation potential of lysine-rich regions of tau upon carbamylation which do not aggregate in their native form. Using an array of biophysical and microscopic analyses such as ThT kinetic assay, fluorescence microscopy, Congo-red staining, and scanning electron microscopy we demonstrate that peptides derived from 4 out of 5 such regions exhibit robust fibrillar amyloid formation. These regions are found in the N-terminal projection domain that encompasses proline-rich domain (148-153, and 223-230), repeat domain R1 (253-260), as well as fibrillary core region (368-378) and, can be described as hidden aggregation hot spots which become activated upon carbamylation. We have further compared the impact of carbamylation with acetylation on the aggregation propensity of lysine-rich peptide (254KKVAVV259) using biophysical experiments and MD simulations and deduced that carbamylation is a much stronger driver of aggregation than acetylation. Our findings may offer more insight into amyloid fibrils' interaction with hidden aggregation-prone nucleating sequences that act as hotspots for inducing tau fibrillation.