The formation of amyloid β (Aβ) fibrils is crucial in initiating the cascade of pathological events that culminates in Alzheimer's disease. In this study, we investigated the mechanism of Aβ fibril formation from hydrodynamically well defined species under controlled aggregation conditions. We present a detailed mechanistic model that furnishes a novel insight into the process of Aβ42 fibril formation and the molecular basis for the different structural transitions in the amyloid pathway. Our data reveal the structure and polymorphism of Aβ fibrils to be critically influenced by the oligomeric state of the starting materials, the ratio of monomeric-to-aggregated forms of Aβ42 (oligomers and protofibrils), and the occurrence of secondary nucleation. We demonstrate that monomeric Aβ42 plays an important role in mediating structural transitions in the amyloid pathway, and for the first time, we provide evidences that Aβ42 fibrillization occurs via a combined mechanism of nucleated polymerization and secondary nucleation. These findings will have significant implications to our understanding of the molecular basis of amyloid formation in vivo, of the heterogeneity of Aβ pathology (e.g., diffuse versus amyloid plaques), and of the structural basis of Aβ toxicity.

Herein, we used protein semisynthesis to investigate, for the first time, the effect of lysine acetylation and phosphorylation, as well as the crosstalk between these modifications on the structure and aggregation of mutant huntingtin exon1 (Httex1). Our results demonstrate that phosphorylation at T3 stabilizes the α-helical conformation of the N-terminal 17 amino acids (Nt17) and significantly inhibits the aggregation of mutant Httex1. Acetylation of single lysine residues, K6, K9 or K15, had no effect on Httex1 aggregation. Interestingly, acetylation at K6, but not at K9 or K15, reversed the inhibitory effect of T3 phosphorylation. Together, our results provide novel insight into the role of Nt17 post-translational modifications in regulating the structure and aggregation of Httex1 and suggest that its aggregation and possibly its function(s) are controlled by regulatory mechanisms involving crosstalk between different PTMs.

The natural enzymes involved in regulating many of the posttranslational modifications (PTMs) within the first 17 residues (Nt17) of Huntingtin exon 1 (Httex1) remain unknown. A semisynthetic strategy that allows the site-specific introduction of PTMs within Nt17 by using expressed protein ligation (EPL) was developed. This strategy was used to produce untagged wild-type (wt) and T3-phosphorylated (pT3) Httex1 containing 23 glutamine residues (Httex1-23Q). Our studies show that pT3 significantly slows the oligomerization and fibrillization of Httex1-23Q and that Httex1 variants containing polyQ repeats below the pathogenic threshold readily aggregate and form fibrils in vitro. These findings suggest that crossing the polyQ pathogenic threshold is not essential for Httex1 aggregation. The ability to produce wt or site-specifically modified tag-free Httex1 should facilitate determining its structure and the role of N-terminal PTMs in regulating the functions of Htt in health and disease.

No abstract

The first exon of the Huntingtin protein (Httex1) is one of the most actively studied Htt fragments because its overexpression in R6/2 transgenic mice has been shown to recapitulate several key features of Huntington disease. However, the majority of biophysical studies of Httex1 are based on assessing the structure and aggregation of fusion constructs where Httex1 is fused to large proteins, such as glutathione S-transferase, maltosebinding protein, or thioredoxin, or released in solution upon in situ cleavage of these proteins. Herein, we report an inteinbased strategy that allows, for the first time, the rapid and efficient production of native tag-free Httex1 with polyQ repeats ranging from 7Q to 49Q. Aggregation studies on these proteins enabled us to identify interesting polyQ-length-dependent effects on Httex1 oligomer and fibril formation that were previously not observed using Httex1 fusion proteins or Httex1 proteins produced by in situ cleavage of fusion proteins. Our studies revealed the inability of Httex1-7Q/15Q to undergo amyloid fibril formation and an inverse correlation between fibril length and polyQ repeat length, suggesting possible polyQ length-dependent differences in the structural properties of the Httex1 aggregates. Altogether, our findings underscore the importance of working with tag-free Httex1 proteins and indicate that model systems based on non-native Httex1 sequences may not accurately reproduce the effect of polyQ repeat length and solution conditions on Httex1 aggregation kinetics and structural properties. Huntington disease (HD)2 is a fatal neurodegenerative disorder caused by a CAG expansion within the first exon (Exon 1) of the huntingtin gene, IT15 (1). The CAG repeat length ranges between 6 and 35 in healthy subjects, whereas patients with HD exhibit lengths of 36 or greater resulting in the synthesis of a mutant Huntingtin protein (Htt) with an expanded polyglutamine (polyQ) domain (2). The length of the polyQ tract directly correlates with disease severity and is inversely correlated with disease age of onset (3). HD patients suffer from motor impairments, cognitive decline, and depression due to neurodegeneration in the striatum and cortex (4 -8).The accumulation of N-terminal fragments of mutant Htt in the nucleus and cytoplasm of striatal neurons led to the hypothesis that these fragments play causative roles in the neurodegeneration and pathology observed in HD (9 -13). This hypothesis is supported by the observation that the overexpression of the expanded Exon 1 in R6/2 transgenic mice recapitulates several key symptoms of HD (14). Subsequent studies demonstrated that aberrant splicing in HttQ150 knock-in mice gave rise to a short mRNA, which translates into the Huntingtin Exon 1 protein (Httex1), thus linking the genetic cause of HD to the generation of a highly toxic N-terminal Htt fragment (15). Together, these findings highlight the importance of this region in HD pathogenesis and underscore the critical importance of investigating the structure, aggregation, a...