Synthesis of pathological and nonpathological human exon 1 huntingtin

Abstract

Huntington's disease (HD) is a neurodegenerative disorder that affects approximately 1 in 10 000 individuals. The underlying gene mutation was identified as a CAG-triplet repeat expansion in the gene huntingtin. The CAG sequence codes for glutamine, and in HD, an expansion of the polyglutamine (poly-Q) stretch above 35 glutamine residues results in pathogenicity. It has been demonstrated in various animal models that only the expression of exon 1 huntingtin, a 67-amino acid-long polypeptide plus a variable poly-Q stretch, is sufficient to cause full HD-like pathology. Therefore, a deeper understanding of exon 1 huntingtin, its structure, aggregation mechanism and interaction with other proteins is crucial for a better understanding of the disease. Here, we describe the synthesis of a 109-amino acid-long exon 1 huntingtin peptide including a poly-Q stretch of 42 glutamines. This microwave-assisted solid phase peptide synthesis resulted in milligram amounts of peptide with high purity. We also synthesized a nonpathogenic version of exon 1 huntingtin (90-amino acid long including a poly-Q stretch of 23 glutamine residues) using the same strategy. In circular dichroism spectroscopy, both polypeptides showed weak alpha-helical properties with the longer peptide showing a higher helical degree. These model peptides have great potential for further biomedical analyses, e.g. for large-scale pre-screenings for aggregation inhibitors, further structural analyses as well as protein-protein interaction studies.

An Intein-based Strategy for the Production of Tag-free Huntingtin Exon 1 Proteins Enables New Insights into the Polyglutamine Dependence of Httex1 Aggregation and Fibril Formation

“…The recombinant tag-free Httex1 proteins produced by the intein strategy exhibited similar aggregation and structural properties compared with tag-free Httex1 produced by semisynthesis (50) or microwave-assisted solid-phase peptide synthesis (51,52). The Httex1 proteins derived from all three strategies have a predominantly disordered structure before aggregation and form virtually identical amyloid-like fibrils upon incubation at 37°C.…”

“…3). In an attempt to conduct a direct and thorough comparison of the synthetic and recombinant Httex1-43Q proteins, we sought to produce this protein using the synthetic protocol described by Singer and Sahoo et al (51,52). Despite extensive efforts to follow the published procedure and the use of different synthetic strategies and peptide synthesizers, we were not able to achieve the chemical synthesis of Httex1.…”

“…Singer et al (51) used total chemical synthesis to produce Httex1, whereas our group employed a semisynthetic strategy to produce unmodified and site-specifically phosphorylated Httex1 (50). However, both approaches demand special technical expertise and equipment that are not commonly accessible in many research groups.…”

Aggregation Behavior of Chemically Synthesized, Full-Length Huntingtin Exon1

“…49 The recent availability of chemically synthesized, full-length HTT exon1 peptides 53,73 has offered the possibility that, given sufficient yields of pure material, systematic in vitro studies can now be conducted on molecules more accurately resembling the HTT exon1 fragments generated in the cell. In this paper, we describe in detail characterization of the aggregation properties of such chemically synthesized full-length HTT exon1 containing both benign and pathological polyQ repeat lengths.…”

Chemical Strategies for Controlling Protein Folding and Elucidating the Molecular Mechanisms of Amyloid Formation and Toxicity

“…Recently, microwave-assisted SPPS allowed the synthesis of exon 1 of the Huntingtin protein. 84 To overcome the size limitation of SPPS, Kent et al developed and pioneered approaches for constructing large proteins based on the chemoselective condensation of two or multiple unprotected peptides in aqueous buffers at physiological pH values. The reaction is called NCL and involves the condensation of an N-terminal peptide bearing a C-terminal thioester and a C-terminal peptide containing an N-terminal Cys to form an amide bond with a Cys at the ligation site (Fig.…”