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

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...

Polyglutamine toxicity in yeast uncovers phenotypic variations between different fluorescent protein fusions

“…It is possible that addition of the fluorescent tag changes the biophysical properties of Htt ex1 oligomers, leading to formation of the toxic species similarly to what is observed with the addition of the FLAG tag at the amino terminus end of the protein . Indeed, recent in vitro data indicate that untagged Htt ex1 can behave differently than the tagged versions . In addition, we found the CFP‐tagged proteins produced a very weak fluorescent signal, especially for the soluble 25Q construct.…”

“…42 Indeed, recent in vitro data indicate that untagged Htt ex1 can behave differently than the tagged versions. 45 In addition, we found the CFP-tagged proteins produced a very weak fluorescent signal, especially for the soluble 25Q construct. This signal is so low that it is on par or even below that emanating from red vacuolar pigment due to the ade2-1 mutation in the W303 strain 46 (Figure 1C).…”

N-terminal phosphorylation of Huntingtin: A molecular switch for regulating Htt aggregation, helical conformation, internalization and nuclear targeting

“…Other studies suggest that the Nt17 interaction with the polyQ domain influences the structure of Httex1 in ways that favor the population of aggregation-prone conformational intermediates (17,24,25). Consistent with these studies, N-terminal Htt fragments lacking Nt17 show reduced aggregation rates compared with those containing Nt17 with similar polyQ repeat lengths (19,26). In addition to its role in Htt aggregation, this domain has been implicated in the regulation of full-length protein-protein interactions (21,(27)(28)(29)(30), subcellular localization (10), and toxicity (31).…”

Design and characterisation of mutant and wild-type huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems

“…The resulting HTT open reading frames encoded within this series of constructs have either an Nterminal FLAG-octapeptide between the START methionine and the N-terminal methionine of the HTT amino acid sequence, or have the FLAGoctapeptide linked to the extreme C-terminus of HTT via a Gly-Gly-Ser-Gly linker ( Figure 1C). As subtle changes to the exon 1 amino acid sequence of the HTT protein have been shown to give rise to changes to biophysical properties of the protein (21,22), the C-terminally FLAG-tagged constructs allow expression of a "clean" exon 1 sequence.…”