Does N‐terminal huntingtin function as a ‘holdase’ for inhibiting cellular protein aggregation?

Abstract

Proteolytic cleavage of huntingtin gives rise to N-terminal fragments. While the role of truncated mutant huntingtin is described in Huntington's disease (HD) pathogenesis, the function of N-terminal wild-type protein is less studied. The yeast model of HD is generated by the presence of FLAG tag and absence of polyproline tract as flanking sequences of the elongated polyglutamine stretch. We show that the same sequence derived from wild-type huntingtin exon1 is able to inhibit the aggregation of proteins in vitro and in yeast cells. It is able to stabilize client proteins as varied as luciferase, α-synuclein, and p53 in a soluble but non-native state. This is somewhat similar to the 'holdase' function of small heat shock proteins and 'nonchaperone proteins' which are able to stabilize partially unfolded client proteins in a nonspecific manner, slowing down their aggregation. Mutagenesis studies show this property to be localized at the N17 domain preceding the polyglutamine tract. Distortion of this ordered segment, either by deletion of this segment or mutation of a single residue (L4A), leads to decreased stability and increased aggregation of client proteins. It is interesting to note that the helical conformation of the N17 domain is also essential for aggregation of the N-terminal mutant protein. Our results provide evidence for a novel function for the amphipathic helix derived from exon1 of wild-type huntingtin.

Stabilization of elongated polyglutamine tracts by a helical peptide derived from N‐terminal huntingtin

“…Thioflavin T is a benzothiazole dye that is used as an extrinsic probe to visualize amyloid fibrils 30–32 . An increase in the fluorescence intensity of Thioflavin T in the presence of 51Q‐htt alone was observed in a time‐dependent manner (Figure 2c), indicating the formation of amyloid type of aggregates 22 . Fibrillation occurred quite fast, with an apparent rate constant ( k app ) of 0.060 hr −1 and reached the maximal value within 80 hr of incubation.…”

“…We had shown earlier that the amphipathic peptide derived from the N‐terminal fragment of 25Q‐htt can function as a stabilization aid against a number of proteins like α‐synuclein, p53, and 103Q‐htt 22 as well as the yeast prion protein Rnq1 21 . Different lengths of polyQ containing huntingtin fragments, namely 51Q‐htt, 72Q‐htt, and 103Q‐htt, were selected to understand the effect of 25Q‐htt on aggregation of elongated polyQ tracts.…”

“…Saccharomyces cerevisiae cells were transformed with pYES2‐ FLAG‐25Q‐htt , 22 FLAG‐25Q‐htt‐EGFP , or pYES2 ‐FLAG‐103Q‐htt‐EGFP 27 . Expression of FLAG‐25Q‐htt, FLAG‐25Q‐htt‐EGFP, or FLAG‐103Q‐htt‐EGFP was induced with 2% galactose 27 .…”

“…Expression of FLAG‐25Q‐htt, FLAG‐25Q‐htt‐EGFP, or FLAG‐103Q‐htt‐EGFP was induced with 2% galactose 27 . Cells were lysed by using the glass beads method 20–22 . Purification of proteins was carried out using anti‐FLAG affinity matrix (Sigma) 22 …”

“…We had reported that the expression level of each protein is a critical determinant of its solubility and decides the toxicity in cells harboring such proteins 20 . The N‐terminal fragment of normal huntingtin was shown to inhibit aggregation of a variety of proteins like Rnq1, 21 luciferase, p53, and α‐synuclein 22 . In this work, we have investigated whether this short fragment of N‐terminal huntingtin is capable of differentiating between elongated polyQ tracts of differing lengths and inhibits their aggregation.…”