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. 2016 Jun 3;291(23):12074-86.
doi: 10.1074/jbc.M116.713982. Epub 2016 Mar 21.

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

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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

Sophie Vieweg et al. J Biol Chem. .
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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, maltose-binding protein, or thioredoxin, or released in solution upon in situ cleavage of these proteins. Herein, we report an intein-based 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.

Keywords: Huntington disease; circular dichroism (CD); electron microscopy (EM); fibril; protein aggregation; protein purification; protein structure.

Figures

FIGURE 1.
FIGURE 1.
Steps involved in the intein-based expression and purification strategy for the generation of tag-free Httex1-QN proteins. A, scheme of the intein-based expression and purification strategy. B, the amino acid sequence of the His6-Ssp-Httex1-QN fusion constructs (His6-Ssp in green, Httex1 in orange). The Httex1 proteins are devoid of the first methionine, as it was reported to be cleaved off in vivo (48). C, the expression of His6-Ssp-Httex1–23Q/43Q (arrow) was analyzed by SDS-PAGE. D, the generation of Httex1–23Q/43Q (star) by splicing of the His6-Ssp-Httex1–23Q/43Q fusion proteins (arrow) monitored by WB using monoclonal anti-Htt MAB5492. E, the splicing of His6-Ssp-Httex1–43Q (1) into His6-Ssp (2), and Httex1–23Q/43Q (3) was assessed by analytical RP-UHPLC. AU, absorbance units. F–H, the purity and integrity of Httex1–23Q/43Q was determined using RP-UHPLC (F), SDS-PAGE and WB (G), and LC/MS (H). Expected molecular masses for Httex1–23Q/43Q are 9944 Da and 12506 Da. I, purity analysis of Httex1–7Q/15Q/29Q/37Q/49Q by SDS-PAGE and RP-UHPLC.
FIGURE 2.
FIGURE 2.
The secondary structure of Httex1–23Q is not altered by the RP-HPLC purification or disaggregation procedure. The secondary structure of Httex1–23Q purified by size-exclusion chromatography (SEC) was assessed by CD (gray line) and compared with the structure of HPLC-purified and disaggregated Httex1–23Q (black line).
FIGURE 3.
FIGURE 3.
The aggregation properties of recombinant wild type (23Q) and mutant (43Q) tag-free Httex1. A, the kinetics and extent of Httex1 aggregation were monitored by measuring the amounts of soluble Httex1–23Q/43Q over time using analytical RP-UHPLC. All data (23Q, n = 3–4; 43Q, n = 2–3) were normalized to t0h and are represented as the mean ± S.D. The resulting data points were fitted single exponentially. B, the secondary structure of Httex1–23Q/43Q was analyzed by CD during incubation at 37 °C. C, aggregates formed by Httex1–23Q/43Q were imaged using negative-stain TEM.
FIGURE 4.
FIGURE 4.
Concentration dependence of Httex1–23Q aggregation. A, the aggregation propensity of Httex1–23Q at 15, 30, 60, and 120 μm was monitored by measuring the soluble protein fraction over time using analytical RP-UHPLC. All data were normalized to t0h and are represented as the mean ± S.D. (15/30/120 μm, n = 3; 60 μm, n = 3–5). B, the CD spectra of Httex1–23Q at the final aggregation time points. C, TEM images of fibrils formed by Httex1–23Q at the end point of aggregation.
FIGURE 5.
FIGURE 5.
PolyQ-length dependence of Httex1 aggregation. A, the aggregation propensity of Httex1–23Q (black, n = 3–5), Httex1–15Q (blue, n = 3), and Httex1–7Q (green, n = 3) at 60 μm. The amount of soluble protein was assessed by analytical RP-UHPLC, and all data were normalized to t0h and are represented as the mean ± S.D. B, the CD spectra of Httex1–23Q (black), Httex1–15Q (blue), and Httex1–7Q (green) at the initial and final aggregation time points. C, TEM images of the aggregates formed by Httex1–23Q/15Q/7Q at the final aggregation time points. The white arrow indicates one of the oligomeric structures formed by Httex1–7Q. D, the aggregation propensity of Httex1–23Q (black, n = 3), Httex1–29Q (purple, n = 5), Httex1–37Q (green, n = 5), and Httex1–43Q (blue, n = 5) at 15 μm. All data were normalized to t0h and are represented as the mean ± S.D. E, the CD spectra of Httex1–23Q (black), Httex1–29Q (purple), Httex1–37Q (green), and Httex1–43Q (blue) at the initial and final aggregation time points. F, TEM images of fibrils formed by Httex1–23Q/29Q/37Q/43Q at 15 μm at the end point of aggregation. G, quantification of fibril lengths for Httex1–23Q (black, n = 419), Httex1–29Q (purple, n = 437), Httex1–37Q (green, n = 483), and Httex1–43Q (blue, n = 380) at the end point of aggregation. Solid lines represent Gaussian fits of the fibril lengths.
FIGURE 6.
FIGURE 6.
Aggregation of mutant Httex1 in the presence of MBP and GST. A, the soluble fraction of Httex1–43Q incubated at 37 °C (black) and co-incubated with MBP (dark gray) or GST (light gray) was determined by analytical RP-UHPLC. All data (n = 2) were normalized to t0h and are represented as the mean ± S.D. The resulting data points were fitted single exponentially. B, TEM images of Httex1–43Q aggregates formed after co-incubation with MBP or GST for 24h at 37 °C.

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