Insights into the Aggregation Mechanism of PolyQ Proteins with Different Glutamine Repeat Lengths

Abstract

Polyglutamine (polyQ) diseases, including Huntington's disease, result from the aggregation of an abnormally expanded polyQ repeat in the affected protein. The length of the polyQ repeat is essential for the disease's onset; however, the molecular mechanism of polyQ aggregation is still poorly understood. Controlled conditions and initiation of the aggregation process are prerequisites for the detection of transient intermediate states. We present an attenuated total reflection Fourier-transform infrared spectroscopic approach combined with protein immobilization to study polyQ aggregation dependent on the polyQ length. PolyQ proteins were engineered mimicking the mammalian N-terminus fragment of the Huntingtin protein and containing a polyQ sequence with the number of glutamines below (Q11), close to (Q38), and above (Q56) the disease threshold. A monolayer of the polyQ construct was chemically immobilized on the internal reflection element of the attenuated total reflection cell, and the aggregation was initiated via enzymatic cleavage. Structural changes of the polyQ sequence were monitored by time-resolved infrared difference spectroscopy. We observed faster aggregation kinetics for the longer sequences, and furthermore, we could distinguish β-structured intermediates for the different constructs, allowing us to propose aggregation mechanisms dependent on the repeat length. Q11 forms a β-structured aggregate by intermolecular interaction of stretched monomers, whereas Q38 and Q56 undergo conformational changes to various β-structured intermediates, including intramolecular β-sheets.

Figure 1

Composition of the polyQ construct including the polyglutamine region (Q_N) (violet), the N-terminal fragment of the mammalian Htt protein (dark blue), the SUMO domain (orange), and a His- and a Flag-tag (light blue and gray). The polyglutamine sequences vary from below (polyQ11), close to (polyQ38), and above (polyQ56) the HD disease threshold. The His-tag is used for affinity purification and enables protein immobilization. The SUMO part enhances the solubility of the construct. Enzymatic cleavage with Ulp1 triggers the aggregation of the polyglutamine sequence. The Flag-tag can be used for immunodetection via Western blot. The construct without a polyglutamine sequence was used for control measurements. To see this figure in color, go online.

Figure 2

Schematic representation of the ATR-FTIR experiment shown for polyQ56 as an example. (a) This panel shows the vesicle deposition onto an activated Ge crystal. (b) The formation of the LB containing POPC/DOGS with vacant NTA groups. (c) The complexation of NTA groups by the addition of Ni²⁺ in the lipid buffer is. (d) PolyQ construct immobilization. The construct binds to the Ni²⁺-NTA via the His-tag. The SUMO (yellow β-sheet, red α-helices) and N17-Q56 (dark blue N17, violet Q56 region). (e) The addition of Ulp1 protease. The enzyme cleavage site is located between SUMO and N17 and is shown by the red arrow in (d). The polyQ-containing fragments are released. (f) The formation of the polyQ aggregates. The polyQ-containing fragments undergo conformational changes and form extended β-sheet aggregates, which are monitored spectroscopically in a time-resolved manner. To see this figure in color, go online.

Figure 3

Amide I spectra of the immobilized constructs (a) polyQ11, (b) polyQ38, (c) polyQ56, and (d) control (without glutamines). The constructs were bound to the Ni²⁺-NTA on the LB via the His-tag. The bands at 1633–1638 cm⁻¹ and 1685–1687 cm⁻¹ (green) indicate the β-sheets from SUMO. The band at 1674–1677 cm⁻¹ arises from turn and loops. The band at 1656–1658 cm⁻¹ (yellow) refers to the contributions from α-helices of SUMO and N17, overlapped by glutamine side-chain vibrations. The band at 1644–1647 cm⁻¹ (blue) occurs from the disordered glutamine backbone and is not present in the control. Second-derivative spectra (insets) reveal the frequency positions of the amide I components for curve fitting and reconstruction of the band components. To see this figure in color, go online.

Figure 4

Conformational changes of released N17-Q_N-Flags upon Ulp1 cleavage and aggregation initiation, (a) N17-Q11-Flag, (b) N17-Q38-Flag, and (c) N17-Q56-Flag. The band components at 1626/1627 and 1690/1691 cm⁻¹ are assigned to antiparallel β-structured aggregates, 1644 cm⁻¹ to disordered, and 1662/1668 cm⁻¹ to loop and turn structures within the polyglutamine sequences. The nonchanging contributions of the LB, the immobilized part of the construct (His-tag and SUMO), Ulp1, and H₂O/buffer were subtracted as background. N17 and Flag-tag are monitored as well but are assumed to perform only minor conformational changes upon polyQ aggregation if at all.

Figure 5

Second-derivative spectra reveal structural transitions of N17-Q11-Flag, 10, 20, 40, 60, and 180 min after aggregation initiation. New band components arise in a frequency region (gray) that is characteristic for β-structured aggregates. The frequency positions indicate the formation of intermolecular β-sheets rearranging over time. The corresponding absorption spectra are presented in Fig. S4.

Figure 6

Second-derivative spectra characterize structural transitions of the N17-Q38-Flag, 10, 20, 40, 60, and 180 min after aggregation initiation. The spectra indicate that a predominantly disordered structure (1648 cm⁻¹) with minor contributions of intermolecular β-sheets (1621 cm⁻¹) undergoes conformational changes to intramolecular β-sheets (1632 cm⁻¹) and expanded β-structured aggregates (1626 cm⁻¹). Corresponding absorption spectra are presented in Fig. S5.

Figure 7

Second-derivative spectra of the N17-Q56-Flag characterize aggregate formation after 10, 20, 40, 60, and 180 min. The minima at 1621 and 1632 cm⁻¹ reveal that inter- and intramolecular β-sheets are already formed at early stages and further rearrange into expanded β-structured aggregates (1626 cm⁻¹). The corresponding absorption spectra are presented in Fig. S6.

Figure 8

Proposed aggregation pathways of N17-Q_N-Flag fragments dependent on the glutamine repeat length. N17-Q11-Flag fragments tend to form extended β-sheets with strands that consist of stretched monomers. 11 glutamines are too short to form turns. The β-sheets are stabilized by intermolecular interactions. In contrast, the longer N17-Q38-Flag and N17-Q56-Flag sequences are highly flexible to change conformation and to form turns and loops. Sequence parts assemble to intra- and intermolecular β-structured aggregates, which still have a significant number of disordered regions. The aggregation kinetics of N17-Q56-Flag is faster than that of N17-Q38-Flag. It is likely that the Q56 conformers accumulate further into higher-ordered aggregate morphologies.