The chaperonin TRiC blocks a huntingtin sequence element that promotes the conformational switch to aggregation

Abstract

Aggregation of proteins containing polyglutamine (polyQ) expansions characterizes many neurodegenerative disorders, including Huntington's disease. Molecular chaperones modulate the aggregation and toxicity of the huntingtin (Htt) protein by an ill-defined mechanism. Here we determine how the chaperonin TRiC suppresses Htt aggregation. Unexpectedly, TRiC does not physically block the polyQ tract itself, but rather sequesters a short Htt sequence element, N-terminal to the polyQ tract, that promotes the amyloidogenic conformation. The residues of this element essential for rapid Htt aggregation are directly bound by TRiC. Our findings illustrate how molecular chaperones, which recognize hydrophobic determinants, can prevent aggregation of polar polyQ tracts associated with neurodegenerative diseases. The observation that short endogenous sequence elements can accelerate the switch of polyQ tracts to an amyloidogenic conformation provides a novel target for therapeutic strategies.

Figure 1. Mapping the contact sites between Htt-exon1 and the chaperonin TRiC

(a) TRiC - Htt-Exon1 contacts identified by UV-induced crosslinking. The photoactivatable crosslinker BPIA (benzophenone-4-iodacetamine) was placed at one of three uniquely designed cysteines (A2C, Q40C or S111C) within MBP (maltose binding protein)-Htt-Exon1. (b) Photo-crosslinked adducts were detected by anti-Htt (upper panel) and anti-TRiC subunit 1 immunoblot analyses (lower panel). A major crosslink product is observed when BPIA is positioned at the N-terminus (A2C; asterisk). The minor crosslink with the polyglutamine region (Q40C) is indicated by an open triangle. (c) Direct interaction of TRiC with the N-terminal domain of Htt. Crosslinking to TRiC was tested for an isolated peptide comprising the N-terminal 17 amino acids of Htt (N17). Photo-adducts were detected probing for the biotin moiety of the peptide. Non-specific background signals are labeled with an asterisk. (−): no TRiC control. Results representative of at least three independent experiments are shown.

Figure 2. The N-terminus of huntingtin promotes rapid polyQ aggregation

(a) Htt-Exon1 fragments used to analyze the contribution of individual domains towards aggregation. Formation of SDS-insoluble, heat-stable aggregates was determined by filter-trapping and quantified by infrared Li-Cor imaging as described in Online Methods, (Supplementary Figs. 2 and 3). (b) Aggregation kinetics of Htt-exon1 fragments (Supplementary Fig. 4). Aggregation was initiated by TEV protease addition (time = 0 h) to GST- Htt-exon1 forms. All Htt variants were efficiently cleaved by TEV protease with similar kinetics (Supplementary Fig. 5). (c) Trans addition of N17 peptide enhances Htt-exon1 aggregation kinetics. A synthetic peptide containing 17 amino acids of the N-terminal region (N17^Htt) was added to Htt-ΔN (i) or to full-length Htt-exon1 (ii) and assayed as in (b). Similar results were obtained when N17 was generated by TEV cleavage from a GST fusion (data not shown). Of note, CD and NMR indicate that N17 by itself was fully soluble at the concentrations used here (data not shown).

Figure 3. The N-terminus of huntingtin interacts with N17 and the polyQ region in Htt-exon1

(a) N17 peptide interactions with Htt-Exon1 were assayed by crosslinking. The indicated purified GST-Htt-Exon1 domain deletion fragments (5 µM) or GST alone were incubated with N17 peptide (12.5 µM) carrying the crosslinker BPIA and crosslinked by UV irradiation. Adducts were resolved by SDS-PAGE and probed for the biotin moiety of the peptide. (−): GST only control. (b) (i) Summary of N17 interaction sites within Htt-Exon1; (ii) Predicted model for how N17 inter- and intramolecular interactions within Htt-Exon1 promote aggregation. (c) Dose-dependent effect of N17 addition on polyQ (−): buffer only control. Results representative of at least three independent experiments are shown.

Figure 4. The amphipathic N-terminal helix of Htt is necessary for rapid aggregation

(a) Secondary structure prediction (see Online Methods) identifies amino acids 4 to 12 of Htt-Exon1 [N_4–12^Htt] as alpha-helical, which are projected as a helical wheel to illustrate its amphipathic nature (left panel). Schematic representation of helical variants of Htt-exon1 analyzed for effects on aggregation (right panel). (b) Aggregation kinetics of Htt-exon1 bearing the indicated N-terminal helix variants assessed as in Fig. 2b. The mutants did not affect the efficiency of TEV protease mediated cleavage (Supplementary Fig. 5). (c) Modification of the hydrophobic face of the N-terminal Htt helix reduces aggregation below the levels observed for isolated polyQ only. Results representative of at least three independent experiments are shown. (d) Fluorescence microscopy of helical variants of Qn-GFP (n = 25, 103) expressing HeLa cells. Cells were scored for foci by visual inspection of GFP aggregates. Statistical analysis was performed using the one-sided, paired Student’s t-test: Means + SE of three independent experiments counting at least 200 cells each are shown. Scale bar represents 20 µm.

Figure 5. The hydrophobic surface of the N17 helix is the major Htt binding site for the chaperonin TRiC

(a) Direct interaction of TRiC with N17 requires the hydrophobic face of the helix. Crosslinking to TRiC was tested for the wild type N17 peptide (N17-WT) or with a peptide carrying alanine substitutions in the hydrophobic side of the N17-helix (N17-NA). TRiC- and UV-dependent adducts were detected probing for the biotin moiety of the peptide. Non-specific background signals are labeled with an asterisk. (−): no peptide control. Results representative of at least three independent experiments are shown. (b) The substrate binding apical domain of TRiC subunit 1 mediates binding to the hydrophobic surface of N17. In vitro crosslinking using N17-WT indicate an interaction with the isolated apical domain of TRiC subunit 1. The interaction is based on similar contacts with intact TRiC, as crosslinking was perturbed by the NA mutation within N17. No interaction is observed to the apical domain of TRiC subunit 3. As with other TRiC substrates, specific subsets of TRiC subunits interact with different substrates. (c) Purified TRiC and Apical 1 domain neutralize the aggregation promoting effect of trans- addition of N17 to Htt-ΔN. (−): Ovalbumin non-specific negative control. The lower panel shows the quantification of signal observed on the membrane (upper panel).

Figure 6. Huntingtin polyQ aggregation is controlled by the interplay of positive and negative regulatory sequence elements with the chaperone machinery

(a) Control of polyQ aggregation in huntingtin. The proline-rich domain (P) attenuates polyQ-aggregation propensity, while the N-terminal region (N) promotes the amyloid conformation. In turn, the chaperonin TRiC counteracts the positive effect of the N region, thereby suppressing aggregate formation. (b) Proposed model for the molecular events regulating Htt conformation. The polyQ domain in the Htt monomer populates predominantly a helical, non-amyloidogenic conformation likely stabilized by the proline-rich region. The amphipathic N-terminal helix of Htt-Exon1 interacts with both the polyQ region and itself. N17-PolyQ interactions likely stabilize the amyloidogenic beta-sheet conformation. Homotypic N-terminal interactions may facilitate intermolecular contacts linking amyloidogenic species to higher ordered structures. TRiC sequesters N17 by binding the hydrophobic side of the N17-helix thus blocking amyloid formation and growth.