1. Cellular Toxicity of Polyglutamine Expansion Proteins
Gregor Schaffar, Peter Breuer, Raina Boteva, Christian Behrends, Nikolay Tzvetkov, Nadine Strippel, Hideki Sakahira, Katja Siegers, Manajit Hayer-Hartl, F.Ulrich Hartl 
Molecular Cell 
Volume 15, Issue 1, Pages (July 2004)
DOI: /j.molcel

2. Figure 1
PolyQ-Mediated Interaction of Mutant Htt with TBP and CBP in N2a Cells
(A) Recruitment of TBP, CBP, and Htt20Q into aggregates by Htt96Q. The proteins were transiently expressed in mouse N2a cells for 48 hr. SDS-resistant aggregates were detected in cell lysates by filter assay and immunostaining for Htt constructs with anti-myc (left) and recruited proteins with anti-FLAG or anti-CBP antibody (right).
(B) Recruitment of TBP into mutant Htt aggregates requires ongoing Htt synthesis. myc-Htt96Q was expressed under Tet control. After 24 hr of induction, TBP was transiently expressed either with or without continued expression of Htt96Q (−Dox, +Dox). Aggregated Htt96Q and TBP were determined as above and quantified by phosphorimager analysis. Protein amounts are given in arbitrary units.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2 In Vitro Reconstitution of PolyQ-Dependent Coaggregation
(A) Schematic representation of the recombinant polyQ proteins analyzed. HA, triple HA-tag. PP, cleavage site for PreScission protease.
(B) Aggregation of purified polyQ proteins analyzed by filter assay. Proteins (3 μM GST-Htt53Q and 0.2 μM of recruitment substrates) were incubated in the combinations indicated (see Experimental Procedures) with ovalbumin (OVA) as a non-polyQ control protein. Aggregation reactions were started at 30°C by the addition of PreScission protease and stopped after 8 hr by adding SDS. Aggregated protein was detected by immunostaining as indicated.
(C) Formation of aggregates of polyQ proteins analyzed by centrifugation. Htt53Q was incubated with recruitment substrate as in (B). Pellet and supernatant fractions were analyzed by SDS-PAGE. Amounts of aggregated protein are indicated in arbitrary units.
(D) Time course of Htt53Q (3 μM) and TBP (0.2 μM) coaggregation. Total aggregated protein was analyzed by centrifugation as in (C).
(E) Htt53Q aggregation reactions were divided into supernatant and pellet fractions (10 min; 20,000 × g) at 0, 1.5, 3, and 6 hr after initiating aggregation. TBP was then added to supernatants and resuspended pellets, and recruitment into total aggregates was measured after 24 hr as in (B).
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
Oligomerization and Conformational Rearrangement of Mutant Htt
(A) Oligomerization of Htt53Q measured by intermolecular FRET. Donor (D)- and acceptor (A)-labeled GST-Htt53Q were mixed at the concentrations indicated, and oligomerization was initiated by the addition of PreScission protease. When indicated, TBP (0.1 μM) was also present. FRET was monitored as the decrease in donor fluorescence at 480 nm (see also Supplemental Figure S3). The kinetics of GST-Htt53Q cleavage is shown for comparison (dashed line). Note that no FRET is observed in the absence of PP.
(B) Effect of recruitment substrates on intermolecular FRET. The efficiency of FRET was measured as above after 2 hr of Htt53Q aggregation (0.5 μM each of A- and D-labeled Htt53Q) in the presence of Htt20Q, TBP, or TBPΔQ (0.1 μM each). The inhibition of FRET efficiency is given in %. Averages ± SD of three independent experiments.
(C) Conformational rearrangement of D/A-labeled Htt53Q upon cleavage from GST-Htt53Q (1 μM) measured by intramolecular FRET. TBP (0.1 μM) was present when indicated. The decrease in donor fluorescence was followed upon cleavage of GST-Htt53Q at 425 nm. The kinetics of GST-Htt53Q cleavage, determined in parallel reactions by SDS-PAGE, is also shown. In a control reaction separate molecule populations of GST-Htt53Q (0.5 μM) were labeled with the fluorophores.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
Effects of Hsp70 and Hsp40 on Htt Aggregation and Transcription Factor Recruitment
(A) Hsp70/Hsp40 inhibit recruitment. Aggregation reactions containing Htt53Q (3 μM) and TBP or ΔN-CBP (0.2 μM, each) were performed as in Figure 2C in the presence or absence of Hsp70 (6 μM), Hsp40 (6 μM), or Hsp70/Hsp40 (6 μM/3 μM) with or without ATP (see Experimental Procedures). The efficiency of recruitment of TBP and ΔN-CBP into aggregates in the absence of chaperones is set to 100%.
(B and C) Effect of Hsp70/Hsp40 (1 μM/0.5 μM) on kinetics of intramolecular (B) and intermolecular FRET of Htt53Q (C) in the presence and absence of ATP. FRET measurements were performed as in Figure 3 at a total concentration of 1 μM Htt53Q.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5 Deactivation of TBP by Oligomerization-Competent Mutant Htt
The effect of Htt constructs and of Hsp70/Hsp40 chaperones on the DNA binding activity of TBP was measured by electrophoretic mobility shift assay (EMSA) in vitro.
(A) TBP (0.2 μM) was incubated either alone or with GST-Htt20Q or GST-Htt53Q (3 μM each) and PreScission protease for up to 6 hr. Nonspecific DNA shifts due to DNA self-oligomerization are marked by an asterisk.
(B) Rapid deactivation of TBP by Htt53Q and prevention of deactivation by Hsp70/Hsp40/ATP (see Figure 4A). EMSA data as in (A) were quantified by phosphorimager analysis.
(C) Proteinase K (PK) resistance of TBP after incubation with GST-Htt20Q or GST-Htt53Q and PreScission protease for 1 hr as in (A). PK treatment was performed on ice for 20 min and stopped by the addition of 1 mM PMSF. Reactions were analyzed by SDS-PAGE and immunoblotting with TBP antibody (anti-hTFIID mAB, Promega), directed against an epitope C-terminal to the polyQ repeat. The asterisks mark a fragment of TBP N-terminally truncated by ∼10 kDa.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
PolyQ-Mediated TBP Deactivation in the Nucleus in a Yeast Model System
(A) Growth defect due to deactivation of human TBP by mutant Htt. NLS-Htt constructs were expressed from a copper regulated promoter in yeast strain GSYΔyTBP/hTBP, which contains human TBP as the only source of TBP. Growth was examined by serial dilutions of cells on SC plates after 48 hr at 34°C. The observed growth defect was suppressed upon simultaneous expression of yeast TBP or human Hsp70/Hsp40 under control of galactose-regulated promoters.
(B) The growth defect caused by expression of NLS-Htt96Q in GSYΔyTBP/hTBP cells does not correlate with the formation of detergent-insoluble Htt aggregates. After growth overnight, cultures were adjusted to an OD of ∼0.3, and expression of the Htt constructs indicated was induced. Growth at 30°C in medium containing 50 mM 3-AT was followed over 8 hr (see Experimental Procedures).
(C) The accumulation of SDS-insoluble Htt aggregates was determined in the cells analyzed in (B) by the filter assay. Amounts of aggregates are expressed in arbitrary units (AU).
(D) Solubility of myc-tagged NLS-Htt96Q, Htt96Q, and GST-Htt96Q (100,000 × g, 30 min) in extracts of GSYΔyTBP/hTBP cells after different times of induction, as detected by immunoblotting with anti-myc antibody.
(E) Size exclusion chromatography of soluble fractions from GSYΔyTBP/hTBP cells expressing NLS-Htt96Q or Htt96Q for 1 hr as indicated (see Experimental Procedures). The distribution of NLS-Htt96Q (top panel, anti-myc) and of human TBP (bottom panels, anti-TBP) in the fractions was analyzed by immunoblotting.
(F) Size fractionation of TBP in GSYΔyTBP/hTBP cells expressing NLS-Htt96Q, Htt96Q, and GST-NLS-Htt96Q was analyzed as in (E) and quantified by densitometry.
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7
Working Model for the Deactivation of TBP by a PolyQ Disease Protein
Toxic species of a polyQ-expanded disease protein (monomers and small oligomers) are generated as a result of a conformational change in the polyQ segment, shown schematically as the formation of an intramolecular, cylindrical β sheet (Perutz et al., 2002). See Discussion for details.
Molecular Cell  , DOI: ( /j.molcel )