Journal: Nature Communications

Article Title: Identification of a HTT-specific binding motif in DNAJB1 essential for suppression and disaggregation of HTT

doi: 10.1038/s41467-022-32370-5

Figure Lengend Snippet: a Schematic representation of class B J-domain protein DNAJB1 wt (in teal) and class A J-domain protein DNAJA1 wt (in orange) and their domains. DNAJA1 possesses a similar HBM in its hinge region as DNAJB1. H and K residues are conserved (black squares). To create DNAJA1 JB1ized , DNAJA1 ZFLR was deleted, and the short G/F-rich domain of DNAJA1 was substituted by the longer G/F-rich domain of DNAJB1. b FRET assay of HTTExon1Q 48 aggregation and the effect of the addition of Hsc70, Apg2, and DNAJB1 wt , DNAJA1 wt or variant DNAJA1 JB1ized . The graph is a representative result of three independent experiments, and a one-way ANOVA analysis of the half-life (T 1/2 ) of HTTExon1Q 48 aggregation under the tested conditions is depicted on the right. Bars represent the mean value and error bars correspond to the mean SD. **** P ≤ 0.0001; ** P ≤ 0.01; * P ≤ 0.05; ns not significant. c Left, the crystal structure of DNAJB1 protomer (pdb: 3agz) showing in orange the amino acids identified as the binding site for Hsc70 , and in teal the huntingtin binding motif (HBM) identified in our study. Right, crystal structure of Hsp110-Hsc70 complex (pdb: 3c7n). Hsp110 is displayed in dark gray, and Hsc70 is shown in light blue. The huntington binding site of Hsc70 identified by our cross-linking-MS experiments (Fig. ) is shown in magenta.

Article Snippet: pGEX-6P1-HttExon1Q 48 -CyPet, and pGEX-HttExon1Q 48 -YPet were obtained as described previously . pGEX-6P1-HttExon1Q 48 ∆N17-CyPet/YPet were previously generated . pET-6His-Smt3-Apg2, pET-6His-Smt3-Hsc70, and pET-6His-Smt3-DNAJB1 were obtained from the Bukau lab . From Addgene, we obtained: pET-Sac-Abeta(M-42) (from Dominic Walsh, #71875), pcDNA5/FRT/TO V5 DNAJA1 (from Harm Kampinga, #19518), mRFP-FKBP12 (from Tamas Balla, #67514) and pGEX-2T-FRB (from Jie Chen, #26607). pCDNA3-HTTExon1Q 97 -EGFP and pCDNA3-DNAJB1 were obtained as described previously .

Techniques: Variant Assay, Binding Assay

Journal: Nature Communications

Article Title: Identification of a HTT-specific binding motif in DNAJB1 essential for suppression and disaggregation of HTT

doi: 10.1038/s41467-022-32370-5

Figure Lengend Snippet: a Schematic representation of the disaggregation assay. HTTExon1Q 48 -CyPet fibrils were incubated with the indicated chaperones and ATP for 24 h. The resolubilized moiety was separated from fibrils by centrifugation, and CyPet fluorescence of the supernatant was measured as a readout of the resolubilized HTTExon1Q 48 . b Top, schematic representation of the HTT fibril-binding assay. Preformed HTTExon1Q 48 fibrils were incubated with DNAJB1 wt or DNAJB1 H244A and their association with the fibrils assessed by a sedimentation analysis and subsequent Western blot. Bottom, Western blot of the intensities of DNAJB1 and DNAJB1 H244A bound to HTTExon1Q 48 fibrils. Quantification of three independent experiments is depicted on the right. Bars represent the mean value, and error bars correspond to the mean SD. ** P ≤ 0.01. c – e Fluorescence level measurements of three independent experiments of disaggregated HTTExon1Q 48 or HTTExon1Q 48 ΔP2 (only c ) by Hsc70, Apg2 and DNAJB1 wt ( c ) or variants DNAJB1 H244A , DNAJB1 K242A , DNAJB1 H244F , DNAJB1 ΔDD ( d ) or DNAJB1 ΔG/F , DNAJB1 JA1-G/F , DNAJA1 wt , DNAJA1 ΔZFLR , and DNAJA1 JB1ized ( e ). The significance was determined by a one-way ANOVA analysis. Bars represent the mean value and error bars correspond to the mean SD. **** P ≤ 0.0001; *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; ns not significant. f Schematic representation of the DNAJB1 and DNAJA1 variants analyzed in ( e ).

Article Snippet: pGEX-6P1-HttExon1Q 48 -CyPet, and pGEX-HttExon1Q 48 -YPet were obtained as described previously . pGEX-6P1-HttExon1Q 48 ∆N17-CyPet/YPet were previously generated . pET-6His-Smt3-Apg2, pET-6His-Smt3-Hsc70, and pET-6His-Smt3-DNAJB1 were obtained from the Bukau lab . From Addgene, we obtained: pET-Sac-Abeta(M-42) (from Dominic Walsh, #71875), pcDNA5/FRT/TO V5 DNAJA1 (from Harm Kampinga, #19518), mRFP-FKBP12 (from Tamas Balla, #67514) and pGEX-2T-FRB (from Jie Chen, #26607). pCDNA3-HTTExon1Q 97 -EGFP and pCDNA3-DNAJB1 were obtained as described previously .

Techniques: Incubation, Centrifugation, Fluorescence, Binding Assay, Sedimentation, Western Blot

Journal: Nature Communications

Article Title: Identification of a HTT-specific binding motif in DNAJB1 essential for suppression and disaggregation of HTT

doi: 10.1038/s41467-022-32370-5

Figure Lengend Snippet:

Article Snippet: pGEX-6P1-HttExon1Q 48 -CyPet, and pGEX-HttExon1Q 48 -YPet were obtained as described previously . pGEX-6P1-HttExon1Q 48 ∆N17-CyPet/YPet were previously generated . pET-6His-Smt3-Apg2, pET-6His-Smt3-Hsc70, and pET-6His-Smt3-DNAJB1 were obtained from the Bukau lab . From Addgene, we obtained: pET-Sac-Abeta(M-42) (from Dominic Walsh, #71875), pcDNA5/FRT/TO V5 DNAJA1 (from Harm Kampinga, #19518), mRFP-FKBP12 (from Tamas Balla, #67514) and pGEX-2T-FRB (from Jie Chen, #26607). pCDNA3-HTTExon1Q 97 -EGFP and pCDNA3-DNAJB1 were obtained as described previously .

Techniques: Construct, Sequencing