Journal: bioRxiv
Article Title: HAP40 functions as a proteostasis regulator by controlling huntingtin interactions and its release into the extracellular space
doi: 10.64898/2026.03.31.715351
Figure Lengend Snippet: A) Schematic representation of the BRET assay workflow. Expression vectors encoding c-myc-NL and PA-mCit tagged proteins were co-transfected into HEK293 cells. Binary interactions were detected in vivo using in-cell bioluminescence resonance energy transfer (BRET). B) BRET ratios of binary interactions between NanoLuc- and mCitrine-tagged full-length HTT Q23 , HAP40, and respective controls. Data are presented as mean ± SD (n=2), each with three technical replicates. Statistical significance was determined via two-way ANOVA followed by Bonferroni’s multiple-comparisons test. C) Left: Cryo-EM structure of HTT-HAP40 (6X9O). HAP40 is shown in red and HTT in blue. Right: Zoomed-in view displaying the five conserved HAP40 amino acid residues (gold: E316, E317, E331, D333, E335) and the three HAP40 mutant variants generated based on the number of amino acids converted into lysine. D) BRET ratios of binary interactions between c-myc-NL-HTT Q23 and PA-mCit-HAP40 (wild-type and mutants). Data are presented as mean ± SD, n=2, each with three technical replicates. Statistical significance was determined via two-way ANOVA followed by Dunnett’s multiple-comparisons test. E) Top: Linear schematic representation of resolved (blue) and unresolved (red) segments of the Cryo-EM structure of HTT-HAP40 (6X9O), indicating the mCitrine (mCit) insertion site. Below: schematic representation of the HTT intramolecular BRET sensor (NL-HTT Q23 (2686-mCit) ), displaying the tagging positions of NanoLuc at the N-terminus and mCitrine between amino acids 2686 and 2687 of human HTT. F) Cryo-EM structures of apo-HTT (6RMH) and HTT-HAP40 (6EZ8), illustrating the distances between resolved residues near the mCitrine insertion site and the N-terminus of HTT. Distances are shown in angstroms (Å). The relative positions of NanoLuc and mCitrine are indicated, along with predicted differences in BRET signal based on donor-acceptor distance. G) BRET saturation assay with co-transfection of the HTT intramolecular BRET sensor (150 ng, pcDNA-NL-HTT Q23 (2686-mCit) ) and increasing amounts of pcDNA-c-myc-HAP40 (wild-type; 0-10 ng) in HEK293-HAP40KO cells. The plot displays BRET ratio values (dashed line) and luminescence values (bar graph). Data are presented as mean ± SD (n=2). Statistical significance was determined via one-way ANOVA followed by Dunnett’s multiple-comparisons test. Corresponding immunoblots related to BRET assay using anti-c-myc, anti-GFP, and anti-TIM23 (loading control) antibodies are shown below. H) BRET saturation assay using the HTT intramolecular BRET sensor with increasing amounts of c-myc-HAP40 (5M mutant). Experimental conditions and statistical analysis were performed as described in G .
Article Snippet: The following primary antibodies were applied overnight at 4°C: rabbit anti-HAP40 (Atlas Antibodies, HPA046960, 1:1000), anti-HTT D7F7 antibody (Cell Signaling, 5656, 1:1000), anti-HTT MAB2166 antibody (Millipore, MAB2166, 1:500), anti-HTT MW1 antibody (DSHB, MW1, 1:500), mouse anti-SQSTM1(p62) (abcam, ab56416, 1:1000), rabbit anti-LC3B (abcam, ab192890, 1:1000), rabbit anti-UBB (PTG, 10201-2-AP, 1:1000), rabbit anti-Cathepsin D (Cell Signaling 69854, 1:1000), rabbit anti-STX17 (PTG 17815-1-AP, 1:1000), mouse anti-FLAG (Sigma, F3165, 1:1000), mouse anti-mNeonGreen (Chromotek, 32F6, 1:200), rabbit anti-GFP (Abcam, ab290, 1:2500), mouse anti-c-Myc (Merck, M4439, 1:2000), mouse anti-Actin (abcam, ab8224, 1:1000), mouse anti-Tubulin (Sigma, T6074, 1:80,000), mouse anti-TIM23 (BD Biosciences, 611223, 1:1000), and mouse anti-GAPDH (Santa Cruz, sc-47724, 1:1000).
Techniques: Bioluminescence Resonance Energy Transfer, Expressing, Transfection, In Vivo, Cryo-EM Sample Prep, Mutagenesis, Generated, Saturation Assay, Cotransfection, Western Blot, Control
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