Fig 1: CG8134 encodes Drosophila Hap40 homologue.(A) Sequence alignment between CG8134/dHap40 and the following vertebrate HAP40 homologs: human (hHAP40/F8a1), mouse (mHAP40/F8a), xenopus (xHAP40) and zebra fish Danio rerio (DrHAP40). Genebank ID of these proteins were indicated. The scale bar above the alignment refers to amino acid position from human HAP40 (NP_036283). Amino acid similarities are annotated in color in the following order in the alignment: (1) black box highlights amino acids that are identical to that in CG8134/dHap40; (2) green boxes are those identical to those in human HAP40; (3) Yellow boxes are those with similar chemical property as those in human HAP40; (4) blue box highlights amino acids that are with similar chemical property as in CG8134/dHap40. The red bars above the alignment cover the sequences of the predicted 14 α-helices and the green bars those invisible in the Cryo-EM model. (B) Schematics of the predicted secondary structures in human HAP40 (top) and CG8134/dHap40 (bottom) proteins using the Garnier-Robson structure prediction module (Protean program by DNASTAR), drawn in scale (top) of human HAP40. Red boxes represent the predicted α-helices. BΦ: the N-terminal α-helices enriched with basic and hydrophobic amino acids, which is part of the N-terminal invisible region in the Cryo-EM model (see Fig 3C below); CC: the α-helices containing two conserved cysteines; EARFL: α-helices with a conserved stretch of E,A,R,F and L amino acids; LR: α-helices with leucine-repeat; P: proline linker after the LR α-helices. (C) Schematics of the secondary structures in human HAP40 protein predicted from the Cryo-EM study. Red rectangles correspond to the predicted 14 α-helices and the green rectangles are regions invisible in the Cryo-EM model. (D) Schematics of genomic structure of cg8134/dhap40 gene and the mutant alleles created in the study. dhap40 is a X-linked gene composed of one intron (solid line) and two coding exons (grey boxes: untranslated regions; black boxes: coding regions), with arrowhead indicting the orientation of the encoded protein from 5’ to 3’ ends. Star annotates the locations of the molecular lesions for each of the characterized dhap40 alleles. Blank area indicates the region of small insertions and deletions and shaded area the induced frameshift in the encoded protein. (E) SDS-PAGE and Coomassie blue staining of protein lysates from E. coli. A protein product of 40kDa size was produced only in cells transformed with a pET protein expression plasmid containing full-length cg8134 cDNA without (lane 2) or with IPTG induction (lane 3) but not in control transformed with an empty protein expression vector alone (lanes 1). (F) Western Blot analysis of CG8134/dHap40 expression in adult flies from wildtype (WT), four dhap40 mutant (ko3, ko7, ko8 and ko9) alleles, and a dHap40 overexpression line (OE) carrying a UAS-cg8134 transgene directed by a strong Actin-Gal4 driver. Note that a 40kDa band (left red arrow) corresponding to endogenous CG81344/dHap40 was detected in WT and present at significantly higher levels in the OE line (right red arrow), but was completely absent in the four dhap40 mutant lines. (G) Western Blot analysis of ectopically expressed human HAP40, detected as a 40kDa protein, from homogenates of two fly lines carrying a UAS-F8A1/HAP40 transgene direct by daughterless-Gal4 driver. (H) co-IP of endogenous dHtt by dHap40 antibodies. Fly homogenates from dHtt-eGFP flies were incubated with two independent anti-dHap40 (anti-CG8134 # 1 and #2) antibodies, or with their corresponding pre-immunization sera (#1 Pre and #2 Pre) as controls. The immunoprecipitates were probed with anti-GFP or anti-dHap40 antibodies after SGS-PAGE separation, as indicated.
Fig 2: The levels of HAP40 protein are lower in HD cells.Western blot assays and quantification of endogenous HTT and HAP40 proteins in normal and HD (A and B) mouse striatal precursor cells and (C-E) human fibroblast cells, as indicated. The human fibroblast cell lines are: GM04729 (WT-1), GM05539 (HD-1), GM21757 (HD-2. CAG repeats reported as 66 and 16), GM02189 (WT-2), GM09197 (HD-3. 180 CAG repeat in affected HD allele), GM04723 (HD-4), GM04787 (WT-3) and GM21756 (HD-5. CAG repeats reported as 70 and 15). (A) Note that for each mutant HD cell lines in these SDS-PAGE gels, mutant HTT proteins (HTT-Q111, indicated by red *) were reduced and migrated slower than wildtype HTT (wtHTT or HTT-Q7, indicated by blue *) in a polyQ length-dependent manner. (C) The membrane was co-probed with both anti-total HTT (D7F7,) and mutant HTT specific (MW1, red) antibodies. In both (B) and (D), N = 6 for quantification of HAP40 protein levels and N = 4 for quantification of HTT. (E) Average relative ratio of the normalized HTT and HAP40 protein levels in wildtype (WT) and HD cells, quantified from the eight wildtype and 13 mutant HD fibroblast cells lines tested in repeats experiments (see S8 Fig for additional data). *p< = 0.05, **p< = 0.01, ***p< = 0.001 (student’s t-test). n.s., no significance. β-Actin served as loading and normalization controls in all the experiments.
Fig 3: Mild effect of HAP40 on neurodegeneration of Drosophila HD models.(A-F) Representative (A-C) confocal images of whole-mount retina with phalloidin staining, dissected from 1-day-old adult female flies, or (D-F) bright-field image of 30-day-old adult fly eyes with eye-specific expression of (A and D) control wildtype fl-HTT-23Q, or (B, C, E and F) human mutant fl-HTT-145Q in (B and E) normal or (C and F) dhap40ko3 background, all directed by eye-specific GMR-Gal4 driver. Compared with (A) the control of the eye expressing wildtype HTT-23Q, which showed regular composition and patterning of the seven photoreceptors within each ommatidium unit, (B) mutant HTT-145Q already caused severe degeneration phenotypes, showing prominent loss of photoreceptor cells and disintegration of ommatidia structure, while (C) in the absence of endogenous dhap40, the eye degeneration phenotypes were significantly suppressed. Similarly, the loss of eye pigmentation induced by fl-HTT-145Q (E A), indicating underlying eye degeneration, was suppressed by the absence of dhap40 (F). Genotypes: (A and D) GMR-Gal4/+ >UAS-hHTT-23Q/+. (B and E) GMR-Gal4/+ >UAS-hHTT-145Q/+. (C and F) dhap40ko3/dhap40ko3; GMR-Gal4/+ >UAS-hHTT = 145Q/+. Flies with the same genotype showed relatively similar phenotypes at the same age (examples in S9B–S9D Fig). More than 4 fly eyes were examined and imaged in (A-C) and 30 flies were examined in (D-F) for each of the genotypes. (G-J) Quantification of photoreceptor cell degeneration and viability phenotypes of flies with neuronal-specific expression of fl-HTT-128Q either (G and H) in dhap40 null background or (I and J) with co-expression of human HAP40, all directed by pan-neuronal nsyb-Gal4 and with respective controls, as indicated. (G and I) Bar chart presentation of the average number of intact photoreceptor cells (PRC) per ommatidium in 7-day-old flies of the following designated genotypes. (G)“nsyb-Gal4/UAS-fl-HTT-128Q”: 4.4 PRC/ommatidium (n = 360 ommatidia from 20 flies); “dhap40 ko3; nsyb-Gal4/UAS-fl-HTT-128Q”: 5.3 PRC/ommatidium (n = 360 ommatidia from 20 flies); The difference between the two was significance (p<0.001, Mann-Whitney rank-sum test). Control flies expressing Fl-HTT-16Q in both normal and dhap40-ko backgrounds showed normal seven photoreceptor cells (blue bars). (I) “UAS-HAP40; nsyb-Gal4 >UAS-fl-HTT-128Q” flies: 5.1 PRC/ommatidium (n = 331 ommatidia from 20 flies); control “UAS-Luciferase-dsRNA; nsyb-Gal4 >UAS-fl-HTT-128Q” flies: 4.5 PRC/ommatidium (n = 320 ommatidia from 20 flies). The difference between the two is significance (p<0.001 by Mann-Whitney rank-sum test). Control flies co-expressing HTT-16Q with HAP40 or with luciferase (green bars) all had seven intact photoreceptor cells. (H and J) Bar chart presentation of the average life spans of the adult flies expressing fl-HTT-128Q. (H) Average life span was 17 days for “dhap40 ko3; nsyb-Gal4/UAS-fl-HTT-128Q” flies (n = 179) and 15 days for control “nsyb-Gal4/UAS-fl-HTT-128Q” flies (n = 229). The difference between the two is significance (p<0.001. Log-rank test). (J) Average life span was 24.3 days for flies co-expressing HTT-Q128 with HAP40 (genotype: “nsyb-Gal4>UAS-HAP40/+; UAS-HTT-128Q/+”. n = 141), and 24.5 days for control flies co-expressing HTT-Q128 with luciferase-dsRNA (“nsyb-Gal4> UAS-luciferase-RNAi/+; UAS-HTT-128Q/+”, n = 174), with no significant difference between the two genotypes (p = 0.9 by Log-rank test). (K-M) Western blot assays for the ectopically expressed human HTT protein in (K and L) flies and (M) human cells, and quantifications from three independent repeat experiments show in bar charts below. (K) The levels of human HTT expressed from the same UAS-HTT transgene was ~60% lower (** p< = 0.01 (student’s t-test), N = 5 repeats) in dhap40-ko (lane 3) than in normal (WT, lane 1) background, and was absent in control flies lacking nSyb-Gal4 driver (lane 2). (L) The levels of HTT were about four times higher and HAP40 about three times higher in flies co-expressing HTT and HAP40 (lane 2) than flies expressing HTT (lane 3) or HAP40 (lane 1) alone, all driven by nSyb-Gal4. (M) Western blot assays for HTT and HAP40 levels in HEK293T cells with simultaneous co-expression of FLAG-tagged HTT-23Q and HTT-145Q, ran as three repeat experiments (N = 3) probed with anti-FLAG and anti-HAP40 antibodies, as indicated. Co-transfection with HAP40 led to an average of about two-fold increase of HAP40 and three- to five-fold increase of HTT-23Q and HTT-145Q (compare lane 4–6 with lanes 1–3). *** p< 0.001 (student’s t-test), which were calculated as the fold changes in HAP40 and HTT levels after co-transfection with HAP40 as compared to before HAP40 transfection.
Fig 4: Proteasome mediates HAP40 degradation.Western blot assays and quantifications for endogenous HAP40 and HTT proteins in HTT-KO, HAP40-KO or wildtype (WT) HEK293 cells under different treatments, as indicated. (B and D) Normalized levels of (B) HAP40 or (D) HTT proteins from three repeat experiments, corresponding to (A) and (C), respectively. Treatment with proteasome inhibitor MG132 for 5 hours partially but significantly restored the levels of depleted HAP40 protein in HTT-KO cells (A and B, N = 5 repeats for all the experiments, p = 0.002 for HTT-KO #1 line, p = 0.018 for HTT-KO #2 line), but showed no clear effect on the levels of endogenous HTT protein in HAP40-KO cells (C and D, N = 4 repeats for all the experiments). Autophagy/lysosome inhibitor ammonium (NH4+) and CQ, also treated for 5 hours. CQ showed partially but significant rescue of HTT levels in HAP40-KO cells (N = 4 repeats for all the experiments. p = 0.016 for HAP40-KO #1 line, p = 0.002 for HAP40-KO #2 line). NH4+ behaved similarly as DMSO mock treatment in both HTT-KO and HAP40-KO cells. * p< 0.05, ** p< 0.01 (student’s t-test). n.s., no significance. α-Tubulin served as loading and normalization controls in all the experiments.
Fig 5: dhap40 mutants show similar loss-of-functions phenotypes as dhtt null flies.(A) Survival curves (top) and bar-chart panels on average life span (bottom) as well as (B) climbing assays (top) and bar-chart panels on climbing ability (bottom) for two dhap40 null alleles ko3 and ko7 (green and purple lines, respectively), together with wildtype control (blue line) and null dhtt-ko mutants (red line), as indicated. Note that both dhap40 mutant alleles manifested similar, albeit significant weaker, phenotypes than dhtt-ko flies. (A) dhap40-ko had an average life span of 40 days (n = 230 total), differing significantly (p<0.001, log-rank test) from both wildtype (59 days, n = 253) and null dhtt-ko mutants (33 days, n = 209). (B) Climbing assays for 28-day-old adult flies of the indicated genotypes, with dhap40 flies (n = 180) showing significant differences (** p<0.01 and *** p<0.001, t-test) from both wildtype (n = 200) and dhtt-ko mutants (n = 180). (C and D) Survival curves (top) and bar-chart panels on average life span (bottom) for rescue experiments on the longevity deficit of dhap40ko3 flies, restored by ectopically expressed (C) fly dHap40 from the UAS-dhap40 or (D) human HAP40 from UAS-HAP40 transgenes, all directed by a ubiquitous da-Gal4 driver (red lines. Genotype: “w1118, dhap40ko3; UAS-dhap40/da-Gal4” (C, n = 300) or “w1118, dhap40ko3; UAS-HAP40/da-Gal4” (D, n = 307)). The survival curves of the rescued flies in (C) and (D) were statistically indistinguishable from wildtype (blue lines, p>0.5, log-rank test n = 253. Genotype: “w1118”), but significantly different from the two controls: (1) dhap40ko3 flies carrying da-Gal4 driver alone (purple lines. *** p<0.001, log-rank test. Genotypes: “w1118, dhap40ko3; da-Gal4/+”. n = 308); and (2) dhap40ko3 flies carrying (C) UAS-dhap40 (Genotype: “w1118, dhap40ko3; UAS-dhap40/+”. n = 355) or (D) UAS-HAP40 (Genotype: “w1118, dhap40ko3; UAS-HAP40/+”. n = 337) transgene alone (green lines, *** p<0.001, log-rank test), as indicated. The same set of data from wildtype (blue lines) and “dhap40ko3; da-Gal4/+” (purple lines) were used in both (C) and (D) as shared controls. (E and F) co-IP experiments between (E) transfected HTT and dHap40 in HEK293 cells or between (F) endogenous HTT and HAP40, as indicated. Note that the pulldown efficiency by HTT was significantly higher against (F) endogenous HAP40 than (E) dHap40.
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