Fig 1: psmc3 morphants and F0 mosaic zebrafish exhibit cataract and show abnormalities during the semicircular canal development in the ear AScheme of a zebrafish eye.B, B'Cataract detection revealed abnormal lens reflection in psmc3 morpholino (MO)-mediated knockdown but not in controls (uninj, ctrl-mo). Similarly, abnormal lens reflection was also observed in embryos injected with sgRNA + Cas9 but not in sgRNA-injected embryos without Cas9 (sgRNA2). Co-injection of wt psmc3 mRNA with either psmc3-mo or sgRNA2 + Cas9 reduced the number of embryos presenting abnormal lens reflection. Scale bar = 50 µm. (B') Quantification of embryos with abnormal lens reflection.CRepresentative image of a zebrafish ear at 4 dpf. kc = kinocilia.D, D'Brightfield images of inner ear development (lateral position). (D) Epithelial projections were fused and formed canal pillars in 4-day-old uninjected and control-injected fish (ctrl-mo, sgRNA2) but not in morphants (mo) and crispants (sgRNA2 + Cas9). Co-injection of wt psmc3 mRNA with psmc3-mo or sgRNA + Cas9 reduced the number of embryos presenting abnormal ear phenotype. Black asterisks indicate fused pillars. Red arrowheads mark unfused projections. Scale bar = 100 µm. (D') Quantification of embryos with abnormal projection outgrowth.E, E'An anti-acetylated tubulin antibody (green) staining revealed an abnormal amount of kinocilia in psmc3 crispants (sgRNA2 + Cas9) compared to uninjected and control-injected embryos (sgRNA2). Nuclei are stained in blue with DAPI. Representative images show kinocilia of the lateral cristae. Scale bar = 20 µm. (E') Quantification of embryos with an abnormal amount of kinocilia.
Fig 2: Schematic diagram depicting TCF11/Nrf1 processing pathway in response to proteasome dysfunction and/or proteotoxic stress in patient carrying the deep intronic homozygous PSMC3 variation (left) and in healthy subjects (right)Under normal conditions, TCF11/Nrf1 is a short-lived ER membrane (endoplasmic reticulum)-resident protein, which is rapidly subjected to proteasome-mediated degradation following retro-translocation to the cytosol by ER-associated degradation machinery (ERAD). In case of proteasome dysfunction (i.e. proteotoxic stress), the half-life of TCF11/Nrf1 is prolonged and become then a substrate for the NGLY1 and DDI2, thereby giving rise to a C-terminal cleaved fragment that enters into the nucleus. After nuclear translocation, cleaved TCF11/Nrf1 associates with Maf and promotes the expression of proteasomes genes, so that protein homeostasis can be restored. In patients carrying the c.1127 + 337A>G homozygous PSMC3 variation, defective proteasomes promote a constitutive activation of TCF11/Nrf1, thereby resulting in increased assembly of newly synthetized non-functional proteasomes, which in turn activate TCF11/Nrf1 again. Over-activation of TCF11/Nrf1 results in pathway exhaustion thus rendering patient cells incapable of responding to further proteotoxic stress.
Fig 3: Fibroblasts derived from patient carrying the c.1127 + 337A>G homozygous PSMC3 variation exhibit an increased amount of both proteasome complexes and ubiquitin–protein conjugates AWhole-cell lysates from control and patient (case index, CI) fibroblasts were assessed by 3–12% native-PAGE gradient gels with proteasome bands (30S, 26S and 20S complexes) visualized by their ability to cleave the Suc-LLVY-AMC fluorogenic peptide.BTen micrograms of control and patient cell lysates was tested for their chymotrypsin-like activity by incubating them with 0.1 mM of the Suc-LLVY-AMC substrate at 37°C over a 180-h period of time in quadruplicates on a 96-well plate. Indicated on the y-axis are the raw fluorescence values measured by a microplate reader and reflecting the AMC cleavage from the peptide. Bars show the mean of 4 independent experiments ± SD.CProteasome complexes from control and patient fibroblasts separated by native-PAGE were subjected to Western blotting using antibodies specific for a6, Rpt5 (PSMC3) and PA28-a, as indicated.DProteins extracted from control and CI PSMC3 were separated by 10 or 12.5% SDS–PAGE prior to Western blotting using primary antibodies directed against ubiquitin and several proteasome subunits and/or components including a6, ß1, ß; ß5, ß5i, Rpt2 (PSMC1), Rpt5 (PSMC3), Rpt3 (PSMC4), Rpt4 (PSMC6) and PA28-a, as indicated. For the PSMC3 staining, two exposure times are shown. Arrow indicates an additional PSMC3 species corresponding to the expected size of the truncated PSMC3 variant. Equal protein loading between samples was ensured by probing the membrane with an anti-a-Tubulin antibody. Source data are available online for this figure.
Fig 4: Family pedigree and cDNA analysis AFamily pedigree. Variant segregation analysis of PSMC3. Electropherogram of a part of intron 10 of PSMC3 encompassing the identified variation (c.[1127 + 337A>G];[1127 + 337A > G], p.[(Ser376Arg15*)];[(Ser376Arg15*)]) in the affected individuals, their unaffected parents and siblings. The variation was found at the homozygous state in the affected individuals (II.2, II.4, II.7) and at the heterozygous state in the parents (I.1, I.2, I.3, I.4, I.5, I.6) and was either at the heterozygous state (II.5) or absent in the unaffected siblings (II.1, II.3, II.6).BFace (up) and profile (down) photographs for patients II.4 (a: 8 yo, b: 16 yo), II.2 (c: 6 yo, d: 14 yo) and II.7 (e: 1 yo, f: 7 yo) over time. One can observe prominent supraciliary arches, synophrys, sunken cheeks, short philtrum and retrusion in the malar region.CSubcutaneous calcifications found only on knees (g: 9 yo and h: 16 yo) and on elbows (i: 9 yo) of patient II.4. White hair were present only on the legs of the 3 patients as illustrated for patient II.4 (j: 16 yo).DTemporal bone CT scan from patient II.7 (left column) and a control (right column) showing malformation of the semicircular canal. The left ear is shown on the upper panels while the right ear on the lower panels.EAmplification of the cDNA fragment between exons 9–10 and 11 of PSMC3 showing the abnormally spliced RNA fragment. One band at 180 bp representing the normal allele is seen for the control and two bands for the individual II.4 (pathologic allele at 300 bp).FSchematic representation for the incorporation of the 114 bp intronic sequence resulting from the c.1127 + 337A > G deep intronic variation on the mRNA. Sanger sequencing of the fragment between exons 9–10 and 11 of the PSMC3 cDNA obtained from patient II.4 fibroblasts’ RNA, showing the insertion of the 114 bp cryptic exon. As a comparison, the schematic representation and sequence from a control individual are shown above. Source data are available online for this figure.
Fig 5: Effect of the deep intronic PSMC3 variant on the proteasome function AAnti-ubiquitin Western blot in control and patient fibroblasts and total amount of proteins loaded (stainfree) showing increased ubiquitination in patient cells (lane 4).BHistogram showing the quantification of ubiquitin with Western blot assays. The data shown correspond to the sum of all bands detected by the anti-ubiquitin antibody expressed as a percentage of the amount of ubiquitin in “Control 1” cells. Bars show mean of ten independent experiments ± SD (n = 10, t-test *P < 0.01, **P < 0.05).CMass spectrometry results from the co-immunoprecipitation with PSMC3 are displayed as the normalized total number of spectra count of each protein computed as the mean from 3 controls (x axis) vs. the mean of patient II.4 triplicate. Proteasome subcomplexes are coloured according to the displayed legend, and standard ratio lines are drawn.
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