EMBO/SourceData huggingface dataset:
Figure 6C: Relative mRNA expression of senescence markers (p16 and p21) and SASP molecules (PAI-1, IL6, IL8) in GEnC at early (p9) or late (p17) passage. n = 5 independent experiments. Data information: Data are means ± SEM. Statistical analysis: t-student testE: Immunofluorescence of paxilin (green) and phalloidin (red) in podocytes stimulated with control medium, supernatant (SN) from glomerular endothelial cells from early (p9) or late (p17) passage and treated either with vehicle or Tiplaxtinin, a PAI-1 inhibitor (upper panel). Quantification of focal adhesions (lower panel) was performed by counting the number of paxilin-positive spots in 30 cells from three independent experiments. Original magnification X1000. Scale bar = 20 μm. Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer test
Figure 7A: PAI-1 staining (left panel) and quantification (right panel) in glomeruli of kidney biopsies from young (< 40 years of age, n = 8) or old (> 80 years of age, n = 10) kidney donors at time of transplantation (M0). Original magnification X400. Scale bar = 20 μm. B: p16 staining (left panel) and quantification (right panel) in glomeruli of kidney biopsies from young (< 40 years of age, n = 8) or old (> 80 years of age, n = 10) kidney donors at time of transplantation (M0). The arrows point to the staining. Original magnification X400. Scale bar = 20 μm. Data information: Data are means ± SEM. Statistical analysis: t-student testF: Quantification of the glomerulosclerosis score at time of (M0), or 12 months after (M12) kidney transplantation from elderly donors in patients displaying a negative (PAI-1-, n = 17) or positive (PAI-1+, n = 13) glomerular PAI-1 staining at time of transplantation (M0). Data information: Data are means ± SEM. Statistical analysis: ANOVA followed by the Tukey-Kramer testH: Urinary PAI-1 measured by ELISA in a cohort of elderly patients with (n = 48) or without (n = 48) chronic kidney disease. Data information: Data are means ± SEM. Statistical analysis: t-student test
Figure 1Coomassie-stained SDS-PAGE gel showing purified protein from the predicted bacterial OTU constructs.Ub-PA activity-based probe assay for wild-type (WT) and catalytic Cys-to-Ala mutants (CA). Strong, Cys-dependent reactivity is indicated with asterisks.Ub-KG(TAMRA) cleavage assay monitored by fluorescence polarization at the indicated DUB concentrations. Note that BurkOTU displays an increase in fluorescence polarization, indicative of noncovalent binding.Heatmap representation of DUB activity against the Ub-KG(TAMRA) substrate shown in G., including the WT enzyme and Ala substitutions at the predicted catalytic Cys, general base His, or acidic position. Substrate remaining at the end of the assay is reported after correction against an initial reading from an equivalent assay performed with the catalytically inactive CA mutants.
Figure 2Ub/Ub-like specificity assay measuring activity of WT and inactive Cys-to-Ala wMelOTU toward the Ub-, ISG15-, NEDD8-, and SUMO1-KG(TAMRA) substrates. Ub/Ub-like specificity assay measuring activity of WT and inactive Cys-to-Ala BurkOTU toward the Ub-, ISG15-, NEDD8-, and SUMO1-KG(TAMRA) substrates. Note that the rise in fluorescence polarization signal, indicative of a noncovalent interaction, is specific to the Ub substrate.Heatmap representation of corrected OTU activities toward the Ub and Ub-like fluorescent substrates. In the reactions marked by an asterisk, an unusually high level of noise in fluorescence polarization signal was observed, likely a result of high OTU concentration.Ub chain specificity assay measuring wMelOTU activity toward the eight diUb linkages. Reaction samples were quenched at the indicated timepoints, resolved by SDS-PAGE, and visualized by Coomassie staining.Ub chain specificity assay measuring BurkOTU activity toward the eight diUb linkages. Reaction samples were quenched at the indicated timepoints, resolved by SDS-PAGE, and visualized by Coomassie staining.Heatmap representation of WT bacterial OTU activities toward the eight diUb linkages at the indicated timepoints.
Figure 3Ub-KG(TAMRA) cleavage assay monitoring the effects of structure-guided wMelOTU mutations. These data were collected in parallel with those presented in Fig. 1G and the WT dataset is shown again for reference.
Figure 4Ub-KG(TAMRA) cleavage assay monitoring the effects of structure-guided EschOTU mutations. These data were collected in parallel with those presented in Fig. 1G and the WT dataset is shown again for clarity.
Figure 5Ub-PA activity-based probe assay for WT vOTU and sequence-permutated vOTUP. Strong reactivity is indicated with asterisks.Ub-KG(TAMRA) cleavage assay monitored by fluorescence polarization for WT vOTU and sequence-permutated vOTUP.
Figure 1About 25 μg of membrane‐enriched lysate from the mass spectrometry experiments was immunoblotted with anti‐PINK1 antibody. TOMM40 and GAPDH serve as markers for mitochondria and cytoplasm, respectively.
Figure 3A-CConfirmation by mass spectrometry that Rab8A (A), Rab8B (B) and Rab13 (C) Ser111 is phosphorylated upon PINK1 activation after CCCP treatment. Flp‐In T‐Rex HEK293 cells expressing empty‐FLAG, WT PINK1‐FLAG and KI (D384A) PINK1‐FLAG were transfected either with HA‐Rab8A (A), HA‐Rab8B (B) or HA‐Rab13 (C) induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole‐cell lysates (10 mg) were immunoprecipitated with anti‐HA agarose, resolved by SDS-PAGE and stained with colloidal Coomassie blue (second panel). Coomassie‐stained bands migrating with expected molecular mass of HA‐Rabs were excised, in‐gel digested with trypsin and subjected to high‐performance liquid chromatography with LC‐MS/MS on an LTQ‐Orbitrap mass spectrometer. Upper panel shows the extracted ion chromatogram (XIC) analysis of Ser111‐containing phosphopeptides (8A, NIEEHApSADVEK; 8B, NIEEHApSSDVER; 13, SIKENApSAGVER) with the combined signal intensity of the 2+ and 3+ forms of the peptide indicated on the y‐axis. Note that the Ser111 phosphopeptide was only detected in samples from WT PINK1‐FLAG‐expressing cells following CCCP stimulation.DCharacterisation of Rab8A, Rab8B and Rab13 phospho‐Ser111 antibodies. Flp‐In T‐Rex HEK293 cells expressing empty‐FLAG, WT PINK1‐FLAG and KI (D384A) PINK1‐FLAG were transfected with either WT or Ser111Ala‐mutant (S111A) HA‐Rab8A, HA‐Rab8B or HA‐Rab13, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole‐cell lysates (0.25 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rab8A, Rab8B or Rab13 phospho‐Ser111 antibodies. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls.
Figure 4PINK1 is essential for CCCP‐mediated Rab8A Ser111 phosphorylation. WT and PINK1 KO HeLa cells were transfected with either WT or Ser111Ala (S111A) mutant constructs of HA‐Rab8A, HA‐Rab8B or HA‐Rab13. Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1‐3xFLAG or KI (D384A) PINK1‐3xFLAG as indicated. After transfection for at least 24 h, cells were treated with DMSO as a vehicle control or CCCP for 20 h. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rab8A, Rab8B or Rab13 phospho‐Ser111 antibody. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls. For the lower panel, whole‐cell lysates (30 μg) were immunoblotted with total PINK1 antibody to confirm PINK1 expression and with GAPDH as loading controls.Endogenous Rab8A Ser111 phosphorylation is PINK1 dependent. WT and PINK1 KO HeLa cells were treated with DMSO as a vehicle control or CCCP for 20 h. Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1‐3xFLAG or KI (D384A) PINK1‐3xFLAG as indicated for at least 24 h before CCCP treatment. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A pre‐bound with protein A agarose followed by immunoblot with Rab8A phospho‐Ser111 antibody. Part of the immunoprecipitates was used to immunoblot with anti‐total Rab8A antibody as loading controls.
Figure 5Absence of Rab8A Ser111 phosphorylation in human mutant PINK1 patient fibroblasts. Primary skinfibroblasts were derived from a patient with homozygous PINK1 Q456X mutation or unaffected control. Cells were incubated with DMSO or CCCP for 20 h, and whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho‐Ser111 antibody. Lysates (1 mg) were also subjected to immunoprecipitation with polyclonal anti‐PINK1 antibody and immunoblotted with monoclonal PINK1 antibody. Equal loading of protein extracts was confirmed by GAPDH.Absence of Rab8A Ser111 phosphorylation in PINK1 knockout MEFs. MEFs were derived from PINK1 knockout embryos or wild‐type controls (see Materials and Methods). Cells were incubated with DMSO or CCCP for 20 h, and whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho‐Ser111 antibody. Lysates (1 mg) were also subjected to immunoprecipitation with a polyclonal anti‐mouse‐specific PINK1 antibody and immunoblotted with a different anti‐mouse‐specific PINK1 antibody. Equal loading of protein extracts was confirmed by GAPDH.
Figure 6Rab8A is not required for the activation of Parkin E3 ligase activity at mitochondria in response to PINK1 activation by CCCPWild‐type (WT) or Rab8A knockout (KO) HeLa cells were transfected with WT or Cys431Phe (C341F)‐mutant Parkin. After transfection for 24 h, cells were treated with DMSO as a vehicle control or 10 μM CCCP for 6 h. Mitochondrial enriched extracts (mitochondrial lysate) were incubated with ubiquitin‐binding resins derived from his‐halo‐ubiquilin1 UBA domain tetramer (UBAUBQLN1). Captured ubiquitylated proteins were subject to immunoblotting with anti‐CISD1 and anti‐mitofusin 2 antibodies. Mitochondrial lysate and total lysate were also subjected to immunoblotting with indicated antibodies for loading and protein expression controls. Phospho‐Ser111 Rab8A was detected after Rab8A immunoprecipitation from 200 μg of mitochondrial lysate with anti‐Rab8A antibody.
Figure 7In vitro phosphorylation analysis of Rab8A by PINK1. WT or S111A‐mutant Rab8A (1.2 μg) was incubated in the presence of MBP‐fused WT or KI (D359A) TcPINK1 (1.1 μg) and Mg2+‐[γ‐32P] ATP for the indicated time. Samples were subjected to SDS-PAGE, and proteins were detected by colloidal Coomassie blue staining (lower panel). The [γ‐32P] incorporation to substrate was detected by autoradiography (upper panel). Cerenkov counting was used to calculate the stoichiometry of substrate phosphorylation as mol of [γ‐32P] incorporation/mol of substrate. Ubiquitin was used as a positive control of the TcPINK1 substrate.
Figure 8Time‐course comparison of PINK1‐mediated Rab8A, Rab8B and Rab13 Ser111 phosphorylation vs. Parkin Ser65 phosphorylation. Flp‐In T‐Rex HEK293 cells expressing WT PINK1‐FLAG were transfected with either WT or Ser111Ala‐(S111A) mutant HA‐Rab8A, HA‐Rab8B or HA‐Rab13, induced with doxycycline and stimulated with CCCP for the indicated time. In parallel, Flp‐In T‐Rex HEK293 cells expressing WT PINK1‐FLAG were transfected with either WT or Ser65 Ala (S65A)‐mutant Parkin. Whole‐cell lysates (0.25 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with indicated phospho‐Ser111 antibodies. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls. For the lower panel, whole‐cell lysates (30 μg) were immunoblotted with indicated antibodies.Time‐course comparison of endogenous PINK1‐mediated Rab8A, Rab8B and Rab13 Ser111 phosphorylation vs. Parkin Ser65 phosphorylation. HeLa cells were transfected with either WT or S111A‐mutant HA‐Rab8A, HA‐Rab8B or HA‐Rab13 for at least 24 h before CCCP treatment for the indicated time. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with indicated phospho‐Ser111 antibodies. In parallel, HeLa cells were transfected with either WT or S65A‐mutant Parkin and whole‐cell lysates (30 μg) were immunoblotted with indicated antibodies.
Figure 9Rabin8‐catalysed mant‐GDP release from mant‐GDP loaded WT, S111A and S111E mutants of Rab8A. The Rab proteins (1 μM) were incubated with 100 μM GDP in buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 1 mM MgCl2, 2 mM DTE), and the reaction was started by the addition of 0.5 μM Rabin8. The decrease in mant fluorescence was used as a measure of mantGDP release.Rab8A Ser111 phosphorylation impairs Rabin8 interaction in cells. Rab8A KO HeLa cells were transfected with wild‐type (WT), S111E or S111A HA‐Rab8A. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rabin8 or anti‐HA antibody. Lysates were immunoblotted with Rabin8 or anti‐HA antibody to confirm equivalent expression of Rabin8 and WT and mutant HA‐Rab8A in extracts.
Figure 1B Mass spectrometry analysis of proteins co-purified with Ccq1HA. Shown is a volcano plot for proteins significantly enriched in Ccq1HA relative to untagged Ccq1 (left panel: WT cells; right panel: rik1∆ cells). Members of the CLRC complex and shelterin are highlighted in red and blue, respectively. Bottom panel displays proteins enriched (log2 ≥ 5 or pval ≤ 0.01)ChIP-qPCR analysis of epitope-tagged shelterin components in WT and mutant strains as indicated (negative control: untagged strain). Positions on x-axis denote distances relative to telomeric repeats (telomere-proximal PCR primer) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEMChIP-qPCR analysis of epitope-tagged shelterin components in WT and mutant strains as indicated (negative control: untagged strain). Positions on x-axis denote distances relative to telomeric repeats (telomere-proximal PCR primer) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEMChIP-qPCR analysis of epitope-tagged shelterin components in WT and mutant strains as indicated (negative control: untagged strain). Positions on x-axis denote distances relative to telomeric repeats (telomere-proximal PCR primer) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEMF ChIP analysis of Ccq1HA and FLAG-TetR-Clr4* (see scheme). Expression and tethering of FLAG-TetR-Clr4* are controlled by thiamine/AHT addition (red dots indicate induced/tethered FLAG-TetR-Clr4*; empty circles indicate non-induced/non-bound controls) (n = 3 independent experiments) Data information data are normalized to input and represented as mean ± SEM
Figure 2A Left: Schemes of CLRC and the shelterin complex. Middle and right: Scheme depicting the position of the ura4+ reporter gene and heterochromatin islands relative to telomeric repeats and TAS regions; note that the minichromosomesome Ch16 does not contain TAS. B-C ChIP-qPCR analyses of H3K9me2 (B) and H3 (C) in WT and ccq1Δ cells (n = 3 independent experiments. D Fold change of WT over ccq1Δ for H3K9me2 and H3 level Data information: In (B, C), data are normalized to input and to the average of three euchromatic loci (EC) as internal contro and represented as mean ± SEM
Figure 3ChIP-qPCR analyses of H3 in WT and mutants as indicated (n = 3 independent experiments). Data information data are represented as mean ± SEChIP-qPCR analyses of H3 in WT and mutants as indicated (n = 3 independent experiments). Data information data are represented as mean ± SEChIP-qPCR analyses of H3K9me2 in WT and mutants as indicated (n = 3 independent experiments). Data information data are represented as mean ± SE
Figure 4A ChIP-seq reads mapped to TEL2R (shown in reverse orientation for consistency)
Figure 5A ChIP-qPCR analysis of H3 at endogenous TAS and a fragment spanning the TAS region from 115-905 bp (relative to telomeric repeat) inserted into the leu1+ locus (see scheme). The TAS fragment was inserted into a strain that lacks endogenous TA Shown are ChIP analyses for WT (left) and ccq1∆ (right; note different the scale of the y-axis) (n = 9-10 independent experiments except for ectopic TAS in ccq1∆ strain where n = 3) Data information data are represented as mean ± SEB ChIP-qPCR analysis of H3 at reporter genes (ura4+ and his3+) at various chromosomal locations (see schemes) in WT cells. TAS1, TAS2, and TAS3 correspond to position 116 bp, 2851 bp and 6291 bp (relative to telomeric repeats), respectively (n = 3 independent experiments). Data information data are represented as mean ± SE
Figure 6A, B RT-qPCR analysis of transcript levels of TERRA (A) and tlh1+ (B) in WT strain and mutants as indicated (double mutants with ccq1Δ are indicated by blue dot). Data information: In (A, B), data is represented as mean ± SEM from 3 independent experiments and shown relative to WT leve
Figure 7A Genomic copy number of his3+, ura4+ and TAS1 in indicated strains harboring the reporter genes tel1L::his3+ and tel2L::ura4+. Cultures from individual WT and freshly generated knockout clones (ccq1Δ, n = 16; clr4Δ, n = 5; ccq1Δ clr4Δ, n = 5) were pre-grown on selective media for several days and inoculated at day 0 to grow in liquid media with regular back-dilution (every 24 hours, approximately 7 generations). Samples were taken at indicated harvest times, and relative copy numbers of genomic regions were assessed by qPCR (normalization against intrachromosomal loci). Black and rainbow color lines indicate WT strains and individual ccq1+ deletion mutants (clones), respectivelyB qPCR analysis as in (A) but with strains harboring the minichromosome Ch16 m23::ura4+ (ccq1Δ, n = 14). Since Ch16 is unstable in ccq1Δ cell Note that Ch16 does not harbor TAS sequences
Figure 3Representative HDX-MS curves showing the protected regions in CD53 and CD2. The HDX experiments were performed (manually or with a LEAP automation system) in triplicate. Error bars show the standard deviation.D Representative HDX-MS curves showing the protected regions in CD81 and CD19. The HDX experiments were manually performed in triplicate. Error bars show the standard deviation.
Figure 5A CD81 knockout of 293T cells. The knockout cell line was generated by CRISPR-Cas9. The immunoblots using CD81 antibody and actin antibody were conducted on the same film.B CD81 facilitates CD19 maturation. CD81 and CD19 (300 ng DNA) were transfected into the CD81-knockout 293T cells. Anti-FLAG immunoblot was performed for CD19 with a C-FLAG tag. The deglycosylation was performed by digesting the cell lysate with PNGase F or Endo H, following the manufacturers' instructions. The deglycosylation pattern of CD19 species (p: precursor, m and m': two mature forms) differs because the glycosylation content of these species is differentC Trypsin digestion of CD19. The digestion was performed at 37 °C with intact cells. The mature CD19 forms (m and m') can be digested because they are surface expressed, whereas the intracellular precursor form (p) is protected from the digestion.E EC1 mutants of CD81 interfere with the accurate glycosylation of CD19. Chol: mutations at the cholesterol-binding site of CD81. CD81 constructs and CD19 (300 ng DNA) were co-transfected into the CD81-knockout 293T cells. Top, Anti-FLAG immunoblot was performed for CD19 with a C-FLAG tag. In presence of wildtype CD81 (wt), most CD19 is in the properly glycosylated mature form (m), and the overglycosylated m' form is not observed. With the EC1 mutants (A4 in particular), however, m' emerges and m shifts to high MW, indicating an alteration in the CD19 glycosylation. The level of intracellular p form is low with the wildtype CD81 or with the mutants. Bottom, Control experiment showing similar expression level of wild-type CD81 and CD81 mutants. Anti-V5 immunoblotting was conducted for the CD81 constructs with a C-V5 tag, and anti-actin immunoblotting was conducted on the same film.
Figure 6A Migration of naïve Cd53-/- cultured primary pre-B cells to 100 ng/mL CXCL12 compared to WT littermate controls (n = 5 mice per group) over three independent experiments. Bars and error bars represent mean ± standard error of the mean. **p < 0.01 by two-way ANOVA.B Migration of Cd53-/- pre-B cells transduced with empty, control, or mutant constructs in response to CXCL12 gradient, 3 days after transduction (n = 2-5 mice per group) over two independent experiments. Bars and error bars represent mean ± standard error of the mean. **p < 0.01 and ***p < 0.001 by two-way ANOVA.
Figure 1A. LN229 and U87 cells treated with Wnt 3a (0, 10, 20, 50, 100 ng/ml) for 4 hr. The indicated proteins were analyzed by Western blotting.B. LN229 cells treated with or without Wnt3a (50 ng/ml) were pulsed with [35S] methionine for 30 min and chased for the times indicated. Protein extracts were used for IP with FoxM1 antibodies and subjected to SDS-PAGE and autoradiography. The intensities of the FoxM1 bands in autoradiography were then quantified by using NIH Image software, and the densities of the FoxM1 bands at time 0 were set as 100%. Values are mean ± SD from two independent experiments.C. Levels of FoxM1 were analyzed by Western blotting in LN229 and U87 cells treated with Wnt inhibitor DKK1 (100 ng/ml) for indicated times. Noted that the basal levels of FoxM1 and β-catenin in (A) and (C) are similar but look different due to variations on the exposure time of the Western blots.D. FoxM1 expression levels were determined in LN229 cells treated with or without GSK3 inhibitor LiCl (10 mM) and CHX (100 µg/ml) for indicated times.E. GSK3 wild-type (WT) and knockout (KO) cells were treated with CHX (100 µg/ml) for indicated times, and FoxM1 and GSK3α/β expression levels were determined by Western blotting.F. Flag-FoxM1 and GSK3βKA or GSK3βKD plasmids were co-transfected into 293T cells for 48 hr. The cells were then treated with CHX (100 µg/ml) for indicated times. Exogenous Flag-FoxM1 expression level was determined by Western blotting.
Figure 2B. Lysates from 293T cells expressing Flag-FoxM1 or its mutants were subjected to IP using mouse anti-Flag antibody, followed by IB with GSK3α/β antibody (upper panel) and rabbit anti-Flag antibody (middle panel).D. HA-ubiquitin and Flag-tagged FoxM1 (WT) or mutants FoxM1S228A, FoxM1T309A, or Flag-FoxM1S474A plasmid were co-transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag antibody, followed by IB with Flag and HA antibody.E. HA-ubiquitin was co-transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with IgG or FoxM1 phospho-S474 antibody, followed by IB with FoxM1 and HA antibody.F. HA-tagged K48-only ubiquitin or K63-only ubiquitin construct was transfected into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with IgG or FoxM1 antibody, followed by IB with FoxM1 and HA antibody.G. GSK3βCA or vector and Flag-tagged FoxM1 (WT), mutants S474A or S478A were co-transfected into 293T cells. Lysates of the cells were subjected to IP with Flag antibody and followed by IB with FoxM1 phospho-S474 antibody.