BAP1 regulates IP3R3-mediated Ca2+ flux to mitochondria suppressing cell transformation

Abstract

BRCA1-associated protein 1 (BAP1) is a potent tumour suppressor gene that modulates environmental carcinogenesis1,2,3. All carriers of inherited heterozygous germline BAP1-inactivating mutations (BAP1+/−) developed one and often several BAP1−/− malignancies in their lifetime4, mostly malignant mesothelioma, uveal melanoma2,5, and so on6,7,8,9,10. Moreover, BAP1-acquired biallelic mutations are frequent in human cancers8,11,12,13,14. BAP1 tumour suppressor activity has been attributed to its nuclear localization, where it helps to maintain genome integrity15,16,17. The possible activity of BAP1 in the cytoplasm is unknown. Cells with reduced levels of BAP1 exhibit chromosomal abnormalities and decreased DNA repair by homologous recombination18, indicating that BAP1 dosage is critical. Cells with extensive DNA damage should die and not grow into malignancies. Here we discover that BAP1 localizes at the endoplasmic reticulum. Here, it binds, deubiquitylates, and stabilizes type 3 inositol-1,4,5-trisphosphate receptor (IP3R3), modulating calcium (Ca2+) release from the endoplasmic reticulum into the cytosol and mitochondria, promoting apoptosis. Reduced levels of BAP1 in BAP1+/− carriers cause reduction both of IP3R3 levels and of Ca2+ flux, preventing BAP1+/− cells that accumulate DNA damage from executing apoptosis. A higher fraction of cells exposed to either ionizing or ultraviolet radiation, or to asbestos, survive genotoxic stress, resulting in a higher rate of cellular transformation. We propose that the high incidence of cancers in BAP1+/− carriers results from the combined reduced nuclear and cytoplasmic activities of BAP1. Our data provide a mechanistic rationale for the powerful ability of BAP1 to regulate gene–environment interaction in human carcinogenesis.

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Figure 1: BAP1 localizes at the ER and modulates Ca2+ signalling and apoptosis.
Figure 2: IP3R3 and BAP1 interaction.
Figure 3: BAP1 deubiquitylates IP3R3.
Figure 4: Reduced nuclear and cytoplasmic BAP1 levels increase survival from DNA damage, resistance to apoptosis, and foci formation.

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Acknowledgements

We are grateful to the members of the L and W families who donated their cells to our research. We acknowledge K. Dixon for advice on ultraviolet radiation studies, M. Pagano for advice on ubiquitylation assays, H. Yu for advice on DNA repair studies, I. Pagano for review of all statistical analyses, and G. Khan for technical support. This work was supported by grants National Cancer Institute (NCI) R01 CA198138 to M.C.; by NCI R01 CA160715, DOD CA120355 to H.Y.; by the University of Hawai’i Foundation, which received unrestricted donations to support mesothelioma research from Honeywell International, to M.C.; by The Riviera United 4-a Cure to M.C. and H.Y.; and by the Italian Association for Cancer Research (AIRC) (IG-18624, MFAG13521) and the Italian Ministry of Health to P.P. and C.G. P.P. thanks C. degli Scrovegni for support.

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Authors

Contributions

M.C. conceived the study. A.B. led the experiments and prepared the figures. M.C., S.Pas. and H.Y. built pedigrees. M.N. and M.T. genotyped patients and controls. P.M., G.S. and L.K.F. performed skin biopsies. A.B., C.G. and K.V. established fibroblast cell cultures. A.B., C.G., S.Pat. and V.S. independently conducted and reproduced cell death assays. D.L. performed flow cytometry experiments. A.R. and C.T. performed electron microscopy studies. A.B. and C.G. performed subcellular fractionation studies. A.B. performed western blot, co-immunoprecipitation, aequorin-based Ca2+ measurements, and in vitro cell transformation assays. C.G. and S.Pat. performed single-cell Ca2+ measurements. C.G., S.Pat. and F.O. performed immunofluorescence studies. A.D. performed proximity ligation assay studies. A.B., with the help of S.K., performed ubiquitylation assays. L.P. performed qRT–PCR studies. A.B., C.G. and S.Pat., with the help of E.B.A., performed ionizing radiation and ultraviolet radiation studies. A.N., S.Pas., G.G. and H.I.P. discussed the results. M.C., H.Y. and P.P. coordinated the study and oversaw the results. A.B. and M.C. wrote the manuscript with help from co-authors.

Corresponding authors

Correspondence to Haining Yang or Paolo Pinton or Michele Carbone.

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Competing interests

M.C. has pending patent applications on BAP1. M.C. provides consultation for mesothelioma diagnosis. The authors have no other potential competing financial interests.

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Reviewer Information Nature thanks M. Campanella, N. Hayward, K. D. Wilkinson and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Fibroblasts from BAP1 germline mutation carriers have reduced BAP1 protein levels, do not show differences in growth or cell cycle progression, and are protected from apoptosis.

ad, Western blot: amounts of wild-type BAP1 in total cell lysates of W (a) and L (c) fibroblasts, matched by gender and age (see Supplementary Fig. 1; that is, W: 26_BAP1WT and 21_BAP1+/; and so on, as shown in subsequent figures); decimals indicate the amounts of BAP1 relative to α-tubulin as per densitometry. b, d, Densitometric analyses. BAP1 protein levels normalized to α-tubulin in fibroblast cell cultures from W (b) and L (d) family members matched by gender and age; data shown as mean ± s.e.m. of n = 4 (b) and n = 3 (d) biological replicates per condition, representative of three or more independent experiments. e, AlamarBlue assay was used to measure cell growth at the indicated time points, in fibroblasts from W (left) and L (right) family members; data shown as mean ± s.d. of n = 6 technical replicates per data point, representative of three or more independent experiments in biological replicates. f, Flow cytometry analyses showing percentage of cells in different phases of the cell cycle; data shown as mean ± s.e.m. of n = 4 biological replicates per condition. No differences were observed between BAP1WT and BAP1+/− fibroblasts from either W or L family members. g, Representative flow cytometry dot plots assessing annexin V/FITC and propidium iodide staining in fibroblasts from L (upper panels, n = 4 independent experiments: three biological replicates per condition, one culture replicate) and W (lower panels, n = 4 biological replicates per condition) family members treated with 100 μM H2O2 for 6 h. h, Late apoptotic cells calculated as the percentage of gated cells in the top right quadrant (Q2: annexin V+/PI+); data shown as mean ± s.e.m. of n = 4 biological replicates per condition. il, Cleaved caspase-3 levels measured by western blot in fibroblast cell cultures from W (i) and L (k) family members, matched by gender and age, treated with H2O2. Decimals indicate the densitometrically determined amounts of cleaved caspase-3 relative to α-tubulin. j, l, Cleaved caspase-3 densitometry of bands in BAP1+/− fibroblasts expressed relatively to the amounts detected in BAP1WT fibroblasts (100%); data shown as mean ± s.e.m. of n = 4 (j) and n = 3 (l) biological replicates per condition, representative of at least three independent experiments. m, Primary human mesothelial cells were transfected with control scrambled siRNA, or siRNAs–BAP1 (siBAP1#1 and siBAP1#5). After 24 h, cells were treated with 200 μM H2O2 for 6 h. Total cell lysates were prepared and analysed by western blot to compare cleaved caspase-3 levels. Decimals indicate densitometrically determined cleaved caspase-3 levels normalized to α-tubulin. Similar results were obtained in three separate human mesothelial cell primary cultures from different donors. Black, BAP1WT; red, BAP1+/−; *P < 0.05; ***P < 0.001, calculated using two-tailed unpaired Student’s t-tests. For western blot source images, see Supplementary Fig. 2.

Extended Data Figure 2 Subcellular fractionation and immunofluorescence showing BAP1 localization at the ER.

a, Western blot showing the amounts of BAP1 in the subcellular fractions of primary fibroblasts, human mesothelial cells, PPM-Mill (a human malignant mesothelioma cell line), and HEK293 (human embryonic kidney cells). H, homogenate; M, mitochondria; C, cytosol; N, nuclei. Markers: mitochondria (VDAC), ER (IP3R3), nuclei (Lamin B1), cytosol (α-tubulin). b, c, Immunofluorescence: BAP1 localization in WT fibroblasts (b) and human mesothelial cells (c). Cells were immunostained for BAP1 (green) and PDI (ER marker, red). Merged images show the overlapping yellow signal between BAP1 and PDI. Inserts show magnified merged images. BAP1, besides its nuclear localization, shows a diffuse pattern of punctate hyper-fluorescent spots in the cytoplasm that co-localized with the ER, both in BAP1WT fibroblasts (b) and in human mesothelial cells (c). Representative immunofluorescence images from n = 10 fields of view; scale bar, 10 μm. d, e, The specificity of BAP1 immunofluorescence staining was confirmed by complete disappearance of this immunofluorescence pattern when BAP1 was downregulated using two different siRNAs, but not in cells transfected with scrambled siRNA. Immunofluorescence in wild-type fibroblasts (d) and human mesothelial cells (e) after BAP1 silencing. Cells were transfected with control scrambled siRNA or siRNAs–BAP1 (siBAP1#1 and siBAP1#5). After 24 h, the cells were immuno-stained using monoclonal antibodies for BAP1 (green) and PDI (ER marker, red). Merged images show the overlapping signal (yellow) between BAP1 and PDI. Representative immunofluorescence images from n = 5 fields of view per condition; scale bar, 10 μm. f, Percentages of nuclear (N) and ER-localized BAP1 in BAP1WT and BAP1+/− fibroblasts, related to Fig. 1d. Densitometric analysis of the intensity of the bands was performed using ImageJ, and the amounts of nuclear and ER-localized BAP1 were normalized on the respective markers, lamin B1 (nuclei) and IP3R1 (ER). The total, combined, amount of nuclear and ER BAP1 was reduced by 47.9% in BAP1+/− fibroblasts, depicted by the smaller size of the pie chart. Percentages of BAP1 in the nuclear or ER fractions are relative to the total amount of nuclear and ER BAP1. g, Immunogold BAP1 labelling of cryo-sections in BAP1WT and BAP1+/− fibroblasts. Note reduced detection of BAP1 associated with the ER (arrows) in BAP1+/− cells; scale bar 200 nm. h, Immunofluorescence showing reduced cytoplasm/nucleus (C/N) ratio intensity in BAP1+/− fibroblasts. Cells were stained with DAPI (nuclei, blue) and BAP1 (red). Representative images from n = 20 fields of view per condition. Rainbow RGB LUT (red, green, blue look-up table) mask shows colour-coded contrast and pseudocolouring according to an arbitrary colour LUT (see pseudocolour scale, numbers on the scale are 0, black; 64, blue; 128, green; 191, light red; 255, darker red). Scale bar, 10 μm. The bar graph shows the mean ± s.e.m. of the cytoplasm/nucleus ratio intensity measured in random regions of interest (ROIs) of the acquired images: the areas (regions of interest) where the intensities of fluorescence were measured are indicated by white and green circles in the cytoplasm and nucleus respectively (n = 40 cells for BAP1WT; n = 46 cells for BAP1+/−); ***P < 0.001. P value calculated using two-tailed unpaired Student’s t-test. For western blot and electron microscopy source images, see Supplementary Figs 2 and 3.

Extended Data Figure 3 BAP1 modulates intracellular Ca2+ homeostasis.

a, BAP1+/− W-fibroblasts displayed reduced Ca2+ release from the ER after stimulation with 1 μM bradykinin compared with wild-type fibroblasts (see also Fig. 1e). b, c, BAP1+/− L (b) and W (c) fibroblasts showed reduced Ca2+ release from the ER after stimulation with H2O2. d, e, BAP1+/− L (d) and W (e) fibroblasts had reduced cytosolic Ca2+ concentrations ([Ca2+]c) after stimulation with 1 μM bradykinin. f, BAP1+/− W-fibroblasts, stimulated with 1 μM bradykinin displayed reduced mitochondrial Ca2+ concentrations ([Ca2+]m) (see also Fig. 1f). g, Representative traces of single-cell Ca2+ measurements using mitochondrial-targeted cameleon (4mtD3cpv) showing reduced mitochondrial Ca2+ in BAP1+/− fibroblasts upon treatment with 1 μM bradykinin. h, i, BAP1+/− L (h) and W (i) fibroblasts showed reduced cytosolic Ca2+ after stimulation with H2O2. j, k, BAP1+/− L (j) and W (k) fibroblasts displayed reduced [Ca2+]m after stimulation with H2O2. l, m, Representative time-lapse traces of single-cell Ca2+ measurements using 4mtD3cpv showing reduced mitochondrial Ca2+ in BAP1+/− L (l) and W (m) fibroblasts after stimulation with 100 μM H2O2 for 20 min. n, Reduced intracellular Ca2+ levels in stimulated BAP1+/− fibroblasts are independent of extracellular Ca2+ influx from the plasma membrane. BAP1+/− and matched wild-type fibroblasts were loaded with Fura-2/AM in Ca2+-free Krebs-Ringer buffer supplemented with 0.1 mM EGTA, then dynamic measurements of intracellular Ca2+ levels were conducted after addition of 1 μM bradykinin to the same buffer. Changes in intracellular Ca2+ responses after bradykinin stimulation are displayed as the ratio of fluorescence at 340/380 nm. o, p, Human mesothelial cells silenced for BAP1 displayed reduced [Ca2+]c (o) and [Ca2+]m (p) after stimulation with 1 μM bradykinin. Human mesothelial cell cultures were transfected with control scrambled siRNA or siRNAs–BAP1 (siBAP1#1 and siBAP1#5). After 24 h, cells were stimulated with 1 μM bradykinin and [Ca2+]c (o) and [Ca2+]m (p) were measured using targeted aequorin probes. q, r, MAFs derived from Bap1+/− mice displayed reduced [Ca2+]c (q) and [Ca2+]m (r) compared with Bap1WT mice, and thus they reproduce the human condition. Representative changes in [Ca2+]c (q) and [Ca2+]m (r) in MAF cells after agonist stimulation (100 μM bradykinin). For source data, see Supplementary Tables 1 and 2.

Extended Data Figure 4 BAP1 targeting to ER, nucleus, and cytoplasm differentially affects mitochondrial Ca2+ homeostasis.

a, Localization of Myc-tagged BAP1 chimaeras. Immunofluorescence: BAP1 localization in wild-type fibroblasts. ER chimaera (Myc–BAP1-ER): BAP1 was fused to the ER targeting sequence from the yeast UBC6 protein to target BAP1 to the cytosolic face of the ER membrane. Nuclear chimaera (Myc–BAP1-Nu): BAP1 was fused to a sequence derived from the glucocorticoid receptor NR3C1. Cytoplasmic chimaera (Myc–BAP1-Cyt): point mutations were introduced in the NLS region of BAP1 to prevent its nuclear localization (*stop codon). BAP1 localization is shown in green using a Myc-Tag antibody; the nuclei and the ER are shown in red using histone H3 (nucleus) and PDI (ER) antibodies. The merged signal is shown in yellow. Note the specific localization of the three chimaeras to the ER, nuclei, and cytoplasm. Mock, empty vector (control). Representative immunofluorescence images from n = 5 fields of view per condition; scale bar, 10 μm. b, Representative traces of single-cell Ca2+ measurements in BAP1+/− fibroblasts co-transfected with mitochondrial-targeted 4mtD3cpv and either BAP1 or targeted BAP1-ER, BAP1-Nu, BAP1-Cyt; mock, co-transfection with an empty vector. Mitochondrial Ca2+ uptake was followed over time after stimulation with 1 μM bradykinin. Descriptive statistics are shown in Supplementary Table 1.

Extended Data Figure 5 BAP1 stabilizes IP3R3.

a, Defining the linear dynamic range of BAP1 and IP3R3 detection by western blot. The top panel shows a representative western blot performed on a twofold dilution series (from 60 to 0.9375 μg) of total cell homogenates from BAP1+/− and matched control BAP1WT fibroblasts. Protein levels were determined using the following primary antibodies: BAP1 (C-4), Santa Cruz Biotechnology, catalogue number sc-28383, 1:300; IP3R3, BD Biosciences, catalogue number 610312, 1:500; α-tubulin (4G1), Santa Cruz Biotechnology, catalogue number sc-58666, 1:15,000. A horseradish peroxidase-conjugated secondary antibody (Stabilized Peroxidase Conjugated Goat Anti-Mouse, Thermo Scientific, catalogue number 32430; 1:1,000 for BAP1 and IP3R3, 1:5,000 for α-tubulin) was used to generate the chemiluminescent signal captured on autoradiography film. Digital images were acquired, then densitometric analysis of the intensity of the bands was performed using ImageJ, and expressed as arbitrary optical densitometry units (AU). The lower three panels show the arbitrary optical densitometry units for IP3R3, BAP1, and α-tubulin, plotted against the protein load. The dotted lines define the linear dynamic range of protein load for BAP1, IP3R3, and α-tubulin detection. b, c, Densitometric analysis of IP3R3 protein levels normalized to α-tubulin in fibroblasts from W (b) and L (c) family members, related to Fig. 2A, B. Densitometry of bands in BAP1+/− fibroblasts is expressed relative to BAP1WT fibroblasts (100%), matched by gender and age as indicated in Supplementary Fig. 1a, b; data shown as mean ± s.e.m. of n = 4 (b) and n = 3 (c) biological replicates per condition, representative of three independent experiments; *P < 0.05 calculated using two-tailed paired Student’s t-tests. d, Malignant mesothelioma cell lines with mutated BAP1 contained reduced amounts of IP3R3: PPM-Mill (wild-type BAP1), Phi (mutated BAP1 with shorter splicing isoform), HMESO (mutated BAP1 alternative splicing, shorter protein), and Rob (BAP1 null). e, Quantitative PCR analysis of ITPR3, the gene that codes for IP3R3. mRNA expression levels were normalized using the geometrical mean of B2M, 18S, and ACTB reference genes in BAP1WT and BAP1+/ fibroblasts. mRNA expression levels in BAP1+/− fibroblasts are expressed relative to BAP1WT. Data shown as mean ± s.e.m. of n = 6 technical replicates, representative of three independent experiments in biological replicates. For western blot source images, see Supplementary Fig. 2.

Extended Data Figure 6 BAP1 binds IP3R3.

a, Immunofluorescence, reduced BAP1 colocalization with IP3R3 in BAP1+/− fibroblasts compared with BAP1WT fibroblasts. Cells were immunostained for BAP1 (green) and IP3R3 (red). Images were processed with ImageJ software equipped with the Colocalization Highlighter plugin; the colocalization signal is shown in white. Scale bar, 10 μm. The bar graph depicts the decreased colocalization of BAP1 and IP3R3 in BAP1+/− fibroblasts, expressed as (%) mean ± s.e.m.; **P < 0.01 (n = 9 cells per condition), P value calculated using two-tailed unpaired Student’s t-tests. b, Co-immunoprecipitation (CoIP) of IP3R3 and BAP1 from HEK293 cells stably expressing Flag–HA–BAP1. c, Co-immunoprecipitation of endogenous IP3R3 and Myc-tagged wild-type BAP1 or the catalytically inactive BAP1(C91S). Washes (first and third) show loss of bound proteins that washed out during three sequential washes of the immuno-complexes. BAP1(C91S) retains the ability to bind IP3R3. d, The dominant negative effect of NT-IP3R3 overexpression on endogenous IP3R3 levels is counteracted by concomitant BAP1 overexpression, findings indicating that BAP1 binds IP3R3 and prevents its degradation. e, Schematic representation of BAP1 domains, truncated W and L mutants (see also ref. 5), and fragments. Domains of the 729-amino-acid BAP1 protein (1–729 amino acids), consisting of an N-terminal UCH domain (1–240 amino acids), a non-regular secondary structure (NORS) domain (240–598 amino acids), a C-terminal (CTD) domain (598–699 amino acids), and NLS (699–729). Numbers refer to amino-acid positions. BAP1(W) and BAP1(L) are the predicted truncations of BAP1 resulting from the germline mutations in W and L families, respectively. f, Mapping of the BAP1 region interacting with IP3R3. HEK293 cells were co-transfected with Flag–IP3R3(NT) and the indicated Myc-tagged BAP1 fragments expression vectors; cell extracts were used for co-immunoprecipitation with anti-Myc resin. The BAP1 region UCH–NORS had the highest binding affinity to Flag–IP3R3(NT), and the CTD–NLS region contributed to the binding, while the UCH region alone showed no interaction. For western blot source images, see Supplementary Fig. 2.

Extended Data Figure 7 Effects of BAP1 silencing on IP3R3 protein levels, mitochondrial Ca2+ uptake, and apoptosis.

a, Western blot of BAP1 and IP3R3 protein levels in BAP1WT fibroblasts silenced for BAP1. b, [Ca2+]m measurements after stimulation with 1 μM bradykinin in BAP1WT fibroblasts transfected with control scrambled siRNA or siRNAs–BAP1 (siBAP1#1 and siBAP1#5). c, Representative traces of single-cell Ca2+ measurements in BAP1WT fibroblasts transfected with mitochondrial-targeted 4mtD3cpv and control scrambled siRNA, or siBAP1#1 and siBAP1#5; mitochondrial Ca2+ uptake was followed over time after stimulation with 1 μM bradykinin. d, Reduced sensitivity to apoptosis after treatment with 100 μM H2O2 for 6 h in BAP1WT fibroblasts after BAP1 silencing. eh, IP3R3 silencing in BAP1WT fibroblasts (e) leads to decreased mitochondrial Ca2+ uptake after stimulation with 1 μM bradykinin—as shown both by cell-population experiments with mitochondrial-targeted aequorin (f) or single-cell experiments with 4mtD3cpv (g)—and protection from apoptosis (h). i, j, BAP1 or IP3R3 silencing in primary human mesothelial cells leads to reduced IP3R3 protein levels (i), and decreased [Ca2+]m after stimulation with 1 μM bradykinin (j and Extended Data Fig. 3p). In a, e, and f decimals indicate the amounts of IP3R3 or BAP1 relative to α-tubulin, as per densitometry. In d and h decimals indicate the amounts of cleaved caspase-3 relative to α-tubulin, as per densitometry. For western blot source images, see Supplementary Fig. 2. For source data in b, c, f, g, j, see Supplementary Tables 1 and 2.

Extended Data Figure 8 Effects of BAP1 rescue on IP3R3 protein levels, mitochondrial Ca2+ uptake, apoptosis, and IP3R3 deubiquitylation.

ac, Phi (human malignant mesothelioma cell line with mutated BAP1, see Extended Data Fig. 5d) cells were transiently transduced with wild-type BAP1 (Ad-BAP1), catalytically inactive BAP1(C91S) mutant, or control (Ad-GFP). Wild-type BAP1 stabilizes IP3R3 (a), increases [Ca2+]m after stimulation with 1 μM bradykinin (b), and enhances apoptosis (c), while the catalytically inactive BAP1(C91S) mutant was less effective. In c, cells were treated with 500 μM H2O2 for 6 h and total cell lysates were prepared and analysed by western blot to compare cleaved caspase-3 levels. df, Stable clones of HMESO (see Extended Data Fig. 5d), in which we reintroduced wild-type BAP1, the catalytically inactive BAP1(C91S), or an empty vector (mock), showed that wild-type BAP1—but not the catalytically inactive BAP1(C91S) mutant—stabilizes IP3R3 (d), and increases [Ca2+]m after stimulation with 1 μM bradykinin (e), and enhances apoptosis in cells treated with 100 μM H2O2 for 3 h (f). Total cell lysates were analysed by western blot to compare cleaved caspase-3 levels. g, h, Human mesothelial cells containing wild-type BAP1 were first silenced for BAP1 using siRNA and subsequently transduced with wild-type BAP1 (Ad-BAP1), catalytically inactive BAP1(C91S) mutant, or control (Ad-GFP). Wild-type BAP1 but not the catalytically inactive BAP1(C91S) mutant, (g) stabilizes IP3R3 and (h) increases [Ca2+]m after stimulation with 1 μM bradykinin. a, c, d, f, g, Decimals indicate the amounts of IP3R3 or cleaved caspase-3 relative to α-tubulin, as per densitometry. i, BAP1+/− fibroblasts were transduced with wild-type BAP1 (Ad-BAP1), truncated BAP1(W) and BAP1(L), or control (Ad-GFP), and [Ca2+]m was measured after stimulation with 1 μM bradykinin. j, Representative traces of single-cell Ca2+ measurements in BAP1+/− fibroblasts co-transfected with mitochondrial-targeted 4mtD3cpv and either BAP1 or the catalytically inactive BAP1(C91S) mutant; mock, co-transfection with an empty vector. Mitochondrial Ca2+ uptake was followed over time after stimulation with 1 μM bradykinin. k, Ubiquitylation assay showing that wild-type BAP1 (Myc–BAP1), but not BAP1(C91S) mutant, deubiquitylates the N terminus of IP3R3. HEK293 cells were co-transfected with Flag–IP3R3(NT) and either Myc–BAP1, Myc–BAP1(C91S), or empty vector. Ub, Ubiquitin. l, Ubiquitylation/deubiquitylation assay to monitor BAP1 deubiquitylation of Flag–IP3R3(NT). Either immunopurified wild-type Myc–BAP1 or catalytically inactive Myc–BAP1(C91S) were incubated in vitro with ubiquitylated Flag–IP3R3(NT). Protein levels were analysed by western blot with the indicated antibodies. The ladder of bands with a relative molecular mass of ≥90 kDa corresponds to ubiquitylated Flag–IP3R3(NT). Decimals indicate the amounts of ubiquitylated Flag–IP3R3(NT) normalized on total co-immunoprecipitated Flag–IP3R3(NT) at 90 kDa. For western blot source images, see Supplementary Fig. 2. For source data in b, e, hj, see Supplementary Tables 1 and 2.

Extended Data Figure 9 BAP1+/− fibroblasts exposed to ionizing or ultraviolet radiation show increased survival despite increased DNA damage.

a, Representative images of comet assays. W and L family-derived fibroblasts were irradiated and analysed at the indicated time points (see also Fig. 4a). Representative results showing the rejoining of the DNA damage measured as the percentage of the tail moment at the indicated time points, after BAP1+/− fibroblasts and matched controls were irradiated with 6 Gy ionizing radiation. The length of the tail of the comet is proportional to the DNA damage. Note the increased tail length in BAP1+/− cells. b, Clonogenic assay showing a higher number of colonies in BAP1+/− fibroblasts after irradiation at the indicated amounts (see Fig. 4c). ce, Reduced intracellular Ca2+ levels in BAP1+/− fibroblasts after ultraviolet radiation with UVA (340 nm) or UVB (312 nm). Dynamic measurements of cytosolic Ca2+ response were performed using the fluorescent Ca2+ indicator Fura-RED. c, Control; no changes in dynamic intracellular Ca2+ levels were detected over time in non-irradiated BAP1+/− fibroblasts and matched controls. d, e, Dynamic measurements of intracellular Ca2+ levels in BAP1+/− fibroblasts and matched controls after UVA (d) or UVB (e). Changes in intracellular Ca2+ responses over time are displayed as the ratio of fluorescence at 406/494 nm. Descriptive statistics are shown in Supplementary Table 1. f, Delayed DNA repair after UVB radiation in BAP1+/− fibroblasts. γ-H2A.X kinetics: BAP1WT and BAP1+/− fibroblasts were exposed to UVB, and γ-H2A.X amounts were measured in cell lysates collected at the indicated time points. Total levels of H2A.X are shown as control. Densitometry: decimals indicate the amounts of γ-H2A.X relative to H2A.X. g, Clonogenic assay at 2 weeks after ultraviolet radiation: higher numbers of colonies in BAP1+/− fibroblasts after UVA or UVB exposure. Plating: untreated, 250,000 cells per well; UVA treated, 250,000 per well; UVB treated, 600,000 per well to accommodate for the higher potency of UVB that caused extensive cell death. Cells were exposed to 25 mJ cm−2; see also Fig. 4d, e. Higher doses of 50, 75, and 100 mJ cm−2 killed all the cells within 2 weeks from exposure. For western blot source images, see Supplementary Fig. 2.

Extended Data Figure 10 Human mesothelial cells and macrophages with reduced levels of BAP1 or IP3R3 are resistant to asbestos-induced apoptosis.

a, Primary human mesothelial cells transfected with either scrambled siRNA, siBAP1, or siIP3R3, and exposed to glass, displayed no changes in intracellular Ca2+ concentrations (control for Fig. 4f). b, Flow cytometric analyses of human mesothelial cells silenced for BAP1, IP3R3, or scrambled control, and exposed to crocidolite asbestos for 24 h. Note that human mesothelial cells silenced for BAP1 or IP3R3 show a reduction in the percentage of apoptotic cells compared with scrambled control. c, d, BAP1 silencing in human THP-1 cells differentiated into macrophages leads to decreased IP3R3 protein levels (c), reduced mitochondrial Ca2+ uptake after stimulation with 100 μM ATP (d), and protection from apoptosis after treatment with 5 μg cm−2 crocidolite asbestos (e). In d, THP-1 cells were treated with 20 μM TPA for 24 h to induce monocyte differentiation into macrophages; subsequently, cells were transduced with WT mitochondrial-targeted aequorin (mtAEQ) for 24 h in the presence of TPA, and then transfected with control scrambled siRNA or siRNAs–BAP1 (siBAP1#1 and siBAP1#5) for an additional 24 h before Ca2+ measurements. In e, cells were treated with 20 μM TPA for 48 h, transfected with control scrambled siRNA, siBAP1#1, and siBAP1#5 for 24 h, and treated with 5 μg cm−2 crocidolite asbestos for an additional 24 h; total cell lysates were analysed by western blot to compare cleaved caspase-3 levels. In c and e, decimals indicate densitometrically determined IP3R3 or cleaved caspase-3 levels normalized to α-tubulin. For western blot source images, see Supplementary Fig. 2. a, d, For source data, see Supplementary Tables 1 and 2.

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This file contains Supplementary Figure 1 (Pedigrees of W and L family members), Supplementary Figure 2 (Uncropped scans with size marker indications) and Supplementary Figure 3 (Uncropped EM images). (PDF 12727 kb)

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Bononi, A., Giorgi, C., Patergnani, S. et al. BAP1 regulates IP3R3-mediated Ca2+ flux to mitochondria suppressing cell transformation. Nature 546, 549–553 (2017). https://doi.org/10.1038/nature22798

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