EMBO/SourceData huggingface dataset:
Figure 1A, Distribution and classification of MAP4K3/GLK-positive cells in 20 ml BALFs of all 12 individuals (A), Φ denotes macrophages; N denotes neutrophils; T denotes T cells; E denotes epithelial cells.3 healthy controls (HC, #1 - #3), 3 mild COVID-19 patients (#1 - #3), and 6 severe COVID-19 patients (#1 - #6) (B) from Cohort #1. Data were shown in UMAP. Single-cell gene expression of MAP4K3 (GLK) in individual cells was shown in color scale.C, D The percentages of MAP4K3/GLK-positive KRT18+ epithelial (C) or KRT18+ TPPP3+ ciliated epithelial cells (D) in BALFs from Cohort #1. Data information: In (C, D), *, P value < 0.05 (ANOVA test); §, P value < 0.05 (Dunnett's test).E, F GLK mRNA levels in KRT18+ epithelial (E) or KRT18+ TPPP3+ ciliated epithelial cells (F) of Cohort #1. n (cell number) = 3, 14, 33 for HC; n = 65, 43, 74 for mild COVID-19 patients; n = 164, 62, 10, 4, 74, 66 for severe COVID-19 patients (E). n = 25, 43, 36 for HC; n = 209, 244, 141 for mild COVID-19 patients; n = 189, 223, 330, 792, 80, 243 for severe COVID-19 patients (F). Data information: In (E, F), *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001; ****, P value < 0.0001 (Kruskal-Wallis test).
Figure 2Immunoblotting analysis of the endogenous GLK and vinculin proteins from the lysates of HCC827 lung epithelial cells infected with SARS-CoV-2 pseudovirus (A) Data information: Data shown are representative results of three (A, independent experiments.Real-time PCR analysis of mouse GLK mRNA levels in HCC827 lung epithelial cells infected with SARS-CoV-2 pseudovirus (B) The mRNA levels of GLK were normalized to GAPDH mRNA levels. n = 2 (technical replicates) per group. Data information: In (B data are presented as means. Data shown are representative results of three B, independent experiments.Endogenous GLK levels were increased by the SARS-CoV-2 spike protein. Immunoblotting analysis of the endogenous GLK and vinculin proteins from the lysates of HCC827 lung epithelial cells treated with the SARS-CoV-2 spike protein (C). Data information: Data shown are representative results of three C, independent experiments.Real-time PCR analysis of mouse GLK mRNA levels in HCC827 lung epithelial cells treated with the SARS-CoV-2 spike protein (D). The mRNA levels of GLK were normalized to GAPDH mRNA levels. n = 2 (technical replicates) per group. Data information: In D), data are presented as means. Data shown are representative results of three D) independent experiments.
Figure 3A ZetaView nanoparticle tracking analysis of particle numbers and sizes of CD63+ extracellular vesicles (EVs) isolated from the supernatants of HCC827 lung epithelial cancer cells, which were transfected with GLK wild-type or GLK kinase-dead (K45E) mutant. EVs were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns. Data information: Data shown are representative results of two (A) independent experiments.B Immunoblotting of ACE2 and CD63 proteins in exosomes isolated from GLK wild-type- or GLK (K45E) kinase-dead-overexpressing HCC827 cells. Exosomes were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns. Arrowhead denotes glycosylated ACE2 proteins; asterisk denotes non-glycosylated ACE2 proteins. Data information: Data shown are representative results of three (B, independent experiments.C Immunoblotting of ACE2 and CD63 proteins in serum exosomes isolated from the sera of 4 healthy controls (HC) and 7 COVID-19 patients from NHRI Biobank for COVID-19 patients in Taiwan (Cohort #3). Serum collection days from the onset are shown; treatment of oxygen therapy on the patient is also indicated. Exosomes were isolated sequentially using ExoQuick kits and then ExoQuick ULTRA columns. Arrowhead denotes glycosylated ACE2 proteins; asterisk denotes non-glycosylated ACE2 proteins.D Confocal microscopy analysis of Flag-tagged ACE2 proteins (red) in recipient cells after incubation with exosomes for 72 h. Exosomes were isolated sequentially from the supernatants of Flag-ACE2-overexpressing or GFP-GLK plus Flag-ACE2-coexpressing HCC827 cells using ExoQuick kits and then ExoQuick ULTRA columns. Arrows denote Flag-tagged ACE2 proteins. Original magnification, ×630; scale bars, 10 μm. Data information: Data shown are representative results of three D, independent experiments.E Confocal microscopy analysis of SARS-CoV-2 spike (S) protein (green) and RFP-labeled early endosomes (red) in exosome-recipient HCC827 epithelial cells. After incubation with exosomes for 72 h, recipient cells were treated with the spike protein (S) for another 24 h. Yellow color suggests the localization of S protein in early endosomes. Original magnification, ×630; scale bars, 25 μm. Data information: Data shown are representative results of three E, independent experiments.F Cell entry eﬃciencies of SARS-CoV-2 pseudovirus into exosome-recipient HCC827 epithelial cells were measured by luciferase activity and presented as relative light units (RLU) at 24 h post infection. n = 3 (biological replicates). Data information: In (F), data are presented as means ± SEM. **, P value < 0.01 (ANOVA test). Data shown are representative results of three F) independent experiments.
Figure 4B Co-immunoprecipitation of Flag-tagged ACE2 with Myc-tagged GLK proteins from lysates of HEK293T transfectants. NS, normal serum. Data information: Data shown are representative results of three (B, independent experiments.C In situ PLA assays of the interaction between ACE2 and Flag-tagged GLK proteins in HEK293T cells. Nuclei were stained with DAPI. Red dots represent direct interaction signals. Original magnification, x200. Scale bars, 50 μm. Data information: Data shown are representative results of three C, independent experiments.D Immunoblotting analyses of the endogenous ACE2, ACE1, CD147, and tubulin proteins, as well as the transfected Flag-GLK proteins in HCC827 lung epithelial cells, which were transfected with either GLK wild-type or kinase-dead (K45E) mutant. Arrowhead denotes glycosylated ACE2 proteins; asterisk denotes non-glycosylated ACE2 proteins. Data information: Data shown are representative results of three D, independent experiments.E Immunoblotting of Flag-tagged ACE2 (anti-Flag), Myc-tagged GLK (anti-GLK), and tubulin proteins from HEK293T cells co-transfected with Flag-ACE2 plus increasing amounts of Myc-GLK plasmids. Data information: Data shown are representative results of three E, independent experiments.F Immunoblotting of ACE2, GLK, and tubulin proteins in the lung tissues of wild-type or GLK-deficient mice. Arrowhead denotes glycosylated ACE2 proteins; asterisk denotes non-glycosylated ACE2 proteins. Data information: Data shown are representative results of three F, independent experiments.G Cycloheximide pulse-chase experiments in HEK293T cells. Immunoblotting of Flag-tagged ACE2 (anti-Flag), Myc-tagged GLK (anti-GLK), and tubulin proteins from HEK293T cells co-transfected with Flag-ACE2 and Myc-GLK. Transfected cells were treated with 100 μg/ml cycloheximide (CHX) for up to 24 h. Data information: Data shown are representative results of three G, independent experiments.H Flag-tagged ACE2 proteins were immunoprecipitated from lysates of HEK293T cells co-transfected with Flag-ACE2 and Myc-GLK, followed by immunoblotting with anti-Lsy48-linked ubiquitination or anti-Flag antibody. Cells were treated with 25 μM MG132 for 2 h before being harvested. NS, normal serum. Data information: Data shown are representative results of three H, independent experiments.I, J Cell entry eﬃciencies of SARS-CoV-2 pseudovirus (I) or VSV-G pseudovirus (J) into GLK wild-type- or GLK (K45E)-overexpressing HCC827 epithelial cells were measured by luciferase activity and presented as relative light units (RLU) at 24 h post infection. n = 3 (biological replicates). Data information: In (I, J), data are presented as means ± SEM. **, P value < 0.01 (ANOVA test); §§, P value < 0.01 (Dunnett's test). Data shown are representative results of three I, J) independent experiments.
Figure 5A Immunoblotting analyses of ACE2 and Flag-tagged GLK in HCC827 cells transfected with ether Flag-GLK or Flag-GLK (K45E) kinase-dead mutant. For immunoblotting of phosphorylated ACE2, Phos-tagged SDS-PAGE gel (Phos-tagTM) was used, followed by immunoblotting with anti-ACE2 antibody. Asterisk denotes unglycosylated ACE2 proteins. Data information: Data shown are representative results of three (A independent experiments.B In vitro kinase assays using purified proteins. ACE2 serine phosphorylation, Flag-tagged GLK (anti-GLK), Flag-tagged GLK (K45E) kinase-dead mutant (anti-GLK), and Flag-tagged ACE2 proteins (anti-ACE2) were detected by immunoblotting. Data information: Data shown are representative results of three B, independent experiments.C Mass spectrometry analysis of GLK-phosphorylated ACE2 proteins after in vitro kinase assays. ACE2 Ser776 and Ser783 residues were phosphorylated by GLK wild-type but not GLK (K45E) kinase-dead mutant.D Mass spectrometry analysis of the ACE2 peptides from the serum exosomes of COVID-19 patients. The ACE2 protein peptide sequences containing phospho-Ser776 or phospho-Ser783 residue of ACE2 proteins detected in the serum exosomes of COVID-19 patients (Cohort #3) are shown. Exosomes were isolated using ExoQuick kits, and soluble proteins were removed by ExoQuick ULTRA columns. Exosomes were further purified by immunoprecipitation using a combination of anti-CD9, anti-CD63, and CD81 magnetic beads.E In vitro kinase assays using immunoprecipitated Flag-tagged ACE2 or Flag-tagged GLK immunocomplexes. ACE2 serine phosphorylation, ACE2, and GLK were detected by immunoblotting using anti-phospho-serine, anti-ACE2, and anti-GLK antibodies, respectively. Data information: Data shown are representative results of three E, independent experiments.F Immunoblotting analyses of Myc-tagged GLK, Flag-tagged ACE2, and tubulin proteins in HEK293T cells co-transfected with Flag-ACE2 wild-type or individual ACE2 (S776/783A, S776A, or S783A) mutants plus either empty vector or Myc-GLK. Data information: Data shown are representative results of three F, independent experiments.G Immunoblotting analyses of Flag-tagged ACE2 and tubulin proteins in HEK293T cells transfected with Flag-ACE2 wild-type or individual phosphomimetic ACE2 (S776/783E, S776E, or S783E) mutants. Data information: Data shown are representative results of three G, independent experiments.H Flag-tagged ACE2 proteins were immunoprecipitated from lysates of HEK293T cells transfected with Flag-ACE2 wild-type or a phospho-mimetic ACE2 (S776/783E) mutant, followed by immunoblotting with anti-Lsy48-linked ubiquitination or anti-Flag antibody. Cells were treated with 25 μM MG132 for 2 h before being harvested. Data information: Data shown are representative results of three H) independent experiments.
Figure 6B Immunoblotting of Myc-tagged ACE2 (anti-ACE2), Flag-tagged UBR4 (anti-Flag), and tubulin proteins from HEK293T cells co-transfected with Myc-ACE2 and Flag-UBR4. Arrowhead denotes Myc-tagged ACE2 proteins; asterisk denotes endogenous ACE2 proteins. Data information: Data shown are representative results of three B, independent experiments.C Immunoblotting of Flag-tagged ACE2 (anti-Flag), Myc-tagged UBR4 (anti-Myc), and tubulin proteins from HEK293T cells co-transfected with Flag-ACE2 and Myc-UBR4. The co-transfected cells were treated with 25 μM MG132 for 2 h before being harvested. Data information: Data shown are representative results of three C, independent experiments.D Flag-tagged ACE2 proteins were immunoprecipitated from lysates of HEK293T cells co-transfected with Flag-ACE2 and Myc-UBR4, followed by immunoblotting with anti-Lsy48-linked ubiquitination, anti-Myc, or anti-Flag antibody. Cells were treated with 25 μM MG132 for 2 h before being harvested. Data information: Data shown are representative results of three D, independent experiments.E In situ PLA assays of the interaction between Flag-tagged ACE2 (wild-type or mutant) and Myc-tagged UBR4 proteins in HEK293T cells treated with 25 μM MG132 for 2 h. Nuclei were stained with DAPI. Red dots represent direct interaction signals. Original magnification, x200. Scale bars, 20 μm. Data information: Data shown are representative results of three E, independent experiments.F Mass spectrometry analysis of the ACE2 peptides containing ubiquitination residues from Flag-ACE2-transfected HEK293T cells treated with 25 μM MG132 for 2 h.G Immunoblotting analyses of Flag-tagged ACE2, Myc-tagged UBR4, and tubulin proteins in HEK293T cells co-transfected with Flag-ACE2 wild-type or individual (K26R, K94R, K112R, or K114R) mutants plus either empty vector or Myc-UBR4. Data information: Data shown are representative results of three G, independent experiments.H Flag-tagged ACE2 proteins were immunoprecipitated from lysates of HEK293T cells co-transfected with Flag-ACE2 wild-type or Flag-ACE2 (K26/112/114R) mutant plus Myc-UBR4 plasmids, followed by immunoblotting with anti-Lsy48-linked ubiquitination or anti-Flag antibody. Cells were treated with 25 μM MG132 for 2 h before being harvested. Data information: Data shown are representative results of three H) independent experiments.
figf1(a) Wild-type (WT), atg17Δ, atg7Δ and atg19Δ strains containing RPL25-GFP were grown to mid log phase, starved in SD-N for 48 hours and examined under a fluorescence microscope (left panel). The arrows indicate vacuolar accumulation of the GFP signal. CVT, cytoplasm-to-vacuole-targeting pathway. After the times indicated, at least 100 cells per sample were scored for the presence of Rpl25p-GFP in the vacuole (right panel). Scale bar, 5 μm.(b) Wild-type and atg7Δ strains containing RPL25-GFP or GFP-ATG8 were grown and starved as in a. Samples were taken at indicated time points and GFP cleavage was analysed by western blotting.(c) The levels of endogenous Rps3p (top) and Rpl3p (middle) were analysed by immunblotting in wild-type and atg7Δ cells. Ponceau staining (bottom) controlled for equal loading. Uncropped images of blots are shown in Supplementary Information, Fig. S6. Starv, starvation.(d) Wild-type and pep4Δ cells expressing RPL25-GFP were analysed as in b; 0.1 mM of PMSF was added to pep4Δ cells at the time of starvation to block all vacuolar proteases completely. The LI-COR imaging system was used to quantify the ratio between cleaved GFP and full-length protein as described in Methods. The last time point of wild-type cells containing RPL25-GFP was set to 1 arbitrary unit (a.u.). One of two independent experiments is shown.(e) Wild-type cells containing either RPL25-GFP or control GFP (HOG1-GFP) were analysed as in d. The average of four independent experiments is shown, and the standard deviation was less than 10% (Supplementary Information, Table S3).
figf2(a) Wild-type, ubp3Δ or bre5Δ strains containing RPL25-GFP and either a control plasmid (pRS315), or plasmids expressing Ubp3p or catalytically inactive Ubp3p-C469A from the endogenous promoter, were grown and starved as described in Fig. 1a. Cells were revealed by phase contrast (right), and the localization of Rpl25p-GFP was analysed by GFP microscopy (left). At least 100 cells per sample were analysed. Scale bar, 5 μm.(b) Ribosomes from wild-type and ubp3Δ cells grown as indicated for 16 h under rich or starvation (Starv) conditions were enriched through a high-salt cushion (pellet) and the levels of endogenous Rpl3p were analysed by immunoblotting. Ponceau staining of the blot revealed the total amount of proteins (bottom panel). Uncropped images of blots are shown in Supplementary Information, Fig. S6.c) Wild-type and ubp3Δ strains containing RPL25-GFP were grown and starved as in Fig. 1a, and GFP cleavage was analysed by western blotting at the times indicated (hours).(d, e) GFP cleavage of Rpl25p-GFP was analysed in wild-type, ubp3Δ, atg9Δ and bre5Δ strains containing the indicated plasmids, and quantified at the times indicated as described in Fig. 1d. One of three representative experiments is shown for d. The average of three independent experiments is shown in e, and the statistical analysis showed that the standard deviation was less than 13% (Supplementary Information, Table S3).
figf3(a) The vacuolar localization of GFP-Atg8p was analysed in more than 100 wild-type, ubp3Δ, bre5Δ and atg7Δ cells containing GFP-ATG8 after 10-15 h of nitrogen starvation (right panel). Corresponding GFP and phase contrast images of representative cells are shown in the left panel.(b) Vacuolar cleavage of GFP-Atg8p was examined at the indicated time points (hours) by anti-GFP western blotting.(c) Wild-type, ubp3Δ and bre5Δ strains containing APE1-RFP were analysed by RFP microscopy (left) and phase contrast (right) 15 h after nitrogen starvation. Scale bars, 5 μm.
figf4(a) Wild-type, ubp3Δ or bre5Δ strains containing either a control plasmid (pRS315) or plasmids expressing Ubp3p or catalytically inactive Ubp3p-C469A from the endogenous promoter were spotted in serial dilutions onto YPD plates with (left) or without (right) 5 nM rapamycin. The plates were photographed after 2 days at 30 °C.(b) Induction of a GCN4-LacZ reporter was measured 0, 2 and 4 h after amino-acid starvation, and is plotted as Miller units.(c) Polysome profiles of exponentially growing wild-type and ubp3Δ cells were determined in rich medium or 3 h after nitrogen starvation. The arrows mark the 60S peak.(d) Wild-type, atg7Δ and ubp3Δ cells containing either RPL25-GFP, control GFP (HOG1-GFP) or RPS2-GFP were analysed as in Fig. 1c. One of three representative experiments is shown.(e) Ribosomes and associated proteins were immunopurified from starved wild-type and ubp3Δ cells containing (RPL25-GFP) and (9 × MYC-UBI) as indicated. The GFP immunoprecipitates (IP; left) and total extracts (TCA, right) were analysed by immunoblotting with anti-Myc antibodies to reveal ubiquitinated proteins (top). Ub, ubiquitin. Bottom: immunoblotting with anti-GFP antibodies controls for the presence of Rpl25p-GFP, and anti-Rpl1p antibodies confirm equal loading of the extract. The asterisk marks protein G on the beads recognized by the secondary antibody.
f1(a) Structure of the GBA1 gene on chromosome 1 and schematic overview of gene correction using ZFNs. Black boxes represent exons. (b) Mutated and gene-corrected (GC) iPSCs after sequencing (upper lanes: iPSCs with heterozygous mutations; lower lanes: iPSCs with homozygous wt-GBA1 sequences as indicated by arrows).
f2Immunostaining of indicated differentiated iPSC cultures at DIV65. Cells were stained for TH (red) and β-TubIII, FOXA2, NURR1, GIRK2 and VMAT2 (all green). Cell nuclei were counterstained with Hoechst (blue). (Bars, 20 μm).
f3a) Gating strategy used for neuronal cell sorting. Live cells as determined by FSC/SSC gating were selected for CD184-APC-negative/low surface expression and subsequently separated for CD24-PE versus CD15/CD29/CD44 expression, detected in the FITC channel as indicated in the dot plots. Post-sort reanalysis shows representative samples of both fractions immediately after FACS enrichment, which were then plated for comparative in vitro analysis (phase contrast images. Bars, 50 μm).(b,c) Immunocytochemistry at DIV2 post-sort for TH (green) and the neuronal marker MAP2 (red) performed on the neuronal (b) and mixed (c) populations of a representative control and GBA-PD line. Nuclei were counterstained with Hoechst (blue). FACS enriches for both TH and neuronal marker positivity in the neuronal population compared with the non-neuronal population. (Bars, 50 μm.)
f4(a) Ganglioside composition of human fibroblasts, iPSCs, differentiated iPSCs and enriched iPSC-derived neurons (after FACS) from control (line C1-1), GBA-PD (line PD1-1) and GD (line GD1-1) subjects. Cell lipids were metabolically labelled with [1-3H]sphingosine and visualized by digital autoradiography.(b) Activities of glycohydrolases (GCase, GBA2, β-galactosidase, β-hexosaminidase) in control iPSCs and iPSC-derived enriched neurons at DIV65. Enzyme activities are expressed as pmoles mg−1 h−1. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.01; Student's t-test.c) GBA1, GBA2, β-galactosidase and HEXA and HEXB gene expression levels in control iPSCs and iPSC-derived enriched neurons at DIV65. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.05; **P0.01, Student's t-test.(d) Representative western blots showing GCase, GBA2, β-galactosidase and HEXA and HEXB protein levels in control iPSCs and corresponding iPSC-derived neurons (DIV65).
f5(a) LC-MS/MS analysis showing the level of Glc-Cer in control, GBA-PD, isogenic controls and GD iPSC-derived neurons. Data are represented as mean+s.d.; n=3, *P0.01, One-way ANOVA.(b) Control iPSCs (line C1-1) were differentiated for 65 days and exposed to the GCase inhibitor CBE (25 μM) for 48 h. Representative western blot showing increased levels of α-syn in CBE-treated iPSC-derived neurons compared to untreated neurons.(c,d) Representative western blots showing the protein levels of α-syn in control, GBA-PD, gene-corrected isogenic controls and GD iPSC-derived neurons.(c,d) Representative western blots showing the protein levels of α-syn in control, GBA-PD, gene-corrected isogenic controls and GD iPSC-derived neurons.(e) Optical densities of α-syn bands normalized by the averaged value of GAPDH and expressed as a percentage of the control (CTRL) line value. Data are represented as mean+s.d.; experiments were independently repeated five times. *P0.01, one-way ANOVA. (f) Average α-syn protein levels normalized by the averaged value of GAPDH and expressed as a percentage of the respective isogenic mutant lines. Data are represented as mean+s.d.; experiments were independently repeated five times. *P0.01, Student's t-test.
f6(a) Glycohydrolase enzyme activities (GCase, GBA2, β-galactosidase, β-hexosaminidase) were measured in enriched neurons from control, GBA-PD, isogenic gene-corrected control and GD iPSC-derived neurons at DIV65. Enzyme activities are expressed as pmoles mg−1 h−1. Data are represented as mean+s.d.; experiments were independently repeated three times in triplicate. *P0.05; **P0.01; ***P0.001 compared with controls, one-way ANOVA.(b,c) Representative western blots of GCase protein levels in control, GBA-PD, corresponding gene-corrected and GD iPSC-derived neurons. Data are represented as mean+s.d.; experiments were independently repeated three times. *P0.05; **P0.01, one-way ANOVA and Student's t-test.
f7CSF glycohydrolase activities were measured in patients with idiopathic PD (iPD), GBA-PD patients and age-matched unaffected individuals (CTRL). Enzyme activities were normalized to total protein concentrations and expressed as pmoles mg−1 h−1. Data are represented as mean+s.e.m.; CTRL, n=14; iPD, n=15; GBA-PD, n=15. Experiments were performed in triplicate. *P0.05; **P0.01, Mann-Whitney U test.
f8(a) Immunostaining of differentiated iPSC cultures at DIV65 under basal conditions. Cells were stained for TH (green), β-TubIII (magenta) and LAMP1 (red). Cell nuclei were counterstained with Hoechst (blue). High magnification insets are shown in the right lane. (Bars, 20 μm and 10 μm in the insets.)(b) Average size and number of LAMP1+ particles in TH+ neurons were quantified by ImageJ. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01, **P0.001, one-way ANOVA.(c) Western blot analysis for LC3 in iPSC-derived neuronal cultures at DIV65, untreated (−) or treated with 200 μM leupeptin and 20 mM NH4Cl for 4 h (+).(d) Quantification of the basal levels of LC3-II relative to β-actin. Data are represented as mean+s.e.m.; experiments were independently repeated three times. *P0.01; **P0.001, one-way ANOVA.(e) Quantification of LC3 flux normalized to actin. Data are represented as mean+s.e.m.; experiments were independently repeated three times in triplicate. *P0.01; **P0.001, one-way ANOVA.