Fig 2: ACE2 receptor is specifically conjugated with SUMO3.a Lysates prepared from HEK293T cells transfected with vector encoding Flag-tagged ACE2 or Flag-tagged empty vector as negative control were immunoprecipitated with a-FLAG M2 beads. The precipitated proteins were subjected to mass spectrometry analysis. This experiment was performed once with two replicates per condition. Gene Ontology (GO) analysis of the proteome after purified with ACE2 receptor as bait. The color code displays the value of (-log10FDR) of the respective GO terms. b Schematic diagram of the conjugation of small ubiquitin-like modifier (SUMO) with target proteins. c Extracts of Calu-3 cells were immunoprecipitated with anti-SUMO and immunoblotted with indicated antibodies. d Lysates of 293 T cells transfected with plasmids of Flag-ACE2 and HA-tagged SUMO, together with Myc-UBC9 were immunoprecipitated with a-FLAG M2 beads and immunoblotted with anti-HA. e Immunoprecipitation and immunoblot analysis of 293T cells transfected with expressing vectors of Flag-ACE2 and HA-SUMO3 and its indicated mutants. f Illustration of the inhibitory site of ML-792. g HeLa cells transfected with Flag-ACE2 in the absence or presence of ML-792 (10 µM), followed by labeling of ACE2 (green) and SUMO2/3 (red) with specific antibodies. Scale bar, 20 µm. A representative experiment out of three is shown. h Schematic representation of the five predicted SUMO-conjugation motifs (?KXE) in ACE2 protein. Details of the SUMO-conjugation motifs and summary of prediction software scores are depicted underneath. Analysis using Ron Hay’s website (https://www.lifesci.dundee.ac.uk/groups/ron_hay/pages/SumomotifQuery.html) provides motif types: S = strict, R = relaxed, and RR = relaxed reverted. i Immunoprecipitation and immunoblot analysis of 293 T cells transfected with vectors encoding HA-SUMO3 and wild-type (WT) Flag-ACE2 or its indicated mutants. j 293 T cells transfected with vectors expressing Flag-ACE2 (WT or K187R) and HA-SUMO3 along with SARS-CoV-2 (MOI = 0.5) infection for indicated time points, followed by immunoprecipitated with a-FLAG M2 beads and immunoblot analysis. For c–e, i, j, similar results are obtained by three independent biological experiments. Source data are provided as a Source data file.

Fig 3: SUMO3 conjugation attenuates ACE2 degradation through autophagy.a 293 T cells were transfected with plasmid encoding Flag-ACE2 together with Myc-PIAS4 vector treated with MG132 (10 µM), 3-methyladenine (10 mM), or bafilomycin A1 (0.2 µM) for 6 h. The cell extracts were analyzed by immunoblotting. b Lysates of Calu-3 cells transfected with scramble or PIAS4-specific siRNA and treated with rapamycin (250 nM) for 24 h, were collected for immunoblot analysis. c hNEPCs were treated with rapamycin (250 nM) (up) or EBSS (down) with indicated time points, and the proteins were harvested for immunoblot analysis. d 293 T cells transfected with scramble siRNA along with RB1CC1- or ATG13-specific siRNAs were transfected with plasmids encoding Flag-ACE2 and Myc-PIAS4, the lysates were analyzed by immunoblotting. e Wild-type (WT), BECN1- and ATG5-knockout (KO) 293 T cells were transfected with vectors of Flag-ACE2 and Myc-PIAS4, the lysates were subjected to immunoblot analysis. f Calu-3 cells were treated with ML-792 (10 µM) (up) or ginkgolic acid (GA) (5 µM) (down) together with 3-MA (10 mM), chlorpromazine (CPZ) (10 µM), methyl-ß-cyclodextrin (MßCD) (1 mM), or cytochalasin D (CD) (2 µM), and the proteins were harvested for immunoblot analysis. g Immunoblot analysis of Calu-3 cells transfected with scramble or PIAS4-specific siRNAs and treated with chlorpromazine (CPZ) (10 µM), methyl-ß-cyclodextrin (MßCD) (1 mM), or cytochalasin D (CD) (2 µM). h HeLa cells transfected with Flag-ACE2 along with ML-792 (10 µM) or ginkgolic acid (GA) (5 µM) treatment, followed by labeling of ACE2 (green) with specific antibodies. Scale bar, 20 µm. Representative confocal microscopy images from n = 3 independent biological samples for each group. i Total, cytosolic, and membrane fractions of Calu-3 cells pretreated with DMSO or ML-792 (10 µM) were immunoblotted with antibodies directed against ACE2, a cytosol marker (a-tubulin), or a PM marker (E-cadherin). In a–g and i, similar results are obtained by three independent biological experiments. Source data are provided as a Source data file.

Fig 4: Manipulation ACE2 SUMOylation contributes to the SARS-CoV-2 infection.a The strategy to acquire SARS-CoV-2 pseudovirus based on the HIV-1 three-plasmid packaging system. b Immunoblotting of 293 T cells transfected with plasmids of Flag-ACE2 and HA-SUMO3 in the presence of 2-D08 (200 µM), ML-792 (10 µM), GA (5 µM), or TAK-981 (5 µM). c Luciferase activity of Calu-3 cells treated with SARS-CoV-2 S pseudotyped virus for 48 hr, along with SUMOylation inhibitor treatment. d Immunoprecipitation and immunoblot analysis of human alveolar epithelial cells (AECs) treated with ML-792 (10 µM) or GA (5 µM). e Luciferase activity of SARS-CoV-2 S pseudotyped virus-infected AECs, along with ML-792 (10 µM) or GA (5 µM) treatment. f Schematic illustration of the adenoviral transduction-based mouse model to study SARS-CoV-2 infection. g Ad5-hACE2-transduced BALB/c mice were intranasally infected with SARS-CoV-2 (1 × 105 PFU) in 50 mL of DMEM for 3 days. The lungs of mice were harvested for viral plaque assay. Titers are expressed as PFU/g lung tissue. h GSEA analysis of differentially expressed genes (DEGs) in Calu-3 cells infected with SARS-CoV-2 (MOI = 0.5) for 24 h with or without 2-D08 (100 µM) treatment. FDR (q-value) was shown. i Dot-plot visualization of enriched GO terms showing the enriched genes upregulated by SARS-CoV-2 with or without 2-D08 treatment comparing with negative control. The ordinate is the GO term description. Bubble size represents the number of DEGs in GO classification; the enrichment P-value is calculated by Fisher exact test; and different color represents different P-value. j Heatmap showing the transcription levels of DEGs belonging to GO annotations for cytokine activity and chemokine activity (GO: 0005125 and GO: 0008009) upregulated by SARS-CoV-2. In c and e, all error bars, mean values ± SEM, P-values are determined by unpaired two-tailed Student’s t-test of n = 3 independent biological experiments. In g, all error bars, mean values ± SD, P-values are determined by unpaired two-tailed Student’s t-test (n = 8 independent biological mice per group). For b, d, similar results are obtained by three independent biological experiments. Source data are provided as a Source data file.

Fig 5: SENP3 induced the deSUMOylation of c-Jun(A) The SUMOylation level of c-Jun was determined by Ni2+-NTA pull down. HA-c-Jun, His-SUMO1/2/3, and Flag-Ubc9 were co-transduced into HEK293T cells. Cell lysates were used for Ni2+-NTA resin pull down and determined by immunoblot analysis.(B) The SUMOylation of endogenous c-Jun in primary microglia treated with OGD/R or not was examined by co-immunoprecipitation (co-IP) assay.(C) SENP3 downregulates the SUMOylation level of c-Jun. HA-c-Jun, His-SUMO2, and Flag-Ubc9 were co-transduced into HEK293T cells along with Myc-SENP3 or Myc-SENP3 C532A mutant. The SUMOylation level of c-Jun was examined by Ni2+-NTA pull down and analyzed by immunoblot.(D) Representative Ni2+-NTA pull-down results demonstrating the SUMOylation level of c-Jun in HEK293T cells when SENP3 is knocked down by specific shRNA.(E and F) The interaction of ectopically expressed c-Jun with SENP3 was determined by co-IP. Flag-SENP3 and HA-c-Jun were co-transduced into HEK293T cells, and cell lysates were subjected to co-IP.(G and H) The binding of endogenous SENP3 with c-Jun in primary microglia subjected to OGD/R or not was detected by co-IP. All the experiments were repeated three times.