Angiotensin converting enzyme 2 is a novel target of the γ-secretase complex

Figures

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  Figure 1. ACE2 is targeted by γS after ectodomain shedding. (A) Schematic and scaled representation of γS targets JAGGED1 or NOTCH1, with ACE2, ACE2-C9 and ACE2-GFP. Domains and regions targeted by sheddases are depicted. Predicted molecular weight of ACE2ΔE after TMPRSS2/ADAM17-mediated cleavage is shown. (B) Western blots from 293 T cells transfected with ACE2-C9 with or without TMPRSS2, then treated with DBZ (dibenzazepine 100 nM) ( +), or DMSO (−). Mobility consistent with full length (FL) and ACE2 lacking its ectodomain (ACE2ΔE) indicated. ACE2ΔE generated in the absence of TMPRSS2 has a higher molecular weight, which corresponds to ADAM17-mediated shedding. (C) Western blots from 293 T cells transfected with ACE2-C9 with or without PMA (200 nM, 15 h) treatment. (D) Western blots from 293 T cells transfected with ACE-C9, with or without TMPRSS2, or treated with PMA, with or without two γS inhibitors [DBZ (dibenzazepine, 100 nM) or CmpE (compound E, 40 nM)]. (E) Western blots from 293 T cells transfected with ACE2-GFP, with or without TMPRSS2, then treated with DBZ and/or MG132 (1 µM, 15 h). (F) Western blots from 293 T cells transfected with ACE2-GFP, with or without PMA, DBZ and/or…

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  Figure 2. γS-deficient cells cannot process ACE2ΔE. (A) Western blots from Presenilin 1/2 double KO (Psen1/2 dKO) or Nicastrin KO (Ncstn KO) MEFs and their wild type (WT) controls. Cells were transfected with ACE2-C9 with or without TMPRSS2. ACE2ΔE generated in the absence of TMPRSS2 has a higher molecular weight, which corresponds to ADAM17-mediated shedding. (B) Representative immunofluorescence images of WT MEFs transfected with ACE2-C9. (C) Representative immunofluorescence images of WT, Psen1/2 dKO and Ncstrn KO MEFs transfected with ACE2-C9 and TMPRSS2. (D) GFP fluorescence in WT or Ncstrn KO MEFs co-expressing ACE2-GFP and TMPRSS2. (E) Representative immunofluorescence images of WT MEFs transfected with ACE2-C9 and TMPRSS2 in the presence of GSI. Scale bars: 10 µm.

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  Figure 3. γS inhibition targets endogenous ACE2. (A) Western blots of Caco-2 cells treated with DBZ ( +) or DMSO (−). (ns) indicates non-specific bands. (B) Western blots of VeroE6 cells treated with DBZ ( +) or DMSO (−). (C) Western blots of VeroE6 cells treated with DBZ compound E (CmpE) or DMSO (−). D, Gene expression in VeroE6after 24 or 72 h treatment with DBZ, showing means ± SD. (E) Gene expression in Caco-2 cells after 24 or 72 h treatment with DBZ, showing means ± SD.

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  Figure 4. γS inhibition does not prevent SARS-CoV-2 S-protein mediated cell entry. Inhibition of SARS-CoV-2 pseudovirus by DBZ at the indicated concentrations tested on VeroE6 or Caco-2 cells. A SARS-CoV-2 neutralizing antibody, 2–15, tested on VeroE6 was used as a positive control. Triplicates are presented as means ± SEM.

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  Figure 5. Model of ACE2 cleavage. Model showing the sequential processing of full length ACE2 by ADAM17/TMPRSS2 and γS, rendering ACE2ΔE and ACE2-ICD, respectively. ACE2-ICD is then rapidly degraded in the proteasome. The functional consequence of ACE2-ICD is not yet known.