Degradation of SARS-CoV-2 receptor ACE2 by the E3 ubiquitin ligase Skp2 in lung epithelial cells

doi:10.1007/s11684-021-0837-6

Front. Med.

2021, Vol. 15

Issue (2) : 252-263 https://doi.org/10.1007/s11684-021-0837-6

RESEARCH ARTICLE

Degradation of SARS-CoV-2 receptor ACE2 by the E3 ubiquitin ligase Skp2 in lung epithelial cells

Guizhen Wang^1,², Qun Zhao^1,³, Hui Zhang⁴, Fan Liang^1,², Chen Zhang^1,², Jun Wang⁵, Zhenyin Chen^1,⁶, Ran Wu^1,⁶, Hong Yu⁷, Beibei Sun¹, Hua Guo¹, Ruie Feng⁴, Kaifeng Xu⁵, Guangbiao Zhou¹(

)

¹. State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China
². State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences & University of Chinese Academy of Sciences, Beijing 100101, China
³. Hubei University of Medicine, Shiyan 442000, China
⁴. Department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China
⁵. Department of Pulmonary and Critical Care Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China
⁶. Guizhou University School of Medicine, Guiyang 550025, China
⁷. School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China

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Abstract

An unexpected observation among the COVID-19 pandemic is that smokers constituted only 1.4%−18.5% of hospitalized adults, calling for an urgent investigation to determine the role of smoking in SARS-CoV-2 infection. Here, we show that cigarette smoke extract (CSE) and carcinogen benzo(a)pyrene (BaP) increase ACE2 mRNA but trigger ACE2 protein catabolism. BaP induces an aryl hydrocarbon receptor (AhR)-dependent upregulation of the ubiquitin E3 ligase Skp2 for ACE2 ubiquitination. ACE2 in lung tissues of non-smokers is higher than in smokers, consistent with the findings that tobacco carcinogens downregulate ACE2 in mice. Tobacco carcinogens inhibit SARS-CoV-2 spike protein pseudovirions infection of the cells. Given that tobacco smoke accounts for 8 million deaths including 2.1 million cancer deaths annually and Skp2 is an oncoprotein, tobacco use should not be recommended and cessation plan should be prepared for smokers in COVID-19 pandemic.

Keywords SARS-CoV-2 tobacco smoke benzo(a)pyrene ACE2 Skp2

Corresponding Author(s): Guangbiao Zhou

Just Accepted Date: 18 December 2020 Online First Date: 03 February 2021 Issue Date: 23 April 2021

Cite this article:

Guizhen Wang,Qun Zhao,Hui Zhang, et al. Degradation of SARS-CoV-2 receptor ACE2 by the E3 ubiquitin ligase Skp2 in lung epithelial cells[J]. Front. Med., 2021, 15(2): 252-263.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-021-0837-6
https://academic.hep.com.cn/fmd/EN/Y2021/V15/I2/252

Fig.1 Effects of tobacco carcinogens on ACE2 expression in lung epithelial cells. (A) 16HBE cells were treated with CSE at indicated protocols, lysed, RNA was extracted, and ACE2 expression at mRNA level was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR). P values, Student’s t-test, * P<0.05. (B, C) 16HBE cells were treated with CSE at indicated protocols, and ACE2 expression at protein level was detected by Western blot. The relative ACE2 levels were determined by densitometry analysis of immunoblot bands and normalized to Actin expression levels. P values, Student’s t-test, *P<0.05, **P<0.01. (D, E) 16HBE cells were treated with BaP at indicated concentrations for 48 h (D) or at indicated protocols (E), lysed, and subjected to Western blot using indicated antibodies. The relative ACE2 levels were determined as described above. P values, Student’s t-test, *P<0.05, **P<0.01, *** P<0.001. (F) Beas-2B cells were treated with BaP, and ACE2 expression at protein level was detected by Western blot using cell lysates and indicated antibodies. Numbers under the Western blot bands are the relative expression values to Actin determined by densitometry analysis. (G) 16HBE cells were treated with NNK, lysed, and subjected to Western blot using indicated antibodies. Numbers under the Western blot bands are the relative ACE2 expression values to Actin.

Fig.2 BaP induces ACE2 degradation. (A) 16HBE cells were treated with BaP, lysed, RNA was extracted, and ACE2 expression at mRNA level was evaluated by quantitative RT-PCR. P values, Student’s t-test, *P<0.05. (B) The HLF cells were transfected with ACE2 promoter-luciferase reporter construct, treated with BaP for 48 h, and assessed by the luciferase assay. (C) 16HBE cells were treated with cycloheximide (50 µg/mL) in the absence or presence of BaP (5 µmol/L), lysed, and subjected to Western blot assays. The relative ACE2 levels were determined as described above. P values, Student’s t-test. (D) 16HBE cells were pre-treated with BaP (5 µmol/L) for 24 h, subsequently co-incubated with epoxomicin (Epo; 10 µmol/L) or MG132 (10 µmol/L) for 12 h, and then lysed for Western blot analyses. (E) 16HBE cells were treated with chloroquine (CQ; 40 µmol/L) and BaP (5 µmol/L) for 36 h, and lysed for Western blot analyses. (F) 293T cells were transfected with HA-Ub for 24 h, treated with BaP (5 µmol/L) for additional 48 h, lysed, and subjected to immunoprecipitation and immunoblot using indicated antibodies. Ub, ubiquitin. (G) 16HBE cells were treated with BaP (5 µmol/L) for 48 h, lysed, and subjected to immunoprecipitation and immunoblot using indicated antibodies.

Fig.3 BaP induces an AhR-dependent Skp2 upregulation. (A) 16HBE cells were treated with BaP at indicated concentrations for 48 h, lysed, and subjected to immunoblot using indicated antibodies. (B) 16HBE cells were treated with BaP as indicated, lysed, RNA were extracted, and analyzed by quantitated RT-PCR. Student’s t-test, * P<0.05; ** P<0.01. Error bars, SD. (C) ACE2 is a target gene of AhR. The upper panel shows the AhR binding site of Skp2 promoter. TSS, transcription start site. Lower panel, 16HBE cells were transfected with the XRE element of Skp2 promoter-luciferase reporter construct, treated with BaP for 48 h, and assessed by the luciferase assays. Student’s t-test, *** P<0.001. Error bars, SD. (D–G) 16HBE cells were treated with BaP in the absence or presence of AhR antagonist ANF (D, E) or CH223191 (F, G), lysed, and analyzed by quantitative RT-PCR (D, F) or Western blot (E, G). Student’s t-test, *P<0.05. Error bars, SD. (H) The expression of ACE2 and Skp2 in 16HBE cells treated with vehicle control (DMSO) or BaP at 10 µmol/L for 48 h. The cells were harvested and detected by immunofluorescence assays using indicated antibodies.

Fig.4 Skp2 mediates ubiquitination and degradation of ACE2 induced by BaP. (A) 293T-ACE2 cells were treated with BaP (5 µmol/L) for 48 h, lysed, and subjected to immunoprecipitation and immunoblot using indicated antibodies. Lysates of 16HBE cells were subjected to immunoprecipitation and immunoblot assays using indicated antibodies (right panel). (B, C) Schematic representation of Skp2 truncated mutants (B), which were transfected into 293T-ACE2 cells for protein purification and subsequent immunoblotting assays using indicated antibodies (C). (D) 16HBE and 293T-ACE2 cells were transfected with Flag-Skp2 and HA-Ub for 48 h, lysed, and subjected to immunoprecipitation and immunoblot using indicated antibodies. (E) 16HBE cells were transfected with Flag-Skp2, treated with CHX, lysed, and subjected to immunoblot using indicated antibodies. (F) 16HBE cells were transfected with siSkp2 for 24 h, followed by co-incubation with BaP (5 µmol/L) for 48 h, lysed, and subjected to immunoblot using indicated antibodies. (G) 16HBE and 293T-ACE2 cells were transfected with siSkp2 and HA-Ub for 48 h, lysed, and subjected to immunoprecipitation and immunoblot using indicated antibodies. (H) 293T-ACE2 cells were transfected with siSkp2 and HA-Ub for 48 h, followed by treatment with BaP at 5 µmol/L for additional 48 h. The cells were lysed, and subjected to immunoprecipitation and immunoblot using indicated antibodies. (I) In vitro ubiquitination assay using Flag-SCF^Skp2 and Flag-ACE2 purified from 293T cells. (J) In vitro ubiquitination assay using recombinant carrier free ACE2 and SCF^Skp2 proteins bought from R&D Systems.

Fig.5 Tobacco smoke and BaP induce downregulation of ACE2 in vivo. (A, B) Immunohistochemistry analysis of lung biopsy samples of patients with benign disease (A), and immunoreactivity score of ACE2 and Skp2 was calculated (B). (C, D) Western blot assays of normal lung tissues harvested 5 cm away from tumor tissues of patients with lung cancer (C) and quantification of ACE2 and Skp2 determined by densitometry analyses of immunoblot bands (D). (E) Spearman correlation analysis of ACE2 and Skp2 relative expression levels of the patients using the results of (D). (F, G) Immunohistochemistry analyses of lung tissues of mice exposed to clean air or tobacco smoke (F), and immunoreactivity score of ACE2 and Skp2 was calculated (G). Arrow, ciliated cells also express ACE2. (H) Immunofluorescence assays of lung tissues of mice exposed to clean air or tobacco smoke. (I, J) Western blot assays of lung tissues of mice exposed to clean air or tobacco smoke (I) and quantification of ACE2 and Skp2 determined by densitometry analyses of immunoblot bands (J). (K, L) Immunohistochemistry analyses of lung tissues of mice treated with vehicle control, BaP, or NNK (K). Immunoreactivity score of ACE2 was calculated (L).

Fig.6 Inhibition of SARS-CoV-2 S protein pseudoviron entry by CSE, BaP, and NNK. (A) Entry of SARS-CoV-2 S protein pseudovirions into 16HBE cells treated with indicated agents. (B) Entry of SARS-CoV-2 S protein pseudovirions into 293T-ACE2 cells treated with indicated agents. (C) Schematic representation of tobacco smoke-induced ACE2 degradation.

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