ACE2 binding to viral S-protein leads to ACE2 dysregulation

This KER summarises the evidence for dysregulation of ACE2 (KE 1854) as a result of binding of the viral spike (S) protein of SARS-COV and SARS-COV2 (KE1739). This is likely an important aspect of COVID19 pathogenesis as an initial step it is a potential target for intervention at (re)infection but also for treatment of the disease. In addition, it is important to summarise the exiting evidence for this KER as the majority of hypotheses on the pathogenesis of COVID19 and potential treatments consider S protein binding leads to down-regulation of ACE2.

The evidence include in this KER was identified in the period between October 2020 and March 2021 with a Pubmed search syntax……, and additional targeted searches for specific aspects to ACE2 regulation. The searches were aimed to identify the evidence for the effect of SARS-COV or SARS-COV2 on any aspect ofACE2 regulation i.e. effect on expression of ACE2 at mRNA or protein level, ACE2 shedding and enzymatic activity, all described in KE 1854.

The studies generally use SARS-COV or SARS-COV2 Virus like Particles (pseudovirus), recombinant or transiently expressed S protein from SARS-COV and SARS-COV2 (in Table 1 designated S protein (1) and S-protein (2), respectively) in various cell lines e.g. Vero E6 and Huh7-that express endogenous ACE2 or HEK293T transfected with ACE2 expressing vectors. Concurrent increase of the soluble form of ACE2 (sACE2) and/or enzymatic (peptidase) activity is monitored in some of the studies (see Table in empirical evidence).

The evidence mostly relates to evaluation of direct binding of S protein to ACE2, but some evidence is indirect and can potentially relate to dysregulation resulting from infection progression/viral replication and reinfection of new cells in the context of the parallel inflammatory processes [authors note: eventually this KER may be split to more, depending on the context of developing KEs and AOPs].

Overall, studies that focus on the expression level of the membrane bound form of ACE2 strongly suggest that interaction of, SARS-COV S protein with ACE2 result in the cleavage (shedding) of the N-terminal enzymatic domain of ACE2 and hence down-regulation of membrane full length ACE2 on the surface of the cells (Glowacka 2010, Haga 2008, Haga 2010, Kuba, 2005).

Evidence for ACE2 protein down-regulation with SARS-COV2 S-protein (2) appears more limited.  Patra et al., 2020 show endogenous ACE2 protein reduction in cell lysates treated by SARS-COV2 pseudovirus or recombinant S protein in vitro.

It should be noted that ACE2 shedding mediated by the SARS-COV S-proteins (1 and 2) is a mechanistically and consequentially different process from the endogenous TACE/ADAM17 mediated cleavage of ACE2 (Iwata et al., 2009, Heurich et all., 2014, Hoffmann et al., 2020, Senapati et al., 2021). However, it remains to be elucidated if the two types of shedding, reduction of membrane flACE2 and potential increase of circulating sACE2 are associated with significant physiological effect. Furthermore, it appears that binding of S-protein to ACE2 also affects the function of the endogenous ACE2 sheddase TACE/ADAM17 (Haga et al., 2008), and therefore potentially its endogenous function on ACE2 shedding or its role in inflammation as a Tumour necrosis factor-alpha converting enzyme (e.g. Reddy et al, 2000). [please include a good review if you have].

A recent preprint by Lei et al., 2020, using primary cell culture of human pulmonary vascular endothelial cells treated with recombinant S-protein (2) appears to also indicate down-regulation of the ACE2 cellular protein level mediated by the binding to the viral protein, but this study remains to be reviewed.

Reduction of ACE2 protein level has also been observed in vivo in mouse lung tissue following intraperitoneal treatment with SARS-COV S-protein (Kuba et al., 2005) and in the myocardial tissue of SARS-COV infected mouse and human patients following death and autopsy (Oudit et al., 2009).

Down-regulation of ACE2 mRNA expression has been predicted for SARS-COV2-S protein interaction, by a study based on Master Regulator Analysis (Guzzi, et al., 2020) of transcriptomics data from lung tissue of SARS/MERS patients compared to SARS-COV2 modelled interactome in a different study (Srinivasan 2020). [note to fellow reviewers: feel free to comment on any of the studies, but please comment on this one. I am also not sure about the implications of the modelled interactome in Srinivasan 2020 and experimentally informed one 10.3390/v12040360 where HEK923T cells were used to express the viral bite proteins.]

Cleavage of ACE2 (shedding) is critical for SARS-COV and SARS-COV2 cell entry mediated by the binding of the S protein (KE-XXX) and it is likely that ACE2 activity is down-regulated on the infected cell membrane by the shedding, or when endocytic pathway of entry is utilised by the virus and ACE2 is likely internalised together with the viral particles (Zhu et al., 2021). Internalisation and intracellular co-localisation of ACE2 and S-protein has been demonstrated with SARS-COV S-protein (Inoue et al., 2007) and with SARS-COV pseudovirus in vitro (Wang H. et al., 2008, Wnag S et al., 2008 [fellow authors: please include evidence for SAERS-COV2 S protein and ACE2 co-internalising, I could't find ]).

However, shedding does not appear to abrogate the enzymatic activity of the sACE2 as evident from the studies that monitor its activity in the cell supernatants after shedding mediated by the interaction with the S-protein (See Table). In addition, there is evidence that high affinity binding of S-protein from the SARS-COV2 can enhance ACE2 enzymatic activity against some model peptide substrates in vitro (Jinghua Lu 2020), indicating that S-mediated up-regulation of ACE2 activity in vivo with physiological substrates cannot be excluded. In this study ACE2 peptidase activity was increased ~3-10 fold against model peptide substrates, such as caspase-1 substrate and Bradykinin-analogue.

Interestingly, increased ACE2 activity has been reported in the plasma of SARS-COV2 positive patients median 36 days after positive PCR test, and remained increased up to median 114 days (last measured). The level of the increase of ACE2 activity in the plasma correlated with severity of the disease (although the number of severe patients was much lower compared to those with milder symptoms) (Patel et al., 2021). Information on medications and comorbid conditions were also collected and considered in this study, and effect of any treatments was excluded. Although this is study does not directly address binding of the S-protein to ACE2, it demonstrates that the level of sACE2 is dysregulated (potentially increased) overtime in the context of SARS-COV2 infection in vivo and that this dysregulation is correlated with the severity of disease, suggesting that ACE2 dysregulation may be one of the KE of COVID19 pathogenesis.

Studies focusing on mRNA measurement or analysis generally suggest up-regulation of ACE2 mRNA as a result of interaction with SARS-COV and SARS-COV2, but also MERS-COV that does not use ACE2 as a receptor.

However, considering the supporting evidence for ACE2 up-regulation by interferon and inflammation in humans (See KE 1854) it is possible that the ACE2 mRNA increase apparent in a number of transcriptomics studies (Lieberman 2020; Feng 2020; Garvin, 2020) and also quantitative RT-PCR (Zhuang et al., 2020) in BALF or Nasopahrungeal swabs from SARS-COV2 infected patients may reflect stimulation of ACE2 transcription by the interferon component of the inflammatory process after the initial infection at later stages of the COVID19 pathogenesis. Nevertheless, ACE2 mRNA up-regulation was also observed in vitro after infection of primary cultures of human bronchial epithelial cells with SARS-COV2 (Li et al., 2020,) as well as SARS-COV and MERS-COV (Smith, 2020; Zhuang et al., 2020).

Rockx et al., (2009) examined the time course of change of ACE2 mRNA levels in the lung tissue of mice exposed to strains of SARS-COV variants of different lethality.  They monitored mRNA levels by quantitative RT-PCR from infected and control mice (young and aged ) up to 72 hours and conclude that expression of ACE2 mRNA is down-regulated in lethal infection of aged compared to young mice. However, the time course of the fold change for ACE2 mRNA during individual viral strain infections reveals more complex dysregulation of the ACE2 mRNA synthesis. For example, in the mice infected with the least lethal strain Urbani, ACE2 mRNA shows increasing trend over time compared to the mock controls, in both young and aged mice. Furthermore, transcriptomic monitoring of mRNA levels in infected tissue in this study shows differential expression over time of a number of genes related to the inflammatory response, lymphocyte mediated immunity and apoptosis, with progressive differences in adult and young mice infected with SARS-COV (Rockx et all, 2009).

Overall the empitical evidence is high that ACE2 expression (both protein and mRNA) and activity is significantly dysregulated as a consequence of the interaction with SARS-COV S-proteins (1 and 2). In addition, evidence suggests that the S-protein mediated ACE2 dysregulation is a rather complex and would require careful consideration of the timing of the replication process together with other aspects of progression of the disease in order to be targeted with drug interventions effectively (e.g.  interaction with parallel KEs, KERs in other related AOPs, e.g. increased interferon synthesis, inflammation, cell transition differentiation, hypoxia, etc..).