Binding to ACE2 leads to ACE2 dysregulation

Angiotensin-converting enzyme 2 (ACE2) is a transmembrne protein which under normal physiological conditions acts as a carboxypeptidase with broad substrate specificity. Angiotensin II is recognised as the main substrate of ACE2, but ACE2 hydrolyses a number of other biologically active peptides including DABK, apelin-13, neurotensin(1–11), dynorphin A-1–13), β-casomorphin-(1–7), and ghrelin (Vickers 2002, Humming, 2007). Hence, ACE2 is one of the major regulating enzymes in the local tissues and systemic plasma RAS and KKs systems, mainly balancing the action of its homologue ACE.

In the gut, ACE2 interacts with the Broad Neutral (0) Amino Acid Transporter 1 (B⁰AT1) (Slc6a19) and chaperons its membrane expression. As B⁰AT1 mediates the uptake of neutral dietary amino acids, such as tryptophan and glutamine, into intestinal cells (Camargo, 2009), ACE2 is indirectly involved in regulating intestinal amino acid homeostasis, expression of antimicrobial peptides, and the gut microbiome (Camargo, 2020, Hashimoto, 2012).  

Considering the above, it is plausible that interference with any aspect of the ACE2 expression, it’s enzymatic or B⁰AT1s-shaperone function represents a potential target for dysregulation with a wide range of downstream effects.

ACE2 is not known to act as a classical receptor that would itself initiate intracellular signalling cascades after binding of ligands or substrates and very few selective inhibitors or stimulators of its activity are available to study the effects of potential stressors and modulators of ACE2.  However, binding of SARS-CoV and SARS-CoV2 spike protein to ACE2 has been demonstrated (refs from Event: 1739) and this interaction represents an essential step for viral entry into the cells (refs from Event: 1738).

How interaction of ACE2 with the spike protein (KE upstream) perturbs ACE2 function (KEdownstream) is evaluated in the empirical evidence section of this KER.

At a molecular level, it is plausible that binding of the spike protein to the extracellular domain of ACE2 may perturb the catalytic activity of both, the cell membrane mACE2 or the cleaved catalytically active ectodomain sACE2, by allosteric interaction with the catalytic site (Liu & Nussinov 2016; Dutta 2022; Wang DS 2022; Trozzi 2022).

Interaction with spike protein may also perturb the shedding of ACE2 catalytic ectodomain. ACE2 is constitutively cleaved (shed) mainly by another membrane bound metalloprotease ADAM17, also known as TACE, TNFalpha Converting Enzyme (Zunke 2017) in a number of cells and tissues (Guy 2008 in human cardiac myofibroblasts; Peng Jia, 2009 in human lung epithelial cells and BALF; Werner, 2005 in canine epithelial polarised kidney MDCKII cells stabilly expressing ACE2; Iwata, 2009 in Chinese Hamster Ovary Cells). The exact role of constitutive ACE2 shedding is not well understood, but proteolytc ectodomain shedding of membrane proteins is a fundamental post-translational regulatory mechanism of the activity/function of a wide variety of proteins, including growth factors, cytokines, receptors and cell adhesion proteins (Lichtenthaler et al., 2018).

Binding of the spike (S) protein from both SARS-CoV has been associated with cleavage of the ACE2 ecodomain by TACE/ ADAM17 (Haga 2008, Haga 2010, Jocher, 2022), but also by another host protease TMPRASS2 (Heurich, 2014; Shula 2011) which also cleaves the Spike protein itself thus mediating viral entry by fusion of the cellular and viral membranes.  Spike protein mediated ADAM17 shedding of ACE2 has not been demonstrated for SARS-CoV2 (to my knowledge). However, the interplay between spike and these proteases has been suggested as critical and linked events based on analogy and also some structure-function considerations (Schreiber 2020; Heasly 2022; Zipeto 2020).  It is plausible then, that the spike induced shedding of ACE2 (via ADAM17 or other proteases) perturbs the distribution of the catalytically active mACE2/sACE2 and consequently differentially dysregulates ACE2 function/activity locally and systematically (Wysocki 2010). This in turn would have significant implications for the development of therapies that would target ACE2 activity and the RAS system appropriately in terms of delivery and timing.

Additionally, it is plausible that membrane bound ACE2 is down-regulated due to endocytosis of viral particles as suggested by earlier studies with SARS-CoV spike protein (Inoue, 2007; Wang H., 2008; Wang S., 2008) but also with SARS-CoV2 spike protein (Raghavan 2021, Gorshkov 2022). The relative contribution of the membrane fusion versus endocyitic route varies for the two SARS-CoV viruses (CoV1 and CoV2). This difference has been suggested as an important aspect of the differential infectivity and transmissibility of the two viruses (Zhu, 2021). It may also affect the relative magnitude and direction of mACE2 and sACE2 dyregulation.

Finally, transcriptional dysregulation of ACE2 mRNA synthesis is also plausible, but the process is not likely to be an immediate consequence of spike binding to ACE2, but rather requires some downstream signalling (potentially other intermediate KEs)and time for the biosynthesis of the mRNA and protein. Consistent with this, there is evidence that Ace2 (human but not the mouse), is an Interferon Stimulated Gene (ISG) activated also by some other viruses, inflammation, hypoxia and Synthetic dsRNA, poly(I:C) (Ziegler, 2020, Smith, 2020; Salka 2021, Zhuang 2020). However, it emerges that interferons and viruses stimulate a particular truncated form of ACE2, the dACE2 and not the full length ACE2 (Onabajo 2020; Scagnolari, 2021; Blume, 2021; Oliveto, 2022). The significance of this (post?)transcriptional modulation of Ace2 mRNA is not clear, and the enzymatic activity of the product of the alternatively spliced or, Ace2 mRNA initiated from a different promotor (which contains the catalytic domain but not the spike protein binding site) has not been examined. In addition, there is some evidence that S-protein binding can also lead to (direct?) suppression of ACE2 and Type I Interferon synthesis. To facilitate the analysis of the WoE for relevant perturbation of ACE2 as related to mRNA synthesis following the interaction with SARS-CoV and SARS-CoV2 spike protein, existing empirical evidence is outlined in the KER highlighting considerations for stressor type and time concordance between the two KEs.

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 included 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 (Table).

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.

Details of the evidence for dysregulation of ACE2 at mRNA and protein level (including enzymatic activity) as a result of interaction with SARS-COV 1 and 2 S proteins:  : https://aopwiki.org/system/dragonfly/production/2022/02/12/8oiauj0w7m_Empirical_evidence_ACE2_dysregulation_120222.pdf

GI tract specific evidence for dysregulation at mRNA level

Evidence Collection Strategy

The literature search is not comprehensive or systematic and needs continuous improvement.

In all of the studies reviewed here, ACE2 dysregulation following exposure to recombinant S-protein and replicating virus is considered as equivalent for the prpose of evaluationg ACE2 dysregulation. However, it should be noted that the literature screening step showed that inflammation and exposure to other stressors (nanomaterials and other viruses, like influenza) were also associated with ACE2 dysregulation, indicating that aspects of the stressor multiplication and/or persistency, additional to the S-protein binding, may be responsible for the observed ACE2 dyregulation. These references were tagged but not further analysed and not included in this KER that is focused on S-protein binding. They can be informative if this KER is eventually modified and potentially split into two: (i) direct/adjacent KER (studies examining ACE2 dysregulation after direct and short term exposure to S-protein binding or non-replicating pseudoviruses) and (ii) indirect/nonadjacent KER (studies with replication virus and persistent infection).

Focus on GI tract relevant evidence (published in https://doi.org/10.3390/jcm11185400)

Both ACE2 down and up regulation are observed in the gut as in the other test systems (see gut unreated evidence table). The apparent inconsistences regarding the direction and magnitude of ACE2 dysregulation in the different studies may reflect the dynamic, temporal components of the dysregulation driven not only by the interaction of ACE2 with the surface viral components i.e. S-protein binding, but also by the interaction of the replicating viral components with the innate immunity response elements, particularly in the test systems with replicating viruses used with the GI-tract-derived organoids.

ACE2 mRNA down-regulation in SARS-COV2 treated GI-derived organoids is reported in one study using scRANseq analysis (Triana et al., (2021). The down-regulation was specific to enterocytes actively replication the virus. Another study Nataf & Pays (2021) also reports profound but transient ACE mRNA down regulation.

Evidence for SARS-COV2 mediated up-regulation of ACE2 mRNA in GI-tract derived organoids is also available (Lamers et al., 2020; Lee at all (2020); Heuberger et al., 2021) and consistent with the similar studies in many other tissue/organ systems (see evidence table for other organs above). This is also consistent with the finding that ace2 is an Interferon Stimulated Gene (IFG) in airway epithelial cells (Ziegler, 2020) and also in colon enterocytes (Heuberger et al., 2021). In fact, all there studies also demonstrate a time concordance of ACE2 mRNA up-regulation with stimulation of ISG response in the infected organoids (Lamers et al., 2020; Lee at all (2020); Heuberger et al., 2021). Interestingly, even the scRNAseq study by Triana et al., 2021 which zoomed on specific cells within the organoid found that SARS-COV2 treatment induced distinct proinflammatory and ISG expression profiles in infected and bystander cells in the organoid. Namely, expression of interferon-stimulated genes was pronounced in bystander cells, while the infected cells showed strong NFkB/TNF-mediated pro-inflammatory response but a limited production of ISGs, suggesting that while SARS-CoV-2 may activate ISG by paracrine signaling, it may suppress the autocrine action of interferon i.e inducton of IGS including ACE2 in infected cells. This would be consistent with down-regulation of ACE2 in the infected cells observed in this study. In addition, this may explain why in some studies down-regulation of ACE2 mRNA can be observed under certain conditions (e.g. bulk mRNA measurements) and at some (earlier) time points of replication. Furthermore, the relationship of an observed increase of ACE2 mRNA to dysregulation at protein and enzymatic level remains to be elucidated. Indeed, most recently Harnik et al. 2021 (10.1038/s42255-021-00504-6) examined the spatial discordances between mRNAs and proteins in the intestinal epithelium and their significance for interpretation of transcriptomic data. Such apparent discordances have also been reported in the heart and lung tissue in mice and human (KE1854).

Identification of alternative forms of ACE2 mRNA and protein, an N-terminus truncated dACE2, which appears to have distinct transcriptional regulation profile compared to flACE2 (Onabajo et al, 2020, Janakowski et al (2021) - 0.1016/ j.isci.2021.102928) may also account for some of the observed inconsistences. However, a detailed analysis of experimental conditions in past, and careful design of probes and primers in future studies is necessary. Interestingly, concomitant down and up regulation of 97kD and 80kD anti-ACE2 polyclonal Ab-reacting proteins has been detected in differentiating human colon adenocarcinoma cell line HT29 (Bártová et al. (2020) 10.18632/aging.202221). Considering only one form of ACE2 relevant (97KD the only form detected in HEK293 and A549), the authors conclude that ACE2 is down-regulated in mature differntiatiatiated enterocytes compared to undifferentiated ones. This is in contrast to all mRNA and even cytoimmunostaining studies described above which demonstrate that a highest level of ACE2, both mRNA and protein is detected in the mature enterocytes and at the brush borders of the intestine and 3D organoids (eg. Lamers et all., 2020, Lee et al., 2020; Hauberge et al., 2021; Triana et al., 2021). Notably, the initial analysis by Onabajo et al, 2020 found that dACE2 mRNA is enriched in in squamous tumors of the respiratory, gastrointestinal and urogenital tracts.

The uncertainties and uncosnstances disused above illustrate clearly the need for careful characterization of the test systems to facilitate robust interpretation of the results.

In addition, we note that to date (to our knowledge), the majority of studies related to SARS-COV2-mediated ACE2 dysregulation, focus on ACE2 mRNA expression while structural/protein and functional studies are lacking, particularly in the gastro intestinal system. The novel gut-derived organoid systems can help address this gap by monitoring level and cell distribution of ACE2 protein as well as its function as B⁰AT1chaperon, via monitoring the membrane expression and/or the transporter function of B⁰AT1 itself. In addition, treatment with S-protein and/or non-replicating SARS-COV2 pseudo-viruses [Minghai & Zhang (2021) 10.7150/ijbs.59184], may help address better any potential direct effect of S-binding on ACE2 dysregulation. Finally, a development of more complex organoid systems that would also include microbiota or elements of the immune and/or vascular system are needed to better examine ACE2 dysregulation by SARS-COV2 but also the effects of such dysreulation at higher organizational level and in conjunction with the other elements of the RAS system.

Finally, evidence on up- or down-regulation of ACE2 in the GI tract of SARS-COV infected patients is not available to our knowledge. Examining the GI specific transcriptomic, proteomic and biomarker databases of COVID19 patents may help address some of these uncertainties.