SARS-CoV-2 relies on its spike (S) protein to bind to cellular receptors to enter host cells. SARS-S, the SARS spike protein, uses ACE2 as an entry receptor into cells. Then the virus utilizes the serine protease TMPRSS2 found within the cell to enhance entry and prime future S proteins. SARS‐CoV‐2 is thought to be more transmissible than others CoVs because its S proteins have a greater affinity for ACE2.

ACE2 as a Receptor for SARS-CoV-2

ACE2 is known to be expressed in a variety of different tissues, including:

- Heart, kidneys, gastrointestinal (GI) tract, lungs, brain, testes, bladder, adipose tissue, vascular system, cholangiocytes (epithelial cells of the oral and nasal mucosa)

ACE2 expression may explain the SARS-CoV-2 organ localization.

Expression of ACE2 in Tissue and COVID-19

Some believe that the body may initiate an autoimmune response when SARS-CoV-2 is attached to an ACE2 receptor.  The body may consider this complex antigenic, meaning it produces antibodies.  However, these antibodies would target both the virus and ACE2.  This theory is substantiated by:

- the patterns of lung damage in patients with Pulmonary Hypertension secondary to Scleroderma, which is hallmarked by high levels of anti-ACE2 antibodies
- the severity of COVID-19 immune responses in those with cardiovascular disease and hypertension, who would be likely to have increased levels of ACE2 receptors in their blood serum

Measuring ACE2 levels in serum could be a way of predicting at-risk groups for ARDS (acute respiratory distress syndrome) and other hyperimmune responses.  

Autoimmunity to ACE2 Theory in the Pathogenesis of COVID-19

COVID-19 results in the down-regulation of ACE2 receptors by the virus binding to transcription factors at ACE2 regulatory regions on the gene.  This results in increased concentrations of Ang II, which would normally be converted to Ang 1-7 by ACE2.  This may contribute to the hyper-inflammation seen in COVID-19 patients.  

SARS-CoV-2 Down-Regulates ACE2 Receptors

- Neuronal ACE2 Expression as a Risk Factor for COVID-19
- Neuronal ACE2 Internalization in COVID-19
- ACE2 Expression in BBB
- ACE2 Expression in neuroglial cells
- ACE2 Expression in the ENS and Choroid Plexas

ACE2 and its Implications in SARS-CoV2 infection of the CNS

- Increased Concentrations of Circulating ACE2 is Associated with Mild COVID-19 Symptoms 
- Circulating ACE2 expression compared to circulating ACE

Expression of Circulating ACE2 and COVID-19

Three primary observations have been made about ACE2 and its relation to SARS/COVID-19:

- ACE2 acts as a functional receptor for SARS-CoV-2 to enter host cells
- Increased ACE2 expression may elevate the risk of a SARS-CoV-2 infection as well as the development of COVID-19
- Reduced ACE2 expression may lead to acute respiratory distress syndrome

SARS‐CoV‐2 may be able to infect other tissues that express ACE2 using viral RNA that circulates in the blood.  

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Retrospective epidemiological studies using clinical data and experimental molecular biologies studies have revealed biomarkers of COVID-19.  These elements not only reveal how COVID-19 mechanistically affects the body, but they may also be risk factors for developing severe symptoms and act as targets for pharmacological intervention.  They include:

- A1AT protection
- ACE2 expression and activity
- ADAM17 sheddase activity
- Furin protease activity
- Irregular respiratory function
- Pro-inflammatory cytokine concentration in the plasma
- TMPRSS2 serine protease genetic variation
- Variation in genetic susceptibility to SARS-CoV-2
- Viral interactions with glycosaminoglycans
- Nsp (Non-specific Protein) Activity
- Neuropilin-1 expression


Pathogenesis of COVID-19

ACE2 is a homologue of ACE.  Both enzymes are carboxypeptidases, meaning they cleave amino acids from the c-terminal of the peptide.  However, these homologues cleave the amino acid sequences at different points which leads to their different products.  ACE2 has four main physiological roles:

- Regulating blood pressure via its role in the renin-angiotensin-aldosterone system (RAAS)

- Limiting the amount of ACE substrate by converting Ang I into Ang 1-9

- Degrading Ang II in order to minimize its harmful effects

- Forming Ang 1-7 in order to maximize its cardioprotective effects

Angiotensin-converting enzyme 2 (ACE2)

Klhůfek, J. (2020). The role of angiotensin-converting enzyme 2 in the pathogenesis of COVID-19: the villain or the hero?. Acta Clinica Belgica, 1-8. https://www.tandfonline.com/doi/full/10.1080/17843286.2020.1786324
doi: 10.1080/17843286.2020.1786324

The role of angiotensin-converting enzyme 2 in the pathogenesis of COVID-19: the villain or the hero?

Tan, H. W., Xu, Y. M., & Lau, A. T. Angiotensin‐converting enzyme 2: The old door for new severe acute respiratory syndrome coronavirus 2 infection. Reviews in Medical Virology, e2122. https://onlinelibrary.wiley.com/doi/full/10.1002/rmv.2122

Angiotensin‐converting enzyme 2: The old door for new severe acute respiratory syndrome coronavirus 2 infection

TMPRSS2 is a human serine protease, an enzyme that cleaves a peptide chain via hydrolysis.  This enzyme has been shown to activate SARS-Cov-2 S protein by cleaving the S1 and S2 subunits of the S protein in order to expose the S2 subunit.  This allows the viral membrane to fuse with the cellular membrane.  Consequently, this process helps facilitate infection.  TMPRSS2 is also responsible for cleaving ACE2 receptors to further enhance SARS-CoV-2 entry.  This serine protease does not have a known, relevant function in human physiology and therefore could be a promising therapeutic target.  

TMPRSS2 in the Pathogenesis of COVID-19

SARS-CoV-2 S protein has two functional domains that regulate entry into a host cell:

- receptor binding domain (RBD)/ACE2 domain
- second domain (S2)

The S1 and S2 subunits of the S protein are connected by a polybasic furin cleavage site.  When furin acts on this site, the RBD and S2 domain are exposed which facilitate viral entry.    

Furin in the Pathogenesis of COVID-19

- European Wide Cohort Study Examines Expression Levels of COVID-19 Susceptible Biomarkers in Patients with Severe Asthma
- Review Investigating the Immunopathogenesis of COVID-19
- Structures and distributions of SARS-CoV-2 spike proteins on intact virions
- Implications of sex differences in immunity for SARS-CoV-2 pathogenesis and design of therapeutic interventions
- Natural killer cell immunotypes related to COVID-19 disease severity

Studies Related to the Pathogenesis of COVID-19

ACE2 in the Pathogenesis of COVID-19

Differences in COVID-19 inter-individual variability and lethality, along with differences among countries, raises the consideration of the role that predisposed genetics may play. Genetic variation may explain why some individuals without COVID-19 risk factors develop severe COVID-19.  Functional polymorphisms in the following genes are of interest due to their coded proteins' role in the pathogenesis of COVID-19:

- ACE2
- TMPRSS2

Understanding these variations in different populations could lead to more individualized treatment in COVID-19 patients.

Genetic Susceptibility to SARS-CoV-2 Infection in the Pathogenesis of COVID-19

Many viruses, including beta coronaviruses, have been shown to interact with human glycosaminoglycans, which are negatively-charged polysaccharides.  Studies suggest that SARS-CoV-2 can bind to the glycosaminoglycans heparan sulphate and heparin.  Additionally, it is thought. that heparin can prevent the virus from entering host cells while heparan sulphates facilitate infection by keeping SARS-CoV-2 S protein in its open conformation which allows it to interact with ACE2 receptors and TMPRSS2.

Glycosaminoglycans in the Pathogenesis of COVID-19

- Heparan sulfate proteoglycans are crucial for SARS-CoV-2 binding and infection 
- Low molecular weight heparins inhibit SARS-CoV-2 infection
- SARS-CoV-2 infection of epithelial cells is blocked by UF heparin and LMWH
- Heparan sulfates are required for transmission by primary dendritic cells
- Heparan sulfate proteoglycans Syndecan 1 and 4 are important for SARS-CoV-2 binding and infection.

 Heparan Sulfates and Low Molecular Weight Heparins in the Pathogenesis of COVID-19

While the mechanism remains unclear, it has been hypothesized that this type I sheddase transmembrane protein is involved in the pathogenesis of COVID-19.  Evidence that suggests that ADAM17 may be involved include:

- its known role in SARS-CoV viral uptake into host cells
- activating TNF‐α through cleave in cases of lung inflammation
- an association between high ADAM17 tissue expression and TNF‐α in an animal model of COPD, a known risk factor of COVID-19
- elevated ADAM17 gene expression in type II diabetic patients with diabetic low‐density lipoprotein (LDL)
- suggested contribution of ADAM17 to inflammation by stimulating IL-8 release
- proposed role in lung fibrosis and COPD through its ability to cleave IL-6R

ADAM17 in the Pathogenesis of COVID-19

A1AT is thought to be involved in the pathogenesis of COVID-19 because of its role as an innate immune system regulator, its function in lung homeostasis, and its potential relevance to the RAS system.  It may have a protective effect against SARS-CoV-2 infections by inhibiting the function of TMPRSS2 and TMPRSS2.  By inhibiting the serine protease ADAM17, which is involved in the viral uptake, the virus may be less efficient in infecting cells or may be prevented from infecting cells.  By inhibiting the sheddase ADAM17,  ACE2 cleavage would be controlled and may protect certain tissues from RAS system imbalance.  Additionally, inhibiting ADAM17 would prevent TNF‐α and IL6R cleavage which would have an anti-inflammatory effect which could ameliorate COVID-19 cytokine storm.  A1AT may also inhibit neutrophil elastase (NE), an enzyme that may be involved in SARS-CoV-2 S protein cleavage.  

A1AT in the Pathogenesis of COVID-19

In the innate immune response to SARS-CoV-2 infection, the host cells' pattern recognition receptors (PRR) recognise evolutionarily conserved molecular structures on the virus, called pathogen associated molecular patterns (PAMPS). When PAMPs bind to PRRs, the inflammatory response begins. PRRs activate several transcription factors to regulate the expression of genes responsible for inflammatory cytokine, interferon, chemokine, and adhesion molecule production.

Onset of Inflammatory Response in COVID-19

Researchers have found that COVID-19 virus is able to enter the body using another receptor, apart from ACE-2 receptor, called Neuropilin-1. This receptor is abundant in many tissues of the human body including respiratory tract, blood vessels and neurons. ACE-2 is expressed in very low levels on cell surfaces, the presence of Neuropilin in larger number may be one of the factor that attribute to its fast spread. 

Neuropilin-1 a receptor for COVID-19 entry 

Nsp stands for non-structural protein. This group of proteins are found in the SARS-CoV-2 virus. The SARS-CoV-2 virus contains Nsp1-16. These Nsp proteins help the virus replicate once it reaches its host cell. Some non-specific proteins play a larger role in cell entry and viral replication than others. 


Nsp proteins that play a significant role in the viral replication of the SARS-CoV-2 virus:

- Nsp1 (Non-structural protein 1)
- Nsp3 (Non-structural protein 3)
- Nsp9 (Non-structural protein 9)
- Nsp12 (Non-structural protein 12)
- Nsp15 (Non-structural protein 15)

Nsp's (Non-structural proteins) in the Pathogenesis of COVID-19

Other Type II Transmembrane Serine Proteases in the Pathogenesis of COVID-19

The nine amino acid long peptide that is formed when ACE 2 acts on Ang 1.  Ang 1-9 and Ang 1-7 are negative regulators of RAS.  

Ang 1-9

The seven amino acid long pepetide that is formed when ACE 2 acts on Ang II.  Ang 1-9 and Ang 1-7 are negative regulators of RAS.  Ang 1-7 is biologically active with MasR being its receptor.  This molecule alleviates the effects of Ang II/AT1R and has cardioprotective properties.  

Ang 1-7

- Inhibition of the binding between the spike protein present on the surface of the virus and the ACE-2 receptor present in the membrane of the host cell. This interaction mediates the entry of the virus in the cell. 
- Because ACE 2 plays a central role in the pathogenesis of COVID-19, it is thought that it could be an efficient drug target that could either treat or prevent infection. 

ACE2 normal role:
-Critical to regulate processes such as pressure, wound healing, and inflammation. 


-It helps modulate ANGII, a protein that increases blood pressure and inflammation, increasing damage to blood vessel lining and tissue. 

COVID-19:
-In response to COVID-19, ANGII has increased inflammation and the death of alveoli cells which are critical for bringing oxygen into the body...ACE2 prevents this.

-When COVID-19 virus binds to ACE2, it inhibits its action, thus increasing the effects of ANGII
 

ACE 2 Receptor as a Pharmacological Target for Treating or Preventing COVID-19

This virus uses angiotensin-converting enzyme 2 (ACE2) as a functional cellular receptor.
The virus also binds to the C-type lectin CD209L (also known as L-SIGN) and DC-SIGN

SARS-CoV cellular receptor

ACE2 is located on the X chromosome (Xp22), and belongs to a subset of genes that escapes X-inactivation, showing a heterogenous sex bias and evolutionary characteristics of tissue specificity and expression (Tukiainen et. al). However, expression differences in distinct tissues suggest presence of X-inactivation, though incomplete.

The figure below shows ACE2 preferential expression in males (blue). 

ACE2 Chromosomal Localization and Activation.

Intestinal ACE2 controls the expression of anti-microbial peptides (AMPs) and regulates intestinal homeostasis through its role in dietary amino acid uptake.  A lack of tryptophan, specifically, is thought to lead to adverse effects as it and its metabolites influence microbial ecology, microbial metabolism, and the interaction between the microbiota and its host.  Animal studies have shown that ACE2 knock outs had altered gut microbiotas, decreased AMP expression, and impaired amino acid uptake.  

ACE2 Modulates Gut Microbiota Composition

ACE2 structure components:
- signal peptide
- single metalloprotease active site
- transmembrane domain

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