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

A serine protease that has a variety of physiological and pathophysiological substrates.  Dysregulation of furin has been shown to lead to the promotion of infectious diseases and certain types of cancer.  Furin can also be called paired basic amino acid cleaving enzyme (PACE).

Michigan State University

Serine proteases are a type of enzyme that are diversely spread throughout all kingdoms of life.  Their function is to cleave a bond in a peptide via hydrolysis.  Typically, they are endoproteases, meaning they work in the middle of a peptide chain.  Additionally, they are allosteric enzymes.  This means that their function and activity can be controlled by an effector molecule that induces a conformational shape change in the enzyme.  Their name is derived from the serine amino acid and its side chain that plays an important role in its function that resides in the enzyme active site.

Serine Proteases

Several cellular proteins have been reported to be involved in the early steps of beta coronavirus infections in humans.  ACE2, a membrane bound protein that is involved in the cellular entry of many beta coronaviruses, is often supported by the function of other proteins.  Some facilitate the shedding of ACE2 from the cell surface, and others cleave viral S proteins to enhance the fusion of the two membranes.  They include:

- ACE2
- ADAM10
- ADAM17
- TMPRSS2
- TMPRSS11A
- TMPRSS11D
- furin
- CTSL
- PI4KB

Cellular Proteins Related to Beta Coronavirus Infections

Braun, E., & Sauter, D. (2019). Furin‐mediated protein processing in infectious diseases and cancer. Clinical & translational immunology, 8(8), e1073.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682551/
doi: 10.1002/cti2.1073

Furin‐mediated protein processing in infectious diseases and cancer

Kermani, N., Song, W. J., Lunt, A., Badi, Y., Versi, A., Yike, G. U. O., ... & Sterk, P. (2020). Airway expression of SARS-CoV-2 receptor, ACE2, and proteases, TMPRSS2 and furin, in severe asthma. medRxiv.
https://www.medrxiv.org/content/10.1101/2020.06.29.20142091v1

Airway expression of SARS-CoV-2 receptor, ACE2, and proteases, TMPRSS2 and furin, in severe asthma

Furin

Transmembrane protease serine 2 (TMPRSS2) is a serine protease.  Its gene is located on chromosome 21 at 21q22.3.  Androgen signaling controls its expression upstream of the gene's transcriptional start site.  

TMPRSS2

Type II Transmembrane Serine Proteases (TTSPs) are a large family of serine proteases.  They can be further categorized based off of the amino acid sequence of their stem region, which exists between a transmembrane signal anchor and the serine protease domain, as well as the chromosomal location of their genes.  Subfamilies include:

- matriptase
- hepsin/transmembrane protease serine (hepsin/TMPRSS) 
- corin
- human airway trypsin-like protease/differentially expressed in squamous cell carcinoma (HAT/DESC)

Type II Transmembrane Serine Proteases (TTSPs) 

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)

18,263 small airway epithelia cells from nonsmokers (NS) and 16,678 cells from smokers (S) were analyzed using single-cell 10x transcriptome.  Differences in gene expression between smokers/nonsmokers as well as between cell type were all less than 10%, and there were no significant results.  Notably, there was no change in ACE2 expression between smokers and nonsmokers.  This may reflect a limitation of using single-cell RNA sequencing compared to bulk RNA sequencing as the results depend on the size and shape of the cell.  However, these results are unique in that they elucidate the gene expression of individual cell types from a mixed population of cells.  

Expression of Cellular Proteins Related to Beta Coronavirus Infections in the Small Airway Epithelium (SAE) by Cell Type

mRNA expression of proteins related to the early steps in a B-coronavirus infection, excluding AC2, were determined in the small airway epithelium (SAE) of smokers (gray circles) and nonsmokers (white circles.)  Expression levels were quantified using RNA sequencing (Illumina HiSeq 2500), and the data values are reported in FPKM (fragments per kilobase of exon per million fragments sequenced.)  Only TMPRSS2 expression was modulated by smoking: its expression was significantly increased (p < 0.01) in smokers compared to nonsmokers.  

Expression of Cellular Proteins Related to Beta Coronavirus Infections in the Small Airway Epithelium (SAE)

ADAM10 belongs to the A Disintegrin And Metalloproteinases (ADAMs) family of proteases.  ADAM10 facilitated ectodomain shedding has been shown to influence the ability of cell adhision molecules and regulate paracrine, autocrine, and juxtacrine signaling.  

ADAM10

ADAM17 belongs to the A Disintegrin And Metalloproteinases (ADAMs) family of proteases.  It regulates various cell processes through its wide range of substrates, including TNFR1, TNFR2, and IL-6R (interleukin-6 receptor.)  ADAM17 notably is involved in the activation of the epidermal growth factor receptor (EGFR) in development through the shedding of TGFα, amphiregulin, HB-EGF, and epiregulin in the presence of phorbol 12-myristate 13-acetate (PMA).  

ADAM17

TMPRSS11A is a member of the HAT/DESC subfamily of TTSPs.  It is notably expressed in the respiratory system as well as on cell surfaces.  Additionally, it has been shown to facility the entry of the virus SARS-CoV-1 into human cells.  

TMRPSS11A

HAT, or TMPRS11D, is a member of the HAT/DESC subfamily of TTSPs.  It was initially discovered in the sputum of chronic respiratory disease patients, and it is thought to have a variety of physiological roles in epithelial tissue.  Proposed functions of this protease include:

- suppressing coagulation through fibrinogenolysis
- activating the protease activated receptor (PAR)-2
- influencing cell migration and adhesion via urokinase plasminogen activator cleavage

TMPRSS11D has been shown to activate the S protein of SARS-CoV-1 by cleaving it into its S1 and S2 subunits  This facilitates endosomal entry of the virus into a host human cell.  

HAT (TMPRSS11D)

Cathespin L is a member of the cathepsin family of proteases.  It is expressed throughout the body, and it is known to be involved in various pathologies, including:

- liver fibrosis
- type I and II diabetes
- cardiac and bone diseases
- immune disorders
- kidney disorders

Cathespin L (CTSL)

PI4KB is a PI4K that forms phosphatidylinositol 4-phosphate (PI4P).  It is primarily located in the Golgi and trans-Golgi network, and consequently, it is involved in cellular processes including lipid transport, membrane trafficking, cytokinesis, and maintaining lysosomal identity.  PI4KB and its isoform PI4KA are common viral targets because they can be used to create PI4P rich organelles that are important to viral reproduction. 

PI4KB

BCi-NS1.1 cells were derived from a single basal cell from the large airway epithelium (LAE) of a healthy nonsmoker, and hSABCi-NS1.1 cells were derived from a single basal cell from the small airway epithelium (SAE) of a healthy nonsmoker.  Expression levels were determined using HiSeq 4000 RNA-seq data at day 0 (baseline) and after 28 days on an air-liquid interface (ALI).  Notable findings include:

- Both cell lines showed an increase in ACE2 expression after 28 days of differentiation
- ADAM10, ADAM17, FURIN, CTSL, and PI4KB expression was relatively maintained throughout differentiation in both cell lines
- TMPRSS2 was notably less expressed in undifferentiated LAE basal cells than in SAE basal cells, and their expression increased.  However, its expression was maintained in SAE basal cells.  
- TPRSS11A and TMPRSS11D expression remained low in both LAE and SAE cells throughout differentiation

Because these cell lines are immortal, they may continue to be useful in SARS-CoV-2 research - especially for early drug screening.  

ACE2 and Other Cellular Proteins Related to B-Coronavirus Infection Expression Increases in Basal Cells from the Large Airway Epithelium Throughout Differentiation

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