Protein Function

List of Major Functions of ACE2:

28, 29, 30

1.) Regulation of intestinal neutral amino acid transport

2.) Receptor for the Severe Acute Respiratory Syndrome Coronaviruses, SARS-CoV and SARS-CoV-2.

3.) Carboxypeptidase activity

4.) Regulation of cytokine production

5.) Endopeptidase activity.

6.) Regulation of Insulin Secretion

7.) Protein binding

8.) Peptidyl-dipeptidase activity

9.) Metallocarboxypeptidase activity

10.) Zinc Ion Binding

Two Most Common Functions:

1.) Carboxypeptidase Activity

Figure 1: Carboxypeptidase Activity of ACE2

(Diagram by Jeffrey Joseph)

The ACE2 receptor is known to function as a carboxypeptidase. According to Uniprot, a carboxypeptidase is an enzyme that catalyzes the hydrolysis of a single C-terminal amino acid residue from a polypeptide chain (14). ACE2 has the ability to convert angiotensin I into angiotensin (1-9) or angiotensin II into angiotensin (1-7). Figure 1 displays ACE2 functioning as a carboxypeptidase and converting angiotensin II into angiotensin (1-7). ACE2 receptors are primarily found on the alveolar epithelial cells and enterocytes of organs such as the lungs and small intestine respectively (31). As angiotensin II passes through the blood within these organs and others, the C-terminal end of angiotensin II can bind to the catalytic domain on the extracellular surface of ACE2 (32). This binding will cause a conformational change to occur with ACE2, thus leading to the cleavage of a peptide bond and removal of the amino acid residue phenylalanine from the C-terminal end. As a result, the active peptide angiotensine (1-7), containing 7 amino acid residues, is produced which is able to conduct s cardioprotective functions such as vasodilation (32).

2.) Regulation of Intestinal Neutral Amino Acid Transport

Figure 2: Regulation of Intestinal Neutral Amino Acid Transport

(Diagram by Jeffrey Joseph)

Another important function of ACE2 is that it helps regulate the B0AT1 protein in conducting neutral amino transport and production of antimicrobial peptides specifically in the small intestine. B0AT1 is part of the Solute Carrier 6 (SLC6) family of neutral amino acid transporting proteins, which aid organs such as the kidney and small intestine in the process of reabsorption of certain amino acids back into circulation within the body (33).

B0AT1 is found on the apical membrane of the small intestine along with ACE2. The role of ACE2 is to stabilize B0AT1 on the apical membrane and enhance B0AT1’s function in transporting amino acids (33). Figure 2, shows ACE2 binded to B0AT1 on the apical surface of the small intestine, which allows for an amino acid such as tryptophan to pass through. As B0AT1 continues to function with the help of ACE2 and the uptake of tryptophan increases in transportation, the mTOR (Mammalian Target of Rapamycin) pathway is activated, hence increasing the secretion of antimicrobial peptides (AMPs) (33). Antimicrobial peptides are essential in order to regulate homeostasis of the intestinal microbiota and prevent issues such as inflammation of the small and large intestinal linings.

Overall, the presence of ACE2 on the apical surface of the small intestine is vital to regulate the proper flow of amino acids back into the bloodstream and aid in the production of antimicrobial peptides.

List of Major Biochemical Pathways:

4, 34

1.) Renin-Angiotensin-Aldosterone System

2.) Peptide Hormone Metabolism

3.) SARS-Cov-2 Pathway

4.) Collagen Chain Trimerization

5.) Protein Metabolism

6.) A-beta Plaque Formation and APP Metabolism

7.) A-beta Uptake and Degradation

8.) mTOR Signaling Pathway

9.) ACE Inhibitor Pathway

10.)TGF-β pathway

Two Major Biochemical Pathways:

1.) Renin-Angiotensin-Aldosterone System (RAAS)

Figure 3: Diagram of the Renin-Angiotensin-Aldosterone Pathway

The Renin-Angiotensin-Aldosterone System (RAAS) is involved in regulating blood pressure and overall aiding in cardiac and renal functioning. It begins with the production of the protein hormone angiotensinogen by hepatocytes and the circulation of angiotensinogen through the bloodstream. Specifically under conditions where blood pressure levels drop and a sufficient amount of blood is not received by the kidneys for proper filtering functions, the enzyme renin is released into the bloodstream from the kidneys. Renin then targets angiotensinogen in the blood and cleaves it to form angiotensin I (37). Following the formation of angiotensin I, the angiotensin converting enzyme (ACE), that is found primarily in lung and kidney tissues, cleaves angiotensin I and converts it into the active angiotensin II (Physiology). Angiotensin II then binds to either angiotensin II type I and type II receptors (AT1R and AT2R), which are a class of G-coupled receptors. Specifically, the binding of angiotensin II to the AT1R receptor is known for causing vasoconstriction and stimulating aldosterone to be released from the adrenal glands.The release of aldosterone causes salt reabsorption in the kidneys to occur, thus elevating blood pressure (36) and (37)..

However, when blood pressure levels are too high in accordance with a high amount of angiotensin I or II in the blood, the angiotensin converting enzyme II (ACE2) works as a high blood pressure counterbalancer by converting angiotensin I into angiotensin (1-9) and angiotensin II into angiotensin (1-7). The main takeaway from this pathway in terms of ACE2’s function is that it eventually converts angiotensin II into angiotensin (1-7) which when binded to the AT2R or MAS receptors cause vasodilation and anti-inflammatory actions to occur (36).

2.) SARS-CoV-2 Pathway

Figure 4: Diagram of the SARS-CoV-2 Pathway

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The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) has led currently to the detrimental COVID-19 pandemic. The first outbreak of SARS-CoV occurred in 2003, eventually leading to the outbreak of the second SARS-CoV virus this past year. It was found that SARS-CoV contains a single, positive strand of viral RNA and is made up of four main structural proteins such as the spike glycoprotein, small envelope glycoprotein, membrane glycoprotein, and nucleocapsid protein (39). It was discovered that the spike (S) glycoprotein surrounding the outer membrane of the virus is responsible for binding to the ACE2 receptor found in cells of the lungs, liver, kidneys, GI tract, blood vessels, and several other parts of the body.

According to Figure 4, SARS-CoV-2 initially binds with one or multiple of its spike (S) glycoproteins to ACE2 receptors on the target cell. The host cell then uses specialized proteases to cleave the S-glycoproteins which then allows the S2 binding domain of the S-glycoprotein to undergo fusion with the host cell’s membrane. Once the virus fuses to the target cell, the virus releases its RNA into the cytoplasm which has the capability of being translated by certain ribosomes (38). Replicase proteins can then be generated to proliferate the viral genome. Additionally, the different proteins mentioned earlier that make up SARS-CoV-2 are transferred to the endoplasmic reticulum and Golgi apparatus. Lastly, the active virion is packaged into vesicles and released from the cell to other parts of the body through exocytosis (38)

ACE2 Expression in Major Tissues: 34,40

This list primarily takes into consideration the expression of ACE2 isoform 1

Small intestine
Kidney
Liver
Lungs
Testis
Heart
Colon
Esophagus
Artery
Stomach

Bladder
Pancreas
Adrenal Gland
Lymph Node
Brain
Thyroid
Salivary Gland
Uterus
Skeletal Muscle
Breast

Isoform 4 Specific Expression: 13

Lung
Gastrointestinal Tract
Urogenital Tract

Key Fact: Expression of ACE2 is upregulated in the lungs and airway epithelial cells with increased cigarette smoking and viral infections. (Uniprot main)

Developmental Stages: (4)

Human Fetal Development

Week 10: heart, intestine, kidney, stomach
Week 11: heart, intestine
Week 15: intestine
Week 16: kidney
Week 17: heart, intestine
Week 18: heart, stomach
Week 20: heart, intestine, kidney

After Birth

The expression of ACE2 (primarily Isoform 1) in further developmental stages can be viewed on the Bgee site.

Sub-cellular Localization:

(41)

Figure 5: Subcellular Localization of ACE2 (34)

Eukaryotic cells are divided into compartments (organelles) in order to carry out specialized, efficient functions. Subcellular localization indicates where a macromolecule such as a protein is expressed in particular locations of the cell. According to the Uniprot and GeneCards databases, a confidence level between 1 and 5 is assigned to a compartment, depending on the amount of the particular protein in that location. Moreover, a score of 5 represents high confidence (dark green) while a score of 0 represents low confidence (white).

In terms of looking at the locations of ACE2, the plasma membrane and extracellular space were given scores of 5 and are found with a dark green color on the cell map. This indicates that ACE2 is highly present in the plasma membrane and the extracellular space of a cell. On the other hand, a lower confidence score of 2 was mainly given to compartments within the cell such as the cytoskeleton, nucleus, and cytosol, and an even lower score of 1 in the Golgi apparatus. (34).

ACE2 is known to be a transmembrane protein, thus located within the membranes of cells. It’s metallopeptidase domain extends into the extracellular space of endothelial cells where ACE2 can function by cleaving the peptide hormones angiotensin I and angiotensin II as the peptide hormones pass through the bloodstream (42). In addition, ACE2’s localization in the plasma membrane allows it to bind and engulf the SARS-CoV viruses within the cell.

ACE2 contains an N-terminus signal peptide which allows it to be embedded into the plasma membrane of the cell after undergoing the secretory pathway. Like most proteins containing a signal peptide, signal recognition particles (SRPs) bind to the signal peptide of ACE2 in the initial stages of polypeptide synthesis by cytosolic ribosomes. The SRPS are then able to transport the ACE2 polypeptide with its bound ribosome to the endoplasmic reticulum where the polypeptide will continue to grow and fold into the ACE2 protein. Moreover, this explains why there is a presence of ACE2 in the endoplasmic reticulum. After the protein is formed, the protein is then properly packaged and modified through the Golgi apparatus and sent to a specific location within the cell. This explains why there is a low presence of ACE2 within the Golgi apparatus, since ACE2 is destined to be localized mainly in the plasma membrane of the cell.

Protein Interactions:

Figure 6: Protein Interactions of ACE2 (34)

1. Angiotensinogen (AGT):

Angiotensinogen is a protein hormone that is secreted by the liver. It is known to initiate the cascade of events involved in the Renin-Angiotensin-Aldosterone System, thus regulating blood pressure and fluid retention. In specific, angiotensinogen is cleaved by the enzyme renin to form the inactive peptide hormone angiotensin I. Eventually, angiotensin I is converted into the active peptide hormone angiotensin II by the angiotensin converting enzyme (ACE). ACE2 is then able to convert angiotensin II into the vasodilator angiotensin (1-7). (43)

2. Neurotensin/neuromedin N (NTS):

Neurotensin is found primarily within the linings of the intestine and is responsible for stimulating pancreatic secretion (44). In addition, neurotensin takes part in the regulation of fat metabolism and conducts smooth muscle contraction in the GI tract (45). Similar to how ACE2 cleaves angiotensin II and converts it to angiotensin (1-7), ACE2 can also cleave an amino acid residue at the C-terminal end of neurotensin as well. This cleavage by ACE2 gives neurotensin the ability to bind to the neurotensin receptor 1 (NTSR1) on endothelial cells, thus creating vasodilating effects like how angiotensin (1-7) does (44).

3.) Catalase (CAT):

Catalase is an enzyme that is found in the peroxisomes of a cell and is responsible for converting hydrogen peroxide (H2O2) into water and oxygen molecules. Thus, catalase acts as a protector of the cell against the toxicity from hydrogen peroxide and aids in immunity through promotion of T-cell and B-cell production (46). Catalase overall helps to reduce the amount of Radical Oxygen Species (ROS), such as hydrogen peroxide, to prevent destruction of cellular parts. Moreover, when ACE2 converts angiotensin II into angiotensin (1,7), particularly in the GI tract, catalase can be activated which then inhibits ROS production in the area. Additionally, as ACE2 helps to lower hypertension, oxidative stress in cells such as cardiomyocytes is reduced through activation of catalase (47).

4.) Spike Glycoprotein (S)

Despite not being listed within the GeneCards’s top five protein interactions, the ACE2 receptor’s interaction with the spike glycoprotein found on the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is an important and current interaction that is being studied. The spike glycoprotein binds to the ACE2 receptor with the S1 domain serving as the receptor binding domain of the spike glycoprotein and the S2 domain causing the virus and the receptor to fuse (48), (49). This binding causes a conformational change and leads to the virus being endocytosed into the target cell.

5.) Kininogen-1 (KNG1)

Kininogen-1 is alternatively spliced into two forms: high molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) (50). In particular, HMWK is involved with the blood coagulation process and serves as a precursor to the peptide bradykinin. Bradykinin is important for cardioprotective events in regulation of blood pressure, mediating inflammation, and having antimicrobial properties (50). The ACE2 receptor is involved with the kininogen-1 and bradykinin interaction by cleaving an amino acid off of the C-terminal end in bradykinin (14). In relation to the SARS-CoV-2 virus, ACE2 receptors are blocked from proper regulation of bradykinin, thus leading to an increase in inflammation and tissue damage (50).

Angio-Associated Migratory Cell Protein (AAMP) and Inositol-3-phosphate synthase 1: Although GeneCards lists AAMP and ISYNA1 as part of the top five interactions with ACE2, there only have been limited studies plus insufficient amount of data showing their definite relationship with one another. Thus, as of now it is unclear of the significance of the protein interactions of ACE2 with AAMP and ISYNA1

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Major Functions

Common Function

Major Pathways

Two Most Common Pathways

Tissue Expression

Developmental Stages

Sub-cell. Localization

Interactions