Review TMPRSS2 - Vitória Luíza Damasceno

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The serine protease activity of TMPRSS2 on the disease dynamics by SARS-CoV-2, Influenza and prostate cancer: a literature review. 
Vitória Luíza Santos Damasceno (vitoria.damasceno@ufabc.edu.br); Márcia Aparecida Sperança (marcia.speranca@ufabc.edu.br). 
Abstract: Transmembrane serine protease type II (TMPRSS2) is widely expressed in epithelial cells of the gastrointestinal and respiratory system, prostate and various organs. The physiological role of this protease remains unknown even with several endogenous substrates found. TMPRSS2 is used as a gateway for SARS-CoV-2 and several other viruses that affect the respiratory system. In this review, we provide an overview of the TMPRSS2 enzyme and as it relates to coronavirus, influenza and prostate cancer.
Keywords: TMPRSS2; coronavirus; SARS-CoV-2. 
The gene encoding the human transmembrane serine protease – TMPRSS2 – was discovered in 1997 by Paoloni-Giacobino et al, is located at chromosome 21 (21q22.3), and presents 15 exons which express an open reading frame of 492 amino acids with four protein domains and a 5’ upstream region (UTR) element responsive to androgen [1]. Transcription of TMPRSS2 gene is stimulated through binding of testosterone and dihydrotestosterone to the androgen receptor. The four domains of TMPRSS2 correspond to a type II transmembrane, an LDL receptor class A (LDLRA) that contains a calcium binding site, a scavenger receptor cysteine-rich (SRCR) associated to cell surface binding to others cells or to extracellular molecules, and a serine protease from S1 family [1,2] (Figure 1).
Processing of TMPRSS2 mRNA can give rise to a second isoform, having an elongated cytoplasmic tail consisting of 37 amino acids, however, there is no evidence of difference in enzymatic activity or different roles between the isoforms. Therefore, the vast majority of studies are concentrated in the form of 492 amino acids [2,3].
Figure 1. TMPRSS2 protein layout. TM: transmembrane domain; LDLRA: low density lipoprotein receptor class A domain; SRCR: scavenger receptor cysteine-rich domain; H, D, S: catalytic triad - His, Asp, Ser. N and C: N and C-terminus. Numbers indicate amino acid position according to UniProt 015393. Figure created with BioRender (accessed on 09 April 2022). 
Proteases are enzymes responsible for cleavage of peptide bonds. The process of proteolytic cleavage is a regular mechanism of activation or inactivation of proteins [4]. Serine proteases have an active site with three conserved amino acids - Ser, His and Asp - forming the catalytic triad of the active site [5]. TMPRSS2 is a trypsin-like enzyme, belonging to S1 family and to the Hepsin/TMPRSS subfamily of Type II Transmembrane Serine Proteases (TTSPs), that cleaves at arginine or lysine residues [6]. 
Serine proteases are known to participate in many physiological and pathological processes. TMPRSS2 is also responsible for facilitating the entry of viruses into host cells, being able to cleave proteolytically and activate glycoproteins of the viral envelope. Viruses that use this protein to enter human cells include influenza viruses and human coronaviruses HCOV-229E, MERS-CoV, SARS-CoV and SARS-CoV-2 [7].
TMPRSS2 expression and function
The exact physiological function of TMPRSS2 remains unknown. In vivo tests, TMPRSS2-deficient mice did not demonstrate physiological abnormalities such as infertility or specific diseases. It has been suggested that TMPRSS2 is redundant and probably contributes to specialised but non-vital functions that would become evident under specific conditions [8]. 
The six conserved cysteine residues within the TTSP form three intradomain disulfide bonds characteristic of the S1 family protease folding, having affinity for substrates containing an arginine or a lysin in the P1 position. TTPS intermolecular activation takes place by other members of the S1 family or via self-activation following the C-terminal protease activity cellular compartments or on the cell surface, where it is linked to the N-terminal TMPRSS2 domain through conserved disulfide bonds [9]. 
Expression of TMPRSS2 was detected in membrane cells of several tissues including the prostate luminal epithelial cells of mouse and human, in renal tubules, epididymis and ducts of pancreas, in gastrointestinal tract, in epithelial cells of upper airways, bronchi and pons [10]. It is associated with physiological processes such as digestion, tissue remodelling, blood clotting, inflammatory responses, tumour cell invasion, apoptosis and pain [11]. Furthermore, it has proteolytic action on the hemagglutinin A (HA) of influenza viruses in human airway and pulmonary cells [5].
TMPRSS2 expression in human prostate epithelium is regulated by androgenic hormones [12] and seems to participate in the proteolytic cascades that result in the activation of a characteristic prostate antigen, the protease with an enzymatic reaction in the seminal fluid analogous to fibrinolytic and blood clotting [13]. The presence of TMPRSS2 in proteasomes provides an increase in male reproductive ability [12]. Recent studies have also suggested that TMPRSS2 plays a mediating role when anchored in the cell membrane when there is pain, mainly related to cancer [14]. 
Bertram et al. (2012) suggested that TMPRSS2 has reduced expression in type II alveolar cells and in alveolar macrophages when compared to bronchial epithelial cells, not being found in type I alveolar cells, during viral infection [31]. TMPRSS2 single nucleotide polymorphisms (SNPs) are associated with modification of expression rates [5].
TMPRSS2 in Coronavirus Infection.
In December 2019, a new infectious respiratory disease, caused by a new coronavirus, emerged in the city of Wuhan, located in Hubei Province, central region of the People's Republic of China, spreading rapidly, causing a health crisis of international proportions and drastic consequences. The clinical disease that has been named COVID-19 is caused by SARS-CoV-2 [3]. 
The research conducted by Lu et al. (2020) [15] determined SARS-CoV-2 genomic and epidemiological characteristics, reporting about 80% of SARS-CoV-2 genome sequence identity with SARS-CoV, the etiological agent of the SARS pandemic that presented cases between 2002 and 2004.
SARS-CoV-2 is a genome-wide enveloped virus composed only of a single-stranded, non-segmented, positively oriented RNA molecule. Viral particles are spherical, although pleomorphic, with approximately 80-220 nanometers. The SARS-CoV-2 RNA has approximately 30kb, making it among the largest RNA viruses identified to date [16]. The RNA genome is responsible for synthesising numerous copies of nucleoproteins projected from the formation of the nucleocapsid and viral envelope. The envelop consists of a lipid bilayer where the Spike (S), membrane (M) and envelope (E) proteins are anchored. The SARS-CoV-2 S protein is structured in trimers and forms the crown appearance of its outer surface, when observed under electron microscopy [17] (Figure 2).
Protein sequence analysis revealed that SARS-CoV-2 has seven conserved non-structural domains, as does SARS-CoV, demonstrating that both coronaviruses are substantially related, and even with amino acid variation at some specific residues, SARS-CoV-2 still has the receptor-binding domain structure analogous to SARS-CoV [15].
The entry mechanism of SARS-CoV-2 into human cells involves the Angiotensin Converting Enzyme 2 (ACE2) cell receptor and TMPRSS2. After binding to ACE2, the S protein is cleaved by TMPRSS2, facilitating viral activation, being one of the main factors for the pathogenicity of SARS-CoV-2 [9]. Zhou et al. (2020) elucidated the process of SARS-CoV-2 entry into human cells, identifying angiotensin-converting enzyme 2 (ACE2) as an entry receptor. Besides TMPRSS2, in addition to the endosomal cysteine proteases, cathepsin B and L (CatB/L), the activation of viruses can also be mediated by other proteases such as TMPRSS4, TMPRSS11A, TMPRSS11D and TMPRSS11E1 [18]. However, the activity of TMPRSS2is the only one fundamental for the entry of viruses into the cell and, consequently, the viral pathogenesis [9]. Other viruses that use TMPRSS2 as entry into cells include the influenza virus and human coronaviruses SARS-CoV, HCOV-229E and MERS-CoV.
 
Figure 2. SARS-CoV-2 schematic structure. The ssRNA (positive-sense single-stranded RNA) virus particle expresses the Spike (S) glycoprotein on its surface, being responsible for binding and infecting host cells. Highlight in the three-dimensional structure of the prefusion spike glycoprotein (S1). Figure created with BioRender (accessed on 09 April 2022).
The glycoprotein S is divided into two subunits, S1 and S2, where S1 is the receptor binding region, classified into three domains called A, B and C. The A domain of the S1 subunit binds to the host receptors [19]. The S2 subunit performs the function of the viral particle with the cell membrane [20]. SARS-CoV-2 enters the target cell through direct interaction with domain B. This domain binds to the human ACE2 receptor [2].
TMPRSS2 is directly responsible for facilitating SARS-CoV and SARS-CoV-2 coronavirus infections in humans through two types of independent mechanisms: proteolytic cleavage of the ACE2 receptor that promotes viral uptake and cleavage of the Spike glycoprotein present in the coronavirus, which allows the entry of the virus into the host cells [13]. 
The type II transmembrane serine proteases, including TMPRSS2, are anchored in the cytoplasmic membranes of cells, so the protease, when in lung tissue, cleaves protein S in several places, facilitating fusion between the membranes of the virus and the host cell. This event causes a decrease in viral sensitivity, leading to inhibition through a neutralising action of antibodies, conferring resistance to the viral infection process [21].
TMPRSS2 exists anchored to lung cells and in other regions, as previously mentioned, this fact may end up contributing to the extrapulmonary spread of the virus, justifying the range of studies on this enzyme, as a potential limiting target of viral spread and infection [22]. Tests In vitro and in vivo were performed, demonstrating that the inhibition of TMPRSS2 leads to a decrease in SARS-CoV-2 infection in host cells [22,23]. 
The discovery of the participation of TMPRSS2 in coronavirus infection was first described in 2011 with SARS-CoV. Glowacka et al (2011) evaluated the behavior of TMPRSS2 and the proteolytic cleavage capacity of the SARS-CoV S protein in cis and in trans. Western blot tests revealed that the S protein was cleaved into several fragments after TMPRSS2 co-expression (cis-cleavage) and after contact between TMPRSS2-positive S-expressing cells (trans-cleavage). Cis-cleavage resulted in the release of S fragments on the cell surface and inhibition of antibody neutralization. In trans-cleavage, S fragments contributed to fusion with target cells, allowing easy viral entry into targets treated with lysosomotropic agents or cathepsin inhibitors [24].
TMPRSS2 in Influenza infection. 
The viral replication cycle begins with the entry of the virus into the cell and when we observe the activity of enveloped viruses in host cells, this entry requires, in the vast majority of cases, virions binding to cell surface receptors and fusing to the membrane of the host cell. Viral envelope glycoproteins act in the control of both processes and the entry into the cell depends on a coordinated process of binding to the receptor that implies changes in these proteins [25]. 
The class I viral fusion protein of the SARS-CoV-2 virus, glycoprotein S, shares several structural features with the hemagglutinin (HA), the principal glycoprotein of influenza virus [26]. Studies have shown that the TMPRSS2 enzyme participates in the process of cleavage of influenza virus HA glycoproteins at an arginine residue. The human airway trypsin-like proteases (HATs) act, in turn, by cleaving the newly synthesised HA during the release of the progeny viruses, the HA of the viruses that arrive before being incorporated into the host cells [27, 28]. 
The TMPRSS2 only cleaves HA from within host cells and is not involved in the proteolytic activation of the HAs of the incoming virions. TMPRSS2 is able to cleave only the HA that originates within host cells and a direct involvement with the proteolytic activation of HA present in invading viruses is not observed and its soluble form, it is speculated, would have a minimal enzymatic activity that does not would be able to support the cleavage of a hemagglutinin [29].
Research in animal models has shown that after infection with H7N9 (A/Anhui/1/13) and H1N1 (A/PR/8/34) influenza strains in wild-type mice, it caused severe disease in the models, with mortality rates of 100% and 20% respectively; whereas in TMPRSS2 deficient mice, the viruses were only pathogenic. High tolerance to H7N9 infection and H1N1 adapted to A/California/04/09 (maCA04) mice was observed in TMPRSS2 knockout mice with lethal doses of 50% (LD50) for WT mice. The results present that TMPRSS2 plays an essential role in the tropism and pathogenicity of the H7N9 variant of the influenza virus and some subtypes of H1N1 in vivo models [30].
TMPRSS2 in Prostate Cancer 
TMPRSS2 is upregulated in responses to androgens [26] and in prostate cancer cells, it is highly expressed which enabled its first characterization after cloning the gene [4]. Increased expression of TMPRSS2 in tumoral cells takes place in the cell lumen alongside prostate epithelial tissue [31]. Occurrence of the 5'UTR region of the TMPRSS2 gene fusion to the oncogene erythroblast-specific-related (ERG) is the most common chromosomal aberration in cases of prostate cancer [26]. ERG is overexpressed in up to 50% of primary prostate cancers because of the androgen-dependent genomic rearrangements at 21q22.2-22.3 locus responsible for fusion production [26]. The mutation is due to chromosomal translocation or intergenic deletion with both genes that are located in the same arm on chromosome 21, resulting in overexpression of ERG chimeric mRNA [32]. The fused gene can lead to high expression of ERG due to the conduction of the androgen reaction element (ARE) of TMPRSS2, playing a role in the regulation of cell growth, apoptosis, and differentiation [33]. 
After Tomlin et al. (2005) reported the TMPRSS2 and ERG genes fusion for the first time, several studies indicated genetic associated differences in the TMPRSS2/ERG fusion genes frequency in the human population [34]. The frequency of this type of fusion was 50% in Caucasian American men, 31% in African American [35] and 18.5% in Asian [36]. This study also points out that the TMPRSS2/ERG fusion may be a driver of prostate cancer progression, leading to a number of oncogenic effects [37].
TMPRSS2 can contribute to the development of prostate cancer not only by increasing its expression but also by inducing intracellular alterations related to the epithelial polarity of transformed cells [37,38]. This abnormality may allow the protease to gain inappropriate access or activate other types of cancer. Men with androgen receptor transcription activities are more likely to develop prostate cancer with the TMPRSS2/ERG fusion. This tumour type also leads to increased signalling of insulin/insulin-like production factors, leading to changes in hormonal factors such as obesity and risk of metastasis [38].
Conclusion
TMPRSS2 transmembrane serine protease increased activity is associated with Influenza and SARS-CoV-2 viral entry, being also associated with a badly behaved prognostic of prostate cancer. Studies in animals revealed that this protein is not essential, and rather has a redundant role in human homeostasis. These characteristics make TMPRSS2 an interesting target to inhibit drug development in order to decrease viral infection and to treat cancer types where the enzyme is overexpressed. 
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