LitCovid-sentences  

PMC:7556165 / 2579-78705 JSONTXT 18 Projects

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Id Subject Object Predicate Lexical cue
T19 0-12 Sentence denotes Introduction
T20 13-191 Sentence denotes Angiotensin-converting enzyme (ACE) has an important role in the metabolism of several peptides and proteins, including chemical messengers, such as angiotensin I and bradykinin.
T21 192-465 Sentence denotes The importance assumed by research focused on ACE biochemistry, physiology and pharmacology is closely linked to the clinical effectiveness showed by ACE inhibitors in the treatment of cardiovascular disease, particularly arterial hypertension and congestive heart failure.
T22 466-546 Sentence denotes At present, ACE inhibitors are among the most widely prescribed drugs worldwide.
T23 547-618 Sentence denotes In 2000 a novel ACE-like enzyme, eventually named ACE2, was discovered.
T24 619-718 Sentence denotes It showed a different substrate selectively, and in particular it could not activate angiotensin I.
T25 719-785 Sentence denotes At the same time, it was not a target of classical ACE inhibitors.
T26 786-985 Sentence denotes Investigations were therefore started, aiming at determining the role of ACE2, and the general hypothesis that ACE2 may counteract several physiological consequences of ACE activation was introduced.
T27 986-1158 Sentence denotes In 2003 ACE2 was also identified as the initial cellular target of the SARS-CoV virus, since its spike glycoprotein was found to be a high affinity ligand of membrane ACE2.
T28 1159-1373 Sentence denotes Similar properties are shared by SARS-CoV-2 virus, and the huge impact of the recent COVID-19 pandemic has triggered novel interest in ACE2, particularly in the regulation of its expression in different cell types.
T29 1374-1615 Sentence denotes Hypertension has been suggested to have a major impact on COVID-19 susceptibility and prognosis, therefore the elusive link between hypertension, anti-hypertensive drugs, and ACE2 expression has soon become the object of a major controversy.
T30 1616-1795 Sentence denotes On the other hand, treatments able to interfere with ACE2 expression, or ACE2 availability for SARS-CoV-2 binding, have been identified as a novel goal of pharmaceutical research.
T31 1796-1903 Sentence denotes The purpose of the present review is to summarize the available knowledge on ACE2 biochemistry and biology.
T32 1904-2074 Sentence denotes We have also tried to point out some crucial open questions, which should be addressed to provide a better background for future experimental and clinical investigations.
T33 2076-2105 Sentence denotes ACE System and ACE2 Discovery
T34 2106-2329 Sentence denotes Human Angiotensin-converting enzyme (ACE) belongs to the M2 gluzincin family of metalloproteinases and (Ehlers and Riordan, 1989; Masuyer et al., 2014) exists in two forms, namely somatic ACE (sACE) and germinal ACE (tACE).
T35 2330-2552 Sentence denotes Both are derived from the same gene, controlled by alternative promotors. sACE is an integral membrane protein, which can be also cleaved by ACE secretases to produce a circulating form of the enzyme (Natesh et al., 2003).
T36 2553-2789 Sentence denotes sACE, hereafter referred to simply as ACE, has been extensively studied, because of its crucial role in the homeostasis of renin-angiotensin-aldosterone (RAAS) system and in cardiovascular diseases (Takimoto-Ohnishi and Murakami, 2019).
T37 2790-3023 Sentence denotes The two extracellular domains N and C domains of ACE (Wei et al., 1991; Jaspard et al., 1993; Natesh et al., 2003; Riordan, 2003) can both hydrolase two crucial peptides, namely angiotensin I and bradykinin, with the same efficiency.
T38 3024-3244 Sentence denotes Indeed, ACE carries out the cleavage of two amino acids (dipeptidase action) from the C-terminal part of angiotensin I to generate angiotensin II, which exerts a potent vasopressor, proliferative, and profibrotic effect.
T39 3245-3356 Sentence denotes Moreover, ACE mediates the cleavage and inactivation of bradykinin, which is a vasodilator hypotensive peptide.
T40 3357-3484 Sentence denotes The pivotal role of ACE in the RAAS system allows a refined blood pressure control and salt homeostasis (Sayer and Bhat, 2014).
T41 3485-3673 Sentence denotes Following the ACE discovery in mid-1950s, despite intense research in the field, no human homologs of the enzyme have been found for more than 50 years (Isaac et al., 1998; Riordan, 2003).
T42 3674-3825 Sentence denotes It was only in 2000 that two independent research groups identified, almost simultaneously, a new human ACE-like enzyme, with two different approaches.
T43 3826-4005 Sentence denotes Tipnis et al. (2000) searched for new metalloproteases in an expressed sequence tag (EST) database, finding an ACE homolog (ACEH) with a single domain, similar to that of insects.
T44 4006-4069 Sentence denotes Subsequently they cloned it from a human lymphoma cDNA library.
T45 4070-4258 Sentence denotes Interestingly ACEH showed high homology (40% identity and 60% similarity) with ACE, particularly around the HEXXH sequence and highly conserved glutamate residue, involved in zinc binding.
T46 4259-4351 Sentence denotes Moreover, they demonstrated the presence of seven glycosylation sites (Tipnis et al., 2000).
T47 4352-4672 Sentence denotes In the same year, Donoghue et al. (2000b) were searching for new genes involved in heart failure and identified a human cDNA ACE homolog, named ACE2, among 19,000 5’end sequences by RACE (rapid amplification of cDNA ends) in the heart ventricle cDNA library, obtained from a woman with idiopathic dilated cardiomyopathy.
T48 4673-4775 Sentence denotes ACE2 showed a transmembrane domain, a zinc catalytic domain 42% identical to ACE and a signal peptide.
T49 4776-4833 Sentence denotes Like ACE, ACE2 seemed to be an ectoenzyme type I protein.
T50 4834-5002 Sentence denotes The authors identified ACE2 transcripts quite exclusively in the heart and in the kidney, suggesting a role for ACE2 in the local RAAS control (Donoghue et al., 2000b).
T51 5003-5142 Sentence denotes In the following years, ACE2 was intensively studied, its structure and function were enlightened, and tentative inhibitors were developed.
T52 5144-5149 Sentence denotes ACE2:
T53 5150-5206 Sentence denotes Structure, Expression, Tissue Distribution, and Function
T54 5208-5225 Sentence denotes Structure of ACE2
T55 5226-5347 Sentence denotes ACE2 is a 40 kb gene and it is positioned on chromosome Xp22, differently from ACE gene that is located on chromosome 17.
T56 5348-5405 Sentence denotes The 18 exons of ACE2 are remarkably similar to ACE exons.
T57 5406-5695 Sentence denotes The ACE2 gene depicts a large polymorphism and several novel polymorphisms of ACE2, with specific geographical distribution, have been described and associated with susceptibility to hypertension and cardiovascular disease (Burrell et al., 2013; Patel et al., 2014; Pinheiro et al., 2019).
T58 5696-5816 Sentence denotes The ACE2 gene codifies for a typical zinc-metallopeptidase of 805 amino acids (120 kDa), with a unique catalytic domain.
T59 5817-5927 Sentence denotes Despite the high resemblance of ACE and ACE2, considerable differences exist in their substrates and products.
T60 5928-6017 Sentence denotes While ACE acts as dipeptidase, ACE2 removes only a single amino acid from its substrates.
T61 6018-6269 Sentence denotes Therefore, ACE2 is not active in transforming angiotensin I to angiotensin II and in inactivating bradykinin; moreover, ACE2 is insensitive to ACE inhibitors, like lisinopril and captopril (Tipnis et al., 2000; Rice et al., 2004; Turner et al., 2004).
T62 6270-6367 Sentence denotes These differences depend on variances in the three-dimensional structure (3D) of the two enzymes.
T63 6368-6476 Sentence denotes Comparative homology modeling and crystallography contributed to shed light on ACE2 3D structure (Figure 1).
T64 6477-6629 Sentence denotes Prabakaran et al. (2004) clarified the major characteristic of ACE2, which is a deep channel on the summit of the protein, hosting the catalytic domain.
T65 6630-6778 Sentence denotes Specific loops, like the long loop N210-Q221 that is exclusive of ACE2, α helices and a portion of β-sheet are located around the catalytic channel.
T66 6779-6939 Sentence denotes The negative charge of the channel and the presence of distinct hydrophobic regions contribute to the specificity of the binding site (Prabakaran et al., 2004).
T67 6940-7202 Sentence denotes The determination of the crystal structure of the extracellular domain to 2.2-3-A resolution from Towler et al. (2004) and the model from Guy et al. (2003) showed that the catalytic domain of ACE and ACE2 are very conserved and have similar mechanisms of action.
T68 7203-7474 Sentence denotes The main difference stems from the smaller ACE2 pocket, thereby lodging only a single amino acid: the crucial substitution of the Gln281 in ACE binding pocket with Arg273 in ACE2 is likely to be responsible for the steric conflict (Guy et al., 2003; Towler et al., 2004).
T69 7475-7711 Sentence denotes Another surprise of the ACE2 structure was its C-terminal domain, which—differently from ACE—revealed high homology with collectrin, a renal protein, which is involved in amino acids trafficking through the membrane (Yang et al., 2017).
T70 7712-7748 Sentence denotes FIGURE 1 Crystal structure of ACE2.
T71 7749-7864 Sentence denotes The peptidase domain (PD) is in green, whereas the collectrin homology domain is enclosed in the light cyan square.
T72 7865-8010 Sentence denotes The active zinc ion is showed enclosed in a red circle, whereas the glycosylation moieties are showed as cyan cubes and denoted by dashed arrows.
T73 8011-8104 Sentence denotes The structures have been drawn from PDB 1R42 (Towler et al., 2004) by Mol on the PDB website.
T74 8106-8186 Sentence denotes Transcriptional, Post-transcriptional, and Post-translational Regulation of ACE2
T75 8187-8470 Sentence denotes The location of the ACE2 gene on the X chromosome questions whether one of the two ACE2 is silenced in females, to balance female/male expression dosage (X chromosome inactivation or XCI), or otherwise belongs to the class of “escape genes” which are transcribed on both chromosomes.
T76 8471-8659 Sentence denotes Interestingly, a wide survey of XCI in several individuals and tissues showed that ACE2 is a heterogeneous escape gene, because it has a tissue-dependent sex bias (Tukiainen et al., 2017).
T77 8660-8812 Sentence denotes A growing number of recent findings point to an important role of epigenetic mechanisms associated with several human diseases (Surguchov et al., 2017).
T78 8813-8987 Sentence denotes In this context, several authors highlighted the regulatory role of 17β-estradiol (E2), a primary female sex steroid, in the expression of ACE2 in a tissue-dependent fashion.
T79 8988-9136 Sentence denotes Liu et al. (2010) and Stelzig et al. (2020) found out that E2 downregulates ACE2 in kidney and differentiated airway epithelial cells, respectively.
T80 9137-9419 Sentence denotes The latter result is particularly important, as the male-bias of ACE2 expression in the lung could account for the alleged higher susceptibility of males to COVID-19 symptoms following ACE2-dependent SARS-CoV-2 infection (section Links Between ACE2 and COVID-19) (Jin et al., 2020).
T81 9420-9592 Sentence denotes Yet, Bukowska et al. (2017) observed that E2 increases ACE2 transcription and expression in human atrial tissue, while at the same time depressing the level of ACE protein.
T82 9593-9804 Sentence denotes This mechanism attenuates the renin-angiotensin system and, in tandem with anti-inflammatory and anti-oxidative effects, enables a stronger response to myocardial stress and contributes to antiarhythmic effects.
T83 9805-9951 Sentence denotes The upregulation of ACE2 (and downregulation of ACE) was clearly linked to binding of E2 to Estrogen Receptor alpha (ERα) (Bukowska et al., 2017).
T84 9952-10192 Sentence denotes The E2-ERα complex might migrate to the nucleus to bind to estrogen response elements, although the actual mechanism is still obscure and should likely involve other co-factors to take into account the observed tissue bias of E2 regulation.
T85 10193-10442 Sentence denotes Nonetheless, it was demonstrated that the Estrogen Related Receptor alpha (ERRα), which likewise ERα recognizes the estrogen response element in target genes, binds to ACE2 promoter to repress transcription (Tremblay et al., 2010; Lee et al., 2012).
T86 10443-10545 Sentence denotes Hopefully, future studies will shed more light on the intriguing role of estrogens in ACE2 regulation.
T87 10546-10708 Sentence denotes A recent analysis of public genomic and transcriptomic data outlined the role of histone methylation, a classical epigenetic mark, to regulate ACE2 transcription.
T88 10709-10870 Sentence denotes Indeed, Li Y. et al. (2020) showed that transcription of ACE2 was significantly upregulated when the histone mutant H3K27M was overexpressed to inhibit H3K27me3.
T89 10871-10953 Sentence denotes Conversely, overexpression of mutant H3K4/9/36M did not change ACE2 transcription.
T90 10954-11109 Sentence denotes Trimethylation of K27 on H3 is catalyzed by the polycomb groups (PcG), a group of conserved transcriptional gene repressors (Schuettengruber et al., 2017).
T91 11110-11172 Sentence denotes PcG proteins assemble into two major complexes: PRC1 and PRC2.
T92 11173-11297 Sentence denotes The simplest model of PcG activity involves trimethylation of H3 by PRC2 at target gene promoters (Blackledge et al., 2015).
T93 11298-11546 Sentence denotes These epigenetic marks recruit PRC1 on DNA, which in turn acts as E3-ligase and ubiquitinates nearby H2A histones (Storti et al., 2019), triggering silencing of gene transcription by local and reversible compaction of chromatin (Illingworth, 2019).
T94 11547-11612 Sentence denotes The catalytic subunit of PRC2 is constituted by the EZH2 protein.
T95 11613-11780 Sentence denotes In agreement with the inverse correlation between ACE2 level and H3K27me3, ACE2 expression in human ESCs was upregulated following EZH2 knock-out (Li Y. et al., 2020).
T96 11781-11844 Sentence denotes On the other side, recovery of EZH2 restored basal ACE2 levels.
T97 11845-12038 Sentence denotes Chromatin immunoprecipitation sequencing (ChIP-seq) showed that EZH2 depletion induced H3K27me3 decrease, with concomitant H3K27ac increase, at ACE2 promoter in human ESCs (Li Y. et al., 2020).
T98 12039-12325 Sentence denotes The role of H3 methylation and acetylation in the epigenetic regulation of ACE2 was also hypothesized by Pinto et al., who demonstrated that co-morbidities such as hypertension, diabetes, and chronic obstructive lung disease increase ACE2 transcription in the lung (Pinto et al., 2020).
T99 12326-12407 Sentence denotes Histone methylation does not appear to exhaust the epigenetic regulation of ACE2.
T100 12408-12556 Sentence denotes Notably, the NAD+-dependent deacetylase SIRT1 binds to ACE2 promoter favoring its transcription during cellular energy stress (Clarke et al., 2014).
T101 12557-12637 Sentence denotes Two recent unrefereed preprints highlighted other epigenetic mechanisms at play.
T102 12638-12842 Sentence denotes Corley et al.1 pointed out that DNA methylation across three CpG islands in the ACE2 promoter was lower in lung epithelial cells compared to other cell types, suggesting high transcription in lung tissue.
T103 12843-13045 Sentence denotes These findings are in excellent agreement with the reported inverse correlation between ACE2 transcription and promoter methylation in tumors, which will be discussed in section ACE2 and Other Diseases.
T104 13046-13228 Sentence denotes This correlation is also supported by the observation that in children ACE2 is normally hypermethylated and poorly expressed either in the lung and in other organs (Pruimboom, 2020).
T105 13229-13450 Sentence denotes Glinsky (2020) addressed the epigenetic role of Vitamin D on ACE2 expression, showing by gene set enrichment analysis that the Vitamin D receptor (VDR) should be involved in a set of regulatory pathways conveying on ACE2.
T106 13451-13660 Sentence denotes More specifically, VDR activation would downregulate ACE2, thus affording a potential reason for the alleged beneficial role of Vitamin D in COVID-19 (section ACE2 and the Inflammatory Response to Sars-CoV-2).
T107 13661-13780 Sentence denotes Finally, the strict homeostatic balance of ACE/ACE2 activities suggests transcriptional co-regulation of both proteins.
T108 13781-14076 Sentence denotes Remarkably, Yang et al. (2016) have demonstrated that a subtle regulatory mechanism acts in cardiac endothelial cells, where the Brg1 chromatin remodeler and the FoxM1 transcription factor cooperate to determine the ACE2/ACE expression ratio, particularly under cardiac hypertrophy of the heart.
T109 14077-14121 Sentence denotes Further regulation occurs at the mRNA level.
T110 14122-14247 Sentence denotes From putative microRNA-binding sites identified in vitro, Lambert et al. (2014) demonstrated that miR-421 downregulates ACE2.
T111 14248-14395 Sentence denotes According to the hypothesized mechanism, miR-421 modulates ACE2 expression by hampering translation rather than by degradation of mRNA transcripts.
T112 14396-14611 Sentence denotes Beside undergoing post-translational modifications by glycosylation and phosphorylation, ACE2 is also post-translationally regulated by shedding from cell membrane through the action of the metalloproteinase ADAM17.
T113 14612-14782 Sentence denotes The proteolysis of ACE2 releases a soluble, enzymatically active form which corresponds to the ACE2 ectodomain (Jia et al., 2009; Xiao et al., 2014; Conrad et al., 2016).
T114 14783-14900 Sentence denotes The function, if any, of soluble ACE2 is still obscure, but the shedding mechanism is under strict molecular control.
T115 14901-15063 Sentence denotes Lambert et al. (2008) highlighted that calmodulin associates with ACE2 to prevent its shedding, while calmodulin inhibitors increase the cellular release of ACE2.
T116 15064-15113 Sentence denotes Patel identified a positive feedback in the RAAS:
T117 15114-15224 Sentence denotes Ang II activates ADAM17, thereby increasing the release of ACE2, its negative regulator (Patel et al., 2016b).
T118 15225-15356 Sentence denotes It is worth noting that high levels of plasma-soluble ACE2 have been associated with myocardial dysfunction (Epelman et al., 2009).
T119 15357-15488 Sentence denotes The potential pathophysiological role of ADAM17 is further discussed in paragraph ACE2 and the Inflammatory Response to Sars-CoV-2.
T120 15489-15609 Sentence denotes Figure 2 summarizes the known transcriptional, post-transcriptional, and post translational regulation pathways of ACE2.
T121 15610-15711 Sentence denotes FIGURE 2 Regulation pathways of transcription, translation, and post-translational shedding of ACE2.
T122 15712-15744 Sentence denotes Red text: non-molecular factors.
T123 15745-15775 Sentence denotes Black text: molecular factors.
T124 15777-15804 Sentence denotes Tissue Distribution of ACE2
T125 15805-15945 Sentence denotes Detectable quantities of ACE2 protein have been found almost ubiquitously in tissues across mammalian species, using immunostaining methods.
T126 15946-16163 Sentence denotes ACE2 is predominantly located in the cardiovascular system and kidney, where it probably plays a role in the maintenance of hydro electrolyte homeostasis (section “Mechanism of Viral Entry Mediated by the S Protein”).
T127 16164-16382 Sentence denotes In fact, ACE2 is pervasively expressed throughout the vasculature, at the level of the arteries and veins, mainly in smooth muscle cells of the media and in the endothelium (Hamming et al., 2004; Burrell et al., 2005).
T128 16383-16473 Sentence denotes Such signal from vessels also delivers part of the expression detected in specific organs.
T129 16474-16812 Sentence denotes Indeed, ACE2 is evident in: coronary vessels and myocardial capillaries (Wiener et al., 2007; Garabelli et al., 2008); lung microvascular endothelial cells (Wiener et al., 2007; Chen et al., 2013); kidney interlobular arteries (Lely et al., 2004); endothelial and smooth muscle cells in the brain (Hamming et al., 2004; Kar et al., 2010).
T130 16813-16990 Sentence denotes Notably, the mesangium and glomerular endothelium in the kidney, and the endothelial lining of the sinusoids in the liver are allegedly negative for ACE2 (Hamming et al., 2004).
T131 16991-17178 Sentence denotes On the contrary, ACE2 is virtually absent from the lymphatic system, and human hemato-lymphoid organs (i.e., spleen, lymph nodes, and bone marrow) (Hamming et al., 2004; Li et al., 2007).
T132 17179-17326 Sentence denotes In blood cells, it has been observed in platelets and macrophages, but not in B and T lymphocytes (Hamming et al., 2004; Fraga-Silva et al., 2011).
T133 17327-17670 Sentence denotes Expression of ACE2 was originally identified in rodent heart (Donoghue et al., 2000a), where it was observed to occur in both atrium an ventricle (Gembardt et al., 2005), and, cellularly, in cardiomyocytes and in specialized cells of the sinoatrial node (Burrell et al., 2005; Garabelli et al., 2008; Ferreira et al., 2011; Wang et al., 2017).
T134 17671-17863 Sentence denotes In human heart, ACE2 has been found in the stromal area in spongiosa layer in aortic valves (Peltonen et al., 2011), where it is expressed in myofibroblasts and fibroblasts (Guy et al., 2008).
T135 17864-18151 Sentence denotes High levels of ACE2 protein expression have been detected in mammalian, including human, kidney (Gembardt et al., 2005; Koka et al., 2008; Reich et al., 2008; Giani et al., 2012; Mitani et al., 2014; Grobe et al., 2015; Shi et al., 2015; Larouche-Lebel et al., 2019; Alawi et al., 2020).
T136 18152-18490 Sentence denotes Strong signals were reported in the brush border of the proximal tubular cells, whereas weak to moderate signals could be found in the glomeruli, Henle’s loop, distal tubules, and collecting duct (Hamming et al., 2004; Lely et al., 2004; Kamilic et al., 2010; Giani et al., 2012; Bae et al., 2015; Cao et al., 2017; Errarte et al., 2017).
T137 18491-18685 Sentence denotes In the respiratory tract of primates, positive labeling for ACE2 has been reported at multiple sites, from the nasal and oral mucosa, to the larynx, trachea, bronchi and lung (Liu et al., 2011).
T138 18686-18971 Sentence denotes Whether ACE2 is expressed in human nasal and oral epithelium remains unclear, as contradictory results have been reported by studies using immunohistochemistry (Hamming et al., 2004; Bertram et al., 2012), in face of positive single-cell RNA sequencing findings (Sungnak et al., 2020).
T139 18972-19142 Sentence denotes This point is of great interest to understand the role of those tissues in SARS-CoV-2 initial infection, spread and clearance (section “Links Between ACE2 and COVID-19”).
T140 19143-19300 Sentence denotes In the upper respiratory tract ACE2 is expressed in the epithelial lining and lamina propria, in some muscle cells and in the salivary gland duct epithelium.
T141 19301-19595 Sentence denotes In the lung, an intense signal for ACE2 protein has been consistently observed in type I and II pneumocytes in several species, including mouse, rat, cat, ferret, monkey and human (Wiener et al., 2007; van den Brand et al., 2008; Liu et al., 2011; Wong et al., 2012; Chen et al., 2013; Zhang B.
T142 19596-19613 Sentence denotes N. et al., 2019).
T143 19614-19778 Sentence denotes Data from rodents suggest an age- and gender-dependent pattern of expression, with a more rapid decline with age in males as compared to females (Xie et al., 2006).
T144 19779-20057 Sentence denotes Although some ACE2 signal has been observed in the liver, it appears to mainly come from small vessel endothelium, and occasionally bile duct epithelial cells, while negligible expression is observed in hepatocytes (Hamming et al., 2004; Paizis et al., 2005; Guan et al., 2020).
T145 20058-20247 Sentence denotes ACE2 protein is abundantly expressed in the brush border of enterocytes of all parts of the small intestine, including the duodenum, jejunum, and ileum, but not in enterocytes of the colon.
T146 20248-20457 Sentence denotes Other organs of the digestive tract, such as the stomach and colon, did not show brush border staining, but rather a positive signal in the muscolaris mucosae and the muscolaris propria (Hamming et al., 2004).
T147 20458-20624 Sentence denotes In rodents, ACE2 is also expressed in both exocrine and endocrine pancreatic tissue, particularly in the islets of Langerhans (Niu et al., 2008; Fang and Yang, 2010).
T148 20625-20715 Sentence denotes ACE2 distribution is widespread in the mouse brain, from the telencephalon to the medulla.
T149 20716-21122 Sentence denotes As expected, ACE2 is found in brain areas involved in the regulation of cardiovascular function and fluid balance, such as the vascular organ of lamina terminalis, subfornical organ, magnocellular neurons in the hypothalamic paraventricular nucleus, area postrema, nucleus of the solitary tract, dorsal motor nucleus of the vagus, nucleus ambiguous, and rostral ventrolateral medulla (Doobay et al., 2007).
T150 21123-21406 Sentence denotes However, significant expression had also been reported in brain areas not engaged in the classical functions of the RAAS, namely the piriform cortex, hippocampus, caudate putamen, hypoglossal nucleus and primary motor cortex (Doobay et al., 2007; Lin et al., 2008; Liu et al., 2014).
T151 21407-21544 Sentence denotes ACE2 immunostaining was identified in neurons as well as astrocytes (Gallagher et al., 2006; Doobay et al., 2007; Yamazato et al., 2007).
T152 21545-21715 Sentence denotes Furthermore, ACE2 has been documented in the retina, predominantly in the inner nuclear layer but also in photoreceptors (Tikellis et al., 2004; Senanayake et al., 2007).
T153 21716-21824 Sentence denotes With regard to the endocrine system, ACE2 expression was found in both male and female reproductive systems.
T154 21825-21962 Sentence denotes In human testis, ACE2 was localized to the Leydig and Sertoli cells, and might be involved in testicular function (Douglas et al., 2004).
T155 21963-22230 Sentence denotes At present, no data about ACE2 protein expression is human ovaries is available, although evidence of expression in stroma, theca, and granulosa cells has been reported in other species (Tonellotto dos Santos et al., 2012; Barreta et al., 2015; Pereira et al., 2015).
T156 22231-22344 Sentence denotes In rodent bone, ACE2 is expressed in osteoblasts and osteoclasts, as well as in epithelial cells and fibroblasts.
T157 22345-22449 Sentence denotes However, a similar expression in human samples still awaits clarification (Queiroz-Junior et al., 2019).
T158 22450-22575 Sentence denotes In human skin, ACE2 was present in the basal cell layer of the epidermis extending to the basal cell layer of hair follicles.
T159 22576-22657 Sentence denotes Smooth muscle cells surrounding the sebaceous glands were also positive for ACE2.
T160 22658-22722 Sentence denotes Weak cytoplasmic staining was observed in sebaceous gland cells.
T161 22723-22807 Sentence denotes A strong granular staining pattern for ACE2 was seen in cells of the eccrine glands.
T162 22808-22988 Sentence denotes Positive staining for ACE2 was also noted in the membrane of human fat cells in various organs, including the epicardial adipose tissue (Hamming et al., 2004; Patel et al., 2016a).
T163 22989-23099 Sentence denotes Globally, ACE2 is chiefly bound to cell membranes, while negligible levels can be detected in the circulation.
T164 23101-23143 Sentence denotes ACE2, the RAAS System and Cardioprotection
T165 23144-23408 Sentence denotes After the initial discovery of ACE2 in the heart and kidney, it is now clear that it is widely distributed in tissues (section Tissue Distribution of ACE2), where it exerts many physiological effects and may be involved in pathophysiological events (Turner, 2015).
T166 23409-23615 Sentence denotes The effect of ACE2 which has been more extensively investigated is the regulation of the RAAS system, where ACE2 counter-balances ACE, limiting the potent vasoconstrictive effect of angiotensin II (Ang-II).
T167 23616-23842 Sentence denotes The first evidence that ACE2 was involved in RAAS control came from the transgenic knockout mouse model (ACE2–/–), which was characterized by severe reduction of cardiac contractility and thinning of the left ventricular wall.
T168 23843-23974 Sentence denotes Interestingly, in this knockout model disruption of the ACE pathway could rescue the myocardial phenotype (Crackower et al., 2002).
T169 23975-24110 Sentence denotes In another study, a selective ACE2 knockout model showed high blood pressure, worsened by the infusion of Ang-II (Gurley et al., 2006).
T170 24111-24249 Sentence denotes As a matter of fact, ACE2 displays its carboxypeptidase activity converting Ang-II to a heptapeptide, namely Ang1–7 (Turner et al., 2004).
T171 24250-24458 Sentence denotes ACE2 can also convert angiotensin I (Ang-I) to the non apeptide Ang1–9, which is in turn converted into Ang1–7 by ACE, competing with Ang-I and thus further decreasing Ang-II (Arendse et al., 2019; Figure 3).
T172 24459-24664 Sentence denotes Ang1–7 has been demonstrated to bind to the MasR receptor, which was initially regarded as an orphan receptor, since the use of a MasR antagonist caused inhibition of Ang1–7 effects (Alenina et al., 2008).
T173 24665-24898 Sentence denotes FIGURE 3 ACE2 signalling pathways: ACE2 displays its carboxypeptidase activity converting Ang-II (ANG II) to ANG1–7 and can also convert angiotensin I (ANG-I) to the nonapeptide Ang1–9, which is in turn converted into ANG1–7 by ACE.
T174 24899-25002 Sentence denotes ANG1-7 binds the MasR receptor to exert its effects on target organs (primarily heart, vessels, lungs).
T175 25003-25141 Sentence denotes ACE2 might also act via the bradykinin-DABK/BKB1R axis: the increased activity of the DABK axis triggers a proinflammatory cytokine storm.
T176 25142-25493 Sentence denotes In the last years the ACE2/Ang1–7/MasR system has been intensively studied: physiological effects on cardiomyocytes include modulation of Ca++ signaling and cytokine production, stimulation of cardiomyocytes progenitors and prevention of uncontrolled cell growth (Grobe et al., 2006; Flores-Muñoz et al., 2012; Souza et al., 2013; Chang et al., 2016).
T177 25494-25741 Sentence denotes Through the Ang1–7 pathway ACE2 produces endothelial antithrombotic effects, vasodilation, nitric oxide release, and inhibits vascular smooth muscle cells (VSMC) proliferation (Loot et al., 2002; Sampaio et al., 2007a,b; Fraga-Silva et al., 2008).
T178 25742-25929 Sentence denotes In preclinical studies Ang1–7 displayed antifibrotic effects, protecting from deleterious myocardial hypertrophy and modulating left ventricle remodeling after myocardial infarction (MI).
T179 25930-26264 Sentence denotes In animal models, Ang1–7 also showed an antiarrhythmic action (Ferreira et al., 2001; Santos et al., 2004; Grobe et al., 2006; Gomes et al., 2010), while compensatory ACE2 upregulation has been observed in explanted human hearts, in patients affected by ischemic or dilated cardiomyopathy (Goulter et al., 2004; Burrell et al., 2005).
T180 26265-26330 Sentence denotes Additional players contribute to ACE2-mediated cardio-protection.
T181 26331-26525 Sentence denotes Another substrate of ACE2 carboxypeptidase activity is Angiotensin A (Ang-A), identified in 2008, which has a similar, although less potent, vasopressor effect as AngII (Jankowski et al., 2007).
T182 26526-26669 Sentence denotes The product of the enzymatic reaction catalyzed by ACE2 is the peptide Ala-Ang (1–7) also known as alamandine (Lautner et al., 2013; Figure 3).
T183 26670-26805 Sentence denotes Alamandine receptor is a Mas-related G-protein coupled receptor, known as MrgD, which is expressed in cardiomyocytes and blood vessels.
T184 26806-26948 Sentence denotes MrgD knockout animals develop a severe cardiopathy and alamandine showed a cardioprotective effect in a model of sepsis (Santos et al., 2019).
T185 26949-27179 Sentence denotes Unlike ACE, ACE2 is not active on bradykinin, but it can degrade the active bradykinin metabolite des-Arg9 bradykinin (DABK), blocking the signaling pathway of the B1 bradykinin receptor (Vickers et al., 2002; Sodhi et al., 2018).
T186 27180-27287 Sentence denotes Other substrates of ACE2 include apelin-13/17, neurotensin, dynorphin A (1–13), and ghrelin (Turner, 2015).
T187 27288-27490 Sentence denotes Interestingly peptides of apelin family have been demonstrated to upregulate ACE2 expression in physiological condition and more actively during heart failure in in vivo experiments (Sato et al., 2013).
T188 27491-27755 Sentence denotes ACE2 in turn is able to regulate apelin bioavailability, establishing a negative feedback loop, and the crosstalk between RAAS, ACE2 and Apelin system might play a significant role in the pathophysiology of hypertension (Kalea and Batlle, 2010; Chen et al., 2015).
T189 27756-28110 Sentence denotes On the whole these findings suggest that ACE2 might exert antihypertensive and/or cardioprotective effects by different mechanisms, namely: (i) limiting the availability of ACE substrates, (ii) degrading Ang-II, (iii) activating the Ang1-7/MasR and/or Alamandine/MrgD pathways, (iv) interfering with other substrates, such as DABK and apelins (Figure 3).
T190 28112-28132 Sentence denotes ACE2 and Lung Injury
T191 28133-28267 Sentence denotes ACE2 is highly expressed in type I and II alveolar epithelial cells and in pulmonary small vessels (either endothelial cells or VSMC).
T192 28268-28574 Sentence denotes The hypothesis that the ACE2 arm of the RAAS system could be of benefit in lung disease derives from the observation that ACE and angiotensin II are upregulated in acute lung injury (ALI), pulmonary fibrosis, pulmonary hypertension, and acute respiratory distress syndrome (ARDS) (Imai et al., 2005, 2010).
T193 28575-28796 Sentence denotes ARDS is an overly aggressive form of ALI and it is the final mechanism of lung injury in many diseases, including sepsis, acid aspiration, pancreatitis, anthrax and virus infections (Spanish flu, H5N1 avian flu and SARS).
T194 28797-28942 Sentence denotes Imai et al. developed three ACE2 knockout mice models with severe ARDS induced by acid aspiration, endotoxin administration or peritoneal sepsis.
T195 28943-29164 Sentence denotes They showed that ARDS was accompanied by increased vessel permeability, lung oedema, and infiltration of inflammation cells, with consequent impairment of respiratory function (Imai et al., 2005, 2007; Kuba et al., 2006).
T196 29165-29309 Sentence denotes The phenotype was dramatically improved and rescued either administering ACE2 recombinant analogs or AT1Ra inhibitors (Imai et al., 2005, 2008).
T197 29310-29560 Sentence denotes ACE2 may also reduce lung inflammation via Ang1–7/MasR, since treatment with recombinant Ang1–7 in a model of allergic asthma reduced eosinophil mobilization, peri-bronchial inflammation, fibrosis and goblet cells metaplasia (El-Hashim et al., 2012).
T198 29561-29759 Sentence denotes An anti-inflammatory effect with reduction of airway remodeling has also been demonstrated in another model of chronic asthma, after administration of Ang1–7 analog (Rodrigues-Machado et al., 2013).
T199 29760-29982 Sentence denotes The underlying mechanism seems to be the modulation of the so-called cytokine storm and particularly the inhibition of transforming growth factor β (TGF- β) and NFkB signaling pathways (Li et al., 2015; Meng et al., 2015).
T200 29983-30061 Sentence denotes ACE2 might also modulate lung inflammation via the bradykinin-DABK/BKB1R axis.
T201 30062-30281 Sentence denotes In fact, decreased ACE2 function in mouse lungs caused increased activity of the DABK axis and triggered a proinflammatory cytokine storm (CXCL5, MIP2, KC, and TNF-α), leading to pulmonary collapse (Sodhi et al., 2018).
T202 30282-30345 Sentence denotes In human, ACE/ACE2 imbalance may be related to genetic factors.
T203 30346-30570 Sentence denotes In particular, a specific polymorphism of the ACE gene, namely ACE D, which determines increased ACE activity and decreased ACE2 activity has been correlated to ARDS susceptibility and mortality rate (Marshall et al., 2002).
T204 30571-30791 Sentence denotes More recently, high levels of angiotensin II have been reported in patients infected with avian influenza viruses H5N1 and H7N9, and they were strongly predictive of a poor outcome (Huang et al., 2014; Zou et al., 2014).
T205 30793-30824 Sentence denotes Links Between ACE2 and COVID-19
T206 30825-31073 Sentence denotes A “second life” for ACE2 was discovered in 2003, when a novel respiratory infective disease, known as severe acute respiratory syndrome (SARS), appeared in China and spread all over Asia and Canada, with a lethality rate of 10% (Rota et al., 2003).
T207 31074-31223 Sentence denotes The responsible pathogen was identified in a positive strand RNA virus belonging to the coronavirus family and named SARS-CoV (Ksiazek et al., 2003).
T208 31224-31444 Sentence denotes The virus genome was sequenced and this allowed the identification of the spike glycoprotein (S), whose N-terminal portion, or S1-domain, was found to mediate the high affinity binding to host cells (Marra et al., 2003).
T209 31445-31732 Sentence denotes The group of Farzan et al. succeeded in identifying the cell receptor: they showed that SARS-CoV efficiently infected African Monkey kidney cell line Vero E6 and co-immunoprecipitated a glycoprotein responsible for virus binding and entry, which was identified as ACE2 (Li et al., 2003).
T210 31733-31839 Sentence denotes SARS-CoV caused the SARS epidemic in 2002–2003, with over 8,000 infections and a fatality rate around 10%.
T211 31840-32036 Sentence denotes In late 2019, another coronavirus emerged as a human pathogen in the city of Wuhan in China, producing symptoms such as fever, severe respiratory impairment, and pneumonia (Petersen et al., 2020).
T212 32037-32215 Sentence denotes This new coronavirus has been denominated SARS-CoV-2 for its genetic resemblance with SARS-CoV (∼80%), and its related disease has been named COVID-19 (COronaVIrus Disease 2019).
T213 32216-32376 Sentence denotes Owing to its high reproduction number R0 (2–3), SARS-CoV-2 has rapidly diffused in several countries and is currently posing a significant global health threat.
T214 32377-32450 Sentence denotes On March 11, 2020, the WHO has declared the COVID-19 outbreak a pandemic.
T215 32451-32645 Sentence denotes The early discovery that SARS-CoV-2 also engages ACE2 as entry door for cell infection has prompted an intense research effort to elucidate the biochemical determinants of CoV-ACE2 interactions.
T216 32647-32697 Sentence denotes Mechanism of Viral Entry Mediated by the S Protein
T217 32698-32823 Sentence denotes A coronavirus contains four structural proteins, namely spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins.
T218 32824-32950 Sentence denotes These proteins assemble around a lipid bilayer to provide the shell enclosing the viral genome (Figure 4A; Tang et al., 2020).
T219 32951-33142 Sentence denotes Homotrimers of S protrude from the viral surface, and are densely decorated by N-linked glycans, creating the “crown” (“Corona” in Latin) that christens this virus group (Walls et al., 2016).
T220 33143-33297 Sentence denotes S is a ∼180 kDa glycoprotein anchored in the viral membrane, which plays the most important roles in viral attachment, fusion and entry (Ou et al., 2020).
T221 33298-33448 Sentence denotes Sequence analysis has shown that SARS-CoV-2 S protein shares 76% of the primary sequence with the corresponding S of human SARS-CoV (Ou et al., 2020).
T222 33449-33579 Sentence denotes Accordingly, it has been early proposed that SARS-CoV-2 utilizes a similar cell entry mechanism as SARS-CoV, pivoted on S protein.
T223 33580-33650 Sentence denotes This hypothesis has been confirmed from an experimental point of view.
T224 33651-33863 Sentence denotes By using pseudotyped virus bearing SARS-CoV S or SARS-CoV-2 S, it was shown that a large panel of cell lines allows comparable entry of SARS-CoV or SARS-CoV-2 viruses (Hoffmann et al., 2020b; Walls et al., 2020).
T225 33864-33996 Sentence denotes FIGURE 4 (A) Structure of a Coronavirus. (B) Functional motifs in the sequence of the S “spike” protein of SARS-CoV and SARS-CoV-2.
T226 33997-34278 Sentence denotes The S protein consists of ∼1300 aminoacids and it is composed by a N-terminal “S1”subunit (∼700 aa) and a C-terminal “S2”subunit (∼600 aa); binding to the host receptor is mediated by S1, whereas S2 induces fusion of the viral envelope with cellular membranes (Walls et al., 2017).
T227 34279-34405 Sentence denotes S1 and S2 can be further subdivided in functional segments with different roles in viral entry (Figure 4B; Tang et al., 2020).
T228 34406-34506 Sentence denotes The S1 subunit contains two subdomains, the N-terminal domain (NTD) and the C-terminal domain (CTD).
T229 34507-34710 Sentence denotes In SARS-CoV (Li, 2015) and SARS-CoV-2 (Wang et al., 2020) CTD encloses the receptor-binding domain (RBD), and the RBD section that directly contacts the receptor is named as receptor-binding motif (RBM).
T230 34711-34864 Sentence denotes The N-region of S2 contains a fusion peptide (FP), two heptapeptide repeat domains (HR1, HR2), a transmembrane domain (TM), and cytoplasmic peptide (CP).
T231 34865-35032 Sentence denotes FP is a short segment composed of mostly hydrophobic residues, such as glycine (G) or alanine (A), which inserts in the host cell membrane to trigger the fusion event.
T232 35033-35216 Sentence denotes HR1 and HR2 are composed of a repetitive heptapeptide with HPPHCPC sequence, where H represents hydrophobic or bulky residues, P polar or hydrophilic residues, and C charged residues.
T233 35217-35315 Sentence denotes HR regions typically fold into α-helices with a hydrophobic interface that drives membrane fusion.
T234 35316-35513 Sentence denotes On the basis of the strong similarity between the S proteins of SARS-CoV and SARS-CoV-2, many researchers early set-out to demonstrate whether both viruses target the same host cell receptor, ACE2.
T235 35514-35672 Sentence denotes Zhou et al. (2020) highlighted that the virus was able to infect cell lines only when they expressed human, bat, civet, and pig (but not mouse) ACE2 receptor.
T236 35673-35864 Sentence denotes Hoffmann et al. (2020b), Ou et al. (2020), and Walls et al. (2020) elegantly outlined that the BHK cell line could be infected by pseudotyped SARS-CoV-2 or SARS-CoV only upon ACE2 expression.
T237 35865-36070 Sentence denotes Conversely, the expression of different human receptors used by other CoVs (hDPP4 and APN, used by MERS CoV and HCoV-229E, respectively) did not enable pseudovirus access to cells (Hoffmann et al., 2020b).
T238 36071-36165 Sentence denotes Taken together, these findings are solid evidence that SARS-CoV-2 engages ACE2 for cell entry.
T239 36166-36389 Sentence denotes Nonetheless, the two viruses were demonstrated by Xia to share also the membrane fusion mechanism, as strongly suggested by the impressive 89.9% sequence identity of S2 between SARS-CoV and SARS-CoV-2 (Xia et al., 2020a,b).
T240 36390-36615 Sentence denotes To date, the cell entry mechanism of SARS-CoV and SARS-CoV-2 has been understood in its general details and it is based on a concerted action of receptor binding and proteolysis of the S protein (Figure 5; Tang et al., 2020).
T241 36616-36807 Sentence denotes Ultrastructural studies showed a metastable “prefusion” V-shaped trimer composed by three S1 heads sitting on top of a trimeric S2 stalk anchored into the virus membrane (Walls et al., 2016).
T242 36808-37025 Sentence denotes The RBD constantly switches between a standing-up (“open”) position for receptor binding and a lying-down (“closed”) configuration, the latter allowing immune evasion (Figure 6; Song et al., 2018; Wrapp et al., 2020).
T243 37026-37144 Sentence denotes Yet only one of the three RBD in trimeric S can flip up at a time and interact with the receptor (Wrapp et al., 2020).
T244 37145-37307 Sentence denotes The second key feature of the fusion mechanism is “priming” by host proteases, which recognize and cleave a short peptide motif at the S1/S2 boundary (Figure 4B).
T245 37308-37671 Sentence denotes This cleavage does not disassemble S1 from S2 in pre-fusion conditions (Belouzard et al., 2009), but the binding interaction of RBD with its receptor, accompanied by a further cleavage in a second site in S2 (S2’site, upstream of FP, Figure 4B), triggers the possible dissociation of S1 and the irreversible refolding of S2 into a “post-fusion” state (Figure 4B).
T246 37672-37907 Sentence denotes In detail, HR1 undergoes a dramatic “jack-knife” conformational change, converting four helical stretches that run in an antiparallel fashion into a single long (∼130 aa) α-helix (Heald-Sargent and Gallagher, 2012; Walls et al., 2017).
T247 37908-38022 Sentence denotes At first, three of these helices assemble into a homotrimeric bundle and stick the FP into the host cell membrane.
T248 38023-38177 Sentence denotes Then, HR2 (one for each S2 chain) fold backward and bind to HR1, yielding the “six-helix bundle fusion core” (6-HB) of post-fusion S2 (Song et al., 2018).
T249 38178-38410 Sentence denotes This conformational foldback brings the FP (at N-terminus of HR1) and the TM (at the C terminus of HR2) close to each other, so that the viral and host cell membranes approach until their outer leaflets merge (hemifusion, Figure 5).
T250 38411-38621 Sentence denotes Eventually the inner leaflets merge (pore formation), enabling a connection between the interior of the virus and the host cell cytoplasm, that allows the delivery of viral genome (Figure 5; Tang et al., 2020).
T251 38622-38671 Sentence denotes FIGURE 5 Coronavirus viral fusion pathway model.
T252 38672-38735 Sentence denotes Initially, the S protein is in the pre-fusion native state (1).
T253 38736-39034 Sentence denotes Then S undergoes priming of the S1 subunit at S1/S2 by local proteases yielding the pre-fusion metastable state (2); note that priming at S1/S2 could also happen upon virus formation in releasing cell: in such a case the virus attaches to a host cell already in the pre-fusion metastable state (2).
T254 39035-39214 Sentence denotes Subsequent triggering by a protease on S2’ enables the FP to insert in the host membrane upon the “jack-knife” transition of HR1 and HR2 yielding the pre-hairpin intermediate (3).
T255 39215-39329 Sentence denotes The pre-hairpin folds back on itself due to HR1 and HR2 interactions eventually forming the post-fusion (6) state.
T256 39330-39500 Sentence denotes During the S protein foldback, the two membranes approach each other until the outer leaflets merge (hemifusion) and eventually the inner leaflets merge (pore formation).
T257 39501-39622 Sentence denotes Note that cell membrane may refer to plasma membrane (direct fusion) or endosomal membrane (fusion in endocytic vesicle).
T258 39623-39655 Sentence denotes Adapted from Tang et al. (2020).
T259 39656-39732 Sentence denotes FIGURE 6 Trimeric S protein of SARS-CoV-2 in the ”Closed” and “Open” forms.
T260 39733-39821 Sentence denotes Note the single RBD protruding out of the V-shaped conformation of the protein assembly.
T261 39822-39870 Sentence denotes The structures have been drawn from PDB 6X2C (R.
T262 39871-39934 Sentence denotes Henderson, 10.1101/2020.05.18.10208) by Mol on the PDB website.
T263 39935-40104 Sentence denotes Although not directly related to ACE2, the role of S “priming” by host cell proteases deserves particular attention in the context of SARS-CoV-2 virus entry and tropism.
T264 40105-40317 Sentence denotes Possibly, the most notable feature of SARS-CoV-2 genome, as compared to SARS-CoV and some related bat coronaviruses, is a four basic aminoacid insert (PRRA) at the S1/S2 junction (Figure 4B; Jaimes et al., 2020).
T265 40318-40473 Sentence denotes This site is potentially cleavable by the protease furin, a proprotein convertase widely recognized to activate the fusion machinery of viral glycoprotein.
T266 40474-40706 Sentence denotes Indeed, many authors showed that pseudoviruses bearing SARS-CoV-2 S were already “primed” (i.e., cleaved) at the S1/S2 boundary by furin upon assembly in the cell, at odds with pseudoviruses bearing SARS-CoV S (Shang et al., 2020a).
T267 40707-41100 Sentence denotes SARS-COV-2 shows a large flexibility with regard to protease priming, which may independently occur by a) furin and furin-like proteases intracellularly, b) trypsin-like proteases such as TMPRSS2 that are present on the host cell membrane (particularly on airway epithelial cells), and 3) endosomal cathepsins activated by a drop in pH (e.g., cathepsin L) (Figure 7; Hoffmann et al., 2020a,b).
T268 41101-41252 Sentence denotes This flexibility could be the crucial factor that explain SARS-CoV-2 cell tropism and the peculiar features of COVID-19 symptoms (Jaimes et al., 2020).
T269 41253-41438 Sentence denotes Additionally, the kind of protease “priming” may determine whether the membrane fusion process occur directly at the plasma membrane or at endosomal level (Tang et al., 2020; Figure 7).
T270 41439-41524 Sentence denotes FIGURE 7 Relevance of S S1/S2 “priming” by host proteases for viral fusion to cells.
T271 41525-41685 Sentence denotes The left cells produce viruses that can be “primed” by endogenous proteases such as furin (blue scissors); other viruses are not primed when they exit the cell.
T272 41686-41911 Sentence denotes The primed viruses (marked by a yellow internal shadow) reach another cell (pathway A), where a membrane protease (e.g., TMPRSS2) may cleave the S2’ site (see Figure RB1b) leading to membrane fusion and delivery of viral RNA.
T273 41912-42222 Sentence denotes Non-primed viruses can deliver their genome by two routes: in B, the virus reaches the cell, is primed on the membrane at both S1/S2 and S2’ by a local protease and then fuse with the plasma membrane; alternatively, in C the virus is internalized by endocytosis and priming/fusion occurs in endocytic vesicles.
T274 42223-42333 Sentence denotes Note that also “primed” viruses may undergo pathway C, depending on their interaction with the recipient cell.
T275 42335-42372 Sentence denotes SARS-CoV-2 RBD and Its Receptor, ACE2
T276 42373-42520 Sentence denotes Since SARS-CoV-2 and SARS-CoV share the same host cell receptor, it was early questioned whether SARS-CoV-2 retains the same RBD motif of SARS-CoV.
T277 42521-42597 Sentence denotes SARS-CoV RBD corresponds to residues 306–527 of S protein (Li et al., 2005).
T278 42598-42737 Sentence denotes Sequence analysis shows that residues 319–541 of SARS-CoV-2 (S319–341) share 73.9% sequence identity with SARS-CoV RBD (Wang et al., 2020).
T279 42738-42868 Sentence denotes Accordingly, Wang et al. (2020) clearly demonstrated that S319–341 corresponds to SARS-CoV-2 RBD by immunofluorescence microscopy.
T280 42869-43072 Sentence denotes Indeed SARS-CoV-2 S1 and S319–341 positively colocalized with GFP-tagged ACE2 expressed on cell surface of HEK cells, whereas this interaction did not occur with membrane-expressed hDPP4 (MERS receptor).
T281 43073-43202 Sentence denotes Additionally, soluble ACE2 inhibited the interaction between viral proteins and ACE2-expressing cells in a dose-dependent manner.
T282 43203-43279 Sentence denotes The SARS-CoV-2 RBD sequence was further investigated by structural analysis.
T283 43280-43677 Sentence denotes X-ray crystallography showed that SARS-CoV-2 RBD folds into two structural domains (Figure 8): (1) the core subdomain with five antiparallel β-strands (β1, β2, β3, β4, β7), (2) the external subdomain, which inserts between β4 and β7, and it is characterized by the two small β5 and β6 strands [β1’ and β2’ in Wang et al. (2020)] connected by a disulfide bond (Lan et al., 2020; Wang et al., 2020).
T284 43678-43945 Sentence denotes In keeping with their high sequence homology, the 3D structure of SARS-COV-2 and SARS-CoV RBD nearly superimpose (RMSD = 0.475 Å for 128 Cα atoms; Wang et al., 2020) with the exception of the β5/β6 loop, which actually entailed the larger primary sequence difference.
T285 43946-44180 Sentence denotes FIGURE 8 Crystal structure of SARS-CoV-2 spike receptor-binding domain bound with ACE2. (A) Cartoon representation. (B) Gaussian surface representation. hACE2 is in green, the core of SARS-CoV-2 RBD is in red, and the RBM is in blue.
T286 44181-44253 Sentence denotes The β1-β7 typical motifs of RBD (Lan et al., 2020) are indicated in (A).
T287 44254-44344 Sentence denotes The structures have been drawn from PDB 6MOJ (Lan et al., 2020) by Mol on the PDB website.
T288 44345-44422 Sentence denotes Several researchers investigated the interaction of SARS-CoV-2 RBD with ACE2.
T289 44423-44509 Sentence denotes Unfortunately, each group committed to slightly different sequences of SARS-CoV-2 RBD.
T290 44510-44670 Sentence denotes To avoid confusion, we will always report the actual sequence with respect to the S protein when the RBD under study differs from the canonical 319–541 stretch.
T291 44671-44785 Sentence denotes In vitro affinity studies revealed dissociation constants of the ACE2-RBD complex in the 1–100 nM range (Table 1).
T292 44786-44934 Sentence denotes Non univocal data are attributable to the dissimilar sequences that were investigated and/or to the immobilization procedures (Shang et al., 2020b).
T293 44935-45126 Sentence denotes In spite of this variability, SARS-CoV-2 RBD was always found to bind ACE2 4–10 fold stronger than SARS-CoV RBD (Lan et al., 2020; Shang et al., 2020b; Walls et al., 2020; Wang et al., 2020).
T294 45127-45344 Sentence denotes The affinity difference in vitro was confirmed also in vivo by the stronger binding of SARS-CoV-2 S331–524 to ACE2 expressed on cells (SARS-CoV-2: EC50 = 0.08 μg/ml vs. SARS-CoV: EC50 = 0.96 μg/ml) (Tai et al., 2020).
T295 45345-45522 Sentence denotes Paradoxically, however, it has been shown that the ACE2 binding affinity for the entire SARS-CoV-2 S protein is lower than or comparable to that of SARS S (Shang et al., 2020a).
T296 45523-45766 Sentence denotes This surprising result suggests that SARS-CoV-2 RBD, albeit more potent, is less efficiently exposed than SARS-CoV RBD by the dynamic transition between the “closed” and “open” states, probably in order to escape the immune system of the host.
T297 45767-46093 Sentence denotes Thus, the non-identical S1 sequences of SARS-CoV-2 and SARS-CoV reflect the molecular evolution of SARS-CoV-2 toward: (1) much stronger affinity toward ACE2, (2) reduced antigenicity of the RBD region (which is one of the most antigenic segments in the S protein), (3) greater and less specific cleavability by host proteases.
T298 46094-46211 Sentence denotes Taken together, these properties account for the sophisticated strategy exploited by SARS-CoV-2 to invade host cells.
T299 46212-46305 Sentence denotes TABLE 1 Binding affinity between SARS-CoV-2 spike (S) protein and S subset regions and ACE2.
T300 46306-46343 Sentence denotes Sequence KD (nM) Method References
T301 46344-46383 Sentence denotes SARS-2 S 14.7 SPR Wrapp et al., 2020
T302 46384-46421 Sentence denotes SARS-2 S 11.2 SPR Lei et al., 2020
T303 46422-46471 Sentence denotes SARS-2 S20–685 (S1) 94.6 SPR Wang et al., 2020
T304 46472-46524 Sentence denotes SARS-2 S319–541 (RBD) 133.3 SPR Wang et al., 2020
T305 46525-46574 Sentence denotes SARS-2 S319–541 (RBD) 4.7 SPR Lan et al., 2020
T306 46575-46622 Sentence denotes SARS-2 S319–529 44.2 SPR Shang et al., 2020b
T307 46623-46689 Sentence denotes SARS-2 S319–591 34.6 Biolayer interferometry Wrapp et al., 2020
T308 46690-46755 Sentence denotes SARS-2 S328–533 1.2 Biolayer interferometry Walls et al., 2020
T309 46757-46790 Sentence denotes Structure of the ACE2-RBD Complex
T310 46791-47024 Sentence denotes Previous studies showed that the extracellular Peptidase Domain (PD) of ACE2, which adopts a claw-like morphology, is the interaction site of SARS-CoV RBD (Towler et al., 2004; Li, 2015; Kirchdoerfer et al., 2018; Song et al., 2018).
T311 47025-47199 Sentence denotes More specifically, ACE2 engages SARS-CoV RBD by establishing contacts with the 424–494 residue domain, which is referred to as Receptor Binding Motif (RBM) (Li et al., 2005).
T312 47200-47480 Sentence denotes In spite of the recent emergence of SARS-CoV-2, multiple authors have already highlighted the structure of ACE2/SARS-CoV-2 RBD complex by either X-ray (Lan et al., 2020; Shang et al., 2020b; Wang et al., 2020) or cryo-EM (Walls et al., 2020; Wrapp et al., 2020; Yan et al., 2020).
T313 47481-47591 Sentence denotes Pleasantly, all data converged to a consistent tridimensional arrangement of the receptor (Wang et al., 2020).
T314 47592-47709 Sentence denotes In the “closed” conformation of S protein, the RBD is buried at the interface between protomers (Walls et al., 2020).
T315 47710-47892 Sentence denotes Only in the “open” S conformation, RBD engages PD of ACE2 (Wrapp et al., 2020), and the complex may involve a dimeric ACE2 that accommodates two S protein trimers (Yan et al., 2020).
T316 47893-48082 Sentence denotes In keeping with their sequence similarity, strong structural homology was found between ACE2/SARS-CoV RBD and ACE2/SARS-CoV-2 RBD (Lan et al., 2020; Shang et al., 2020b; Wang et al., 2020).
T317 48083-48271 Sentence denotes SARS-CoV-2 RBM spans from residue 438–506 of S sequence and, likewise SARS-CoV RBM, it approaches the outer surface of ACE2 by a gently concave surface with a ridge on one side (Figure 8).
T318 48272-48437 Sentence denotes The concave surface is made up by the two short β5 and β6 sheets of the external RBD subdomain, whereas the ridge contains the β5/β6 loop (loop 1: residues 474–489).
T319 48438-48539 Sentence denotes A second smaller loop (loop 2: residues 498–505) is visible on the other side of the concave surface.
T320 48540-48805 Sentence denotes Inspection of the complex structure and molecular dynamics (MD) highlighted that the motifs 453–456 (in β5), 484–489 (in the loop 1), and 500–505 (in the loop 2) are at the basis of the largest differences between SARS-CoV-2 and SARS-CoV RBM interactions with ACE2.
T321 48806-49060 Sentence denotes SARS-CoV RBM ridge contains a Pro-Pro-Ala motif that is replaced by Gly-Val-Glu-Gly in SARS-CoV-2 (residues 482–485), yielding a more compact loop able to engage more interactions with proximal ACE2 residues (e.g., Ser19 and Gln24) (Shang et al., 2020b).
T322 49061-49269 Sentence denotes Additionally, Phe486 of SARS-CoV-2 RBM (which replaces Ile of SARS) inserts into a hydrophobic pocket on the receptor surface, establishing strong aromatic interactions with Tyr83 of ACE2 (Wang et al., 2020).
T323 49270-49365 Sentence denotes Asn501 in loop 2 further engages recognized hotspots on the ACE2 surface (Shang et al., 2020b).
T324 49366-49517 Sentence denotes Consistently, MD studies confirmed that loop 1 and 2 are much more rigid in RBM-ACE2 complex of SARS-CoV-2 with respect to SARS (Brielle et al., 2020).
T325 49518-49638 Sentence denotes These subtle structural differences probably account for the higher affinity of SARS-CoV-2 for ACE2 (Wang et al., 2020).
T326 49639-49848 Sentence denotes Interestingly, MD simulations suggest that the difference in affinity is largely due to the solvation energy, emphasizing the relevant role of hydrophobic patches in RBM/ACE2 binding surface (He et al., 2020).
T327 49849-50116 Sentence denotes It is worth noting that the RBD-receptor engagement is the crucial effector of viral-host interaction, which eventually determines viral host range, and in tandem with the host proteases is responsible for virus tropism and pathogenicity (Millet and Whittaker, 2015).
T328 50117-50351 Sentence denotes Structure-guided sequence analysis has suggested that several mammals, including pets such as cats and dogs, host ACE2 receptors that could bind effectively to SARS-CoV-2 S protein and propagate COVID-19 infection (Luan et al., 2020).
T329 50352-50434 Sentence denotes Yet, no correlation between genetic distance and the S/ACE2 interaction was found.
T330 50435-50646 Sentence denotes In this context, a third human CoV, hCoV-NL63, has been previously found to use ACE2 for cell entry (Hofmann et al., 2005), although its S1 sequence is rather dissimilar from SARS (23.4%) and SARS-CoV-2 (29.2%).
T331 50647-50819 Sentence denotes In spite of this, the structures of hCoV-NL63 (Wu et al., 2009) and SARS-CoV RBD (Li et al., 2005) were found to engage some sterically overlapping sites in ACE (Li, 2015).
T332 50820-50980 Sentence denotes This homology can be transitively extended to SARS-CoV-2, suggesting these three CoVs have evolved to recognize a “hotspot” region in ACE2 for receptor binding.
T333 50981-51174 Sentence denotes This might represent a critical feature for the appearance of novel CoV able to infect humans in the future, as it is known that at least three more bat CoVs bind ACE2 (Hoffmann et al., 2020b).
T334 51175-51398 Sentence denotes Indeed, bat RaTG13 CoV binds ACE2 and contains a similar four-residue motif in the ACE2 binding ridge of RBM, suggesting that SARS-CoV-2 could have evolved from RaTG13 or a yet-unknown related bat CoV (Shang et al., 2020b).
T335 51400-51441 Sentence denotes The Controversial Role of ACE2 Expression
T336 51442-51682 Sentence denotes Epidemiological data consistently show that the COVID-19 patients at highest risk of a poor prognosis are males older than 60 years with chronic underlying diseases, mostly hypertension, cardiovascular diseases and type-2 diabetes mellitus.
T337 51683-51865 Sentence denotes Clinical reports have been rapidly delivered from all over the world, and meta-analyses assessing the prevalence of comorbidities and their impact on prognosis are already available.
T338 51866-52113 Sentence denotes A meta-analysis pooling data from seven studies following a total number of 1,576 infected patients from hospitals in China found that the most prevalent comorbidities were hypertension (21.1%), diabetes (9.7%), and cardiovascular diseases (8.4%).
T339 52114-52322 Sentence denotes These increased the risk of developing a more serious disease (i.e., requiring intensive care treatment), with odds ratios ranging from 2.4 (hypertension) to 3.4 (cardiovascular disease) (Yang et al., 2020b).
T340 52323-52598 Sentence denotes These findings have been confirmed in the analysis performed by the Chinese Center for Disease Control and Prevention in a huge sample of 72314 COVID-19 cases (Epidemiology Working Group for Ncip Epidemic Response and Chinese Center for Disease Control and Prevention, 2020).
T341 52599-52752 Sentence denotes A study with 1591 Italian patients, similarly, reported a significant association between hypertension and mortality in Intensive Care Unit (63 vs. 40%).
T342 52753-52897 Sentence denotes This series reported an even higher prevalence of hypertension (49%), diabetes (17%), and cardiovascular disease (21%) (Grasselli et al., 2020).
T343 52898-53086 Sentence denotes Diabetes has been reported to predict the occurrence of ARDS (HR = 1.44), acute kidney injury (HR = 3.01), septic shock (HR = 1.95), and all-cause mortality (HR = 1.70) (Zhu et al., 2020).
T344 53087-53245 Sentence denotes Notably, poor glycemic control was significantly associated with worse clinical outcomes, namely multi-organ injuries and higher mortality (Zhu et al., 2020).
T345 53246-53327 Sentence denotes Obesity has also emerged as an important factor in determining COVID-19 severity.
T346 53328-53596 Sentence denotes Indeed, obesity was more frequent in patients admitted to critical care for SARS-CoV-2 as compared to the general population; moreover, the BMI was positively related to the need for invasive mechanical ventilation and mortality (Drucker, 2020; Simonnet et al., 2020).
T347 53597-53739 Sentence denotes The RAAS system is the target of widely used anti-hypertensive drugs, such as ACE-inhibitors (ACEI) and Ang-II type 1 receptor blockers (ARB).
T348 53740-53952 Sentence denotes Several experimental studies reported that, although not directly affecting ACE2 activity, these drugs are able to upregulate its expression (Ishiyama et al., 2004; Ferrario et al., 2005; Gallagher et al., 2008).
T349 53953-54185 Sentence denotes Increased expression of ACE2 has also been reported to facilitate SARS-CoV infection in several experimental models and postulated to act in the same way for SARS-CoV-2 (Li et al., 2003; Hofmann et al., 2004; Perrotta et al., 2020).
T350 54186-54356 Sentence denotes Hence, following the SARS-CoV-2 outbreak, it has been speculated that the use of ACEI and ARB could increase viral invasion and should therefore be temporarily suspended.
T351 54357-54825 Sentence denotes This topic has been abundantly debated in the last few months (March–May 2020), with contradictory views (Bavishi et al., 2020; Buckley et al., 2020; Danser et al., 2020; Fang et al., 2020; Huang Z. et al., 2020; Kreutz et al., 2020; Kuster et al., 2020; Mourad and Levy, 2020; Park et al., 2020; Rico-Mesa et al., 2020; Sommerstein et al., 2020; South et al., 2020; Tignanelli et al., 2020; Vaduganathan et al., 2020; Verdecchia et al., 2020b; Zhang P. et al., 2020).
T352 54826-55215 Sentence denotes As a consequence of this debate, several clinical societies have stated that suspension of ACEI and ARB is not justified on the basis of the present scientific evidence, although a recent BMJ editorial (Aronson and Ferner, 2020) suggested to consider stopping ACE inhibitors or angiotensin receptor blockers in patients with mild hypertension who are at high risk of coronavirus infection.
T353 55216-55441 Sentence denotes On the other hand, a different viewpoint is based on the intriguing observation that several conditions increasing the risk of viral infection and disease severity are all characterized by a certain degree of ACE2 deficiency.
T354 55442-55629 Sentence denotes As discussed above (section “Structure of ACE2”) ACE2 deficiency has been suggested to play a significant role in the pathophysiology of hypertension, cardiovascular disease and diabetes.
T355 55630-55827 Sentence denotes The role of ACE2 expression in the panel of comorbidities correlated to SARS-CoV-2 is a matter of intense still unsolved debate, as brilliantly revised by Shyh et al. (2020) in a very recent paper.
T356 55828-55967 Sentence denotes On the whole, there is still uncertainty about the relationship between ACE2 density on cell membrane and the fate of SARS-CoV-2 infection.
T357 55968-56309 Sentence denotes An interesting speculation which could reconcile the two position comes from the hypothesis that while ACE2 is for sure the entry door for the virus, once the infection has evolved, there is the subsequent downregulation of ACE2, responsible for the precipitating of respiratory distress, as showed also in animal models (Kuba et al., 2005).
T358 56310-56482 Sentence denotes It should be pointed out that the great clinical impact of this issue has encouraged discussions that are largely based on indirect speculations rather than objective data.
T359 56483-56776 Sentence denotes Data from appropriate clinical trials are still lacking, although a retrospective multicenter study on more than 1,000 patients with COVID-19 and hypertension treated with ACEI or ARB revealed that the use of these drugs was associated with lower mortality from all cause (Bosso et al., 2020).
T360 56777-57181 Sentence denotes A definite conclusion would require specific prospective clinical trials, which are still running: https://clinicaltrials.gov/ct2/show/NCT04312009 is a multi-centered double blind trial with the aim to test the role of ARB in patients with COVID-19 and https://clinicaltrials.gov/ct2/show/NCT04331574 is another trial evaluating the outcome of patients with COVID-19 in therapy with either ACEI and ARBS.
T361 57182-57737 Sentence denotes In the meanwhile, partial but important information might be obtained by circumscribed experimental and clinical investigations focused some on crucial issues, namely: fate of SARS-CoV-2 infection in human cells manipulated in order to modify ACE2 density; correlation of ACE2 density (in different cell types) and clinical course in human patients; potential role of additional proteins interacting with ACE2, such as the membrane transporter SIT1, which appears to be associated with COVID-19 prognosis and is also affected by anti-hypertensive therapy.
T362 57739-57787 Sentence denotes ACE2 and the Inflammatory Response to SARS-CoV-2
T363 57788-57954 Sentence denotes Differently from other flu-like viral infections, COVID19 showed a relatively high lethality, although the latter was quite different in different geographical areas.
T364 57955-58114 Sentence denotes The appearance of ARDS and acute severe lung injury was initially regarded as the main insult, and patients underwent the typical treatment developed for ARDS.
T365 58115-58430 Sentence denotes However, more recent evidence from autopsy series revealed that COVID-19 is a systemic disease, in which interstitial pneumonia is associated with extensive microvascular damage, which is not limited to the lungs and leads to fibrin deposition, neutrophils trapping in microvasculature, and arteriolar microthrombi.
T366 58431-58585 Sentence denotes Moreover, cardiovascular involvement has been observed, occasionally leading to myocardial injury and sudden death after weeks from the initial infection.
T367 58586-58757 Sentence denotes These findings are crucial to optimize the treatment of COVID-19 patients, hopefully improving the prognosis (Buja et al., 2020; Carsana et al., 2020; Magro et al., 2020).
T368 58758-58906 Sentence denotes Another determinant of the clinical picture and a negative prognostic factor is an aggressive proinflammatory response of the host to the infection.
T369 58907-59045 Sentence denotes Huang described the clinical features of 41 patients admitted to their Institute in January of 2020 with confirmed COVID-19 and pneumonia.
T370 59046-59144 Sentence denotes In 29% (n = 12) of patients ARDS occurred and in 10% invasive mechanical ventilation was required.
T371 59145-59270 Sentence denotes These patients clearly showed increased production of IL-1β, IFN-γ, IP-10, MCP-1, IL-4, and IL-10 compared to healthy people.
T372 59271-59586 Sentence denotes Moreover, the patients who required access to the intensive care unit had significantly higher levels of proinflammatory cytokines compared to those who did not require intensive care, suggesting that this “cytokine storm” may trigger the development of the most severe forms of the disease (Huang C. et al., 2020).
T373 59587-59705 Sentence denotes The molecular mechanisms involved in the cytokine storm and the role of ACE2 are currently still not fully understood.
T374 59706-59919 Sentence denotes As discussed above, virus binding to the ACE2 is the event that initiates viral replication in susceptible cells, such as alveolar epithelial cells, vascular cells and immune system cells (macrophages, monocytes).
T375 59920-60048 Sentence denotes This has been suggested to trigger the primary inflammatory response, which involves apoptosis and pyroptosis (Fu et al., 2020).
T376 60049-60299 Sentence denotes The apoptosis pattern indices cell death to avoid viral replication in the absence of overt inflammation, whereas, pyroptosis is a violent form of programmed cell death, followed by an inflammatory storm (Cookson and Brennan, 2001; Liu et al., 2016).
T377 60300-60546 Sentence denotes In the standard pyroptosis model, when the pathogen enters the host cell, some specific structures on the pathogen surface (PAMPs–pathogen associated molecular patterns) are identified by pattern recognition receptors (PRRs) on the host membrane.
T378 60547-60992 Sentence denotes One common PRR is NOD-like receptors protein 3 (NLRP3), which forms together with a protein caspase activating protein (ASC or apoptosis-associated speck-like protein containing a caspase recruitment domain) and pro-caspase 1, the inflammasome unit capable of recruiting proinflammatory cytokines and inducing cell lysis with further inflammation signals (Fernandes-Alnemri et al., 2007; Schroder and Tschopp, 2010; Wree et al., 2014; Figure 9).
T379 60993-61100 Sentence denotes FIGURE 9 Possible role of ACE2 in the molecular mechanisms involved in the “cytokine storm” of SARS-CoV-2.
T380 61101-61322 Sentence denotes In the SARS-CoV-2 infection, pyroptosis has been demonstrated to be active in macrophages, being viroporin 3a a trigger for NLRP3 with subsequent production of proinflammatory cytokines, such as IL-1β (Chen et al., 2019).
T381 61323-61461 Sentence denotes In COVID-19 patients, the pyroptosis activation has been indirectly demonstrated by the high serum level of IL-1β (Huang C. et al., 2020).
T382 61462-61649 Sentence denotes Moreover, the severe leucopoenia and lymphopenia, commonly showed in COVID-19 pneumonia and associated with poor prognosis, are likely due to lymphocyte injury by pyroptosis (Yang, 2020).
T383 61650-61775 Sentence denotes Another proposed mechanism that triggers the proinflammatory response to SARS-CoV-2 is ACE/ACE2 imbalance in the RAAS system.
T384 61776-62072 Sentence denotes It has been speculated that when ACE2 is occupied by the virus, its effects in limiting the amount and activity of Ang-II is decreased; Ang-II is consequently increased and it has been reported to induce a proinflammatory effect via the AT1R receptor (Marchesi et al., 2008; Eguchi et al., 2018).
T385 62073-62417 Sentence denotes The Ang-II/AT1R system activates disintegrin and the metalloprotease ADAM 17 (also known as TNFα cleavage enzyme TACE), which lead to the intracellular production of epidermal growth factor ligands (EGFR) and TNFα production, with subsequent stimulation of the transcription factor NF-K B, a pivotal player in proinflammatory cytokines release.
T386 62418-62657 Sentence denotes Additionally, ADAM 17 activation induces the production of the soluble form of IL-6Rα, active on IL-6-STAT3 pathway, which in turn amplifies NF-K B signaling (Murakami et al., 2019; Takimoto-Ohnishi and Murakami, 2019; Palau et al., 2020).
T387 62658-62754 Sentence denotes The convergence on hyperactivation of NF-K B seems to be crucial in inducing the cytokine storm.
T388 62755-62951 Sentence denotes The mechanism is self-feeding, given that NF-K B induces the expression of the angiotensinogen gene, amplifying the Ang-II inflammatory response (Costanzo et al., 2003; Hirano and Murakami, 2020).
T389 62952-63040 Sentence denotes ACE2 shedding is an additional mechanism contributing to inflammation (Fu et al., 2020).
T390 63041-63294 Sentence denotes ACE2 shedding consists in proteolytic cleavage of the protein ectodomain and leads to the release of enzymatically active soluble ACE2 (sACE), whose action is not completely understood, but might enhance the pro-inflammatory response (Guy et al., 2005).
T391 63295-63510 Sentence denotes ADAM 17 and disintegrin are able to shed not only TNFα but also a variety of membrane-anchored enzymes, including ACE2 (Black et al., 1997; Palau et al., 2020), as demonstrated by Lambert in 2005 (Guy et al., 2005).
T392 63511-63827 Sentence denotes In their experiment, human embryonic kidney cells (HEK293) expressing human ACE2 (HEK-ACE2) were treated with phorbol myristate acetate stimulation and the production of a polypeptide of 105 kDa was demonstrated by western blot analysis, suggesting the occurrence of a proteolytic shedding in the ectodomain of ACE2.
T393 63828-64048 Sentence denotes Subsequently, they demonstrated that the cleavage was performed by ADAM17, incubating cells with a specific hydroxamic acid-based metalloproteinase inhibitor, namely TAPI-1 (TNFα protease inhibitor 1) (Guy et al., 2005).
T394 64049-64166 Sentence denotes ACE2 shedding seems to be inducible by specific stimuli produced either by a viral infection or by the immune system.
T395 64167-64280 Sentence denotes Jia et al. (2009) confirmed ACE2 shedding in differentiated primary airway epithelial cells and Calu-3-cell line.
T396 64281-64489 Sentence denotes Furthermore, they demonstrated that the incubation with IL-1β at the dosage of 100 ng/ml and TNF α 100 ng/ml induced ACE2 shedding and sACE release, with a maximum after 18 h of incubation (Jia et al., 2009).
T397 64490-64625 Sentence denotes These findings suggest that an inflammatory response is likely to modulate ACE2 expression and shedding, also during a viral infection.
T398 64626-64810 Sentence denotes Following this data, ADAM17 inhibition was proposed as a possible pharmacological target in SARS-CoV-2, but further studies are needed to evaluate this hypothesis (Palau et al., 2020).
T399 64811-64970 Sentence denotes Other conditions have been assumed to be involved in the susceptibility to SARS-CoV-2 and among them vitamin D deficiency was proposed as a credible candidate.
T400 64971-65132 Sentence denotes The interesting candidate could be identified as a modifiable risk factor (hypovitaminosis D) and a potential tool in COVID-19 prevention or ancillary treatment.
T401 65133-65789 Sentence denotes The rationale has been summarized by Grant et al. in a recent review on the evidence supporting a possible correlation between vitamin D and SARS-CoV-2 risk: (i) the seasonal flare of SARS-CoV-2 which coincides with the nadir of vitamin D levels, (ii) the association between hypovitaminosis D and pulmonary infections together with the demonstrated protective role in acute respiratory infections, in adults (iii) the anti-inflammatory role of vitamin D which could be of benefit against the so called “cytokine storm,” which seems to be a major player in SARS-CoV-2 morbidity and mortality (Martineau et al., 2017; Zhou et al., 2019; Grant et al., 2020).
T402 65791-65819 Sentence denotes ACE2 as a Therapeutic Target
T403 65820-65908 Sentence denotes As evidence builds up, ACE2 rapidly emerged as a specific target for COVID-19 treatment.
T404 65909-66213 Sentence denotes Since this enzyme was identified as the SARS-CoV-2 receptor (Zhou et al., 2020), several approaches to address ACE2 mediated infection have been described (Li Y. et al., 2020; Zhang H. et al., 2020), with the aim to prevent host cell entry and subsequent viral replication, as well as severe lung injury.
T405 66214-66267 Sentence denotes Potential therapeutic approaches include (Figure 10):
T406 66268-66379 Sentence denotes FIGURE 10 Schematic representation of the potential therapeutic approaches to address ACE2 mediated infection.
T407 66380-66584 Sentence denotes Exogenous administration of soluble recombinant human ACE 2 sequesters circulating viral particles, while specific ACE2 blockers bind the receptor, impeding the S-protein interaction with the host target.
T408 66585-66587 Sentence denotes 1.
T409 66589-66603 Sentence denotes ACE2 blockers;
T410 66604-66606 Sentence denotes 2.
T411 66608-66641 Sentence denotes Exogenous administration of ACE2.
T412 66643-66656 Sentence denotes ACE2 Blockers
T413 66657-66907 Sentence denotes Blockade of ACE2 receptor could be achieved through specific antibodies (Li et al., 2003), rationally designed small molecules (Dales et al., 2002; Huentelman et al., 2004; Gross et al., 2020; Pillaiyar et al., 2020) or peptides (Huang et al., 2003).
T414 66908-67096 Sentence denotes Although their efficacy needs to be confirmed, some of these agents are currently available on the market and have been show to effectively block SARS-CoV invasion (Li S.-R. et al., 2020).
T415 67097-67424 Sentence denotes For instance, the small synthetic inhibitor N-(2-aminoethyl)-1aziridine-ethanamine (NAAE) binds ACE2 active site in its closed conformation; this contact triggers the shifting of SARS-CoV S binding residues preventing the molecular interaction with targeted enzyme and the subsequent cell-cell fusion (Huentelman et al., 2004).
T416 67425-67623 Sentence denotes Therefore, although ACE2 catalytic site is distinct from the S-protein-binding domain, NAAE exerts dual inhibitory effects on ACE2 catalytic activity and SARS binding (Adedeji and Sarafianos, 2014).
T417 67624-67920 Sentence denotes However, since a protecting role for ACE2 receptor against virus-induced acute lung injury in infections with SARS coronavirus has not been excluded (Imai et al., 2005; Kuba et al., 2005; Li S.-R. et al., 2020), the choice of ACE2 inhibition as therapeutic approach should be carefully evaluated.
T418 67922-67954 Sentence denotes Exogenous Administration of ACE2
T419 67955-68363 Sentence denotes The administration of a large amount of soluble form of ACE2 could represent an intriguing opportunity, since excessive ACE2 may exert dual functions: (a) competitively bind SARS-CoV-2 to neutralize the virus and/or slow viral entry in the host cell; (b) rescue cellular ACE2 activity, which negatively regulates RAAS and may theoretically exert a protective effect in lung injury (Verdecchia et al., 2020a).
T420 68364-68543 Sentence denotes A pilot clinical study is currently investigating the efficiency of a recombinant human angiotensin-converting enzyme 2 (rhACE2) in patients with COVID-19 (Zhang H. et al., 2020).
T421 68544-68748 Sentence denotes Recombinant human ACE 2, rhACE2 (hrsACE2, APN01, GSK2586881), sequesters circulating viral particles interfering with S-protein binding to its host target, beside its role in regulating the systemic RAAS.
T422 68749-69104 Sentence denotes Taken together, these activities may offer therapeutic benefits in COVID-19 patients, although the large molecular weight of the protein may potentially limit its effects on local RAAS (Gheblawi et al., 2020). rhACE2 has already undergone phase 1 and 2 clinical trials in healthy volunteers and in a small cohort of patients with ARDS (Khan et al., 2017).
T423 69105-69311 Sentence denotes Moreover, it has been demonstrated that rhACE2 can significantly block the early stages of SARS-CoV-2 infections in engineered human blood vessel organoids and human kidney organoids (Monteil et al., 2020).
T424 69312-69473 Sentence denotes In this context, Procko (2020) was able to engineer hACE2 sequences to obtain soluble receptors able to sequester SARS-CoV-2 RBD and inhibit its cell attachment.
T425 69474-69680 Sentence denotes Remarkably, combinatorial mutants enhanced ACE2 binding to SARS-CoV-2 RBD by an order of magnitude, as compared to the wild type receptor form, and targeted ACE2 mutations might provide further improvement.
T426 69681-69910 Sentence denotes Additionally, the availability of ACE2 nanoparticles applied to nose filters, chewing gums, clothes, filters and gloves could be of help in sequestering the virus thus preventing its entry into the host (Aydemir and Ulusu, 2020).
T427 69911-70143 Sentence denotes Prevention virus transmission could represent a more convenient strategy than therapeutic interventions on viral infection, avoiding interference with ACE2 and disturbance of the finely regulated RAAS axis (Aydemir and Ulusu, 2020).
T428 70145-70168 Sentence denotes ACE2 and Other Diseases
T429 70169-70223 Sentence denotes ACE2 is a multiform protein (Feng et al., 2010, 2011).
T430 70224-70414 Sentence denotes As discussed above (section Structure of ACE2) its C-terminal domain is similar to collectrin, a kidney protein involved in amino acids trafficking and insulin secretion (Kuba et al., 2013).
T431 70415-70527 Sentence denotes Alterations in ACE2 have been demonstrated in Hartnup’s disease due to a disturbance in amino acids homeostasis.
T432 70528-70645 Sentence denotes Indeed, ACE2 has been proposed to modulate amino acid transport in bowel and gut microbiome (Hashimoto et al., 2012).
T433 70646-70743 Sentence denotes Moreover, ACE2 participates in the regulation of metabolism, particularly of glucose homeostasis.
T434 70744-70851 Sentence denotes In the pancreas, activation of the ACE2-Ang1-7-MasR pathway improves insulin secretion (Yuan et al., 2013).
T435 70852-71038 Sentence denotes Obesity and high-fat diets cause a reduction in ACE2 expression in the adipose tissue, which in turn results in increased blood pressure (Gupte et al., 2008, 2012; Carsana et al., 2020).
T436 71039-71184 Sentence denotes A role for ACE2 is emerging in Alzheimer disease, since it has been shown that ACE2 can hydrolyse Beta amyloid peptides (Zou et al., 2007, 2013).
T437 71185-71268 Sentence denotes There is also increasing evidence that the RAAS system may be implicated in cancer.
T438 71269-71469 Sentence denotes ACE2 was found to inhibit cancer cell growth, metastasis, and angiogenesis in breast (Yu et al., 2016; Zhang Q. et al., 2019), pancreatic (Zhou et al., 2011), and colon cancer (Bernardi et al., 2012).
T439 71470-71679 Sentence denotes Another study pointed out that hepatocellular carcinoma patients with higher levels of ACE2 had longer survival times, suggesting a positive link between ACE2 expression and better prognosis (Ye et al., 2015).
T440 71680-71846 Sentence denotes Studies on human xenografts in mice clearly indicated that ACE2 inhibited tumor growth by suppressing invasion and angiogenesis in Non-Small Cell Lung Cancer (NSCLC).
T441 71847-72120 Sentence denotes Remarkably, the same group demonstrated that overexpression of ACE2 promotes the expression of E-cadherin at expenses of mesenchymal markers such as vimentin, and thereby inhibits the epithelial-mesenchymal transition in NSCLC models (Feng et al., 2010; Qian et al., 2013).
T442 72121-72327 Sentence denotes The discovery that lung cancer patients that harbor COVID-19 display more severe symptoms (Liang et al., 2020) set out intensive research on the possible connection between malignancies and ACE2 expression.
T443 72328-72445 Sentence denotes A thorough bioinformatics analysis of the TCGA dataset on several kinds of cancer (Chai et al., 2020) has shown that:
T444 72446-72514 Sentence denotes (1) Mutation and amplification of ACE2 gene are frequent in cancer.
T445 72515-72620 Sentence denotes Yet, hot-spot mutation sites were never observed, as ACE2 mutations were distributed across all 18 exons.
T446 72621-72877 Sentence denotes (2) ACE2 transcription was upregulated in six tumors: colon adenocarcinoma (COAD), kidney renal papillary cell carcinoma (KIRP), pancreatic adenocarcinoma (PAAD), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), and lung adenocarcinoma (LUAD).
T447 72878-72965 Sentence denotes Interestingly, ACE2 transcription was unchanged in lung squamous cell carcinoma (LUSC).
T448 72966-73110 Sentence denotes (3) ACE2 transcription was downregulated in three tumors: testicular germ cell (TGCT), thyroid carcinoma (THCA), and kidney chromophobe (KICH).
T449 73111-73266 Sentence denotes (4) Changes of ACE2 transcription were epigenetic in nature, as both mutation and copy variation of ACE2 did not correlate with its up- or downregulation.
T450 73267-73371 Sentence denotes (5) In most cases, changes of ACE2 transcription strongly correlated with methylation in ACE2 promoter.
T451 73372-73502 Sentence denotes More specifically, decreased methylation levels correlated with upregulation, whereas increased methylation led to downregulation.
T452 73503-73590 Sentence denotes (6) No prognostic role of ACE2 expression on patient’s survival could be demonstrated.
T453 73591-73711 Sentence denotes These data confirm the remarkable role of methylation in determining the ACE2 expression, as described in paragraph 2.2.
T454 73712-74057 Sentence denotes Interestingly, a second bioinformatics study on Oncomine and TCGA databases gave slightly different results in terms of tumor-associated ACE2 expression changes, but confirmed the role of promoter hypomethylation in KIRP and uterine corpus endometrial carcinoma (UCEC) where ACE2 transcription was significantly upregulated (Yang et al., 2020a).
T455 74059-74069 Sentence denotes Conclusion
T456 74070-74193 Sentence denotes From the structural point of view ACE2 is closely related to ACE, and its discovery was a by-product of ACE investigations.
T457 74194-74308 Sentence denotes However, this macromolecule has soon showed peculiar functional properties and has attracted increasing attention.
T458 74309-74560 Sentence denotes The expression of ACE2 in the heart, kidney and vascular system pointed to a role in the regulation of blood pressure and cardiovascular homeostasis, possibly as a counterbalance to ACE activation, but this hypothesis now appears to be too simplistic.
T459 74561-74759 Sentence denotes Additional functions of ACE2 are strongly suggested by its capability to modulate cytokine metabolism and the inflammatory reaction, as well as by its remarkable expression in the respiratory tract.
T460 74760-74970 Sentence denotes The recent COVID-19 pandemic has put ACE2 in the spotlight, since it has been identified as the initial target of the SARS-Cov and SARS-Cov2 viruses, representing the receptor responsible for cellular adhesion.
T461 74971-75204 Sentence denotes Recent investigations have clarified the molecular details of the interaction of ACE2 with the spike glycoprotein present in this class of coronaviruses, and have partly unraveled the mechanism of protein cleavage and cellular entry.
T462 75205-75503 Sentence denotes ACE2 density on cell surface and ACE2-modulated inflammatory reactions have been suggested to be important determinants of COVID-19 susceptibility and clinical course, while drugs interfering with ACE2 expression, availability and processing are under investigation as potential therapeutic agents.
T463 75504-75710 Sentence denotes However, several crucial issues concerning the link between ACE2 and COVID-19 pathophysiology are still unclear, pointing to the need for focused experimental investigations and prospective clinical trials.
T464 75712-75732 Sentence denotes Author Contributions
T465 75733-75835 Sentence denotes FS, GR, SS, LB, BS, and RB wrote the first draft of the manuscript and prepared the table and figures.
T466 75836-75871 Sentence denotes RZ revised and integrated the text.
T467 75872-75931 Sentence denotes All authors decided upon the general outline of the review.
T468 75933-75953 Sentence denotes Conflict of Interest
T469 75954-76126 Sentence denotes The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.