PubAnnotation

Id	Subject	Object	Predicate	Lexical cue
T1	0-30	Sentence	denotes	ACE2 in the Era of SARS-CoV-2:
T2	31-67	Sentence	denotes	Controversies and Novel Perspectives
T3	69-77	Sentence	denotes	Abstract
T4	78-206	Sentence	denotes	Angiotensin-converting enzyme 2 (ACE2) is related to ACE but turned out to counteract several pathophysiological actions of ACE.
T5	207-295	Sentence	denotes	ACE2 exerts antihypertensive and cardioprotective effects and reduces lung inflammation.
T6	296-418	Sentence	denotes	ACE2 is subjected to extensive transcriptional and post-transcriptional modulation by epigenetic mechanisms and microRNAs.
T7	419-548	Sentence	denotes	Also, ACE2 expression is regulated post-translationally by glycosylation, phosphorylation, and shedding from the plasma membrane.
T8	549-666	Sentence	denotes	ACE2 protein is ubiquitous across mammalian tissues, prominently in the cardiovascular system, kidney, and intestine.
T9	667-955	Sentence	denotes	ACE2 expression in the respiratory tract is of particular interest, in light of the discovery that ACE2 serves as the initial cellular target of severe acute respiratory syndrome (SARS)-coronaviruses, including the recent SARS-CoV2, responsible of the COronaVIrus Disease 2019 (COVID-19).
T10	956-1102	Sentence	denotes	Since the onset of the COVID-19 pandemic, an intense effort has been made to elucidate the biochemical determinants of SARS-CoV2-ACE2 interaction.
T11	1103-1377	Sentence	denotes	It has been determined that SARS-CoV2 engages with ACE2 through its spike (S) protein, which consists of two subunits: S1, that mediates binding to the host receptor; S2, that induces fusion of the viral envelope with the host cell membrane and delivery of the viral genome.
T12	1378-1647	Sentence	denotes	Owing to the role of ACE2 in SARS-CoV2 pathogenicity, it has been speculated that medical conditions, i.e., hypertension, and/or drugs, i.e., ACE inhibitors and angiotensin receptor blockers, known to influence ACE2 density could alter the fate of SARS-CoV-2 infection.
T13	1648-1792	Sentence	denotes	The debate is still open and will only be solved when results of properly designed experimental and clinical investigations will be made public.
T14	1793-1919	Sentence	denotes	An interesting observation is, however that, upon infection, ACE2 activity is reduced either by downregulation or by shedding.
T15	1920-2032	Sentence	denotes	These events might precipitate the so-called “cytokine storm” that characterizes the most severe COVID-19 forms.
T16	2033-2142	Sentence	denotes	As evidence accumulates, ACE2 appears a druggable target in the attempt to limit virus entry and replication.
T17	2143-2346	Sentence	denotes	Strategies aimed at blocking ACE2 with antibodies, small molecules or peptides, or at neutralizing the virus by competitive binding with exogenously administered ACE2, are currently under investigations.
T18	2347-2577	Sentence	denotes	In this review, we will present an overview of the state-of-the-art knowledge on ACE2 biochemistry and pathophysiology, outlining open issues in the context of COVID-19 disease and potential experimental and clinical developments.
T19	2579-2591	Sentence	denotes	Introduction
T20	2592-2770	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	2771-3044	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	3045-3125	Sentence	denotes	At present, ACE inhibitors are among the most widely prescribed drugs worldwide.
T23	3126-3197	Sentence	denotes	In 2000 a novel ACE-like enzyme, eventually named ACE2, was discovered.
T24	3198-3297	Sentence	denotes	It showed a different substrate selectively, and in particular it could not activate angiotensin I.
T25	3298-3364	Sentence	denotes	At the same time, it was not a target of classical ACE inhibitors.
T26	3365-3564	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	3565-3737	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	3738-3952	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	3953-4194	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	4195-4374	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	4375-4482	Sentence	denotes	The purpose of the present review is to summarize the available knowledge on ACE2 biochemistry and biology.
T32	4483-4653	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	4655-4684	Sentence	denotes	ACE System and ACE2 Discovery
T34	4685-4908	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	4909-5131	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	5132-5368	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	5369-5602	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	5603-5823	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	5824-5935	Sentence	denotes	Moreover, ACE mediates the cleavage and inactivation of bradykinin, which is a vasodilator hypotensive peptide.
T40	5936-6063	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	6064-6252	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	6253-6404	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	6405-6584	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	6585-6648	Sentence	denotes	Subsequently they cloned it from a human lymphoma cDNA library.
T45	6649-6837	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	6838-6930	Sentence	denotes	Moreover, they demonstrated the presence of seven glycosylation sites (Tipnis et al., 2000).
T47	6931-7251	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	7252-7354	Sentence	denotes	ACE2 showed a transmembrane domain, a zinc catalytic domain 42% identical to ACE and a signal peptide.
T49	7355-7412	Sentence	denotes	Like ACE, ACE2 seemed to be an ectoenzyme type I protein.
T50	7413-7581	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	7582-7721	Sentence	denotes	In the following years, ACE2 was intensively studied, its structure and function were enlightened, and tentative inhibitors were developed.
T52	7723-7728	Sentence	denotes	ACE2:
T53	7729-7785	Sentence	denotes	Structure, Expression, Tissue Distribution, and Function
T54	7787-7804	Sentence	denotes	Structure of ACE2
T55	7805-7926	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	7927-7984	Sentence	denotes	The 18 exons of ACE2 are remarkably similar to ACE exons.
T57	7985-8274	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	8275-8395	Sentence	denotes	The ACE2 gene codifies for a typical zinc-metallopeptidase of 805 amino acids (120 kDa), with a unique catalytic domain.
T59	8396-8506	Sentence	denotes	Despite the high resemblance of ACE and ACE2, considerable differences exist in their substrates and products.
T60	8507-8596	Sentence	denotes	While ACE acts as dipeptidase, ACE2 removes only a single amino acid from its substrates.
T61	8597-8848	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	8849-8946	Sentence	denotes	These differences depend on variances in the three-dimensional structure (3D) of the two enzymes.
T63	8947-9055	Sentence	denotes	Comparative homology modeling and crystallography contributed to shed light on ACE2 3D structure (Figure 1).
T64	9056-9208	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	9209-9357	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	9358-9518	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	9519-9781	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	9782-10053	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	10054-10290	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	10291-10327	Sentence	denotes	FIGURE 1 Crystal structure of ACE2.
T71	10328-10443	Sentence	denotes	The peptidase domain (PD) is in green, whereas the collectrin homology domain is enclosed in the light cyan square.
T72	10444-10589	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	10590-10683	Sentence	denotes	The structures have been drawn from PDB 1R42 (Towler et al., 2004) by Mol on the PDB website.
T74	10685-10765	Sentence	denotes	Transcriptional, Post-transcriptional, and Post-translational Regulation of ACE2
T75	10766-11049	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	11050-11238	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	11239-11391	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	11392-11566	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	11567-11715	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	11716-11998	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	11999-12171	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	12172-12383	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	12384-12530	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	12531-12771	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	12772-13021	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	13022-13124	Sentence	denotes	Hopefully, future studies will shed more light on the intriguing role of estrogens in ACE2 regulation.
T87	13125-13287	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	13288-13449	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	13450-13532	Sentence	denotes	Conversely, overexpression of mutant H3K4/9/36M did not change ACE2 transcription.
T90	13533-13688	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	13689-13751	Sentence	denotes	PcG proteins assemble into two major complexes: PRC1 and PRC2.
T92	13752-13876	Sentence	denotes	The simplest model of PcG activity involves trimethylation of H3 by PRC2 at target gene promoters (Blackledge et al., 2015).
T93	13877-14125	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	14126-14191	Sentence	denotes	The catalytic subunit of PRC2 is constituted by the EZH2 protein.
T95	14192-14359	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	14360-14423	Sentence	denotes	On the other side, recovery of EZH2 restored basal ACE2 levels.
T97	14424-14617	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	14618-14904	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	14905-14986	Sentence	denotes	Histone methylation does not appear to exhaust the epigenetic regulation of ACE2.
T100	14987-15135	Sentence	denotes	Notably, the NAD+-dependent deacetylase SIRT1 binds to ACE2 promoter favoring its transcription during cellular energy stress (Clarke et al., 2014).
T101	15136-15216	Sentence	denotes	Two recent unrefereed preprints highlighted other epigenetic mechanisms at play.
T102	15217-15421	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	15422-15624	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	15625-15807	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	15808-16029	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	16030-16239	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	16240-16359	Sentence	denotes	Finally, the strict homeostatic balance of ACE/ACE2 activities suggests transcriptional co-regulation of both proteins.
T108	16360-16655	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	16656-16700	Sentence	denotes	Further regulation occurs at the mRNA level.
T110	16701-16826	Sentence	denotes	From putative microRNA-binding sites identified in vitro, Lambert et al. (2014) demonstrated that miR-421 downregulates ACE2.
T111	16827-16974	Sentence	denotes	According to the hypothesized mechanism, miR-421 modulates ACE2 expression by hampering translation rather than by degradation of mRNA transcripts.
T112	16975-17190	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	17191-17361	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	17362-17479	Sentence	denotes	The function, if any, of soluble ACE2 is still obscure, but the shedding mechanism is under strict molecular control.
T115	17480-17642	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	17643-17692	Sentence	denotes	Patel identified a positive feedback in the RAAS:
T117	17693-17803	Sentence	denotes	Ang II activates ADAM17, thereby increasing the release of ACE2, its negative regulator (Patel et al., 2016b).
T118	17804-17935	Sentence	denotes	It is worth noting that high levels of plasma-soluble ACE2 have been associated with myocardial dysfunction (Epelman et al., 2009).
T119	17936-18067	Sentence	denotes	The potential pathophysiological role of ADAM17 is further discussed in paragraph ACE2 and the Inflammatory Response to Sars-CoV-2.
T120	18068-18188	Sentence	denotes	Figure 2 summarizes the known transcriptional, post-transcriptional, and post translational regulation pathways of ACE2.
T121	18189-18290	Sentence	denotes	FIGURE 2 Regulation pathways of transcription, translation, and post-translational shedding of ACE2.
T122	18291-18323	Sentence	denotes	Red text: non-molecular factors.
T123	18324-18354	Sentence	denotes	Black text: molecular factors.
T124	18356-18383	Sentence	denotes	Tissue Distribution of ACE2
T125	18384-18524	Sentence	denotes	Detectable quantities of ACE2 protein have been found almost ubiquitously in tissues across mammalian species, using immunostaining methods.
T126	18525-18742	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	18743-18961	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	18962-19052	Sentence	denotes	Such signal from vessels also delivers part of the expression detected in specific organs.
T129	19053-19391	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	19392-19569	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	19570-19757	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	19758-19905	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	19906-20249	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	20250-20442	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	20443-20730	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	20731-21069	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	21070-21264	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	21265-21550	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	21551-21721	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	21722-21879	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	21880-22174	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	22175-22192	Sentence	denotes	N. et al., 2019).
T143	22193-22357	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	22358-22636	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	22637-22826	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	22827-23036	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	23037-23203	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	23204-23294	Sentence	denotes	ACE2 distribution is widespread in the mouse brain, from the telencephalon to the medulla.
T149	23295-23701	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	23702-23985	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	23986-24123	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	24124-24294	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	24295-24403	Sentence	denotes	With regard to the endocrine system, ACE2 expression was found in both male and female reproductive systems.
T154	24404-24541	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	24542-24809	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	24810-24923	Sentence	denotes	In rodent bone, ACE2 is expressed in osteoblasts and osteoclasts, as well as in epithelial cells and fibroblasts.
T157	24924-25028	Sentence	denotes	However, a similar expression in human samples still awaits clarification (Queiroz-Junior et al., 2019).
T158	25029-25154	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	25155-25236	Sentence	denotes	Smooth muscle cells surrounding the sebaceous glands were also positive for ACE2.
T160	25237-25301	Sentence	denotes	Weak cytoplasmic staining was observed in sebaceous gland cells.
T161	25302-25386	Sentence	denotes	A strong granular staining pattern for ACE2 was seen in cells of the eccrine glands.
T162	25387-25567	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	25568-25678	Sentence	denotes	Globally, ACE2 is chiefly bound to cell membranes, while negligible levels can be detected in the circulation.
T164	25680-25722	Sentence	denotes	ACE2, the RAAS System and Cardioprotection
T165	25723-25987	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	25988-26194	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	26195-26421	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	26422-26553	Sentence	denotes	Interestingly, in this knockout model disruption of the ACE pathway could rescue the myocardial phenotype (Crackower et al., 2002).
T169	26554-26689	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	26690-26828	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	26829-27037	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	27038-27243	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	27244-27477	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	27478-27581	Sentence	denotes	ANG1-7 binds the MasR receptor to exert its effects on target organs (primarily heart, vessels, lungs).
T175	27582-27720	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	27721-28072	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	28073-28320	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	28321-28508	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	28509-28843	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	28844-28909	Sentence	denotes	Additional players contribute to ACE2-mediated cardio-protection.
T181	28910-29104	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	29105-29248	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	29249-29384	Sentence	denotes	Alamandine receptor is a Mas-related G-protein coupled receptor, known as MrgD, which is expressed in cardiomyocytes and blood vessels.
T184	29385-29527	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	29528-29758	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	29759-29866	Sentence	denotes	Other substrates of ACE2 include apelin-13/17, neurotensin, dynorphin A (1–13), and ghrelin (Turner, 2015).
T187	29867-30069	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	30070-30334	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	30335-30689	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	30691-30711	Sentence	denotes	ACE2 and Lung Injury
T191	30712-30846	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	30847-31153	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	31154-31375	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	31376-31521	Sentence	denotes	Imai et al. developed three ACE2 knockout mice models with severe ARDS induced by acid aspiration, endotoxin administration or peritoneal sepsis.
T195	31522-31743	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	31744-31888	Sentence	denotes	The phenotype was dramatically improved and rescued either administering ACE2 recombinant analogs or AT1Ra inhibitors (Imai et al., 2005, 2008).
T197	31889-32139	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	32140-32338	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	32339-32561	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	32562-32640	Sentence	denotes	ACE2 might also modulate lung inflammation via the bradykinin-DABK/BKB1R axis.
T201	32641-32860	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	32861-32924	Sentence	denotes	In human, ACE/ACE2 imbalance may be related to genetic factors.
T203	32925-33149	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	33150-33370	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	33372-33403	Sentence	denotes	Links Between ACE2 and COVID-19
T206	33404-33652	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	33653-33802	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	33803-34023	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	34024-34311	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	34312-34418	Sentence	denotes	SARS-CoV caused the SARS epidemic in 2002–2003, with over 8,000 infections and a fatality rate around 10%.
T211	34419-34615	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	34616-34794	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	34795-34955	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	34956-35029	Sentence	denotes	On March 11, 2020, the WHO has declared the COVID-19 outbreak a pandemic.
T215	35030-35224	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	35226-35276	Sentence	denotes	Mechanism of Viral Entry Mediated by the S Protein
T217	35277-35402	Sentence	denotes	A coronavirus contains four structural proteins, namely spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins.
T218	35403-35529	Sentence	denotes	These proteins assemble around a lipid bilayer to provide the shell enclosing the viral genome (Figure 4A; Tang et al., 2020).
T219	35530-35721	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	35722-35876	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	35877-36027	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	36028-36158	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	36159-36229	Sentence	denotes	This hypothesis has been confirmed from an experimental point of view.
T224	36230-36442	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	36443-36575	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	36576-36857	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	36858-36984	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	36985-37085	Sentence	denotes	The S1 subunit contains two subdomains, the N-terminal domain (NTD) and the C-terminal domain (CTD).
T229	37086-37289	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	37290-37443	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	37444-37611	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	37612-37795	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	37796-37894	Sentence	denotes	HR regions typically fold into α-helices with a hydrophobic interface that drives membrane fusion.
T234	37895-38092	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	38093-38251	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	38252-38443	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	38444-38649	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	38650-38744	Sentence	denotes	Taken together, these findings are solid evidence that SARS-CoV-2 engages ACE2 for cell entry.
T239	38745-38968	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	38969-39194	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	39195-39386	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	39387-39604	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	39605-39723	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	39724-39886	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	39887-40250	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	40251-40486	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	40487-40601	Sentence	denotes	At first, three of these helices assemble into a homotrimeric bundle and stick the FP into the host cell membrane.
T248	40602-40756	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	40757-40989	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	40990-41200	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	41201-41250	Sentence	denotes	FIGURE 5 Coronavirus viral fusion pathway model.
T252	41251-41314	Sentence	denotes	Initially, the S protein is in the pre-fusion native state (1).
T253	41315-41613	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	41614-41793	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	41794-41908	Sentence	denotes	The pre-hairpin folds back on itself due to HR1 and HR2 interactions eventually forming the post-fusion (6) state.
T256	41909-42079	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	42080-42201	Sentence	denotes	Note that cell membrane may refer to plasma membrane (direct fusion) or endosomal membrane (fusion in endocytic vesicle).
T258	42202-42234	Sentence	denotes	Adapted from Tang et al. (2020).
T259	42235-42311	Sentence	denotes	FIGURE 6 Trimeric S protein of SARS-CoV-2 in the ”Closed” and “Open” forms.
T260	42312-42400	Sentence	denotes	Note the single RBD protruding out of the V-shaped conformation of the protein assembly.
T261	42401-42449	Sentence	denotes	The structures have been drawn from PDB 6X2C (R.
T262	42450-42513	Sentence	denotes	Henderson, 10.1101/2020.05.18.10208) by Mol on the PDB website.
T263	42514-42683	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	42684-42896	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	42897-43052	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	43053-43285	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	43286-43679	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	43680-43831	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	43832-44017	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	44018-44103	Sentence	denotes	FIGURE 7 Relevance of S S1/S2 “priming” by host proteases for viral fusion to cells.
T271	44104-44264	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	44265-44490	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	44491-44801	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	44802-44912	Sentence	denotes	Note that also “primed” viruses may undergo pathway C, depending on their interaction with the recipient cell.
T275	44914-44951	Sentence	denotes	SARS-CoV-2 RBD and Its Receptor, ACE2
T276	44952-45099	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	45100-45176	Sentence	denotes	SARS-CoV RBD corresponds to residues 306–527 of S protein (Li et al., 2005).
T278	45177-45316	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	45317-45447	Sentence	denotes	Accordingly, Wang et al. (2020) clearly demonstrated that S319–341 corresponds to SARS-CoV-2 RBD by immunofluorescence microscopy.
T280	45448-45651	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	45652-45781	Sentence	denotes	Additionally, soluble ACE2 inhibited the interaction between viral proteins and ACE2-expressing cells in a dose-dependent manner.
T282	45782-45858	Sentence	denotes	The SARS-CoV-2 RBD sequence was further investigated by structural analysis.
T283	45859-46256	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	46257-46524	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	46525-46759	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	46760-46832	Sentence	denotes	The β1-β7 typical motifs of RBD (Lan et al., 2020) are indicated in (A).
T287	46833-46923	Sentence	denotes	The structures have been drawn from PDB 6MOJ (Lan et al., 2020) by Mol on the PDB website.
T288	46924-47001	Sentence	denotes	Several researchers investigated the interaction of SARS-CoV-2 RBD with ACE2.
T289	47002-47088	Sentence	denotes	Unfortunately, each group committed to slightly different sequences of SARS-CoV-2 RBD.
T290	47089-47249	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	47250-47364	Sentence	denotes	In vitro affinity studies revealed dissociation constants of the ACE2-RBD complex in the 1–100 nM range (Table 1).
T292	47365-47513	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	47514-47705	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	47706-47923	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	47924-48101	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	48102-48345	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	48346-48672	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	48673-48790	Sentence	denotes	Taken together, these properties account for the sophisticated strategy exploited by SARS-CoV-2 to invade host cells.
T299	48791-48884	Sentence	denotes	TABLE 1 Binding affinity between SARS-CoV-2 spike (S) protein and S subset regions and ACE2.
T300	48885-48922	Sentence	denotes	Sequence KD (nM) Method References
T301	48923-48962	Sentence	denotes	SARS-2 S 14.7 SPR Wrapp et al., 2020
T302	48963-49000	Sentence	denotes	SARS-2 S 11.2 SPR Lei et al., 2020
T303	49001-49050	Sentence	denotes	SARS-2 S20–685 (S1) 94.6 SPR Wang et al., 2020
T304	49051-49103	Sentence	denotes	SARS-2 S319–541 (RBD) 133.3 SPR Wang et al., 2020
T305	49104-49153	Sentence	denotes	SARS-2 S319–541 (RBD) 4.7 SPR Lan et al., 2020
T306	49154-49201	Sentence	denotes	SARS-2 S319–529 44.2 SPR Shang et al., 2020b
T307	49202-49268	Sentence	denotes	SARS-2 S319–591 34.6 Biolayer interferometry Wrapp et al., 2020
T308	49269-49334	Sentence	denotes	SARS-2 S328–533 1.2 Biolayer interferometry Walls et al., 2020
T309	49336-49369	Sentence	denotes	Structure of the ACE2-RBD Complex
T310	49370-49603	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	49604-49778	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	49779-50059	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	50060-50170	Sentence	denotes	Pleasantly, all data converged to a consistent tridimensional arrangement of the receptor (Wang et al., 2020).
T314	50171-50288	Sentence	denotes	In the “closed” conformation of S protein, the RBD is buried at the interface between protomers (Walls et al., 2020).
T315	50289-50471	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	50472-50661	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	50662-50850	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	50851-51016	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	51017-51118	Sentence	denotes	A second smaller loop (loop 2: residues 498–505) is visible on the other side of the concave surface.
T320	51119-51384	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	51385-51639	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	51640-51848	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	51849-51944	Sentence	denotes	Asn501 in loop 2 further engages recognized hotspots on the ACE2 surface (Shang et al., 2020b).
T324	51945-52096	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	52097-52217	Sentence	denotes	These subtle structural differences probably account for the higher affinity of SARS-CoV-2 for ACE2 (Wang et al., 2020).
T326	52218-52427	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	52428-52695	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	52696-52930	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	52931-53013	Sentence	denotes	Yet, no correlation between genetic distance and the S/ACE2 interaction was found.
T330	53014-53225	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	53226-53398	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	53399-53559	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	53560-53753	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	53754-53977	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	53979-54020	Sentence	denotes	The Controversial Role of ACE2 Expression
T336	54021-54261	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	54262-54444	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	54445-54692	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	54693-54901	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	54902-55177	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	55178-55331	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	55332-55476	Sentence	denotes	This series reported an even higher prevalence of hypertension (49%), diabetes (17%), and cardiovascular disease (21%) (Grasselli et al., 2020).
T343	55477-55665	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	55666-55824	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	55825-55906	Sentence	denotes	Obesity has also emerged as an important factor in determining COVID-19 severity.
T346	55907-56175	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	56176-56318	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	56319-56531	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	56532-56764	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	56765-56935	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	56936-57404	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	57405-57794	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	57795-58020	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	58021-58208	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	58209-58406	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	58407-58546	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	58547-58888	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	58889-59061	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	59062-59355	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	59356-59760	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	59761-60316	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	60318-60366	Sentence	denotes	ACE2 and the Inflammatory Response to SARS-CoV-2
T363	60367-60533	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	60534-60693	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	60694-61009	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	61010-61164	Sentence	denotes	Moreover, cardiovascular involvement has been observed, occasionally leading to myocardial injury and sudden death after weeks from the initial infection.
T367	61165-61336	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	61337-61485	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	61486-61624	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	61625-61723	Sentence	denotes	In 29% (n = 12) of patients ARDS occurred and in 10% invasive mechanical ventilation was required.
T371	61724-61849	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	61850-62165	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	62166-62284	Sentence	denotes	The molecular mechanisms involved in the cytokine storm and the role of ACE2 are currently still not fully understood.
T374	62285-62498	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	62499-62627	Sentence	denotes	This has been suggested to trigger the primary inflammatory response, which involves apoptosis and pyroptosis (Fu et al., 2020).
T376	62628-62878	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	62879-63125	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	63126-63571	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	63572-63679	Sentence	denotes	FIGURE 9 Possible role of ACE2 in the molecular mechanisms involved in the “cytokine storm” of SARS-CoV-2.
T380	63680-63901	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	63902-64040	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	64041-64228	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	64229-64354	Sentence	denotes	Another proposed mechanism that triggers the proinflammatory response to SARS-CoV-2 is ACE/ACE2 imbalance in the RAAS system.
T384	64355-64651	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	64652-64996	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	64997-65236	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	65237-65333	Sentence	denotes	The convergence on hyperactivation of NF-K B seems to be crucial in inducing the cytokine storm.
T388	65334-65530	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	65531-65619	Sentence	denotes	ACE2 shedding is an additional mechanism contributing to inflammation (Fu et al., 2020).
T390	65620-65873	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	65874-66089	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	66090-66406	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	66407-66627	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	66628-66745	Sentence	denotes	ACE2 shedding seems to be inducible by specific stimuli produced either by a viral infection or by the immune system.
T395	66746-66859	Sentence	denotes	Jia et al. (2009) confirmed ACE2 shedding in differentiated primary airway epithelial cells and Calu-3-cell line.
T396	66860-67068	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	67069-67204	Sentence	denotes	These findings suggest that an inflammatory response is likely to modulate ACE2 expression and shedding, also during a viral infection.
T398	67205-67389	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	67390-67549	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	67550-67711	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	67712-68368	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	68370-68398	Sentence	denotes	ACE2 as a Therapeutic Target
T403	68399-68487	Sentence	denotes	As evidence builds up, ACE2 rapidly emerged as a specific target for COVID-19 treatment.
T404	68488-68792	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	68793-68846	Sentence	denotes	Potential therapeutic approaches include (Figure 10):
T406	68847-68958	Sentence	denotes	FIGURE 10 Schematic representation of the potential therapeutic approaches to address ACE2 mediated infection.
T407	68959-69163	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	69164-69166	Sentence	denotes	1.
T409	69168-69182	Sentence	denotes	ACE2 blockers;
T410	69183-69185	Sentence	denotes	2.
T411	69187-69220	Sentence	denotes	Exogenous administration of ACE2.
T412	69222-69235	Sentence	denotes	ACE2 Blockers
T413	69236-69486	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	69487-69675	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	69676-70003	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	70004-70202	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	70203-70499	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	70501-70533	Sentence	denotes	Exogenous Administration of ACE2
T419	70534-70942	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	70943-71122	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	71123-71327	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	71328-71683	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	71684-71890	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	71891-72052	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	72053-72259	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	72260-72489	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	72490-72722	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	72724-72747	Sentence	denotes	ACE2 and Other Diseases
T429	72748-72802	Sentence	denotes	ACE2 is a multiform protein (Feng et al., 2010, 2011).
T430	72803-72993	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	72994-73106	Sentence	denotes	Alterations in ACE2 have been demonstrated in Hartnup’s disease due to a disturbance in amino acids homeostasis.
T432	73107-73224	Sentence	denotes	Indeed, ACE2 has been proposed to modulate amino acid transport in bowel and gut microbiome (Hashimoto et al., 2012).
T433	73225-73322	Sentence	denotes	Moreover, ACE2 participates in the regulation of metabolism, particularly of glucose homeostasis.
T434	73323-73430	Sentence	denotes	In the pancreas, activation of the ACE2-Ang1-7-MasR pathway improves insulin secretion (Yuan et al., 2013).
T435	73431-73617	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	73618-73763	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	73764-73847	Sentence	denotes	There is also increasing evidence that the RAAS system may be implicated in cancer.
T438	73848-74048	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	74049-74258	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	74259-74425	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	74426-74699	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	74700-74906	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	74907-75024	Sentence	denotes	A thorough bioinformatics analysis of the TCGA dataset on several kinds of cancer (Chai et al., 2020) has shown that:
T444	75025-75093	Sentence	denotes	(1) Mutation and amplification of ACE2 gene are frequent in cancer.
T445	75094-75199	Sentence	denotes	Yet, hot-spot mutation sites were never observed, as ACE2 mutations were distributed across all 18 exons.
T446	75200-75456	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	75457-75544	Sentence	denotes	Interestingly, ACE2 transcription was unchanged in lung squamous cell carcinoma (LUSC).
T448	75545-75689	Sentence	denotes	(3) ACE2 transcription was downregulated in three tumors: testicular germ cell (TGCT), thyroid carcinoma (THCA), and kidney chromophobe (KICH).
T449	75690-75845	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	75846-75950	Sentence	denotes	(5) In most cases, changes of ACE2 transcription strongly correlated with methylation in ACE2 promoter.
T451	75951-76081	Sentence	denotes	More specifically, decreased methylation levels correlated with upregulation, whereas increased methylation led to downregulation.
T452	76082-76169	Sentence	denotes	(6) No prognostic role of ACE2 expression on patient’s survival could be demonstrated.
T453	76170-76290	Sentence	denotes	These data confirm the remarkable role of methylation in determining the ACE2 expression, as described in paragraph 2.2.
T454	76291-76636	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	76638-76648	Sentence	denotes	Conclusion
T456	76649-76772	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	76773-76887	Sentence	denotes	However, this macromolecule has soon showed peculiar functional properties and has attracted increasing attention.
T458	76888-77139	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	77140-77338	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	77339-77549	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	77550-77783	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	77784-78082	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	78083-78289	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	78291-78311	Sentence	denotes	Author Contributions
T465	78312-78414	Sentence	denotes	FS, GR, SS, LB, BS, and RB wrote the first draft of the manuscript and prepared the table and figures.
T466	78415-78450	Sentence	denotes	RZ revised and integrated the text.
T467	78451-78510	Sentence	denotes	All authors decided upon the general outline of the review.
T468	78512-78532	Sentence	denotes	Conflict of Interest
T469	78533-78705	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.
T470	78707-78715	Sentence	denotes	Funding.
T471	78716-78799	Sentence	denotes	This work is supported by the PRA 2020-22 to GR, granted by the University of Pisa.
T472	78800-78854	Sentence	denotes	1 https://www.preprints.org/manuscript/202003.0295/v1

T1

0-30

Sentence

denotes

ACE2 in the Era of SARS-CoV-2:

T2

31-67

Sentence

denotes

Controversies and Novel Perspectives

T3

69-77

Sentence

denotes

Abstract

T4

78-206

Sentence

denotes

Angiotensin-converting enzyme 2 (ACE2) is related to ACE but turned out to counteract several pathophysiological actions of ACE.

T5

207-295

Sentence

denotes

ACE2 exerts antihypertensive and cardioprotective effects and reduces lung inflammation.

T6

296-418

Sentence

denotes

ACE2 is subjected to extensive transcriptional and post-transcriptional modulation by epigenetic mechanisms and microRNAs.

T7

419-548

Sentence

denotes

Also, ACE2 expression is regulated post-translationally by glycosylation, phosphorylation, and shedding from the plasma membrane.

T8

549-666

Sentence

denotes

ACE2 protein is ubiquitous across mammalian tissues, prominently in the cardiovascular system, kidney, and intestine.

T9

667-955

Sentence

denotes

ACE2 expression in the respiratory tract is of particular interest, in light of the discovery that ACE2 serves as the initial cellular target of severe acute respiratory syndrome (SARS)-coronaviruses, including the recent SARS-CoV2, responsible of the COronaVIrus Disease 2019 (COVID-19).

T10

956-1102

Sentence

denotes

Since the onset of the COVID-19 pandemic, an intense effort has been made to elucidate the biochemical determinants of SARS-CoV2-ACE2 interaction.

T11

1103-1377

Sentence

denotes

It has been determined that SARS-CoV2 engages with ACE2 through its spike (S) protein, which consists of two subunits: S1, that mediates binding to the host receptor; S2, that induces fusion of the viral envelope with the host cell membrane and delivery of the viral genome.

T12

1378-1647

Sentence

denotes

Owing to the role of ACE2 in SARS-CoV2 pathogenicity, it has been speculated that medical conditions, i.e., hypertension, and/or drugs, i.e., ACE inhibitors and angiotensin receptor blockers, known to influence ACE2 density could alter the fate of SARS-CoV-2 infection.

T13

1648-1792

Sentence

denotes

The debate is still open and will only be solved when results of properly designed experimental and clinical investigations will be made public.

T14

1793-1919

Sentence

denotes

An interesting observation is, however that, upon infection, ACE2 activity is reduced either by downregulation or by shedding.

T15

1920-2032

Sentence

denotes

These events might precipitate the so-called “cytokine storm” that characterizes the most severe COVID-19 forms.

T16

2033-2142

Sentence

denotes

As evidence accumulates, ACE2 appears a druggable target in the attempt to limit virus entry and replication.

T17

2143-2346

Sentence

denotes

Strategies aimed at blocking ACE2 with antibodies, small molecules or peptides, or at neutralizing the virus by competitive binding with exogenously administered ACE2, are currently under investigations.

T18

2347-2577

Sentence

denotes

In this review, we will present an overview of the state-of-the-art knowledge on ACE2 biochemistry and pathophysiology, outlining open issues in the context of COVID-19 disease and potential experimental and clinical developments.

T19

2579-2591

Sentence

denotes

Introduction

T20

2592-2770

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

2771-3044

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

3045-3125

Sentence

denotes

At present, ACE inhibitors are among the most widely prescribed drugs worldwide.

T23

3126-3197

Sentence

denotes

In 2000 a novel ACE-like enzyme, eventually named ACE2, was discovered.

T24

3198-3297

Sentence

denotes

It showed a different substrate selectively, and in particular it could not activate angiotensin I.

T25

3298-3364

Sentence

denotes

At the same time, it was not a target of classical ACE inhibitors.

T26

3365-3564

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

3565-3737

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

3738-3952

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

3953-4194

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

4195-4374

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

4375-4482

Sentence

denotes

The purpose of the present review is to summarize the available knowledge on ACE2 biochemistry and biology.

T32

4483-4653

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

4655-4684

Sentence

denotes

ACE System and ACE2 Discovery

T34

4685-4908

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

4909-5131

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

5132-5368

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

5369-5602

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

5603-5823

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

5824-5935

Sentence

denotes

Moreover, ACE mediates the cleavage and inactivation of bradykinin, which is a vasodilator hypotensive peptide.

T40

5936-6063

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

6064-6252

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

6253-6404

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

6405-6584

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

6585-6648

Sentence

denotes

Subsequently they cloned it from a human lymphoma cDNA library.

T45

6649-6837

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

6838-6930

Sentence

denotes

Moreover, they demonstrated the presence of seven glycosylation sites (Tipnis et al., 2000).

T47

6931-7251

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

7252-7354

Sentence

denotes

ACE2 showed a transmembrane domain, a zinc catalytic domain 42% identical to ACE and a signal peptide.

T49

7355-7412

Sentence

denotes

Like ACE, ACE2 seemed to be an ectoenzyme type I protein.

T50

7413-7581

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

7582-7721

Sentence

denotes

In the following years, ACE2 was intensively studied, its structure and function were enlightened, and tentative inhibitors were developed.

T52

7723-7728

Sentence

denotes

ACE2:

T53

7729-7785

Sentence

denotes

Structure, Expression, Tissue Distribution, and Function

T54

7787-7804

Sentence

denotes

Structure of ACE2

T55

7805-7926

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

7927-7984

Sentence

denotes

The 18 exons of ACE2 are remarkably similar to ACE exons.

T57

7985-8274

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

8275-8395

Sentence

denotes

The ACE2 gene codifies for a typical zinc-metallopeptidase of 805 amino acids (120 kDa), with a unique catalytic domain.

T59

8396-8506

Sentence

denotes

Despite the high resemblance of ACE and ACE2, considerable differences exist in their substrates and products.

T60

8507-8596

Sentence

denotes

While ACE acts as dipeptidase, ACE2 removes only a single amino acid from its substrates.

T61

8597-8848

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

8849-8946

Sentence

denotes

These differences depend on variances in the three-dimensional structure (3D) of the two enzymes.

T63

8947-9055

Sentence

denotes

Comparative homology modeling and crystallography contributed to shed light on ACE2 3D structure (Figure 1).

T64

9056-9208

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

9209-9357

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

9358-9518

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

9519-9781

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

9782-10053

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

10054-10290

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

10291-10327

Sentence

denotes

FIGURE 1 Crystal structure of ACE2.

T71

10328-10443

Sentence

denotes

The peptidase domain (PD) is in green, whereas the collectrin homology domain is enclosed in the light cyan square.

T72

10444-10589

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

10590-10683

Sentence

denotes

The structures have been drawn from PDB 1R42 (Towler et al., 2004) by Mol on the PDB website.

T74

10685-10765

Sentence

denotes

Transcriptional, Post-transcriptional, and Post-translational Regulation of ACE2

T75

10766-11049

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

11050-11238

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

11239-11391

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

11392-11566

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

11567-11715

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

11716-11998

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

11999-12171

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

12172-12383

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

12384-12530

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

12531-12771

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

12772-13021

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

13022-13124

Sentence

denotes

Hopefully, future studies will shed more light on the intriguing role of estrogens in ACE2 regulation.

T87

13125-13287

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

13288-13449

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

13450-13532

Sentence

denotes

Conversely, overexpression of mutant H3K4/9/36M did not change ACE2 transcription.

T90

13533-13688

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

13689-13751

Sentence

denotes

PcG proteins assemble into two major complexes: PRC1 and PRC2.

T92

13752-13876

Sentence

denotes

The simplest model of PcG activity involves trimethylation of H3 by PRC2 at target gene promoters (Blackledge et al., 2015).

T93

13877-14125

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

14126-14191

Sentence

denotes

The catalytic subunit of PRC2 is constituted by the EZH2 protein.

T95

14192-14359

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

14360-14423

Sentence

denotes

On the other side, recovery of EZH2 restored basal ACE2 levels.

T97

14424-14617

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

14618-14904

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

14905-14986

Sentence

denotes

Histone methylation does not appear to exhaust the epigenetic regulation of ACE2.

T100

14987-15135

Sentence

denotes

Notably, the NAD+-dependent deacetylase SIRT1 binds to ACE2 promoter favoring its transcription during cellular energy stress (Clarke et al., 2014).

T101

15136-15216

Sentence

denotes

Two recent unrefereed preprints highlighted other epigenetic mechanisms at play.

T102

15217-15421

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

15422-15624

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

15625-15807

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

15808-16029

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

16030-16239

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

16240-16359

Sentence

denotes

Finally, the strict homeostatic balance of ACE/ACE2 activities suggests transcriptional co-regulation of both proteins.

T108

16360-16655

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

16656-16700

Sentence

denotes

Further regulation occurs at the mRNA level.

T110

16701-16826

Sentence

denotes

From putative microRNA-binding sites identified in vitro, Lambert et al. (2014) demonstrated that miR-421 downregulates ACE2.

T111

16827-16974

Sentence

denotes

According to the hypothesized mechanism, miR-421 modulates ACE2 expression by hampering translation rather than by degradation of mRNA transcripts.

T112

16975-17190

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

17191-17361

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

17362-17479

Sentence

denotes

The function, if any, of soluble ACE2 is still obscure, but the shedding mechanism is under strict molecular control.

T115

17480-17642

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

17643-17692

Sentence

denotes

Patel identified a positive feedback in the RAAS:

T117

17693-17803

Sentence

denotes

Ang II activates ADAM17, thereby increasing the release of ACE2, its negative regulator (Patel et al., 2016b).

T118

17804-17935

Sentence

denotes

It is worth noting that high levels of plasma-soluble ACE2 have been associated with myocardial dysfunction (Epelman et al., 2009).

T119

17936-18067

Sentence

denotes

The potential pathophysiological role of ADAM17 is further discussed in paragraph ACE2 and the Inflammatory Response to Sars-CoV-2.

T120

18068-18188

Sentence

denotes

Figure 2 summarizes the known transcriptional, post-transcriptional, and post translational regulation pathways of ACE2.

T121

18189-18290

Sentence

denotes

FIGURE 2 Regulation pathways of transcription, translation, and post-translational shedding of ACE2.

T122

18291-18323

Sentence

denotes

Red text: non-molecular factors.

T123

18324-18354

Sentence

denotes

Black text: molecular factors.

T124

18356-18383

Sentence

denotes

Tissue Distribution of ACE2

T125

18384-18524

Sentence

denotes

Detectable quantities of ACE2 protein have been found almost ubiquitously in tissues across mammalian species, using immunostaining methods.

T126

18525-18742

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

18743-18961

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

18962-19052

Sentence

denotes

Such signal from vessels also delivers part of the expression detected in specific organs.

T129

19053-19391

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

19392-19569

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

19570-19757

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

19758-19905

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

19906-20249

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

20250-20442

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

20443-20730

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

20731-21069

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

21070-21264

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

21265-21550

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

21551-21721

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

21722-21879

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

21880-22174

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

22175-22192

Sentence

denotes

N. et al., 2019).

T143

22193-22357

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

22358-22636

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

22637-22826

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

22827-23036

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

23037-23203

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

23204-23294

Sentence

denotes

ACE2 distribution is widespread in the mouse brain, from the telencephalon to the medulla.

T149

23295-23701

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

23702-23985

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

23986-24123

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

24124-24294

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

24295-24403

Sentence

denotes

With regard to the endocrine system, ACE2 expression was found in both male and female reproductive systems.

T154

24404-24541

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

24542-24809

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

24810-24923

Sentence

denotes

In rodent bone, ACE2 is expressed in osteoblasts and osteoclasts, as well as in epithelial cells and fibroblasts.

T157

24924-25028

Sentence

denotes

However, a similar expression in human samples still awaits clarification (Queiroz-Junior et al., 2019).

T158

25029-25154

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

25155-25236

Sentence

denotes

Smooth muscle cells surrounding the sebaceous glands were also positive for ACE2.

T160

25237-25301

Sentence

denotes

Weak cytoplasmic staining was observed in sebaceous gland cells.

T161

25302-25386

Sentence

denotes

A strong granular staining pattern for ACE2 was seen in cells of the eccrine glands.

T162

25387-25567

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

25568-25678

Sentence

denotes

Globally, ACE2 is chiefly bound to cell membranes, while negligible levels can be detected in the circulation.

T164

25680-25722

Sentence

denotes

ACE2, the RAAS System and Cardioprotection

T165

25723-25987

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

25988-26194

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

26195-26421

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

26422-26553

Sentence

denotes

Interestingly, in this knockout model disruption of the ACE pathway could rescue the myocardial phenotype (Crackower et al., 2002).

T169

26554-26689

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

26690-26828

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

26829-27037

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

27038-27243

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

27244-27477

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

27478-27581

Sentence

denotes

ANG1-7 binds the MasR receptor to exert its effects on target organs (primarily heart, vessels, lungs).

T175

27582-27720

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

27721-28072

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

28073-28320

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

28321-28508

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

28509-28843

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

28844-28909

Sentence

denotes

Additional players contribute to ACE2-mediated cardio-protection.

T181

28910-29104

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

29105-29248

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

29249-29384

Sentence

denotes

Alamandine receptor is a Mas-related G-protein coupled receptor, known as MrgD, which is expressed in cardiomyocytes and blood vessels.

T184

29385-29527

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

29528-29758

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

29759-29866

Sentence

denotes

Other substrates of ACE2 include apelin-13/17, neurotensin, dynorphin A (1–13), and ghrelin (Turner, 2015).

T187

29867-30069

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

30070-30334

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

30335-30689

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

30691-30711

Sentence

denotes

ACE2 and Lung Injury

T191

30712-30846

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

30847-31153

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

31154-31375

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

31376-31521

Sentence

denotes

Imai et al. developed three ACE2 knockout mice models with severe ARDS induced by acid aspiration, endotoxin administration or peritoneal sepsis.

T195

31522-31743

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

31744-31888

Sentence

denotes

The phenotype was dramatically improved and rescued either administering ACE2 recombinant analogs or AT1Ra inhibitors (Imai et al., 2005, 2008).

T197

31889-32139

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

32140-32338

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

32339-32561

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

32562-32640

Sentence

denotes

ACE2 might also modulate lung inflammation via the bradykinin-DABK/BKB1R axis.

T201

32641-32860

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

32861-32924

Sentence

denotes

In human, ACE/ACE2 imbalance may be related to genetic factors.

T203

32925-33149

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

33150-33370

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

33372-33403

Sentence

denotes

Links Between ACE2 and COVID-19

T206

33404-33652

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

33653-33802

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

33803-34023

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

34024-34311

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

34312-34418

Sentence

denotes

SARS-CoV caused the SARS epidemic in 2002–2003, with over 8,000 infections and a fatality rate around 10%.

T211

34419-34615

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

34616-34794

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

34795-34955

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

34956-35029

Sentence

denotes

On March 11, 2020, the WHO has declared the COVID-19 outbreak a pandemic.

T215

35030-35224

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

35226-35276

Sentence

denotes

Mechanism of Viral Entry Mediated by the S Protein

T217

35277-35402

Sentence

denotes

A coronavirus contains four structural proteins, namely spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins.

T218

35403-35529

Sentence

denotes

These proteins assemble around a lipid bilayer to provide the shell enclosing the viral genome (Figure 4A; Tang et al., 2020).

T219

35530-35721

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

35722-35876

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

35877-36027

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

36028-36158

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

36159-36229

Sentence

denotes

This hypothesis has been confirmed from an experimental point of view.

T224

36230-36442

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

36443-36575

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

36576-36857

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

36858-36984

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

36985-37085

Sentence

denotes

The S1 subunit contains two subdomains, the N-terminal domain (NTD) and the C-terminal domain (CTD).

T229

37086-37289

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

37290-37443

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

37444-37611

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

37612-37795

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

37796-37894

Sentence

denotes

HR regions typically fold into α-helices with a hydrophobic interface that drives membrane fusion.

T234

37895-38092

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

38093-38251

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

38252-38443

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

38444-38649

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

38650-38744

Sentence

denotes

Taken together, these findings are solid evidence that SARS-CoV-2 engages ACE2 for cell entry.

T239

38745-38968

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

38969-39194

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

39195-39386

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

39387-39604

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

39605-39723

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

39724-39886

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

39887-40250

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

40251-40486

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

40487-40601

Sentence

denotes

At first, three of these helices assemble into a homotrimeric bundle and stick the FP into the host cell membrane.

T248

40602-40756

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

40757-40989

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

40990-41200

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

41201-41250

Sentence

denotes

FIGURE 5 Coronavirus viral fusion pathway model.

T252

41251-41314

Sentence

denotes

Initially, the S protein is in the pre-fusion native state (1).

T253

41315-41613

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

41614-41793

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

41794-41908

Sentence

denotes

The pre-hairpin folds back on itself due to HR1 and HR2 interactions eventually forming the post-fusion (6) state.

T256

41909-42079

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

42080-42201

Sentence

denotes

Note that cell membrane may refer to plasma membrane (direct fusion) or endosomal membrane (fusion in endocytic vesicle).

T258

42202-42234

Sentence

denotes

Adapted from Tang et al. (2020).

T259

42235-42311

Sentence

denotes

FIGURE 6 Trimeric S protein of SARS-CoV-2 in the ”Closed” and “Open” forms.

T260

42312-42400

Sentence

denotes

Note the single RBD protruding out of the V-shaped conformation of the protein assembly.

T261

42401-42449

Sentence

denotes

The structures have been drawn from PDB 6X2C (R.

T262

42450-42513

Sentence

denotes

Henderson, 10.1101/2020.05.18.10208) by Mol on the PDB website.

T263

42514-42683

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

42684-42896

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

42897-43052

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

43053-43285

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

43286-43679

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

43680-43831

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

43832-44017

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

44018-44103

Sentence

denotes

FIGURE 7 Relevance of S S1/S2 “priming” by host proteases for viral fusion to cells.

T271

44104-44264

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

44265-44490

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

44491-44801

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

44802-44912

Sentence

denotes

Note that also “primed” viruses may undergo pathway C, depending on their interaction with the recipient cell.

T275

44914-44951

Sentence

denotes

SARS-CoV-2 RBD and Its Receptor, ACE2

T276

44952-45099

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

45100-45176

Sentence

denotes

SARS-CoV RBD corresponds to residues 306–527 of S protein (Li et al., 2005).

T278

45177-45316

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

45317-45447

Sentence

denotes

Accordingly, Wang et al. (2020) clearly demonstrated that S319–341 corresponds to SARS-CoV-2 RBD by immunofluorescence microscopy.

T280

45448-45651

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

45652-45781

Sentence

denotes

Additionally, soluble ACE2 inhibited the interaction between viral proteins and ACE2-expressing cells in a dose-dependent manner.

T282

45782-45858

Sentence

denotes

The SARS-CoV-2 RBD sequence was further investigated by structural analysis.

T283

45859-46256

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

46257-46524

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

46525-46759

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

46760-46832

Sentence

denotes

The β1-β7 typical motifs of RBD (Lan et al., 2020) are indicated in (A).

T287

46833-46923

Sentence

denotes

The structures have been drawn from PDB 6MOJ (Lan et al., 2020) by Mol on the PDB website.

T288

46924-47001

Sentence

denotes

Several researchers investigated the interaction of SARS-CoV-2 RBD with ACE2.

T289

47002-47088

Sentence

denotes

Unfortunately, each group committed to slightly different sequences of SARS-CoV-2 RBD.

T290

47089-47249

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

47250-47364

Sentence

denotes

In vitro affinity studies revealed dissociation constants of the ACE2-RBD complex in the 1–100 nM range (Table 1).

T292

47365-47513

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

47514-47705

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

47706-47923

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

47924-48101

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

48102-48345

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

48346-48672

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

48673-48790

Sentence

denotes

Taken together, these properties account for the sophisticated strategy exploited by SARS-CoV-2 to invade host cells.

T299

48791-48884

Sentence

denotes

TABLE 1 Binding affinity between SARS-CoV-2 spike (S) protein and S subset regions and ACE2.

T300

48885-48922

Sentence

denotes

Sequence KD (nM) Method References

T301

48923-48962

Sentence

denotes

SARS-2 S 14.7 SPR Wrapp et al., 2020

T302

48963-49000

Sentence

denotes

SARS-2 S 11.2 SPR Lei et al., 2020

T303

49001-49050

Sentence

denotes

SARS-2 S20–685 (S1) 94.6 SPR Wang et al., 2020

T304

49051-49103

Sentence

denotes

SARS-2 S319–541 (RBD) 133.3 SPR Wang et al., 2020

T305

49104-49153

Sentence

denotes

SARS-2 S319–541 (RBD) 4.7 SPR Lan et al., 2020

T306

49154-49201

Sentence

denotes

SARS-2 S319–529 44.2 SPR Shang et al., 2020b

T307

49202-49268

Sentence

denotes

SARS-2 S319–591 34.6 Biolayer interferometry Wrapp et al., 2020

T308

49269-49334

Sentence

denotes

SARS-2 S328–533 1.2 Biolayer interferometry Walls et al., 2020

T309

49336-49369

Sentence

denotes

Structure of the ACE2-RBD Complex

T310

49370-49603

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

49604-49778

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

49779-50059

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

50060-50170

Sentence

denotes

Pleasantly, all data converged to a consistent tridimensional arrangement of the receptor (Wang et al., 2020).

T314

50171-50288

Sentence

denotes

In the “closed” conformation of S protein, the RBD is buried at the interface between protomers (Walls et al., 2020).

T315

50289-50471

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

50472-50661

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

50662-50850

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

50851-51016

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

51017-51118

Sentence

denotes

A second smaller loop (loop 2: residues 498–505) is visible on the other side of the concave surface.

T320

51119-51384

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

51385-51639

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

51640-51848

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

51849-51944

Sentence

denotes

Asn501 in loop 2 further engages recognized hotspots on the ACE2 surface (Shang et al., 2020b).

T324

51945-52096

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

52097-52217

Sentence

denotes

These subtle structural differences probably account for the higher affinity of SARS-CoV-2 for ACE2 (Wang et al., 2020).

T326

52218-52427

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

52428-52695

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

52696-52930

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

52931-53013

Sentence

denotes

Yet, no correlation between genetic distance and the S/ACE2 interaction was found.

T330

53014-53225

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

53226-53398

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

53399-53559

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

53560-53753

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

53754-53977

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

53979-54020

Sentence

denotes

The Controversial Role of ACE2 Expression

T336

54021-54261

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

54262-54444

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

54445-54692

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

54693-54901

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

54902-55177

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

55178-55331

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

55332-55476

Sentence

denotes

This series reported an even higher prevalence of hypertension (49%), diabetes (17%), and cardiovascular disease (21%) (Grasselli et al., 2020).

T343

55477-55665

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

55666-55824

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

55825-55906

Sentence

denotes

Obesity has also emerged as an important factor in determining COVID-19 severity.

T346

55907-56175

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

56176-56318

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

56319-56531

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

56532-56764

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

56765-56935

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

56936-57404

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

57405-57794

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

57795-58020

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

58021-58208

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

58209-58406

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

58407-58546

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

58547-58888

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

58889-59061

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

59062-59355

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

59356-59760

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

59761-60316

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

60318-60366

Sentence

denotes

ACE2 and the Inflammatory Response to SARS-CoV-2

T363

60367-60533

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

60534-60693

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

60694-61009

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

61010-61164

Sentence

denotes

Moreover, cardiovascular involvement has been observed, occasionally leading to myocardial injury and sudden death after weeks from the initial infection.

T367

61165-61336

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

61337-61485

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

61486-61624

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

61625-61723

Sentence

denotes

In 29% (n = 12) of patients ARDS occurred and in 10% invasive mechanical ventilation was required.

T371

61724-61849

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

61850-62165

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

62166-62284

Sentence

denotes

The molecular mechanisms involved in the cytokine storm and the role of ACE2 are currently still not fully understood.

T374

62285-62498

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

62499-62627

Sentence

denotes

This has been suggested to trigger the primary inflammatory response, which involves apoptosis and pyroptosis (Fu et al., 2020).

T376

62628-62878

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

62879-63125

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

63126-63571

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

63572-63679

Sentence

denotes

FIGURE 9 Possible role of ACE2 in the molecular mechanisms involved in the “cytokine storm” of SARS-CoV-2.

T380

63680-63901

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

63902-64040

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

64041-64228

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

64229-64354

Sentence

denotes

Another proposed mechanism that triggers the proinflammatory response to SARS-CoV-2 is ACE/ACE2 imbalance in the RAAS system.

T384

64355-64651

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

64652-64996

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

64997-65236

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

65237-65333

Sentence

denotes

The convergence on hyperactivation of NF-K B seems to be crucial in inducing the cytokine storm.

T388

65334-65530

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

65531-65619

Sentence

denotes

ACE2 shedding is an additional mechanism contributing to inflammation (Fu et al., 2020).

T390

65620-65873

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

65874-66089

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

66090-66406

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

66407-66627

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

66628-66745

Sentence

denotes

ACE2 shedding seems to be inducible by specific stimuli produced either by a viral infection or by the immune system.

T395

66746-66859

Sentence

denotes

Jia et al. (2009) confirmed ACE2 shedding in differentiated primary airway epithelial cells and Calu-3-cell line.

T396

66860-67068

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

67069-67204

Sentence

denotes

These findings suggest that an inflammatory response is likely to modulate ACE2 expression and shedding, also during a viral infection.

T398

67205-67389

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

67390-67549

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

67550-67711

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

67712-68368

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

68370-68398

Sentence

denotes

ACE2 as a Therapeutic Target

T403

68399-68487

Sentence

denotes

As evidence builds up, ACE2 rapidly emerged as a specific target for COVID-19 treatment.

T404

68488-68792

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

68793-68846

Sentence

denotes

Potential therapeutic approaches include (Figure 10):

T406

68847-68958

Sentence

denotes

FIGURE 10 Schematic representation of the potential therapeutic approaches to address ACE2 mediated infection.

T407

68959-69163

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

69164-69166

Sentence

denotes

1.

T409

69168-69182

Sentence

denotes

ACE2 blockers;

T410

69183-69185

Sentence

denotes

2.

T411

69187-69220

Sentence

denotes

Exogenous administration of ACE2.

T412

69222-69235

Sentence

denotes

ACE2 Blockers

T413

69236-69486

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

69487-69675

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

69676-70003

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

70004-70202

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

70203-70499

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

70501-70533

Sentence

denotes

Exogenous Administration of ACE2

T419

70534-70942

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

70943-71122

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

71123-71327

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

71328-71683

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

71684-71890

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

71891-72052

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

72053-72259

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

72260-72489

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

72490-72722

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

72724-72747

Sentence

denotes

ACE2 and Other Diseases

T429

72748-72802

Sentence

denotes

ACE2 is a multiform protein (Feng et al., 2010, 2011).

T430

72803-72993

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

72994-73106

Sentence

denotes

Alterations in ACE2 have been demonstrated in Hartnup’s disease due to a disturbance in amino acids homeostasis.

T432

73107-73224

Sentence

denotes

Indeed, ACE2 has been proposed to modulate amino acid transport in bowel and gut microbiome (Hashimoto et al., 2012).

T433

73225-73322

Sentence

denotes

Moreover, ACE2 participates in the regulation of metabolism, particularly of glucose homeostasis.

T434

73323-73430

Sentence

denotes

In the pancreas, activation of the ACE2-Ang1-7-MasR pathway improves insulin secretion (Yuan et al., 2013).

T435

73431-73617

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

73618-73763

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

73764-73847

Sentence

denotes

There is also increasing evidence that the RAAS system may be implicated in cancer.

T438

73848-74048

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

74049-74258

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

74259-74425

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

74426-74699

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

74700-74906

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

74907-75024

Sentence

denotes

A thorough bioinformatics analysis of the TCGA dataset on several kinds of cancer (Chai et al., 2020) has shown that:

T444

75025-75093

Sentence

denotes

(1) Mutation and amplification of ACE2 gene are frequent in cancer.

T445

75094-75199

Sentence

denotes

Yet, hot-spot mutation sites were never observed, as ACE2 mutations were distributed across all 18 exons.

T446

75200-75456

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

75457-75544

Sentence

denotes

Interestingly, ACE2 transcription was unchanged in lung squamous cell carcinoma (LUSC).

T448

75545-75689

Sentence

denotes

(3) ACE2 transcription was downregulated in three tumors: testicular germ cell (TGCT), thyroid carcinoma (THCA), and kidney chromophobe (KICH).

T449

75690-75845

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

75846-75950

Sentence

denotes

(5) In most cases, changes of ACE2 transcription strongly correlated with methylation in ACE2 promoter.

T451

75951-76081

Sentence

denotes

More specifically, decreased methylation levels correlated with upregulation, whereas increased methylation led to downregulation.

T452

76082-76169

Sentence

denotes

(6) No prognostic role of ACE2 expression on patient’s survival could be demonstrated.

T453

76170-76290

Sentence

denotes

These data confirm the remarkable role of methylation in determining the ACE2 expression, as described in paragraph 2.2.

T454

76291-76636

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

76638-76648

Sentence

denotes

Conclusion

T456

76649-76772

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

76773-76887

Sentence

denotes

However, this macromolecule has soon showed peculiar functional properties and has attracted increasing attention.

T458

76888-77139

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

77140-77338

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

77339-77549

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

77550-77783

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

77784-78082

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

78083-78289

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

78291-78311

Sentence

denotes

Author Contributions

T465

78312-78414

Sentence

denotes

FS, GR, SS, LB, BS, and RB wrote the first draft of the manuscript and prepared the table and figures.

T466

78415-78450

Sentence

denotes

RZ revised and integrated the text.

T467

78451-78510

Sentence

denotes

All authors decided upon the general outline of the review.

T468

78512-78532

Sentence

denotes

Conflict of Interest

T469

78533-78705

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.

T470

78707-78715

Sentence

denotes

Funding.

T471

78716-78799

Sentence

denotes

This work is supported by the PRA 2020-22 to GR, granted by the University of Pisa.

T472

78800-78854

Sentence

denotes

1 https://www.preprints.org/manuscript/202003.0295/v1

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