ACE for all – a molecular perspective

Angiotensin-I converting enzyme (ACE, EC 3.4.15.1) is a zinc dependent dipeptidyl carboxypeptidase with an essential role in mammalian blood pressure regulation as part of the renin-angiotensin aldosterone system (RAAS). As such, it has long been targeted in the treatment of hypertension through the use of ACE inhibitors. Although ACE has been studied since the 1950s, only recently have the full range of functions of this enzyme begun to truly be appreciated. ACE homologues have been found in a host of other organisms, and are now known to be conserved in insects. Insect ACE homologues typically share over 30 % amino acid sequence identity with human ACE. Given that insects lack a mammalian type circulatory system, they must have crucial roles in other physiological processes. The first ACE crystal structures were reported during the last decade and have enabled these enzymes to be studied from an entirely different perspective. Here we review many of these key developments and the implications that they have had on our understanding of the diverse functions of these enzymes. Specifically, we consider how structural information is being used in the design of a new generation of ACE inhibitors with increased specificity, and how the structures of ACE homologues are related to their functions. The Anopheles gambiae genome is predicted to code for ten ACE homologues, more than any genome studied so far. We have modelled the active sites of some of these as yet uncharacterised enzymes to try and infer more about their potential roles at the molecular level.     

Interaction with angiotensin-converting enzyme-encoding gene in female infertility: Insertion and deletion polymorphism studies

Angiotensin-converting enzyme (ACE), a key enzyme in the renin– angiotensin–aldosterone system, converts angiotensin I to angiotensin II. Ethnic origin should be carefully considered in studies pertaining to ACE I/D genotype and disease etiology. This study was evaluated between the ACE gene I/D polymorphism and female infertility in the Saudi population. Out of a A total of 300 women who participated in this study genomic DNA samples from the 150 infertile and 150 fertile women’s were isolated who has participated in this study. Genomic DNA was isolated using an Invitrogen kit according to the manufacturer’s protocol, and D allele specific primers were used for amplification by polymerase chain reaction. Electrophoresis was carried out on a 2% agarose gel. The mean age and BMI of the cases and controls were similar (p > 0.05), and a significant association was noted between the family history and female infertility (p = 0.0001). The D allele (OR: 1.67 [95% CI: 1.18–2.35], p = 0.003), DD genotype (OR: 2.46 [95% CI: 1.20–5.02], p = 0.01) and dominant model (OR: 1.97 [95% CI: 1.00–3.88], p = 0.04) were significantly associated with female infertility or fertility. The results of this study show that the ACE polymorphism plays an important role in female infertility in the Saudi population.     

The molybdate-binding protein (ModA) of the plant pathogen Xanthomonas axonopodis pv. citri

The modABC operon of phytopathogen Xanthomonas axonopodis pv. citri (X. citri) encodes a putative ABC transporter involved in the uptake of the molybdate and tungstate anions. Sequence analyses showed high similarity values of ModA orthologs found in X. campestris pv. campestris (X. campestris) and Escherichia coli. The X. citri modA gene was cloned in pET28a and the recombinant protein, expressed in the E. coli BL21 (DE3) strain, purified by immobilized metal affinity chromatography. The purified protein remained soluble and specifically bound molybdate and tungstate with K(d) 0.29+/-0.12 microM and 0.58+/-0.14 microM, respectively. Additionally binding of molybdate drastically enhanced the thermal stability of the recombinant ModA as compared to the apoprotein. This is the first characterization of a ModA ortholog expressed by a phytopathogen and represents an important tool for functional, biochemical and structural analyses of molybdate transport in Xanthomonas species.     

ACE I/D polymorphism in Alzheimer’s disease

Angiotensin-converting enzyme (ACE) has been reported to show altered activity in patients with neurological diseases. The recent studies found that a 287 bp insertion/deletion (I/D) polymorphism of the ACE gene may be associated with susceptibility to Alzheimer’s disease (AD) but the results have been heterogenous between studies in Europe. In the present study we examined for the first time the association of ACE I/D polymorphism along with APOE genotype in 70 sporadic AD and 126 control subjects in Slovak Caucasians (Central Europe). An increased risk for AD was observed in subjects with at least one APOE*E4 allele (OR=3.99, 95% CI=1.97–8.08). No significant differences for the genotype distribution or the allele frequency were revealed comparing controls and patients for ACE gene. Gene-gene interaction analysis showed increase of the risk to develop AD in subjects carrying both the ACE DD genotype and the APOE*E4 allele (OR=10.32, 95% C.I. 2.67–39.81).     

The isolation of angiotensin-converting enzyme cDNA

Angiotensin-converting enzyme (ACE) is an Zn(II)-containing dipeptidyl carboxypeptidase that converts angiotensin I to the potent vasoconstrictor, angiotensin II. Using oligonucleotide probes based on the amino acid sequence of mouse kidney ACE, cDNA encoding this protein has been isolated. One cDNA, ACE.31, encodes the N-terminal 332 amino acids of mouse ACE, a portion of the protein containing a putative 34-amino acid leader sequence and the N terminus of the mature protein. Northern analyses with cloned ACE cDNA revealed that both mouse kidney and lung express two ACE mRNAs, one of 4900 and another of 4150 bases. Southern analysis suggests that cDNA ACE.31 is the product of a single gene, and thus these data add evidence to the hypothesis that the converting enzymes produced by epithelial and endothelial cells are identical.     

Biochemical and structural insights into intramembrane metalloprotease mechanisms

Intramembrane metalloproteases are nearly ubiquitous in living organisms and they function in diverse processes ranging from cholesterol homeostasis and the unfolded protein response in humans to sporulation, stress responses, and virulence of bacteria. Understanding how these enzymes function in membranes is a challenge of fundamental interest with potential applications if modulators can be devised. Progress is described toward a mechanistic understanding, based primarily on molecular genetic and biochemical studies of human S2P and bacterial SpoIVFB and RseP, and on the structure of the membrane domain of an archaeal enzyme. Conserved features of the enzymes appear to include transmembrane helices and loops around the active site zinc ion, which may be near the membrane surface. Extramembrane domains such as PDZ (PSD-95, DLG, ZO-1) or CBS (cystathionine-β-synthase) domains govern substrate access to the active site, but several different mechanisms of access and cleavage site selection can be envisioned, which might differ depending on the substrate and the enzyme. More work is needed to distinguish between these mechanisms, both for enzymes that have been relatively well-studied, and for enzymes lacking PDZ and CBS domains, which have not been studied. This article is part of a Special Issue entitled: Intramembrane Proteases.     

Purification of angiotensin I converting enzyme from pig lung using concanavalin-A sepharose chromatography

Angiotensin I converting enzyme (ACE) plays a major role in blood pressure regulation, catalyzing the conversion of angiotensin I to the vasoconstrictor angiotensin II. In this report we describe a two-step affinity chromatography method for preparative purification of ACE from pig lung using Concanavalin-A Sepharose 4B and affinity chromatography on Lisinopril Sepharose 6B. The same purification scheme was used to obtain Cobalt-ACE, where zinc ion located at the active site is replaced by cobalt. Cobalt-ACE visible spectrum shows a characteristic broad peak from 500 to 600 nm. The shape and maximum absorptivity of this peak changes in presence of ACE inhibitors that bind at the catalytic site.     

High-Resolution Crystal Structures of Drosophila melanogaster Angiotensin-Converting Enzyme in Complex with Novel Inhibitors and Antihypertensive Drugs

Angiotensin I-converting enzyme (ACE), one of the central components of the renin-angiotensin system, is a key therapeutic target for the treatment of hypertension and cardiovascular disorders. Human somatic ACE (sACE) has two homologous domains (N and C). The N- and C-domain catalytic sites have different activities toward various substrates. Moreover, some of the undesirable side effects of the currently available and widely used ACE inhibitors may arise from their targeting both domains leading to defects in other pathways. In addition, structural studies have shown that although both these domains have much in common at the inhibitor binding site, there are significant differences and these are greater at the peptide binding sites than regions distal to the active site. As a model system, we have used an ACE homologue from Drosophila melanogaster (AnCE, a single domain protein with ACE activity) to study ACE inhibitor binding. In an extensive study, we present high-resolution structures for native AnCE and in complex with six known antihypertensive drugs, a novel C-domain sACE specific inhibitor, lisW-S, and two sACE domain-specific phosphinic peptidyl inhibitors, RXPA380 and RXP407 (i.e., nine structures). These structures show detailed binding features of the inhibitors and highlight subtle changes in the orientation of side chains at different binding pockets in the active site in comparison with the active site of N- and C-domains of sACE. This study provides information about the structure-activity relationships that could be utilized for designing new inhibitors with improved domain selectivity for sACE.     

New potent inhibitors of angiotensin converting enzyme

Using an earlier model of the favoured orientation of binding functions of angiotensin converting enzyme (ACE) inhibitors, it has been possible to postulate a new, 7,6-bicyclic system, based on hexahydropyridazine, which might be expected to have high potency. Some members of this system which have been synthesised have been shown to be very active ACE inhibitors, in vitro and in vivo.     

Angiotensin Converting Enzyme-2 (ACE2) and its Possible Roles in Hypertension, Diabetes and Cardiac Function

Angiotensin converting enzyme-2 (ACE2) is a recently described homologue of the vasoactive peptidase, angiotensin converting enzyme (ACE). Like ACE, ACE2 is an integral (type I) membrane zinc metallopeptidase, which exists as an ectoenzyme. ACE2 is less widely distributed than ACE in the body, being expressed at highest concentrations in the heart, kidney and testis. ACE2 also differs from ACE in its substrate specificity, functioning exclusively as a carboxypeptidase rather than a peptidyl dipeptidase. A key role for ACE2 appears to be emerging in the conversion of angiotensin II to angiotensin (1–7), allowing it to act as a counter-balance to the actions of ACE. ACE2 has been localised to the endothelial and epithelial cells of the heart and kidney where it may have a role at the cell surface in hydrolysing bioactive peptides such as angiotensin II present in the circulation. A role for ACE2 in the metabolism of other biologically active peptides also needs to be considered. ACE2 also serendipitously appears to act as a receptor for the severe acute respiratory syndrome (SARS) coronavirus. Studies using ace2 ^-/- mice, and other emerging studies in vivo and in vitro, have revealed that ACE2 has important functions in cardiac regulation and diabetes. Together with its role as a SARS receptor, ACE2 is therefore likely to be an important therapeutic target in a diverse range of disease states.     

New insights and perspectives on intrarenal renin-angiotensin system: focus on intracrine/intracellular angiotensin II

Although renin, the rate-limiting enzyme of the renin-angiotensin system (RAS), was first discovered by Robert Tigerstedt and Bergman more than a century ago, the research on the RAS still remains stronger than ever. The RAS, once considered to be an endocrine system, is now widely recognized as dual (circulating and local/tissue) or multiple hormonal systems (endocrine, paracrine and intracrine). In addition to the classical renin/angiotensin I-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II receptor (AT₁/AT₂) axis, the prorenin/(Pro)renin receptor (PRR)/MAP kinase axis, the ACE2/Ang (1-7)/Mas receptor axis, and the Ang IV/AT₄/insulin-regulated aminopeptidase (IRAP) axis have recently been discovered. Furthermore, the roles of the evolving RAS have been extended far beyond blood pressure control, aldosterone synthesis, and body fluid and electrolyte homeostasis. Indeed, novel actions and underlying signaling mechanisms for each member of the RAS in physiology and diseases are continuously uncovered. However, many challenges still remain in the RAS research field despite of more than one century's research effort. It is expected that the research on the expanded RAS will continue to play a prominent role in cardiovascular, renal and hypertension research. The purpose of this article is to review the progress recently being made in the RAS research, with special emphasis on the local RAS in the kidney and the newly discovered prorenin/PRR/MAP kinase axis, the ACE2/Ang (1-7)/Mas receptor axis, the Ang IV/AT₄/IRAP axis, and intracrine/intracellular Ang II. The improved knowledge of the expanded RAS will help us better understand how the classical renin/ACE/Ang II/AT₁ receptor axis, extracellular and/or intracellular origin, interacts with other novel RAS axes to regulate blood pressure and cardiovascular and kidney function in both physiological and diseased states.     

Crystal structure of plant asparaginase

In plants, specialized enzymes are required to catalyze the release of ammonia from asparagine, which is the main nitrogen-relocation molecule in these organisms. In addition, K+-independent plant asparaginases are also active in splitting the aberrant isoaspartyl peptide bonds, which makes these proteins important for seed viability and germination. Here, we present the crystal structure of potassium-independent L-asparaginase from yellow lupine (LlA) and confirm the classification of this group of enzymes in the family of Ntn-hydrolases. The alpha- and beta-subunits that form the mature (alphabeta)2 enzyme arise from autoproteolytic cleavage of two copies of a precursor protein. In common with other Ntn-hydrolases, the (alphabeta) heterodimer has a sandwich-like fold with two beta-sheets flanked by two layers of alpha-helices (alphabetabetaalpha). The nucleophilic Thr193 residue, which is liberated in the autocatalytic event at the N terminus of subunit beta, is part of an active site that is similar to that observed in a homologous bacterial enzyme. An unusual sodium-binding loop of the bacterial protein, necessary for proper positioning of all components of the active site, shows strictly conserved conformation and metal coordination in the plant enzyme. A chloride anion complexed in the LlA structure marks the position of the alpha-carboxylate group of the L-aspartyl substrate/product moiety. Detailed analysis of the active site suggests why the plant enzyme hydrolyzes asparagine and its beta-peptides but is inactive towards substrates accepted by similar Ntn-hydrolases, such as taspase1, an enzyme implicated in some human leukemias. Structural comparisons of LlA and taspase1 provide interesting insights into the role of small inorganic ions in the latter enzyme.     

The levels of renin-angiotensin related components are modified in the hippocampus of rats submitted to pilocarpine model of epilepsy

We previously showed that patients with temporal lobe epilepsy (TLE) present an increased expression of angiotensin II (AngII) AT1 and AT2 receptors in the hippocampus, supporting the idea of an upregulation of renin-angiotensin system (RAS) in this disease. This study aimed to verify the relationship between the RAS and TLE during epileptogenesis. Levels of the peptides angiotensin I (AngI), angiotensin II (AngII) and angiotensin 1-7 (Ang 1-7), were detected by HPLC assay. Angiotensin AT1 and AT2 receptors, Mas mRNA receptors and angiotensin converting enzyme (ACE), tonin and neutral endopeptidase (NEP) mRNA were also quantified at the hippocampus of Wistar rats by real time PCR, during acute (n=10), silent (n=10) and chronic (n=10) phases of pilocarpine-induced epilepsy. We observed an increased peptide level of Ang1-7 into acute and silent phases, decreasing importantly (p≤0.05) in the chronic phase, suggesting that AngI may be converted into Ang 1-7 by NEP, which is present in high levels in these periods. Our results also showed increased peptide level of AngII in the chronic phase of this model. In contraposition, the ACE expression is reduced in all periods. These data suggest that angiotensinogen or AngI may be cleaved to AngII by tonin, which showed increased expression in all phases. We found changes in AT1, AT2 and Mas mRNA receptors levels suggesting that Ang1-7 could act at Mas receptor during the silent period. Herein, we demonstrated for the first time, changes in angiotensin-related peptides, their receptors as well as the releasing enzymes in the hippocampus of rats during pilocarpine-induced epilepsy.     

In search of synaptosomal Na+, K+-ATPase regulators

The arrival of the nerve impulse to the nerve endings leads to a series of events involving the entry of sodium and the exit of potassium. Restoration of ionic equilibria of sodium and potassium through the membrane is carried out by the sodium/potassium pump, that is the enzyme Na⁺,K⁺-ATPase. This is a particle-bound enzyme that concentrates in the nerve ending or synaptosomal membranes. The activity of Na⁺,K⁺-ATPase is essential for the maintenance of numerous reactions, as demonstrated in the isolated synaptosomes. This lends interest to the knowledge of the possible regulatory mechanisms of Na⁺,K⁺-ATPase activity in the synaptic region. The aim of this review is to summarize the results obtained in the author's laboratory, that refer to the effect of neurotransmitters and endogenous substances on Na⁺,K⁺-ATPase activity. Mention is also made of results in the field obtained in other laboratories. Evidence showing that brain Na⁺,K⁺-ATPase activity may be modified by certain neurotransmitters and insulin have been presented. The type of change produced by noradrenaline, dopamine, and serotonin on synaptosomal membrane Na⁺,K⁺-ATPase was found to depend on the presence or absence of a soluble brain fraction. The soluble brain fraction itself was able to stimulate or inhibit the enzyme, an effect that was dependent in turn on the time elapsed between preparation and use of the fraction. The filtration of soluble brain fraction through Sephadex G-50 allowed the separation of two active subfractions: peaks I and II. Peak I increased Na⁺,K⁺- and Mg²⁺-ATPases, and peak II inhibited Na⁺,K⁺-ATPase. Other membrane enzymes such as acetylcholinesterase and 5′-nucleotidase were unchanged by peaks I or II. In normotensive anesthetized rats, water and sodium excretion were not modified by peak I but were increased by peak II, thus resembling ouabain effects.³H-ouabain binding was unchanged by peak I but decreased by peak II in some areas of the CNS assayed by quantitative autoradiography and in synaptosomal membranes assayed by a filtration technique. The effects of peak I and II on Na⁺,K⁺-ATPase were reversed by catecholamines. The extent of Na⁺,K⁺-ATPase inhibition by peak II was dependent on K⁺ concentration, thus suggesting an interference with the K⁺ site of the enzyme. Peak II was able to induce the release of neurotransmitter stored in the synaptic vesicles in a way similar to ouabain. Taking into account that peak II inhibits only Na⁺,K⁺-ATPase, increases diuresis and natriuresis, blocks high affinity³H-ouabain binding, and induces neurotransmitter release, it is suggested that it contains an ouabain-like substance.     

Stress-Induced Changes in the Activity of Angiotensin-Converting Enzyme in Structures of the Hypothalamo-Hypophyseal-Adrenocortical System of Adrenalectomized Rats

We studied the effects of stress induced by different influences (immobilization and compulsory swimming) on the activity of angiotensin-converting enzyme (ACE, an enzyme of the proteolytic conversion of angiotensin II) in structures of the hypothalamo-hypophyseal-adrenocortical system (HHAS) of unilaterally adrenalectomized (hemiadrenalectomized, HAE) rats. The pattern of stress-induced changes in the activity of ACE depended on the type of stress; rigid daily immobilization of rats for 1 h resulted in more significant shifts. Post-immobilization stress changes in the activity of ACE in the HHAS structures of HAE rats (with a lower basal activity of the endogenous angiotensin system in their hypothalamus) differed from the stress-induced reaction of the enzyme in intact rats. In HAE rats, we also observed inhibition of the activity of a glucocorticoid link of the stress system, as compared with that in intact animals. An inhibitor of ACE, captopril, and a stable analog of leucine-enkephalin, dalargin, when injected before stressing, were capable of decreasing the stress-induced ACE reaction in the hypothalamus and adenohypophysis and of limiting manifestations of the reaction of the adrenals to immobilization. This is interpreted as a proof of the involvement of the components of the angiotensin and enkephalin systems in the formation of the HHAS system to stressing of HAE rats.     

Identification and cloning of a gene encoding tannase (tannin acylhydrolase) from Lactobacillus plantarum ATCC 14917(T)

The gene tanLpl, encoding a novel tannase enzyme (TanLpl), has been cloned from Lactobacillus plantarum ATCC 14917(T). This is the first report of a tannase gene cloned from a bacterial source other than from Staphylococcus lugdunensis, which has been reported elsewhere. The open reading frame of tanLpl, spanning 1410 bp, encoded a 469-amino-acid protein that showed 28.8% identity to the tannase of S. lugdunensis with several commonly conserved sequences. These sequences could not be found in putative tannases reported for other bacteria and fungi. TanLpl was expressed in Escherichia coli DH5alpha from a pGEM-T expression system and purified. SDS-PAGE analysis indicated that purified TanLpl was a monomer polypeptide of approximately 50 kDa in size. Subsequent enzymatic characterization revealed that TanLpl was most active in an alkaline pH range at 40 degrees C, which was quite different from that observed for a fungal tannase of Aspergillus oryzae. In addition, the Michaelis-Menten constant of TanLpl was markedly lower than that of A. oryzae tannase. The evidence suggests that TanLpl should be classified into a novel family of tannases.     

Identifying the regulatory element for human angiotensin-converting enzyme 2 (ACE2) expression in human cardiofibroblasts

Angiotensin-converting enzyme 2 (ACE2) has been proposed as a potential target for cardioprotection in regulating cardiovascular functions, owing to its key role in the formation of the vasoprotective peptides angiotensin-(1-7) from angiotensin II (Ang II). The regulatory mechanism of ace2 expression, however, remains to be explored. In this study, we investigated the regulatory element within the upstream of ace2. The human ace2 promoter region, from position −2069 to +20, was cloned and a series of upstream deletion mutants were constructed and cloned into a luciferase reporter vector. The reporter luciferase activity was analyzed by transient transfection of the constructs into human cardiofibroblasts (HCFs) and an activating domain was identified in the −516/−481 region. Deletion or reversal of this domain within ace2 resulted in a significant decrease in promoter activity. The nuclear proteins isolated from the HCFs formed a DNA-protein complex with double stranded oligonucleotides of the −516/−481 domain, as detected by electrophoretic mobility shift assay. Site-directed mutagenesis of this region identified a putative protein binding domain and a potential binding site, ATTTGGA, homologous to that of an Ikaros binding domain. This regulatory element was responsible for Ang II stimulation via the Ang II-Ang II type-1 receptor (AT1R) signaling pathway, but was not responsible for pro-inflammatory cytokines TGF-β1 and TNF-α. Our results suggest that the nucleotide sequences −516/−481 of human ace2 may be a binding domain for an as yet unidentified regulatory factor(s) that regulates ace2 expression and is associated with Ang II stimulation.