Testis Angiotensin-converting Enzyme Research Articles

Testicular ACE regulates sperm metabolism and fertilization through the transcription factor PPARγ

Testis angiotensin-converting enzyme (tACE) plays a critical role in male fertility, but the mechanism is unknown. By using ACE C-domain KO (CKO) mice which lack tACE activity, we found that ATP in CKO sperm was 9.4-fold lower than WT sperm. Similarly, an ACE inhibitor (ACEi) reduced ATP production in mouse sperm by 72%. Metabolic profiling showed that tACE inactivation severely affects oxidative metabolism with decreases in several Krebs cycle intermediates including citric acid, cis-aconitic acid, NAD, α-ketoglutaric acid, succinate, and L-malic acid. We found that sperms lacking tACE activity displayed lower levels of oxidative enzymes (CISY, ODO1, MDHM, QCR2, SDHA, FUMH, CPT2, and ATPA) leading to a decreased mitochondrial respiration rate. The reduced energy production in CKO sperms leads to defects in their physiological functions including motility, acrosine activity, and fertilization in vitro and in vivo. Male mice treated with ACEi show severe impairment in reproductive capacity when mated with female mice. In contrast, an angiotensin II receptor blocker (ARB) had no effect. CKO sperms express significantly less peroxisome proliferators-activated receptor gamma (PPARγ) transcription factor, and its blockade eliminates the functional differences between CKO and WT sperms, indicating PPARγ might mediate the effects of tACE on sperm metabolism. Finally, in a cohort of human volunteers, in vitro treatment with the ramipril or a PPARγ inhibitor reduced ATP production in human sperm and hence its motility and acrosine activity. These findings may have clinical significance since millions of people take ACEi daily, including men who are reproductively active.

AN IN VITRO STUDY OF CINNAMOMUM ZEYLANICUM AS NATURAL INHIBITOR OF ANGIOTENSIN-CONVERTING ENZYME (ACE) ON SHEEP (OVIS ARIES) TISSUES

ABSTRACTObjective: The present study was aimed to find the angiotensin-converting enzyme (ACE) inhibitory activity using the methanolic extract ofCinnamomum zeylanicum (as a natural inhibitor) on sheep tissues as the enzyme source.Methods: Hippuryl-histidyl-leucine (HHL) as a substrate, tissue ACE activity was measured spectrophotometrically at 228 nm. For an incubationperiod of 30 minutes at 37°C, the linearity of ACE activity of kidney, lung, and testis enzyme was established. A known medicinal plant C. zeylanicumwas used as natural inhibitor of ACE. In this enzyme assay, inhibitory effect of methanolic extract of C. zeylanicum on kidney, lung and testicular ACEwas determined. ACE activity was confirmed by captopril, a standard inhibitor of ACE.Results: In the presence of a methanolic extract of C. zeylanicum (10:1), ACE activity was determined and this has inhibited ACE activity verysignificantly. C. zeylanicum leaves extract has reduced sheep kidney, lung, and testis ACE activity by 70.06%, 12.63%, and 20.23%, respectively.Conclusion: Significant inhibition was observed in the kidney ACE than in lung and testis ACE activity. This can propose that there may be a possiblerole in controlling blood pressure or reduction in cardiovascular diseases. Some plants with the great medicinal property may be considered aspromising sources of natural inhibitors of ACE for medicine and commercial uses. This comprehensive study may show numerous beneficial effects asa potential therapeutic agent for lowering blood pressure.Keywords: Angiotensin-converting enzyme, Natural angiotensin-converting enzyme inhibitor, Kinetic assay, Hippuryl-histidyl-leucine, Cinnamomumzeylanicum, Cardiovascular diseases.

Identification of Angiotensin Converting Enzyme Inhibitor: An In Silico Perspective

Angiotensin Converting Enzyme (ACE) regulates blood pressure and electrolyte balance by converting angiotensin I into angiotensin II, which acts as vasoconstrictor and aldosterone-stimulating peptide. ACE is also known to inactivate vasodilators, bradykinin and angiotensin 1–7 peptide. Thus, the down-regulation of ACE activity would be very beneficial in various cardiovascular diseases. In present in silico approach, virtual screening was performed to identify possible novel inhibitors of testis ACE (tACE) from ZINC database using Dockblaster. All screened compounds were filtered through Lipinski’s rules and other properties. PyRx tool was then implemented to dock selected compounds. Finally, ligand ZINC48251687 re-docked using Autodock 4.2 with His 383, His 387 and Glu 411 as flexible residues of tACE. Molecular Dynamics simulation was then carried out to investigate the binding stability of the screened ligand in a dynamic environment. It has been observed that tACE-ligand complex was quite stable over the entire simulation run. We found that ligand was stabilized by strong hydrogen bonding interactions with Ala 354, Ala 356, Tyr 523 and Zn2+ of tACE. Thus, ZINC48251687 could be used as starting point for the development of new non-peptidic inhibitor of tACE and for designing drug against cardiovascular and related renal diseases.

Catalytic Mechanism of Angiotensin-Converting Enzyme and Effects of the Chloride Ion

The angiotensin-converting enzyme (ACE) exhibits critical functions in the conversion of angiotensin I to angiotensin II and the degradation of bradykinin and other vasoactive peptides. As a result, the ACE inhibition has become a promising approach in the treatment of hypertension, heart failure, and diabetic nephropathy. Extending our recent molecular dynamics simulation of the testis ACE in complex with a bona fide substrate molecule, hippuryl-histidyl-leucine, we presented here a detailed investigation of the hydrolytic process and possible influences of the chloride ion on the reaction using a combined quantum mechanical and molecule mechanical method. Similar to carboxypeptidase A and thermolysin, the promoted water mechanism is established for the catalysis of ACE. The E384 residue was found to have the dual function of a general base for activating the water nucleophile and a general acid for facilitating the cleavage of amide C-N bond. Consistent with experimental observations, the chloride ion at the second binding position is found to accelerate the reaction rate presumably due to the long-range electrostatic interactions but has little influence on the overall substrate binding characteristics.

Inhibitor and Substrate Binding by Angiotensin-Converting Enzyme: Quantum Mechanical/Molecular Mechanical Molecular Dynamics Studies

Angiotensin-converting enzyme (ACE) is an important zinc-dependent hydrolase responsible for converting the inactive angiotensin I to the vasoconstrictor angiotensin II and for inactivating the vasodilator bradykinin. However, the substrate binding mode of ACE has not been completely understood. In this work, we propose a model for an ACE Michaelis complex based on two known X-ray structures of inhibitor-enzyme complexes. Specifically, the human testis angiotensin-converting enzyme (tACE) complexed with two clinic drugs were first investigated using a combined quantum mechanical and molecular mechanical (QM/MM) approach. The structural parameters obtained from the 550 ps molecular dynamics simulations are in excellent agreement with the X-ray structures, validating the QM/MM approach. Based on these structures, a model for the Michaelis complex was proposed and simulated using the same computational protocol. Implications to ACE catalysis are discussed.

Characterization of domain-selective inhibitor binding in angiotensin-converting enzyme using a novel derivative of lisinopril

Human ACE (angiotensin-converting enzyme) (EC 3.4.15.1) is an important drug target because of its role in the regulation of blood pressure via the renin-angiotensin-aldosterone system. Somatic ACE comprises two homologous domains, the differing substrate preferences of which present a new avenue for domain-selective inhibitor design. We have co-crystallized lisW-S, a C-domain-selective derivative of the drug lisinopril, with human testis ACE and determined a structure using X-ray crystallography to a resolution of 2.30 A (1 A=0.1 nm). In this structure, lisW-S is seen to have a similar binding mode to its parent compound lisinopril, but the P2' tryptophan moiety takes a different conformation to that seen in other inhibitors having a tryptophan residue in this position. We have examined further the domain-specific interactions of this inhibitor by mutating C-domain-specific active-site residues to their N domain equivalents, then assessing the effect of the mutation on inhibition by lisW-S using a fluorescence-based assay. Kinetics analysis shows a 258-fold domain-selectivity that is largely due to the co-operative effect of C-domain-specific residues in the S2' subsite. The high affinity and selectivity of this inhibitor make it a good lead candidate for cardiovascular drug development.

Investigating the Domain Specificity of Phosphinic Inhibitors RXPA380 and RXP407 in Angiotensin-Converting Enzyme

Human somatic angiotensin-converting enzyme (ACE) is a membrane-bound dipeptidyl carboxypeptidase that contains two extracellular domains (N and C). Although highly homologous, they exhibit different substrate and inhibition profiles. The phosphinic inhibitors RXPA380 and RXP407 are highly selective for the C- and N-domains, respectively. A number of residues, implicated by structural data, are likely to contribute to this selectivity. However, the extent to which these different interactions are responsible for domain selectivity is unclear. In this study, a series of C- and N-domain mutants containing conversions to corresponding domain residues were used to scrutinize the contribution of these residues to selective inhibitor binding. Enzyme kinetic analyses of the purified mutants indicated that the RXPA380 C-selectivity is particularly reliant on the interaction between the P2 substituent and Phe 391 (testis ACE numbering). Moreover, a C-domain mutant in which Phe 391 has been changed to a Tyr residue, in addition to containing an N-domain S2' pocket (S2'F/Y), displayed the greatest shift toward a more N-domain-like Ki. None of the single mutations within the N-domain caused a large shift in RXP407's affinity for these enzymes. However, the double mutant containing the Tyr 369 to Phe change as well as Arg 381 to Glu displayed a 100-fold decrease in binding affinity, confirming that the S2 pocket plays a major role in RXP407 selectivity. Taken together, these data advance our understanding regarding the molecular basis for the remarkable ACE domain selectivity exhibited by these inhibitors.

Probing the Basis of Domain-Dependent Inhibition Using Novel Ketone Inhibitors of Angiotensin-Converting Enzyme

Human angiotensin-converting enzyme (ACE) has two homologous domains, the N and C domains, with differing substrate preferences. X-ray crystal structures of the C and N domains complexed with various inhibitors have allowed identification of active site residues that might be important for the molecular basis of this selectivity. However, it is unclear to what extent the different residues contribute to substrate domain selectivity. Here, cocrystal structures of human testis ACE, equivalent to the C domain, have been determined with two novel C domain-selective ketomethylene inhibitors, (5 S)-5-[( N-benzoyl)amino]-4-oxo-6-phenylhexanoyl- l-tryptophan (kAW) and (5 S)-5-[( N-benzoyl)amino]-4-oxo-6-phenylhexanoyl- l-phenylalanine (kAF). The ketone groups of both inhibitors bind to the zinc ion as a hydrated geminal diolate, demonstrating the ability of the active site to catalyze the formation of the transition state. Moreover, active site residues involved in inhibitor binding have been mutated to their N domain counterparts, and the effect of the mutations on inhibitor binding has been determined. The C domain selectivity of these inhibitors was found to result from interactions between bulky hydrophobic side chain moieties and C domain-specific residues F391, V518, E376, and V380 (numbering of testis ACE). Mutation of these residues decreased the affinity for the inhibitors 4-20-fold. T282, V379, E403, D453, and S516 did not contribute individually to C domain-selective inhibitor binding. Further domain-selective inhibitor design should focus on increasing both the affinity and selectivity of the side chain moieties.

Development of Domain‐Selective Angiotensin I‐Converting Enzyme Inhibitors

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Homologous substitution of ACE C-domain regions with N-domain sequences: effect on processing, shedding, and catalytic properties

Angiotensin-converting enzyme (ACE) exists as two isoforms: somatic ACE (sACE), comprised of two homologous N and C domains, and testis ACE (tACE), comprised of the C domain only. The N and C domains are both active, but show differences in substrate and inhibitor specificity. While both isoforms are shed from the cell surface via a sheddase-mediated cleavage, tACE is shed much more efficiently than sACE. To delineate the regions of tACE that are important in catalytic activity, intracellular processing, and regulated ectodomain shedding, regions of the tACE sequence were replaced with the corresponding N-domain sequence. The resultant chimeras C1-163Ndom-ACE, C417-579Ndom-ACE, and C583-623Ndom-ACE were processed to the cell surface of transfected Chinese hamster ovary (CHO) cells, and were cleaved at the identical site as that of tACE. They also showed acquisition of N-domain-like catalytic properties. Homology modelling of the chimeric proteins revealed structural changes in regions required for tACE-specific catalytic activity. In contrast, C164-416Ndom-ACE and C191-214Ndom-ACE demonstrated defective intracellular processing and were neither enzymatically active nor shed. Therefore, critical elements within region D164-V416 and more specifically I191-T214 are required for the processing, cell-surface targeting, and enzyme activity of tACE, and cannot be substituted for by the homologous N-domain sequence.

Development of Domain‐Selective Angiotensin I‐Converting Enzyme Inhibitors

Somatic angiotensin-converting enzyme (ACE) is an essential component of the renin-angiotensin system and consequently plays a key role in blood pressure and electrolyte homeostasis. Thus, ACE inhibitors are widely used in the treatment of cardiovascular disease, causing a decrease in the production of angiotensin II and an increase in the circulating vasodilator bradykinin. The ectodomain of ACE consists of two parts (N and C domains), each bearing an active site that differs in substrate and inhibitor specificity. Advances in the elucidation of the functional roles of these two domains and an expanded view of the renin-angiotensin system underscore the need for the next generation of domain-selective inhibitors with improved pharmacologic profiles. Moreover, recent breakthroughs in determining the crystal structure of testis ACE (identical to the C domain) and its homologue ACE2 provide new mechanistic insights into the interactions of ACE inhibitors and substrates with active site pockets. This review summarizes the structural basis and recent synthetic chemistry approaches to the development of novel domain-selective inhibitors.

Structural details on the binding of antihypertensive drugs captopril and enalaprilat to human testicular angiotensin I-converting enzyme.

Angiotensin converting enzyme (ACE) plays a critical role in the circulating or endocrine renin-angiotensin system (RAS) as well as the local regulation that exists in tissues such as the myocardium and skeletal muscle. Here we report the high-resolution crystal structures of testis ACE (tACE) in complex with the first successfully designed ACE inhibitor captopril and enalaprilat, the Phe-Ala-Pro analogue. We have compared these structures with the recently reported structure of a tACE-lisinopril complex [Natesh et al. (2003) Nature 421, 551-554]. The analyses reveal that all three inhibitors make direct interactions with the catalytic Zn(2+) ion at the active site of the enzyme: the thiol group of captopril and the carboxylate group of enalaprilat and lisinopril. Subtle differences are also observed at other regions of the binding pocket. These are compared with N-domain models and discussed with reference to published biochemical data. The chloride coordination geometries of the three structures are discussed and compared with other ACE analogues. It is anticipated that the molecular details provided by these structures will be used to improve the binding and/or the design of new, more potent domain-specific inhibitors of ACE that could serve as new generation antihypertensive drugs.

Role of the N-terminal Catalytic Domain of Angiotensin-converting Enzyme Investigated by Targeted Inactivation in Mice

Angiotensin-converting enzyme (ACE) produces the vasoconstrictor angiotensin II. The ACE protein is composed of two homologous domains, each binding zinc and each independently catalytic. To assess the physiologic significance of the two ACE catalytic domains, we used gene targeting in mice to introduce two point mutations (H395K and H399K) that selectively inactivated the ACE N-terminal catalytic site. This modification does not affect C-terminal enzymatic activity or ACE protein expression. In addition, the testis ACE isozyme is not affected by the mutations. Analysis of homozygous mutant mice (termed ACE 7/7) showed normal plasma levels of angiotensin II but an elevation of plasma and urine N-acetyl-Ser-Asp-Lys-Pro, a peptide suggested to inhibit bone marrow maturation. Despite this, ACE 7/7 mice had blood pressure, renal function, and hematocrit that were indistinguishable from wild-type mice. We also studied compound heterozygous mice in which one ACE allele was null (no ACE expression) and the second allele encoded the mutations selectively inactivating the N-terminal catalytic domain. These mice produced approximately half the normal levels of ACE, with the ACE protein lacking N-terminal catalytic activity. Despite this, the mice have a phenotype indistinguishable from wild-type animals. This study shows that, in vivo, the presence of the C-terminal ACE catalytic domain is sufficient to maintain a functional renin-angiotensin system. It also strongly suggests that the anemia present in ACE null mice is not due to the accumulation of the peptide N-acetyl-Ser-Asp-Lys-Pro.

Seasonal changes in angiotensin converting enzyme activity in male and female frogs ( Rana esculenta)

Gonad, lung, kidney and serum angiotensin converting enzyme (ACE) activities were determined by specific substrate hydrolysis in male and female Rana esculenta over 1 year. Ovary ACE activity showed the highest values among the different tissues, with a significant peak (223±52 nmol min −1 mg protein −1) in late winter-early spring. Testis ACE activity followed a significant seasonal cycle, increasing from September to peak in April (2.5±0.8 nmol min −1 mg protein −1) and then decreased in the post-reproductive period. Lung and kidney ACE activities were not correlated with the annual reproductive cycle phases. In serum a peak of activity was present in the post-reproductive period both in male and female frogs. The present data show a correlation between ACE and the annual reproductive cycle of R. esculenta.

Deletion of the cytoplasmic domain increases basal shedding of angiotensin-converting enzyme

Ectodomain shedding generates soluble isoforms of cell-surface proteins, including angiotensin-converting enzyme (ACE). Increasing evidence suggests that the juxtamembrane stalk of ACE, where proteolytic cleavage-release occurs, is not the major site of sheddase recognition. The role of the cytoplasmic domain has not been completely defined. We deleted the cytoplasmic domain of human testis ACE and found that this truncation mutant (ACE-ΔCYT) was shed constitutively from the surface of transfected CHO-K1 cells. Phorbol ester treatment produced only a slight increase in shedding of ACE-ΔCYT, unlike the marked stimulation seen with wild-type ACE. However, for both wild-type ACE and ACE-ΔCYT, shedding was inhibited by the peptide hydroxamate TAPI and the major cleavage site was identical, indicating the involvement of similar or identical sheddases. Cytochalasin D markedly increased the basal shedding of wild-type ACE but had little effect on the shedding of ACE-ΔCYT. These data suggest that the cytoplasmic domain of ACE interacts with the actin cytoskeleton and that this interaction is a negative regulator of ectodomain shedding.

Structure-function discrimination of the N- and C- catalytic domains of human angiotensin-converting enzyme: implications for Cl- activation and peptide hydrolysis mechanisms.

Human somatic angiotensin I-converting enzyme (sACE) has two active sites present in two sequence homologous protein domains (ACE_N and ACE_C) possessing several biochemical features that differentiate the two active sites (i.e. chloride ion activation). Based on the recently solved X-ray structure of testis angiotensin-converting enzyme (tACE), the 3D structure of ACE_N was modeled. Electrostatic potential calculations reveal that the ACE_N binding groove is significantly more positively charged than the ACE_C, which provides a first rationalization for their functional discrimination. The chloride ion pore for Cl2 (one of the two chloride ions revealed in the X-ray structure of tACE) that connects the external solution with the inner part of the protein was identified on the basis of an extended network of water molecules. Comparison of ACE_C with the X-ray structure of the prokaryotic ClC Cl(-) channel from Salmonella enterica serovar typhimurium demonstrates a common molecular basis of anion selectivity. The critical role for Cl2 as an ionic switch is emphasized. Sequence and structural comparison between ACE_N and ACE_C and of other proteins of the gluzincin family highlights key residues that could be responsible for the peptide hydrolysis mechanism. Currently available mutational and substrate hydrolysis data for both domains are evaluated and are consistent with the predicted model.

Ace revisited: a new target for structure-based drug design.

Current-generation angiotensin-converting enzyme (ACE) inhibitors are widely used for cardiovascular diseases, including high blood pressure, heart failure, heart attack and kidney failure, and have combined annual sales in excess of US $6 billion. However, the use of these ACE inhibitors, which were developed in the late 1970s and early 1980s, is hampered by common side effects. Moreover, we now know that ACE actually consists of two parts (called the N- and C-domains) that have different functions. Therefore, the design of specific domain-selective ACE inhibitors is expected to produce next-generation drugs that might be safer and more effective. Here we discuss the structural features of current inhibitors and outline how next-generation ACE inhibitors could be designed by using the three-dimensional molecular structure of human testis ACE. The ACE structure provides a unique opportunity for rational drug design, based on a combination of in silico modelling using existing inhibitors as scaffolds and iterative lead optimization to drive the synthetic chemistry.

Deglycosylation, processing and crystallization of human testis angiotensin-converting enzyme.

Angiotensin I-converting enzyme (ACE) is a highly glycosylated type I integral membrane protein. A series of underglycosylated testicular ACE (tACE) glycoforms, lacking between one and five N-linked glycosylation sites, were used to assess the role of glycosylation in tACE processing, crystallization and enzyme activity. Whereas underglycosylated glycoforms showed differences in expression and processing, their kinetic parameters were similar to that of native tACE. N-glycosylation of Asn-72 or Asn-109 was necessary and sufficient for the production of enzymically active tACE but glycosylation of Asn-90 alone resulted in rapid intracellular degradation. All mutants showed similar levels of phorbol ester stimulation and were solubilized at the same juxtamembrane cleavage site as the native enzyme. Two mutants, tACEDelta36-g1234 and -g13, were successfully crystallized, diffracting to 2.8 and 3.0 A resolution respectively. Furthermore, a truncated, soluble tACE (tACEDelta36NJ), expressed in the presence of the glucosidase-I inhibitor N -butyldeoxynojirimycin, retained the activity of the native enzyme and yielded crystals belonging to the orthorhombic P2(1)2(1)2(1) space group (cell dimensions, a=56.47 A, b=84.90 A, c=133.99 A, alpha=90 degrees, beta=90 degrees and gamma=90 degrees ). These crystals diffracted to 2.0 A resolution. Thus underglycosylated human tACE mutants, lacking O-linked oligosaccharides and most N-linked oligosaccharides or with only simple N-linked oligosaccharides attached throughout the molecule, are suitable for X-ray diffraction studies.

Activity of testis angiotensin converting enzyme (ACE) in ejaculated human spermatozoa.

Testicular angiotensin converting enzyme (ACE) isozyme is likely to play important functional roles in male reproduction. Several studies have shown that ACE is released from human spermatozoa during capacitation and that ACE is associated with reduced sperm motility. Recently, we established an assay to detect testicular ACE activity in human spermatozoa. The purpose of this study was to determine if testicular ACE activity is related to sperm motility in human ejaculates. Semen samples were collected from 80 infertile patients. According to the semen characteristics, they were divided into four (WHO) categories. Enzyme activities of ACE in spermatozoa (testicular ACE) and seminal plasma (somatic ACE) were spectrophotometrically determined. Total testicular ACE activity in spermatozoa was measured by solubilization of spermatozoa with Triton X-100. Membrane testicular ACE activity was measured in a sperm : PBS suspension. Sperm concentration and sperm motility were 136.6 +/- 154.1 x 10(6)/mL and 58.6 +/- 23.4%, respectively (mean +/- SD). Enzyme activities of membrane testicular ACE, total testicular ACE and somatic ACE were 0.273 +/- 1.219 microU/10(6) spermatozoa, 0.35 +/- 1.34 microU/10(6) spermatozoa and 684.7 +/- 226.6 mU/mL, respectively. A negative correlation was observed between sperm motility and membrane testicular ACE activity (p < 0.05). Membrane testicular ACE activity in 44 normal semen samples was 0.04 +/- 0.02 microU/10(6) spermatozoa, whilst that in 36 abnormal semen samples was 0.24 +/- 0.42 microU/10(6) spermatozoa. There was a significant difference between these two groups (p < 0.01). Membrane testicular ACE in sperm samples from normozoospermic men was significantly lower than that from oligoasthenozoospermic men (p < 0.05). These findings suggest that testicular ACE is released from normal functional spermatozoa for them to have fertilizing ability.

Assay of testicular angiotensin-converting enzyme activity in human spermatozoa.

The testicular isozyme of angiotensin-converting enzyme (ACE) is associated with male fertility. Spermatozoa from mice lacking ACE showed defects in transport within the oviducts and in binding to zonae pellucidae although the animals had normal sperm count, morphology and motility. In fact, unexplained infertility is difficult to be predicted by conventional parameters such as sperm count. We measured membrane testicular ACE activity in a sperm suspension in PBS and total testis ACE activity in spermatozoa by solubilization with Triton X-100. Total testis ACE activity and membrane testis ACE activity of the same subject were compared in 12 control subjects. We demonstrated that testicular ACE is stable in spermatozoa and the assay of testicular ACE activity is possible. Total testicular ACE activity was approximately twice the membrane testicular ACE activity in all of the subjects tested. The assay of testicular ACE activity in human spermatozoa could be a new method for the assessment of sperm function.