Browsing by Author "Schwager, Sylva L U"
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- ItemOpen AccessCharacterisation of the structural motifs Involved in the cleavage and secretion of human angiotensin-converting enzyme(2014) Conrad, Nailah; Sturrock, Edward D; Schwager, Sylva L UAngiotensin converting enzyme is an ectoprotein prone to regulated proteolytic solubilisation by an as yet unknown protease or sheddase. Proteolytic cleavage of membrane proteins is an essential cellular process that controls their expression and function, and modulates cellular and physiological processes. Testis ACE (tACE) is shed at a higher rate than somatic ACE and it has been proposed that regions in its ectodomain direct its shedding. Discrete secondary structures on the surface of the distal ectodomain of tACE were replaced with their N-domain counterparts to determine their role in the ectodomain shedding of ACE. None of the regions investigated proved to be an absolute requirement for shedding, but the mutant ACE proteins were subject to variations in shedding compared to wild-type tACE. To investigate the role of the proximal ectodomain in shedding the residues H610-L614 were mutated to alanines, causing a decrease in shedding. An extension of this mutation on the N-terminal side to seven alanines resulted in a reduction in ACE activity and, more importantly, it affected the processing of the protein to the membrane, resulting in expression of an underglycosylated form of ACE. When E608-H614 was mutated to the homologous region of the N-domain, processing was normal and shedding only marginally reduced. These data suggest that this region is more crucial for the processing of ACE than is for regulating shedding. Construction of a P628L mutation in tACE showed an increase in shedding. Furthermore, MALDI analysis of a tryptic digest established that the putative glycosylation site N620WT became glycosylated. Further mutagenesis of the P628L mutant to remove the newly formed glycosylation site, resulted in an even greater increase in shedding. Soluble fluorogenic peptides mimicking the ACE stalk were used in a cell-based assay to characterise the contribution of the stalk to ACE shedding. Hydrolysis of the wild-type peptide Abz-NSARSEGPQ-EDDnp was not responsive to phorbol ester or the hydroxamate inhibitor (TAPI), however, it was inhibited by EDTA. The aminopeptidase inhibitor bestatin did not inhibit cleavage or alter the cleavage site. Therefore the protease involved in the cleavage of the ACE stalk peptides is likely different to the sheddase responsible for ACE shedding. Substitution of the P1 and P1' sites of the peptides did not significantly influence the rate of cleavage. All the peptides were cleaved at the E-G bond, which is C-terminal to the physiological R-S cleavage site. Removal of the fluorogenic capping groups resulted in no cleavage of the peptides and lengthening of the peptide did not result in cleavage. This confirms the need for the ACE sheddase and its substrate to be anchored in the membrane and suggests the use of soluble peptide substrates in a cell assay has limited application for investigating the ectodomain shedding of ACE.
- ItemRestrictedCrystal structure of the N domain of human somatic angiotensin I-converting enzyme provides a structural basis for domain-specific inhibitor design(Elsevier, 2006) Corradi, Hazel R; Schwager, Sylva L U; Nchinda, Aloysius T; Sturrock, Edward D; Acharya, K RaviHuman somatic angiotensin I-converting enzyme (sACE) is a key regulator of blood pressure and an important drug target for combating cardiovascular and renal disease. sACE comprises two homologous metallopeptidase domains, N and C, joined by an inter-domain linker. Both domains are capable of cleaving the two hemoregulatory peptides angiotensin I and bradykinin, but differ in their affinities for a range of other substrates and inhibitors. Previously we determined the structure of testis ACE (C domain); here we present the crystal structure of the N domain of sACE (both in the presence and absence of the antihypertensive drug lisinopril) in order to aid the understanding of how these two domains differ in specificity and function. In addition, the structure of most of the inter-domain linker allows us to propose relative domain positions for sACE that may contribute to the domain cooperativity. The structure now provides a platform for the design of “domain-specific” second-generation ACE inhibitors.
- ItemRestrictedDeletion of the cytoplasmic domain increases basal shedding of angiotensin-converting enzyme(Elsevier, 2004) Chubb, Anthony J; Schwager, Sylva L U; van der Merwe, Elizabeth; Ehlers, Mario R W; Sturrock, Edward DEctodomain 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.
- ItemOpen AccessProtein-protein interactions of human somatic angiotensin-converting enzyme(2011) Gordon, Kerry; Sturrock, Edward D; Schwager, Sylva L UIn this study, novel disulphide bridges were engineered into the linker region of ACE [Angiotensin-converting enzyme] in an attempt to limit inter-domain movement, thereby producing a candidate for crystallisation and to determine the effect of these bridges on inter-domain movement.
- ItemRestrictedStructural details on the binding of antihypertensive drugs captopril and enalaprilat to human testicular angiotensin I-converting enzyme(American Chemical Society, 2004) Natesh, Ramanathan; Schwager, Sylva L U; Evans, Hazel R; Sturrock, Edward D; Acharya, K RaviAngiotensin 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 Zn2+ 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.