Browsing by Author "Ehlers, Mario R W"

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    Angiotensin-converting enzyme - new insights into structure, biological significance and prospects for domain-selective inhibitors.
    (Bentham Science Publishers, 2009) Watermeyer, Jean M; Kröger, Wendy L; Sturrock, Edward D; Ehlers, Mario R W
    Somatic angiotensin-converting enzyme (ACE) - well known for its role in cardiovascular pathophysiology - has an unusual, two-domain, double active-site structure. The two domains (designated N and C) are 55% identical and each contains a similar active site with overlapping but distinct substrate preferences. While both convert angiotensin I to angiotensin II in vitro, current evidence suggests the C domain site predominates in this role in vivo. The N domain site inactivates a hemoregulatory and antifibrotic peptide, AcSDKP, in vivo, although the significance of this remains unclear. However, differences in the characteristics of the two domains may result in different context-dependent activities, as is the case with other enzymes containing tandem repeats. The N domain may also have a role in modulating C domain activity, through a combination of inter-domain cooperativity and structural stabilization. Comparison of ACE with its structural homologues reveals conservation of peptidase activity and a tendency to hinge about the active-site cleft. Recent work on ACE active-site mutants containing one or more key residues replaced by their cognate residues from the other domain, synthesis of domain-selective inhibitors, and co-crystal structures of each domain with such inhibitors, has led to a better resolution of the basis for domain selectivity and should enable the design of next-generation, domain-selective inhibitors with distinct pharmacological profiles.
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    Binding of Mycobacterium tuberculosis to complement receptor type 3 expressed in mammalian cells : dependence on serum opsonins
    (1996) Cywes, Colette; Ehlers, Mario R W
    Nonopsonic invasion of mononuclear phagocytes by Mycobacterium tuberculosis (M. tb.) is likely important in the establishment of a primary infection in the lung. M. tb. binds to a variety of phagocyte receptors, of which the mannose receptor and the complement receptor type 3 (CR3) may support nonopsonic binding. CR3, a β₂ integrin, is a target for diverse intracellular pathogens, but its role in nonopsonic binding remains uncertain. We have examined the binding of M. tb. to human CR3 heterologously expressed in Chinese hamster ovary (CHO) cells, thereby circumventing the problems of competing receptors and endogenously synthesised complement, which are inherent in studies with mononuclear phagocytes. The surface expression and functional activity of CR3 were confirmed by rosetting with beads coupled to anti-CR3 monoclonal antibodies (MAbs) and with C3bi-coated microspheres, respectively. We found thatM. tb. binds 4-7-fold more avidly to CR3- expressing CHO cells than to wild-type cells, and importantly, that this binding is very similar in the presence of fresh or heat-inactivated human or bovine sera, or no serum. The binding of M. tb. to the transfected CHO cells is CR3-specific, as it is inhibited by anti-CDllb and anti-CD18 MAbs; interestingly, binding is not inhibited by a MAb (2LPM19c) specific for the C3bi-binding site on CDI lb. Electron micrographs of infected CR3-expressing CHO cells reveal the presence of intracellular bacteria enclosed in well-defined, membrane-bound vacuoles. We conclude that the binding of M. tb. to CR3 is nonopsonic and that the organism likely expresses a ligand that directly binds to CR3.
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    Deletion 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 D
    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.
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    Identification and characterisation of proteases in Mycobacterium tuberculosis
    (1999) Dave, Joel Alex; Ehlers, Mario R W; Sturrock, Edward D
    Virulence determinants of M. tuberculosis remain largely unknown. Of key interest has been the ability of the bacterium to survive intracellularly within its host cell, the macrophage, and its ability to cause extensive tissue necrosis. Exported proteases are commonly associated with virulence in bacterial pathogens, yet their role in Mycobacterium tuberculosis has virtually not been studied. Preliminary experiments showed M. tuberculosis culture filtrates contained a proteolytic activity inhibited by mixed serine/cysteine protease inhibitors and activated by Ca²⁺, features typical of some serine proteases, notably subtilisins, and possibly metalloproteases. Purification attempts were unsuccessful. A family of five genes that encode putative, secreted, serine proteases has recently been described in M. tuberculosis. These proteases share 36-47% sequence identity and are all encoded with putative signal peptides, suggesting that they are translocated across the cytoplasmic membrane. One member, mycP1, was selected for further study. The gene product, mycosin-1, was 30-35% identical to bacterial subtilisin-like serine proteases and contained the classic catalytic triad and oxyanion hole. Mycosin-1 also contained a typical signal peptide, a likely propeptide, and a Cterminal hydrophobic sequence with a high transmembrane potential. Topology analyses predicted mycosin-1 to be a type I ectoprotein. Consistent with this, expression of mycosin-1 in M. tuberculosis and in Mycobacterium smegmatis transformed with mycP1 (M. smegmatis-P1) was limited strictly to the cell envelope, as seen by Western blotting, and immunogold electron microscopy. Only full-length, 50-kDa mycosin-1 was observed by Western blotting in broth-grown M. tuberculosis and M. smegmatis-P1 lysates, whereas a 40-kDa species was detected in 6-week M. tuberculosis culture filtrates. A similar 40-kDa immunoreactive band was also observed in lysates of macrophages infected with M. tuberculosis, consistent with robust transcription of the mycP 1 gene during growth in macrophages. Since putative mature mycosin-1 has a molecular weight of 38.6 kDa, the 40-kDa protein may represent activated mycosin-1 after propeptide cleavage. In conclusion, mycosin-1 is an exported, cell envelopeassociated subtilisin homolog that is expressed during growth of M. tuberculosis in vitro and in macrophages.