Vasoactive Peptidases
One of the major areas of our research is concerned with functional/structural studies of numerous cell-surface peptidases that are involved in the metabolism of peptide hormones and other bioactive peptides. These peptidases are integral proteins of predominantly the plasma membrane, asymmetrically oriented, with the catalytic site exposed at the extracytoplasmic surface. Currently we have projects focusing on the following enzymes:
Angiotensin Converting Enzyme and its Homologue ACE2
Angiotensin converting enzyme (ACE) is a Type I integral membrane zinc metallopeptidase that plays a key role in blood pressure regulation and fluid and electrolyte homeostasis. ACE converts the inactive angiotensin I into the vasocontrictor peptide angiotensin II, and inactivates the vasodilator bradykinin. Inhibitors of the enzyme such as captopril and enalapril are currently used as anti-hypertensive therapeutics.
Although ACE exists primarily as a membrane-bound form, a soluble form is present in plasma, amniotic fluid and other body fluids. This soluble form is derived from the membrane-bound form by a post-translational proteolytic cleavage/shedding event (Figure 6). We have characterized the secretase/sheddase involved as an integral membrane zinc metalloprotease, probably a member of the ADAM (a disintegrin and metalloprotease) family of zinc metalloproteases.
Figure 6. Model for the proteolytic shedding of ACE (pink) from the membrane (green) by its cognate sheddase/secretase (blue). The sheddase cleaves the juxtamembrane stalk of ACE between the Arg and Ser residues.
In 2000 we identified, cloned, sequenced and expressed a novel enzyme that may provide a new therapeutic target for the treatment of hypertension and other heart conditions. This new enzyme, termed ACE2 (ACEH) (Figure 7), is the first human homologue of the well-characterised angiotensin converting enzyme (ACE), which is a key target for anti-hypertensive drugs such as captopril. Current projects are focusing on the molecular and cellular characterisation of ACE2.
Figure 7. Schematic of the various members of the ACE family. Each protein is a type 1 integral membrane protein with a large extracellular domain containing the catalytic site (HEMGH), a transmembrane region (blue in ACE, red in ACE2) and a short cytoplasmic domain.
Aminopeptidase P
Aminopeptidase P (X-Pro aminopeptidase, EC 3.4.11.9) releases N-terminal amino acids from peptides with a penultimate proline, such as bradykinin, substance P, β-casomorphin, and peptides of the pancreatic polypeptide family. Aminopeptidase P may act in concert with angiotensin converting enzyme in the physiological inactivation of the vasodilator bradykinin. We were the first to demonstrate that aminopeptidase P was a GPI-anchored protein, and subsequently to purify, clone and sequence its cDNA.
Although aminopeptidase P is a zinc metallopeptidase its amino acid sequence contains none of the known zinc binding motifs. Residues important in binding the zinc ion and in catalysis have been identified through molecular modelling and site-directed mutagenesis (Figure 8). Current projects are concerned with characterising the promoter region of the human aminopeptidase P gene and identifying and characterising polymorphisms in the gene.
Figure 8. Model of the active site of aminopeptidase P showing the pita bread fold and the 2 bound metal ions (yellow spheres).
The Neprilysin Family
Neprilysin (endopeptidase-24.11), a plasma membrane zinc peptidase was discovered in Leeds and shown to play a key role in the metabolism of regulatory peptides, especially enkephalins, tachykinins and atrial natriuretic peptide. Much of our work has focused on structure, localisation and functions of this peptidase and has led to a model of how it serves to terminate peptide signals in the nervous system.
More recently, we have focused on other members of this family, designated ECE, KELL, PEX and XCE. In particular, ECE (endothelin converting enzyme) plays a critical role in the biosynthesis of the potent vasoconstrictor peptide, endothelin and is a potent mitogen. Thus it is a potential target in hypertension, atherosclerosis and certain forms of cancer.
Our current studies are directed towards understanding the trafficking of ECE between plasma membrane and the trans-Golgi network through mutagenesis of targeting signals in the protein and the localization of the different isoforms of ECE by immunochemical methods.
We also have carried out extensive mutagenesis of the active site of the enzyme to gain an understanding of its mechanism and have shown it to hydrolyse a range of biologically active peptides.
Three members of this family of previously unknown function are also beginning to be characterised. KELL is an erythrocyte protein; PEX is a protein linked with the disease, X-linked hypophosphataemic rickets; and SEP(NEP2) exists in a secreted, soluble form.
X-converting enzyme (XCE) is a homologue expressed mainly in CNS and probably involved in the central control of respiration. It is identical with damage-induced neuronal endopeptidase (DINE). All are putative peptidases and the availability of their cDNAs now allows their expression and characterization.