Endothelin-converting enzyme-2 (ECE-2) is a membranebound zinc-dependent metalloprotease that shares a high degree of sequence homology with ECE-1, but displays an acidic pH optimum characteristic of maturing enzymes acting late in the secretory pathway. Although ECE-2, like ECE-1, can cleave the big endothelin intermediate to produce the vasoconstrictive endothelin peptide, its true physiological function remains to be elucidated, a task that is hampered by the lack of specific tools to study and discriminate ECE-2 from ECE-1, i.e. specific substrates and/or specific inhibitors. To fill this gap, we searched for novel ECE-specific peptide substrates. To this end, peptides derived from the big endothelin intermediate were tested using ECE-1 and ECE-2, leading to the identification of an ECE-1-specific substrate. Moreover, screening of our proprietary fluorigenic peptide Fluofast libraries using ECE-1 and ECE-2 allowed the identification of Ac-SKG-Pya-F-W-Nop-GGK-NH 2 (PL405), as a specific and high affinity ECE-2 substrate. Indeed, ECE-2 cleaved PL405 at the Pya-F amide bond with a specificity constant (k cat /K m ) of 8.1 ؎ 0.9 ؋ 10 3 M ؊1 s ؊1 . Using this novel substrate, we also characterized the first potent (K i ؍ 7.7 ؎ 0.3 nM) and relatively selective ECE-2 inhibitor and developed a quantitative fluorigenic ECE-2 assay. The assay was used to study the ex vivo ECE-2 activity in wild type and ECE-2 knockout tissues and was found to truly reflect ECE-2 expression patterns. The PL405 assay is thus the first tool to study ECE-2 inhibition using high throughput screening or for ex vivo ECE-2 quantification.Endothelin-converting enzyme-2 (ECE-2) 2 is a zinc-dependant metalloprotease belonging to the M13 family, in which it shares the highest degree of sequence homology with ECE-1 (EC 3.4.24.71) (1, 2). The family is also constituted by neprilysin (NEP, EC 3.4.24.11) (3); damage-induced neuronal endopeptidase, also known as endothelin converting enzyme like (ECEL) (4, 5); the Kell blood group antigen (6); phosphate-regulating neutral endopeptidase on the X chromosome (7); and neprilysin 2 (NEP2) (8 -11). These peptidases are type II integral membrane glycoproteins composed of a small cytosolic tail followed by a transmembrane region and a large C-terminal core that is typically exposed at the cell surface. The active site of the enzyme is located within this last domain and is characterized by two highly conserved zinc-binding consensus motifs HEXXH and EXXXD (where X is any amino acid) (12). The structure of the human ECE-1 enzyme co-crystallized with phosphoramidon, a generic inhibitor of the family, was recently reported (13) showing the conserved "thermolysin-like" arrangement of its catalytic site (14, 15).The endothelin-converting enzymes were first characterized as cleaving the big endothelin inactive intermediate at its Trp 21 -Val 22 bond to produce the highly vasoconstrictive endothelin peptide (1, 2). It was thereafter found that ECE-1 can also hydrolyze smaller peptides in vitro such as big endothel...
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