Cell-specific activity of neprilysin 2 isoforms and enzymic specificity compared with neprilysin

Rose, Christiane; Voisin, Stéphanie; Gros, Claude P.; Schwartz, Jean‐Charles; Ouimet, Tanja

doi:10.1042/0264-6021:3630697

Cited by 32 publications

(63 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The NEP2 isoforms, NEP2(m) and NEP2(s), are identical in their large C-terminal core, possess identical enzymatic properties, and are inhibited by the same compounds with comparable potencies (28). To ease the study and production of NEP, a secreted form of this enzyme was engineered (secNEP) (44) and was found to have the same enzymatic properties as the natural enzyme.…”

Section: Resultsmentioning

confidence: 99%

“…Site-directed Mutagenesis of rNEP2(s)-The rNEP2(s)-expressing plasmid, pcDNA-rNEP2(s), has been previously described (28). Doublestranded mutagenesis of rNEP2(s) was carried out using the Transformer site-directed mutagenesis kit (Clontech) following the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…Incubations were allowed to proceed for 60 min at 37°C followed by a 60-min incubation with an excess of aminopeptidase M (Pierce) to release fluorescent AMC. The enzyme concentrations were quantified by densitometric analysis of immunoblots using the known amounts of purified NEP2 standards (28).…”

Section: Expression Of Wild-type and Mutated Forms Of Rnep2(s) In Att20mentioning

confidence: 99%

“…All of the transition state inhibitors of NEP also inhibit NEP2 activity. Thus, whereas the generic compound phosphoramidon binds both proteases with comparably high affinity (nanomolar range), thiorphan, a specific inhibitor of NEP also inhibits NEP2 at nanomolar concentrations but with an affinity 30 times higher for NEP than for NEP2 (28).…”

mentioning

confidence: 99%

“…The second isoform, mainly expressed in CNS, does not include the subtilisin-type cleavage site and thus leads to a membranebound protein (1,28). Expression of these isoforms in the endocrine corticotrope cell line AtT20 shows that both secreted and membrane-bound isoforms have identical catalytic activities and that they resemble the catalytic activity of NEP insofar, as they have been shown to cleave many of the same substrates, such as tachykinins and enkephalins, at the same amide bonds and with comparable kinetic parameters (28). All of the transition state inhibitors of NEP also inhibit NEP2 activity.…”

mentioning

confidence: 99%

See 4 more Smart Citations

A Three-dimensional Model of the Neprilysin 2 Active Site Based on the X-ray Structure of Neprilysin

Voisin

Rognan

Gros

et al. 2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Neprilysin 2 (NEP2), a recently identified member of the M13 subfamily of metalloproteases, shares the highest degree of homology with the prototypical member of the family neprilysin. Whereas the study of the in vitro enzymatic activity of NEP2 shows that it resembles that of NEP as it cleaves the same substrates often at the same amide bonds and binds the same inhibitory compounds albeit with different potencies, its physiological role remains elusive because of the lack of selective inhibitors. To aid in the design of these novel compounds and better understand the different inhibitory patterns of NEP and NEP2, the x-ray structure of NEP was used as a template to build a model of the NEP2 active site. The results of our modeling suggest that the overall structure of NEP2 closely resembles that of NEP. The model of the active site reveals a 97% sequence identity with that of NEP with differences located within the S 2 subsite of NEP2 where Ser 133 and Leu 739 replace two glycine residues in NEP. To validate the proposed model, site-directed mutagenesis was performed on a series of residues of NEP2, mutants expressed in AtT20 cells, and their ability to bind various substrates and inhibitory compounds was tested. The results confirm the involvement of the conserved Arg 131 and Asn 567 in substrate binding and catalytic activity of NEP2 and further show that the modifications in its S 2 pocket, particularly the presence therein of Leu 739 , account for a number of differences in inhibitor binding between NEP and NEP2.Neprilysin 2 (SEP and NL-1) is a recently identified type II membrane-bound zinc-dependent metalloprotease (1-3). It is part of the M13 family of metalloproteases, which also comprises neprilysin (NEP, 1 EC 3.4.24.11) (4 -6), the endothelinconverting enzymes, ECE-1 (EC 3.4.24.71) (7) and ECE-2 (8), the Kell blood group antigen (9), the phosphate-regulating neutral endopeptidase on the X chromosome (10) and X-converting enzyme (11). Not only do all of the members of this family of metalloproteases share considerable sequence identities, suggesting a common ancestor as well as a common fold, NEP2, by far the closest homologue of NEP displays over 50% of overall protein sequence identity with NEP, a degree of homology that also suggests a common function (12).The best characterized member of the family, NEP (13, 14), was first identified as a kidney brush-border neutral endopeptidase slowly hydrolyzing [ 125 I]iodoinsulin B chain (15) and rediscovered as an "enkephalinase" of cerebral membranes hydrolyzing the Gly 3 -Phe 4 bond of enkephalins (16). Thereafter, it was shown to hydrolyze a number of biologically active peptides into inactive fragments in vitro, such as tachykinins (17), bradykinin (18), and atrial natriuretic peptides (19,20). The physiological implication of NEP in the inactivation of these messenger peptides was confirmed in vivo by the design and use of selective and potent inhibitors, e.g. thiorphan (21-24). These functions of NEP and the design of specific inhibitors thereof are of ...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Expression Of Wild-type and Mutated Forms Of Rnep2(s) In Att20mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

A Three-dimensional Model of the Neprilysin 2 Active Site Based on the X-ray Structure of Neprilysin

Voisin

Rognan

Gros

et al. 2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

The tripartite architecture of the eukaryotic integral membrane protein zinc metalloprotease Ste24

2019

View full text Add to dashboard Cite

Ste24 enzymes, a family of eukaryotic integral membrane proteins, are zinc metalloproteases (ZMPs) originally characterized as “CAAX proteases” targeting prenylated substrates, including a‐factor mating pheromone in yeast and prelamin A in humans. Recently, Ste24 was shown to also cleave nonprenylated substrates. Reduced activity of the human ortholog, HsSte24, is linked to multiple disease states (laminopathies), including progerias and lipid disorders. Ste24 possesses a unique “α‐barrel” structure consisting of seven transmembrane (TM) α‐helices encircling a large intramembranous cavity (~14 000 Å3). The catalytic zinc, coordinated via a HExxH…E/H motif characteristic of gluzincin ZMPs, is positioned at one of the cavity's bases. The interrelationship between Ste24 as a gluzincin, a long‐studied class of soluble ZMPs, and as a novel cavity‐containing integral membrane protein protease has been minimally explored to date. Informed by homology to well‐characterized soluble, gluzincin ZMPs, we develop a model of Ste24 that provides a conceptual framework for this enzyme family, suitable for development and interpretation of structure/function studies. The model consists of an interfacial, zinc‐containing “ZMP Core” module surrounded by a “ZMP Accessory” module, both capped by a TM helical “α‐barrel” module of as yet unknown function. Multiple sequence alignment of 58 Ste24 orthologs revealed 38 absolutely conserved residues, apportioned unequally among the ZMP Core (18), ZMP Accessory (13), and α‐barrel (7) modules. This Tripartite Architecture representation of Ste24 provides a unified image of this enzyme family.

show abstract

Computer modeling and nanosecond simulation of the enzyme?substrate complex of the common lymphoblastic leukemia antigen (neprilysin) indicates shared residues at the primary specificity pocket (S1') with matrix metalloproteases

Manzetti¹

2003

Journal of Molecular Modeling

View full text Add to dashboard Cite

The common acute lymphoblastic leukemia antigen (neprilysin, CD10, neutral endopeptidase 24.11) is a member of the neprilysin family, and projects functions in signaling pathways in pathophysiological processes such as cancer, Alzheimer's disease and hypertension. Given its pathophysiological importance, an investigation of the natural substrate specificity of this metalloprotease is presented here through the application of enzyme-substrate modeling and molecular dynamics simulations. The results show that the substrate modeled, LATAC downward arrow FG, satisfies a complementary backbone H-bonding with Ala543-Tyr545, thereby suggested to be the putative substrate-binding beta-sheet, analogously to matrix metalloproteases. The modeling further suggests that phenylalanine at the P1' position (substrate) is directed in the same fashion as the synthetic inhibitor of the reference crystal structure and that this enzyme does not bind the P3'/P4' positions of a substrate, as other metalloproteases do. After a specific comparison with one member of the matrix metalloproteases, MMP-3, a common conserved valine residue at the primary S1' subsite was found to be shared between these two otherwise different proteases. These results may prove useful for selective drug design for neprilysin, and lay a foundation for future subsite analysis for other members of the neprilysin family.

show abstract

Cell-specific activity of neprilysin 2 isoforms and enzymic specificity compared with neprilysin

Cited by 32 publications

References 39 publications

A Three-dimensional Model of the Neprilysin 2 Active Site Based on the X-ray Structure of Neprilysin

A Three-dimensional Model of the Neprilysin 2 Active Site Based on the X-ray Structure of Neprilysin

The tripartite architecture of the eukaryotic integral membrane protein zinc metalloprotease Ste24

Computer modeling and nanosecond simulation of the enzyme?substrate complex of the common lymphoblastic leukemia antigen (neprilysin) indicates shared residues at the primary specificity pocket (S1') with matrix metalloproteases

Contact Info

Product

Resources

About