Expression of NEP2, a soluble neprilysin-like endopeptidase, during embryogenesis in Drosophila melanogaster

Members of the neprilysin family of neutral endopeptidases (M13) are typically membrane-bound enzymes known to be involved in the extra-cellular metabolism of signalling peptides and have important roles during mammalian embryogenesis. In this study we show that membranes prepared from embryos of Drosophila melanogaster possess neprilysin-like activity that is inhibited by phosphoramidon and thiorphan, both inhibitors of mammalian neprilysin. Unexpectedly, we also found strong neprilysin-like neutral endopeptidase activity in a soluble embryo fraction, which we identify as NEP2 by Western blot and immunoprecipitation experiments using NEP2 specific antibodies. NEP2 is a soluble secreted member of the neprilysin family that has been shown previously to be expressed in larval and adult Malpighian tubules and in the testes of adult males. In situ hybridization studies reveal expression at stage 10-11 in a pattern similar to that previously described for stellate cell progenitors of the caudal visceral mesoderm. In later stages of embryogenesis, some of these cells appear to migrate into the growing Malpighian tubule. Recombinant NEP2 protein is N-glycosylated and displays optimum endopeptidase activity at neutral pH, consistent with a role as an extracellular peptidase. The recombinant enzyme hydrolyses Drosophila tachykinin peptides (DTK) at peptide bonds N-terminal to hydrophobic residues. DTK2, like Locusta tachykinin-1, was cleaved at the penultimate peptide bond (Gly(7)-Leu(8)), whereas the other Drosophila peptides were cleaved centrally at Xxx-Phe bonds. However, the rates of hydrolysis of the latter substrates were much slower than the hydrolysis rates of DTK2 and Locusta tachykinin-1, suggesting that the interaction of the bulky side-chain of phenylalanine at the S'(1) sub-site is less favorable for peptide bond hydrolysis. The secretion of NEP2 from tissues during embryogenesis suggests a possible developmental role for this endopeptidase in peptide signalling in D. melanogaster.     

Characterisation of neutral endopeptidase 3.4.24.11 (NEP) in the kidney: comparison between normotensive, genetically hypertensive and experimentally hypertensive rats.

Neutral endopeptidase 3.4.24.11 (NEP) has been identified as the major atrial natriuretic factor (ANF) degrading enzyme in rat kidney, therefore, suggesting a possible role for this enzyme in blood volume and pressure regulation. Various experimentally induced and genetically hypertensive rat models have been used to test NEP inhibitors. The presence of different isoforms of NEP in the various hypertensive rat models would have relevance when searching for novel NEP inhibitors. Therefore, we compared the properties of NEP in kidney cortex homogenates in order to test for possible differences in the following hypertensive rat models and their appropriate controls: spontaneously hypertensive rats (SHR), Wistar Kyoto strain (WKY), DOCA-salt hypertensive rats, and Sprague Dawley control rats (SD). No relevant differences were found when comparing the following parameters: (1) specific activity (mean: 204 U/mg protein), (2) Michaelis constant (mean: 280 microM), (3) IC50 of thiorphan (mean: 6.5 nM) and phosphoramidon (mean: 54 nM), (4) pH profiles (optimum at pH 8.0), (5) heat inactivation profiles (half-life 20 min at 65 degrees C), (6) immunotitration of kidney cortex homogenates, (7) molecular weight as determined by gel filtration (92,000 Dalton) and (8) affinity chromatography with concanavalin A. Without evidence for the presence of different NEP isoforms, it is unlikely that divergent findings in DOCA-salt rats and SHR using a given NEP inhibitor are due to isoforms of NEP.     

Characterisation of Neutral Endopeptidase 3.4.24.11 (NEP) in the Kidney: Comparison Between Normotensive,Genetically Hypertensive and Experimentally Hypertensive Rats

Abstract

Neutral endopeptidase 3.4.24.11 (NEP) has been identified as the major atrial natriuretic factor (ANF) degrading enzyme in rat kidney, therefore, suggesting a possible role for this enzyme in blood volume and pressure regulation. Various experimentally induced and genetically hypertensive rat models have been used to test NEP inhibitors. The presence of different isoforms of NEP in the various hypertensive rat models would have relevance when searching for novel NEP inhibitors. Therefore, we compared the properties of NEP in kidney cortex homogenates in order to test for possible differences in the following hypertensive rat models and their appropriate controls: spontaneously hypertensive rats (SHR), Wistar Kyoto strain (WKY). DOCA-salt hypertensive rats, and Sprague Dawley control rats (SD). No relevant differences were found when comparing the following parameters,: (1) specific activity (mean: 204 U/mg protein), (2) Michaelis constant (mean: 280μM), (3) IC₅₀ of thiorphan (mean: 6.5 nM) and phosphoramidon (mean: 54 nM), (4) pH profiles (optimum at pH8.0), (5) heat inactivation profiles (half-life 20min at 65°C), (6) immunotitration of kidney cortex homogenates, (7) molecular weight as determined by gel filtration (92,000 Dalton) and (8) affinity chromatography with concanavalin A. Without evidence for the presence of different NEP isoforms, it is unlikely that divergent findings in DOCA-salt rats and SHR using a given NEP inhibitor are due to isoforms of NEP.     

A three-dimensional model of endothelin-converting enzyme (ECE) based on the X-ray structure of neutral endopeptidase 24.11 (NEP).

Endothelin-converting enzyme 1 (ECE-1, EC 3.4.24.71) is a zinc-dependent type II mammalian membrane protein comprising the active site in the ectodomain. It exists in multiple splice variants that all catalyze the last and rate-limiting step in the activation of preproendothelin to the highly potent vasoconstrictor endothelin. There is high interest in finding small and potent inhibitors for this enzyme that could be used in numerous indications, e.g. hypertension. Since there is no structural information available for this important enzyme, we built a model of the complete ectodomain using the recently solved structure of human NEP as template. The naturally derived metalloproteinase inhibitor phosphoramidon was docked in the active site of this model and comparisons with the respective NEP complex were made.     

Influenza B and C Virus NEP (NS2) Proteins Possess Nuclear Export Activities

Nucleocytoplasmic transport of viral ribonucleoproteins (vRNPs) is an essential aspect of the replication cycle for influenza A, B, and C viruses. These viruses replicate and transcribe their genomes in the nuclei of infected cells. During the late stages of infection, vRNPs must be exported from the nucleus to the cytoplasm prior to transport to viral assembly sites on the cellular plasma membrane. Previously, we demonstrated that the influenza A virus nuclear export protein (NEP, formerly referred to as the NS2 protein) mediates the export of vRNPs. In this report, we suggest that for influenza B and C viruses the nuclear export function is also performed by the orthologous NEP proteins (formerly referred to as the NS2 protein). The influenza virus B and C NEP proteins interact in the yeast two-hybrid assay with a subset of nucleoporins and with the Crm1 nuclear export factor and can functionally replace the effector domain from the human immunodeficiency virus type 1 Rev protein. We established a plasmid transfection system for the generation of virus-like particles (VLPs) in which a functional viral RNA-like chloramphenicol acetyltransferase (CAT) gene is delivered to a new cell. VLPs generated in the absence of the influenza B virus NEP protein were unable to transfer the viral RNA-like CAT gene to a new cell. From these data, we suggest that the nuclear export of the influenza B and C vRNPs are mediated through interaction between NEP proteins and the cellular nucleocytoplasmic export machinery.     

人类疾病研究的新焦点:中性肽链内切酶(NEP)——NEP生理功能及其潜在调控因子研究之进展

 Neprilysin（ NEP）是M13锌金属蛋白酶家族的一种Ⅱ型整合细胞膜糖蛋白.自从1974年首次被定义以来,NEP作为一种神经肽的降解酶,已经被发现广泛地存在于中枢神经系统以及外周各种组织中,并具有众多组织特异性功能.其中, NEP具有两大重要生理功能：一是NEP通过抑制细胞迁移增殖并诱导细胞程序性死亡具有抗肿瘤作用,这依赖它的酶学特性以及与涉及细胞信号转导通路的关键分子直接的蛋白-蛋白间相互作用;另外, NEP具有神经保护作用,这主要通过阻止神经毒物——淀粉样蛋白Aβ在大脑中的聚集来实现.通过研究各种人类疾病的进展,目前发现这些疾病经常伴随有NEP表达的削弱和活性下降.基于这些发现,调控NEP在病理细胞中的不同水平正在逐渐被认可为具有治疗应用前景的一种策略,从而达到恢复正常的细胞功能并缓解病症的目的.现阶段的研究重点,主要在于揭示涉及NEP的表达以及活性的一些调控机制,并且获得了大量鼓舞人心的研究成果.例如,男性荷尔蒙已知是前列腺中NEP的转录调节因子.除它之外,最新研究结果还发现,女性雌激素,柳栎的水合萃取物,绿茶的一些组分,各种神经肽比如韩蛙皮素和生长激素抑制素以及淀粉样前体蛋白的胞内区,也都对NEP的表达和活性有刺激作用.     

Independent signals determine the subcellular localization of NEP in prostate cancer cells

NEP (Neutral endopeptidase 24.11) is a cell surface enzyme that hydrolyzes bioactive neuropeptides implicated in the transition from androgen-dependent prostate cancer (PC) to androgen-independent PC. We report the cloning and sequence analyses of NEP cDNAs from human androgen-responsive LNCaP PC cells and prostatic stromal cells. To investigate the functional role of a nuclear localization sequence (NLS) detected within the N-terminus and of an endoplasmic reticulum retention signal within the C-terminus, NEP-GFP expression vectors were constructed containing the whole NEP gene, fragments encoding the N-terminus/C-terminus of the protein (5(')NEP-GFP/3(')NEP-GFP), and 5(')NEP-GFP constructs lacking the NLS. 3(')NEP-GFP transfected cells showed plasma membrane/cytoplasmic fluorescence whereas the 5(')NEP-GFP fusion protein was also detected in the nucleus. The omission of the NLS resulted in no reduction in nuclear and an increase in cytoplasmic staining. The results suggest that the analyzed structural motifs determine the subcellular distribution of NEP in epithelial LNCaP PC cells and stromal prostatic cells and therefore could be responsible for the altered cellular localization of NEP observed in PC.     

The importance of disulfide bridges in human endopeptidase (enkephalinase) after proteolytic cleavage

The neutral endopeptidase (NEP) is a membrane-bound enzyme, which is solubilized by treatment with the protease, papain. Papain did not affect the apparent catalytic activity or the molecular mass of the purified human enzyme in SDS-PAGE. When NEP was treated with a reducing agent after papain digestion, it dissociated into smaller, lower molecular mass fragments. Amino acid analysis and s-carboxymethylation of the half cystine residues indicated that NEP contains four S-S bridges. We concluded that, although covalent bonds appear to be cleaved in NEP by papain, its activity and structure are sustained by S-S bridges.     

Human renal 11beta-hydroxysteroid dehydrogenase 1 functions and co-localizes with COX-2

The local renal metabolism of glucocorticoids (GCs) by isoforms of 11beta-hydroxysteroid dehydrogenase (11beta-HSD1 and 11beta-HSD2) determines their biological effects. 11beta-HSD2, located in collecting duct epithelial cells of the mammalian and human kidney, serves as a putative "guardian" preventing GCs from binding to mineralocorticoid receptors. Various investigators have shown that both isoforms are present in kidney tissue from the rat, dog and other mammals. There is controversy as to whether 11beta-HSD1 exists and functions in human kidney. The current studies examine the locale and function of both isoforms in human kidney. The expression of 11beta-HSD1 was similar to that of 11beta-HSD2 by Western blot. Two distinct Lineweaver Burke plots could be drawn providing enzyme kinetics for both isoforms. The apparent Km for the NADP dependent 11beta-HSD1 enzyme was 0.42 muM while the apparent Km for the NAD dependent 11beta-HSD2 enzyme was 10.2 nM. Human renal 11beta-HSD1 appears to function as a dehydrogenase with no significant "reverse" reductase activity. Using immuno-histochemistry and Western blot analysis, 11beta-HSD1 was found to co-localize with COX-2 in proximal tubule cells; COX-2 was not seen with 11beta-HSD2 in cortical collecting duct. Thus, normal human kidney contains active 11beta-HSD1 and 11beta-HSD2. 11beta-HSD1 co-localizes with COX-2 in proximal tubule cells.     

Identification of glutamic acid 646 as a zinc-coordinating residue in endopeptidase-24.11.

Neutral endopeptidase (EC 3.424.11, NEP) is a membrane-bound zinc-metallopeptidase. The substrate specificity and catalytic activity of NEP resemble those of thermolysin, a bacterial zinc-metalloprotease. Comparison of the primary structure of both enzymes suggests that several amino acids present in the active site of thermolysin are also found in NEP. Using site-directed mutagenesis of the cDNA encoding the NEP sequence, we have already shown that His residues 583 and 587 are two of the three zinc ligands. In order to identify the third zinc ligand, we have substituted Val or Asp for Glu616 or Glu646. Val616 NEP showed the same kinetic parameters as the non-mutated NEP. In contrast, the mutant Val646 NEP was almost completely devoid of catalytic activity and unable to bind the tritiated inhibitor [3H]N-[2(R,S)-3-hydroxyaminocarbonyl-2-benzyl-1-oxypropyl]gl ycine, the binding of which is dependent on the presence of the zinc ion. Replacing Glu for Asp at position 646 conserved the negative charge, and the mutant enzyme exhibited the same Km value as the non-mutated enzyme, but kCat was decreased to less than 3% of the value of the non-mutated enzyme. When compared to the non-mutated enzyme Asp646 NEP showed a higher susceptibility to chelating agents, but bound the tritiated inhibitor with the same affinity. Taken together, these observations strongly suggest that Glu646 of NEP is the third zinc-coordinating residue and is equivalent to Glu166 in thermolysin.     

Zinc Inhibits Apoptosis and Maintains NEP Downregulation, Induced by Ropivacaine, in HaCaT Cells

Zinc (Zn), a cell-protective metal against various toxic compounds, is the key agent for neutral endopeptidase (NEP) functional structure. NEP is a zinc metalloenzyme which degrades endogenous opioids and is expressed in human keratinocytes (HaCaT). Ropivacaine, a widely used opiate local anaesthetic, exerts cell toxic and apoptotic effects against HaCaT cells. The aim of the present study is to investigate whether zinc modulates the effects of ropivacaine on proliferation, viability, apoptosis and NEP expression in HaCaT cells. To investigate the role of ropivacaine in NEP function, HaCaT cells overexpressing NEP were generated via cell transfection with plasmids carrying NEP cDNA. Ropivacaine's anti-proliferative effect was tested by Neubauer's chamber cell counting, and induction of cell death was demonstrated by trypan blue exclusion assay. Apoptosis due to ropivacaine was tested via DNA fragmentation and poly-ADP-ribose-polymerase (PARP) cleavage. NEP and PARP expression was performed by western blot analysis. Results showed that zinc (15????) inhibited proliferation and cell death induction by ropivacaine (0.5, 1 and 2?mM) (p?<?0.05) as well as apoptosis induced by the drug (0.5 and 1?mM) in HaCaT cells. Ropivacaine (1.0, 2.0 and 5.0?mM) downregulated NEP expression in the presence of zinc (15????) while NEP overexpression enhanced ropivacaine's apoptotic effect. In conclusion, the abilities of zinc to inhibit the toxic and apoptotic effects of ropivacaine, to maintain NEP downregulation induced by the drug and, consequently, to enhance its anaesthetic result suggest that zinc may have a significant role in pain management and tissue protection.     

Properties of a fetal multipotent neural stem cell (NEP cell)

Multipotent neural stem cells (NSCs) present in the developing neural tube (E10.5, neuroepithelial cells; NEP) were examined for the expression of candidate stem cell markers, and the expression of these markers was compared with later appearing precursor cells (E14.5) that can be distinguished by the expression of embryonic neural cell adhesion molecule (E-NCAM) and A2B5. NEP cells possess gap junctions, express connexins, and appear to lack long cilia. Most candidate markers, including Nestin, Presenilin, Notch, and Numb, were expressed by both NEP cells as well as other cell populations. Fibroblast growth factor receptor 4 (FGFR4), Frizzled 9 (Fz9), and SRY box-containing gene 2 (Sox2) as assessed by immunocytochemistry and in situ hybridization are markers that appear to distinguish NSCs from other precursor cells. Neither Hoechst 33342 nor rhodamine-123 staining, telomerase (Tert) expression, telomerase activity, or breakpoint cluster region protein 1 (Bcrp1) transporter expression could be used to distinguish NEP stem cells from other dividing cells. NEP cells, however, lacked expression of several lineage markers that are expressed by later appearing cells. These included absence of expression of CD44, E-NCAM, A2B5, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor-alpha (PDGFR alpha), suggesting that negative selection using cell surface epitopes could be used to isolate stem cell populations from mixed cultures of cells. Using mixed cultures of cells isolated from E14.5 stage embryos, we show that NEP cells can be enriched by depleting differentiating cells that express E-NCAM or A2B5 immunoreactivity. Overall, our results show that a spectrum of markers used in combination can reliably distinguish multipotent NSCs from other precursor cells as well as differentiated cells present in the CNS.     

Correlation between neutral endopetidase (NEP) 3.4.24.11 in serum and the degree of the bronchial hyperreactivity

Neutral endopeptidase (NEP) is described in airways as the major degrading enzyme of tachykinins such as neurokinin A (NKA) and substance P (SP). Due to its localization and mode of action NEP may play a role in the pathophysiology of bronchial reactivity (BR) especially under the aspect of neurogenic inflammation. Serum NEP concentrations were measured by ELISA to investigate if there is a correlation between serum NEP and the degree of bronchial reactivity expressed by PC20-FEV1 histamine(.). PC20-FEV1 histamine was determined in 31 asthmatic patients [age 31.9+/-1.3 years (mean+/-SEM) FEV1=92.1+/-2.4% (mean+/-SEM) 16 females/15 males]. Prior to the histamine challenge blood samples were obtained and stored at -70 degrees C until determination using ELISA. A significant correlation between serum NEP and the PC20-FEV1 (n=31, r=0.49, P<0.01) was found. The results suggest that serum NEP is modulating neuropeptide-induced effects in the pathophysiology of airway responsiveness.     

Synthesis and enzymatic evaluation of novel partially fluorinated thiol dual ACE/NEP inhibitors

A novel family of peptidomimetics incorporating fluoroalkyl groups, mainly a trifluoromethyl, in α-position to a zinc(II)-binding thiol function, was synthesized in solution as well as in solid-phase. These compounds showed inhibitory potency in the nanomolar range against both angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP), whereas no inhibition of endothelin-converting enzyme-1 (ECE-1) was observed. The trifluoromethyl-derivatives were more potent than the parent unfluorinated analogues in the case of ACE, and less potent in the case of NEP.     

Structural plasticity in influenza virus protein NS2 (NEP).

The cellular nuclear transport machinery relies on the assembly of specialized transport complexes between soluble transport receptors, transport substrates, and additional accessory proteins. This study focuses on the structural characteristics of influenza virus protein NS2 (NEP), which interacts with the nuclear export machinery during viral replication, and has been proposed to act as an adapter molecule between the nuclear export machinery and the viral ribonucleoprotein complex. For this purpose, we have purified recombinant NS2 under nondenaturing conditions, and have investigated its structure and aggregation state using optical spectroscopy, differential scanning calorimetry, as well as hydrodynamic techniques. Our results indicate that isolated NS2 exists as a monomer in solution, and adopts a compact, but very flexible conformation, which shows characteristics of the molten globule state under near physiological conditions. Proteolytic sensitivity suggests that, despite its overall plasticity, the structure of NS2 is heterogeneous. While the C terminus of the protein adopts a relatively rigid conformation, its N terminus, which is recognized by the nuclear export machinery, exists in a highly mobile and exposed state. It is proposed that the flexibility observed in the nuclear export domain of NS2 is an important element in the recognition of substrate proteins by the nuclear export machinery.     

Influenza A virus NEP (NS2 protein) downregulates RNA synthesis of model template RNAs

The influenza A virus NEP (NS2) protein is an structural component of the viral particle. To investigate whether this protein has an effect on viral RNA synthesis, we examined the expression of an influenza A virus-like chloramphenicol acetyltransferase (CAT) RNA in cells synthesizing the four influenza A virus core proteins (nucleoprotein, PB1, PB2, and PA) and NEP from recombinant plasmids. Influenza A virus NEP inhibited drastically, and in a dose-dependent manner, the level of CAT expression mediated by the recombinant influenza A virus polymerase. This inhibitory effect was not observed in an analogous artificial system in which expression of a synthetic CAT RNA is mediated by the core proteins of an influenza B virus. This result ruled out the possibility that inhibition of reporter gene expression was due to a general toxic effect induced by NEP. Analysis of the virus-specific RNA species that accumulated in cells expressing the type A recombinant core proteins and NEP showed that there was an important reduction in the levels of minireplicon-derived vRNA, cRNA, and mRNA molecules. Taken together, the results obtained suggest a regulatory role for NEP during virus-specific RNA synthesis, and this finding is discussed regarding the biological implications for the virus life cycle.     

New Function for an Old Enzyme: NEP Deficient Mice Develop Late-Onset Obesity

Background

According to the World Health Organization (WHO) there is a pandemic of obesity with approximately 300 million people being obese. Typically, human obesity has a polygenetic causation. Neutral endopeptidase (NEP), also known as neprilysin, is considered to be one of the key enzymes in the metabolism of many active peptide hormones.

Methodology/Principal Findings

An incidental observation in NEP-deficient mice was a late-onset excessive gain in body weight exclusively from a ubiquitous accumulation of fat tissue. In accord with polygenetic human obesity, mice were characterized by deregulation of lipid metabolism, higher blood glucose levels, with impaired glucose tolerance. The key role of NEP in determining body mass was confirmed by the use of the NEP inhibitor candoxatril in wild-type mice that increased body weight due to increased food intake. This is a peripheral and not a central NEP action on the switch for appetite control, since candoxatril cannot cross the blood-brain barrier. Furthermore, we demonstrated that inhibition of NEP in mice with cachexia delayed rapid body weight loss. Thus, lack in NEP activity, genetically or pharmacologically, leads to a gain in body fat.

Conclusions/Significance

In the present study, we have identified NEP to be a crucial player in the development of obesity. NEP-deficient mice start to become obese under a normocaloric diet in an age of 6–7 months and thus are an ideal model for the typical human late-onset obesity. Therefore, the described obesity model is an ideal tool for research on development, molecular mechanisms, diagnosis, and therapy of the pandemic obesity.     

Intracranial Injection of AAV Expressing NEP but Not IDE Reduces Amyloid Pathology in APP+PS1 Transgenic Mice

The accumulation of β-amyloid peptides in the brain has been recognized as an essential factor in Alzheimer’s disease pathology. Several proteases, including Neprilysin (NEP), endothelin converting enzyme (ECE), and insulin degrading enzyme (IDE), have been shown to cleave β-amyloid peptides (Aβ). We have previously reported reductions in amyloid in APP+PS1 mice with increased expression of ECE. In this study we compared the vector-induced increased expression of NEP and IDE. We used recombinant adeno-associated viral vectors expressing either native forms of NEP (NEP-n) or IDE (IDE-n), or engineered secreted forms of NEP (NEP-s) or IDE (IDE-s). In a six-week study, immunohistochemistry staining for total Aβ was significantly decreased in animals receiving the NEP-n and NEP-s but not for IDE-n or IDE-s in either the hippocampus or cortex. Congo red staining followed a similar trend revealing significant decreases in the hippocampus and the cortex for NEP-n and NEP-s treatment groups. Our results indicate that while rAAV-IDE does not have the same therapeutic potential as rAAV-NEP, rAAV-NEP-s and NEP-n are effective at reducing amyloid loads, and both of these vectors continue to have significant effects nine months post-injection. As such, they may be considered reasonable candidates for gene therapy trials in AD.     

Catabolism of calcitonin gene-related peptide and substance P by neutral endopeptidase.

Calcitonin gene-related peptide (CGRP) and substance P (SP) are released from sensory nerves upon exposure to irritating stimuli. Neutral endopeptidase (NEP), a membrane-bound peptidase, cleaves many peptides including SP, thereby limiting their biological actions. Recombinant NEP cleaved CGRP1 approximately 88-fold less rapidly than it cleaved SP. The slow cleavage by NEP of CGRP compared to SP suggests that this enzyme is likely to have weaker physiologic effects on CGRP than have been demonstrated for SP.