Nardilysin Research Articles

Associations among plasma markers for N-methyl-d-aspartate receptor hypofunction, redox dysregulation, and insufficient myelination in patients with schizophrenia

BackgroundSeveral hypotheses regarding the pathomechanisms of schizophrenia have been proposed. If schizophrenia is a unitary disease, then these pathological processes must be linked; however, if such links do not exist, schizophrenia may best be considered a group of disorders. Only a few studies have examined the relationships among these pathomechanisms. Herein, we examined the relationships among deficient myelination, NMDA receptor hypofunction, and metabolic dysregulation by measuring various plasma markers and examining their correlations. MethodsPlasma samples were collected from 90 patients with schizophrenia and 68 healthy controls. Concentrations of nardilysin (N-arginine dibasic convertase, NRDC), a positive regulator of myelination, the NMDA receptor co-agonist d-serine and glycine, various additional amino acids related to NMDA receptor transmission (glutamate, glutamine, and l-serine), and homocysteine (Hcy), were measured. Concentrations were compared using independent samples t-test or logistic regression, and associations were evaluated using Pearson's correlation coefficients. ResultsPlasma glycine (t = 2.05, p = 0.042), l-serine (t = 2.25, p = 0.027), and homocysteine (t = 3.71, p < 0.001) concentrations were significantly higher in patients with schizophrenia compared to those in healthy controls. Logistic regression models using age, sex, smoking status, glutamine, glutamate, glycine, l-serine, d-serine, homocysteine, and NRDC as independent variables revealed significantly lower plasma d-serine (p = 0.024) and NRDC (p = 0.028), but significantly higher l-serine (p = 0.024) and homocysteine (p = 0.001) in patients with schizophrenia. Several unique correlations were found between NMDA receptor-related amino acids and NRDC in patients with schizophrenia compared to those in healthy controls, while no correlations were found between plasma homocysteine and other markers. No associations were found between plasma marker concentrations and disease status or cognitive function in patients with schizophrenia, except for a significant correlation between plasma glycine and full intelligence quotient. ConclusionReduced myelination and NMDA receptor hypofunction may be related to pathological mechanisms in schizophrenia, while homocysteine dysregulation appears to be an independent pathological process. These results suggest that schizophrenia may be a group of disorders with unique or partially overlapping etiologies.

Serum Nardilysin as a Prognostic Biomarker in Pancreatic Ductal Adenocarcinoma.

Background: Nardilysin, (N-arginine dibasic convertase, NRDC) has been reported to play an important role in cancer progression, and is associated with tumor proliferation signals and inflammatory signals, such as tumor necrosis factor-a (TNF-a) and heparin-binding epidermal growth factor-like growth factor (HB-EGF), through the activation of disintegrin and metalloproteinase (ADAM) proteases. NRDC has recently been revealed to be involved in the tumorigenesis of various types of cancer, including intrahepatic cholangiocarcinoma, malignant cerebral infarction, esophageal squamous cell carcinoma, and gastric cancer. However, the expression profiles and biological relevance of NRDC in pancreatic ductal adenocarcinoma have rarely been reported. Methods: We analyzed the NRDC expression profile in pancreatic ductal adenocarcinoma by enzyme-linked immunosorbent assay (ELISA) and identified NRDC as a circulating biomarker in the serum of 112 pancreatic ductal adenocarcinoma patients. The diagnostic value of NRDC was analyzed by the area under the curve (AUC) and the receiver operating characteristic (ROC) test. Results: Our results demonstrated that the clinical prognosis significance of NRDC with the clinical characteristics in pancreatic ductal adenocarcinoma (PDAC). NRDC was notably decreased in PDAC patient serum compared with the control group (p < 0.001). Furthermore, the present study found that the NRDC expression level was correlated with T grade (p < 0.001), metastasis(p < 0.001), differentiation(p < 0.001), and TNM stage (p = 0.011). Further bioinformatics analysis revealed that NRDC correlated with proliferation and migration pathways; in particular, it mediated cell-matrix adhesion-dependent activation in pancreatic ductal adenocarcinoma. Conclusions: Serum NRDC may play a useful diagnostic biomarker to evaluate the aggressive clinical features in PAAD patients.

Environmentally relevant concentrations of fluconazole alter the embryonic development, oxidative status, and gene expression of NRF1, NRF2, WNT3A, WNT8A, NRD1, and NRD2 of Danio rerio embryos

Up to date, there is little information published concerning fluconazole (FCZ) toxicity at environmentally relevant concentrations. Bearing in mind the above background of FCZ, we aimed to evaluate the embryotoxic effects environmentally relevant concentrations of FCZ (800-1000 ng/L) may induce in Danio rerio. Moreover, we also wanted to prove whether these FCZ concentrations could generate oxidative stress and alter the expression of several genes related to the antioxidant mechanisms, sterol and retinol biosynthesis, and embryogenesis. Our findings demonstrate that FCZ, at all concentrations, induced pericardial edema, yolk sac deformation, scoliosis, and tail malformation in embryos. Moreover, we also demonstrated this drug altered the redox equilibrium of fish, promoting the production of lipoperoxidation level, hydroperoxide content, and protein carbonyl content in a concentration-dependent manner. Concerning gene expression, FCZ downregulated wingless-type MMTV integration site family member 3a (WNT3A), wingless-type MMTV integration site family member 8a (WNT8A), N-arginine dibasic convertase 1 (NRD1), and N-arginine dibasic convertase 2 (NRD2) and upregulated cytochrome P450 family 26 subfamily a member 1 (CYP26A1), cytochrome P450 family 26 subfamily a member 1B (CYP261B), nuclear respiratory factor 1 (NRF1), and nuclear respiratory factor 2 (NRF2) in D. rerio larvae. Collectively, our results point out that FCZ, at low concentrations, may alter the embryogenesis, oxidative status, and expression of several genes in D. rerio embryos via an impairment in sterol and retinol biosynthesis. Thus, our results provide some of the first evidence that FCZ, even at environmentally relevant concentrations, is harmful to aquatic species.

P2560Metalloprotease nardilysin controls heart rate through the transcriptional regulation of ion channels critical for sinus automaticity

Abstract Nardilysin (NRDC; N-arginine dibasic convertase) is a metalloprotease of the M16 family. We have demonstrated that NRDC in the extracellular space enhances ectodomain cleavage of multiple membrane proteins. We also reported that nuclear NRDC regulates transcription of several target genes as a transcriptional coregulator. These results indicated that NRDC is a protease having localization-dependent multiple functions. NRDC-deficient mice (Nrdc−/−) showed wide range of phenotypes such as hypomyelination, hypothermia, and bradycardia. In this study, we have explored a role of NRDC in the regulation of heart rate. (1) Pharmacological blocking of autonomic nervous system revealed that an intrinsic heart rate of Nrdc−/− was significantly reduced compared with that of wild-type mice. (2) In Nrdc−/− hearts, mRNA levels of Cav3.1 and HCN1/4, ion channels responsible for sinus automaticity, were significantly reduced. (3) Funny (If) current and T-type Ca current measured in the sinus node cells were markedly reduced in Nrdc−/− cells, indicating that the functions of Cav3.1 and HCN4 are impaired. (4) Gene knockdown of NRDC in primary rat ventricular myocyte led to the reduction of mRNA level of HCN4. (5) Chromatin immunoprecipitation-PCR analysis showed that NRDC binds to the promoter region of Cav3.1 and HCN4, suggesting the direct involvement of NRDC in transcriptional regulation of these ion channels. (6) Atrium-specific Nrdc−/− (Sarcolipin-Cre) showed mild bradycardia and reduced Cav3.1 mRNA expression. Together, our results indicated that NRDC in cardiomyocyte controls heart rate through the transcriptional regulation of ion channels critical for sinus automaticity. Acknowledgement/Funding KAKENHI (17K09575)

Increased serum nardilysin is associated with worse long-term outcome of ST-elevation myocardial infarction.

Nardilysin (N-arginine dibasic convertase, NRDC) is a kind of metalloendopeptidase associated with several inflammatory diseases. NRDC is reported to be eligible for early detection of acute coronary syndrome. However, the availability and effectiveness of NRDC in predicting the prognosis of patients with ST-elevation myocardial infarction has never been investigated. From January 2010 to January 2012, the prospective observational cohort study enrolled a total of 396 STEMI patients, who were sampled with blood within 24 hours after admission. A long-term follow-up was conducted to record the primary endpoint of all-cause mortality, as well as secondary endpoint of myocardial infarction, stroke, emergent revascularization and admission due to heart failure. Hazard ratio (HR) related to the serum NRDC level was estimated by Cox regression model. The enrolled patients completed the follow-up with an average of 4.6 ± 0.5 years, of whom 24 patients died (primary endpoint, 6.1%), while 89 episodes of secondary endpoints occurred (22.5%). Patients with highest quartile level of NRDC (Q4 level, > 2041 pg/ml) were subjected to higher risk of all-cause death [HR 3.973, 95% CI (1.648-9.575), p = 0.002] compared to patients with lower three quartiles level of NRDC (Q1 to Q3, < 2041 pg/ml), while there was no difference in adverse events (p = 0.403). The increased serum NRDC level at admission is associated with a higher risk of all-cause mortality for ST-elevation myocardial infarction patients.

Nardilysin controls intestinal tumorigenesis through HDAC1/p53-dependent transcriptional regulation.

Colon cancer is a complex disease affected by a combination of genetic and epigenetic factors. Here we demonstrate that nardilysin (N-arginine dibasic convertase; NRDC), a metalloendopeptidase of the M16 family, regulates intestinal tumorigenesis via its nuclear functions. NRDC is highly expressed in human colorectal cancers. Deletion of the Nrdc gene in ApcMin mice crucially suppressed intestinal tumor development. In ApcMin mice, epithelial cell-specific deletion of Nrdc recapitulated the tumor suppression observed in Nrdc-null mice. Moreover, epithelial cell-specific overexpression of Nrdc significantly enhanced tumor formation in ApcMin mice. Notably, epithelial NRDC controlled cell apoptosis in a gene dosage-dependent manner. In human colon cancer cells, nuclear NRDC directly associated with HDAC1, and controlled both acetylation and stabilization of p53, with alterations of p53 target apoptotic factors. These findings demonstrate that NRDC is critically involved in intestinal tumorigenesis through its epigenetic regulatory function, and targeting NRDC may lead to a novel prevention or therapeutic strategy against colon cancer.

Insulin-like growth factor-1 activates different catalytic subunits p110 of PI3K in a cell-type-dependent manner to induce lipogenesis-dependent epithelial-mesenchymal transition through the regulation of ADAM10 and ADAM17.

The activation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) is critical for the induction of epithelial-mesenchymal transition (EMT) by growth factors, including insulin-like growth factor 1 (IGF-1). The activation of intracellular lipogenesis provides proliferative and survival signals for cancer cells. In this study, we investigated the connection between lipogenesis-related EMT processes and IGF-1-mediated PI3K p110 isoform activation in primary (SW480 cells) and metastatic (SW620) colon carcinoma cells. We also examined the underlying signaling pathway that promotes fatty acid synthesis in IGF-1-activated colon cancer cells. IGF-1 stimulation upregulated the expression of lipogenic enzymes as well as the activation of Nardilysin (N-arginine dibasic convertase, NRD1) and its downstream targets, a disintegrin and metalloproteases 10 (ADAM10) and ADAM17. The upregulation of the Lyn/Syk-mediated PI3K p110δ isoform in SW480 cells and the Lyn-dependent PI3K p110α isoform in SW620 cells triggered fatty acid production and cell motility in IGF-1-activated colon cancer cells. Pharmacological inhibition with A66 (PI3K p110α specific inhibitor) and CAL-101 (PI3K p110δ specific inhibitor) efficiently inhibited EMT in colon cancer cells by blocking the NRD1/ADAM family protein signaling pathway. Gene silencing of NRD1 and ADAM family proteins attenuated the generation of intracellular fatty acid and the migratory activity of colon cancer cells. Our results suggest that the different isoforms of the PI3K p110 subunit could be therapeutic targets for primary and metastatic colon cancer and that regulation of the NRD1/ADAM signaling pathway controls lipogenesis-mediated EMT in IGF-1-stimulated colon cancer cells.

Nardilysin regulates inflammation, metaplasia, and tumors in murine stomach

Chronic inflammation contributes to a wide variety of human disorders. In the stomach, longstanding gastritis often results in structural alterations in the gastric mucosa, including metaplastic changes and gastric cancers. Therefore, it is important to elucidate factors that are involved in gastric inflammation. Nardilysin (N-arginine dibasic convertase; Nrdc) is a metalloendopeptidase of the M16 family that promotes ectodomain shedding of the precursor forms of various growth factors and cytokines by enhancing the protease activities of a disintegrin and metalloproteinase (ADAM) proteins. Here, we have demonstrated that Nrdc crucially regulates gastric inflammation caused by Helicobacter felis infection or forced expression of prostaglandin E2 in K19-C2mE mice. Metaplastic changes following gastric inflammation were suppressed by the deletion of Nrdc. Furthremore, the deletion of Nrdc significantly suppressed N-methyl-N-nitrosourea (MNU)-induced gastric tumorigenesis in the murine stomach. These data may lead to a global therapeutic approach against various gastric disorders by targeting Nrdc.

Nardilysin Is Required for Maintaining Pancreatic β-Cell Function.

Type 2 diabetes (T2D) is associated with pancreatic β-cell dysfunction, manifested by reduced glucose-stimulated insulin secretion (GSIS). Several transcription factors enriched in β-cells, such as MafA, control β-cell function by organizing genes involved in GSIS. Here we demonstrate that nardilysin (N-arginine dibasic convertase; Nrd1 and NRDc) critically regulates β-cell function through MafA. Nrd1(-/-) mice showed glucose intolerance and severely decreased GSIS. Islets isolated from Nrd1(-/-) mice exhibited reduced insulin content and impaired GSIS in vitro. Moreover, β-cell-specific NRDc-deficient (Nrd1(delβ)) mice showed a diabetic phenotype with markedly reduced GSIS. MafA was specifically downregulated in islets from Nrd1(delβ) mice, whereas overexpression of NRDc upregulated MafA and insulin expression in INS832/13 cells. Chromatin immunoprecipitation assay revealed that NRDc is associated with Islet-1 in the enhancer region of MafA, where NRDc controls the recruitment of Islet-1 and MafA transcription. Our findings demonstrate that NRDc controls β-cell function via regulation of the Islet-1-MafA pathway.

Loss of Peripheral Protection in Pancreatic Islets by Proteolysis-Driven Impairment of VTCN1 (B7-H4) Presentation Is Associated with the Development of Autoimmune Diabetes.

Ag-specific activation of T cells is an essential process in the control of effector immune responses. Defects in T cell activation, particularly in the costimulation step, have been associated with many autoimmune conditions, including type 1 diabetes (T1D). Recently, we demonstrated that the phenotype of impaired negative costimulation, due to reduced levels of V-set domain-containing T cell activation inhibitor 1 (VTCN1) protein on APCs, is shared between diabetes-susceptible NOD mice and human T1D patients. In this study, we show that a similar process takes place in the target organ, as both α and β cells within pancreatic islets gradually lose their VTCN1 protein during autoimmune diabetes development despite upregulation of the VTCN1 gene. Diminishment of functional islet cells' VTCN1 is caused by the active proteolysis by metalloproteinase N-arginine dibasic convertase 1 (NRD1) and leads to the significant induction of proliferation and cytokine production by diabetogenic T cells. Inhibition of NRD1 activity, alternatively, stabilizes VTCN1 and dulls the anti-islet T cell responses. Therefore, we suggest a general endogenous mechanism of defective VTCN1 negative costimulation, which affects both lymphoid and peripheral target tissues during T1D progression and results in aggressive anti-islet T cell responses. This mechanism is tied to upregulation of NRD1 expression and likely acts in two synergistic proteolytic modes: cell-intrinsic intracellular and cell-extrinsic systemic. Our results highlight an importance of VTCN1 stabilization on cell surfaces for the restoration of altered balance of immune control during T1D.

Deletion of nardilysin prevents the development of steatohepatitis and liver fibrotic changes.

Nonalcoholic steatohepatitis (NASH) is an inflammatory form of nonalcoholic fatty liver disease that progresses to liver cirrhosis. It is still unknown how only limited patients with fatty liver develop NASH. Tumor necrosis factor (TNF)-α is one of the key molecules in initiating the vicious circle of inflammations. Nardilysin (N-arginine dibasic convertase; Nrd1), a zinc metalloendopeptidase of the M16 family, enhances ectodomain shedding of TNF-α, resulting in the activation of inflammatory responses. In this study, we aimed to examine the role of Nrd1 in the development of NASH. Nrd1+/+ and Nrd1−/− mice were fed a control choline-supplemented amino acid-defined (CSAA) diet or a choline-deficient amino acid-defined (CDAA) diet. Fatty deposits were accumulated in the livers of both Nrd1+/+ and Nrd1−/− mice by the administration of the CSAA or CDAA diets, although the amount of liver triglyceride in Nrd1−/− mice was lower than that in Nrd1+/+ mice. Serum alanine aminotransferase levels were increased in Nrd1+/+ mice but not in Nrd1−/− mice fed the CDAA diet. mRNA expression of inflammatory cytokines were decreased in Nrd1−/− mice than in Nrd1+/+ mice fed the CDAA diet. While TNF-α protein was detected in both Nrd1+/+ and Nrd1−/− mouse livers fed the CDAA diet, secretion of TNF-α in Nrd1−/− mice was significantly less than that in Nrd1+/+ mice, indicating the decreased TNF-α shedding in Nrd1−/− mouse liver. Notably, fibrotic changes of the liver, accompanied by the increase of fibrogenic markers, were observed in Nrd1+/+ mice but not in Nrd1−/− mice fed the CDAA diet. Similar to the CDAA diet, fibrotic changes were not observed in Nrd1−/− mice fed a high-fat diet. Thus, deletion of nardilysin prevents the development of diet-induced steatohepatitis and liver fibrogenesis. Nardilysin could be an attractive target for anti-inflammatory therapy against NASH.

Nardilysin prevents amyloid plaque formation by enhancing α-secretase activity in an Alzheimer's disease mouse model

Amyloid beta (Aβ) peptide, the main component of senile plaques in patients with Alzheimer's disease (AD), is derived from proteolytic cleavage of amyloid precursor protein (APP) by β- and γ-secretases. Alpha-cleavage of APP by α-secretase has a potential to preclude the generation of Aβ because it occurs within the Aβ domain. We previously reported that a metalloendopeptidase, nardilysin (N-arginine dibasic convertase; NRDc) enhances α-cleavage of APP, which results in the decreased generation of Aβ in vitro. To clarify the in vivo role of NRDc in AD, we intercrossed transgenic mice expressing NRDc in the forebrain with an AD mouse model. Here we demonstrate that the neuron-specific overexpression of NRDc prevents Aβ deposition in the AD mouse model. The activity of α-secretase in the mouse brain was enhanced by the overexpression of NRDc, and was reduced by the deletion of NRDc. However, reactive gliosis adjacent to the Aβ plaques, one of the pathological features of AD, was not affected by the overexpression of NRDc. Taken together, our results indicate that NRDc controls Aβ formation through the regulation of α-secretase.

Abstract LB-91: Transcriptome analysis of esophageal squamous cell carcinoma by SAGE identifies a new biomarker, NRD1

Abstract Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide. Because most ESCC is diagnosed at advanced stages, the overall 5-year survival rate of ESCC still remains low in spite of the use of various treatment modalities. Therefore, identification of new diagnostic markers and therapeutic targets for ESCC is important. Serial Analysis of Gene Expression (SAGE) is one of the common comprehensive methods to analyze transcript expression levels, and enables the quantitative and simultaneous analysis of a large number of transcripts. To identify potential diagnostic markers and therapeutic targets for ESCC, the SAGE library was generated from one ESCC sample. A total of 14,430 tags were generated, including 5,765 that were unique. By comparing SAGE tags from the ESCC sample with those from normal human squamous esophagus, several genes that is upregulated in ESCC compared with normal squamous esophagus in ESCC were identified. Among these, we focused on the nardilysin (N-arginine dibasic convertase, NRD1) gene. NRD1 has been reported to be associated with ectodomein shedding of TNF-α through activation of ADAM proteases. There is only one report in the literature mentioning the association of NRD1 with cancer (breast cancer), however, there is no report that links expression of NRD1 and ESCC. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed a high level of NRD1 expression in three of five esophageal cancer cell lines and seven of 14 ESCC samples, but not in 14 normal systemic tissues. Western blot analysis also showed upregulation of NRD1 protein in total five esophageal cancer cell lines. Immunohistochemical analysis revealed expression of NRD1 in cytoplasm of 43 out of 109 ESCC cases (39%), as well as heart, skeletal muscle, and testis in agreement with previous reports. In clinicopathological analysis, expression of NRD1 correlated with the advanced T grade, N grade and tumor stage, and was an independent predictor of survival in patients with ESCC. Knockdown of NRD1 in TE5 cells inhibited cell invasion ability. Furthermore, qRT-PCR analysis showed a reduction of matrix metalloproteinase-2 (MMP-2) expression in NRD1 knocked down cells. In conclusion, NRD1 is an independent predictor of survival, and contribute to cell invasion through regulation of MMP-2 in ESCC. NRD1 could be a novel diagnostic and therapeutic target for ESCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-91. doi:1538-7445.AM2012-LB-91

Retinoic acid-induced upregulation of the metalloendopeptidase nardilysin is accelerated by co-expression of the brain-specific protein p42 IP4 (centaurin α1; ADAP1) in neuroblastoma cells

The mainly neuronally expressed protein p42 IP4 (centaurin α1; ADAP1), which interacts with the metalloendopeptidase nardilysin (NRD) was found to be localized in neuritic plaques in Alzheimer disease (AD) brains. NRD was shown to enhance the cleavage of the amyloid precursor protein (APP) by α-secretases, thereby increasing the release of neuroprotective sAPPα. We here investigated in vitro the biochemical interaction of p42 IP4 and NRD and studied the physiological interaction in SH-SY5Y cells. NRD is a member of the M16 family of metalloendopeptidases. Some members of this M16 family act bi-functionally, as protease and as non-enzymatic scaffold protein. Here, we show that p42 IP4 enhances the enzymatic activity of NRD 3–4 times. However, p42 IP4 is not a substrate for NRD. Furthermore, we report that differentiation of SH-SY5Y cells by stimulation with 10 μM retinoic acid (RA) results in upregulation of NRD protein levels, with a 6-fold rise after 15 days. NRD is expressed in the neurites of RA-stimulated SH-SY5Y cells, and localized in vesicular structures. Since p42 IP4 is not expressed in untreated SH-SY5Y cells, we could use this cell system as a model to find out, whether there is a functional interaction. Interestingly, SH-SY5Y cells, which we stably transfected with GFP-tagged-p42 IP4 showed an enhanced NRD protein expression already at an earlier time point after RA stimulation.

Nardilysin regulates axonal maturation and myelination in the central and peripheral nervous system

Axonal maturation and myelination are essential processes for establishing an efficient neuronal signaling network. We found that nardilysin (N-arginine dibasic convertase, also known as Nrd1 and NRDc), a metalloendopeptidase enhancer of protein ectodomain shedding, is a critical regulator of these processes. Nrd1-/- mice had smaller brains and a thin cerebral cortex, in which there were less myelinated fibers with thinner myelin sheaths and smaller axon diameters. We also found hypomyelination in the peripheral nervous system (PNS) of Nrd1-/- mice. Neuron-specific overexpression of NRDc induced hypermyelination, indicating that the level of neuronal NRDc regulates myelin thickness. Consistent with these findings, Nrd1-/- mice had impaired motor activities and cognitive deficits. Furthermore, NRDc enhanced ectodomain shedding of neuregulin1 (NRG1), which is a master regulator of myelination in the PNS. On the basis of these data, we propose that NRDc regulates axonal maturation and myelination in the CNS and PNS, in part, through the modulation of NRG1 shedding.

Ectodomain shedding of TNF-α is enhanced by nardilysin via activation of ADAM proteases

Tumor necrosis factor-α (TNF-α) is released from cells by proteolytic cleavage of a membrane-anchored precursor. The TNF-α-converting enzyme (TACE/ADAM17) is the major sheddase for ectodomain shedding of TNF-α. At present, however, it is poorly understood how its catalytic activity is regulated. Here, we show that nardilysin ( N-arginine dibasic convertase; NRDc) enhanced TNF-α shedding. In a cell-based shedding assay, expression of NRDc synergistically enhanced TACE-induced TNF-α shedding. A peptide cleavage assay in vitro showed that recombinant NRDc enhances the cleavage of TNF-α induced by TACE. Notably, co-incubation of NRDc completely reversed the inhibitory effect of a physiological concentration of salt on TACE’s activity in vitro. Overexpression of NRDc in TACE-deficient fibroblasts resulted in an increase in the amount of TNF-α released. Co-expression of NRDc with ADAM10 promoted the release compared with the sole expression of ADAM10. These results suggested that NRDc enhances TNF-α shedding through activation of not only TACE but ADAM10. Our results indicate the involvement of NRDc in ectodomain shedding of TNF-α, which may be a novel target for anti-inflammatory therapies.

Enhancement of α‐secretase cleavage of amyloid precursor protein by a metalloendopeptidase nardilysin

Amyloid-beta (Abeta) peptide, the principal component of senile plaques in the brains of patients with Alzheimer's disease, is derived from proteolytic cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. Alternative cleavage of APP by alpha-secretase occurs within the Abeta domain and precludes generation of Abeta peptide. Three members of the ADAM (a disintegrin and metalloprotease) family of proteases, ADAM9, 10 and 17, are the main candidates for alpha-secretases. However, the mechanism that regulates alpha-secretase activity remains unclear. We have recently demonstrated that nardilysin (EC 3.4.24.61, N-arginine dibasic convertase; NRDc) enhances ectodomain shedding of heparin-binding epidermal growth factor-like growth factor through activation of ADAM17. In this study, we show that NRDc enhances the alpha-secretase activity of ADAMs, which results in a decrease in the amount of Abeta generated. When expressed with ADAMs in cells, NRDc dramatically increased the secretion of alpha-secretase-cleaved soluble APP and reduced the amount of Abeta peptide generated. A peptide cleavage assay in vitro also showed that recombinant NRDc enhances ADAM17-induced cleavage of the peptide substrate corresponding to the alpha-secretase cleavage site of APP. A reduction of endogenous NRDc by RNA interference was accompanied by a decrease in the cleavage by alpha-secretase of APP and increase in the amount of Abeta generated. Notably, NRDc is clearly expressed in cortical neurons in human brain. Our results indicate that NRDc is involved in the metabolism of APP through regulation of the alpha-secretase activity of ADAMs, which may be a novel target for the treatment of Alzheimer's disease.

Miniglucagon (MG)-Generating Endopeptidase, which Processes Glucagon into MG, Is Composed of N-Arginine Dibasic Convertase and Aminopeptidase B

Miniglucagon (MG), the C-terminal glucagon fragment, processed from glucagon by the MG-generating endopeptidase (MGE) at the Arg17-Arg18 dibasic site, displays biological effects opposite to that of the mother-hormone. This secondary processing occurs in the glucagon- and MG-producing alpha-cells of the islets of Langerhans and from circulating glucagon. We first characterized the enzymatic activities of MGE in culture media from glucagon and MG-secreting alphaTC1.6 cells as made of a metalloendoprotease and an aminopeptidase. We observed that glucagon is a substrate for N-arginine dibasic convertase (NRDc), a metalloendoprotease, and that aminopeptidase B cleaves in vitro the intermediate cleavage products sequentially, releasing mature MG. Furthermore, immunodepletion of either enzyme resulted in the disappearance of the majority of MGE activity from the culture medium. We found RNAs and proteins corresponding to both enzymes in different cell lines containing a MGE activity (mouse alphaTC1.6 cells, rat hepatic FaO, and rat pituitary GH4C1). Using confocal microscopy, we observed a granular immunostaining of both enzymes in the alphaTC1.6 and native rat alpha-cells from islets of Langerhans. By immunogold electron microscopy, both enzymes were found in the mature secretory granules of alpha-cells, close to their substrate (glucagon) and their product (MG). Finally, we found NRDc only in the fractions from perfused pancreas that contain glucagon and MG after stimulation by hypoglycemia. We conclude that MGE is composed of NRDc and aminopeptidase B acting sequentially, providing a molecular basis for this uncommon regulatory process, which should be now addressed in both physiological and pathophysiological situations.

Post-ischemic administration of heparin-binding epidermal growth factor-like growth factor (HB-EGF) reduces infarct size and modifies neurogenesis after focal cerebral ischemia in the rat.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a hypoxia-inducible, neuroprotective protein that also stimulates proliferation of neuronal precursor cells. Accordingly, HB-EGF may contribute to recovery from cerebral injury through direct neuroprotective effects, by enhancing neurogenesis, or both. When administered by the intracerebroventricular route 1-3 days after focal cerebral ischemia in adult rats, HB-EGF decreased the volume of the resulting infarcts and reduced post-ischemic neurological deficits. HB-EGF also increased the incorporation of bromodeoxyuridine into cells expressing the immature neuronal marker protein TUC-4 in the dentate subgranular and rostral subventricular zones, consistent with increased proliferation of neuronal precursors. However, HB-EGF decreased the number of newborn neurons that migrated into the ischemic striatum, perhaps partly because reduction of infarct size by HB-EGF also reduced the stimulus to migration. To determine if HB-EGF might also directly inhibit migration of neuronal precursors, we co-cultured subventricular zone (SVZ) explants treated with HB-EGF or vehicle together with hypoxic cerebral cortical explants, and measured cell migration from the former toward the latter. HB-EGF reduced directed migration of SVZ cells toward the cortical explants, possibly due to a local chemoattractant effect on neuronal precursor cells, which may be mediated through the HB-EGF-specific receptor, N-arginine dibasic convertase. The delayed neuroprotective effect of HB-EGF may have implications for efforts to prolong the therapeutic window for intervention in stroke.

Nardilysin cleaves peptides at monobasic sites.

Nardilysin (N-arginine dibasic convertase, EC 3.4.24.61) was first identified on the basis of its ability to cleave peptides containing an arginine dibasic pair, i.e., Arg-Arg or Arg-Lys. However, it was observed that an aromatic residue adjacent to the dibasic pair (i.e., Phe-Arg-Lys) could alter the cleavage site. In this study we determined whether nardilysin can cleave peptides at a single basic residue. Nardilysin cleaves beta-endorphin at the monobasic site, Phe(17)-Lys(18), with a k(cat)/K(m) of 2 x 10(8) M(-)(1) min(-)(1). This can be compared to a k(cat)/K(m) of 8.5 x 10(8) M(-)(1) min(-)(1) for cleavage between a dibasic pair in dynorphin B-13. Nardilysin also cleaves calcitonin at His-Arg and somatostatin-14 at Cys-Lys. We examined the hydrolysis of fluorogenic peptides based on the beta-endorphin 12-24 sequence, Abz-T-P-L-V-T-L-X(1)-X(2)-N-A-I-I-K-Q-EDDnp. Nardilysin hydrolyzes the peptides when X(1)-X(2) = F-K, F-R, W-K, M-K, Y-K, and L-K. The kinetics of cleavage at F-K and F-R are similar; however, K-F is not hydrolyzed. Nardilysin cleaves at two monobasic sites M-K and F-R of the kallidin model peptide Abz-MISLMKRPPGFSPFRSSRI-NH(2), releasing desArg(10) kallidin (KRPPGFSPF). However, nardilysin does not release desArg(10) kallidin from the physiological precursor low molecular weight kininogen. These studies extend the range of potential substrates for nardilysin and further substantiate that nardilysin is a true peptidase.