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.

Like other members of the epidermal growth factor family, heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a transmembrane protein that can be shed enzymatically to release a soluble growth factor. Ectodomain shedding is essential to the biological functions of HB-EGF and is strictly regulated. However, the mechanism that induces the shedding remains unclear. We have recently identified nardilysin (N-arginine dibasic convertase (NRDc)), a metalloendopeptidase of the M16 family, as a protein that specifically binds HB-EGF (Nishi, E., Prat, A., Hospital, V., Elenius, K., and Ectodomain shedding is an irreversible post-translational modification that releases the extracellular domain of membrane-anchored proteins through proteolysis. A broad spectrum of membrane proteins are susceptible to ectodomain shedding. Ectodomain shedding of most proteins occurs constitutively in resting cells, but can be rapidly and markedly induced by cell activation. However, the mechanism that induces shedding remains unclear (1-4).Heparin-binding epidermal growth factor-like growth factor (HB-EGF) 2 is synthesized as a transmembrane protein (pro-HB-EGF) that can be shed proteolytically to release a soluble growth factor (5-8). Metalloproteinases have been implicated as sheddases of pro-HB-EGF because various metalloproteinase inhibitors inhibit HB-EGF ectodomain shedding efficiently. MMP3 (matrix metalloproteinase-3), MMP7, ADAM9 (a disintegrin and metalloproteinase-9), ADAM10, ADAM12, and ADAM17 (tumor necrosis factor-␣-converting enzyme (TACE)) have all been suggested as the proteases responsible (9 -14). Ectodomain shedding of pro-HB-EGF is induced by various stimuli, including phorbol esters, calcium ionophore, and lysophosphatidic acid (6,15,16). With respect to the mechanism of the induction, however, very little is known.Ectodomain shedding of HB-EGF is indispensable for G-protein-coupled receptor-induced epidermal growth factor receptor (EGFR) transactivation, which plays critical roles in biological consequences of G-protein-coupled receptor activation (17). The physiological significance of HB-EGF ectodomain shedding was further underscored by the findings that knock-in mice harboring an uncleavable mutant construct of HB-EGF show phenotypes very similar to those of HB-EGF knock-out mice (e.g. hypertrophied cardiac valve and dilated heart) and that knock-in mice with the soluble mutant have even more severe phenotypes (18 -20). Notably, a similarly defective valvulogenesis was observed in both EGFR-and TACE-deficient mice, suggesting that HB-EGF-induced EGFR activation and TACE-induced HB-EGF shedding are required for valvulogenesis (19,21). In other words, ectodomain shedding of pro-HB-EGF is required for the activation of EGFR by HB-EGF. The same conclusion was obtained in a study that showed that most of the biological effects of EGFR ligands in cell culture are blocked by metalloprotease inhibitors (22). 2 The abbreviations used are: HB-EGF, heparin-binding epidermal growth factor-like...

The tumor suppressor protein p53 is known to induce either apoptosis or growth arrest depending on cellular background. We have previously reported that a bacterial redox protein azurin induces apoptosis in J774 cell line-derived macrophages whereas a site-directed mutant M44KM64E azurin shows very little cytotoxicity and fails to induce apoptosis in J774 cells. We now report that purified M44KM64E mutant azurin protein can enter both J774 cells as well as the human breast cancer MCF-7 cells. Entry of M44KM64E mutant azurin in J774 cells causes strong inhibition of cell-cycle progression at the G1 to S phase and a higher level of transcription of the p21 gene. Corresponding to high p21 levels, the levels of cyclins and cyclin-dependent kinases were greatly lowered in M44KM64E mutant azurin-treated J774 cells. Interestingly, M44KM64E mutant azurin protein failed to elicit inhibition of cell-cycle progression in MCF-7 cells, presumably because of mutation at the retinoblastoma tumor suppressor protein that allows functional E2F formation in MCF-7 cells even in the presence of high intracellular p21 level. Thus, the WT azurin induces apoptosis but little inhibition of cell-cycle progression whereas the M44KM64E mutant azurin is deficient in the induction of apoptosis but mediates strong inhibition of cell-cycle progression, demonstrating the role of a single bacterial protein and its hydrophobic patch in modulating two important functions of p53.

No abstract

Amyloid-b (Ab) 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 b-and c-secretases. Alternative cleavage of APP by a-secretase occurs within the Ab domain and precludes generation of Ab peptide. Three members of the ADAM (a disintegrin and metalloprotease) family of proteases, ADAM9, 10 and 17, are the main candidates for a-secretases. However, the mechanism that regulates a-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 a-secretase activity of ADAMs, which results in a decrease in the amount of Ab generated. When expressed with ADAMs in cells, NRDc dramatically increased the secretion of a-secretase-cleaved soluble APP and reduced the amount of Ab peptide generated. A peptide cleavage assay in vitro also showed that recombinant NRDc enhances ADAM17-induced cleavage of the peptide substrate corresponding to the a-secretase cleavage site of APP. A reduction of endogenous NRDc by RNA interference was accompanied by a decrease in the cleavage by a-secretase of APP and increase in the amount of Ab 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 a-secretase activity of ADAMs, which may be a novel target for the treatment of Alzheimer's disease. Keywords: a disintegrin and metalloprotease proteases, a-secretase, Alzheimer's disease, amyloid-b, ectodomain shedding, nardilysin. Alzheimer's disease (AD), a progressive neurodegenerative disorder, is characterized by a variety of pathological features in the brain such as extracellular senile plaques and intracellular neurofibrillary tangles. The main component of the extracellular senile plaques is amyloid b (Ab) peptide, which is derived from amyloid precursor protein (APP), a type I transmembrane protein, by two sequential proteolytic cleavages. The initial cleavage is mediated by b-secretase, b-site APP-cleaving enzyme 1, at the N-terminus of the Ab domain, which generates a soluble N-terminal fragment (bsecretase-cleaved soluble APP; sAPPb) and a transmembrane C-terminal fragment (C99). C99 is susceptible to a second intramembrane cleavage by c-secretase, releasing the 4-kDa