Biochemical analysis of neutral endopeptidase activity reveals independent catabolism of atrial and brain natriuretic peptide

Recent reports presented contradictory results regarding the catabolism of mature atrial (ANP) and brain (BNP) natriuretic peptides in circulation. Especially the role of neutral endopeptidase (NEP) in BNP degradation was conversely discussed. Our present in vitro-studies characterize the NEP-dependent metabolism of ANP and BNP in different tissues via HPLC-analysis using NEP-deficient mice and specific NEP inhibitors. Our results show a strong tissue-dependent degradation pattern of both peptides, which are not only due to the different NEP activities in these tissues. Whereas NEP rapidly degraded ANP, it had no influence in BNP-metabolism. Additional experiments with purified NEP confirmed this result. Moreover, we describe a degradation of ANP and BNP in NEP-deficient- and NEP-inhibited membranes. Consequently, we postulate the existence of at least one further natriuretic peptide (NP) degrading enzyme, which has not been characterized yet. Thus, the commonly accepted model of the natriuretic peptide system with NEP as the central degrading peptidase has to be partly revised. Moreover, the NEP-independent BNP degradation provides an effective means for achieving a beneficial BNP increase in cardiovascular pathology by inhibiting the assumed novel NP-degrading peptidase(s).     

An insect brain peptide as a member of insulin family

Amino acid sequencing of bombyxin (previously called 4K-PTTH) isolated from the heads of the silkmoth Bombyx mori has disclosed sequence homology of this insect neuropeptide with insulin. Immunohistochemistry using an antibody against a synthetic bombyxin fragment detected 4 pairs of immunoreactive neurosecretory cells in the dorso-medial region of the Bombyx brain. The same cells were reactive to bovine insulin antibody.     

Loss of proteostasis induced by amyloid beta peptide in brain endothelial cells

Abnormal accumulation of amyloid-β (Aβ) peptide in the brain is a pathological hallmark of Alzheimer's disease (AD). In addition to neurotoxic effects, Aβ also damages brain endothelial cells (ECs) and may thus contribute to the degeneration of cerebral vasculature, which has been proposed as an early pathogenic event in the course of AD and is able to trigger and/or potentiate the neurodegenerative process and cognitive decline. However, the mechanisms underlying Aβ-induced endothelial dysfunction are not completely understood. Here we hypothesized that Aβ impairs protein quality control mechanisms both in the secretory pathway and in the cytosol in brain ECs, leading cells to death. In rat brain RBE4 cells, we demonstrated that Aβ_1–40 induces the failure of the ER stress-adaptive unfolded protein response (UPR), deregulates the ubiquitin–proteasome system (UPS) decreasing overall proteasome activity with accumulation of ubiquitinated proteins and impairs the autophagic protein degradation pathway due to failure in the autophagic flux, which culminates in cell demise. In conclusion, Aβ deregulates proteostasis in brain ECs and, as a consequence, these cells die by apoptosis.     

Neonatal lethality in mice deficient in XCE, a novel member of the endothelin-converting enzyme and neutral endopeptidase family.

XCE, a new member of the endothelin-converting enzyme and neutral endopeptidase family, is preferentially expressed in specific areas of the central nervous system including spinal chord and medulla. To elucidate the importance and function of XCE, we disrupted its gene in mouse embryonic stem cells by homologous recombination and created mice deficient in XCE. The resulting phenotype is characterized by neonatal lethality. All XCE -/- homozygous mice died of respiratory failure shortly after birth, and in most cases their lungs were never ventilated. Apart from the atelectasis, anatomical and histological examinations of embryonic day 18.5 XCE -/- embryos and newborn homozygotes did not reveal any obvious abnormalities in organs and tissues. Malformations that are related to the knock-out were also not found in the skeletons of XCE -/- mice. In addition, XCE knock-out animals showed no deficiency of pulmonary surfactant proteins and had normal heart beat frequencies. Taken together, our results demonstrate that XCE is an essential gene. The phenotype of the XCE-deficient mice together with the central nervous system-specific expression further suggest that XCE may play a vital role in the control of respiration.     

Autoantibody-catalyzed hydrolysis of amyloid beta peptide

We describe IgM class human autoantibodies that hydrolyze amyloid beta peptide 1-40 (Abeta40). A monoclonal IgM from a patient with Waldenstr?m's macroglobulinemia hydrolyzed Abeta40 at the Lys-28-Gly-29 bond and Lys-16-Ala-17 bonds. The catalytic activity was inhibited stoichiometrically by an electrophilic serine protease inhibitor. Treatment with the catalytic IgM blocked the aggregation and toxicity of Abeta40 in neuronal cell cultures. IgMs purified from the sera of patients with Alzheimer disease (AD) hydrolyzed Abeta40 at rates superior to IgMs from age-matched humans without dementia. IgMs from non-elderly humans expressed the least catalytic activity. The reaction rate was sufficient to afford appreciable degradation at physiological Abeta and IgM concentrations found in peripheral circulation. Increased Abeta concentrations in the AD brain are thought to induce neurodegenerative effects. Peripheral administration of Abeta binding antibodies has been suggested as a potential treatment of AD. Our results suggest that catalytic IgM autoantibodies can help clear Abeta, and they open the possibility of using catalytic Abs for AD immunotherapy.     

Investigation of a peptide responsible for amyloid fibril formation of beta 2-microglobulin by achromobacter protease I.

To obtain insight into the mechanism of amyloid fibril formation from beta(2)-microglobulin (beta2-m), we prepared a series of peptide fragments using a lysine-specific protease from Achromobacter lyticus and examined their ability to form amyloid fibrils at pH 2.5. Among the nine peptides prepared by the digestion, the peptide Ser(20)-Lys(41) (K3) spontaneously formed amyloid fibrils, confirmed by thioflavin T binding and electron microscopy. The fibrils composed of K3 peptide induced fibril formation of intact beta2-m with a lag phase, distinct from the extension reaction without a lag phase observed for intact beta2-m seeds. Fibril formation of K3 peptide with intact beta2-m seeds also exhibited a lag phase. On the other hand, the extension reaction of K3 peptide with the K3 seeds occurred without a lag phase. At neutral pH, the fibrils composed of either intact beta2-m or K3 peptide spontaneously depolymerized. Intriguingly, the depolymerization of K3 fibrils was faster than that of intact beta2-m fibrils. These results indicated that, although K3 peptide can form fibrils by itself more readily than intact beta2-m, the K3 fibrils are less stable than the intact beta2-m fibrils, suggesting a close relation between the free energy barrier of amyloid fibril formation and its stability.     

Somatostatin regulates brain amyloid beta peptide Abeta42 through modulation of proteolytic degradation

Expression of somatostatin in the brain declines during aging in various mammals including apes and humans. A prominent decrease in this neuropeptide also represents a pathological characteristic of Alzheimer disease. Using in vitro and in vivo paradigms, we show that somatostatin regulates the metabolism of amyloid beta peptide (Abeta), the primary pathogenic agent of Alzheimer disease, in the brain through modulating proteolytic degradation catalyzed by neprilysin. Among various effector candidates, only somatostatin upregulated neprilysin activity in primary cortical neurons. A genetic deficiency of somatostatin altered hippocampal neprilysin activity and localization, and increased the quantity of a hydrophobic 42-mer form of Abeta, Abeta(42), in a manner similar to presenilin gene mutations that cause familial Alzheimer disease. These results indicate that the aging-induced downregulation of somatostatin expression may be a trigger for Abeta accumulation leading to late-onset sporadic Alzheimer disease, and suggest that somatostatin receptors may be pharmacological-target candidates for prevention and treatment of Alzheimer disease.     

C-type natriuretic peptide (CNP): a new member of natriuretic peptide family identified in porcine brain

Two types of natriuretic peptide, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), very similar to each other in structure and in pharmacological effect, are known to be present in mammalian heart and brain. In our present survey for unidentified peptides in porcine brain extracts, we found a new peptide of 22 amino acid residues, eliciting a potent relaxant activity on chick rectum. The amino acid sequence determined for the peptide shows remarkable similarity to those of ANP and BNP, especially in the 17-residue sequences flanked by two cysteine residues. The peptide shows a pharmacological spectrum similar to ANP and BNP. Thus, the peptide was designated "C-type natriuretic peptide (CNP)", the third member to join the natriuretic peptide family. In contrast to ANP and BNP, CNP terminates in the second cysteine residue, lacking a further C-terminal extension.     

Degradation of brain natriuretic peptide by neutral endopeptidase: species specific sites of proteolysis determined by mass spectrometry

Brain natriuretic peptide (BNP) from 3 different species was cleaved by neutral endopeptidase (NEP) and the products separated by HPLC. The newly formed products were identified by fast atom bombardment or nebulizer-assisted electrospray mass spectrometry to elucidate the sites of proteolysis. Porcine BNP was cleaved at the Arg8-Leu9 and Ser14-Leu15 bonds. Rat BNP was cleaved at the Arg23-Leu24 and Arg30-Leu31 bonds. Human BNP was cleaved at the Pro2-Lys3, Met4-Val5 and Arg17-Leu18 bonds. The Cys-Phe bond which is present in all species of BNP is not cleaved by NEP.     

Lysophosphatidylcholine modulates fibril formation of amyloid beta peptide

Phospholipids are known to influence fibril formation of amyloid beta (Aβ) peptide. Here, we show that lysophosphatidylcholine (LPC), a polar phospholipid, enhances Aβ(1-42) fibril formation, by decreasing the lag time and the critical peptide concentration required for fibril formation, and increasing the fibril elongation rate. Conversely, LPC did not have an enhancing effect on Aβ(1-40) fibril formation, and appeared to be inhibitory. Tyrosine fluorescence spectroscopy showed that LPC altered the fluorescence spectra of Aβ(1-40) and Aβ(1-42) in opposite ways. Further, 8-anilino-1-naphthalene sulfonic acid fluorescence spectroscopy showed that LPC significantly increased the hydrophobicity of Aβ(1-42), but not of Aβ(1-40). Tris-tricine gradient SDS/PAGE revealed that LPC increased the formation of higher-molecular-weight species of Aβ(1-42), including trimers and tetramers. LPC had no such effect on Aβ(1-40), and thus may specifically influence the oligomerization and nucleation processes of Aβ(1-42) in a manner dependent on its native structure. Dot-blot assays confirmed that LPC induced Aβ(1-42) oligomer formation at an early time point. Thus our results indicate that LPC specifically enhances the formation of Aβ(1-42) fibrils, the main component of senile plaques in Alzheimer's disease patients, and may be involved in Alzheimer's disease pathology.     

Copper complexing decreases the ability of amyloid beta peptide to cross the BBB and enter brain parenchyma

The amyloid hypothesis states that amyloid beta protein (Abeta) plays a major causal role in the onset of Alzheimer's disease. Toxicity of Abeta can be modified by metal ions. Two mechanisms by which such Abeta and metal ions could interact are by enhanced oxidative stress or by altered fibrillation. Specifically, Abeta fibrillation is increased by aluminum (Al) and copper (Cu) and Al also increases Abeta uptake into brain. Here, we determined whether chelation with Cu would alter uptake of the human or rat 1-42 form of Abeta (Abeta42) by brain or alter Abeta-induced oxidative stress in an immortalized line of rat brain endothelial cells (RBE4). We found that Cu enhanced cytotoxicity of rat, but not of human Abeta, had no effect on glutathione (GSH) or cysteine (CYS) levels. Cu significantly decreased homocysteine (HCYS) levels when complexed with Abeta. Cu chelation did not alter Abeta uptake into brain or other tissues (except for kidney) or alter clearance from blood or brain in vivo, but did increase efflux in an in vitro model of the blood-brain barrier (BBB). Chelation to Cu also impaired the capillary to brain transport of Abeta, an effect opposite to that previously found for chelation of Abeta to Al. These results show that metal ions have varied effects on Abeta uptake by brain and that Cu could be protective against the neurotoxic effects of circulating Abeta.     

Biochemical characterisation of the SWI/SNF family member HLTF

HLTF is highly similar in domain organisation to yeast Rad5. We identify PTIP and RPA70, both involved in DNA replication and DNA repair, as HLTF-interacting proteins although cells depleted of HLTF did not show defects in cellular responses to DNA damage. In vitro, HLTF has ATPase activity and E3 ubiquitin ligase activity with a range of E2 ubiquitin conjugating enzymes. HLTF expression is severely reduced in a range of cancer cells, and we suggest that the HLTF antibodies generated in this study could be used for cancer diagnostic purposes.     

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.     

Detecting bioactive amyloid beta peptide species in Alzheimer's disease

Amyloid beta peptide (A beta) is believed to play a central role in the pathogenesis of Alzheimer's disease (AD). However, the form of A beta that induces neurodegeneration in AD, defined here as bioactive A beta, is not clear. Preventing the formation of bioactive A beta or inactivating previously formed bioactive A beta should be a promising approach to treat AD. We have previously developed a cell-based assay for the detection of bioactive A beta species. The assay is based upon the correlation between the ability of an A beta sample to induce a unique form of cellular MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] formazan exocytosis, and its ability to activate glia and induce neurotoxicity. Here, we show that this cell-based assay is not only useful for a cellular model of A beta amyloidogenesis but is also able to detect bioactive A beta species in a transgenic mouse model of AD, as well as in post-mortem cortex samples from AD patients. There is a good correlation between the extent of glia activation and the level of bioactive A beta species in the mouse brain. A promising deuteroporphyrin that can inactivate bioactive A beta species was also identified using this assay. These novel insights and findings should have important implications for the treatment of AD.     

Degradation of the Alzheimer's amyloid beta peptide by endothelin-converting enzyme

Deposition of beta-amyloid (Abeta) peptides in the brain is an early and invariant feature of all forms of Alzheimer's disease. As with any secreted protein, the extracellular concentration of Abeta is determined not only by its production but also by its catabolism. A major focus of Alzheimer's research has been the elucidation of the mechanisms responsible for the generation of Abeta. Much less, however, is known about the mechanisms responsible for Abeta removal in the brain. In this report, we describe the identification of endothelin-converting enzyme-1 (ECE-1) as a novel Abeta-degrading enzyme. We show that treatment of endogenous ECE-expressing cell lines with the metalloprotease inhibitor phosphoramidon causes a 2-3-fold elevation in extracellular Abeta concentration that appears to be due to inhibition of intracellular Abeta degradation. Furthermore, we show that overexpression of ECE-1 in Chinese hamster ovary cells, which lack endogenous ECE activity, reduces extracellular Abeta concentration by up to 90% and that this effect is completely reversed by treatment of the cells with phosphoramidon. Finally, we show that recombinant soluble ECE-1 is capable of hydrolyzing synthetic Abeta40 and Abeta42 in vitro at multiple sites.     

The redox chemistry of the Alzheimer's disease amyloid beta peptide

There is a growing body of evidence to support a role for oxidative stress in Alzheimer's disease (AD), with increased levels of lipid peroxidation, DNA and protein oxidation products (HNE, 8-HO-guanidine and protein carbonyls respectively) in AD brains. The brain is a highly oxidative organ consuming 20% of the body's oxygen despite accounting for only 2% of the total body weight. With normal ageing the brain accumulates metals ions such iron (Fe), zinc (Zn) and copper (Cu). Consequently the brain is abundant in antioxidants to control and prevent the detrimental formation of reactive oxygen species (ROS) generated via Fenton chemistry involving redox active metal ion reduction and activation of molecular oxygen. In AD there is an over accumulation of the Amyloid beta peptide (Abeta), this is the result of either an elevated generation from amyloid precursor protein (APP) or inefficient clearance of Abeta from the brain. Abeta can efficiently generate reactive oxygen species in the presence of the transition metals copper and iron in vitro. Under oxidative conditions Abeta will form stable dityrosine cross-linked dimers which are generated from free radical attack on the tyrosine residue at position 10. There are elevated levels of urea and SDS resistant stable linked Abeta oligomers as well as dityrosine cross-linked peptides and proteins in AD brain. Since soluble Abeta levels correlate best with the degree of degeneration [C.A. McLean, R.A. Cherny, F.W. Fraser, S.J. Fuller, M.J. Smith, K. Beyreuther, A.I. Bush, C.L. Masters, Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease, Ann. Neurol. 46 (1999) 860-866] we suggest that the toxic Abeta species corresponds to a soluble dityrosine cross-linked oligomer. Current therapeutic strategies using metal chelators such as clioquinol and desferrioxamine have had some success in altering the progression of AD symptoms. Similarly, natural antioxidants curcumin and ginkgo extract have modest but positive effects in slowing AD development. Therefore, drugs that target the oxidative pathways in AD could have genuine therapeutic efficacy.     

Characterization of member of DUF1888 protein family, self-cleaving and self-assembling endopeptidase

The crystal structure of SO1698 protein from Shewanella oneidensis was determined by a SAD method and refined to 1.57 Å. The structure is a β sandwich that unexpectedly consists of two polypeptides; the N-terminal fragment includes residues 1–116, and the C-terminal one includes residues 117–125. Electron density also displayed the Lys-98 side chain covalently linked to Asp-116. The putative active site residues involved in self-cleavage were identified; point mutants were produced and characterized structurally and in a biochemical assay. Numerical simulations utilizing molecular dynamics and hybrid quantum/classical calculations suggest a mechanism involving activation of a water molecule coordinated by a catalytic aspartic acid.     

Seeding-dependent maturation of beta2-microglobulin amyloid fibrils at neutral pH

Beta2-microglobulin (beta2-m) is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Recent studies have focused on the mechanism by which amyloid fibrils are formed under physiological conditions, which had been difficult to reproduce quantitatively. Yamamoto et al. (Yamamoto, S., Hasegawa, K., Yamaguchi, I., Tsutsumi, S., Kardos, J., Goto, Y., Gejyo, F. & Naiki, H. (2004) Biochemistry 43, 11075-11082) showed that a combination of seed fibrils prepared under acidic conditions and a low concentration of sodium dodecyl sulfate below its critical micelle concentration enabled extensive fibril formation at pH 7.0. Here, we found that repeated self-seeding at pH 7.0 with fibrils formed at the same pH causes a marked acceleration of growth, indicating the maturation of fibrils. The observed maturation can be simulated by assuming the existence of two types of fibrils with different growth rates. Importantly, some mutations of beta2-m or the addition of a low concentration of urea, both destabilizing the native conformation, were not enough to extend the fibrils at pH 7.0, and a low concentration of sodium dodecyl sulfate (i.e. 0.5 mM) was essential. Thus, even though the first stage fibrils in patients are unstable and require stabilizing factors to remain at neutral pH, they can adapt to a neutral pH with repeated self-seeding, implying a mechanism of development of amyloid deposition after a long latent period in patients.