pepstatin and Alzheimer-Disease

Although several ADAMs (A disintegrin-like and metalloproteases) have been shown to contribute to the amyloid precursor protein (APP) metabolism, the full spectrum of metalloproteases involved in this metabolism remains to be established. Transcriptomic analyses centred on metalloprotease genes unraveled a 50% decrease in ADAM30 expression that inversely correlates with amyloid load in Alzheimer's disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aβ peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30(mut)) did not affect Aβ secretion. Proteomics/cell-based experiments showed that ADAM30-dependent regulation of APP metabolism required both cathepsin D (CTSD) activation and APP sorting to lysosomes. Accordingly, in Alzheimer-like transgenic mice, neuronal ADAM30 over-expression lowered Aβ42 secretion in neuron primary cultures, soluble Aβ42 and amyloid plaque load levels in the brain and concomitantly enhanced CTSD activity and finally rescued long term potentiation alterations. Our data thus indicate that lowering ADAM30 expression may favor Aβ production, thereby contributing to Alzheimer's disease development.

Topics: ADAM Proteins; Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Animals; Brain; Cathepsin D; Cell Line, Tumor; Down-Regulation; HEK293 Cells; Humans; Lysosomes; Macrolides; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Fluorescence; Patch-Clamp Techniques; Pepstatins; RNA Interference; RNA, Small Interfering

To develop a novel cerebrospinal fluid (CSF) beta-secretase-1 activity assay and evaluate beta-secretase-1 (BACE-1) activity as a potential biomarker in human Alzheimer's disease.. The assay consisted of an enzymatic reaction of CSF samples with an optimized beta-secretase peptide substrate and the cleavage products were detected using a neo-epitope specific antibody.. The CSF BACE-1 activity assay described exhibits time, temperature, dose, and pH dependence, with sensitivity down to <1 pM of recombinant BACE-1 enzyme, and is completely blocked by BACE-1 inhibitors. The endogenous BACE-1 enzyme in CSF appears to exist as a c-terminally truncated protein, based on both western blotting and capture-based activity assays. In a small cohort of human subjects, an age-dependent increase in CSF BACE activity was observed (~1.0 pM/year, p<0.05). In Alzheimer's disease subjects, a significant decline in age-adjusted CSF BACE activity was observed compared to controls (56% in the log-transformed scale, p=0.02).. We have developed a robust assay to measure CSF BACE-1 activity which could serve as a potential biomarker in human Alzheimer's disease subjects.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Alzheimer Disease; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Enzyme-Linked Immunosorbent Assay; Female; Humans; Macaca mulatta; Male; Middle Aged; Pepstatins; Protease Inhibitors; Sensitivity and Specificity

Proteolysis of apolipoprotein E (apoE) may be involved in the pathogenesis of Alzheimer's disease (AD). We previously identified aspartic protease(s) as possibly contributing to the proteolysis of apoE in human brain homogenates. The current study used biochemical and immunohistochemical methods to examine whether cathepsin D (catD) and cathepsin E (catE), candidate aspartic proteases, may be involved in apoE proteolysis. CatD was found to proteolyze both lipid-free recombinant full-length human apoE and lipidated human plasma full-length apoE (apoE4/dipalmitoylphosphatidylcholine-reconstituted discs). CatE was found to proteolyze lipid-free recombinant human apoE to a much greater extent than lipidated apoE. This proteolysis, as well as proteolysis of human apoE added to brain homogenates from apoE-deficient mice, was inhibited by pepstatin A (an aspartic protease inhibitor), but not by phenylmethanesulfonyl fluoride (a serine protease inhibitor). The major apoE fragment obtained with catD included the receptor-binding domain and had an apparent molecular weight similar to that found in human brain homogenates. There was little immunoreactivity for catE in AD brain tissue sections. In contrast, qualitative and quantitative analyses of immunostained sections of the frontal cortex revealed that catD and apoE are colocalized in a subset of predominantly dense-core neuritic plaques and in some neurofibrillary tangles. A positive correlation was observed between estimated duration of illness and the percentage of apoE-positive plaques that were also catD-positive. These results suggest that aspartic proteases, catD in particular, may be involved in proteolysis of apoE and perhaps contribute to the generation of apoE fragments previously implicated in AD pathology.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Animals; Apolipoproteins E; Blotting, Western; Brain; Cathepsin D; Cathepsin E; Drug Interactions; Female; Humans; Immunohistochemistry; Male; Mice; Mice, Knockout; Middle Aged; Neurofibrillary Tangles; Pepstatins; Phenylmethylsulfonyl Fluoride; Phosphopyruvate Hydratase; Plaque, Amyloid; Postmortem Changes; Protease Inhibitors; Time Factors

An effective approach for enhancing the selectivity of beta-site amyloid precursor protein cleaving enzyme (BACE 1) inhibitors is developed based on the unique features of the S1' pocket of the enzyme. A series of low molecular weight (<600) compounds were synthesized with different moieties at the P1' position. The selectivity of BACE 1 inhibitors versus cathepsin D and renin was enhanced 120-fold by replacing the hydrophobic propyl group with a hydrophilic propionic acid group.

Topics: Alzheimer Disease; Amyloid Precursor Protein Secretases; Aspartic Acid Endopeptidases; Cathepsin D; Drug Design; Endopeptidases; Enzyme Inhibitors; Humans; Molecular Structure; Molecular Weight; Propionates; Renin; Substrate Specificity

Alzheimer's disease is characterized by the deposits of the 4-kDa amyloid beta peptide (A beta). The A beta protein precursor (APP) is cleaved by beta-secretase to generate a C-terminal fragment, CTF beta, which in turn is cleaved by gamma-secretase to generate A beta. Alternative cleavage of the APP by alpha-secretase at A beta 16/17 generates the C-terminal fragment, CTFalpha. In addition to A beta, endoproteolytic cleavage of CTF alpha and CTF beta by gamma-secretase should yield a C-terminal fragment of 57-59 residues (CTF gamma). However, CTF gamma has not yet been reported in either brain or cell lysates, presumably due to its instability in vivo. We detected the in vitro generation of A beta as well as an approximately 6-kDa fragment from guinea pig brain membranes. We have provided biochemical and pharmacological evidence that this 6-kDa fragment is the elusive CTF gamma, and we describe an in vitro assay for gamma-secretase activity. The fragment migrates with a synthetic peptide corresponding to the 57-residue CTF gamma fragment. Three compounds previously identified as gamma-secretase inhibitors, pepstatin-A, MG132, and a substrate-based difluoroketone (t-butoxycarbonyl-Val-Ile-(S)-4-amino-3-oxo-2, 2-difluoropentanoyl-Val-Ile-OMe), reduced the yield of CTF gamma, providing additional evidence that the fragment arises from gamma-secretase cleavage. Consistent with reports that presenilins are the elusive gamma-secretases, subcellular fractionation studies showed that presenilin-1, CTF alpha, and CTF beta are enriched in the CTF gamma-generating fractions. The in vitro gamma-secretase assay described here will be useful for the detailed characterization of the enzyme and to screen for gamma-secretase inhibitors.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Brain; Caspase 3; Caspases; Cells, Cultured; Detergents; Endopeptidases; Guinea Pigs; Hydrogen-Ion Concentration; Membrane Proteins; Molecular Weight; Pepstatins; Peptide Fragments; Phenanthrolines; Protease Inhibitors; Protein Processing, Post-Translational; Solubility; Subcellular Fractions

gamma-Secretase is a membrane-associated protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C termini of the two Abeta peptide isoforms, Abeta40 and Abeta42. Here we report the detergent solubilization and partial characterization of gamma-secretase. The activity of solubilized gamma-secretase was measured with a recombinant substrate, C100Flag, consisting largely of the C-terminal fragment of amyloid precursor protein downstream of the beta-secretase cleavage site. Cleavage of C100Flag by gamma-secretase was detected by electrochemiluminescence using antibodies that specifically recognize the Abeta40 or Abeta42 termini. Incubation of C100Flag with HeLa cell membranes or detergent-solubilized HeLa cell membranes generates both the Abeta40 and Abeta42 termini. Recovery of catalytically competent, soluble gamma-secretase critically depends on the choice of detergent; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) but not Triton X-100 is suitable. Solubilized gamma-secretase activity is inhibited by pepstatin and more potently by a novel aspartyl protease transition-state analog inhibitor that blocks formation of Abeta40 and Abeta42 in mammalian cells. Upon gel exclusion chromatography, solubilized gamma-secretase activity coelutes with presenilin 1 (PS1) at an apparent relative molecular weight of approximately 2.0 x 10(6). Anti-PS1 antibody immunoprecipitates gamma-secretase activity from the solubilized gamma-secretase preparation. These data suggest that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Aspartic Acid Endopeptidases; Carbamates; Cell Fractionation; Cell Membrane; Cholic Acids; Detergents; Dipeptides; Endopeptidases; HeLa Cells; Humans; Membrane Proteins; Neoplasm Proteins; Pepstatins; Presenilin-1; Protease Inhibitors; Recombinant Fusion Proteins; Solubility; Substrate Specificity

Previous studies established that the populations of neurons that frequently degenerate in Alzheimer's disease exhibit robust up-regulation of the lysosomal system. In this study, we investigated alterations of the lysosomal system during different forms of experimental injury in rat hippocampal neurons in culture, utilizing a combination of immunocytochemical and biochemical methods. Using triple-label immnocytochemistry for activated caspase-3, fragmentation of DNA and the microtubule-associated protein-2, we characterized treatment paradigms as models of the apoptotic (staurosporine, camptothecin), the oncotic (high-dose menadione, glutamate), and the mixed apoptotic and oncotic (low-dose menadione) pathways of neuronal death. Slowly developing apoptotic or slowly developing mixed apoptotic and oncotic forms of neuronal injury were associated with substantial increases in the number and size of cathepsin D-positive vesicles (late endosomes and mature lysosomes) as determined by immunocytochemistry, and elevated levels of cathepsin D by western blotting. In agreement with our previous findings in Alzheimer's disease, where lysosomal system activation was not restricted to overtly degenerating neurons, up-regulation of this system was also detected quite early during the course of experimental neuronal injury, preceding the development of dystrophic neurites, nuclear segmentation or fragmentation of DNA. These findings implicate lysosomal system activation, both in Alzheimer's disease and in experimental models of neuronal injury, as an important event associated with early stages of neurodegeneration.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Astrocytes; Blotting, Western; Cathepsin D; Female; Hippocampus; Humans; Lysosomes; Male; Middle Aged; Neurons; Pepstatins; Up-Regulation; Wounds and Injuries

To investigate the mechanism of regulation of Ass production by familial Alzheimer's disease (FAD)-linked presenilin 1 (PS1), we used a cell-free system that allows de novo Ass generation to examine whether PS1 participates directly in the gamma-secretase reaction. Optimal Ass generation in vitro was achieved at mildly acidic pH and could be inhibited by the aspartyl protease inhibitor pepstatin A, consistent with the suggestion that gamma-secretase is an aspartyl protease. Dominant negative mutations of the critical transmembrane aspartates in PS1 or full deletion of PS1 did not alter the maturation of APP in the secretory pathway. Instead, PS1 had a direct effect on the inhibition of Ass production by a designed peptidomimetic inhibitor: the inhibition was significantly less effective in cells expressing FAD-causing mutations in either APP or PS1 than in cells expressing the wild-type proteins. Taken together, these findings suggest that PS1 participates physically in a complex with APP during the gamma-secretase cleavage event.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Binding Sites; Cell Fractionation; Cell-Free System; CHO Cells; Cricetinae; Endopeptidases; Enzyme Inhibitors; Genes, Dominant; Golgi Apparatus; Hydrogen-Ion Concentration; Macromolecular Substances; Membrane Proteins; Microsomes; Mutation, Missense; Pepstatins; Presenilin-1; Protein Binding; Protein Processing, Post-Translational

A recent study has shown mutations in CLN2 gene, that encodes a novel lysosomal pepstatin-insensitive proteinase (LPIP), in the pathophysiology of late-infantile neuronal ceroid lipofuscinosis (LINCL). We have measured the LPIP activities in brains from various forms of human neuronal ceroid lipofuscinoses (NCL), canine ceroid lipofuscinosis and other neurodegenerative disorders with a highly sensitive assay using a tetrapeptide Gly-Phe-Phe-Leu-amino-trifluoromethyl coumarin (AFC) as substrate. Brain LPIP has a pH optimum of 3.5 and an apparent km of 100 microM for the crude enzyme. The enzyme activity is totally absent in LINCL patients. Pronounced increase in the LPIP activity was seen in patients suffering from infantile (INCL), juvenile (JNCL) and adult (ANCL) forms of neuronal ceroid lipofuscinoses. LPIP activity was also found to be increased about two-fold in Alzheimer's disease when compared with normal or age-matched controls, while in globoidal-cell leukodystrophy (Krabbe's disease) it was similar to the normal controls. Although mannose-6-phosphorylated LPIP is increased 13-fold in brains of patients with JNCL, this form of LPIP did not have any enzyme activity. The mechanism by which LPIP activities are increased in a wide range of neurodegenerative diseases is unknown, although neuronal loss, followed by gliosis are common characteristics of these diseases.

Topics: Adult; Aged; Aged, 80 and over; Aging; Alzheimer Disease; Aminopeptidases; Animals; Brain; Child; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; Dog Diseases; Dogs; Endopeptidases; Female; Humans; Leukodystrophy, Globoid Cell; Male; Mannose; Middle Aged; Neurodegenerative Diseases; Neuronal Ceroid-Lipofuscinoses; Pepstatins; Peptide Hydrolases; Reference Values; Serine Proteases; Tripeptidyl-Peptidase 1

The formation of A beta and A beta-containing fragments is likely a key event in the process of neural degeneration in Alzheimer's disease. The N-terminal residue (Asp-1) of A beta and its C-terminally extended sequences is liberated from the beta-amyloid precursor protein (beta APP) by beta-secretase(s). This activity appears highly increased by the presence (N-terminally to Asp-1) of a double-mutation (KM-->NL) found in several Swedish families affected by early onset Alzheimer's disease. By means of synthetic peptides encompassing the "normal' (N peptide) and mutated (delta NL peptide) sequences targeted by beta-secretase(s), we have detected a human brain protease displaying preferred efficiency for the delta NL peptide than for the non-mutated analog. This activity is sensitive to pepstatin, maximally active at acidic pH and hydrolyses the two peptides at the expected M/D or L/D cleavage sites. Such acidic activity is also detected in rat brain, PC12 cells and primary cultured astrocytes. The pepstatin sensitivity and pH maximum of the brain activity that appeared reminiscent of those displayed by the acidic protease cathepsin D led us to examine this enzyme as a putative beta-secretase-like candidate. Purified cathepsin D displays higher catalytic parameters for the delta NL peptide than for the non-mutated peptide, cleaves these two substrates at the expected M/D or L/D sites, and is maximally active at acidic pH. However, cathepsin D does not cleave peptides bearing mutations that were previously shown to drastically lower or fully block A beta secretion by transfected cells. Furthermore, cathepsin D hydrolyses recombinant baculoviral delta NL beta APP751 at a 6-fold higher rate than beta APP751 and gives rise to a 12-kDa C-terminal product that is recognized by antibodies fully specific of the N-terminus of A beta. Altogether, our study indicates that cathepsin D displays several in vitro beta-secretase-like properties that suggests that this protease could fulfill such a role, at least in the Swedish genetic form of Alzheimer's disease.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Astrocytes; Brain; Cathepsin D; Cell Line; Endopeptidases; Humans; Kinetics; Mice; Mutagenesis, Site-Directed; Oligopeptides; PC12 Cells; Pepstatins; Point Mutation; Protease Inhibitors; Rats; Recombinant Proteins; Spodoptera; Substrate Specificity; Transfection

Two proteases, denoted beta- and gamma-secretase, process the beta-amyloid peptide precursor (APP) to yield the Abeta peptides involved in Alzheimer's disease. A third protein, alpha-secretase, cleaves APP near the middle of the Abeta sequence and thus prevents Abeta formation. These enzymes have defied identification. Because of its similarity to the systems of mammalian cells the yeast secretory system has provided important clues for finding mammalian processing enzymes. When expressed in Saccharomyces cerevisiae APP is processed by enzymes that possess the specificity of the alpha-secretases of multicellular organisms. APP processing by alpha-secretases occurred in sec1 and sec7 mutants, in which transport to the cell surface or to the vacuole is blocked, but not in sec17 or sec18 mutants, in which transport from the endoplasmic reticulum to the Golgi is blocked. Neutralization of the vacuole by NH4Cl did not block alpha-secretase action. The time course of processing of a pro-alpha-factor leader-APP chimera showed that processing by Kex2 protease, a Golgi protease that removes the leader, preceded processing by alpha-secretase. Deletions of the genes encoding the GPI-linked aspartyl proteases Yap3 and Mkc7 decreased alpha-secretase activity by 56 and 29%, respectively; whereas, the double deletion decreased the activity by 86%. An altered form of APP-695, in which glutamine replaced Lys-612 at the cleavage site, is cleaved by Yap3 at 5% the rate of the wild-type APP. Mkc7 protease cleaved APP (K612Q) at about 20% the rate of wild-type APP. The simplest interpretation of these results is that Yap3 and Mkc7 proteases are alpha-secretases which act on APP in the late Golgi. They suggest that GPI-linked aspartyl proteases should be investigated as candidate secretases in mammalian tissues.

Topics: Alzheimer Disease; Amino Acid Sequence; Ammonium Chloride; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Aspartic Acid Endopeptidases; beta-Fructofuranosidase; Cloning, Molecular; Endopeptidases; Fungal Proteins; Glycoside Hydrolases; Glycosylation; Molecular Sequence Data; Mutagenesis, Site-Directed; Pepstatins; Plasmids; Proprotein Convertases; Protein Processing, Post-Translational; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Subtilisins; Temperature; Transformation, Genetic; Tunicamycin