alpha-chymotrypsin and Alzheimer-Disease

Peptides derived from proteolytic degradation of the amyloid precursor protein, e.g., amyloid beta (A beta), are considered to be central to the pathology of Alzheimer's disease (AD). Soluble A beta is present in measurable concentrations in cerebrospinal fluid and blood. There are indications that soluble A beta present in circulation can cross the blood-brain barrier via transcytosis mediated by brain capillary endothelial cells. It implies that A beta originating from circulation may contribute to vascular and parenchymal A beta deposition in AD. Enhancing of A beta catabolism mediated by proteolytic degradation or receptor-mediated endocytosis could be a key mechanism to maintain low concentrations of soluble A beta. To launch A beta clearance we have exploited the A beta-degrading activity of diverse alpha 2-macroglobulin (alpha 2-M)-proteinase complexes. Complexes with trypsin, alpha-chymotrypsin, and bromelain strongly degrade (125)I-A beta 1--42 whereas complexes with endogenous proteinases, e.g., plasmin and prostate-specific antigen, were not effective. A beta degradation by the complexes was not inhibited by alpha 1-antichymotrypsin and soybean trypsin inhibitor which normally would inactivate the free serine proteinases. A prerequisite for A beta degradation is its binding to specific binding sites in alpha 2-M that may direct A beta to the active site of the caged proteinase. Ex vivo, enhanced degradation of (125)I-A beta 1--42 in blood could be achieved upon oral administration of high doses of proteinases to volunteers. These results suggest that up-regulation of A beta catabolism could probably reduce the risk of developing AD by preventing A beta accumulation in brain and vasculature.

Topics: alpha-Macroglobulins; Alzheimer Disease; Amyloid beta-Peptides; Binding, Competitive; Blood Proteins; Bromelains; Chymotrypsin; Drug Combinations; Electrophoresis, Polyacrylamide Gel; Humans; Macromolecular Substances; Peptide Fragments; Rutin; Trypsin

Mass spectrometry coupled with bioaffinity separation techniques is considered a powerful tool for studying protein interactions. This work is focused on epitope analysis of tau protein, which contains two VQIXXK aggregation motifs regarded as crucial elements in the formation of paired helical filaments, the main pathological characteristics of Alzheimer's disease. To identify major immunogenic structures, the epitope extraction technique utilizing protein fragmentation and magnetic microparticles functionalized with specific antibodies was applied. However, the natural adhesiveness of some newly generated peptide fragments devalued the experimental results. Beside presumed peptide fragment specific to applied monoclonal anti-tau antibodies, the epitope extraction repeatedly revealed inter alia tryptic fragment 299-HVPGGGSVQIVYKPVDLSK-317 containing the fibril-forming motif 306-VQIVYK-311. The tryptic fragment pro-aggregation and hydrophobic properties that might contribute to adsorption phenomenon were examined by Thioflavin S and reversed-phase chromatography. Several conventional approaches to reduce the non-specific fragment sorption onto the magnetic particle surface were performed, however with no effect. To avoid methodological complications, we introduced an innovative approach based on altered proteolytic digestion. Simultaneous fragmentation of tau protein by two immobilized proteases differing in the cleavage specificity (TPCK-trypsin and α-chymotrypsin) led to the disruption of motif responsible for undesirable adhesiveness and enabled us to obtain undistorted structural data.

Topics: Adhesiveness; Adsorption; Alzheimer Disease; Amino Acid Motifs; Antibodies, Monoclonal; Benzothiazoles; Biomarkers; Chymotrypsin; Epitopes; Humans; Magnetics; Mass Spectrometry; Proteolysis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; tau Proteins; Thiazoles; Trypsin

Alzheimer's disease (AD) is the most common cause of dementia in the elderly. Very little is known about the causes of AD, except that its end stages involve extensive neuronal loss and the appearance of distinctive neuropathological features. This study was under taken to investigate the role of α-chymotrypcin (α-ch) in management of AD-induced in ovariectomized rats.. Sixty female Sprague Dawley rats were divided into four groups n=15, (1) normal control group (con), (2) group underwent surgery to remove ovaries (ovx control group), (3) ovx group received aluminum chloride in a dose of 17 mg/kg daily for 2 months to induce AD (AD group), (4) AD group treated with α-chymotrypcin (α-ch) at dose (8.1 unit/rat/day) which is equivalent to the recommended human dose (α-ch-treated group) for three months. At the end of the experimental period, rats were sacrificed; brain samples were obtained for different biochemical analyses and histopathological examination. The biochemical analyses included determination of tumor necrosis factor-α (TNF- α), IL-18, monocyte chemo attractant protein-1 MCP-1, FAS, B-cell lymphoma 2 (Bcl2).. In comparison with normal control group, the ovx control group recorded significant increase in the brain levels of TNF-α, IL-18, MCP-1 and FAS. On the other hand, the brain level of Bcl2 was significantly decreased. Also, AD group showed a significant increase in TNF-α, IL-18, MCP-1 and FAS levels in brain tissue. In contrast, significant decrease in brain Bcl2 level was detected in AD group as compared to the ovx control group. However, the treatment of AD group with α-chymotrypcin caused an improvement in the most studied biochemical parameters as indicated by decreased brain levels of TNF-α, IL-18, MCP-1 and FAS accompanied with significant increase in the level of Bcl2 compared to AD group. Histopathological investigation of brain tissue of ovx rats administered with aluminum (AD group) showed AD plaques. While, AD group treated with α-chymotrypcin showed great improvement in the brain morphological structure with the disappearance of amyloid plaques.. This study revealed that α-chymotrypcin significantly ameliorates the neuroinflammation characterizing Alzheimer's disease in ovariectomized rats due to it's proteolytic activity as well as it's anti-inflammatory effect.

Topics: Aluminum Chloride; Aluminum Compounds; Alzheimer Disease; Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Apoptosis Regulatory Proteins; Brain; Chlorides; Chymotrypsin; Cytokines; Female; Ovariectomy; Rats; Rats, Sprague-Dawley

Alzheimer's disease (AD) is a severe chronic neurodegenerative disease. During aging and neurodegeneration, misfolded proteins accumulate and activate the ubiquitin-proteasome system. The aim of the present study is to explore whether ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, ubiquitin or proteasome activity are affected in peripheral blood mononuclear cells (PBMC) of AD, mild cognitive impairment (MCI) and Parkinson's disease (PD) patients compared to healthy subjects. PBMCs were isolated from EDTA blood samples and extracts were analyzed by Western Blot. Proteasome activity was measured with fluorogenic substrates. When compared to healthy subjects, the concentration of enzyme E1 was increased in PBMCs of AD patients, whereas the concentration of the enzyme E2 was decreased in these same patients. Ubiquitin levels and proteasome activity were unchanged in AD patients. No changes in enzyme expression or proteasome activity was observed in MCI patients compared to healthy and AD subjects. In PD patients E2 levels and proteasomal activity were significantly reduced, while ubiquitin and E1 levels were unchanged. The present investigation demonstrates the differences in enzyme and proteasome activity patterns of AD and PD patients. These results suggest that different mechanisms are involved in regulating the ubiquitin-proteasomal system in different neurodegenerative diseases.

Topics: Aged; Alzheimer Disease; Animals; Blood Cells; Chymotrypsin; Cognition Disorders; Female; Humans; Male; Mental Status Schedule; Middle Aged; Neuropsychological Tests; Parkinson Disease; Proteasome Endopeptidase Complex; Rats; Statistics, Nonparametric; Ubiquitin; Ubiquitin-Activating Enzymes

Previous studies have suggested a possible relationship between the ubiquitin-proteasome pathway and some pathological manifestations of Alzheimer's disease (AD). This study investigated the possibility that the Abeta peptides interact with the ubiquitin-proteasome pathway inside neuronal cells. The ubiquitin-proteasome activity decreased with age in the brains of Tg2576 mice while the Abeta(1-42) levels increased. In cultured neuronal cells, an extracellular treatment of Abeta markedly decreased the proteasome activity and extracellular treated Abeta peptides were found in the cytoplasmic compartment. These results suggest that the extracellular Abeta peptides enter the cell and inhibit the proteasome activity, which might play a role in the pathogenesis of AD.

Topics: Alzheimer Disease; Amyloid; Animals; Blotting, Western; Brain; Cell Line, Tumor; Cell Survival; Chymotrypsin; Green Fluorescent Proteins; Immunohistochemistry; Mice; Mice, Transgenic; Microscopy, Confocal; Microscopy, Fluorescence; Models, Statistical; Neuroblastoma; Neurodegenerative Diseases; Neurons; Peptides; Proteasome Endopeptidase Complex; Rats; Subcellular Fractions; Time Factors; Transfection; Ubiquitin

Insulysin (IDE) and neprilysin (NEP) were found to be inactivated by oxidation with hydrogen peroxide, an iron-ascorbate oxidation system, and by treatment with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). In each case reaction led to the introduction of protein carbonyl groups as judged by reaction with 2,4-dintrophenylhydrazine. IDE was inactivated by reaction with 4-hydroxy-2-nonenal (HNE) with the concomitant formation of protein adducts. NEP was not inactivated to a significant extent by HNE, but some HNE-adduct formation did occur. Prior reaction with hydrogen peroxide or AAPH led to enhanced formation of HNE adducts. Treatment of IDE with AAHP or hydrogen peroxide increased its susceptibility to proteolysis, while treatment of NEP with iron/ascorbate or hydrogen peroxide increased its susceptibility to proteolysis. Since IDE and NEP play a prominent role in the clearance of amyloid beta peptides, their oxidative inactivation and enhanced proteolysis can contribute to the onset and/or progression of Alzheimer's disease.

Topics: Aldehydes; Alzheimer Disease; Amidines; Amyloid beta-Peptides; Ascorbic Acid; Chlorides; Chymotrypsin; Ferric Compounds; Hydrogen Peroxide; Insulysin; Neprilysin; Oxidation-Reduction; Trypsin

A potential goal in the prevention or therapy of Alzheimer's disease is to decrease or eliminate neuritic plaques composed of fibrillar beta-amyloid (Abeta). In this paper we describe N-methyl amino acid containing congeners of the hydrophobic "core domain" of Abeta that inhibit the fibrillogenesis of full-length Abeta. These peptides also disassemble preformed fibrils of full-length Abeta. A key feature of the inhibitor peptides is that they contain N-methyl amino acids in alternating positions of the sequence. The most potent of these inhibitors, termed Abeta16-22m, has the sequence NH(2)-K(Me-L)V(Me-F)F(Me-A)E-CONH(2). In contrast, a peptide, NH(2)-KL(Me-V)(Me-F)(Me-F)(Me-A)-E-CONH(2), with N-methyl amino acids in consecutive order, is not a fibrillogenesis inhibitor. Another peptide containing alternating N-methyl amino acids but based on the sequence of a different fibril-forming protein, the human prion protein, is also not an inhibitor of Abeta40 fibrillogenesis. The nonmethylated version of the inhibitor peptide, NH(2)-KLVFFAE-CONH(2) (Abeta16-22), is a weak fibrillogenesis inhibitor. Perhaps contrary to expectations, the Abeta16-22m peptide is highly soluble in aqueous media, and concentrations in excess of 40 mg/mL can be obtained in buffers of physiological pH and ionic strength, compared to only 2 mg/mL for Abeta16-22. Analytical ultracentrifugation demonstrates that Abeta16-22m is monomeric in buffer solution. Whereas Abeta16-22 is susceptible to cleavage by chymotrypsin, the methylated inhibitor peptide Abeta16-22m is completely resistant to this protease. Circular dichroic spectroscopy of Abeta16-22m indicates that this peptide is a beta-strand, albeit with an unusual minimum at 226 nm. In summary, the inhibitor motif is that of alternating N-methyl and nonmethylated amino acids in a sequence critical for Abeta40 fibrillogenesis. These inhibitors appear to act by binding to growth sites of Abeta nuclei and/or fibrils and preventing the propagation of the network of hydrogen bonds that is essential for the formation of an extended beta-sheet fibril.

Topics: Alzheimer Disease; Amino Acid Sequence; Amino Acids; Amyloid beta-Peptides; Benzothiazoles; Chymotrypsin; Drug Resistance; Humans; Methylation; Microscopy, Fluorescence; Molecular Sequence Data; Peptide Fragments; Protein Structure, Secondary; Protein Structure, Tertiary; Thiazoles; Ultracentrifugation

It is unclear how and when insoluble beta-amyloid in senile plaques exerts degenerative effects on distant hippocampal neurons in Alzheimer's disease. Racemization of Ser and Asp residues of insoluble beta-amyloid is a typical age-dependent process. In this study, we investigated the fibril formation activity and cytotoxic activity of beta-amyloid 1-40 racemized at the Asp or Ser residue. In contrast to beta-amyloid 1-40 and its derivative substituted with the D-Asp(1, 7 or 23) or D-Ser(8) residue, [D-Ser(26)]beta-amyloid 1-40 was non-toxic to PC12 cells, and did not exhibit significant fibril formation activity making it soluble. However, [D-Ser(26)]beta-amyloid 1-40, but not beta-amyloid 1-40, was converted in vitro to a potent neurotoxic and truncated peptide, [D-Ser(26)]beta-amyloid 25-35 or [D-Ser(26)]beta-amyloid 25-40, by chymotrypsin-like enzymes and aminopeptidase M. Soluble [D-Ser(26)]beta-amyloid 1-40 was injected into rat hippocampus with a non-toxic dose of ibotenic acid, an excitatory amino acid. Nissl staining and microtubule-associated protein-2 immunostaining revealed that [D-Ser(26)]beta-amyloid 1-40, as well as [D-Ser(26)]beta-amyloid 25-35, produced a drastic degeneration of the CA1 neurons with ibotenic acid although [D-Ser(26)]beta-amyloid 1-40 alone or ibotenic acid alone did not exert neuronal damage. This suggests the in vivo conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to the toxic and truncated peptides which enhance the susceptibility of neurons to the excitatory amino acid.These results and the presence of [D-Ser(26)]beta-amyloid 25-35-like antigens in Alzheimer's disease brains suggest that soluble [D-Ser(26)]beta-amyloid 1-40, possibly formed during the aging process, is released from senile plaques, and converted by brain proteinases to truncated [D-Ser(26)]beta-amyloid 25-35(40)-like peptides, which degenerate hippocampal neurons by enhancing the susceptibility to excitatory amino acids in Alzheimer's disease brains. These findings may provide the basis for a new therapeutic approach to prevent the neurodegeneration in Alzheimer's disease.

Topics: Alzheimer Disease; Amino Acid Isomerases; Amino Acid Sequence; Aminopeptidases; Amyloid beta-Peptides; Animals; Aspartic Acid; Chymotrypsin; Coloring Agents; Endopeptidases; Excitatory Amino Acid Agonists; Hippocampus; Ibotenic Acid; Male; Nerve Degeneration; Neurofibrillary Tangles; Neurons; PC12 Cells; Peptide Fragments; Plaque, Amyloid; Rats; Rats, Sprague-Dawley; Serine

Cerebrovascular amyloidosis belongs to the pathological hallmarks of Alzheimer's disease brains. Although definite proof is still lacking, it is very well possible that the amyloid and its associated proteins are produced locally in the brain. In this paper we describe the development of a model system of cultured human brain pericytes to study the mechanisms of microvascular amyloid formation in vitro. These cultured cells may serve to study several aspects of cerebrovascular amyloidosis, which include the production of the amyloid precursor protein and of amyloid beta-protein-associated proteins as well as cytotoxic effects of amyloid beta-protein on perivascular cells. We demonstrated that pericytes produce and metabolize the amyloid precursor protein, and that they produce amyloid beta-protein-associated proteins, such as heparan sulfate proteoglycans, apolipoprotein E, and complement factor C1q. They are also prone to cellular degeneration after treatment with amyloid beta-protein, which is accompanied by increased expression of a number of amyloid beta-protein-associated proteins. This may be an important mechanism to explain the cell death observed in vivo. Our data indicate that this cell culture model of human brain pericytes provides a useful and pathophysiologically relevant tool to study cerebrovascular amyloidosis.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloidosis; Apolipoproteins; Blotting, Western; Brain; Cell Survival; Cells, Cultured; Cerebrovascular Disorders; Chymotrypsin; Heparan Sulfate Proteoglycans; Humans; Immunohistochemistry; In Vitro Techniques; Pericytes; Time Factors

Peptide aldehyde inhibitors of the chymotrypsin-like activity of the proteasome (CLIP) such as N-acetyl-Leu-Leu-Nle-H (or ALLN) have been shown previously to inhibit the secretion of beta-amyloid peptide (A beta) from cells. To evaluate more fully the role of the proteasome in this process, we have tested the effects on A beta formation of a much wider range of peptide-based inhibitors of CLIP than published previously. The inhibitors tested included several peptide boronates, some of which proved to be the most potent peptide-based inhibitors of beta-amyloid production reported so far. We found that the ability of the peptide aldehyde and boronate inhibitors to suppress A beta formation from cells correlated extremely well with their potency as CLIP inhibitors. Thus, we conclude that the proteasome may be involved either directly or indirectly in A beta formation.

Topics: Aldehydes; Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Boronic Acids; Cell Line; Chymotrypsin; Cysteine Endopeptidases; Multienzyme Complexes; Peptide Fragments; Proteasome Endopeptidase Complex; Transfection

alpha1-Antichymotrypsin (ACT) is an acute phase protein expressed in the brain which specifically colocalizes with amyloid-beta during Alzheimer's disease. We analyzed ACT synthesis in cultured human cortical astrocytes in response to various cytokines and growth factors. Oncostatin M (OSM) and interleukin (IL)-1beta were potent stimulators of ACT mRNA expression, whereas tumor necrosis factor-alpha had modest activity, and IL-6 and leukemia inhibitory factor (LIF) were ineffective. The finding that OSM, but not LIF or IL-6, stimulated ACT expression suggests that human astrocytes express a "specific" OSM receptor, but not IL-6 or LIF receptors. However, cotreatment of human astrocytes with soluble IL-6 receptor (sIL-6R).IL-6 complex did result in potent stimulation of ACT expression. When the human ACT gene was cloned, two elements binding STAT1 and STAT3 (signal transducer and activator of transcription) in response to OSM or IL-6.sIL-6R complexes could be identified and characterized. Taken together, these findings indicate that OSM or IL-6.sIL-6 complexes may regulate ACT expression in human astrocytes and thus directly or indirectly contribute to the pathogenesis of Alzheimer's disease.

Topics: Acute-Phase Proteins; Alzheimer Disease; Astrocytes; Base Sequence; Brain; Chymotrypsin; Cloning, Molecular; Cytokines; DNA-Binding Proteins; Gene Expression Regulation; Humans; Interleukin-6; Molecular Sequence Data; Oncostatin M; Peptides; Promoter Regions, Genetic; Receptors, Interleukin-6; RNA, Messenger; Serine Proteinase Inhibitors; Tumor Necrosis Factor-alpha

Topics: Aged; Aged, 80 and over; alpha 1-Antitrypsin; Alzheimer Disease; Chymotrypsin; Humans

The precursor of the non-A beta component of Alzheimer's disease amyloid (NACP) is a presynaptic protein whose function has been suspected to be tightly involved in neuronal biogenesis including synaptic regulations. NACP was suggested to seed the neuritic plaque formation in the presence of A beta during the development of Alzheimer's disease (AD). Recombinant NACP purified through heat treatment, DEAE-Sephacel anion-exchange, Sephacryl S-200 size-exclusion, and S-Sepharose cation-exchange chromatography steps appeared as a single band on SDS-PAGE with Mr of 19 kDa. Its N-terminal amino acid sequence clearly confirmed that the protein was NACP. Interestingly, however, the protein was split into a doublet on a nondenaturing (ND)-PAGE with equal intensities. The doublet was located slightly above a 45-kDa marker protein on a 12.5% ND-PAGE. In addition, the size of NACP was more carefully estimated as 53 kDa with high-performance gel-permeation chromatography using a TSK G3000sw size-exclusion column. Recently, Lansbury and his colleagues (Biochemistry 35, 13709-13715) have reported that NACP exists as an elongated "natively unfolded" structure which would make the protein more actively involved in protein-protein interactions and Kim (Mol. Cells 7, 78-83) has also shown that the natively unfolded protein is extremely sensitive to proteases. Here, we report that the structure of NACP could be altered by certain environmental factors. Aluminum, a suspected risk factor for AD, converged the doublet of NACP into a singlet with slightly lower mobility on ND-PAGE. Spectroscopic analysis employing uv absorption, intrinsic fluorescence, and circular dichroism indicated that NACP experienced the structural alterations in the presence of aluminum such as the secondary structure transition to generate about 33% alpha-helix. This altered structure of NACP became resistant to proteases such as trypsin, alpha-chymotrypsin, and calpain. Therefore, it is suggested that aluminum, which influences two pathologically critical processes in AD such as the protein turnover and the protein aggregation via the structural modifications, could participate in the disease.

Topics: Aluminum; Alzheimer Disease; Amino Acid Sequence; Amyloid; Calpain; Chymotrypsin; Circular Dichroism; Electrophoresis, Polyacrylamide Gel; Humans; Molecular Sequence Data; Molecular Weight; Nerve Tissue Proteins; Neurofibrillary Tangles; Protein Conformation; Protein Precursors; Recombinant Proteins; Sequence Analysis; Spectrometry, Fluorescence; Spectrophotometry; Synucleins; Trypsin

Apolipoprotein J (apoJ) has been found associated with soluble amyloid beta (sA beta) in plasma and cerebrospinal fluid in normal individuals and co-deposited with fibrillar A beta in Alzheimer's cerebrovascular and parenchymal lesions. Although studies in vitro and in vivo indicate that apoJ is a major carrier protein for sA beta, its role in the fibrillogenesis process is not known. We report herein that apoJ in its native high-density lipoprotein lipidic environment is fully active to interact with A beta peptides. Furthermore, apoJ prevents aggregation and polymerization of synthetic A beta in vitro. The interaction was stable for at least 14 days at 37 degrees C in physiologic buffers, and the peptide retrieved after complex dissociation at low pH retained its inherent aggregation properties. In addition, the binding to apoJ protects synthetic A beta from proteolytic degradation; both A beta 1-42 and A beta 1-40 were more resistant to proteolysis by trypsin and chymotrypsin when complexed to apoJ. The data suggest that the interaction may preclude sA beta aggregation in biological fluids and point to a protecting role of apoJ for complexed A beta species.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Apolipoproteins E; Chromatography, Affinity; Chromatography, High Pressure Liquid; Chymotrypsin; Circular Dichroism; Clusterin; Enzyme-Linked Immunosorbent Assay; Glycoproteins; Humans; Hydrogen-Ion Concentration; Immunoelectrophoresis; Lipoproteins, HDL; Molecular Chaperones; Molecular Sequence Data; Peptide Fragments; Prealbumin; Solubility; Trypsin

A growing body of experimental evidence demonstrates that the serpin antichymotrypsin plays a regulatory role in Alzheimer plaque physiology by interacting with the 42 residue beta-amyloid protein, and we have used molecular modeling and energy minimization techniques to study this interaction. Based on the unique plasticity of beta-sheet elements in antichymotrypsin (as well as other serpins), we conclude that the interaction of the two proteins is mediated by insertion of the N-terminus of beta-amyloid into beta-sheet C of antichymotrypsin as a pseudo-strand s1C. This beta-strand insertion requires the displacement of native antichymotrypsin strand s1C, which is known to occur partially or completely at different stages of serpin function. Thus, the association of the two proteins in vivo may be facilitated by a particular functional state of the serpin, e.g., the native or protease-complexed state.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Chymotrypsin; Humans; Protein Binding; Protein Conformation

The recently reported phenomenon that red blood cells (RBC) from Alzheimer disease (AD) patients and normal individuals, which have identical electrophoretic mobilities (EPM) in phosphate-buffered saline (PBS), have different EPM in appropriately selected polymer solutions, has been further explored. Of a number of in vitro treatments to which AD and normal RBC were subjected prior to EPM measurements in bottom phase (from a dextran-poly(ethylene glycol) (PEG) aqueous phase system) only trypsin eliminated the difference. Thus, the differential polymer interaction between AD and normal RBC, thought to be the basis for their dissimilar EPM, can be abolished by appropriate proteolytic modification of the cell surfaces and suggests protein as a source of difference. Because young and old RBC from normal individuals, which have the same EPM in PBS, have different EPM in certain polymer solutions, and the RBC from AD patients have been reported to age abnormally, we also compared the young and old RBC subpopulations from these two sources. By the criterion of cell electrophoresis in polymer solutions the differences between AD and normal RBC and between young and old RBC are distinct. The EPM of AD and normal RBC differ in bottom phase or PEG but not in dextran solution; while the EPM of young and old RBC differ predominantly in dextran. We speculate that since the observed difference in EPM of RBC from AD patients and normals depends on protein(s) yet is anticoagulant-related (being obtained only when blood is collected in citrate or oxalate) it might be the result of an interaction (Ca(2+)-mediated?) between the surfaces of these cells and protein component(s) of their respective, compositionally differing sera.

Topics: Alzheimer Disease; Anticoagulants; Chymotrypsin; Dextrans; Electrophoresis; Erythrocyte Aging; Erythrocytes; Humans; Polyethylene Glycols; Reference Values; Trypsin; Viscosity

Intraneuronal accumulation of ubiquitin conjugates in inclusion bodies and neurofibrillary tangles is a pathological feature of neurodegenerative disorders such as Alzheimer's disease and Down's syndrome and of normal aging of the brain. Amyloid beta-protein (A beta) and its precursor are found in neurofibrillary tangle-containing neurons. A beta is the major component of extracellular plaques. We showed that A beta acts as an inhibitor of the ubiquitin-dependent protein degradation in vitro. We examined the effect of A beta on the steps of this proteolytic pathway that contribute to the level of ubiquitin conjugates in the cell. Neither conjugate formation nor conjugate deubiquitination was affected by the presence of A beta. However, A beta significantly reduced the rate of conjugate degradation. Our results indicate that A beta interacts with the proteolytic step of the ubiquitin degradative pathway. Since this step is performed by the 26 S proteasome, the effect of A beta on the catalytic core of this proteolytic complex, the 20 S proteasome, was determined. We found that A beta selectively inhibits the chymotrypsin-like activity of the 20 S proteasome. Under pathological conditions in the affected neuron, A beta could interfere with ubiquitin-dependent degradation by inhibiting the 26 S proteasome activity. This finding may explain the origin of the accumulation of ubiquitin conjugates.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Chymotrypsin; Cysteine Endopeptidases; Humans; In Vitro Techniques; Kinetics; Multienzyme Complexes; Neurofibrillary Tangles; Peptide Fragments; Proteasome Endopeptidase Complex; Proteins; Rabbits; Reticulocytes; Ubiquitins

Extracellular deposition of amyloid fibrils is responsible for the pathology in the systemic amyloidoses and probably also in Alzheimer disease [Haass, C. & Selkoe, D. J. (1993) Cell 75, 1039-1042] and type II diabetes mellitus [Lorenzo, A., Razzaboni, B., Weir, G. C. & Yankner, B. A. (1994) Nature (London) 368, 756-760]. The fibrils themselves are relatively resistant to proteolysis in vitro but amyloid deposits do regress in vivo, usually with clinical benefit, if new amyloid fibril formation can be halted. Serum amyloid P component (SAP) binds to all types of amyloid fibrils and is a universal constituent of amyloid deposits, including the plaques, amorphous amyloid beta protein deposits and neurofibrillary tangles of Alzheimer disease [Coria, F., Castano, E., Prelli, F., Larrondo-Lillo, M., van Duinen, S., Shelanski, M. L. & Frangione, B. (1988) Lab. Invest. 58, 454-458; Duong, T., Pommier, E. C. & Scheibel, A. B. (1989) Acta Neuropathol. 78, 429-437]. Here we show that SAP prevents proteolysis of the amyloid fibrils of Alzheimer disease, of systemic amyloid A amyloidosis and of systemic monoclonal light chain amyloidosis and may thereby contribute to their persistence in vivo. SAP is not an enzyme inhibitor and is protective only when bound to the fibrils. Interference with binding of SAP to amyloid fibrils in vivo is thus an attractive therapeutic objective, achievement of which should promote regression of the deposits.

Topics: Alzheimer Disease; Amyloid; Amyloid beta-Protein Precursor; Amyloidosis; Chymotrypsin; Galactose; Humans; Kinetics; Neurofibrillary Tangles; Protein Binding; Serum Amyloid A Protein; Spleen; Trypsin

The inhibition of alpha-chymotrypsin induced by cerebrospinal fluid from patients with senile dementia of Alzheimer's type, vascular dementia, and nondemented controls was investigated. We optimized the sensitivity of the assay so that the inhibition of alpha-chymotrypsin could be measured in all samples. The competitive inhibition was proportional to the amount of cerebrospinal fluid (CSF) added to the reaction mixture. After correction for protein concentration, the inhibition was higher with CSF from patients with senile dementia of Alzheimer's type than with those from patients with vascular dementia or controls. Inhibitory activity appeared to be specific for alpha-chymotrypsin because no inhibition for papain was found. Moreover, the depletion of alpha 1-antichymotrypsin from CSF by immunoadsorption revealed that this serpin was responsible for the disappearance of the inhibitory activity. Our findings suggested that the increased alpha-chymotrypsin inhibitory activity might represent an in vivo functional index of an abnormal protease metabolism in patients with Alzheimer's disease.

Topics: Aged; Alzheimer Disease; Chymotrypsin; Dementia, Multi-Infarct; Dementia, Vascular; Female; Humans; Hydrocephalus, Normal Pressure; Male; Plant Proteins; Reference Values

Amyloid deposits in Alzheimer's disease, Down's syndrome and aged brain are composed largely of A beta protein, which is generated by proteolytic processing of beta-amyloid precursor protein. Proteases responsible for liberating the A beta protein from the precursor have not yet been identified. Here, we examined the ability of cathepsin G, a chymotrypsin-like protease, to cleave two protease substrates: (i) a fluorogenic hexapeptide, whose sequence spans the cleavage site in the precursor for generating the A beta NH2-terminus, and (ii) recombinant human beta-amyloid precursor protein purified from a baculovirus expression system. Unlike two other members of the chymotrypsin family, cathepsin G readily degraded the hexapeptide. Furthermore, cathepsin G cleaved the beta-amyloid precursor protein to generate several breakdown products, including a prominent 11,500 mol. wt fragment immunoreactive with antibodies directed against the COOH-terminus of the protein. This COOH-terminal fragment co-migrated using two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis with C-100, a recombinant COOH-terminal segment of the beta-amyloid precursor, whose NH2-terminus is one residue upstream of the NH2-terminus of the A beta domain. We also examined the localization of cathepsin G in human brain. The distribution of cathepsin G-containing cells was examined by immunohistochemistry in the temporal cortex of both Alzheimer's and aged control samples. Cathepsin G-like immunoreactivity was contained specifically within neutrophils. As visualized by double-labeling with antibodies to cathepsin G and Factor VIII, neutrophils were most frequently found within meningeal or cortical blood vessels. In addition, occasional neutrophils could be identified without an apparent vascular surround, in the brain parenchyma. By simultaneous labeling with antibodies to cathepsin G and A beta protein, neutrophils were also sometimes found associated with both parenchymal and vessel amyloid deposits; however, these associations were rare. These findings indicate that cathepsin G is capable of cleaving the beta-amyloid precursor protein to liberate the free NH2-terminus of the A beta protein and may have access to areas where this material is deposited in Alzheimer's disease. However, since there is no physical association between neutrophils and deposited amyloid and no increase in the number of neutrophils in an Alzheimer's brain, cathepsin G seems

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Protein Precursor; Base Sequence; Cathepsin G; Cathepsins; Cerebral Cortex; Chymases; Chymotrypsin; Humans; Molecular Sequence Data; Molecular Weight; Nerve Tissue Proteins; Neutrophils; Peptide Fragments; Recombinant Proteins; Serine Endopeptidases; Temporal Lobe

The brain of Alzheimer disease patients contains plaques that are diagnostic for the disease. The plaques also contain beta-amyloid peptide, alpha 1-antichymotrypsin, and the element aluminum. We present indirect evidence that can relate all three components of plaques to each other in such a way as to suggest their involvement in the etiology of the disease. The beta-amyloid peptide is derived by proteolytic processing from beta-amyloid precursor proteins and some of these proteins contain a domain that is highly homologous to bovine pancreatic trypsin inhibitor. Bovine pancreatic trypsin inhibitor also inhibits alpha-chymotrypsin and we show that aluminum affects both the activity and the inhibition of this enzyme. At pH 6.5, in the presence of aluminum, the enzyme activity is doubled, and the inhibitor is only 1% as effective as in the absence of the metal ion. The inhibition by BX-9, a protease inhibitor prepared from protein components of amyloid plaques, is also reduced by aluminum; so too is that by alpha 1-antichymotrypsin but to a lesser degree. In the Alzheimer brain, we propose that aluminum may accelerate proteolytic processing of the beta-amyloid precursor protein by suppression of the inhibitor domain. Thus, the beta-amyloid peptide may accumulate and initiate plaque formation.

Topics: Aluminum; Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Aprotinin; Brain; Chymotrypsin; Enzyme Activation; Humans; Hydrogen-Ion Concentration; Serine Endopeptidases; Transferrin

Neurofibrillary tangles in Alzheimer's disease have been previously found to be labeled by some neurofilament antibodies that also recognize tau proteins. We have studied the reactivity of two such monoclonal antibodies, RT97 and 8D8, and of an anti-ubiquitin serum with the abnormal paired helical filaments (PHF)-tau (A68) polypeptides known to be the main component of the PHFs constituting the neurofibrillary tangles. 8D8 recognized the three major PHF-tau polypeptides, but RT97 reacted only with the two larger PHF-tau species. PHF-tau polypeptides were labeled by 8D8 and RT97 much more strongly than normal human tau and this labeling was decreased after alkaline phosphatase treatment. Anti-ubiquitin and anti-phosphotyrosine antibodies did not label PHF-tau polypeptides. The immunoreactivity of proteolytic fragments of PHF-tau polypeptides was studied with RT97, 8D8, and a panel of tau antibodies. The epitope for 8D8 on PHF-tau was localized between amino acids 222 and 427 in the carboxyl half of tau. The RT97 epitope on PHF-tau was localized in the amino domain of tau, probably in the 29-amino-acid insertion (insert 1) found towards the amino terminus of some tau isoforms. These results show that the basis for the labeling of neurofibrillary tangles by antibodies 8D8 and RT97 to neurofilament is their ability to react with PHF-tau polypeptides by recognizing sites specifically modified on PHF-tau, including a site specific to some tau isoforms.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Antibodies, Monoclonal; Blotting, Western; Chymotrypsin; Epitopes; Humans; Microscopy, Immunoelectron; Neurofibrillary Tangles; Neurofilament Proteins; Peptide Fragments; Peptide Mapping; Phosphotyrosine; tau Proteins; Trypsin; Tyrosine; Ubiquitins

The pathological findings of Alzheimer's disease include amyloid deposition in cerebral blood vessels and in senile plaques. Both deposits are known to include peptides that contain a common sequence. Both forms of amyloid were isolated and their peptide compositions were determined. The peptides were resolved by size-exclusion chromatography in 70% formic acid, and reverse-phase chromatography in 60% formic acid, 0-40% acetonitrile. Senile plaque amyloid cores contain about 25% protein, about 70% of which is composed of peptides containing the beta-amyloid sequence. Amino-terminal sequencing of the core amyloid peptides (CAPs) revealed extensive amino-terminal heterogeneity, with variable amounts of blocked amino termini. Matrix-assisted, laser-desorption-time-of-flight mass spectrometry of the CAP mixture revealed an array of peptides the molecular weights of which corresponded to peptides beginning with each of the first 11 amino acids of the beta-peptide sequence and ending with Ala-42 of that sequence. The carboxyl-terminal residues were identified by tandem mass spectrometry of chymotrypsin digests. CAP possessed a minor degree of carboxyl-terminal heterogeneity. Cerebrovascular amyloid peptides (CVAPs) possessed minor degrees of both amino- and carboxyl-terminal heterogeneity. The major CVAP commenced at Asp-1 and ended at Val-40. Minor components of CAP possessed masses of 8000-9000 Da and the same amino-terminal residues as the major components of CAP. They may be precursors to the smaller CAPs. The differences in amino termini and carboxyl termini of CAPs and CVAPs suggest that the two types of amyloid form by different pathways, on which they encounter different proteases.

Topics: Alzheimer Disease; Amino Acid Sequence; Amino Acids; Amyloid; Capillaries; Cerebral Cortex; Chromatography, High Pressure Liquid; Chymotrypsin; Humans; Mass Spectrometry; Molecular Sequence Data; Neurofibrillary Tangles; Peptide Fragments; Peptides; Spectrometry, Mass, Fast Atom Bombardment

Highly purified and SDS-soluble paired helical filaments (PHFs) were immunogold labeled and immunoblotted with antibodies to tau: Tau 14 (N-terminal half), AH-1 (microtubule-binding domain), and Tau 46 (C-terminal end). The main component of PHFs was modified tau of 68, 64, and 60 kd, also called A68 or PHF-tau. Trypsin digestion reduced the maximum width of PHFs by 10%-20%, increased aggregation of filaments, and abolished the binding of Tau 14, but had no effect on the binding of AH-1. The smallest tau-reactive tryptic fragments were 13 and 7-8 kd, positive with AH-1, and negative with Tau 46. Our results and the model of Crowther and Wischik suggest that by self-association and anti-parallel arrangement of the microtubule-binding domains, PHF-tau forms the backbone of PHFs.

Topics: Alzheimer Disease; Animals; Blotting, Western; Brain; Chymotrypsin; Epitopes; Humans; Immunohistochemistry; Microtubule-Associated Proteins; Microtubules; Nerve Tissue Proteins; Neurofibrils; Peptide Fragments; tau Proteins; Trypsin

Alz 50, a monoclonal antibody raised against Alzheimer brain homogenate, reacts with neurofibrillary tangles, microtubule-associated proteins tau, and Alzheimer brain proteins of molecular weight 70-60 kDa (A68). To study the relationship between A68 and normal human tau we compared the biochemical properties of these proteins and tested the reactivity of A68 with eight antibodies (Alz 50, Tau 60, Tau-2, Tau 14, Tau-1, Ab 636.7, NP14, Tau 46) that bind to various regions of tau molecule. On Western blots, all tau-reactive antibodies, except Tau-1, recognized A68. Pretreatment with alkaline phosphatase was required for the Tau-1 binding to A68. A68 consisted of three polypeptides of 68, 64, and 60 kDa, while tau contained 4-6 polypeptides of 50-65 kDa. A68 was less heterogenous than tau in the number of pI variants on two-dimensional gels. All A68 variants were more acidic (pI 5.5-6.5) than human tau (pI 6.5-8.5). Phosphatase treatment had only a minor effect on the pI and mobility of A68. Limited proteolysis of A68 with trypsin or chymotrypsin generated large fragments of 56-66 kDa (chymotrypsin) and 40-45 kDa (trypsin). While none of the fragments was recognized by Alz 50, the chymotryptic fragments were reactive with all the other tau antibodies, and the tryptic fragments were positive with five of the antibodies (Tau 14, Tau-1, Ab 636.7, NP14, and Tau 46). The peptide maps of A68 differed from that of tau in the number and the size of the peptide fragments. The differences in biochemical properties of these proteins and the sharing multiple epitopes suggest that A68 is a modified form of tau. The modification in part may be due to phosphorylation, although other changes rendering different isoelectrical properties and susceptibility to proteases need to be considered. The removal of the Alz 50 epitope by a cleavage of a 2-3 kDa fragment which does not contain the most C-terminal epitope (Tau 46) indicates that the Alz 50 epitope is located at the N-terminal periphery of the A68 molecule.

Topics: Alzheimer Disease; Antibodies, Monoclonal; Antibody Specificity; Chymotrypsin; Epitopes; Humans; Microtubule-Associated Proteins; Nerve Tissue Proteins; Peptide Mapping; Phosphoric Monoester Hydrolases; tau Proteins

Protease nexin-II (PN-II) is a protease inhibitor that forms SDS-resistant inhibitory complexes with the epidermal growth factor (EGF)-binding protein, the gamma-subunit of nerve growth factor, and trypsin. The properties of PN-II indicate that it has a role in the regulation of certain proteases in the extracellular environment. Here we describe more of the amino-acid sequence of PN-II and its identity to the deduced sequence of the amyloid beta-protein precursor (APP). Amyloid beta-protein is present in neuritic plaques and cerebrovascular deposits in individuals with Alzheimer's disease and Down's syndrome. A monoclonal antibody against PN-II (designated mAbP2-1) recognized PN-II in immunoblots of serum-free culture medium from human glioblastoma cells and neuroblastoma cells, as well as in homogenates of normal and Alzheimer's disease brains. In addition, mAbP2-1 stained neuritic plaques in Alzheimer's disease brain. PN-II was a potent inhibitor of chymotrypsin with an inhibition constant Ki of 6 x 10(-10)M. Together, these data demonstrate that PN-II and APP are probably the same protein. The regulation of extracellular proteolysis by PN-II and the deposition of at least parts of the molecule in senile plaques is consistent with previous reports that implicate altered proteolysis in the pathogenesis of Alzheimer's disease.

Topics: alpha 1-Antichymotrypsin; Alzheimer Disease; Amino Acid Sequence; Amyloid; Amyloid beta-Protein Precursor; Antibodies, Monoclonal; Chymotrypsin; Humans; Immunoenzyme Techniques; Molecular Sequence Data; Protease Inhibitors; Protein Precursors; Sequence Homology, Nucleic Acid

Two monoclonal antibodies that recognize Alzheimer's neurofibrillary tangles (ANTs), AD10 and AB18, have been characterized by immunoblotting against human and calf spinal cord neurofilament (NF) and calf brain microtubule preparations. Both antibodies bind to the 200-kilodalton (kd) (NF-H) and 160-kd (NF-M) but not to the 68-kd (NF-L) NF triplet proteins. They also bind to high-molecular-weight microtubule-associated proteins (MAPs) and tau. AD10 immunostains MAP2 and MAP1 families, whereas AB18 stains mainly MAP1 bands. Preincubation of intact filament preparation or nitrocellulose strips containing electroblotted NF proteins with Escherichia coli alkaline phosphatase completely blocks AD10 binding and partially blocks binding of AB18. These results suggest that the determinants recognized by these antibodies are phosphorylated. Immunoblotting of peptide fragments generated by limited proteolysis of NF proteins with alpha-chymotrypsin and Staphylococcus aureus V8 protease shows that the localization of the antigenic determinants to AD10 and AB18 in NF-H is approximately 100 and 60 kd, respectively, away from the carboxy terminal, a region previously shown to form the NF projection side arm. In NF-M, the antigenic determinants to both antibodies are located also in the projection side arm, in a 60-kd polypeptide adjacent to the alpha-helical filament core. The results show that ANTs contain at least two phosphorylated antigenic sites that are present in NF and MAPs, a finding suggesting that ANTs may be composed of proteins or their fragments with epitopes shared by cytoskeletal proteins.

Topics: Alzheimer Disease; Animals; Antibodies, Monoclonal; Chymotrypsin; Cytoskeleton; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Epitopes; Fluorescent Antibody Technique; Immunosorbent Techniques; Intermediate Filament Proteins; Intermediate Filaments; Mice; Microtubule-Associated Proteins; Neurofilament Proteins; Serine Endopeptidases