Characterization of endogenous APP processing in a cell-free system.

We have developed a simple in vitro assay using tissue homogenates that allows detection and characterization of several endogenous proteolytic activities which convert Alzheimer's amyloid precursor protein (APP) to the smaller, carboxy-terminal fragments, postulated to be intermediates in the formation of ß-amyloid peptide (Aß). Incubation at 37°C results in the degradation of transmembrane APP and formation of a mixture of carboxy-terminal containing peptides with mass values of 9-12 kDa. Epitope mapping and electrophoretic comparison with a truncated APP standard showed one of these peptides to contain the entire Aß sequence. Analysis of pH dependence shows that formation of this carboxy-terminal product as well as another fragment, that is the likely product of 'secretase' activity, requires acidic pH. This suggests that cleavage of full-length APP to secreted forms may take place in an acidic intracellular compartment.

Selective aggregation of endogenous beta-amyloid peptide and soluble amyloid precursor protein in cerebrospinal fluid by zinc.

Zinc added to buffered solutions of synthetic beta-amyloid peptide (A beta) has been reported to induce accelerated formation of insoluble aggregates. This observation suggests that zinc may play a role in the formation of senile plaques, which contain A beta, in Alzheimer's disease. To test this hypothesis under conditions more representative of the brain, we investigated the ability of zinc to induce aggregation of A beta in freshly drawn canine CSF, which contains the same sequence as human A beta. Aggregates were separated from CSF by ultracentrifugation before and after incubation with zinc and assayed by quantitative western blotting and ELISA. We found that zinc induced the rapid aggregation of endogenous A beta in CSF, with an EC50 of 120-140 microM. The reaction was specific, because most (> or = 95%) CSF protein remained soluble under conditions where most A beta was insoluble, as assayed by scanning densitometry of Coomassie-stained gels. Staining of the precipitated material resulted in the visualization of punctate regions that were thioflavin positive or birefringent when stained with Congo red, suggesting the formation of amyloid-related structures. These results suggest that zinc could play a role in amyloid deposition, because there is overlap between the regions of the brain where zinc concentrations are highest and regions with the highest amyloid content. It is surprising that zinc induced the aggregation of endogenous soluble APP at lower concentrations than required for A beta (EC50 80 microM). The possibility that zinc-induced aggregation of APP may precede the deposition of A beta into plaques is discussed. Investigation of aggregation of A beta in CSF will aid in assessing the biological relevance of other agents that have been reported to accelerate amyloid formation.

Evaluation of cathepsins D and G and EC 3.4.24.15 as candidate beta-secretase proteases using peptide and amyloid precursor protein substrates.

No single protease has emerged that possesses all the expected properties for beta-secretase, including brain localization, appropriate peptide cleavage specificity, and the ability to cleave amyloid precursor protein exactly at the amino-terminus of beta-amyloid peptide. We have isolated and purified a brain-derived activity that cleaves the synthetic peptide substrate SEVKMDAEF between methionine and aspartate residues, as required to generate the amino-terminus of beta-amyloid peptide. Its molecular size of 55-60 kDa and inhibitory profile indicate that we have purified the metalloprotease EC 3.4.24.15. We have compared the sequence specificity of EC 3.4.24.15, cathepsin D, and cathepsin G for their ability to cleave the model peptide SEVKMDAEF or related peptides that contain substitutions reported to modulate beta-amyloid peptide production. We have also tested the ability of these enzymes to form carboxyl-terminal fragments from full-length, membrane-embedded amyloid precursor protein substrate or amyloid precursor protein that contains the Swedish KM --> NL mutation. The correct cleavage was tested with an antibody specific for the free amino-terminus of beta-amyloid peptide. Our results exclude EC 3.4.24.15 as a candidate beta-secretase. Although cathepsin G cleaves the model peptide correctly, it displays poor ability to cleave the Swedish KM --> NL peptide and does not generate carboxy-terminal fragments that are immunoreactive with amino-terminal-specific antiserum. Cathepsin D does not cleave the model peptide or show specificity for wild-type amyloid precursor protein; however, it cleaves the Swedish "NL peptide" and "NL precursor" substrates appropriately. Our results suggest that cathepsin D could act as beta-secretase in the Swedish type of familial Alzheimer's disease and demonstrate the importance of using full-length substrate to verify the sequence specificity of candidate proteases.

Mutagenicity of soluble and insoluble nickel compounds at the gpt locus in G12 Chinese hamster cells.

Nickel is an established human and animal carcinogen, but efforts to demonstrate its mutagenicity in a number of cell types have not been successful. In this report we describe the mutational response to nickel compounds in the G12 cell line, an hprt deficient V79 cell line containing a single copy of the E. coli gpt gene. This cell line has a low spontaneous background, making it suitable for assessment of mutagenic responses to environmental contaminants. When G12 cells were treated with insoluble particles of crystalline nickel sulfide < 5 microns in diameter, a strong, dose-dependent mutagenic response was observed up to 80 times the spontaneous background. Of 48 mutant gpt(-) clones isolated that were induced by insoluble nickel, all were capable of DNA amplification of the gpt sequences by polymerase chain reaction (PCR). The ability to produce full-length PCR products is an indication that large deletions of gene sequences have not occurred. When G12 cells were treated with soluble nickel sulfate, the mutational response was not significantly increased over the spontaneous background. This difference in mutagenic response reflects a large difference in the mutagenic potential of soluble and insoluble nickel compounds, which reflects the carcinogenic potential of these forms of nickel.

The effect of Ni(II) on DNA replication.

The cellular regulation of DNA replication is governed in part by the availability of essential metal ions. A continuous supply of Mg(II) ions is necessary for the efficient and faithful replication of parental strands during S-phase as well as during the repair of DNA damage. A metal ion such as Ni(II) may interfere with the replication process by binding to sites on proteins at which essential ions normally bind. Binding at these sites by a toxic metal ion may produce inappropriate responses from the replication proteins and thus alter the normal balance in one or more of the microsteps comprising DNA synthesis. We have studied the effect of Ni(II) on DNA replication in a reconstituted in vitro system using a HeLa cell extract as a source of polymerase activity on a template of activated calf thymus DNA. Ni(II) has an initial stimulatory effect that is followed by an overall inhibition of the incorporation of DNA precursors. These results suggest that Ni(II), similar to Mg(II) may have more than one binding site, but that the binding of Ni(II) to replication proteins may significantly alter the timing of events in DNA synthesis.

Chromosomal changes in cell lines from mouse tumors induced by nickel sulfide and methylcholanthrene.

Rhabdomyosarcomas were induced in mice by intramuscular injections of crystalline nickel sulfide and 3-methylcholanthrene. At early passage, karyotypes were performed by G-banding for four nickel sulfide cell lines and for three 3-methylcholanthrene cell lines. Six cell lines were near-diploid and one nickel sulfide line was near-tetraploid. Three of the nickel sulfide cell lines were characterized by a rearranged marker chromosome which was present in a majority of the cells of each line. The rearrangements leading to the formation of marker chromosomes were different in each nickel sulfide cell line but involved chromosome 4 in two of the nickel sulfide cell lines. Extra copies of chromosome 15 were present in two nickel sulfide cell lines. Possible rearrangement and/or gene activation was examined for the c-mos oncogene on chromosome 4 and the c-myc oncogene on chromosome 15, but no alteration or activation was observed. None of the 3-methylcholanthrene cell lines contained rearranged marker chromosomes; however, one MCA cell line did contain large numbers of double minutes. In all cell lines, minichromosomes (small atypical acrocentric chromosomes) were observed that contained distinct centromeric regions but no other G-positive bands.

Novel human light chain V kappa segment: serologic and structural analyses of the kappa III-like Bence Jones protein and IgG kappa light chain REE.

Immunochemical and sequence analyses of kappa light chain REE (Bence Jones protein REE and the light chain isolated from IgG kappa myeloma protein REE) revealed antigenic and structural features not previously described for human kappa-chains. Although closely related to proteins of the V kappa III subgroup, light chain REE is readily distinguished from light chains classified serologically as members of the kappa IIIa or kappa IIIb sub-subgroups. Light chains REE (Bence Jones protein REE and light chain REE) are identical in sequence and differ from kappa III proteins by at least 10 uncommon amino acid substitutions in the first three framework regions. Further, kappa-chain REE is unique by virtue of a four-residue deletion in the third complementarity-determining region. The deletion encompasses the three carboxyl-terminal residues in the V kappa-encoded segment and the first residue at the site of V-J recombination. Urine specimens from patient REE also contained a light chain fragment that lacked the first (amino-terminal) 85 residues of the native light chain but otherwise was identical in sequence to the light chain REE. The extensive amino acid differences and unique length of the V kappa segment in light chain REE indicate that this kappa-chain is the product of an unusual V kappa III gene or, alternatively, represents a rarely expressed and novel human V kappa gene.