Oligomerizaiton and fibril asssembly of the amyloid-beta protein.

In this chapter, we attempt to analyze the evolution of the amyloid-beta (Abeta) molecular structure from its inception as part of the Abeta precursor protein to its release by the secretases and its extrusion from membrane into an aqueous environment. Biophysical studies suggest that the Abeta peptide sustains a series of transitions from a molecule rich in alpha-helix to a molecule in which beta-strands prevail. It is proposed that initially the extended C-termini of two opposing Abeta dimers form an antiparallel beta-sheet and that the subsequent addition of dimers generates a helical Abeta protofilament. Two or more protofilaments create a strand in which the hydrophobic core of the beta-sheets is shielded from the aqueous environment by the N-terminal polar domains of the Abeta dimers. Once the nucleation has occurred, the Abeta filament grows in length by the addition of dimers or tetramers.

Small assemblies of unmodified amyloid beta-protein are the proximate neurotoxin in Alzheimer's disease.

Pioneering work in the 1950s by Christian Anfinsen on the folding of ribonuclease has shown that the primary structure of a protein "encodes" all of the information necessary for a nascent polypeptide to fold into its native, physiologically active, three-dimensional conformation (for his classic review, see [Science 181 (1973) 223]). In Alzheimer's disease (AD), the amyloid beta-protein (Abeta) appears to play a seminal role in neuronal injury and death. Recent data have suggested that the proximate effectors of neurotoxicity are oligomeric Abeta assemblies. A fundamental question, of relevance both to the development of therapeutic strategies for AD and to understanding basic laws of protein folding, is how Abeta assembly state correlates with biological activity. Evidence suggests, as argued by Anfinsen, that the formation of toxic Abeta structures is an intrinsic feature of the peptide's amino acid sequence-one requiring no post-translational modification or invocation of peptide-associated enzymatic activity.

Escherichia coli cells lacking methylation-blocking factor (leucine-responsive regulatory protein) have precise timing of initiation of DNA replication in the cell cycle.

A protein that is required for specific methylation inhibition of two GATC sites in the papBA pilin promoter region, known as methylation-blocking factor (Mbf) and recently shown to be identical to the leucine-responsive regulatory protein (Lrp), is not responsible for the delayed methylation at oriC implicated in an eclipse period following initiation of DNA replication. Cells containing a transposon mutation within the mbf (lrp) gene initiate DNA replication at the correct time during the cell cycle, whereas cells with increased amounts of the Dam methyltransferase initiate DNA replication randomly throughout the cell cycle.

RecA protein of Escherichia coli and chromosome partitioning.

Escherichia coli cells deficient in RecA protein frequently contain an abnormal number of chromosomes after completion of ongoing rounds of DNA replication. This suggests that RecA protein may be required for correct timing of initiation of DNA replication; however, we show here that initiation of DNA replication is properly timed in recA mutants. We also find that more than 10% of recA mutant cells contain no DNA. These anucleate cells appear to arise from partitioning of all the DNA into one daughter cell and no DNA into the other daughter cell. Based on these and previously published results, we propose that RecA protein is required for equal partitioning of chromosomes into the two daughter cells.

The nanometer-scale structure of amyloid-beta visualized by atomic force microscopy.

Amyloid-beta (A beta) is the major protein component of neuritic plaques found in Alzheimer's disease. Evidence suggests that the physical aggregation state of A beta directly influences neurotoxicity and specific cellular biochemical events. Atomic force microscopy (AFM) is used to investigate the three-dimensional structure of aggregated A beta and characterize aggregate/fibril size, structure, and distribution. Aggregates are characterized by fibril length and packing densities. The packing densities correspond to the differential thickness of fiber aggregates along a zeta axis (fiber height above the x-y imaging surface). Densely packed aggregates ( > or = 100 nm thick) were observed. At the edges of these densely packed regions and in dispersed regions, three types of A beta fibrils were observed. These were classified by fibril thickness into three size ranges: 2-3 nm thick, 4-6 nm thick, and 8-12 nm thick. Some of the two thicker classes of fibrils exhibited pronounced axial periodicity. Substructural features observed included fibril branching or annealing and a height periodicity which varied with fibril thickness. When identical samples were visualized with AFM and electron microscopy (EM) the thicker fibrils (4-6 nm and 8-12 nm thick) had similar morphology. In comparison, the densely packed regions of approximately > or = 100 nm thickness observed by AFM were difficult to resolve by EM. The small, 2- to 3-nm-thick, fibrils were not observed by EM even though they were routinely imaged by AFM. These studies demonstrate that AFM imaging of A beta fibrils can, for the first time, resolve nanometer-scale, zeta-axis, surface-height (thickness) fibril features. Concurrent x-y surface scans of fibrils reveal the surface submicrometer structure and organization of aggregated A beta. Thus, when AFM imaging of A beta is combined with, and correlated to, careful studies of cellular A beta toxicity it may be possible to relate certain A beta structural features to cellular neurotoxicity.

Morphology and toxicity of Abeta-(1-42) dimer derived from neuritic and vascular amyloid deposits of Alzheimer's disease.

In the course of analyzing the chemical composition of Alzheimer's disease neuritic and vascular amyloid, we have purified stable dimeric and trimeric components of Abeta peptides. These peptides (molecular mass 9.0 and 13.5 kDa) were separated by size exclusion chromatography in the presence of 80% formic acid or 5 guanidine thiocyanate, pH 7.4. The average ratio of monomers, dimers, and trimers was 55:30:15, respectively. Similar structures were produced over time upon incubation of synthetic Abeta-(1-42) at pH 7.4. The stability of these oligomeric forms was also demonstrated by Western blot and mass spectrometry. Atomic force microscopy and electron microscopy rotary shadowing revealed that the monomers polymerized into 8-10-nm filaments, whereas the dimers generated prolate ellipsoids measuring 3-4 nm in diameter. The pathogenic effects of the dimeric Abeta-(1-40/42) were tested in cultures of rat hippocampal neuron glia cells. Only in the presence of microglia did the dimer elicit neuronal killing. It is possible that these potentially pathogenic Abeta-(1-40/42) dimers and trimers from Alzheimer's disease amyloid represent the soluble oligomers of Abeta recently described in Alzheimer's disease brains (Kuo, Y.-M., Emmerling, M. R., Vigo-Pelfrey, C., Kasunic, T. C., Kirkpatrick, J. B., Murdoch, G. H., Ball, M. J., and Roher, A. E. (1996) J. Biol. Chem., 271, 4077-4081).

Clusterin (apoJ) alters the aggregation of amyloid beta-peptide (A beta 1-42) and forms slowly sedimenting A beta complexes that cause oxidative stress.

Clusterin (apoJ), a multifunctional apolipoprotein made by cells in the brain and many other locations, is associated with aggregated amyloid beta-peptide (A beta) in senile and diffuse plaques of Alzheimer's disease (AD). We observed that purified human serum clusterin partially blocked the aggregation of synthetic A beta 1-42, as shown by centrifugal assays (14,000g x 10 min) and by atomic force (scanning probe) microscopy. Slowly sedimenting A beta complexes were formed in the presence of clusterin, which included aggregates > 200 kDa that resist dissociation by low concentrations of SDS. Clusterin enhanced the oxidative stress caused by A beta, as assayed by oxidative stress in PC12 cells with MTT, which is widely used to estimate neurotoxicity. These indications of enhanced neurotoxicity by the MTT assay were observed in the highly aggregated rapidly sedimenting fraction, but also in more slowly sedimenting "soluble" forms. This novel activity of slowly sedimenting A beta may enhance the neurotoxicity of A beta deposits in AD brains, because soluble complexes have a potential for diffusing to damage distal neurons.