The progressive neurodegeneration of Alzheimer's disease has been hypothesized to be mediated, at least in part, by beta-amyloid protein. A relationship between the aggregation state of beta-amyloid protein and its ability to promote degeneration in vitro has been previously suggested. To evaluate this hypothesis and to define a structure-activity relationship for beta-amyloid, aggregation properties of an overlapping series of synthetic beta-amyloid peptides (beta APs) were investigated and compared with beta AP neurotoxic properties in vitro. Using light microscopy, electrophoresis, and ultracentrifugation assays, we found that few beta APs assembled into aggregates immediately after solubilization, but that over time peptides containing the highly hydrophobic beta 29-35 region formed stable aggregations. In short-term neuronal cultures, toxicity was associated specifically with those beta APs that also exhibited significant aggregation. Further, upon the partial reversal of beta 1-42 aggregation, a concomitant loss of toxicity was observed. A synthetic peptide derived from a different amyloidogenic protein, islet amyloid polypeptide, exhibited aggregation but not toxicity, suggesting that beta AP-induced neurotoxicity in vitro is not a nonspecific reaction to aggregated protein. The correlation between beta AP aggregation and neurotoxicity was also observed in long-term neuronal cultures but not in astrocyte cultures. These data are consistent with the hypothesis that beta-amyloid protein contributes to neurodegeneration in Alzheimer's disease.

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The A4 or 13 protein is a peptide that constitutes the major protein component of senile plaques in Alzheimer disease. The A4/13 protein is derived from a larger, transmembrane amyloid precursor protein (APP). The putative abnormal processing events leading to amyloid accumulation are largely unknown. Here we report that a 42-residue synthetic peptide, 181-42, corresponding to one of the longer forms of the A4/13 protein, accumulates in cultured human skin fibroblasts and is stable for at least 3 days. The A4/13 protein is derived from a larger transmembrane protein, the amyloid precursor protein (APP), which is expressed as multiple different mRNA splicing products (3-5).Accumulation of the A4/1B protein is apparently the result of abnormal processing, since the extracellular domain of APP is normally cleaved at residue 16 within the A4/13 region (6, 7). The fact that normal processing precludes A4/13 accumulation suggests that abnormal processing may be the initial step leading to amyloid deposition. Using synthetic peptide analogs of the A4/(3 protein, we have defined some of its intrinsic biochemical and physical properties that are related to its assembly into amyloid-like fibrils and its ability to aggregate (8). We found that assembly into amyloid-like fibrils and aggregation in SDS/polyacrylamide gels are separate and distinct properties of the amyloid peptides. Low pH (pH 3.5-6.5) and high concentrations of peptide are important for promoting assembly of the peptides into amyloid-like fibrils (8). The length of the hydrophobic C terminus (-42 residues) and a high concentration of peptide are critical for the ability of the (31 2 peptide to self-aggregate into multiple discrete bands in SDS/polyacrylamide gels. These intrinsic factors may be important for amyloid deposition in vivo because the acid environment of endosomes and lysosomes and their ability to concentrate solutes would promote amyloid fibril formation and peptide aggregation, which could compromise the ability of the cell to degrade the A4/13 protein.To explore whether cells are able to degrade the A4//3 protein, we examined uptake and degradation of three peptides that corresponded to residues 1-28 (extracellular portion) (p13-28), FRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVG-GVVIA) were synthesized by fluoren-9-ylmethoxycarbonyl chemistry using a continuous-flow semiautomatic instrument, purified by reverse-phase HPLC, and characterized by sequencing and electrospray mass spectrometry (8). Na1251 was obtained from Amersham; chloroglycouracil (lodo-Gen) from Pierce, Bio-Gel P-2 from Bio-Rad, fetal bovine serum from GIBCO, Dulbecco's modified Eagle's medium (DMEM) from Irvine Scientific, and Percoll from Pharmacia. Analytical-grade solvents and other reagents were from various commercial sources (Sigma; Fisher Scientific).Peptide Iodination. Aliquots (10 pg) of each peptide in 40 jul of 1 M Tris (pH 7.4) were radioiodinated to a specific activity of 50,000-150,000 cpm per ng in the presence of 50 u.g of Iodo-Gen at 0C for 20 min (8). Free ...

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We have analyzed the effect of internalized amyloid beta-protein (A beta) 1-42 aggregates on the metabolism of the amyloid precursor protein (APP) in stably transfected 293 cells. The amount of potentially amyloidogenic fragments of APP immunoprecipitated by anti-carboxyl-terminal APP and anti-A beta antibodies is dramatically enhanced by the treatment of the cells with A beta 1-42, which is resistant to degradation, but not A beta 1-28, which does not accumulate in cells. This accumulation of amyloidogenic carboxyl-terminal fragments is specific, since there is relatively little effect of A beta 1-42 on the amount of the nonamyloidogenic alpha-secretase carboxyl-terminal fragment. The amyloidogenic fragments accumulate in the same nonionic detergent-insoluble fraction of the cell that contains the internalized A beta 1-42. Western analysis indicates that a subset of the amyloidogenic fragments react with antibodies that recognize a conformation of A beta that is specifically associated with aggregated forms of A beta, suggesting that the adoption of this aggregation-related conformation may be an early event which precedes the final processing that produces A beta. Pulse-chase analysis of the [35S]Met-labeled 16-kDa amyloidogenic fragment indicates that it is relatively stable in A beta 1-42-treated cells, with a half-life of approximately 50 h. This fragment is degraded with a half-life of 30 min in control cells treated with A beta 1-28. In contrast, the turnover of the nonamyloidogenic alpha-secretase product is not significantly altered by the presence of A beta 1-42. The continuous uptake of A beta 1-42 from the medium is not required for the stimulation of amyloidogenic fragment accumulation, suggesting that the presence of intracellular A beta 1-42 aggregates establishes a new pathway for APP catabolism in cells which leads to the long term stability of the fragments. If these amyloidogenic fragments of APP ultimately give rise to A beta, then the production of A beta may be an autocatalytic, "runaway" process in cells containing A beta 1-42 nuclei. It is conceivable that the accumulation of insoluble APP and amyloidogenic fragments of APP in response to A beta 1-42 aggregates may mimic the pathophysiology of dystrophic neurites, where the accumulation of intracellular APP and APP fragments has been documented by immunohistochemistry.

Protein kinases are known for their highly conserved adenosine triphosphate (ATP)-binding site, rendering the discovery of selective inhibitors a major challenge. In theory, allosteric inhibitors can achieve high selectivity by targeting less conserved regions of the kinases, often with an added benefit of retaining efficacy under high physiological ATP concentration. Although often overlooked in favor of ATP-site directed approaches, performing a screen at high ATP concentration or stringent hit triaging with high ATP concentration offers conceptually simple methods of identifying inhibitors that bind outside the ATP pocket. Here, we applied the latter approach to the With-No-Lysine (K) (WNK) kinases to discover lead molecules for a next-generation antihypertensive that requires a stringent safety profile. This strategy yielded several ATP noncompetitive WNK1-4 kinase inhibitors, the optimization of which enabled cocrystallization with WNK1, revealing an allosteric binding mode consistent with the observed exquisite specificity for WNK1-4 kinases. The optimized compound inhibited rubidium uptake by sodium chloride cotransporter 1 (NKCC1) in HT29 cells, consistent with the reported physiology of WNK kinases in renal electrolyte handling.

Phosphorylation of the nervous system-specific growth cone protein GAP-43 by kinase C in vivo occurs exclusively in growth cones and distal axons, and the onset of this phosphorylation is delayed relative to the onset of axonogenesis, with the delay predicted on the time needed for axons to reach the vicinity of their targets (Meiri et al., 1991). We have used a subcellular fraction of intact growth cones (IGCs) to investigate whether this induction of GAP-43 phosphorylation can be influenced by target-derived substances, and show here that increased phosphorylation of GAP-43 can be both stimulated and maintained by NGF at concentrations of 2 x 10(-10) M. This low concentration of NGF and the subsequent phosphorylation of GAP-43 are both consistent with the interpretation that phosphorylation is due to the binding of NGF to a biologically active high-affinity receptor. Second, we used the monoclonal antibody 2G12 to show that the NGF-stimulated phosphorylation of GAP-43 occurs on serine, the kinase C phosphorylation site, consistent with the results seen in vivo. Levels of phosphorylated GAP-43 in the intact IGCs are also modulated by calcium-stimulated dephosphorylation that could be inhibited by EGTA but not okadaic acid and that therefore resembled the calcineurin-stimulated dephosphorylation reported in vitro. The results suggest that the spatial and temporal regulation of GAP-43 phosphorylation that occurs during axonogenesis in vivo can be regulated by target-derived neurotropic molecules, specifically NGF.