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Biomedical subjects

G A Krafft

Publications and source records attributed to G A Krafft.

At least 19 recordsLinked to original sources
PubMed1998

Involvement of cell surface glycosyl-phosphatidylinositol-linked aspartyl proteases in alpha-secretase-type cleavage and ectodomain solubilization of human Alzheimer beta-amyloid precursor protein in yeast.

Human beta-amyloid precursor protein (APP) introduced into yeast undergoes alpha-secretase-type cleavage, suggesting that yeast have alpha-secretase-like protease(s). Here we report that two structurally and functionally related glycosyl-phosphatidylinositol-linked yeast aspartyl proteases, Mkc7p and Yap3p (collectively termed yapsin), are responsible for alpha-secretase-type cleavage of APP expressed in yeast, resulting in release of soluble APP into the extracellular space. Disruption of MKC7 and YAP3 in a vacuolar protease-deficient strain abolished this APP cleavage/release, and APP cleavage/release could be restored by introduction of MKC7 or YAP3 on a single copy plasmid. Purified Mkc7p cleaved an internally quenched fluorogenic APP peptide substrate at the alpha-secretase cleavage site. Measurement of proteolytic activity either in yeast homogenates or on the yeast cell surface revealed that most Mkc7p and Yap3p activities were localized at the cell surface. These results establish a molecular basis for alpha-secretase-type cleavage in yeast and support the generally held concept that alpha-secretase cleavage of APP occurs at the cell surface.

Amyloid Precursor Protein Secretases↗
PubMed1998

Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins.

Abeta1-42 is a self-associating peptide whose neurotoxic derivatives are thought to play a role in Alzheimer's pathogenesis. Neurotoxicity of amyloid beta protein (Abeta) has been attributed to its fibrillar forms, but experiments presented here characterize neurotoxins that assemble when fibril formation is inhibited. These neurotoxins comprise small diffusible Abeta oligomers (referred to as ADDLs, for Abeta-derived diffusible ligands), which were found to kill mature neurons in organotypic central nervous system cultures at nanomolar concentrations. At cell surfaces, ADDLs bound to trypsin-sensitive sites and surface-derived tryptic peptides blocked binding and afforded neuroprotection. Germ-line knockout of Fyn, a protein tyrosine kinase linked to apoptosis and elevated in Alzheimer's disease, also was neuroprotective. Remarkably, neurological dysfunction evoked by ADDLs occurred well in advance of cellular degeneration. Without lag, and despite retention of evoked action potentials, ADDLs inhibited hippocampal long-term potentiation, indicating an immediate impact on signal transduction. We hypothesize that impaired synaptic plasticity and associated memory dysfunction during early stage Alzheimer's disease and severe cellular degeneration and dementia during end stage could be caused by the biphasic impact of Abeta-derived diffusible ligands acting upon particular neural signal transduction pathways.

Alzheimer Disease↗
PubMed1998

Amyloid-beta peptide activates cultured astrocytes: morphological alterations, cytokine induction and nitric oxide release.

A common feature of many neurodegenerative disorders is an abundance of activated glial cells (astrocytes and microglia). In Alzheimer's disease (AD), activated astrocytes are in close apposition to and surrounding the amyloid plaques. The mechanisms by which the astrocytes become activated in AD and the consequences of reactive astrocytosis to disease progression are not known. We examined the possibility that the amyloid-beta (Abeta) peptide, a major constituent of the amyloid plaque, could act as a stimulus leading to activation. We found that treatment of rat cortical astrocyte cultures with aggregated Abeta 1-42 peptide induces activation, as assessed by reactive morphological changes and upregulation of selective glial mRNA and proteins, such as the inflammatory cytokine interleukin-1beta. Abeta also stimulates inducible nitric oxide synthase (iNOS) mRNA levels and nitric oxide (NO) release. Abeta 1-42, a major form of amyloid associated with neurotoxicity, activated astrocytes in a time- and dose-dependent manner, whereas a scrambled Abeta 1-42 sequence or Abeta 17-42 had little or no effect. We also determined that the Abeta activity can be found in a supernatant fraction containing soluble Abeta oligomers. Our data suggest that Abeta plays a role in the reactive astrocytosis of AD and that the inflammatory response induced upon glial activation is a critical component of the neurodegenerative process.

Amino Acid Sequence↗
PubMed1997

Rapid impact of beta-amyloid on paxillin in a neural cell line.

Beta-amyloid1-42 (Abeta) is a naturally occuring peptide whose accumulation in the brain is putatively coupled to Alzheimer's disease pathogenesis. Deleterious effects of Abeta on neurons have been linked to the inappropriate activation of signaling pathways within the cell (reviewed in Yankner, 1996), including tyrosine phosphorylation of focal adhesion kinase (FAK) (Zhang et al., 1994, 1996a,b). Here we have investigated the effects of Abeta on paxillin in a neural cell line. Paxillin, a substrate for FAK, is thought to act as a signal "integrator," functioning to link other proteins into multi-molecular signaling complexes (reviewed in Turner, 1994). Treatment of the rat central nervous system B103 cell line with aggregates of Abeta was found to induce the tyrosine phosphorylation of paxillin within 30 min, nearly 24 hr prior to significant cell death. Particularly striking was a subsequent "mobilization" of paxillin to the cytoskeleton in Abeta-treated cells. The amount of paxillin associated with the cytoskeleton in Abeta-treated cells was increased 10-fold over controls. The Abeta-induced paxillin accumulation could be visualized immunocytochemically, with an increase in number and size of paxillin-labeled focal contacts upon treatment with Abeta. This effect was specific, in that vinculin, another focal contact protein, was unaffected by Abeta. Disruption of f-actin, which inhibits both Abeta-induced neurotoxicity (Furukawa and Mattson, 1995) and focal contact signaling in B103 cells (Zhang et al., 1996b) was found to block the cytoskeletal paxillin accumulation. The rapid tyrosine phosphorylation and cytoskeletal mobilization of paxillin links Abeta to the activation of focal contact signaling events that may influence neuronal cytoskeletal architecture, gene expression, synaptic plasticity and cell viability.

Actins↗
PubMed1997

Internally consistent libraries of fluorogenic substrates demonstrate that Kex2 protease specificity is generated by multiple mechanisms.

Kex2 protease from the yeast Saccharomyces cerevisiae is the prototype for a family of eukaryotic proprotein processing proteases. To clarify understanding of the interactions responsible for substrate recognition in this family of enzymes, we have carried out a systematic examination of Kex2 substrate specificity using internally consistent sets of substrates having substitutions at only one or two positions. We examined Kex2 sequence recognition for residues at P3, P2, and P1 using two types of fluorogenic peptide substrates, peptidyl-methylcoumarinamides and internally quenched substrates in which cleavage occurs at an actual peptide bond. Kinetic analysis of the two sets of substrates gave comparable data on specificity at these three positions. For the best substrate sequences, high catalytic constants (kCM/KM) of (2-5) x 10(7) M-1 s-1 were seen for cleavage of both peptidyl-methylcoumarinamides and peptide bonds. While no evidence for positive interactions with the P3 residue emerged, Kex2 was found to discriminate against at least one residue Asp. at this position. Specificity at P2 was shown to rely primarily on recognition of a positive charge, although steric constraints on the P2 side chain were also apparent. Kex2 was demonstrated to be exquisitely selective for Arg at P1. Substitutions with similar charge (Lys, ornithine) or similar hydrogen-bonding capability (citrulline) do not confer efficient catalysis. Comparison of otherwise identical substrates having either Arg or citrulline at P1 showed that the positive charge of the Arg guanidinium group stabilizes the transition state by approximately 6.8 kcal/mol.

Arginine↗
PubMed1996

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).

Alzheimer Disease↗
PubMed1996

A beta peptide enhances focal adhesion kinase/Fyn association in a rat CNS nerve cell line.

Neurotoxicity of the amyloid beta protein (A beta) is known to correlate with a selective change in protein tyrosine phosphorylation (Tyr(P)) of focal adhesion kinase (FAK) (Zhang et al., J. Biol. Chem., 269 (1994) 25247-25250). The current work has found that exposure of neuronal cells to A beta upregulates the stable association of FAK with Fyn, a neuronally-enriched protein tyrosine kinase of the Src-family. In cells incubated with aged A beta 1-42, the amount of immunoprecipitable FAK-Fyn complex increased approximately 280%. Equivalent results were obtained whether anti-FAK or anti-Fyn was used to precipitate the complex. Cells incubated with non-toxic A beta 17-42, which makes aggregates and attaches to cells but does not upregulate FAK Tyr(P), exhibited no increase in FAK-Fyn complex. Aberrant Fyn activity due to the A beta evoked association with FAK could play a role in neuronal degeneration and also cause anomalies in synaptic plasticity. These possibilities are of particular significance because of the reported increase in Fyn immunoreactivity in Alzheimer's-afflicted neurons (Shirazi and Wood, NeuroReport, 4 (1993) 435-437).

Amyloid beta-Protein Precursor↗
PubMed1996

Protein kinase C and F-actin are essential for stimulation of neuronal FAK tyrosine phosphorylation by G-proteins and amyloid beta protein.

Focal adhesion kinase (FAK) is a protein tyrosine kinase implicated in signal transduction pathways for integrins, neuropeptides, and lysophosphatidic acid. FAK, first discovered in non-neuronal cells, recently has been reported to occur in neurons, where its tyrosine phosphorylation is upregulated by fibronectin and by the Alzheimer's Abeta peptide. The current work has elucidated molecular events leading to tyrosine phosphorylation of FAK in the rat B103 CNS nerve cell line. Activation of receptor-coupled G-proteins by Mas-7 was found to evoke rapid upregulation of FAK tyrosine phosphorylation (Tyr(P)). Upregulation by Mas-7 was blocked by GF109203X, a potent inhibitor of protein kinase C (PKC). Phorbol ester also upregulated FAK-YP, verifying a role for PKC in the transduction cascade. Upregulation of FAK-YP by activation of G-proteins and PKC was dependent upon intact F-actin, as cytochalasin D abolished stimulation by Mas-7 and by phorbol ester. The relatively slow increase in FAK-YP evoked by chronic exposure to Abeta also was abolished by GF109203X and by cytochalasin D. The results show that tyrosine phosphorylation of FAK in neurons is regulated positively by PKC, functioning down-stream from G-proteins through an F-actin-dependent mechanism. The Alzheimer's Abeta peptide is capable of activating elements of this same signal transduction pathway, via membrane events that remain to be determined.

Actins↗
PubMed1996

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.

Alzheimer Disease↗
PubMed1995

Synthetic chemical diversity: solid phase synthesis of libraries of C2 symmetric inhibitors of HIV protease containing diamino diol and diamino alcohol cores.

Solid phase synthesis of non-oligomeric organic compounds has been pursued for high-efficiency generation of large numbers of structurally diverse compounds for drug screening. Known as chemical diversity libraries or combinatorial libraries (when the synthesis is carried out in a combinatorial fashion), these compounds can be used for de novo discovery of drug leads or for expedient structure--activity relationship (SAR) studies. To expand the scope of solid phase synthesis beyond the capability of the traditional method of solid phase synthesis for peptides, a strategy was developed for bi-directional solid phase synthesis starting with diamino alcohol or diamino diol core structures. The strategy relies on using bifunctional linkers to modify the core structures, simultaneously protecting the hydroxyl group or the diol moiety of the core and providing a carboxyl group for attachment of the modified cores to a solid support. The two NH2 groups of the modified cores attached to the solid support were then deprotected and reacted with a wide variety of amine-reactive reagents (carboxylic acids, sulfonyl chlorides, isocyanates, chloroformates, etc.) to extend the molecule in both directions. This strategy was successfully applied to automated parallel synthesis of a library of C2 symmetric inhibitors of HIV protease containing the known symmetry-based diamino diol and diamino alcohol core structures, thus enabling expedient access of large numbers of analogs in this series. A library of over 300 discrete compounds was synthesized using this methodology in order to identify potent (IC50 < 100 nM) HIV protease inhibitors with reduced size. This paper describes the technical aspects of this technology.

Alcohols↗
PubMed1995

Cathepsin D from Alzheimer's-diseased and normal brains.

An acid protease activity from human brain was found to cleave a fluorogenic peptide substrate encompassing the amino terminus of Alzheimer's amyloid-beta peptide (A beta). The protease was isolated and determined to be cathepsin D based on chromatographic, immunological, and enzymatic data. Analysis of the cleavage sites indicated that cathepsin D hydrolyzed the methionine--aspartate bond generating the in vivo amino terminus of A beta. These data suggested that cathepsin D could be involved in amyloidogenic processing of the amyloid precursor protein. Consequently, cathepsin D from both Alzheimer's-diseased and control brains was compared to determine whether there were any differences which could account for an increase in A beta production in Alzheimer's disease. No differences were detected in isoform composition or tissue content of cathepsin D as measured by 2-D IEF-SDS-PAGE. Enzymological characterization of brain cathepsin D demonstrated that it could undergo a previously undescribed pH-dependent reversible activation. However, that activation appeared identical for both AD and normal brain enzymes. These data demonstrate that concentration, isoform distribution, and several enzymological characteristics of cathepsin D are not distinguishable between AD and normal brain. The pH dependence of cathepsin D activity suggests, however, that its intracellular localization may be important in considering the potential role of cathepsin D in Alzheimer's disease.

Aged↗
PubMed1994

Focal adhesion kinase expressed by nerve cell lines shows increased tyrosine phosphorylation in response to Alzheimer's A beta peptide.

A beta is a 39-43-amino acid peptide that accumulates as extracellular aggregates in Alzheimer's disease-afflicted brain tissue. Contact between these aggregates and neurons is potentially pathogenic, although little is known about the cellular transduction mechanisms. We have investigated the impact of A beta aggregates on the neuronal control of protein tyrosine phosphorylation, which underlies signal transduction for multiple families of growth factor and adhesion receptors. Added to cultures of rat and human nerve cell lines, A beta aggregates evoked a non-desensitizing increase (1.3-3.6-fold) in tyrosine phosphorylation in a band at 118 kDa. The 118-kDa protein was determined by immunoprecipitation to be pp125FAK, not previously documented in cells of neuronal lineage. Immunoblots with anti-focal adhesion kinase (FAK) showed that A beta aggregates had no effect on FAK protein levels. The increase in FAK tyrosine phosphorylation occurred at doses of A beta aggregates that evoked lactate dehydrogenase release; evoked tyrosine phosphorylation preceded the first detectable lactate dehydrogenase release by 4 h. Like degeneration, the FAK response was dependent on A beta aggregation and neuronal differentiation. Since tyrosine phosphorylation of FAK is essential to its activity as a transduction component of integrin-, peptide-, and lysophosphatidic acid-mediated signaling, the data establish a link between A beta aggregates and signal transduction pathways implicated in diverse cell functions including neurite outgrowth, control of the cell cycle, and apoptosis.

Amino Acid Sequence↗
PubMed1994

Cleavage at the amino and carboxyl termini of Alzheimer's amyloid-beta by cathepsin D.

Amyloid beta (A beta) is a 39-43-residue protein that originates from proteolysis of the beta-protein precursor (beta PP) and accumulates in senile plaques in brains of Alzheimer's disease (AD) patients. Mutant beta PP, which incorporates an AD-causing double mutation at positions 687-688, has been shown to enhance A beta production in transfected cells. In this work we investigate the susceptibility of the mutant beta PP sequence to proteolytic cleavage by proteinases from human brain. Internally quenched fluorogenic substrates were used that encompass the NH2-terminal sequence of A beta from wild-type beta PP, the double mutant, and the two single substitutions. Proteinase activity in brain extract cleaved the mutant substrate 100-fold faster than the wild-type substrate and the partial mutants 25-fold faster. The major cleavage site in all substrates was at the amyloidogenic Asp1 site. The brain activity appeared to be cathepsin D (CD), as indicated by similarities to purified CD in 1) the rate and site of substrates cleavage, 2) the pH optima, and 3) the sensitivity to pepstatin A. The increased activity against the mutant substrate was not shared by cathepsins B and C, pepsin, HIV proteinase, and Candida albicans Asp-proteinase. Furthermore, CD cleaved a substrate that incorporates the COOH terminus of A beta at positions equivalent to Thr43 and Ala42, at ratios of 68% and 32%, respectively. CD degraded A beta 1-40 into six fragments but A beta 1-42 was completely resistant to digestion, probably because of its aggregation characteristics. These results indicate that CD is capable of producing the cleavages resulting in A beta production and that it may prove to be a suitable therapeutic target.

Amino Acid Sequence↗
PubMed1994

Complement C1q does not bind monomeric beta-amyloid.

The tendency of both labeled and unlabeled beta-amyloid to bind in solution to C1q, the recognition species in the complement cascade, was examined using both hydrodynamic and spectroscopic methods. Potential binding interactions were evaluated using a purified synthetic beta-amyloid 1-40 sequence, alone, and selectively labeled at the amino terminus with spectroscopic probes. The probes permitted both absorbance and fluorescence analyses of beta-amyloid binding interactions. Under conditions used for the analyses beta-amyloid exists exclusively as a monomer in solution, and C1q retains an intact quaternary structure and is capable of binding to IgM. When mixed together the monomeric beta-amyloid does not bind to, or interact with, the complement C1q at concentrations below approximately 100 microM. The data suggest that if beta-amyloid toxicity is associated with complement activation in Alzheimer's disease then monomeric beta-amyloid is likely not responsible for activation through the classical complement pathway.

Amyloid beta-Peptides↗
PubMed1994

Potential beta PP-processing proteinase activities from Alzheimer's and control brain tissues.

Fluorogenic peptide substrates designed to encompass the reported alpha-secretory and amyloidogenic cleavage sites of the amyloid-beta precursor protein (beta PP) were used to analyze proteinase activities in brain extracts from control patients and those with Alzheimer's disease (AD). Activity against the secretory substrate at pH 7.5 in control and AD brains produced a major endopeptidase cleavage at the Lys687-Leu688 bond (beta PP770 numbering), consistent with the beta PP secretase cleavage. Activity in control brains against the amyloidogenic substrate at pH 7.5 produced one cleavage at the Ala673-Glu674 bond, two residues C-terminal to the amyloidogenic Met-Asp site. However, in three of four AD brains, the major cleavage was at the Asp-Ala bond, one residue from the amyloidogenic site. Both endopeptidase and carboxypeptidase activities in AD brains were lower than in control brains. Proteinase activities against the secretory substrate had a major optimum at pH 3.0-4.0 and another at pH 6.0-7.5. Proteinase activities against the amyloidogenic substrate had a major optimum at or below pH 3.0 and another at pH 6.0. Using both substrates, activities at low pH were higher in AD-brains than in controls, while at pH above 6.5, activities in control brains were higher than in AD. These results indicate that the levels of proteolytic enzymes in AD brains are altered relative to controls.

Aged↗
PubMed1994

Synthetic approaches to continuous assays of retroviral proteases.

This chapter has described a number of approaches for continuous assay of retroviral proteases using either chromogenic or fluorogenic synthetic substrates. The significant progress in this area has been catalyzed by the intense interest in HIV protease as a therapeutic target, but these versatile methods will be used widely in future for studies of many other proteases.

Amino Acid Sequence↗