Biomedical subjects

D Schubert

Publications and source records attributed to D Schubert.

At least 73 records · Page 4Linked to original sources

PubMed1997

Cytoskeleton-membrane connections in the human erythrocyte membrane: band 4.1 binds to tetrameric band 3 protein.

Band 4.1 provides, besides ankyrin, the main linkage between the erythrocyte membrane and its cytoskeleton. Its predominant binding sites in the membrane are located on the glycophorins. However, the cytoplasmic domain of band 3 can also bind band 4.1. We have studied which of the different band 3 oligomers observed (monomers, dimers, tetramers) can act as band 4.1 binding sites, by equilibrium sedimentation experiments on mixtures of purified band 3 and dye-labelled band 4.1 in solutions of a nonionic detergent. At low molar ratios of band 4.1 and band 3, the sedimentation equilibrium distributions obtained could all be perfectly fitted assuming that only two dye-labelled particles were present: uncomplexed band 4.1 and a complex formed between one band 4.1 molecule and one band 3 tetramer. The presence of small amounts of complexes containing band 3 monomers or dimers could not be completely ruled out but is unlikely. On the other hand, stabilized band 3 dimers effectively bound band 4.1. At higher molar band 4.1/band 3 ratio, the band 3 tetramer apparently could bind up to at least four band 4.1 molecules. The band 4.1/band 3 tetramer complex was found to be unstable. The results described, together with those reported previously, point at a prominent role of tetrameric band 3 in ligand binding.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1997

Denaturation and reactivation of dimeric human glutathione reductase--an assay for folding inhibitors.

Human glutathione reductase (GR; which catalyzes the reaction NADPH + GSSG + H+ --> 2 GSH + NADP+) is an obligatory FAD-containing homodimer of known geometry. Native human GR, a potential target of antimalarial and cytostatic agents, cannot be dissociated by dilution or by means of subunit-interface mimetics, similarly to well-studied viral dimeric proteins. However, ab initio folding and/or dimerization of human GR can be inhibited by point mutations or by peptides corresponding to subunit-interface areas, for example synthetic peptide P11, which represents the intersubunit-contact helix H11. The structure of this peptide, which might assist inhibitor design, was solved by high-resolution NMR spectroscopy. Residues 440-453, were found to be alpha helical in the isolated peptide. To quantitate the efficacy of inhibitors such as P11, we developed the following unfolding/reactivation assay. The effects of various guanidine hydrochloride (Gdn/HCl) concentrations were studied by analytical ultracentrifugation. It was shown that human GR denatured by greater than 3 M Gdn/HCl is monomeric and free of FAD. Circular-dichroism experiments at 223 nm indicated a half-life of approximately 20 s at 20 degrees C for the unfolding process. To optimize the reactivation yield, four parameters [protein concentration (x) in the range 0.3-10 microg/ml, cofactor supplementation, temperature (y: 0-32 degrees C), and time (0-72 h)] were varied systematically, and a reactivation score z was given to each constellation of parameters. This type of analysis might be useful to optimize refolding and activation yields for other proteins. For human GR, the highest recovery was found not to occur at one of the corners of the x,y plane, but close to its center. Consequently, the optimal assay conditions for folding and dimerization inhibitors are as follows. The enzyme (at 300 microg/ml) is denatured by 5 M guanidine hydrochloride/5 mM dithiothreitol, then reactivated by dilution to 1 microg/ml at pH 6.9 and 20 degrees C. In the absence of inhibitors, this procedure leads to 70% of the control activity within 8 h. Peptides representing the upper subunit interface (for instance residues 436-478) of human GR were found to inhibit refolding with EC50% values in the micromolar range, whereas fragments from other regions of the protein had no influence on this process. For peptide P11, the EC50% value was 20 microM. In conclusion, hGR, enzyme with a tight intersubunit contact area of 21 nm2, appears to be suitable for studying protein folding, dimerization, and prosthetic-group complexation in the absence and presence of compounds that inhibit these processes. There is a shortage, at least for oligomeric enzymes of eukaryotes, of published systematic studies on protein (re)activation.

Dimerization↗

PubMed1997

A role for 12-lipoxygenase in nerve cell death caused by glutathione depletion.

An early and highly specific decrease in glutathione (GSH) in the substantia nigra is associated with Parkinson's disease, and low levels of GSH lead to the degeneration of cultured dopaminergic neurons. Using immature cortical neurons and a clonal nerve cell line, it is shown that a decrease in GSH triggers the activation of neuronal 12-lipoxygenase (12-LOX), which leads to the production of peroxides, the influx of Ca2+, and ultimately to cell death. The supporting evidence includes: 1) inhibitors of arachidonate metabolism and 12-LOX block cell death induced by GSH depletion; 2) there is an increase in 12-LOX activity and a membrane translocation in HT22 cells, and an induction of the enzyme in primary cortical neurons following the reduction of GSH; 3) 12-LOX is directly inhibited by GSH; and 4) exogenous arachidonic acid potentiates cell death. These data show that the LOX pathway is a critical intermediate in at least some forms of neuronal degeneration.

Animals↗

PubMed1997

Mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction is one of the most frequently used methods for measuring cell proliferation and neural cytotoxicity. It is widely assumed that MTT is reduced by active mitochondria in living cells. By using isolated mitochondria from rat brain and B12 cells, we indeed found that malate, glutamate, and succinate support MTT reduction by isolated mitochondria. However, the data presented in this study do not support the exclusive role of mitochondria in MTT reduction by intact cells. Using a variety of approaches, we found that MTT reduction by B12 cells is confined to intracellular vesicles that later give rise to the needle-like MTT formazan at the cell surface. Some of these vesicles were identified as endosomes or lysosomes. In addition, MTT was found to be membrane impermeable. These and other results suggest that MTT is taken up by cells through endocytosis and that reduced MTT formazan accumulates in the endosomal/lysosomal compartment and is then transported to the cell surface through exocytosis.

Animals↗

PubMed1997

Cytotoxic amyloid peptides inhibit cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction by enhancing MTT formazan exocytosis.

Amyloid beta peptide (A beta) neurotoxicity is believed to play a central role in the pathogenesis of Alzheimer's disease. An early indicator of A beta toxicity is the inhibition of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction to MTT formazan, a widely used assay for measuring cell viability. In this report we show that A beta and other cytotoxic amyloid peptides such as human amylin dramatically enhance MTT formazan exocytosis, resulting in the inhibition of cellular MTT reduction. Only the amyloid peptides that are known to be cytotoxic enhanced MTT formazan exocytosis. Basal MTT formazan exocytosis and amyloid peptide-enhanced MTT formazan exocytosis are blocked by several drugs with diverse known effects. These and other data suggest that MTT formazan exocytosis is a multistep process and that cytotoxic amyloid peptides enhance MTT formazan exocytosis through an intracellular signal transduction pathway.

Amyloid↗

PubMed1997

Serpins inhibit the toxicity of amyloid peptides.

The amyloid plaque in Alzheimer's disease (AD) contains numerous proteins, some of which may be relevant to the pathogenesis of the disease. The serine protease inhibitor alpha1-antichymotrypsin is specifically localized in AD plaques. It is shown here that alpha1-antichymotrypsin and several other serine protease inhibitors (serpins) inhibit the toxicity of amyloid peptides on primary cortical nerve cell cultures as well as a clonal cell line. This inhibition of toxicity is not mediated via the serpin enzyme complex receptor, the transferrin receptor, or by interference with the polymerization of amyloid fibrils. Since a variety of synthetic serine protease inhibitors mimic the effects of serpins on amyloid toxicity, it is likely that the antiprotease activities of serpins are responsible for their biological effects.

Adenoma, Islet Cell↗

PubMed1996

Associations between erythrocyte band 3 protein and aldolase in detergent solution. Determining their stoichiometry by analytical ultracentrifugation.

The cytoplasmic domain of band 3, the predominant polypeptide of the erythrocyte membrane, represents a binding site for certain glycolytic enzymes. We have studied the association between human band 3 protein and aldolase, in order to clarify the role of the different band 3 oligomers as ligand binding sites. The experiments were performed on mixtures of solubilized band 3 and aldolase in solutions of a nonionic detergent, nonaethyleneglycol lauryl ether. The main technique applied was sedimentation equilibrium analysis in an analytical ultracentrifuge. In addition, nonequilibrium centrifugation techniques were used. To facilitate the evaluations, the aldolase was labelled with a dye. The following results were obtained. (1) With unmodified band 3, aldolase is bound exclusively or at least predominantly to the band 3 tetramer (but not to monomers or dimers). (2) The band 3 tetramer can bind up to four aldolase tetramers. (3) The band 3 tetramer/aldolase complex is unstable on the time scale of the techniques used. (4) Stable band 3 dimers (stabilized either covalently or noncovalently) can also associate with aldolase and can bind up to two aldolase tetramers. The results described, together with those reported previously, point at a prominent role of the band 3 tetramer in ligand binding.

Animals↗

PubMed1996

Increased antioxidant enzyme activity in amyloid beta protein-resistant cells.

Clones of the rat pheochromocytoma cell line PC12 were selected for their resistance to amyloid beta protein (A beta). These A beta-resistant cells also survive higher concentrations of exogenously applied peroxides than the parent cells. A beta triggers intracellular H2O2 accumulation in the parent PC12 cells but not in the A beta-resistant cells. The absence of H2O2 accumulation in A beta-resistant cells is not attributable to differences in A beta binding to the cell surface. However, the mRNA and protein levels of catalase and glutathione peroxidase, as well as the corresponding enzyme activities, are highly elevated in A beta-resistant clones. These activities correlate well with the increased resistance of cells to A beta or peroxides. Finally, cells transfected with catalase and glutathione peroxidase are also more resistant to A beta toxicity. These results indicate that increased antioxidant enzyme activities in A beta-resistant cells account for at least part of their resistance to A beta and substantiate further the role of H2O2 in A beta toxicity.

Alzheimer Disease↗

PubMed1996

Characterization of a novel platelet-derived growth factor-associated protein.

A novel mitogen-associated protein was isolated from a rat neural retina cell line. The protein copurified with platelet-derived growth factor (PDGF)-A and was therefore termed PDGF-associated protein (PAP). cDNAs corresponding to the protein were characterized from both rat and human cDNA libraries. PAP binds to PDGF with low affinity and enhances the mitogenic effect of PDGF-A but lowers the mitogenic activity of PDGF-B. PAP mRNA is abundant in the brain of newborn rats and is found in several other tissues.

3T3 Cells↗

PubMed1995

The role of iron in beta amyloid toxicity.

There is evidence that oxidative damage plays a causative role in Alzheimer's disease and amyloid beta protein (A beta) toxicity. Iron is frequently a potent facilitator of free radical production due to its ability to mediate the conversion of H2O2 to hydroxyl radicals via the Fenton reaction or by virtue of hypervalent iron compounds. It is shown here that iron facilitates A beta toxicity to cultured cells.

Alzheimer Disease↗

PubMed1995

The influence of two anion-transport inhibitors, 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate and 4,4'-dibenzoylstilbene-2,2'-disulfonate, on the self-association of erythrocyte band 3 protein.

4,4'-Diisothiocyanatodihydrostilbene-2,2'-disulfonate and 4,4'-dibenzoylstilbene-2,2'-disulfonate potently inhibit the erythrocyte anion transporter. These inhibitors act by binding, with a 1:1 stoichiometry, to the band 3 transport protein. We have studied, by sedimentation equilibrium analysis in an analytical ultracentrifuge, the effect of the two closely related stilbenedisulfonates on the state of association of band 3 in the nonionic detergent nonaethyleneglycol lauryl ether. It was found that covalent binding of 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate to band 3 did not significantly disturb the monomer/dimer/tetramer association equilibrium shown by the unliganded protein. An entirely different result was obtained after addition of 4,4'-dibenzoylstilbene-2,2'-disulfonate to the protein, at both low and high chloride concentrations. The amount of band 3 dimer in the samples increased with increasing inhibitor concentration c1, and for c1 > or = 15 microM virtually all of the protein was present as dimer. After removal of the inhibitor (by gel filtration or dialysis), the original monomer/dimer/tetramer distribution of the band 3 protein was restored. Our data show that the (noncovalent) binding of 4,4'-dibenzoylstilbene-2,2'-disulfonate drastically changes the coupling between band 3 protomers. In addition, a reversible change in the state of association of band 3 induced by ligand binding is demonstrated.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1995

Amyloid peptides are toxic via a common oxidative mechanism.

beta-Amyloid protein (A beta) is a member of a small group of proteins that accumulate as amyloid deposits in various tissues. It has recently been demonstrated that the toxicity of A beta toward some neural cells is caused by oxidative damage. Since all of the amyloid diseases are characterized by protein deposited in the antiparallel beta-sheet conformation, it was asked whether there is a common toxic mechanism. It is shown here that the protein components of other human amyloidoses, including amylin, calcitonin, and atrial natriuretic peptide, are all toxic to clonal and primary cells. The toxicity is mediated via a free radical pathway indistinguishable from that of A beta. Experiments with synthetic peptides suggest that it is the amphiphilic nature of the peptides generated by their beta structure rather than their beta structure per se that causes toxicity. These results tend to rule out the alternative that amyloid toxicity is exclusively mediated via specific cell surface receptors.

Amino Acid Sequence↗

PubMed1995

Schwannoma-derived growth factor interacts with the epidermal growth factor receptor.

Schwannoma-derived growth factor (SDGF) is a potent mitogen and neuronal differentiation factor. Because of its relationship to epidermal growth factor (EGF) and the heregulins, it was asked if SDGF interacts with the EGF receptor or HER2/neu. SDGF binds to and causes the phosphorylation on tyrosine of the EGF receptor but not HER2/neu.

3T3 Cells↗

PubMed1994

The dimerization stability of the HLH-LZ transcription protein family is modulated by the leucine zippers: a CD and NMR study of TFEB and c-Myc.

In the HLH-LZ protein family, the helix-loop-helix DNA-binding dimerization domain is followed in the sequence by a leucine zipper motif. The precise function of this second dimerization domain is still unclear, since the HLH motif of a subset of this family has been shown to be necessary and sufficient for dimerization. However, deletion and mutagenesis studies of the leucine zipper in various HLH-LZ proteins have shown a clear influence of this motif on homo- and heterodimerization. In this paper, we present a structural characterization of synthetic peptides encompassing the leucine zipper sequences of c-Myc and TFEB, using circular dichroism, analytical ultracentrifugation, and nuclear magnetic resonance. We show that the different ability of the synthetic leucine zippers of c-Myc and TFEB to homodimerize at neutral pH reflects the different dimerization properties reported for the entire proteins. The TFEB protein is known to form homodimers. c-Myc, on the other hand, does not homodimerize in vivo, but is mostly found in heterodimeric complexes with Max, another protein of the HLH-LZ family. Accordingly, our results show that the TFEB peptide homodimerizes at neutral pH whereas the Myc peptide dimerizes to a comparable amount only at acidic pH and high ionic strength. Both synthetic peptides are far less stable than leucine zippers of the b-ZIP family. The relative stability of the two leucine zippers and the factors which stabilize the dimer formation are discussed.

Amino Acid Sequence↗

PubMed1994

Erythrocyte band 3 protein strongly interacts with phosphoinositides.

85% of the phosphorus coisolated with band 3 protein during separation of the intrinsic proteins of the human erythrocyte membrane by zonal electrophoresis in high concentrations of acetic acid was found to be derived from phosphoinositides, mainly phosphatidylinositol 4,5-bisphosphate. When native band 3 protein and pyrene-labelled phospholipids were present in micelles of the nonionic detergent nonaethyleneglycol lauryl ether, strong resonance energy transfer was observed between the tryptophan residues and phosphatidylinositol 4,5-bisphosphate and, to a smaller degree, phosphatidylinositol 4-phosphate. We conclude that band 3 protein strongly interacts with phosphoinositides, in particular with phosphatidylinositol 4,5-bisphosphate.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1994

Hydrogen peroxide mediates amyloid beta protein toxicity.

Amyloid beta protein (A beta) is a 40-43 amino acid peptide that is associated with plaques in the brains of Alzheimer's patients and is cytotoxic to cultured neurons. Using both primary central nervous system cultures and clonal cell lines, it is shown that a number of anti-oxidants protect cells from A beta toxicity, suggesting that at least one pathway to A beta cytotoxicity results in free radical damage. A beta causes increased levels of H2O2 and lipid peroxides to accumulate in cells. The H2O2-degrading enzyme catalase protects cells from A beta toxicity. Clonal cell lines selected for their resistance to A beta toxicity also become resistant to the cytolytic action of H2O2. In addition, A beta induces the activity of NF-kappa B, a transcription factor thought to be regulated by oxidative stress. Finally, A beta-induced H2O2 production and A beta toxicity are blocked by reagents that inhibit flavin oxidases, suggesting that A beta activates a member of this class of enzymes. These results show that the cytotoxic action of A beta on neurons results from free radical damage to susceptible cells.

Amino Acid Sequence↗

PubMed1994

Amyloid beta peptide induces necrosis rather than apoptosis.

Amyloid beta peptide (A beta P), a major component of Alzheimer's disease plaques, is toxic to rat pheochromocytoma PC12 cells and to rat cortical neurons. A reduction in cell survival could be detected after 24 h incubation with 0.01 to 20 microM of the 25-35 peptide fragment (beta 25-35) of A beta P. To study the mechanism of cell death induced by A beta P, the morphological as well as the biochemical features of neuronal cell death were analyzed. To distinguish between necrosis and apoptosis, PC12 cell death caused by beta 25-35 was compared to that induced by serum deprivation, a process known to be apoptotic in these cells. The DNA-degradation pattern of A beta P treated cells appeared random rather than at distinct internucleosomal sites as with apoptosis. Electron microscopic studies of NGF-treated PC12 cells and cortical primary cultures exposed to 20 microM beta 25-35 revealed immediate cellular damage such as vacuolization of the cytoplasm, breakdown of Golgi-apparatus and other membrane systems, and neurite disintegration. This was followed by total collapse of the cytoplasm and cell lysis. These data show that A beta P toxicity occurs via a necrotic rather than an apoptotic pathway.

Amyloid beta-Peptides↗

PubMed1994

Peptides containing the RERMS sequence of amyloid beta/A4 protein precursor bind cell surface and promote neurite extension.

Amyloid beta/A4 protein precursor (APP) is secreted into medium by most cultured cells and can function as an autocrine factor. To study the biological function of secreted forms of APP (sAPP) on neurons, we used a clonal CNS neuronal line, B103, which does not synthesize detectable levels of APP. B103 cells transfected with APP construct developed neurites faster than the parent B103 cells when plated in a serum-free defined medium. Neurite outgrowth of B103 cells was promoted by the conditioned medium of APP-695-over-producing cells or by the bacteria-produced sAPP-695 (named KB75). A series of peptides having sequences between Ala-319 and Met-335 of APP-695 also stimulated neurite outgrowth of B103 cells. The sequence of five amino acids, RERMS (APP 328-332), within this stretch of sequence, was the shortest active peptide, although the concentration required for the neuritotropic activity was higher than that of KB75. Binding assay using 125I-labeled APP 17-mer peptide corresponding to Ala-319 to Met-335 of APP-695 as a ligand demonstrated specific and saturable cell-surface binding sites. The predicted KD value was 20 +/- 5 nM and the Bmax value was 80 +/- 8 fmol/10(6) cells. The binding could be displaced with KB75. A 17-mer peptide with reverse sequence neither induced neurite outgrowth nor competed for the binding. A bacteria-produced sAPP fragment lacking the active 17-mer sequence (named KB75 delta) did not compete with 125I-labeled 17-mer for binding or stimulate neurite extension. A peptide of sequence RMSQ (APP 330-333), which partially overlaps the active sequence RERMS, could block the neuritotropic effects of both KB75 and the 17-mer at higher concentrations. APP 17-mer was also found to induce the accumulation of inositol polyphosphates, suggesting that the APP 17-mer effects involve activation of inositol phospholipid signal transduction systems. These data indicate that sAPP induces neurite extension through cell-surface binding and that the domain containing the RERMS sequence (APP 328-332) represents the active site responsible for this function.

Amino Acid Sequence↗