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S J Pollack

Publications and source records attributed to S J Pollack.

16 recordsLinked to original sources
PubMed2001

The non-peptidyl fungal metabolite L-783,281 activates TRK neurotrophin receptors.

Neurotrophin binding to the extracellular surface of the Trk family of tyrosine kinase receptors leads to the activation of multiple signalling cascades, culminating in neuroregenerative effects, including neuronal survival and neurite outgrowth. Since neurotrophins themselves are not ideal drug candidates due to their poor pharmacokinetic behaviour and bioavailability, small molecule neurotrophin mimetics may be beneficial in treating a number of neurodegenerative disorders. The present study demonstrates that L-783,281, a non-peptidyl fungal metabolite, is capable of stimulating TrkA, B and C phosphorylation to various extents in CHO cells stably expressing human Trk receptors. L-783,281 also stimulated Trk phosphorylation in a number of rat and human primary neuronal cultures, whereas the highly similar compound, L-767,827, was without effect. Mechanistic studies utilizing transiently transfected PDGF/TrkA and TrkA/PDGF chimeras, demonstrated that L-783,281 is likely to interact with the intracellular domain of the TrkA receptor. Further investigations suggested that L-783,281 was nevertheless able to instigate receptor dimerization by binding in a non-covalent manner. Although the cytotoxicity of the compound was shown to preclude its effects in neuronal survival and neurite outgrowth assays, it is a prototype for a small molecule neurotrophin mimetic that activates Trk by interacting at a site different from the neurotrophin-binding site.

Animals↗
PubMed1995

Sulphated compounds attenuate beta-amyloid toxicity by inhibiting its association with cells.

Agents that interfere with the toxic effects of beta-amyloid protein may be therapeutically useful against Alzheimer's disease. We reported recently that several sulphated glycosaminoglycans and sulphonated dyes attenuate the toxic effects of beta-amyloid fragments beta 25-35 and beta 1-40 in two clonal cell lines. We now demonstrate that this protective effect is due to interference with beta-amyloid cell association rather than effects on beta-amyloid structure. Using an enzyme-linked immunoabsorbance assay to detect cell-associated beta 1-40, we found in a range of compounds a strong correlation between inhibition of HeLa cell association of beta 1-40 and attenuation of cellular toxicity as measured by inhibition of 3-[4,5-dimethylthia-zol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction. In contrast, effects on peptide structure, as measured by Congo red binding, were generally inconsistent with the attenuating effects of the compounds on cellular toxicity. These results suggest that by binding beta-amyloid these agents prevent its interaction with cells.

Amyloid beta-Peptides↗
PubMed1995

Sulfonated dyes attenuate the toxic effects of beta-amyloid in a structure-specific fashion.

We recently reported that several sulfate-containing glycosaminoglycans, a class of compounds associated with the beta-amyloid plaques of Alzheimer's disease, attenuate the toxic effects of beta-amyloid fragments beta 25-35 and beta 1-40. The amyloid-binding sulfonated dye Congo Red was shown to have a similar effect. Using two clonal cell lines, we now demonstrate that several sulfonated dyes attenuate beta-amyloid toxicity and that the protective effect appears specific for compounds whose sulfonate groups can interact with the beta-pleated structure of aggregated amyloid. These results suggest that by binding beta-amyloid these compounds may prevent toxic interactions of the peptide with cells.

Amyloid beta-Peptides↗
PubMed1995

Structure and mechanism of inositol monophosphatase.

Since lithium inhibits IMPase and modulates phosphatidylinositol (PtdIns) cell signalling at therapeutically relevant concentrations (0.5-1.0 mM), IMPase has attracted attention as a putative molecular target for lithium in the treatment of manic depression. IMPase is a homodimer, with each subunit organised in an alpha beta alpha beta alpha arrangement of alpha-helices and beta-sheets, and this type of structure seems crucial to the two-metal catalysed mechanism in which an activated water molecule serves as a nucleophile. Lithium appears to inhibit the enzyme following substrate hydrolysis by occupying the second metal binding site before the phosphate group can dissociate from its interaction with the site 1 metal. The understanding of IMPase structure and the mechanism of substrate hydrolysis and lithium inhibition should be useful in the development of novel inhibitors which may prove clinically useful in the treatment of manic depression.

Amino Acid Sequence↗
PubMed1995

Sulfated glycosaminoglycans and dyes attenuate the neurotoxic effects of beta-amyloid in rat PC12 cells.

Glycosaminoglycan (GAG)-containing proteoglycans are associated with the neuritic plaques and cerebrovascular beta-amyloid deposits of Alzheimer's disease as well as with the amyloid deposits of prion and other disorders. GAGs and other sulfate-containing compounds have previously been shown to bind beta-amyloid peptide in vitro, suggesting possible effects of beta-amyloid deposition and/or toxicity in vivo. Using reduction of the redox dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to measure beta-amyloid neurotoxicity in rat pheochromocytoma PC12 cells, several polysulfated GAGs and synthetic sulfate-containing compounds were found to attenuate the neurotoxic effects of beta-amyloid fragments beta 25-35 and beta 1-40. These results suggest that by binding beta-amyloid these compounds may prevent toxic interactions of the peptide with cells.

Amyloid beta-Peptides↗
PubMed1995

Inositol monophosphatase--a putative target for Li+ in the treatment of bipolar disorder.

Attenuation of the phosphatidylinositol (PI) signal transduction pathway as a consequence of inhibition of inositol monophosphatase (IMPase) has been proposed as the mechanism for the efficacy of Li+ in the treatment of bipolar disorder. Nevertheless, Li+ also affects other aspects of PI signal transduction, and it is therefore not clear whether modulation of PI responses by Li+ can be attributed solely to inhibition of IMPase. However, inhibitors of IMPase mimic the effects of Li+ on some aspects of PI cell signalling, thus highlighting the potential of IMPase as a target for the treatment of bipolar disorder. The recent description of the three-dimensional structure of IMPase in conjunction with site-directed mutagenesis and kinetic studies has led to the elucidation of the enzyme mechanism. These structural and mechanistic data should prove useful in the development of novel inhibitors of IMPase that might ultimately prove useful clinically.

Antimanic Agents↗
PubMed1994

Structural analysis of inositol monophosphatase complexes with substrates.

The structures of ternary complexes of human inositol monophosphatase with inhibitory Gd3+ and either D- or L-myo-inositol 1-phosphate have been determined to 2.2-2.3 A resolution using X-ray crystallography. Substrate and metal are bound identically in each active site of the phosphatase dimer. The substrate is present at full occupancy, while the metal is present at only 35% occupancy, suggesting that Li+ from the crystallization solvent partially replaces Gd3+ upon substrate binding. The phosphate groups of both substrates interact with the phosphatase in the same manner with one phosphate oxygen bound to the octahedrally coordinated active site metal and another oxygen forming hydrogen bonds with the amide groups of residues 94 and 95. The active site orientations of the inositol rings of D- and L-myo-inositol 1-phosphate differ by rotation of nearly 60 degrees about the phosphate ester bond. Each substrate utilizes the same key residues (Asp 93, Ala 196, Glu 213, and Asp 220) to form the same number of hydrogen bonds with the enzyme. Mutagenesis experiments confirm the interaction of Glu 213 with the inositol ring and suggest that interactions with Ser 165 may develop during the transition state. The structural data suggest that the active site nucleophile is a metal-bound water that is activated by interaction with Glu 70 and Thr 95. Expulsion of the ester oxygen appears to be promoted by three aspartate residues acting together (90, 93, and 220), either to donate a proton to the leaving group or to form another metal binding site from which a second Mg2+ coordinates the leaving group during the transition state.

Crystallography, X-Ray↗
PubMed1994

Mechanism of inositol monophosphatase, the putative target of lithium therapy.

myo-Inositol monophosphatase (myo-inositol-1-phosphate phosphohydrolase, EC 3.1.3.25) is an attractive target for mechanistic investigation due to its critical role in the phosphatidylinositol signaling pathway and the possible relevance of its inhibition by Li+ to manic depression therapy. The x-ray crystallographic structure of human inositol monophosphatase in the presence of the inhibitory metal Gd3+ showed only one metal bound per active site, whereas in the presence of Mn2+, three ions were present with one being displaced upon phosphate binding. We report here modeling, kinetic, and mutagenesis studies on the enzyme, which reveal the requirement for two metal ions in the catalytic mechanism. Activity titration curves with Zn2+ or Mn2+ in the presence or absence of Mg2+ are consistent with a two-metal mechanism. Modeling studies based on the various x-ray crystallographic structures (including those with Gd3+ and substrate bound) further support a two-metal mechanism and define the positions of the two metal ions relative to substrate. While the first metal ion may activate water for nucleophilic attack, a second metal ion, coordinated by three aspartate residues, appears to act as a Lewis acid, stabilizing the leaving inositol oxyanion. In this model, the 6-OH group of substrate acts as a ligand for this second metal ion, consistent with the reduced catalytic activity observed with substrate analogues lacking the 6-OH. Evidence from Tb3+ fluorescence quenching and the two-metal kinetic titration curves suggests that Li+ binds at the site of this second metal ion.

Amino Acid Sequence↗
PubMed1993

Probing the role of metal ions in the mechanism of inositol monophosphatase by site-directed mutagenesis.

Since inhibition of myo-inositol monophosphatase (EC 3.1.3.25) by lithium ions and the resulting attenuation of phosphatidylinositol cycle activity may be the mechanism by which lithium exerts its therapeutic effect in the treatment of manic depression, it is of great interest to understand the mechanism of the enzyme and how lithium and other metals interact with it. Divalent magnesium is essential for enzyme activity, whereas Li+ and high concentrations of Mg2+ act as uncompetitive inhibitors with respect to substrate. From the recently solved crystal structure of the human enzyme, several amino acid residues in the active site were targeted for mutagenesis studies. Nine single-residue substituted mutants were characterized with regard to catalytic parameters, Mg2+ dependence, and Li+ inhibition. In addition, a terbium fluorescence assay was developed to determine the metal binding properties of the wild-type and mutant enzymes. Although none of these mutations affected Km for substrate substantially, the mutations Glu70-->Gln, Glu70-->Asp, Asp90-->Asn and Thr95-->Ala, in which residues within coordinating distance of the active site metal were modified, all resulted in large reductions in catalytic activity. The position of Glu70 in the crystal structure further suggests that this residue may be involved in activating water for nucleophilic attack on the substrate. The mutations Lys36-->Ile, Asp90-->Asn, Thr95-->Ala, Thr95-->Ser, His217-->Gln, and Cys218-->Ala all resulted in parallel reductions in both lithium and magnesium affinity, suggesting that Li+ and Mg2+ share a common binding site.

Base Sequence↗
PubMed1992

Cloning, overexpression and mechanistic studies of carboxyphosphonoenolpyruvate mutase from Streptomyces hygroscopicus.

The enzyme carboxyphosphonoenolpyruvate mutase catalyses the formation of one of the two C-P bonds in bialaphos, a potent herbicide isolated from Streptomyces hygroscopicus. The gene encoding the enzyme has been cloned from a subgenomic library from S. hygroscopicus by colony hybridisation using an exact nucleotide probe. An open reading frame has been identified that encodes a protein of molecular mass 32700 Da, in good agreement with the subunit molecular mass of the carboxyphosphonoenolpyruvate mutase recently isolated from this source [Hidaka, T., Imai, S., Hara, O., Anzai, H., Murakami, T., Nagaoka, K. & Seto, H. (1990) J. Bacteriol. 172, 3066-3072]. The gene shares significant sequence similarity with that of phosphoenolpyruvate mutase, an enzyme that catalyses the related interconversion of phosphoenolpyruvate and phosphonopyruvate. When the carboxyphosphonoenolpyruvate-mutase gene was subcloned into the vector pET11a, the mutase was expressed as about 20% of the total soluble cellular protein in Escherichia coli. The mutase has been purified to homogeneity in three steps in 40% yield. With malate dehydrogenase/NADH, (hydroxyphosphinyl)pyruvate gives (hydroxyphosphinyl)lactate (kcat 164 s-1 and Km 680 microM) and this spectrophotometric assay for the product of the mutase reaction has been employed in the mechanistic studies. The kinetics for the mutase reaction have been evaluated for the substrate, carboxyphosphonoenolpyruvate, and for the putative reaction intermediate carboxyphosphinopyruvate, both of which have been prepared by chemical synthesis. Carboxyphosphonoenolpyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 0.020 s-1 and a Km of 270 microM, and carboxyphosphinopyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 7.6 x 10(-4) s-1 and a Km of 2.2 microM. Although the exogenously added intermediate is not kinetically competent, these results suggest that the mechanism for the mutase reaction involves an initial rearrangement to the intermediate carboxyphosphinopyruvate, followed by decarboxylation to yield the product (hydroxyphosphinyl)pyruvate.

Amino Acid Sequence↗
PubMed1988

Introduction of nucleophiles and spectroscopic probes into antibody combining sites.

A general chemical strategy has been developed whereby antibody combining sites can be selectively derivatized with natural or synthetic molecules, such as catalytic groups, drugs, metals, or reporter molecules. Cleavable affinity labels were used to selectively introduce a thiol into the combining site of the immunoglobulin A MOPC 315. This thiol acted both as a nucleophile to accelerate ester thiolysis 60,000-fold and as a handle for selectively derivatizing the antibody with additional functional groups. For example, derivatization of the antibody with a fluorophore made possible a direct spectroscopic assay of antibody-ligand complexation. This chemistry should not only extend our ability to exploit antibody specificity in chemical catalysis, diagnostics, and therapeutics, but may also prove generally applicable to the functional modification of other proteins for which detailed structural information is unavailable.

Affinity Labels↗
PubMed1986

Selective chemical catalysis by an antibody.

The immunoglobulin MOPC167, which binds the transition state analog p-nitrophenylphosphorylcholine with high affinity, catalyzed the hydrolysis of the corresponding carbonate 1. MOPC167 catalysis displayed saturation kinetics with catalytic constant (kcat) = 0.4 min-1 and Michaelis constant (Km) = 208 microM, showed substrate specificity, and was inhibited by p-nitrophenylphosphorylcholine. The rate of the reaction was first order in hydroxide ion concentration between pH 6.0 and 8.0. The lower limit for the rate of acceleration of hydrolysis by the antibody above the uncatalyzed reaction was 770. This study begins to define the rules for the generation of catalytic antibodies.

Antibodies↗