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H LeVine

Publications and source records attributed to H LeVine.

At least 19 recordsLinked to original sources
PubMed2002

Modeling Alzheimer's disease and other proteopathies in vivo: is seeding the key?

Protein misfolding and aberrant polymerization are salient features of virtually all central neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease, triplet repeat disorders, tauopathies, and prion diseases. In many instances, a single amino acid change can predispose to disease by increasing the production and/or changing the biophysical properties of a specific protein. Possible pathogenic similarities among the cerebral proteopathies suggest that therapeutic agents interfering with the proteopathic cascade might be effective against a wide range of diseases. However, testing compounds preclinically will require disease-relevant animal models. Numerous transgenic mouse models of beta-amyloidosis, tauopathy, and other aspects of AD have now been produced, but none of the existing models fully recapitulates the pathology of AD. In an attempt to more faithfully replicate the human disease, we infused dilute AD-brain extracts into Tg2576 mice at 3-months of age (i.e. 5-6 months prior to the usual onset of beta-amyloid deposition). We found that intracerebral infusion of AD brain extracts results in: 1). Premature deposition of beta-amyloid in eight month-old, beta-amyloid precursor protein ( betaAPP)-transgenic mice (Kane et al., 2000); 2). augmented amyloid load in the injected hemisphere of 15 month-old transgenic mice; 3). evidence for the spread of pathology to other brain areas, possibly by neuronal transport mechanisms; and 4). tau hyperphosphorylation (but not neurofibrillary pathology) in axons passing through the injection site. The seeding of beta-amyloid in vivo by AD brain extracts suggests pathogenic similarities between beta-amyloidoses such as AD and other cerebral proteopathies such as the prionoses, and could provide a new model for studying the proteopathic cascade and its neuronal consequences in neurodegenerative diseases.

Alzheimer Disease↗
PubMed2000

125I-labeled ApoE binds competitively to beta(1-40) fibrils with pathological chaperone proteins.

Radiolabeled Apolipoprotein E (Apo E) was used in a competitive binding filtration assay to amyloid fibrils preformed from beta(1-40) peptide as a probe of the binding sites for proteins either found in senile plaques in Alzheimer's Disease brain or reported to be associated with the soluble peptide. Apo E, Apo J, Apo A-I, Apo B, laminin, complement components C3 and C4, and alpha 1-antichymotrypsin all displayed sub-micromolar apparent affinities for the Apo E binding site on fibrils. Transthyretin, alpha 2-macroglobulin, amyloid P protein, heparan sulfate proteoglycan, complement component C1q, chondroitin sulfate A, and GM1 ganglioside were much less effective. The epsilon 2, epsilon 3, and epsilon 4 isoforms of Apo E showed different affinities for fibrils and lipidation of these lipoproteins made little difference. Other fibrillar beta-peptides also bound Apo E, with A beta 40-A beta 42 > A beta(12-28); A beta(25-35) = 0. A series of soluble beta-peptides and fragments failed to effect Apo E binding. Thus, both conformational and quaternary structural features are important in high affinity binding of Apo E to A beta 40 fibrils. Different amyloid plaque-associated molecules apparently associate with alternative primary and secondary structural features on fibrils.

Alzheimer Disease↗
PubMed1999

Structural features of heterotrimeric G-protein-coupled receptors and their modulatory proteins.

Over the past 20 years, the general mechanism for signaling through 7-transmembrane helix receptors coupled to GTP hydrolysis has been worked out. Although similar in overall organization, subtype variability and subcellular localization of components have built in considerable signaling specificity. Atomic resolution structures for many of the components have delineated the domain organization of these complex proteins and have given physical form to the idea of subtype specificity. This review describes what is known about the physical structures of the 7-transmembrane helix receptors, the heterotrimeric GTP binding coupling proteins, the adenylate cyclase and phospholipase C effector proteins, and signaling modulatory proteins, such as arrestin, phosducin, recoverin-type myristoyl switch proteins, and the pleckstrin homology domain of G-protein receptor kinase-2. These images allow experimenters to contemplate the details of the supramolecular organization of the multiprotein complexes involved in the transmission of signals across the cellular lipid bilayer.

Amino Acid Sequence↗
PubMed1998

The solution structure and dynamics of the pleckstrin homology domain of G protein-coupled receptor kinase 2 (beta-adrenergic receptor kinase 1). A binding partner of Gbetagamma subunits.

The solution structure of an extended pleckstrin homology (PH) domain from the beta-adrenergic receptor kinase is obtained by high resolution NMR. The structure establishes that the beta-adrenergic receptor kinase extended PH domain has the same fold and topology as other PH domains, and there are several unique features, most notably an extended C-terminal alpha-helix that behaves as a molten helix, and a surface charge polarity that is extensively modified by positive residues in the extended alpha-helix and the C terminus. These observations complement biochemical evidence that the C-terminal portion of this PH domain participates in protein-protein interactions with Gbetagamma subunits. This suggests that the C-terminal segment of the PH domain may function to mediate protein-protein interactions with the targets of PH domains.

Amino Acid Sequence↗
PubMed1997

Stopped-flow kinetics reveal multiple phases of thioflavin T binding to Alzheimer beta (1-40) amyloid fibrils.

The benzothiazole dye thioflavin T (ThT) is a classical amyloid stain for senile plaques containing beta/A4 peptide in Alzheimer's disease brain. ThT also binds rapidly and specifically to the anti-parallel beta-sheet fibrils formed from synthetic beta (1-40) peptide, but does not bind to monomer or oligomeric intermediates. The fibrillar beta-sheet-bound dye species undergoes a characteristic 120 nm red shift of its excitation spectrum that may be selectively excited at 450 nm, resulting in a fluorescence signal at 482 nm. Mixing of preformed beta (1-40) amyloid fibrils with ThT in a stopped-flow spectrophotometer, monitoring fluorescence emission at > 475 nm while exciting at 450 nm, distinguished multiple kinetic phases of roughly equivalent amplitude with tau's in the ranges of 0.007, 0.05, 0.75, and 10-20 s. The fastest reaction appears to reflect a bimolecular dye binding event while the remaining reactions are rate-limited by protein tertiary or quaternary conformational changes. The high activation energies of the three slower reactions support this interpretation. The ThT concentration dependence of the reaction rates at different ratios of ThT/beta (1-40) amyloid fibrils rules out a rate-limiting conformational change occurring prior to ligand binding. ThT is a useful probe for the aggregated fibrillar state of beta (1-40) amyloid fibrils as the amyloid-specific fluorescence reports only fibrillar species. The binding of ThT does not interfere with the aggregation of this peptide into amyloid fibrils. The putative conformational changes detected by the ThT fluorescence suggest that small pharmacologic ligands can perturb and possibly dissociate A beta amyloid fibrils.

Alzheimer Disease↗
PubMed1996

An intact N terminus of the gamma subunit is required for the Gbetagamma stimulation of rhodopsin phosphorylation by human beta-adrenergic receptor kinase-1 but not for kinase binding.

Cleavage after lysine 32 in the Ggamma2 subtype and after lysine 36 in the Ggamma3 subtype of purified mixed brain Gbetagamma by endoproteinase Lys-C blocks Gbetagamma-mediated stimulation of phosphorylation of rhodopsin in urea-extracted rod outer segments by recombinant human beta-adrenergic receptor kinase (hbetaARK1) holoenzyme while hbetaARK1 binding to rod outer segments is partially affected. This treatment does not attenuate the binding of the treated Gbetagamma to C-terminal fragments of hbetaARK1 containing the pleckstrin homology domain. Lys-C proteolysis also does not alter the association of the Gbetagamma with phospholipids, its ability to support pertussis toxin-catalyzed Galphao/Galphai ADP-ribosylation, or its ability to inhibit forskolin-stimulated platelet adenylate cyclase. The Gbeta subunit remains noncovalently associated with the cleaved Ggamma fragments. Thus, in addition to recruiting hbetaARK1 to its receptor substrate, Ggamma contributes secondary and/or tertiary structural features to activate the kinase.

Adenylyl Cyclase Inhibitors↗
PubMed1996

Desensitization of the neurokinin 1 receptor is mediated by the receptor carboxy-terminal region, but is not caused by receptor internalization.

The carboxy-terminal cytoplasmic regions of the rat neurokinin 1 (substance P) and neurokinin 2 (neurokinin A) receptors have been exchanged to determine if this region of the neurokinin 1 receptor is involved in its desensitization. When expressed at similar levels in stably transfected Chinese hamster ovary (CHO) cell lines, receptors containing the carboxy-terminal region of the neurokinin 1 receptor desensitized significantly more (as measured by reduction of the inositol 1,4,5-trisphosphate response) when preexposed for 1 min to 1 microM neurokinin, indicating a role for the carboxy-terminal region of the neurokinin 1 receptor in its desensitization. Measurement of receptor internalization using radiolabeled neurokinins (0.3 nM) indicated that approximately 75-80% of the receptors were internalized in each cell line after 10 min at 37 degrees C, with no observable correlation between neurokinin receptor desensitization and internalization. Measurement of loss of receptor surface sites for cell lines CHO NK1 and CHO NK1NK2 following exposure to 1 microM substance P also indicated no obvious relationship between the percent desensitization and percent of receptors internalized. Also, two inhibitors of neurokinin 1 receptor internalization, phenylarsine oxide and hyperosmolar sucrose, did not inhibit neurokinin 1 receptor desensitization. The protein kinase inhibitors Ro 31-8220, staurosporine, and Zn2+ had no effect on neurokinin 1 receptor desensitization, indicating that the kinases affected by these agents are not rate-limiting in neurokinin 1 receptor desensitization in this system.

Animals↗
PubMed1995

Phosphorylation and activation of beta-adrenergic receptor kinase by protein kinase C.

The aim of this study was to test the possible modification of beta-adrenergic receptor kinase (beta ARK) activity by second messengers and/or their downstream components. Using human mononuclear leukocytes (MNL), we found that calcium ionophores could elevate beta ARK activity by about 80% in a protein kinase C (PKC)-dependent manner. This was confirmed by the ability of the PKC activator phorbol 12-myristate 13-acetate (PMA) to produce a similar effect, suggesting a PKC-dependent modulation of beta ARK activity. In vitro experiments with purified proteins showed that PKC could directly phosphorylate beta ARK1 with an apparent Km for beta ARK1 of 6 nM. The ability of beta ARK1 to phosphorylate rhodopsin was 61% greater when it was phosphorylated by PKC. The level of phosphorylation of beta ARK1 immunoprecipitated from MNL and Sf9 cells overexpressing this kinase was enhanced by about 2-3-fold after PMA treatment. Functional significance of PKC-dependent increase in beta ARK activity ws demonstrated by beta-adrenergic receptor (beta AR) homologous desensitization experiments in MNL. beta AR desensitization, as induced by exposure to 10 microM isoproterenol (5 min at 37 degrees C), was increased from 42 +/- 10% in control to 68 +/- 8% in PMA-pretreated MNL. beta ARK inhibitor heparin (160 micrograms/ml) prevented the augmenting effect of PMA on beta AR desensitization. These results show that beta ARK activity can be increased through phosphorylation by PKC, thus indicating that beta ARK can be preconditioned to modulate the subsequent cellular responsiveness to receptor activation, providing the cell with a mechanism by which specific homologous desensitization can be regulated heterologously.

Animals↗
PubMed1995

Structural studies on the PH domains of Db1, Sos1, IRS-1, and beta ARK1 and their differential binding to G beta gamma subunits.

Pleckstrin homology (PH) domains are approximately 110 amino acid residues in length and are structurally conserved in a number of intracellular signaling proteins. A role for these domains has been postulated for beta ARK, which binds to G beta gamma subunits. We have quantified the binding of individual (His)6-tag PH domains of human Db1, human Sos1, rat IRS-1, human beta ARK, and human beta ARK with an extra 33-residue C-terminal extension (beta ARK + C) to G beta gamma subunits. Our in vitro binding studies show that all of the PH domains (apart from Sos1), bind G beta gamma subunits in a dose-dependent manner, but beta ARK + C binds 4 times as much G beta gamma at saturation as the others. The IRS-1 PH domain has a similar half-maximal concentration of G beta gamma binding (18 nM) to beta ARK + C (30 nM), suggesting that the IRS-1 PH domain has sufficient determinants for G beta gamma binding. The beta ARK PH domain alone has a half-maximal value of 45 nM but a drastically reduced extent of G beta gamma binding, suggesting that both the PH domain and the C-terminal 33 residues are necessary for maximal binding. Db1 has a half-maximum concentration of G beta gamma binding of 45 nM and a maximal extent of binding similar to that of beta ARK, but it is difficult to demonstrate saturable binding of G beta gamma to Sos1. Since it was previously predicted that the C-terminal PH domain of Pleckstrin [Tyers, M., et al. (1988) Nature 333, 470-473] contains a potential calcium binding site, we have tested the different PH domains for calcium binding. Only the PH domain of Db1 bound 45Ca2+ with a Kd of 10 microM. CD spectroscopy of the purified recombinant PH domains indicated that they are predominantly beta-sheet structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

A direct interaction between G-protein beta gamma subunits and the Raf-1 protein kinase.

Raf-1 is a serine/threonine protein kinase positioned downstream of Ras in the mitogen-activated protein kinase cascade. Using a yeast two-hybrid strategy to identify other proteins that interact with and potentially regulate Raf-1, we isolated a clone encoding the carboxyl-terminal half of the G beta 2 subunit of heterotrimeric G-proteins. In vitro, purified G beta gamma subunits specifically bound to a GST fusion protein encoding amino acids 1-330 of Raf-1 (Raf/330). Binding assays with truncation mutants of GST-Raf indicate that the region located between amino acids 136 and 239 is a primary determinant for interaction with G beta gamma. In competition experiments, the carboxyl terminus of beta-adrenergic receptor kinase (beta ARK) blocked the binding of G beta gamma to Raf/330; however, the Raf-1-binding proteins, Ras and 14-3-3, had no effect. Scatchard analysis of in vitro binding between Raf/330 and G beta gamma revealed an affinity of interaction (Kd = 163 +/- 36 nM), similar to that seen between G beta gamma and beta ARK (Kd = 87 +/- 24 nM). The formation of native heterotrimeric G alpha beta gamma complexes, as measured by pertussis toxin ADP-ribosylation of G alpha, could be disrupted by increasing amounts of Raf/330, with an EC50 of approximately 200 nM, in close agreement with the estimated binding affinity. In vivo complexes of Raf-1 and G beta gamma were isolated from human embryonic kidney 293-T cells transfected with epitope-tagged G beta 2. The identification and characterization of this novel interaction raises several possibilities for signaling cross-talk between growth factor receptors and those receptors coupled to heterotrimeric G-proteins.

Amino Acid Sequence↗
PubMed1993

Thioflavine T interaction with synthetic Alzheimer's disease beta-amyloid peptides: detection of amyloid aggregation in solution.

Thioflavine T (ThT) associates rapidly with aggregated fibrils of the synthetic beta/A4-derived peptides beta(1-28) and beta(1-40), giving rise to a new excitation (ex) (absorption) maximum at 450 nm and enhanced emission (em) at 482 nm, as opposed to the 385 nm (ex) and 445 nm (em) of the free dye. This change is dependent on the aggregated state as monomeric or dimeric peptides do not react, and guanidine dissociation of aggregates destroys the signal. There was no effect of high salt concentrations. Binding to the beta(1-40) is of lower affinity, Kd 2 microM, while it saturates with a Kd of 0.54 microM for beta(1-28). Insulin fibrils converted to a beta-sheet conformation fluoresce intensely with ThT. A variety of polyhydroxy, polyanionic, or polycationic materials fail to interact or impede interaction with the amyloid peptides. This fluorometric technique should allow the kinetic elucidation of the amyloid fibril assembly process as well as the testing of agents that might modulate their assembly or disassembly.

Amino Acid Sequence↗
PubMed1992

Amino acid sequence determination of ancrod, the thrombin-like alpha-fibrinogenase from the venom of Akistrodon rhodostoma.

The thrombin-like serine protease and antithrombotic agent, Ancrod, was rapidly purified from the crude venom of Akistrodon rhodostoma by agmatine-Sepharose affinity chromatography followed by MonoQ anion exchange chromatography. N-Terminal sequencing and analysis of overlapping proteolytic fragments of purified Ancrod by automated Edman degradation in combination with tandem mass spectroscopy allowed the determination of the 234 amino acid sequence of the protease. Glycosylation sites at all five canonical N-linked glycosylation sites were inferred from the appearance of blank sequencer cycles in the amino acid sequence and were confirmed by mass spectroscopic analysis of the N-glycanase-treated peptides. Monoclonal antibodies raised against the denatured protein and HF-deglycosylated protein recognized Ancrod on Western blots. Sequence comparison to other thrombin-like serine proteases and reptilian fibrinogenases revealed a number of similarities, most notably the catalytic triad and many conserved cysteine positions.

Amino Acid Sequence↗
PubMed1992

Metal ion stabilization of the conformation of a recombinant 19-kDa catalytic fragment of human fibroblast collagenase.

A recombinant 19-kDa human fibroblast collagenase catalytic fragment modeled on a naturally occurring proteolytic product was purified from E. coli inclusion bodies. Following renaturation in the presence of zinc and calcium, the fragment demonstrated catalytic activity with the same primary sequence specificity against small synthetic substrates as the full-length collagenase. Unlike the parent enzyme, it rapidly cleaved casein and gelatin but not native type I collagen. Intrinsic fluorescence of the three tryptophan residues was used to monitor the conformational state of the enzyme, which underwent a 24-nm red shift in emission upon denaturation accompanied by quenching of the fluorescence and loss of catalytic activity. Low concentrations of denaturant unfolded the fragment while the full-length enzyme displayed a shallow extended denaturation curve. Calcium remarkably stabilized the 19-kDa fragment, zinc less so, while together they were synergistically stabilizing. Among divalent cations, calcium was the most effective stabilizer, EC50 approximately 60 microM, and similar amounts were required for substrate hydrolysis. Catalytic activity was more sensitive to denaturation than was tryptophan fluorescence. Least sensitive was the polypeptide backbone secondary structure assessed by CD. These observations suggest that the folding of the 19-kDa collagenase fragment is a multistep process stabilized by calcium.

Circular Dichroism↗
PubMed1992

Determination of endothelin by an immobilized receptor assay utilizing a 96-well format.

Bovine cerebellar membranes immobilized on 96-well microtiter plates provide receptors for 125I-labeled endothelin-1 as the basis for a competitive binding assay. Adsorption of the membranes to a surface does not significantly alter the ligand-receptor interaction and reduces non-specific binding to 3-7% of total binding compared to 10-20% for a filtration technique. Considerable savings in reagents are realized since assays can be performed in 100 microliter volumes with only 10-20 micrograms of membrane protein. The 96-well format allows the rapid quantitation of large numbers of samples, and the assay is especially attractive in that it utilizes readily available reagents and equipment without the need for specific antibodies. The endothelin-receptor-based assay may be used to measure conversion of big endothelin-1 to endothelin-1 in aqueous assays. Since the presence of serum does not affect this method, tissue culture medium may be directly analyzed for endothelin production by cultured cells. All three isoforms of endothelin are detected, and the specificity of the receptor is retained since fragments and precursor forms of endothelin are not recognized. In cases where multiple endothelin isoforms may be present or where specificity of binding is in question, this assay may be used in conjunction with high pressure liquid chromatography to distinguish active peptides.

Animals↗
PubMed1991

Isolation and complete amino acid sequence of two fibrinolytic proteinases from the toxic Saturnid caterpillar Lonomia achelous.

The major toxic and fibrinolytic activity of the saliva and hemolymph of the larval form of Lonomia achelous was purified to homogeneity by a combination of metal chelate and affinity chromatography. Two apparent isozymes, Achelase I (213 amino acids, pIcalc = 10.55) and Achelase II (214 amino acids, pIcalc = 8.51), were sequenced by automated Edman degradation, and their C-termini confirmed by Fourier-transform mass spectrometry. The calculated molecular weights (22,473 and 22,727) correspond well to Mr estimates of 24,000 by SDS-PAGE. No carbohydrate was detected during sequencing. The enzymes degraded all three chains of fibrin, alpha greater than beta much greater than gamma, yielding a fragmentation pattern indistinguishable from that produced by trypsin. Chromogenic peptides S-2222 (Factor Xa and trypsin), S-2251 (plasmin), S-2302 (kallikrein) and S-2444 (urokinase) were substrates while S-2288 (broad range of serine proteinases including thrombin) was not hydrolyzed. Among a range of inhibitors Hg+2, aminophenylmercuriacetate, leupeptin, antipain and E-64 but not N-ethylmaleimide or iodoacetate abolished the activity of the purified isozymes against S-2444. Phenylmethylsulfonyl fluoride, soybean trypsin inhibitor and aprotinin were less effective. The presence of the classic catalytic triad (histidine-41, aspartate-86 and serine-189) suggests that Achelases I and II may be serine proteinases, but with a potentially free cysteine-185 which could react with thiol proteinase-directed reagents.

Amino Acid Sequence↗