Showing papers in "Biochemistry in 1999"
TL;DR: It is shown that human A beta directly produces hydrogen peroxide (H2O2) by a mechanism that involves the reduction of metal ions, Fe(III) or Cu(II), setting up conditions for Fenton-type chemistry.
Abstract: Oxidative stress markers characterize the neuropathology both of Alzheimer's disease and of amyloid-bearing transgenic mice. The neurotoxicity of amyloid Aβ peptides has been linked to peroxide gen...
1,065 citations
TL;DR: The results suggest that the role of the Cys --> Gln beta-ketoacyl synthases found in the loading domains of some modular polyketide synthases likely is to act as malonyl, or methylmalonyL, decarboxylases that provide a source of primer for the chain extension reactions catalyzed by associated modules containing fully competent beta- ketoacyL synthases.
Abstract: beta-Ketoacyl synthases involved in the biosynthesis of fatty acids and polyketides exhibit extensive sequence similarity and share a common reaction mechanism, in which the carbanion participating in the condensation reaction is generated by decarboxylation of a malonyl or methylmalonyl moiety; normally, the decarboxylation step does not take place readily unless an acyl moiety is positioned on the active-site cysteine residue in readiness for the ensuing condensation reaction. Replacement of the cysteine nucleophile (Cys-161) with glutamine, in the beta-ketoacyl synthase domain of the multifunctional animal fatty acid synthase, completely inhibits the condensation reaction but increases the uncoupled rate of malonyl decarboxylation by more than 2 orders of magnitude. On the other hand, replacement with Ser, Ala, Asn, Gly, and Thr compromises the condensation reaction without having any marked effect on the decarboxylation reaction. The affinity of the beta-ketoacyl synthase for malonyl moieties, in the absence of acetyl moieties, is significantly increased in the Cys161Gln mutant compared to that in the wild type and is similar to that exhibited by the wild-type beta-ketoacyl synthase in the presence of an acetyl primer. These results, together with modeling studies of the Cys --> Gln mutant from the crystal structure of the Escherichia coli beta-ketoacyl synthase II enzyme, suggest that the side chain carbonyl group of the Gln-161 can mimic the carbonyl of the acyl moiety in the acyl-enzyme intermediate so that the mutant adopts a conformation analogous to that of the acyl-enzyme intermediate. Catalysis of the decarboxylation of malonyl-CoA requires the dimeric form of the Cys161Gln fatty acid synthase and involves prior transfer of the malonyl moiety from the CoA ester to the acyl carrier protein domain and subsequent release of the acetyl product by transfer back to a CoA acceptor. These results suggest that the role of the Cys --> Gln beta-ketoacyl synthases found in the loading domains of some modular polyketide synthases likely is to act as malonyl, or methylmalonyl, decarboxylases that provide a source of primer for the chain extension reactions catalyzed by associated modules containing fully competent beta-ketoacyl synthases.
1,015 citations
TL;DR: Evidence is found for significant coupling between local and global features of the conformational ensembles adopted in such states and the effective dimensions of the polypeptide chain are found to depend significantly on the level of persistence of regions of secondary structure or features within a conformational ensemble.
Abstract: Pulse field gradient NMR methods have been used to determine the effective hydrodynamic radii of a range of native and nonnative protein conformations. From these experimental data, empirical relationships between the measured hydrodynamic radius (Rh) and the number of residues in the polypeptide chain (N) have been established; for native folded proteins Rh = 4.75N 0.29A and for highly denatured states Rh = 2.21N 0.57A. Predictions from these equations agree well with experimental data from dynamic light scattering and small-angle X-ray or neutron scattering studies reported in the literature for proteins ranging in size from 58 to 760 amino acid residues. The predicted values of the hydrodynamic radii provide a framework that can be used to analyze the conformational properties of a range of nonnative states of proteins. Several examples are given here to illustrate this approach including data for partially structured molten globule states and for proteins that are unfolded but biologically active unde...
967 citations
TL;DR: It was demonstrated that individual peptides varied widely in their ability to depolarize the cytoplasmic membrane potential of E. coli, with certain peptides such as the loop peptide bactenecin and the alpha-helical peptide CP26 being unable to cause depolarization at the minimal inhibitory concentration (MIC), and others like gramicidin S causing maximal depolarized below the MIC.
Abstract: Antimicrobial cationic peptides are prevalent throughout nature as part of the intrinsic defenses of most organisms, and have been proposed as a blueprint for the design of novel antimicrobial agents. They are known to interact with membranes, and it has been frequently proposed that this represents their antibacterial target. To see if this was a general mechanism of action, we studied the interaction, with model membranes and the cytoplasmic membrane of Escherichia coli, of 12 peptides representing all 4 structural classes of antimicrobial peptides. Planar lipid bilayer studies indicated that there was considerable variance in the interactions of the peptides with model phospholipid membranes, but generally both high concentrations of peptide and high transmembrane voltages (usually −180 mV) were required to observe conductance events (channels). The channels observed for most peptides varied widely in magnitude and duration. An assay was developed to measure the interaction with the Escherichia coli cy...
687 citations
TL;DR: The data reported here combined with previous work provide for the first time a complete set of thermodynamic parameters for molecular recognition of DNA by DNA with or without single internal mismatches, useful for primer design and understanding the mechanism of triplet repeat diseases.
Abstract: Thermodynamic measurements are reported for 51 DNA duplexes with A·A, C·C, G·G, and T·T single mismatches in all possible Watson−Crick contexts. These measurements were used to test the applicability of the nearest-neighbor model and to calculate the 16 unique nearest-neighbor parameters for the 4 single like with like base mismatches next to a Watson−Crick pair. The observed trend in stabilities of mismatches at 37 °C is G·G > T·T ≈ A·A > C·C. The observed stability trend for the closing Watson−Crick pair on the 5‘ side of the mismatch is G·C ≥ C·G ≥ A·T ≥ T·A. The mismatch contribution to duplex stability ranges from −2.22 kcal/mol for GGC·GGC to +2.66 kcal/mol for ACT·ACT. The mismatch nearest-neighbor parameters predict the measured thermodynamics with average deviations of ΔG°37 = 3.3%, ΔH° = 7.4%, ΔS° = 8.1%, and TM = 1.1 °C. The imino proton region of 1-D NMR spectra shows that G·G and T·T mismatches form hydrogen-bonded structures that vary depending on the Watson−Crick context. The data reported ...
579 citations
TL;DR: The selectivity in the chemokine-GAG interaction suggests selective disruption of the haptotactic gradient may be an achievable therapeutic approach in inflammatory disease.
Abstract: Chemokines selectively recruit and activate a variety of cells during inflammation. Interactions between cell surface glycosaminoglycans (GAGs) and chemokines drive the formation of haptotactic or ...
571 citations
TL;DR: Although the phosphorylation sites on Ser-Pro or Thr-Pro motifs are the most prominent ones on Alzheimer PHFs (by antibody labeling), they are only weakly inhibitory to PHF assembly, which implies that the hyperphosphorylation of tau in Alzheimer's disease is not directly responsible for the pathological aggregation into PHFs; on the contrary, phosphorylated protects tau against aggregation.
Abstract: One of the hallmarks of Alzheimer's disease is the abnormal state of the microtubule-associated protein tau in neurons. It is both highly phosphorylated and aggregated into paired helical filaments, and it is commonly assumed that the hyperphosphorylation of tau causes its detachment from microtubules and promotes its assembly into PHFs. We have studied the relationship between the phosphorylation of tau by several kinases (MARK, PKA, MAPK, GSK3) and its assembly into PHFs. The proline-directed kinases MAPK and GSK3 are known to phosphorylate most Ser-Pro or Thr-Pro motifs in the regions flanking the repeat domain of tau: they induce the reaction with several antibodies diagnostic of Alzheimer PHFs, but this type of phosphorylation has only a weak effect on tau-microtubule interactions and on PHF assembly. By contrast, MARK and PKA phosphorylate several sites within the repeats (notably the KXGS motifs including Ser262, Ser324, and Ser356, plus Ser320); in addition PKA phosphorylates some sites in the flanking domains, notably Ser214. This type of phosphorylation strongly reduces tau's affinity for microtubules, and at the same time inhibits tau's assembly into PHFs. Thus, contrary to expectations, the phosphorylation that detaches tau from microtubules does not prime it for PHF assembly, but rather inhibits it. Likewise, although the phosphorylation sites on Ser-Pro or Thr-Pro motifs are the most prominent ones on Alzheimer PHFs (by antibody labeling), they are only weakly inhibitory to PHF assembly. This implies that the hyperphosphorylation of tau in Alzheimer's disease is not directly responsible for the pathological aggregation into PHFs; on the contrary, phosphorylation protects tau against aggregation.
514 citations
TL;DR: The sequence and structural selectivity of 15 different DNA binding agents was explored using a novel, thermodynamically rigorous, competition dialysis procedure and selective agents showed in many cases less dramatic binding selectivity than anticipated from published reports that compared their binding to only a few structural forms.
Abstract: The sequence and structural selectivity of 15 different DNA binding agents was explored using a novel, thermodynamically rigorous, competition dialysis procedure. In the competition dialysis method, 13 different nucleic acid structures were dialyzed against a common ligand solution. More ligand accumulated in the dialysis tube containing the structural form with the highest ligand binding affinity. DNA structural forms included in the assay ranged from single-stranded forms, through a variety of duplex forms, to multistranded triplex and tetraplex forms. Left-handed Z-DNA, RNA, and a DNA-RNA hybrid were also represented. Standard intercalators (ethidium, daunorubicin, and actinomycin D) served as control compounds and were found to show structural binding preferences fully consistent with their previously published behavior. Standard groove binding agents (DAPI, distamycin, and netropsin) showed a strong preference for AT-rich duplex DNA forms, along with apparently strong binding to the poly(dA)-[poly(dT)](2) triplex. Thermal denaturation studies revealed the apparent triplex binding to be complex, and perhaps to result from displacement of the third strand. Putative triplex (BePI, coralyne, and berberine) and tetraplex [H(2)TmPyP, 5,10,15, 20-tetrakis[4-(trimethylammonio)phenyl]-21H,23H-porphine, and N-methyl mesoporphyrin IX] selective agents showed in many cases less dramatic binding selectivity than anticipated from published reports that compared their binding to only a few structural forms. Coralyne was found to bind strongly to single-stranded poly(dA), a novel and previously unreported interaction. Finally, three compounds (berenil, chromomycin A, and pyrenemethylamine) whose structural preferences are largely unknown were examined. Pyrenemethylamine exhibited an unexpected and unprecedented preference for duplex poly(dAdT).
504 citations
TL;DR: Glutathionylation of the cysteine sulfenic derivative will prevent the enzyme from further oxidation to its irreversible forms, and constitutes an efficient regulatory mechanism.
Abstract: The reversible regulation of protein tyrosine phosphatase is an important mechanism in processing signal transduction and regulating cell cycle. Recent reports have shown that the active site cysteine residue, Cys215, can be reversibly oxidized to a cysteine sulfenic derivative (Denu and Tanner, 1998; Lee et al., 1998). We propose an additional modification that has implications for the in vivo regulation of protein tyrosine phosphatase 1B (PTP1B, EC 3.1.3.48): the glutathionylation of Cys215 to a mixed protein disulfide. Treatment of PTP1B with diamide and reduced glutathione or with only glutathione disulfide (GSSG) results in a modification detected by mass spectrometry in which the cysteine residues are oxidized to mixed disulfides with glutathione. The activity is recovered by the addition of dithiothreitol, presumably by reducing the cysteine disulfides. In addition, inactivated PTP1B is reactivated enzymatically by the glutathione-specific dethiolase enzyme thioltransferase (glutaredoxin), indicat...
483 citations
TL;DR: These functional Cys-SOHs have roles in diverse cellular processes, including signal transduction, oxygen metabolism and the oxidative stress response, and transcriptional regulation, as well as in the industrial production of acrylamide, and their detailed analyses are beginning to provide the chemical foundation necessary for understanding protein-S OH stabilization and function.
Abstract: While it has been known for more than 20 years that unusually stable cysteine-sulfenic acid (Cys-SOH) derivatives can be introduced in selected proteins by mild oxidation, only recently have chemical and crystallographic evidence for functional Cys-SOH been presented with native proteins such as NADH peroxidase and NADH oxidase, nitrile hydratase, and the hORF6 and AhpC peroxiredoxins. In addition, Cys-SOH forms of protein tyrosine phosphatases and glutathione reductase have been suggested to play key roles in the reversible inhibition of these enzymes during tyrosine phosphorylation-dependent signal transduction events and nitrosative stress, respectively. Substantial chemical data have also been presented which implicate Cys-SOH in redox regulation of transcription factors such as Fos and Jun (activator protein-1) and bovine papillomavirus-1 E2 protein. Functionally, the Cys-SOHs in NADH peroxidase, NADH oxidase, and the peroxiredoxins serve as either catalytically essential redox centers or transient intermediates during peroxide reduction. In nitrile hydratase, the active-site Cys-SOH functions in both iron coordination and NO binding but does not play any catalytic redox role. In Fos and Jun and the E2 protein, on the other hand, a key Cys-SH serves as a sensor for intracellular redox status; reversible oxidation to Cys-SOH as proposed inhibits the corresponding DNA binding activity. These functional Cys-SOHs have roles in diverse cellular processes, including signal transduction, oxygen metabolism and the oxidative stress response, and transcriptional regulation, as well as in the industrial production of acrylamide, and their detailed analyses are beginning to provide the chemical foundation necessary for understanding protein-SOH stabilization and function.
483 citations
TL;DR: Atomic force microscopy allowed the identification of an unanticipated intermediate in in vitro fibril formation, the Aβ amyloid protofibril, which is a precursor species whose formation is linked to fibrillogenesis in vivo.
Abstract: Amyloid fibrils comprising primarily the peptides Aβ40 and Aβ42 are a defining feature of the Alzheimer's disease (AD) brain, and convergent evidence suggests that the process of their formation plays a central role in the AD pathogenic pathway. Elucidation of fibril assembly is critical for the discovery of potential AD diagnostics and therapeutics, since the pathogenic entity is not necessarily the product fibril, but could be a precursor species whose formation is linked to fibrillogenesis in vivo. Atomic force microscopy allowed the identification of an unanticipated intermediate in in vitro fibril formation, the Aβ amyloid protofibril. This manuscript describes studies of the structure of the Aβ40 protofibril and its in vitro assembly and disassembly using atomic force microscopy (AFM). The Aβ40 protofibril has a height of ca. 4.3 ± 0.5 nm and a periodicity of ca. 20 ± 4.7 nm. The rate of its elongation depends on the total concentration of Aβ40, the temperature, and ionic strength of the medium. Aβ4...
TL;DR: SpsA is a glycosyltransferase implicated in the synthesis of the spore coat of Bacillus subtilis, whose homologues include cellulose synthase and many lipopolysaccharide and bacterial O-antigen synthases, and the structure and sequence conservation are determined, which begin to shed light on the mechanism of this ubiquitous family of inverting gly cosyltransferases.
Abstract: The enzymatic formation of glycosidic bonds may be catalyzed by the transfer of the glycosyl moiety from an activated nucleotide-diphospho-sugar donor to a specific acceptor. SpsA is a glycosyltransferase implicated in the synthesis of the spore coat of Bacillus subtilis, whose homologues include cellulose synthase and many lipopolysaccharide and bacterial O-antigen synthases. The three-dimensional crystal structure of SpsA has been determined by conventional MIR techniques at a resolution of 1.5 A. It is a two-domain protein with a nucleotide-binding domain together with an acceptor binding domain which features a disordered loop spanning the active site. The structures of SpsA in complex with both Mg-UDP and Mn-UDP have also been determined at 2.0 and 1.7 A, respectively. These complexes, together with the sequence conservation, begin to shed light on the mechanism of this ubiquitous family of inverting glycosyltransferases.
TL;DR: These results strongly suggest that in vitro HBV assembly is nucleated by a trimer of dimers and proceeds by the addition of individual dimeric subunits, and suggest that HBV capsid assembly could be an important target for antiviral therapeutics.
Abstract: The capsids of most spherical viruses are icosahedral, an arrangement of multiples of 60 subunits. Though it is a salient point in the life cycle of any virus, the physical chemistry of virus capsid assembly is poorly understood. We have developed general models of capsid assembly that describe the process in terms of a cascade of low order association reactions. The models predict sigmoidal assembly kinetics, where intermediates approach a low steady state concentration for the greater part of the reaction. Features of the overall reaction can be identified on the basis of the concentration dependence of assembly. In simulations, and on the basis of our understanding of the models, we find that nucleus size and the order of subsequent "elongation" reactions are reflected in the concentration dependence of the extent of the reaction and the rate of the fast phase, respectively. The reaction kinetics deduced for our models of virus assembly can be related to the assembly of any "spherical" polymer. Using light scattering and size exclusion chromatography, we observed polymerization of assembly domain dimers of hepatitis B virus (HBV) capsid protein. Empty capsids assemble at a rate that is a function of protein concentration and ionic strength. The kinetics of capsid formation were sigmoidal, where the rate of the fast phase had second-power concentration dependence. The extent of assembly had third-power concentration dependence. Simulations based on the models recapitulated the concentration dependences observed for HBV capsid assembly. These results strongly suggest that in vitro HBV assembly is nucleated by a trimer of dimers and proceeds by the addition of individual dimeric subunits. On the basis of this mechanism, we suggest that HBV capsid assembly could be an important target for antiviral therapeutics.
TL;DR: Calculations of Voronoi atomic volumes showed that protein atoms buried at the interface with DNA are on average as closely packed as in the protein interior.
Abstract: We analyzed the atomic models of 75 X-ray structures of protein−nucleic acid complexes with the aim of uncovering common properties. The interface area measured the extent of contact between the protein and nucleic acid. It was found to vary between 1120 and 5800 A2. Despite this wide variation, the interfaces in complexes of transcription factors with double-stranded DNA could be broken up into recognition modules where 12 ± 3 nucleotides on the DNA side contact 24 ± 6 amino acids on the protein side, with interface areas in the range 1600 ± 400 A2. For enzymes acting on DNA, the recognition module is on average 600 A2 larger, due to the requirement of making an active site. As judged by its chemical and amino acid composition, the average protein surface in contact with the DNA is more polar than the solvent accessible surface or the typical protein−protein interface. The protein side is rich in positively charged groups from lysine and arginine side chains; on the DNA side the negative charges from pho...
TL;DR: P pH titrations of popular GFP S65T/H148D variants indicate that the chromophore pKa can be modulated over a broad range from 6 to 8, allowing for pH determination from pH 5 to pH 9.6.
Abstract: The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become a useful tool in molecular and cell biology. Recently, it has been found that the fluorescence spectra of most mu...
TL;DR: The data suggest that destabilization of PrPC is neither a general mechanism underlying the formation of PrPSc nor the basis of disease phenotypes in inherited human TSEs.
Abstract: Transmissible spongiform encephalopathies (TSEs) are caused by a unique infectious agent which appears to be identical with PrPSc, an oligomeric, misfolded isoform of the cellular prion protein, PrPC. All inherited forms of human TSEs, i.e., familial Creutzfeldt−Jakob disease, Gerstmann−Straussler−Scheinker syndrome, and fatal familial insomnia, segregate with specific point mutations or insertions in the gene coding for human PrP. Here we have tested the hypothesis that these mutations destabilize PrPC and thus facilitate its conversion into PrPSc. Eight of the disease-specific amino acid replacements are located in the C-terminal domain of PrPC, PrP(121−231), which constitutes the only part of PrPC with a defined tertiary structure. Introduction of all these replacements into PrP(121−231) yielded variants with the same spectroscopic characteristics as wild-type PrP(121−231) and similar to full-length PrP(23−231), which excludes the possibility that the exchanges a priori induce a PrPSc-like conformation...
TL;DR: Wolfe et al. as discussed by the authors proposed a difluoro ketone peptidomimetic (1) that blocks Aβ production at the γ-secretase level.
Abstract: The amyloid β-protein (Aβ), implicated in the pathogenesis of Alzheimer's disease (AD), is a proteolytic metabolite generated by the sequential action of β- and γ-secretases on the amyloid precursor protein (APP). The two main forms of Aβ are 40- and 42-amino acid C-terminal variants, Aβ40 and Aβ42. We recently described a difluoro ketone peptidomimetic (1) that blocks Aβ production at the γ-secretase level [Wolfe, M. S., et al. (1998) J. Med. Chem. 41, 6−9]. Although designed to inhibit Aβ42 production, 1 also effectively blocked Aβ40 formation. Various amino acid changes in 1 still resulted in inhibition of Aβ40 and Aβ42 production, suggesting relatively loose sequence specificity by γ-secretase. The alcohol counterparts of selected difluoro ketones also lowered Aβ levels, indicating that the ketone carbonyl is not essential for activity and suggesting that these compounds inhibit an aspartyl protease. Selected compounds inhibited the aspartyl protease cathepsin D but not the cysteine protease calpain, ...
TL;DR: A well-defined peptide will be an effective tool for probing the role of 14-3-3 in various signaling pathways, and may lead to the development of high-affinity antagonists with pharmacological applications.
Abstract: The 14-3-3 proteins interact with diverse cellular molecules involved in various signal transduction pathways controlling cell proliferation, transformation, and apoptosis. To aid our investigation of the biological function of 14-3-3 proteins, we have set out to identify high-affinity antagonists. By screening phage display libraries, we have identified a set of peptides which bind 14-3-3 proteins. One of these peptides, termed R18, exhibited a high affinity for different isoforms of 14-3-3 with estimated K(D) values of 7-9 x 10(-)(8) M. Recognition of multiple isoforms of 14-3-3 suggests the targeting of R18 to a structure that is common among 14-3-3 proteins, such as the conserved ligand-binding groove. Indeed, mutations that alter critical residues in the ligand-binding site of 14-3-3 drastically decreased the level of 14-3-3-R18 association. R18 efficiently blocked the binding of 14-3-3 to the kinase Raf-1, a physiological ligand of 14-3-3, and effectively abolished the protective role of 14-3-3 against phosphatase-induced inactivation of Raf-1. The cocrystal structure of R18 in complex with 14-3-3zeta revealed the occupancy of the general binding groove of 14-3-3zeta by R18, explaining the potent inhibitory effect of R18 on 14-3-3-ligand interactions. Such a well-defined peptide will be an effective tool for probing the role of 14-3-3 in various signaling pathways, and may lead to the development of 14-3-3 antagonists with pharmacological applications.
TL;DR: Results support and strengthen results from previous studies suggesting that DRM have a lipid composition that promotes liquid-ordered structure and demonstrate the potential of mass spectrometry for examining the role of membrane structure in receptor signaling and other cellular processes.
Abstract: We recently showed that ligand-mediated cross-linking of FceRI, the high-affinity receptor for immunoglobulin E, on RBL-2H3 mast cells results in its co-isolation with detergent-resistant membranes (DRM) and its consequent tyrosine phosphorylation by the co-localized tyrosine kinase Lyn that is a critical early event in signaling by this receptor [Field et al. (1997) J. Biol. Chem. 272, 4276−4280]. As part of efforts to determine the structural bases for these interactions, we examined the phospholipid composition of DRM vesicles isolated from RBL-2H3 cells under conditions that preserve FceRI association. We used positive and negative mode electrospray Fourier transform ion cyclotron resonance mass spectrometry to compare quantitatively the phospholipid composition of isolated DRM to that of total cell lipids and to a plasma membrane preparation. From these analyses, over 90 different phospholipid species were spectrally resolved and unambiguously identified; more than two-thirds of these were determined...
TL;DR: The ability to generate crystals of the carbon monoxide bound state of the hydrogenase that are isomorphous to those of the native enzyme has allowed for a direct comparison of the crystallographic data and an unambiguous identification of the site ofcarbon monoxide binding at the active site of CpI.
Abstract: A site for the binding of exogenously added carbon monoxide has been identified at the active site of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum. The binding and inhibition of carbon monoxide have been exploited in biochemical and spectroscopic studies to gain mechanistic insights. In the present study, we have taken advantage of the ability to generate an irreversibly carbon monoxide bound state of CpI. The crystallization and structural characterization of CpI inhibited in the presence of carbon monoxide indicates the addition of a single molecule of carbon monoxide. The ability to generate crystals of the carbon monoxide bound state of the hydrogenase that are isomorphous to those of the native enzyme has allowed for a direct comparison of the crystallographic data and an unambiguous identification of the site of carbon monoxide binding at the active site of CpI. Carbon monoxide binds to an Fe atom of the 2Fe subcluster at the site of a terminally bound water molecule in the as crystallized native state of CpI that has been previously suggested to be a potential site of reversible hydrogen oxidation. Binding of carbon monoxide at this site results in an active site that is coordinately saturated with strong ligands (S, CO, and CN), providing a rational potential mechanism for inhibition of reversible hydrogen oxidation at the active site of CpI.
TL;DR: It is proposed that filament side binding by the Arp2/3 complex enhances its activation by WASp WA, and the rate of this nucleation is a function of the concentrations of both Wasp WA and the ArP2/ 3 complex.
Abstract: The 70 C-terminal amino acids of Wiskott-Aldrich syndrome protein (WASp WA) activate the actin nucleation activity of the Arp2/3 complex. WASp WA binds both the Arp2/3 complex and actin monomers, but the mechanism by which it activates the Arp2/3 complex is not known. We characterized the effect of WASp WA on actin polymerization in the absence and presence of the human Arp2/3 complex. WASp WA binds actin monomers with an apparent Kd of 0.4 μM, inhibiting spontaneous nucleation and subunit addition to pointed ends, but not addition to barbed ends. A peptide containing only the WASp homology 2 motif behaves similarly but with a 10-fold lower affinity. In contrast to previously published results, neither WASp WA nor a similar region of the protein Scar1 significantly depolymerizes actin filaments under a variety of conditions. WASp WA and the Arp2/3 complex nucleate actin filaments, and the rate of this nucleation is a function of the concentrations of both WASp WA and the Arp2/3 complex. With excess WASp W...
TL;DR: It is shown that the synthetic fluorescent long-chain fatty acid trans-parinaric acid binds to PPARalpha with high affinity and can be used as a probe to monitor protein-ligand interactions by the receptor and demonstrate the utility of the optical method in examining the ligand-selectivity of PPARs.
Abstract: Peroxisome proliferator-activated receptors (PPAR α, β, and γ) are nuclear hormone receptors that play critical roles in regulating lipid metabolism. It is well established that PPARs are the targets for the hypolipidemic synthetic compounds known as peroxisome proliferators, and it has been proposed that various long-chain fatty acids and metabolites of arachidonic acid serve as the physiological ligands that activate these receptors in vivo. However, a persistent problem is that reported values of the equilibrium dissociation constants (Kds) of complexes of PPARs with these ligands are in the micromolar range, at least an order of magnitude higher than the physiological concentrations of the ligands. Thus, the identity of the endogenous ligands for PPAR remains unclear. Here we report on a fluorescence-based method for investigating the interactions of PPAR with ligands. It is shown that the synthetic fluorescent long-chain fatty acid trans-parinaric acid binds to PPARα with high affinity and can be use...
TL;DR: Abeta-derived peptides of fifteen residues were found to be inhibitory of Abeta polymerization and the activity of these peptides was subsequently enhanced through modification of their amino termini with specific organic reagents.
Abstract: Cellular toxicity resulting from nucleation-dependent polymerization of amyloid beta-peptide (Abeta) is considered to be a major and possibly the primary component of Alzheimer's disease (AD). Inhibition of Abeta polymerization has thus been identified as a target for the development of therapeutic agents for the treatment of AD. The intrinsic affinity of Abeta for itself suggested that Abeta-specific interactions could be adapted to the development of compounds that would bind to Abeta and prevent it from polymerizing. Abeta-derived peptides of fifteen residues were found to be inhibitory of Abeta polymerization. The activity of these peptides was subsequently enhanced through modification of their amino termini with specific organic reagents. Additional series of compounds prepared to probe structural requirements for activity allowed reduction of the size of the inhibitors and optimization of the Abeta-derived peptide portion to afford a lead compound, cholyl-Leu-Val-Phe-Phe-Ala-OH (PPI-368), with potent polymerization inhibitory activity but limited biochemical stability. The corresponding all-D-amino acyl analogue peptide acid (PPI-433) and amide (PPI-457) retained inhibitory activity and were both stable in monkey cerebrospinal fluid for 24 h.
TL;DR: The active sites of aged sarin- and soman-TcAChE were essentially identical and provided structural models for the negatively charged, tetrahedral intermediate that occurs during deacylation with the natural substrate, acetylcholine.
Abstract: Organophosphorus acid anhydride (OP) nerve agents are potent inhibitors which rapidly phosphonylate acetylcholinesterase (AChE) and then may undergo an internal dealkylation reaction (called “aging”) to produce an OP-enzyme conjugate that cannot be reactivated. To understand the basis for irreversible inhibition, we solved the structures of aged conjugates obtained by reaction of Torpedo californica AChE (TcAChE) with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (sarin), or O-pinacolylmethylphosphonofluoridate (soman) by X-ray crystallography to 2.3, 2.6, or 2.2 A resolution, respectively. The highest positive difference density peak corresponded to the OP phosphorus and was located within covalent bonding distance of the active-site serine (S200) in each structure. The OP-oxygen atoms were within hydrogen-bonding distance of four potential donors from catalytic subsites of the enzyme, suggesting that electrostatic forces significantly stabilize the aged enzyme. The active sit...
TL;DR: The role of the highly conserved amino acid residues in blocks I-IV in GPAT activity is examined through chemical modification and site-directed mutagenesis experiments and it is hypothesize that blocks I -IV and, in particular, the invariant amino acids contained within these regions form a catalytically important site in this family of acyltransferases.
Abstract: Alignment of amino acid sequences from various acyltransferases [sn-glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), acyl-CoA:dihydroxyacetone-phosphate acyltransferase (DHAPAT), 2-acylglycerophosphatidylethanolamine acyltransferase (LPEAT)] reveals four regions of strong homology, which we have labeled blocks I-IV. The consensus sequence for each conserved region is as follows: block I, [NX]-H-[RQ]-S-X-[LYIM]-D; block II, G-X-[IF]-F-I-[RD]-R; block III, F-[PLI]-E-G-[TG]-R-[SX]-[RX]; and block IV, [VI]-[PX]-[IVL]-[IV]-P-[VI]. We hypothesize that blocks I-IV and, in particular, the invariant amino acids contained within these regions form a catalytically important site in this family of acyltransferases. Using Escherichia coli GPAT (PlsB) as a model acyltransferase, we examined the role of the highly conserved amino acid residues in blocks I-IV in GPAT activity through chemical modification and site-directed mutagenesis experiments. We found that the histidine and aspartate in block I, the glycine in block III, and the proline in block IV all play a role in E. coli GPAT catalysis. The phenylalanine and arginine in block II and the glutamate and serine in block III appear to be important in binding the glycerol 3-phosphate substrate. Since blocks I-IV are also found in LPAAT, DHAPAT, and LPEAT, we believe that these conserved amino acid motifs are diagnostic for the acyltransferase reaction involving glycerol 3-phosphate, 1-acylglycerol 3-phosphate, and dihydroxyacetone phosphate substrates.
TL;DR: The ability of the arachidonic acid metabolite, lipoxin A4, to bind to and activate the Ah receptor in Hepa-1 cells is reported and established as a new class of Ah receptors ligand, one that differs dramatically from classical Ah receptor ligands.
Abstract: The Ah receptor is a ligand-activated transcription factor that mediates many of the biological actions of a large class of environmental compounds. Support for a role of the Ah receptor in normal physiology also has been reported, but an endogenous regulating ligand has not been identified. We have examined candidate endogenous lipophilic substances and report here the ability of the arachidonic acid metabolite, lipoxin A4, to bind to and activate the Ah receptor in Hepa-1 cells. Lipoxin A4 produced a concentration-dependent response in a DRE-driven CAT reporter construct, with a greater than 10-fold increase in CAT activity at 0.3 μM. Lipoxin A4 transformed the Ah receptor to an active DRE-binding form in a concentration-dependent manner as indicated by gel mobility shift analysis. Results of Ah receptor competitive binding experiments indicated that at a concentration of 100 nM, lipoxin A4 produced a half-maximum displacement (EC50) of [3H]TCDD binding. Results of Northern blot analyses indicated a tra...
TL;DR: The drastic change of the Cu(II) binding mode on going from neutral to weakly acidic conditions suggests that the micro-environmental pH in the brain cell regulates the Cu (II) affinity of the prion protein, which is supposed to undergo pH changes in the pathway from the cell surface to endosomes.
Abstract: The cellular form of prion protein is a precursor of the infectious isoform, which causes fatal neurodegenerative diseases through intermolecular association. One of the characteristics of the prio...
TL;DR: A model of pore formation in which the D2L2 loops move to the membrane-proximal face of the heptamer and interact to form a 14-strand transmembrane beta-barrel is supported, which provides a basis for further exploration of the role of PA(63) in translocation of anthrax toxin's enzymic moieties across membranes.
Abstract: PA63, the active 63 kDa form of anthrax protective antigen, forms a heptameric ring-shaped oligomer that is believed to represent a precursor of the membrane pore formed by this protein. When maintained at pH ≥8.0, this “prepore” dissociated to monomeric subunits upon treatment with SDS at room temperature, but treatment at pH ≤7 (or with β-octylglucoside at pH 8.0) caused it to convert to an SDS-resistant pore-like form. Transition to this form involved major changes in the conformation of loop 2 of domain 2 (D2L2), as evidenced by (i) occlusion of a chymotrypsin site within D2L2 and (ii) excimer formation by pyrene groups linked to N306C within this loop. The pore-like form retained the capacity to bind anthrax toxin A moieties and cell surface receptors, but was unable to form pores in membranes or mediate translocation. Mutant PA63 in which D2L2 had been deleted was inactive in pore formation and translocation but, like the prepore, was capable of forming heptamers that converted to an SDS-resistant f...
TL;DR: The present study demonstrates that the corresponding region in cTnI (residues 147-163) binds to the regulatory domain of cTNC only in the Ca2+-saturated state to form a 1:1 complex, with an affinity approximately six times weaker than that between the skeletal counterparts.
Abstract: The interaction of troponin-C (TnC) with troponin-I (TnI) plays a central role in skeletal and cardiac muscle contraction. We have recently shown that the binding of Ca2+ to cardiac TnC (cTnC) does not induce an “opening” of the regulatory domain in order to interact with cTnI [Sia, S. K., et al. (1997) J. Biol. Chem. 272, 18216−18221; Spyracopoulos et al. (1997) Biochemistry 36, 12138−12146], which is in contrast to the regulatory N-domain of skeletal TnC (sTnC). This implies that the mode of interaction between cTnC and cTnI may be different than that between sTnC and sTnI. In sTnI, a region downstream from the inhibitory region (residues 115−131) has been shown to bind the exposed hydrophobic pocket of Ca2+-saturated sNTnC [McKay, R. T., et al. (1997) J. Biol. Chem. 272, 28494−28500]. The present study demonstrates that the corresponding region in cTnI (residues 147−163) binds to the regulatory domain of cTnC only in the Ca2+-saturated state to form a 1:1 complex, with an affinity approximately six tim...
TL;DR: It is determined by HPLC-MS that the modification of one (out of 24) Cys residue of the sarcoplasmic reticulum (SR) Ca-ATPase isoform SERCA1, Cys(349), by peroxynitrite is sufficient for the modulation of enzyme activity.
Abstract: Skeletal muscle contraction and relaxation is efficiently modulated through the reaction of reactive oxygen-nitrogen species with sarcoplasmic reticulum protein thiols in vivo. However, the exact locations of functionally important modifications are at present unknown. Here, we determine by HPLC-MS that the modification of one (out of 24) Cys residue of the sarcoplasmic reticulum (SR) Ca-ATPase isoform SERCA1, Cys(349), by peroxynitrite is sufficient for the modulation of enzyme activity. Despite the size and nature of the SR Ca-ATPase, a 110 kDa membrane protein, identification and quantitation of Cys modification was achieved through labeling with 4-(dimethylamino)phenylazophenyl-4'-maleimide (DABMI) and/or N-(2-iodoethyl)trifluoroacetamide (IE-TFA) followed by an exhaustive tryptic digestion and on-line HPLC-UV-electrospray MS analysis. The reaction with IE-TFA generates aminoethylcysteine, a new trypsin cleavage site, which allows the production of specific peptide fragments that are diagnostic for IE-TFA labeling, conveniently identified by mass spectrometry. Exposure of the SR Ca-ATPase to low concentrations (0.1 mM) of peroxynitrite resulted in the fully reversible chemical modification of Cys at positions 344, 349, 471, 498, 525, and 614 (nitrosylation of Cys(344) and Cys(349) was seen), whereas higher concentrations of peroxynitrite (0.45 mM) additionally affected Cys residues at positions 636, 670, and 674. When the SR Ca-ATPase was exposed to 0.45 mM peroxynitrite in the presence of 5.0 mM glutathione (GSH), thiol modification became partially reversible and S-glutathiolation was detected for Cys residues at positions 344, 349, 364, 498, 525, and 614. The extent of enzyme inactivation (determined previously) quantitatively correlated with the loss of labeling efficiency (i) of a single Cys residue and (ii) of the tryptic fragment containing both Cys(344) and Cys(349). Earlier results had shown that the independent selective modification of Cys(344) is functionally insignificant [Kawakita, M., and Yamashita, T. (1987) J. Biochem. (Tokyo) 102, 103-109]. Thus, we conclude that modification of only Cys(349) is responsible for the modulation of the SR Ca-ATPase activity by peroxynitrite.