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1.
Oncogene ; 31(41): 4472-83, 2012 Oct 11.
Article in English | MEDLINE | ID: mdl-22266849

ABSTRACT

Elevated levels of the oncoprotein, osteopontin (OPN), are associated with poor outcome of several types of cancers including melanoma. We have previously reported an important involvement of DNAJB6, a member of heat-shock protein 40 (HSP40) family, in negatively impacting tumor growth. The current study was prompted by our observations reported here which revealed a reciprocal relationship between DNAJB6 and OPN in melanoma specimens. The 'J domain' is the most conserved domain of HSP40 family of proteins. Hence, we assessed the functional role of the J domain in activities of DNAJB6. We report that the J domain of DNAJB6 is involved in mediating OPN suppression. Deletion of the J domain renders DNAJB6 incapable of impeding malignancy and suppressing OPN. Our mechanistic investigations reveal that DNAJB6 binds HSPA8 (heat-shock cognate protein, HSC70) and causes dephosphorylation of glycogen synthase kinase 3ß (GSK3ß) at Ser 9 by recruiting protein phosphatase, PP2A. This dephosphorylation activates GSK3ß, leading to degradation of ß-catenin and subsequent loss of TCF/LEF (T cell factor1/lymphoid enhancer factor1) activity. Deletion of the J domain abrogates assembly of this multiprotein complex and renders GSK3ß inactive, thus, stabilizing ß-catenin, a transcription co-activator for OPN expression. Our in-vitro and in-vivo functional analyses show that silencing OPN expression in the background of deletion of the J domain renders the resultant tumor cells less malignant despite the presence of stabilized ß-catenin. Thus, we have uncovered a new mechanism for regulation of GSK3ß activity leading to inhibition of Wnt/ß-catenin signaling.


Subject(s)
Glycogen Synthase Kinase 3/metabolism , HSP40 Heat-Shock Proteins/physiology , Molecular Chaperones/physiology , Nerve Tissue Proteins/physiology , Osteopontin/genetics , Protein Phosphatase 2/metabolism , beta Catenin/metabolism , Animals , Cell Line, Tumor , Down-Regulation , Epithelial-Mesenchymal Transition , Female , Gene Expression Regulation, Neoplastic , Glycogen Synthase Kinase 3 beta , HSC70 Heat-Shock Proteins/metabolism , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/metabolism , Humans , Lymphoid Enhancer-Binding Factor 1/genetics , Lymphoid Enhancer-Binding Factor 1/metabolism , Melanoma/metabolism , Melanoma/secondary , Mice , Mice, Nude , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Neoplasm Transplantation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Okadaic Acid/pharmacology , Oligonucleotide Array Sequence Analysis , Osteopontin/metabolism , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Protein Phosphatase 2/antagonists & inhibitors , Protein Processing, Post-Translational , Protein Structure, Tertiary , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , T Cell Transcription Factor 1/genetics , T Cell Transcription Factor 1/metabolism , Transcription, Genetic , Transcriptome
2.
Oncogene ; 31(28): 3370-80, 2012 Jul 12.
Article in English | MEDLINE | ID: mdl-22056874

ABSTRACT

The Hedgehog (Hh) pathway is well known for its involvement in angiogenesis and vasculogenesis during ontogeny. The ligand, Sonic Hh (SHH), has an important role in vascular formation during development. However, SHH expression is upregulated on tumor cells and can impact the tumor microenvironment. We have investigated the effects of autocrine as well as paracrine Hh signaling on tumor cells as well as on endothelial cells, respectively. Upon constitutive expression of SHH, breast cancer cells showed aggressive behavior and rapid xenograft growth characterized by highly angiogenic tumors that were spontaneously metastatic. In these cells, SHH caused activation of the Hh transcription factor, GLI1, leading to upregulated expression of the potent pro-angiogenic secreted molecule, CYR61 (cysteine-rich angiogenic inducer 61). Silencing of CYR61 from these SHH-expressing Hh activated cells blunted the malignant behavior of the tumor cells and resulted in reduced tumor vasculature and limited hematogenous metastases. Thus, CYR61 is a critical downstream contributor to the Hh influenced pro-angiogenic tumor microenvironment. We also observed concomitant upregulation of SHH and CYR61 transcripts in tumors from patients with advanced breast cancer, further ratifying the clinical relevance of our findings. In summary, we have defined a novel, VEGF-independent, clinically relevant, pro-angiogenic factor, CYR61, that is a transcriptional target of Hh-GLI signaling.


Subject(s)
Breast Neoplasms/blood supply , Breast Neoplasms/pathology , Cysteine-Rich Protein 61/genetics , Hedgehog Proteins/metabolism , Neovascularization, Pathologic/metabolism , Signal Transduction , Up-Regulation , Animals , Autocrine Communication/genetics , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Transformation, Neoplastic , Endothelial Cells/metabolism , Endothelial Cells/pathology , Female , Gene Expression Regulation, Neoplastic , Humans , Mice , Neoplasm Metastasis , Neovascularization, Pathologic/genetics , Paracrine Communication/genetics , Transcription, Genetic
3.
J Mol Biol ; 313(4): 845-59, 2001 Nov 02.
Article in English | MEDLINE | ID: mdl-11697908

ABSTRACT

Rev is a 116 residue basic protein encoded by the genome of human immunodeficiency virus type 1 (HIV-1) that binds to multiple sites in the Rev response element (RRE) of viral mRNA transcripts in nuclei of host cells, leading to transport of incompletely spliced and unspliced viral mRNA to the cytoplasm of host cells in the latter phases of the HIV-1 life cycle. Rev is absolutely required for viral replication. Because Rev aggregates and fibrillizes in solution at concentrations required for crystal growth or liquid state NMR measurements, high-resolution structural characterization of full-length Rev has not been possible. Previously, circular dichroism studies have shown that approximately 50 % of the Rev sequence adopts helical secondary structure, predicted to correspond to a helix-loop-helix structural motif in the N-terminal half of the protein. We describe the application of solid-state NMR techniques to Rev fibrils as a means of obtaining site-specific, atomic-level structural constraints without requiring a high degree of solubility or crystallinity. Solid-state NMR measurements, using the double-quantum chemical shift anisotropy and constant-time double-quantum-filtered dipolar recoupling techniques, provide constraints on the phi and psi backbone dihedral angles at sites in which consecutive backbone carbonyl groups are labeled with (13)C. Quantitative analysis of the solid-state NMR data, by comparison with numerical simulations, indicates helical phi and psi angles at residues Leu13 and Val16 in the predicted helix 1 segment, and at residues Arg39, Arg 42, Arg43, and Arg44 in the predicted helix 2 segment. These data represent the first site-specific structural constraints from NMR spectroscopy on full-length Rev, and support the helix-loop-helix structural model for its N-terminal half.


Subject(s)
Gene Products, rev/chemistry , Gene Products, rev/metabolism , HIV-1/chemistry , Helix-Loop-Helix Motifs , Nuclear Magnetic Resonance, Biomolecular , Amino Acid Sequence , Amyloid/chemistry , Anisotropy , Circular Dichroism , Gene Products, rev/ultrastructure , Microscopy, Electron , Molecular Sequence Data , Protein Binding , Protein Structure, Quaternary , Protein Structure, Secondary , Solubility , rev Gene Products, Human Immunodeficiency Virus
4.
Chem Res Toxicol ; 14(9): 1330-8, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11559050

ABSTRACT

The effect of adduct stereochemistry on the susceptibility to hydrolysis by snake venom (VPD) and bovine spleen (SPD) phosphodiesterases was investigated with short deoxyoligonucleotides containing defined adducts derived from alkylation of the exocyclic 6-amino group of dA by polycyclic aromatic hydrocarbon diol epoxides (DEs). In accordance with several earlier reports, we have found that adducts with R configuration at the site of attachment of dA to the DE moiety derived from either benzo[a]pyrene (BaP) or benzo[c]phenanthrene (BcPh) are generally more resistant to hydrolysis by VPD than are their (S)-diastereomers. The reaction with VPD initially yields a fragment containing the adducted dA residue at its 3'-end, which slowly hydrolyzes to a dimer (pXpA*) with an intact 5'-phosphodiester bond to the adducted dA. With several of the adducts studied, this dimer underwent cleavage to release eventually the monomeric adduct p(dA*). Adducts derived from cis opening of the epoxide ring of both BaP and BcPh DEs were considerably more resistant to VPD than the corresponding trans-opened adducts. Although several previous investigations had suggested that oligonucleotides containing adducts which have S configuration at the site of attachment of the hydrocarbon to adenine are more resistant to cleavage by SPD than are their (R)-diastereomers, the present results with a more extensive set of oligonucleotides indicate that SPD, in contrast to VPD, exhibits little discrimination between adducts with R and S configuration at the site of attachment to the base. Notably, for both enzymes, the most resistant internucleotide linkage (the bond 3'-sugar to phosphate for VPD and 5'-sugar to phosphate for SPD) is between the modified base and the base immediately 5' to it, regardless of the configuration of the adduct.


Subject(s)
DNA Adducts , Epoxy Compounds/chemistry , Exonucleases/metabolism , Phosphoric Diester Hydrolases/metabolism , Polycyclic Aromatic Hydrocarbons/chemistry , Animals , Cattle , Chromatography , Deoxyadenosines/chemistry , Humans , Hydrolysis , Molecular Conformation , Oligonucleotides/chemistry , Phosphodiesterase I
5.
Proc Natl Acad Sci U S A ; 98(19): 10578-83, 2001 Sep 11.
Article in English | MEDLINE | ID: mdl-11526206

ABSTRACT

S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme in the biosynthesis of spermidine and spermine, is first synthesized as a proenzyme, which is cleaved posttranslationally to form alpha and beta subunits. The alpha subunit contains a covalently bound pyruvoyl group derived from serine that is essential for activity. With the use of an Escherichia coli overexpression system, we have purified AdoMetDCs encoded by the E. coli, Saccharomyces cerevisiae, and Salmonella typhimurium genes. Unexpectedly we found by mass spectrometry that these enzymes had been modified posttranslationally in vivo by a mechanism-based "suicide" inactivation. A large percentage of the alpha subunit of each enzyme had been modified in vivo to give peaks with masses m/z = 57 +/- 1 and m/z = 75 +/- 1 daltons higher than the parent peak. AdoMetDC activity decreased markedly during overexpression concurrently with the increase of the additional peaks for the alpha subunit. Sequencing of a tryptic fragment by tandem mass spectrometry showed that Cys-140 was modified with a +75 +/- 1 adduct, which is probably derived from the reaction product. Comparable modification of the alpha subunit was also observed in in vitro experiments after incubation with the substrate or with the reaction product, which is consistent with the in vitro alkylation of E. coli AdoMetDC reported by Diaz and Anton [Diaz, E. & Anton, D. L. (1991) Biochemistry 30, 4078-4081].


Subject(s)
Adenosylmethionine Decarboxylase/metabolism , Protein Processing, Post-Translational , Adenosylmethionine Decarboxylase/chemistry , Adenosylmethionine Decarboxylase/genetics , Adenosylmethionine Decarboxylase/isolation & purification , Binding Sites , Chromatography, Liquid/methods , Escherichia coli/enzymology , Escherichia coli/genetics , Isopropyl Thiogalactoside , Mass Spectrometry/methods , S-Adenosylmethionine/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Salmonella typhimurium/enzymology , Salmonella typhimurium/genetics , Substrate Specificity
6.
Phytochemistry ; 58(1): 153-7, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11524125

ABSTRACT

The organic soluble extract from the leaves of Glycyrrhiza lepidota showed moderate activity in the US National Cancer Institute in vitro anti-HIV-1 bioassay. Chromatographic separation of this extract resulted in the identification of a new diprenylated bibenzyl as the compound responsible for the observed anti-viral activity. Extensive spectroscopic experiments provide the complete 1H NMR and 13C NMR spectral assignments to support the proposed structure. Known compounds glepidotin B and glepidotin A were also isolated from the extract and shown to be inactive in the anti-viral assay.


Subject(s)
Anti-HIV Agents/chemistry , Bibenzyls/chemistry , Glycyrrhiza/chemistry , HIV/drug effects , Plants, Medicinal/chemistry , Stilbenes , Anti-HIV Agents/isolation & purification , Anti-HIV Agents/pharmacology , Bibenzyls/isolation & purification , Bibenzyls/pharmacology , Carbon Isotopes , Cell Line , Humans , Magnetic Resonance Spectroscopy , Plant Leaves/chemistry , Plant Stems/chemistry
7.
J Nat Prod ; 64(2): 249-50, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11430013

ABSTRACT

A 37 amino acid cyclic polypeptide has been isolated from the organic extract of the tropical tree Palicourea condensata. Palicourein (1) is the largest of a growing family of plant peptides that contain a cyclized amino acid backbone cross-linked via three internal disulfide bridges. Palicourein inhibits the in vitro cytopathic effects of HIV-1RF infection of CEM-SS cells with an EC50 value of 0.1 microM and an IC50 value of 1.5 microM.


Subject(s)
Anti-HIV Agents/isolation & purification , Cyclotides , Peptides, Cyclic/isolation & purification , Rubiaceae/chemistry , Amino Acid Sequence , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Cells, Cultured , Chromatography, High Pressure Liquid , Cytopathogenic Effect, Viral/drug effects , Disulfides/analysis , Humans , Molecular Sequence Data , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Protein Conformation , Trees/chemistry
8.
J Nat Prod ; 64(5): 572-7, 2001 May.
Article in English | MEDLINE | ID: mdl-11374946

ABSTRACT

Several new compounds were isolated from the organic extract of the cyanobacterium Microcoleus lyngbyaceus, and their structures were determined by spectroscopic means. Polychlorinated acetamidoalkynes and alkanes were the major metabolites. 6-Acetamido-1,1,1-trichloroundecane, a positional isomer of the naturally occurring 5-acetamido-1,1,1-trichloroundecane, was synthesized in six steps from delta-decanolactone.


Subject(s)
Acetamides/isolation & purification , Acetamides/pharmacology , Anti-HIV Agents/isolation & purification , Cyanobacteria/chemistry , Anti-HIV Agents/pharmacology , Magnetic Resonance Spectroscopy , Solvents , Spectrometry, Mass, Fast Atom Bombardment , Spectrophotometry, Infrared
9.
J Nat Prod ; 64(1): 117-21, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11170684

ABSTRACT

Microspinosamide (1), a new cyclic depsipeptide incorporating 13 amino acid residues, was isolated from extracts of an Indonesian collection of the marine sponge Sidonops microspinosa. Its structure was elucidated by extensive NMR and mass spectral analyses, and by chemical degradation and derivatization studies. The tridecapeptide 1 incorporates numerous uncommon amino acids, and it is the first naturally occurring peptide to contain a beta-hydroxy-p-bromophenylalanine residue. Microspinosamide (1) inhibited the cytopathic effect of HIV-1 infection in an XTT-based in vitro assay with an EC(50) value of approximately 0.2 microg/mL.


Subject(s)
Anti-HIV Agents/isolation & purification , Depsipeptides , Oligopeptides/isolation & purification , Peptides, Cyclic/isolation & purification , Porifera/chemistry , Animals , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Chromatography, Liquid , Cytopathogenic Effect, Viral/drug effects , HIV-1/drug effects , Magnetic Resonance Spectroscopy , Oligopeptides/chemistry , Oligopeptides/pharmacology , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Spectrometry, Mass, Fast Atom Bombardment
10.
Bioorg Chem ; 29(6): 387-97, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11846436

ABSTRACT

The membrane associated iron chelator of Pseudomonas aeruginosa has been extracted from membranes of iron-rich cells with ethanol and purified by reverse phase HPLC. Using 13C NMR and FAB mass spectroscopy, the structure of the chelator has been determined to be 4-hydroxy-2-nonylquinoline. This compound has been previously isolated and named pseudan IX, a name which we use here. We synthesized pseudan IX and show that the spectral properties of the synthesized compound and the purified compound are nearly identical. Also purified from the ethanol extract of membranes is 4-hydroxy-2-heptylquinoline, i.e., pseudan VII. Bacterially purified pseudan IX binds iron as indicated by the incorporation of radiolabeled iron into the chelator and by the formation of pink micelles in a concentrated ethanol extract. The formation of pink micelles upon addition of iron to the synthesized compound indicates that it binds iron.


Subject(s)
Iron Chelating Agents/isolation & purification , Pseudomonas aeruginosa/chemistry , Quinolines/isolation & purification , Quinolones/isolation & purification , Chromatography, High Pressure Liquid , Gas Chromatography-Mass Spectrometry , Iron Chelating Agents/chemical synthesis , Magnetic Resonance Spectroscopy , Micelles , Quinolines/chemical synthesis , Quinolones/chemical synthesis
11.
J Nat Prod ; 63(10): 1411-3, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11076564

ABSTRACT

Two new 3,6-epidioxy-7,10-tetrahydrofurano C(26) unsaturated fatty acids, stolonic acids A (1) and B (2), were isolated from a previously undescribed ascidian species, Stolonica sp. collected off the Maldive Islands in the Indian Ocean. The structures and relative stereochemistry of 1 and 2 were determined using conventional spectroscopic methods. Both compounds exhibited antiproliferative activity against selected human melanoma and ovarian tumor cell lines, with IC(50) values of approximately 0.05-0.1 microg/mL.


Subject(s)
Antineoplastic Agents/isolation & purification , Fatty Acids, Unsaturated/isolation & purification , Furans/isolation & purification , Peroxides/isolation & purification , Urochordata/chemistry , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Drug Screening Assays, Antitumor , Fatty Acids, Unsaturated/chemistry , Fatty Acids, Unsaturated/pharmacology , Furans/chemistry , Furans/pharmacology , Humans , Indian Ocean , Magnetic Resonance Spectroscopy , Molecular Structure , Peroxides/chemistry , Peroxides/pharmacology , Tumor Cells, Cultured
12.
Protein Expr Purif ; 20(2): 186-95, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11049743

ABSTRACT

Interleukin-13 (IL-13) is a pleiotropic cytokine that elicits both proinflammatory and anti-inflammatory immune responses. Recent studies underscore its role in several diseases, including asthma and cancer. Solution studies of IL-13 and its soluble receptors may facilitate the design of antagonists/agonists which would require milligram quantities of specifically labeled protein. A synthetic gene encoding human IL-13 (hIL-13) was inserted into the pMAL-c2 vector with a cleavage site for the tobacco etch virus (TEV) protease. Coexpression of the fusion protein and TEV protease led to in vivo cleavage, resulting in high levels of hIL-13 production. hIL-13, localized to inclusion bodies, was purified and refolded to yield approximately 2 mg per liter of bacteria grown in minimal media. Subsequent biochemical and biophysical analysis of both the unlabeled and (15)N-labeled protein revealed a bioactive helical monomer. In addition, the two disulfide bonds were unambiguously demonstrated to be Cys29-Cys57 and Cys45-Cys71 by a combined proteolytic digestion and mass spectrometric analysis.


Subject(s)
ATP-Binding Cassette Transporters , Escherichia coli Proteins , Interleukin-3/isolation & purification , Interleukin-3/metabolism , Monosaccharide Transport Proteins , Protein Folding , Protein Processing, Post-Translational , Protein Renaturation , Carrier Proteins/genetics , Carrier Proteins/metabolism , Circular Dichroism , Disulfides/metabolism , Electrophoresis, Polyacrylamide Gel , Endopeptidases/metabolism , Escherichia coli , Humans , Interleukin-3/chemistry , Interleukin-3/genetics , Magnetic Resonance Spectroscopy , Maltose-Binding Proteins , Mass Spectrometry , Protein Denaturation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism
13.
Biochem Biophys Res Commun ; 275(3): 962-7, 2000 Sep 07.
Article in English | MEDLINE | ID: mdl-10973828

ABSTRACT

Nitric oxide (NO) has been reported to modulate the oxygen affinity of blood from sickle cell patients (SS), but not that of normal adult blood (AA), with little or no heme oxidation. However, we had found that the NO donor compounds 2-(N, N-diethylamino)-diazenolate-2-oxide (DEANO) and S-nitrosocysteine (CysNO) caused increased oxygen affinity of red cells from both AA and SS individuals and also caused significant methemoglobin (metHb) formation. Rapid kinetic experiments in which HbA(0), AA, or SS erythrocytes were mixed with CysNO or DEANO showed biphasic time courses indicative of initial heme oxidation followed by reductive heme nitrosylation, respectively. Hemolysates treated with CysNO showed by electrospray mass spectrometry a peak corresponding to a 29 mass unit increase (consistent with NO binding) of both the beta(A) and beta(S) chains but not of the alpha chains. Therapeutic use of NO in sickle cell disease may ultimately require further optimization of these competing reactions, i.e., heme reactivity (nitrosylation or oxidation) versus direct S-nitrosation of hemoglobin on the beta-globin.


Subject(s)
Anemia, Sickle Cell/metabolism , Erythrocytes/metabolism , Globins/metabolism , Heme/metabolism , Nitric Oxide/metabolism , S-Nitrosothiols , Cysteine/analogs & derivatives , Cysteine/metabolism , Diethylamines/metabolism , Erythrocytes/pathology , Hemoglobin A/metabolism , Hemoglobin, Sickle/metabolism , Humans , Kinetics , Mass Spectrometry , Methemoglobin/metabolism , Nitrogen Oxides , Nitroso Compounds/metabolism , Oxidation-Reduction , Oxygen/metabolism , Spectrophotometry , Thermodynamics
14.
J Nat Prod ; 63(8): 1170-4, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10978222

ABSTRACT

Aqueous extracts from the African plant Myrianthus holstii potently inhibited the infection of the T-lymphoblastoid cell line, CEM-SS, by human immunodeficiency virus-1(RF) (HIV-1(RF)). The active constituent, M. holstii lectin (MHL), was purified by LH-20 column chromatography and reversed phase HPLC. MHL, a 9284-Da cysteine-rich protein, was characterized by amino acid analysis, N-terminal sequencing, ESIMS, and matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. Pure MHL had anti-HIV activity, with an EC(50) value of 150 nM. Delaying the addition of MHL for up to 8 h after initial exposure of CEM-SS cells to virus did not result in loss of the antiviral activity; however, if addition of the compound was delayed for 16 h or more, there was a marked decrease in the antiviral activity. MHL bound to a virus-free, soluble form of the viral envelope protein gp120 but did not inhibit the subsequent binding to a cell-free, soluble form of the cellular receptor CD4.


Subject(s)
HIV-1/drug effects , Lectins/isolation & purification , Plant Roots/chemistry , Plants/chemistry , Amino Acid Sequence , CD4 Antigens/chemistry , Cell Aggregation/drug effects , Cells, Cultured , Chitinases/analysis , Chromatography, Gel , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Gas Chromatography-Mass Spectrometry , HIV Envelope Protein gp120/chemistry , HIV Infections/drug therapy , Hemagglutination Tests , Humans , Lectins/chemistry , Lectins/pharmacology , Molecular Sequence Data , Plant Lectins , Sequence Alignment , Sequence Analysis, Protein , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tanzania
15.
Proc Natl Acad Sci U S A ; 97(18): 9943-8, 2000 Aug 29.
Article in English | MEDLINE | ID: mdl-10954746

ABSTRACT

To quantify the reactions of nitric oxide (NO) with hemoglobin under physiological conditions and to test models of NO transport on hemoglobin, we have developed an assay to measure NO-hemoglobin reaction products in normal volunteers, under basal conditions and during NO inhalation. NO inhalation markedly raised total nitrosylated hemoglobin levels, with a significant arterial-venous gradient, supporting a role for hemoglobin in the transport and delivery of NO. The predominant species accounting for this arterial-venous gradient is nitrosyl(heme)hemoglobin. NO breathing increases S-nitrosation of hemoglobin beta-chain cysteine 93, however only to a fraction of the level of nitrosyl(heme)hemoglobin and without a detectable arterial-venous gradient. A strong correlation between methemoglobin and plasma nitrate formation was observed, suggesting that NO metabolism is a primary physiological cause of hemoglobin oxidation. Our results demonstrate that NO-heme reaction pathways predominate in vivo, NO binding to heme groups is a rapidly reversible process, and S-nitrosohemoglobin formation is probably not a primary transport mechanism for NO but may facilitate NO release from heme.


Subject(s)
Cysteine , Heme/chemistry , Hemoglobins/chemistry , Hemoglobins/metabolism , Nitric Oxide/blood , Nitroso Compounds/blood , Administration, Inhalation , Heme/metabolism , Humans , Kinetics , Luminescent Measurements , Nitrates/blood , Nitric Oxide/administration & dosage , Nitric Oxide/pharmacokinetics , Nitrites/blood , Ozone , Potassium Cyanide/pharmacokinetics , Reproducibility of Results , Sensitivity and Specificity
16.
Biochemistry ; 39(27): 8007-16, 2000 Jul 11.
Article in English | MEDLINE | ID: mdl-10891082

ABSTRACT

The bifunctional enzyme peptidylglycine-alpha-amidating monooxygenase mediates the conversion of C-terminal glycine-extended peptides to their active alpha-amidated products. Peptidylglycine-alpha-hydroxylating monooxygenase (PHM, EC 1.14.17. 3) catalyzes the first reaction in this two-step process. The olefinic compound 4-phenyl-3-butenoic acid (PBA) is the most potent irreversible, mechanism-based PHM inactivator known. While the details of the inhibitory action of PBA on PHM remain undefined, covalent modification of the protein has been proposed as the underlying mechanism. We report here that, in the process of inactivating PHM, PBA itself serves as a substrate without covalently labeling the enzyme. Approximately 100 molecules of PBA are metabolized per molecule of PHM inactivated, under saturating conditions. The metabolism of PBA by PHM generates two hydroxylated products, 2-hydroxy-4-phenyl-3-butenoic acid and its allylic isomer, 4-hydroxy-4-phenyl-2-butenoic acid. While one enantiomer for each product is significantly favored in the reaction, both are produced. From these observations, we conclude that hydroxylated PBA products are formed by a delocalized free radical mechanism and that the lack of absolute stereospecificity indicates significant freedom of movement within the catalytic site. The ability of PHM to metabolize PBA suggests that the physiological functions of PHM may include the hydroxylation of substrates other than those containing terminal glycines.


Subject(s)
Fatty Acids, Monounsaturated/metabolism , Mixed Function Oxygenases/metabolism , Multienzyme Complexes , Catalytic Domain , Hydroxylation , Mass Spectrometry , Mixed Function Oxygenases/antagonists & inhibitors , Recombinant Proteins/metabolism , Substrate Specificity
17.
J Org Chem ; 65(1): 124-8, 2000 Jan 14.
Article in English | MEDLINE | ID: mdl-10813905

ABSTRACT

Four novel anti-HIV macrocyclic peptides containing 28-31 amino acid residues, named cycloviolins A-D, have been isolated from the hitherto unstudied tropical plant Leonia cymosa. Their primary structure, including amino acid composition and sequence, was determined by a combination of MALDI-TOF and FAB MS and by enzymatic digestion of reduced derivatives, followed by Edman degradation and mass analyses. All of the cycloviolins contain six cysteines, which are present as three intramolecular disulfide bridges. Intriguingly, cycloviolins A-D showed high degrees of sequence homology to the known cyclopsychotride A and circulins A and B from the Rubiaceae family but much less homology to the varv peptides from Viola, a member of the same family (Violaceae).


Subject(s)
Anti-HIV Agents/chemistry , Peptides, Cyclic/chemistry , Plants/chemistry , Amino Acid Sequence , Anti-HIV Agents/isolation & purification , Chromatography, High Pressure Liquid , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Peptides, Cyclic/isolation & purification , Sequence Homology, Amino Acid , Spectrometry, Mass, Fast Atom Bombardment , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
18.
J Nat Prod ; 63(3): 352-6, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10757717

ABSTRACT

Structure elucidation of five components of the actinomycin Z complex (Z(1)-Z(5)) isolated from Streptomyces fradiae is described. The components were separated by Si gel column chromatography and TLC/PLC and analyzed by ESIMS, FABMS, LC-MS of derivatized hydrolysates, and 2D NMR techniques. This permitted determination of the complete structures of actinomycins Z(1)-Z(5). In Z(3) and Z(5,) site 1 of the beta-depsipeptide is occupied by the rare 4-chloro-L-threonine, an amino acid not previously found in an actinomycin. The structural variants of the actinomycin Z complex have the molecular architecture typical of other actinomycins but possess greater structural diversity resulting from the presence of several highly unusual amino acids. Actinomycins Z(3) and Z(5,) but not Z(1), were more potent than actinomycin D in cytotoxicity assays against three tumor cell lines.


Subject(s)
Antibiotics, Antineoplastic/chemistry , Dactinomycin/chemistry , Streptomyces/chemistry , Threonine/analogs & derivatives , Antibiotics, Antineoplastic/pharmacology , Dactinomycin/analogs & derivatives , Dactinomycin/pharmacology , Drug Screening Assays, Antitumor , Humans , Molecular Structure , Spectrum Analysis , Threonine/chemistry , Tumor Cells, Cultured
19.
J Biol Chem ; 275(26): 19877-82, 2000 Jun 30.
Article in English | MEDLINE | ID: mdl-10747981

ABSTRACT

In human immunodeficiency virus (HIV)-infected individuals, the level of the HIV envelope protein gp41 in brain tissue is correlated with neurological damage and dementia. In this paper we show by biochemical methods and electron microscopy that the extracellular ectodomain of purified HIV and simian immunodeficiency virus gp41 (e-gp41) forms a mixture of soluble high molecular weight aggregate and native trimer at physiological pH. The e-gp41 aggregate is shown to be largely alpha-helical and relatively stable to denaturants. The high molecular weight form of e-gp41 is variable in size ranging from 7 to 70 trimers, which associate by interactions at the interior of the aggregate involving the loop that connects the N- and C-terminal helices of the e-gp41 core. The trimers are predominantly arranged with their long axes oriented radially, and the width of the high molecular weight aggregate corresponds to the length of two e-gp41 trimers (approximately 200 A). Using both light and electron microscopy combined with immunohistochemistry we show that HIV gp41 accumulates as an extracellular aggregate in the brains of HIV-infected patients diagnosed with dementia. We postulate that the high molecular weight aggregates of e-gp41 are responsible for HIV-associated neurological damage and dementia, consistent with known mechanisms of encephalopathy.


Subject(s)
AIDS Dementia Complex/genetics , Brain Diseases/virology , HIV Envelope Protein gp41/chemistry , HIV Envelope Protein gp41/metabolism , AIDS Dementia Complex/pathology , Brain/pathology , Brain/ultrastructure , Brain/virology , Brain Diseases/genetics , Chromatography, Gel , Endopeptidases/metabolism , Extracellular Space/metabolism , HIV Envelope Protein gp41/ultrastructure , HIV Seropositivity/complications , Humans , Hydrogen-Ion Concentration , Immunohistochemistry , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Membrane Glycoproteins/ultrastructure , Microscopy, Electron , Protein Binding , Protein Structure, Tertiary , Retroviridae Proteins/chemistry , Retroviridae Proteins/metabolism , Retroviridae Proteins/ultrastructure
20.
J Nat Prod ; 63(2): 176-8, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10691702

ABSTRACT

Four new macrocyclic polypeptides were isolated and identified from an extract of the tropical tree Chassalia parvifolia. Circulins C-F are 29-30 amino acid cyclic peptides in which the entire primary amino acid chain is covalently cyclized via peptide bonds. Their structures were deduced from a combination of FABMS analyses, N-terminal Edman degradation, endoproteinase digestion, and amino acid analyses. All the peptides share a high degree of sequence homology and contain six cysteine residues forming three intramolecular disulfide bridges. Circulins C-F inhibited the cytopathic effects of in vitro HIV-1 infection with EC(50) values of 50-275 nM.


Subject(s)
Anti-Bacterial Agents/analysis , Anti-HIV Agents/analysis , Cyclotides , Plants, Medicinal/chemistry , Alkylation , Amino Acid Sequence , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Anti-HIV Agents/isolation & purification , Anti-HIV Agents/pharmacology , Chromatography, High Pressure Liquid , Cysteine/analysis , HIV-1/drug effects , Humans , Microbial Sensitivity Tests , Molecular Sequence Data , Oxidation-Reduction , Plant Stems/chemistry , Spectrometry, Mass, Fast Atom Bombardment , Tasmania
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