| 12754519 |
Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. |
10.1038/nbt827 |
Nat. Biotechnol. |
Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry.
Abstract
- Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes, or sub-proteomes, are isolated and analyzed. Glycosylation is the most common post-translational modification. Here we describe a method for the selective isolation, identification and quantification of peptides that contain N-linked carbohydrates. It is based on the conjugation of glycoproteins to a solid support using hydrazide chemistry, stable isotope labeling of glycopeptides and the specific release of formerly N-linked glycosylated peptides via peptide- N-glycosidase F (PNGase F). The recovered peptides are then identified and quantified by MS/MS. We applied the approach to the analysis of plasma membrane proteins and proteins contained in human blood serum.
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| 12759905 |
Methyl dynamics in crystalline amino acids: MD and NMR |
10.1002/jcc.10263. |
J Comput Chem |
Methyl dynamics in crystalline amino acids: MD and NMR
Abstract
- Correlation times for rotation of deuterated methyls in crystalline leucine, valine, and cyclo-L-alanyl-L-alanine are calculated with molecular dynamics and compared with NMR data. The simulations distinguish between methyls having different steric environments in the crystal, yielding correlation times differing by a factor of up to 30 for methyls within a given crystal. MD and NMR correlation times agree to within a factor of 2. However, averaging over quivalent methyls can yield correlation functions that, although actually multiexponential, are well fit by single exponentials. This may have significance for interpreting NMR data; previous NMR data did not distinguish between the methyls in these crystals. Adiabatic rotational barriers calculated with the X-ray structure differ from effective barriers during simulation by up to +/-1 kcal/mol; the difference indicates that dynamical effects have a significant role in determining rotational correlation times.
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| 12762800 |
Ulongapeptin, a cytotoxic cyclic depsipeptide from a Palauan marine cyanobacterium Lyngbya sp |
10.1021/np030050s. |
J Nat Prod |
Ulongapeptin, a cytotoxic cyclic depsipeptide from a Palauan marine cyanobacterium Lyngbya sp
Abstract
- Ulongapeptin (1), a cyclic depsipeptide, was isolated from a Palauan marine cyanobacterium Lyngbya sp. The gross structure was elucidated through one-dimensional TOCSY experiments and other spectroscopic techniques. The absolute and relative stereochemistry of the beta-amino acid, 3-amino-2-methyl-7-octynoic acid (AMO), in 1 was determined by synthesis of the saturated alpha-alkyl-beta-amino acid and Marfey's analysis of the acid hydrolysate of tetrahydro-1. Ulongapeptin (1) was cytotoxic against KB cells at an IC(50) value of 0.63 microM.
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| 12771141 |
Structural basis of Synercid (quinupristin-dalfopristin) resistance in Gram-positive bacterial pathogens. |
10.1074/jbc.m303766200 |
J. Biol. Chem. |
Structural basis of Synercid (quinupristin-dalfopristin) resistance in Gram-positive bacterial pathogens.
Abstract
- Synercid, a new semisynthetic streptogramin-derived antibiotic containing dalfopristin and quinupristin, is used in treatment of life-threatening infections caused by glycopeptide-resistant Enterococcus faecium and other bacterial pathogens. However, dissemination of genes encoding virginiamycin acetyltransferases, enzymes that confer resistance to streptogramins, threatens to limit the medical utility of the quinupristin-dalfopristin combination. Here we present structures of virginiamycin acetyltransferase D (VatD) determined at 1.8 A resolution in the absence of ligands, at 2.8 A resolution bound to dalfopristin, and at 3.0 A resolution in the presence of acetyl-coenzyme A. Dalfopristin is bound by VatD in a similar conformation to that described previously for the streptogramin virginiamycin M1. However, specific interactions with the substrate are altered as a consequence of a conformational change in the pyrollidine ring that is propagated to adjacent constituents of the dalfopristin macrocycle. Inactivation of dalfopristin involves acetyl transfer from acetyl-coenzyme A to the sole (O-18) hydroxy group of the antibiotic that lies close to the side chain of the strictly conserved residue, His-82. Replacement of residue 82 by alanine is accompanied by a fall in specific activity of >105-fold, indicating that the imidazole moiety of His-82 is a major determinant of catalytic rate enhancement by VatD. The structure of the VatD-dalfopristin complex can be used to predict positions where further structural modification of the drug might preclude enzyme binding and thereby circumvent Synercid resistance.
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| 12782038 |
Expression of Viola cyclotides by liquid chromatography-mass spectrometry and tandem mass spectrometry sequencing of intercysteine loops after introduction of charges and cleavage sites by aminoethylation |
10.1016/s0003-2697(03)00114-3. |
Anal Biochem |
Expression of Viola cyclotides by liquid chromatography-mass spectrometry and tandem mass spectrometry sequencing of intercysteine loops after introduction of charges and cleavage sites by aminoethylation
Abstract
- The expression of cyclotides-macrocyclic plant peptides-was profiled in six violets, Viola cotyledon, V. biflora, V. arvensis, V. tricolor, V. riviniana, and V. odorata, by LC-MS. All were found to express notably complex mixtures, with single species containing >50 cyclotides. To facilitate their sequencing by MS-MS, an analytical strategy is presented involving aminoethylation of cysteines. This overcomes a number of problems intimately associated with the cyclotide core structure-that is, their joined N and C termini, disulfide knot, and low or clustered content of positively charged amino acids and enzymatic cleavage sites. As a result, charges as well as cleavage sites are introduced at the most conserved part of their sequence, the cysteines. Combined with tryptic digestion, all intercysteine loops are then of suitable size and charge for MS-MS sequencing. The utility of this strategy is shown by the sequencing of two novel cyclotides isolated from V. cotyledon; vico A (cyclo-(AESCVYIPCFTGIAGCSCKNKVCYYNGSIPC)) and vico B (cyclo-(AESCVYIPCITGIAGCSCKNKVCYYNGSIPC)); their complete sequence could be determined by nanospray MS-MS. The strategy for converting conserved cysteines to enzymatic cleavage sites might also benefit the study of other peptides and proteins displaying similar structural problems for MS analysis.
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| 12790623 |
Sulfinyl moiety as an internal nucleophile. 1. Efficient stereoselective synthesis of fragment a of cryptophycin 3 |
10.1021/jo026802p. |
J Org Chem |
Sulfinyl moiety as an internal nucleophile. 1. Efficient stereoselective synthesis of fragment a of cryptophycin 3
Abstract
- A novel, efficient, and stereoselective synthesis of fragment A of cryptophycin 3 is disclosed. The key step involves the regio- and stereoselective transformation of an unsaturated ester to a bromohydrin via anchimeric assistance by the sulfinyl group.
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| 12794352 |
cis,cis-Ceratospongamide N,N-dimethylacetamide hemisolvate in the presence of twinning |
10.1107/s010827010300369x. |
Acta Crystallogr C |
cis,cis-Ceratospongamide N,N-dimethylacetamide hemisolvate in the presence of twinning
Abstract
- Ceratospongamide (CS) is a potent inhibitor of secreted phospholipase A(2), and cis,cis and trans,trans isomers, related with respect to the two proline amide bonds, are known. Crystals of cis,cis-CS were grown from N,N-dimethylacetamide solution, giving the title compound, the cyclic ester of isoleucyloxazolinylphenylalanylprolylthiazolylphenylalanylproline [cyclo(-Ile-Oxz-Phe-Pro-Thz-Phe-Pro-)] N,N-dimethylacetamide hemisolvate, C(41)H(49)N(7)O(6)S.0.5C(4)H(9)NO. The structure is the third example of cis,cis-CS to be investigated and comprises twinned crystals, in which the a and b axes are interchanged. The ratio of co-existing twin crystals is approximately 50%. The peptide has a 'saddle-like' structure and is very similar to previously reported structures of cis,cis-CS, which implies that the structure of cis,cis-CS is very stable in spite of differences in crystallization conditions.
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| 12808242 |
Bioactive constituents from Chinese natural medicines. XI. inhibitors on NO production and degranulation in RBL-2H3 from Rubia yunnanensis: structures of rubianosides II, III, and IV, rubianol-g, and rubianthraqui |
10.1248/cpb.51.654. |
Chem Pharm Bull (Tokyo) |
Bioactive constituents from Chinese natural medicines. XI. inhibitors on NO production and degranulation in RBL-2H3 from Rubia yunnanensis: structures of rubianosides II, III, and IV, rubianol-g, and rubianthraqui
Abstract
- Three new arborinane-type triterpene glycosides, rubianosides II, III, and IV, a new arborinane-type triterpene, rubianol-g, and a new anthraqui, rubianthraqui, were isolated from a Chinese natural medicine, the roots of Rubia yunnanensis. The structures of the new constituents including their absolute configurations were determined on the basis of chemical and physicochemical evidence. The inhibitory effects of the isolated constituents on nitric oxide production in lipopolysaccharide-activated macrophages were examined. Among them, a cyclic peptide constituent, RA-XII and its aglycon, RA-V (deoxybouvadin), potently inhibited overproduction of nitric oxide and induction of inducible nitric oxide synthase. In addition, an anthraqui constituent, 2-methyl-1,3,6-trihydroxy-9,10-anthraqui, was found to show inhibitory effects on the release of beta-hexosaminidase in RBL-2H3 cells.
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| 12821455 |
Synergistic antimicrobial activity of metabolites produced by a nonobligate bacterial predator |
10.1128/AAC.47.7.2113-2117.2003. |
Antimicrob Agents Chemother |
Synergistic antimicrobial activity of metabolites produced by a nonobligate bacterial predator
Abstract
- A naturally occurring, gram-negative, nonobligate predator bacterial strain 679-2, exhibits broad-spectrum antimicrobial activity that is due, in part, to the production of three extracellular compounds. Antimicrobial-activity-directed fractionation of a culture of strain 679-2 against a panel of microorganisms has led to the isolation of three compounds: pyrrolnitrin, maculosin, and a new compound, which we have named banegasine. Although pyrrolnitrin is well known in the literature, it has not been found in cells with the herbicide maculosin. Further, this is the first report of production of maculosin by a prokaryote. Both maculosin and banegasine, which displayed no antimicrobial activities alone, were found to potentiate the antimicrobial activity of pyrrolnitrin. Based on 16S rRNA sequence, cellular fatty acid composition, and biochemical and cultural characteristics, strain 679-2 appears to represent a new genus and species of eubacteria, Aristabacter necator. The potent, broad-spectrum antimicrobial activity of predator strain 679-2 may be due to synergism between metabolites.
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| 12821780 |
Mdm2-dependent ubiquitination and degradation of the insulin-like growth factor 1 receptor |
10.1073/pnas.1431613100. |
Proc Natl Acad Sci U S A |
Mdm2-dependent ubiquitination and degradation of the insulin-like growth factor 1 receptor
Abstract
- Recently, p53 was demonstrated to affect the expression of the insulin-like growth factor 1 receptor (IGF-1R), a receptor tyrosine kinase that plays a crucial role in growth and survival of cancer cells. However, the underlying mechanisms for interaction between p53 and IGF-1R are still not fully understood. One of the challenging questions remaining to be answered is why the wild-type p53, which per se represses the transcription of the IGF-1R gene, in overexpressed form is necessary for a high IGF-1R expression. In this study, we show that inhibition of p53 causes ubiquitination and down-regulation, through increased degradation, of the IGF-1R in human malignant melanoma cells. This effect, which was independent of the p53 status (i.e., wild type or mutated), was prevented if Mdm2 was coinhibited. Similar results were obtained in UV-irradiated human melanocytes (harboring wild-type p53), in which level of the IGF-1R increased after up-regulation of p53. Interestingly, the basal ubiquitination of the IGF-1R in untreated cells also depended on Mdm2. We could prove that Mdm2 physically associates with IGF-1R and that Mdm2 causes IGF-1R ubiquitination in an in vitro assay. Taken together our data provide evidence that Mdm2 serves as a ligase in ubiquitination of the IGF-1R and thereby causes its degradation by the proteasome system. Consequently, by sequestering Mdm2 in the cell nuclei, the level of p53 may indirectly influence the expression of IGF-1R. This role of Mdm2 and p53 represents an unexpected mechanism for the regulation of IGF-1R and cell growth.
|
| 12824025 |
Synthesis and antimicrobial activity of novel globomycin analogues |
10.1016/s0960-894x(03)00432-3. |
Bioorg Med Chem Lett |
Synthesis and antimicrobial activity of novel globomycin analogues
Abstract
- Globomycin, a signal peptidase II inhibitor, and its derivatives show potent antibacterial activity against Gram-negative bacteria. The synthesis and antimicrobial activity of novel globomycin analogues are reported. One of the analogues showed a more potent activity against Gram-negative bacteria than globomycin and also exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA).
|
| 12828459 |
The guineamides, novel cyclic depsipeptides from a Papua New Guinea collection of the marine cyanobacterium Lyngbya majuscula |
10.1021/np020492o. |
J Nat Prod |
The guineamides, novel cyclic depsipeptides from a Papua New Guinea collection of the marine cyanobacterium Lyngbya majuscula
Abstract
- The guineamides (1-6) are novel cyclic depsipeptides isolated and characterized from a Papua New Guinea collection of the marine cyanobacterium Lyngbya majuscula. The planar structures of these new natural products were established using an extensive array of 1D and 2D NMR experiments, including HSQC, TOCSY, and HMBC. Absolute stereochemistry was determined using a combination of chemical manipulations as well as Marfey's method. These metabolites all contain beta-amino or beta-hydroxy carboxylic acid residues, an increasingly common feature in marine cyanobacterial metabolites. The identification of 2,2-dimethyl-3-hydroxyhexanoic acid (Dmhha) in guineamides E (5) and F (6) represents the first report of such a residue in a natural product. In addition, characterization of the unique beta-amino acid 2-methyl-3-aminopentanoic acid (Mapa) in guineamide A (1), which has also been reported as a component of several marine molluscan metabolites, especially from those of Dolabella auricularia, further supports the diet-derived nature of such compounds as isolated from marine invertebrates. Guineamides B (2) and C (3) possess moderate cytotoxicity to a mouse neuroblastoma cell line with IC(50) values of 15 and 16 microM, respectively.
|
| 12837748 |
Acetylated SP3 is a transcriptional activator. |
10.1074/jbc.m305961200 |
J. Biol. Chem. |
Acetylated SP3 is a transcriptional activator.
Abstract
- Sp3 transcription factor can either activate or repress target gene expression. However, the molecular event that controls this dual function is unclear. We previously reported (Ammanamanchi, S., and Brattain, M. G. (2001) J. Biol. Chem. 276, 3348-3352) that unmodified Sp3 acts as a transcriptional repressor of transforming growth factor-beta receptors in MCF-7L breast cancer cells. We now report that histone deacetylase inhibitor trichostatin A (TSA) induces acetylation of Sp3, which acts as a transcriptional activator of transforming growth factor-beta receptor type II (RII) in MCF-7L cells. Mutation analysis indicated the TSA response is mediated through a GC box located on the RII promoter, which was previously identified as an Sp1/Sp3-binding site that was critical for RII promoter activity. Ectopic Sp3 expression in Sp3-deficient MCF-7E breast cancer cells repressed RII promoter activity in the absence of TSA. However, in the TSA-treated MCF-7E cells ectopic Sp3 activated RII promoter. Histone acetyltransferase p300 was shown to acetylate Sp3. Sp3-mediated RII promoter activity was stimulated by wild type p300 but not the histone acetyltransferase domain-deleted mutant p300 in MCF-7L cells, suggesting the positive effect of p300 acetylase activity on Sp3. Consequently, the
Results presented in this manuscript demonstrate that acetylation acts as a switch that controls the repressor and activator role of Sp3.
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| 12842879 |
Solution structure of the recombinant penaeidin-3, a shrimp antimicrobial peptide |
10.1074/jbc.M305450200. |
J Biol Chem |
Solution structure of the recombinant penaeidin-3, a shrimp antimicrobial peptide
Abstract
- Penaeidins are a family of antimicrobial peptides of 47-63 residues isolated from several species of shrimp. These peptides display a proline-rich domain (N-terminal part) and a cysteine-rich domain (C-terminal part) stabilized by three conserved disulfide bonds whose arrangement has not yet been characterized. The recombinant penaeidin-3a of Litopenaeus vannamei (63 residues) and its [T8A]-Pen-3a analogue were produced in Saccharomyces cerevisiae and showed similar antimicrobial activity. The solution structure of the [T8A]-Pen-3a analogue was determined by using two-dimensional 1H NMR and simulated annealing calculations. The proline-rich domain, spanning residues 1-28 was found to be unconstrained. In contrast, the cysteine-rich domain, spanning residues 29-58, displays a well defined structure, which consists of an amphipathic helix (41-50) linked to the upstream and the downstream coils by two disulfide bonds (Cys32-Cys47 and Cys48-Cys55). These two coils are in turn linked together by the third disulfide bond (Cys36-Cys54). Such a disulfide bond packing, which is in agreement with the analysis of trypsin digests by ESI-MS, contributes to the highly hydrophobic core. Side chains of Arg45 and Arg50, which belong to the helix, and side chains of Arg37 and Arg53, which belong to the upstream and the downstream coils, are located in two opposite parts of this globular and compact structure. The environment of these positively charged residues, either by hydrophobic clusters at the surface of the cysteine-rich domain or by sequential hydrophobic residues in the unconstrained proline-rich domain, gives rise to the amphipathic character required for antimicrobial peptides. We hypothesize that the antimicrobial activity of penaeidins can be explained by a cooperative effect between the proline-rich and cysteine-rich features simultaneously present in their sequences.
|
| 12846570 |
Structure of Petunia hybrida defensin 1, a novel plant defensin with five disulfide bonds |
10.1021/bi034379o. |
Biochemistry |
Structure of Petunia hybrida defensin 1, a novel plant defensin with five disulfide bonds
Abstract
- The structure of a novel plant defensin isolated from the flowers of Petunia hybrida has been determined by (1)H NMR spectroscopy. P. hybrida defensin 1 (PhD1) is a basic, cysteine-rich, antifungal protein of 47 residues and is the first example of a new subclass of plant defensins with five disulfide bonds whose structure has been determined. PhD1 has the fold of the cysteine-stabilized alphabeta motif, consisting of an alpha-helix and a triple-stranded antiparallel beta-sheet, except that it contains a fifth disulfide bond from the first loop to the alpha-helix. The additional disulfide bond is accommodated in PhD1 without any alteration of its tertiary structure with respect to other plant defensins. Comparison of its structure with those of classic, four-disulfide defensins has allowed us to identify a previously unrecognized hydrogen bond network that is integral to structure stabilization in the family.
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