| 11594819 |
Atom-economical synthesis of the N(10)-C(17) fragment of cyclotheonamides via a novel Passerini reaction-deprotection-acyl migration strategy |
10.1021/ol0165239. |
Org Lett |
Atom-economical synthesis of the N(10)-C(17) fragment of cyclotheonamides via a novel Passerini reaction-deprotection-acyl migration strategy
Abstract
- [reaction: see text]. A novel variant of the atom-economical Passerini reaction between suitably protected argininal, dipeptide isonitrile, and proline components afforded adduct 13. Orthogonal N-deprotection of 13 led, via a smooth O- to N-acyl migration, to 14, which constitutes the N(10)-C(17) fragment of the cyclotheonamide family of serine protease inhibitors. Each reaction in this three-step protocol proceeds in good yield and under very mild conditions.
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| 11598120 |
Multiple activation loop conformations and their regulatory properties in the insulin receptor's kinase domain |
10.1074/jbc.M107236200. |
J Biol Chem |
Multiple activation loop conformations and their regulatory properties in the insulin receptor's kinase domain
Abstract
- Low catalytic efficiency of protein kinases often results from intrasteric inhibition caused by the activation loop blocking the active site. In the insulin receptor's kinase domain, Asp-1161 and Tyr-1162 in the peptide substrate-like sequence of the unphosphorylated activation loop can interact with four invariant residues in the active site: Lys-1085, Asp-1132, Arg-1136, and Gln-1208. Contributions of these six residues to intrasteric inhibition were tested by mutagenesis, and the unphosphorylated kinase domains were characterized. The mutations Q1208S, K1085N, and Y1162F each relieved intrasteric inhibition, increasing catalytic efficiency but without changing the rate-limiting step of the reaction. The mutants R1136Q and D1132N were virtually inactive. Steric accessibility of the active site was ranked by relative changes in iodide quenching of intrinsic fluorescence, and A-loop conformation was ranked by limited tryptic cleavage. Together these ranked the openness of the active site cleft as R1136Q approximately D1132N > or = D1161A > Y1162F approximately K1085N > Q1208S > or = wild-type. These findings demonstrate the importance of specific invariant residues for intrasteric inhibition and show that diverse activation loop conformations can produce similar steady-state kinetic properties. This suggests a broader range of regulatory properties for the activation loop than expected from a simple off-versus-on switch for kinase activation.
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| 11601906 |
Inactivation of frog virus 3 and channel catfish virus by esculentin-2P and ranatuerin-2P, two antimicrobial peptides isolated from frog skin |
10.1006/viro.2001.1080. |
Virology |
Inactivation of frog virus 3 and channel catfish virus by esculentin-2P and ranatuerin-2P, two antimicrobial peptides isolated from frog skin
Abstract
- While it is clear that some amphibian populations have recently experienced precipitous declines, the causes of those die-offs are complex and likely involve multiple variables. One theory suggests that environmental factors may trigger events that result in depressed immune function and increased susceptibility to infectious disease. Here we examine one aspect of innate immunity in amphibians and show that esculentin-2P (E2P) and ranatuerin-2P (R2P), two antimicrobial peptides isolated from Rana pipiens, inactivate frog virus 3, a potentially pathogenic iridovirus infecting anurans, and channel catfish herpesvirus. In contrast to mammalian antimicrobial peptides, E2P and R2P act within minutes, at temperatures as low as 0 degrees C, to inhibit viral infectivity. Moreover, these compounds appear to inactivate the virus directly and do not act by inhibiting replication in infected cells. This is the first report linking amphibian antimicrobial peptides with protection from an amphibian viral pathogen and suggests that these compounds may play a role in maintaining amphibian health.
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| 11602581 |
Histone deacetylase 1 phosphorylation promotes enzymatic activity and complex formation. |
10.1074/jbc.m105590200 |
J. Biol. Chem. |
Histone deacetylase 1 phosphorylation promotes enzymatic activity and complex formation.
Abstract
- Accessibility of the genome to DNA-binding transcription factors is regulated by proteins that control the acetylation of amino-terminal lysine residues on nucleosomal histones. Specifically, histone deacetylase (HDAC) proteins repress transcription by deacetylating histones. To date, the only known regulatory mechanism of HDAC1 function is via interaction with associated proteins. Although the control of HDAC1 function by protein interaction and recruitment is well precedented, we were interested in exploring HDAC1 regulation by post-translational modification. Human HDAC1 protein was analyzed by ion trap mass spectrometry, and two phosphorylated serine residues, Ser(421) and Ser(423), were unambiguously identified. Loss of phosphorylation at Ser(421) and Ser(423) due to mutation to alanine or disruption of the casein kinase 2 consensus sequence directing phosphorylation reduced the enzymatic activity and complex formation of HDAC1. Deletion of the highly charged carboxyl-terminal region of HDAC1 also decreased its deacetylase activity and protein associations, revealing its requirement in maintaining HDAC1 function. Our
Results reinforce the importance of protein associations in modulating HDAC1 function and provide the first step toward characterizing the role of post-translational modifications in regulating HDAC activity in vivo.
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| 11602604 |
RGS16 function is regulated by epidermal growth factor receptor-mediated tyrosine phosphorylation |
10.1074/jbc.M108862200. |
J Biol Chem |
RGS16 function is regulated by epidermal growth factor receptor-mediated tyrosine phosphorylation
Abstract
- Galpha(i)-coupled receptor stimulation results in epidermal growth factor receptor (EGFR) phosphorylation and MAPK activation. Regulators of G protein signaling (RGS proteins) inhibit G protein-dependent signal transduction by accelerating Galpha(i) GTP hydrolysis, shortening the duration of G protein effector stimulation. RGS16 contains two conserved tyrosine residues in the RGS box, Tyr(168) and Tyr(177), which are predicted sites of phosphorylation. RGS16 underwent phosphorylation in response to m2 muscarinic receptor or EGFR stimulation in HEK 293T or COS-7 cells, which required EGFR kinase activity. Mutational analysis suggested that RGS16 was phosphorylated on both tyrosine residues (Tyr(168) Tyr(177)) after EGF stimulation. RGS16 co-immunoprecipitated with EGFR, and the interaction did not require EGFR activation. Purified EGFR phosphorylated only recombinant RGS16 wild-type or Y177F in vitro, implying that EGFR-mediated phosphorylation depended on residue Tyr(168). Phosphorylated RGS16 demonstrated enhanced GTPase accelerating (GAP) activity on Galpha(i). Mutation of Tyr(168) to phenylalanine resulted in a 30% diminution in RGS16 GAP activity but completely eliminated its ability to regulate G(i)-mediated MAPK activation or adenylyl cyclase inhibition in HEK 293T cells. In contrast, mutation of Tyr(177) to phenylalanine had no effect on RGS16 GAP activity but also abolished its regulation of G(i)-mediated signal transduction in these cells. These data suggest that tyrosine phosphorylation regulates RGS16 function and that EGFR may potentially inhibit Galpha(i)-dependent MAPK activation in a feedback loop by enhancing RGS16 activity through tyrosine phosphorylation.
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| 11606751 |
Gambicin: a novel immune responsive antimicrobial peptide from the malaria vector Anopheles gambiae |
10.1073/pnas.221466798. |
Proc Natl Acad Sci U S A |
Gambicin: a novel immune responsive antimicrobial peptide from the malaria vector Anopheles gambiae
Abstract
- A novel mosquito antimicrobial peptide, gambicin, and the corresponding gene were isolated in parallel through differential display-PCR, an expressed sequence tag (EST) project, and characterization of an antimicrobial activity in a mosquito cell line by reverse-phase chromatography. The 616-bp gambicin ORF encodes an 81-residue protein that is processed and secreted as a 61-aa mature peptide containing eight cysteines engaged in four disulfide bridges. Gambicin lacks sequence homology with other known proteins. Like other Anopheles gambiae antimicrobial peptide genes, gambicin is induced by natural or experimental infection in the midgut, fatbody, and hemocyte-like cell lines. Within the midgut, gambicin is predominantly expressed in the anterior part. Both local and systemic gambicin expression is induced during early and late stages of natural malaria infection. In vitro experiments showed that the 6.8-kDa mature peptide can kill both Gram-positive and Gram-negative bacteria, has a morphogenic effect on a filamentous fungus, and is marginally lethal to Plasmodium berghei ookinetes. An oxidized form of gambicin isolated from the cell line medium was more active against bacteria than the nonoxidized form from the same medium.
|
| 11641403 |
Solution conformation of alpha-conotoxin EI, a neuromuscular toxin specific for the alpha 1/delta subunit interface of torpedo nicotinic acetylcholine receptor |
10.1074/jbc.M107798200. |
J Biol Chem |
Solution conformation of alpha-conotoxin EI, a neuromuscular toxin specific for the alpha 1/delta subunit interface of torpedo nicotinic acetylcholine receptor
Abstract
- A high resolution structure of alpha-conotoxin EI has been determined by (1)H NMR spectroscopy and molecular modeling. alpha-Conotoxin EI has the same disulfide framework as alpha 4/7 conotoxins targeting neuronal nicotinic acetylcholine receptors but antagonizes the neuromuscular receptor as do the alpha 3/5 and alpha A conotoxins. The unique binding preference of alpha-conotoxin EI to the alpha(1)/delta subunit interface of Torpedo neuromuscular receptor makes it a valuable structural template for superposition of various alpha-conotoxins possessing distinct receptor subtype specificities. Structural comparison of alpha-conotoxin EI with the gamma-subunit favoring alpha-conotoxin GI suggests that the Torpedo delta-subunit preference of the former originates from its second loop. Superposition of three-dimensional structures of seven alpha-conotoxins reveals that the estimated size of the toxin-binding pocket in nicotinic acetylcholine receptor is approximately 20 A (height) x 20 A (width) x 15 A (thickness).
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| 11667471 |
Waiakeamide, a Cyclic Hexapeptide from the Sponge Ircinia dendroides(1) |
10.1021/jo960771e. |
J Org Chem |
Waiakeamide, a Cyclic Hexapeptide from the Sponge Ircinia dendroides(1)
Abstract
- From the sponge, Ircinia dendroides, collected in Indonesia we isolated a new cyclic hexapeptide, waiakeamide (1). Its structure, consisting of three proline residues, two methionine sulfoxides, and one thiazolylphenylalanine, was elucidated by spectral analysis and chemical degradation. Isolation and structural elucidation of waiakeamide is described.
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| 11667527 |
Kahalalides: Bioactive Peptides from a Marine Mollusk Elysia rufescens and Its Algal Diet Bryopsis sp.(1) |
10.1021/jo960877+. |
J Org Chem |
Kahalalides: Bioactive Peptides from a Marine Mollusk Elysia rufescens and Its Algal Diet Bryopsis sp.(1)
Abstract
- In addition to the previously reported bioactive kahalalide F six new peptides are described. Six of these, including kahalalide F, are cyclic depsipeptides, ranging from a C(31) tripeptide to a C(75) tridecapeptide isolated from a sacoglossan mollusk, Elysiarufescens. The mollusk feeds on a green alga, Bryopsis sp., which has also been shown to elaborate some of these peptides in smaller yields, in addition to an acyclic analog of F, kahalalide G. The bioassay results of antitumor, antiviral, antimalarial, and OI (activity against AIDS opportunistic infections) tests are reported.
|
| 11675394 |
Homodimeric theta-defensins from rhesus macaque leukocytes: isolation, synthesis, antimicrobial activities, and bacterial binding properties of the cyclic peptides |
10.1074/jbc.M109117200. |
J Biol Chem |
Homodimeric theta-defensins from rhesus macaque leukocytes: isolation, synthesis, antimicrobial activities, and bacterial binding properties of the cyclic peptides
Abstract
- Rhesus theta-defensin 1 (RTD-1) is a unique tridisulfide, cyclic antimicrobial peptide formed by the ligation of two 9-residue sequences derived from heterodimeric splicing of similar 76-amino acid, alpha-defensin-related precursors, termed RTD1a and RTD1b (Tang, Y. Q., Yuan, J., Osapay, G., Osapay, K., Tran, D., Miller, C. J., Ouellette, A. J., and Selsted, M. E. (1999) Science 286, 498-502). The structures of RTD-2 and RTD-3 were predicted to exist if homodimeric splicing of the RTD1a and RTD1b occurs in vivo. Western blotting disclosed the presence of putative theta-defensins, distinct from RTD-1, in leukocyte extracts. Two new theta-defensins, RTD-2 and RTD-3, were purified by reverse-phase high performance liquid chromatography and characterized by amino acid analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, and comparison to the synthetic standards. RTD-2 and RTD-3 are the predicted homodimeric splicing products of RTD1b and RTD1a, respectively. The cellular abundances of RTD-1, -2, and -3 were 29:1:2, indicating that there is a preference for the heterodimeric ligation that generates RTD-1. RTD-1, -2, and -3 had similar antimicrobial activities against Staphylococcus aureus, Candida albicans, and Cryptococcus neoformans, whereas the activity of RTD-2 against Escherichia coli was 2-3-fold less than those of RTD-1 and RTD-3. Equal amounts of each theta-defensin bound to E. coli cells, indicating that the differences in antibacterial activities are the result of post-binding processes.
|
| 11682065 |
Structural study of novel antimicrobial peptides, nigrocins, isolated from Rana nigromaculata |
10.1016/s0014-5793(01)02956-8. |
FEBS Lett |
Structural study of novel antimicrobial peptides, nigrocins, isolated from Rana nigromaculata
Abstract
- Novel cationic antimicrobial peptides, named nigrocin 1 and 2, were isolated from the skin of Rana nigromaculata and their amino acid sequences were determined. These peptides manifested a broad spectrum of antimicrobial activity against various microorganisms with different specificity. By primary structural analysis, it was revealed that nigrocin 1 has high sequence homology with brevinin 2 but nigrocin 2 has low sequence homology with any other known antimicrobial peptides. To investigate the structure-activity relationship of nigrocin 2, which has a unique primary structure, circular dichroism (CD) and homonuclear nuclear magnetic resonance spectroscopy (NMR) studies were performed. CD investigation revealed that nigrocin 2 adopts mainly an alpha-helical structure in trifluoroethanol (TFE)/H(2)O solution, sodium dodecyl sulfate (SDS) micelles, and dodecylphosphocholine micelles. The solution structures of nigrocin 2 in TFE/H(2)O (1:1, v/v) solution and in SDS micelles were determined by homonuclear NMR. Nigrocin 2 consists of a typical amphipathic alpha-helix spanning residues 3-18 in both 50% TFE solution and SDS micelles. From the structural comparison of nigrocin 2 with other known antimicrobial peptides, nigrocin 2 could be classified into the family of antimicrobial peptides containing a single linear amphipathic alpha-helix that potentially disrupts membrane integrity, which would result in cell death.
|
| 11683628 |
Delta-conotoxin structure/function through a cladistic analysis |
10.1021/bi010683a. |
Biochemistry |
Delta-conotoxin structure/function through a cladistic analysis
Abstract
- Delta-conotoxins are Conus peptides that inhibit inactivation of voltage-gated sodium channels. The suggestion that delta-conotoxins might be an essential component of the venoms of fish-hunting cone snails which rapidly immobilize their prey [Terlau, H., Shon, K., Grilley, M., Stocker, M., Stühmer, W., and Olivera, B. M. (1996) Nature 381, 148-151] has not been tested. On the basis of cDNA cloning, all of the fish-hunting Conus analyzed yielded at least one delta-conotoxin sequence. In addition, one delta-conotoxin isolated from the venom of Conus striatus had an amino acid sequence identical to that predicted from cDNA cloning. This new peptide exhibited properties of delta-conotoxins: it targeted sodium channels and potentiated action potentials by slowing channel inactivation. Homologous sequences of delta-conotoxins from two groups (clades) of related fish-hunting Conus species share consensus features but differ significantly from the two known delta-conotoxins from mollusc-hunting Conus venoms. Three large hydrophobic amino acids were conserved; analogues of the previously described delta-conotoxin PVIA with alanine substituted for the conserved amino acids F9 and I12 lost substantial biological activity. In contrast, both the T8A and K13A delta-conotoxin PVIA analogues, where substitutions were at nonconserved loci, proved to be biologically active. Taken together, our results indicate that a cladistic approach can identify amino acids critical for the activity of conotoxins and provide extensive information as to which amino acid substitutions can be made without significant functional consequences.
|
| 11690596 |
Oxytocin and autistic disorder: alterations in peptide forms |
10.1016/s0006-3223(01)01139-8. |
Biol Psychiatry |
Oxytocin and autistic disorder: alterations in peptide forms
Abstract
- Oxytocin (OT) is synthesized as a prohormone that is sequentially processed to peptides. These peptides are the bioactive amidated form (OT) and the C-terminal extended peptides, OT-Gly, OT-Gly-Lys and OT-Gly-Lys-Arg, which are designated together as OT-X. As an extension of our previous study finding decreased plasma OT in autism, studies were conducted to determine whether there were changes in OT peptide forms in autistic children.
Twenty eight male subjects (97 +/- 20 months; range, 70-139 months), diagnosed with DSM-IV autistic disorder through observation and semi-structured interview, were compared with 31 age-matched nonpsychiatric control subjects (106 +/- 22 months; range, 74-140 months). Using OT antisera with different specificity for the peptide forms, we measured plasma OT and OT-X in each group.
T tests showed that there was a decrease in plasma OT (t = 4.4, p <.0001), an increase in OT-X (t = 2.3, p <.03) and an increase in the ratio of OT-X/OT (t = 4.5, p <.0001) in the autistic sample, compared with control subjects.
The results suggest that children with autistic disorder show alterations in the endocrine OT system. Deficits in OT peptide processing in children with autism may be important in the development of this syndrome.
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| 11694888 |
Structure and autoregulation of the insulin-like growth factor 1 receptor kinase |
10.1038/nsb721. |
Nat Struct Biol |
Structure and autoregulation of the insulin-like growth factor 1 receptor kinase
Abstract
- The insulin-like growth factor 1 (IGF1) receptor is closely related to the insulin receptor. However, the unique biological functions of IGF1 receptor make it a target for therapeutic intervention in human cancer. Using its isolated tyrosine kinase domain, we show that the IGF1 receptor is regulated by intermolecular autophosphorylation at three sites within the kinase activation loop. Steady-state kinetic analyses of the isolated phosphorylated forms of the IGF1 receptor kinase (IGF1RK) reveal that each autophosphorylation event increases enzyme turnover number and decreases Km for ATP and peptide. We have determined the 2.1 A-resolution crystal structure of the tris-phosphorylated form of IGF1RK in complex with an ATP analog and a specific peptide substrate. The structure of IGF1RK reveals how the enzyme recognizes peptides containing hydrophobic residues at the P+1 and P+3 positions and how autophosphorylation stabilizes the activation loop in a conformation that facilitates catalysis. Although the nucleotide binding cleft is conserved between IGF1RK and the insulin receptor kinase, sequence differences in the nearby interlobe linker could potentially be exploited for anticancer drug design.
|
| 11695870 |
Separation of WAP-8294A components, a novel anti-methicillin-resistant staphylococcus aureus antibiotic, using high-speed counter-current chromatography |
10.1016/s0021-9673(01)01235-3. |
J Chromatogr A |
Separation of WAP-8294A components, a novel anti-methicillin-resistant staphylococcus aureus antibiotic, using high-speed counter-current chromatography
Abstract
- The WAP-8294A complex was isolated from the fermentation broth of Lysobacter sp. WAP-8294, whose major component, WAP-8294A2, showed a strong activity against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci in vitro, and also exhibited a potent activity against MRSA in vivo. The previous separation procedure using the conventional chromatographic methods was laborious and time-consuming, and the recovery of the desired compound was often unsatisfactory. In the present study, high-speed counter-current chromatography (HSCCC) was applied to the separation of the main components of the WAP-8294A complex. Due to the high polarity of the target compounds, we selected a hydrophilic two-phase solvent system composed of n-butanol-ethyl acetate-aqueous 0.005 M trifluoroacetic acid (1.25:3.75:5, v/v/v) which provided a suitable range of partition coefficient values for these compounds. Although the settling time of this solvent system was much longer than the optimum range, suggesting a low retention of the stationary phase under the standard experimental conditions, the separation was successfully performed at the low flow-rate of 0.5 ml/min. A sample size of 25 mg yielded pure fractions of three components (1-6 mg). The identification of each component was carried out by HPLC and fast atom bombardment mass spectrometry. The method will contribute to the clinical development of WAP-8294A2 as an anti-MRSA agent.
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