| 17933431 |
Identification of six novel T-1 conotoxins from Conus pulicarius by molecular cloning |
10.1016/j.peptides.2007.08.026. |
Peptides |
Identification of six novel T-1 conotoxins from Conus pulicarius by molecular cloning
Abstract
- Cone snails are a group of ancient marine gastropods with highly sophisticated defense and prey strategies using conotoxins in their venom. Conotoxins are a diverse array of small peptides, mostly with multiple disulfide bridges. Using a 3' RACE approach, we identified six novel peptides from the venom ducts of a worm-hunting cone snail Conus pulicarius. These peptides are named Pu5.1-Pu5.6 as their primary structures show the typical pattern of T-1 conotoxin family, a large and diverse group of peptides widely distributed in venom ducts of all major feeding types of Conus. Except for the conserved signal peptide sequences in the precursors and unique arrangement of Cys residues (CC-CC) in mature domains, the six novel T-1 conotoxins show remarkable sequence diversity in their pro and mature regions and are, thus, likely to be functionally diversified. Here, we present a simple and fast strategy of gaining novel disulfide-rich conotoxins via molecular cloning and our detailed sequence analysis will pave the way for the future functional characterization of toxin-receptor interaction.
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| 17935298 |
Planktocyclin, a cyclooctapeptide protease inhibitor produced by the freshwater cyanobacterium Planktothrix rubescens |
10.1021/np0700873. |
J Nat Prod |
Planktocyclin, a cyclooctapeptide protease inhibitor produced by the freshwater cyanobacterium Planktothrix rubescens
Abstract
- The freshwater cyanobacterium Planktothrix rubescens produces the cyclooctapeptide cyclo(Pro-Gly-Leu-Val-Met-Phe-Gly-Val). The chemical structure is new. This homodetic cyclic octapeptide was named planktocyclin ( 1). It consists solely of proteinogenic l-amino acids and is a strong inhibitor of mammalian trypsin and alpha-chymotrypsin and a moderately active inhibitor of human recombinant caspase-8. Mass spectrometric and 2D-NMR spectroscopic data allowed the determination of its structure. Synthetic planktocyclin was identical to the natural product.
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| 17935487 |
Structure and mode of action of the antimicrobial peptide arenicin |
10.1042/BJ20071051. |
Biochem J |
Structure and mode of action of the antimicrobial peptide arenicin
Abstract
- The solution structure and the mode of action of arenicin isoform 1, an antimicrobial peptide with a unique 18-residue loop structure, from the lugworm Arenicola marina were elucidated here. Arenicin folds into a two-stranded antiparallel beta-sheet. It exhibits high antibacterial activity at 37 and 4 degrees C against Gram-negative bacteria, including polymyxin B-resistant Proteus mirabilis. Bacterial killing occurs within minutes and is accompanied by membrane permeabilization, membrane detachment and release of cytoplasm. Interaction of arenicin with reconstituted membranes that mimic the lipopolysaccharide-containing outer membrane or the phospholipid-containing plasma membrane of Gram-negative bacteria exhibited no pronounced lipid specificity. Arenicin-induced current fluctuations in planar lipid bilayers correspond to the formation of short-lived heterogeneously structured lesions. Our results strongly suggest that membrane interaction plays a pivotal role in the antibacterial activity of arenicin.
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| 17936702 |
S6K1-mediated disassembly of mitochondrial URI/PP1gamma complexes activates a negative feedback program that counters S6K1 survival signaling. |
10.1016/j.molcel.2007.08.010 |
Mol. |
S6K1-mediated disassembly of mitochondrial URI/PP1gamma complexes activates a negative feedback program that counters S6K1 survival signaling.
Abstract
- S6 kinase 1 (S6K1) acts to integrate nutrient and growth factor signals to promote cell growth but also cell survival as a mitochondria-tethered protein kinase that phosphorylates and inactivates the proapoptotic molecule BAD. Here we report that the prefoldin chaperone URI represents a mitochondrial substrate of S6K1. In growth factor-deprived or rapamycin-treated cells, URI forms stable complexes with protein phosphatase (PP)1gamma at mitochondria, thereby inhibiting the activity of the bound enzyme. Growth factor stimulation induces disassembly of URI/PP1gamma complexes through S6K1-mediated phosphorylation of URI at serine 371. This activates a PP1gamma-dependent negative feedback program that decreases S6K1 activity and BAD phosphorylation, thereby altering the threshold for apoptosis. These findings establish URI and PP1gamma as integral components of an S6K1-regulated mitochondrial pathway dedicated, in part, to oppose sustained S6K1 survival signaling and to ensure that the mitochondrial threshold for apoptosis is set in accord with nutrient and growth factor availability.
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| 17938991 |
Selection for antimicrobial peptide diversity in frogs leads to gene duplication and low allelic variation |
10.1007/s00239-007-9045-5. |
J Mol Evol |
Selection for antimicrobial peptide diversity in frogs leads to gene duplication and low allelic variation
Abstract
- Antimicrobial peptides are highly diverse pathogen-killing molecules. In many taxa, their evolution is characterized by positive selection and frequent gene duplication. It has been proposed that genes encoding antimicrobial peptides might be subject to balancing selection and/or an enhanced mutation rate, but these hypotheses have not been well evaluated because allelic variation has rarely been studied at antimicrobial peptide loci. We present an evolutionary analysis of novel antimicrobial peptide genes from leopard frogs, Rana. Our results demonstrate that a single genome contains multiple homologous copies, among which there is an excess of nonsynonymous nucleotide site divergence relative to that expected from synonymous site divergence. Thus, we confirm the trends of recurrent duplication and positive selection. Allelic variation is quite low relative to interspecies divergence, indicating a recent positive selective sweep with no evidence of balancing selection. Repeated gene duplication, rather than a balanced maintenance of divergent allelic variants at individual loci, appears to be how frogs have responded to selection for a diverse suite of antimicrobial peptides. Our data also support a pattern of enhanced synonymous site substitution in the mature peptide region of the gene, but we cannot conclude that this is due to an elevated mutation rate.
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| 17963697 |
CR/periphilin is a transcriptional co-repressor involved in cell cycle progression |
10.1016/j.bbrc.2007.10.090. |
Biochem Biophys Res Commun |
CR/periphilin is a transcriptional co-repressor involved in cell cycle progression
Abstract
- CR/periphilin (CR) retards cell cycle progression mainly at the S-phase in part by transcriptionally repressing expression of Cdc7, the key regulator of DNA replication, and in part by unknown mechanisms. In this study, we show that enforced expression of CR inhibits Cdc7 promoter activity. The attachment of the DNA-binding domain of the yeast GAL4 transcription factor to CR that appears without DNA-binding sequences, enables CR to repress GAL4 promoter-mediated transcription in a histone deacetylase (HDAC) activity-dependent manner. CR forms a complex with mSin3A, a common component in transcriptional repressor complexes, as well as with HDAC1, suggesting that CR may behave as a co-repressor by functional interaction with the Sin3/HDAC co-repressor complex. We also demonstrate that an alternatively spliced variant of CR, CR-S, which is without a region encoded by exon 4 of the CR gene and is a weak interactor with HDAC1, shows a suppressing effect on CR activity.
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| 17965019 |
NOM1 targets protein phosphatase I to the nucleolus. |
10.1074/jbc.m706708200 |
J. Biol. Chem. |
NOM1 targets protein phosphatase I to the nucleolus.
Abstract
- Protein phosphatase I (PP1) is an essential eukaryotic serine/threonine phosphatase required for many cellular processes, including cell division, signaling, and metabolism. In mammalian cells there are three major isoforms of the PP1 catalytic subunit (PP1alpha, PP1beta, and PP1gamma) that are over 90% identical. Despite this high degree of identity, the PP1 catalytic subunits show distinct localization patterns in interphase cells; PP1alpha is primarily nuclear and largely excluded from nucleoli, whereas PP1gamma and to a lesser extent PP1beta concentrate in the nucleoli. The subcellular localization and the substrate specificity of PP1 catalytic subunits are determined by their interaction with targeting subunits, most of which bind PP1 through a so-called "RVXF" sequence. Although PP1 targeting subunits have been identified that direct PP1 to a number of subcellular locations and/or substrates, no targeting subunit has been identified that localizes PP1 to the nucleolus. Identification of nucleolar PP1 targeting subunit(s) is important because all three PP1 isoforms are included in the nucleolar proteome, enzymatically active PP1 is present in nucleoli, and PP1gamma is highly concentrated in nucleoli of interphase cells. In this study, we identify NOM1 (nucleolar protein with MIF4G domain 1) as a PP1-interacting protein and further identify the NOM1 RVXF motif required for its binding to PP1. We also define the NOM1 nucleolar localization sequence. Finally, we demonstrate that NOM1 can target PP1 to the nucleolus and show that a specific NOM1 RVXF motif and the NOM1 nucleolar localization sequence are required for this targeting activity. We therefore conclude that NOM1 is a PP1 nucleolar targeting subunit, the first identified in eukaryotic cells.
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| 17974005 |
The full-ORF clone resource of the German cDNA consortium. |
10.1186/1471-2164-8-399 |
BMC Genomics |
The full-ORF clone resource of the German cDNA consortium.
Abstract
- Background: With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes.
Results: Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen). A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure. Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned. Conclusion: The German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci. 177 splice variants were cloned representing 121 of these genes. The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications. As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available in the community.
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| 17985394 |
Structures of cyclic, antimicrobial peptides in a membrane-mimicking environment define requirements for activity |
10.1002/psc.924. |
J Pept Sci |
Structures of cyclic, antimicrobial peptides in a membrane-mimicking environment define requirements for activity
Abstract
- New antimicrobial compounds are of major importance because of the growing problem of bacterial resistance. In this context, antimicrobial peptides have received a lot of attention. Their mechanism of action, however, is often obscure. Here, the structures of two cyclic, antimicrobial peptides from the family of arginine- and tryptophan-rich peptides determined in a membrane-mimicking environment are described. The sequence of the peptides has been obtained from a cyclic parent peptide by scrambling the amino acids. While the activity of the peptides is similar to that of the parent peptide, the structures are not. The peptides do, however, all adopt an amphiphilic structure. A comparison between the structures helps to define the requirements for the activity of these peptides.
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| 17990954 |
Expression profiles of genes encoded by the supernumerary chromosome controlling AM-toxin biosynthesis and pathogenicity in the apple pathotype of Alternaria alternata |
10.1094/MPMI-20-12-1463. |
Mol Plant Microbe Interact |
Expression profiles of genes encoded by the supernumerary chromosome controlling AM-toxin biosynthesis and pathogenicity in the apple pathotype of Alternaria alternata
Abstract
- The apple pathotype of Alternaria alternata produces host-specific AM-toxin and causes Alternaria blotch of apple. Previously, we cloned two genes, AMT1 and AMT2, required for AM-toxin biosynthesis and found that these genes are encoded by small, supernumerary chromosomes of <1.8 Mb in the apple pathotype strains. Here, we performed expressed sequence tag analysis of the 1.4-Mb chromosome encoding AMT genes in strain IFO8984. A cDNA library was constructed using RNA from AM-toxin-producing cultures. A total of 40,980 clones were screened with the 1.4-Mb chromosome probe, and 196 clones encoded by the chromosome were isolated. Sequence analyses of these clones identified 80 unigenes, including AMT1 and AMT2, and revealed that the functions of 43 (54%) genes are unknown. The expression levels of the 80 genes in AM-toxin-producing and nonproducing cultures were analyzed by real-time quantitative polymerase chain reaction (PCR). Most of the genes were found to be expressed in both cultures at markedly lower levels than the translation elongation factor 1-alpha gene used as an internal control. Comparison of the expression levels of these genes between two cultures showed that 21 genes, including AMT1 and AMT2, were upregulated (>10-fold) in AM-toxin-producing cultures. Two of the upregulated genes were newly identified to be involved in AM-toxin biosynthesis by the gene disruption experiments and were named AMT3 and AMT4. Thus, the genes upregulated in AM-toxin-producing cultures contain ideal candidates for novel AM-toxin biosynthetic genes.
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| 17996262 |
I(1)-superfamily conotoxins and prediction of single D-amino acid occurrence |
10.1016/j.toxicon.2007.09.006. |
Toxicon |
I(1)-superfamily conotoxins and prediction of single D-amino acid occurrence
Abstract
- The considerable diversity of Conus peptides in the I(1)-superfamily provides a rare opportunity to define parameters important for the post-translational l- to d-isomerization of amino acids. This subtlest of post-translational modifications is not readily detectable by most techniques, and it would be a considerable advance if one could predict its potential occurrence purely from gene sequences. We previously described three I(1)-conotoxins, iota-RXIA (formerly designated r11a), r11b and r11c, each containing a d-amino acid at the third position from the C-terminus. In this work, we investigated two novel I(1)-superfamily members, r11d and ar11a, which we show have only l-amino acids. Based on these observations and an analysis of cDNA sequences of other group members, we suggest that there is a rule to predict d-amino acids in I(1)-superfamily peptides. Two factors are important: the residue to be modified should be three amino acids from the C-terminus of the precursor sequence, and it should be in a suitable sequence context. We apply the rule to other members of the I(1)-superfamily, to determine a priori which are probably modified.
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| 17996965 |
The orphan nuclear receptor Rev-erbbeta recruits Tip60 and HDAC1 to regulate apolipoprotein CIII promoter |
10.1016/j.bbamcr.2007.09.004. |
Biochim Biophys Acta |
The orphan nuclear receptor Rev-erbbeta recruits Tip60 and HDAC1 to regulate apolipoprotein CIII promoter
Abstract
- Nuclear hormone receptors function as ligand activated transcription factors. Ligand binding and modification such as acetylation have been reported to regulate nuclear hormone receptors. The orphan receptors, Rev-erbalpha and Rev-erbbeta, are members of the nuclear receptor superfamily and act as transcriptional repressors. In this study, the role of recruitment of co-factors by Rev-erbbeta and acetylation of Rev-erbbeta in modulating apolipoprotein CIII (apoCIII) transcription were investigated. Rev-erbbeta was found to transcriptionally repress apoCIII after binding to the apoCIII promoter. Tip60, a histone acetyl-transferase (HAT), was a novel binding partner for Rev-erbbeta and recruited to the apoCIII promoter by Rev-erbbeta. Tip60 was able to acetylate Rev-erbbeta and relieve the apoCIII repression mediated by Rev-erbbeta. This de-repression effect depended on acetylation of Rev-erbbeta at its RXKK motif by Tip60. In addition, histone deacetylase 1 (HDAC1) interacted with Rev-erbbeta and was recruited to the apoCIII promoter by Rev-erbbeta to antagonize Tip60's activity. Taken together, we have provided evidence that Rev-erbbeta modulates the apoCIII gene expression by recruiting different transcription co-activator or co-repressor.
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| 18003733 |
Simian virus 40 DNA replication is dependent on an interaction between topoisomerase I and the C-terminal end of T antigen. |
10.1128/jvi.01314-07 |
J. Virol. |
Simian virus 40 DNA replication is dependent on an interaction between topoisomerase I and the C-terminal end of T antigen.
Abstract
- Topoisomerase I (topo I) is needed for efficient initiation of simian virus 40 (SV40) DNA replication and for the formation of completed DNA molecules. Two distinct binding sites for topo I have been previously mapped to the N-terminal (residues 83 to 160) and C-terminal (residues 602 to 708) regions of T antigen. By mutational analysis, we identified a cluster of six residues on the surface of the helicase domain at the C-terminal binding site that are necessary for efficient binding to topo I in enzyme-linked immunosorbent assay and far-Western blot assays. Mutant T antigens with single substitutions of these residues were unable to participate normally in SV40 DNA replication. Some mutants were completely defective in supporting DNA replication, and replication was not enhanced in the presence of added topo I. The same mutants were the ones that were severely compromised in binding topo I. Other mutants demonstrated intermediate levels of activity in the DNA replication assay and were correspondingly only partially defective in binding topo I. Mutations of nearby residues outside this cluster had no effect on DNA replication or on the ability to bind topo I. These
Results strongly indicate that the association of topo I with these six residues in T antigen is essential for DNA replication. These residues are located on the back edges of the T-antigen double hexamer. We propose that topo I binds to one site on each hexamer to permit the initiation of SV40 DNA replication.
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| 18007294 |
Synthesis of the key precursor of Hirsutellide A |
10.3390/10010259. |
Molecules |
Synthesis of the key precursor of Hirsutellide A
Abstract
- Hexadepsipeptide 2, the precursor of Hirsutellide A (1), was synthesized in an overall yield of 45% from N-Boc-Me-Gly via three coupling reactions using dicyclohexylcarbodiimide (DCC), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyl- uronium hexafluorophosphate (HATU) and bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl), respectively.
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| 18008101 |
"Non-toxic" cyclic peptides induce lysis of cyanobacteria-an effective cell population density control mechanism in cyanobacterial blooms |
10.1007/s00248-007-9336-9. |
Microb Ecol |
"Non-toxic" cyclic peptides induce lysis of cyanobacteria-an effective cell population density control mechanism in cyanobacterial blooms
Abstract
- The presence of planktopeptin BL1125, anabaenopeptin B and anabaenopeptin F, two types of "non-toxic" cyclic peptide produced in bloom forming cyanobacteria, can provoke lysis of different non-axenic Microcystis aeruginosa cell lines via the induction of virus-like particles. The resulting particles are also able to infect the axenic M. aeruginosa cell line without lytic effects. Nevertheless, the presence of "non-toxic" cyclic peptides of cyanobacterial origin can induce lysis of these previously infected cells. This effect implies that a possible role of these peptides in the natural environment is the control of cyanobacterial population density. Lysogenic cyanobacteria can consequently act as hot-spots that, in the presence of cyanobacterial cyclic peptides, release numerous infectious particles. The process can be self-augmented with the simultaneous release of additional cyclic peptides from the producing lysogens, starting a forest fire effect that ends in collapse of cyanobacterial blooms.
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