Maturation and specificity of Plasmodium falciparum subtilisin-like protease-1, a malaria merozoite subtilisin-like serine protease.
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Plasmodium falciparum subtilisin-like protease-1 (PfSUB-1) is a protein belonging to the subtilisin-like superfamily of serine proteases (subtilases). PfSUB-1 undergoes extensive posttranslational proteolytic processing. The primary translation product is converted in the parasite endoplasmic reticulum to p54. This is further processed to p47, which accumulates in secretory organelles within the merozoite. Here, we present a detailed study of this processing. In vitro translated PfSUB-1 showed no capacity to undergo autocatalytic processing. However, parasite extracts contain a protease that cleaves the in vitro translated proprotein between Asp(219) and Asn(220) to form two products of 31 (p31) and 54 kDa; the latter was indistinguishable from authentic p54 and remained complexed with p31 in a noncovalent interaction characteristic of that between a subtilase prodomain and its cognate catalytic domain. Cross-linking studies showed that this complex also exists in the parasite. Expression of PfSUB-1 in recombinant baculovirus also resulted in processing to p54. Mutation of the predicted active site serine abolished processing. Recombinant p54 was secreted in a complex with p31, and could be further converted to p47 in vitro. Conversion required calcium, was an intramolecular autocatalytic process, and involved a second cleavage between Asp(251) and Ala(252). A decapeptide based on sequence flanking Asp(219) was efficiently cleaved by recombinant PfSUB-1. We conclude that PfSUB-1 is a subtilase with an unusual substrate specificity and that it is activated by two autocatalytic processing steps. (+info)
Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR.
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A major inhibitor of diagnostic PCR in human plasma was identified and the mechanism of inhibition was characterized. Human blood was divided by centrifugation into buffy coat, plasma, platelets, and erythrocytes. All these blood fractions were found to be highly inhibitory to a standardized PCR mixture containing the thermostable DNA polymerase AmpliTaq Gold. PCR inhibitors in human plasma were purified by chromatographic procedures and were characterized by a process of elimination, so that the PCR-inhibitory effects of plasma fractions were tested after each purification step. The major inhibitor in human plasma, as determined by size-exclusion chromatography, anion-exchange chromatography, and chromatofocusing, was found to be immunoglobulin G (IgG) on the basis of N-terminal amino acid sequencing and electrophoretic analysis of the purified polypeptide. When different concentrations of purified plasma IgG (PIgG) were added to PCR mixtures containing 11 different thermostable DNA polymerases and 1 ng of Listeria monocytogenes DNA as template DNA, the only polymerase that resisted inhibition was rTth. The inhibitory effect was reduced when PIgG was heated at 95 degrees C before it was added to PCR or after the addition of excess nontarget DNA to the PCR mixture. However, heating of PIgG together with target DNA at 95 degrees C was found to block the amplification. Inhibition by PIgG may be due to an interaction with single-stranded DNA, which makes the target DNA unavailable for 10 of the DNA polymerases tested. The results show the danger of using boiling as a method of sample pretreatment or using a hot start prior to PCR. The effect of plasma PCR inhibition could be removed by mixing plasma with DNA-agarose beads prior to amplification, while plasma PCR inhibitors were found to bind to the DNA-agarose beads. (+info)
Two functionally distinct forms of the RSC nucleosome-remodeling complex, containing essential AT hook, BAH, and bromodomains.
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RSC is an essential 15 protein nucleosome-remodeling complex from S. cerevisiae. We have identified two closely related RSC members, Rsc1 and Rsc2. Biochemical analysis revealed Rsc1 and Rsc2 in distinct complexes, defining two forms of RSC. Genetic analysis has shown that Rsc1 and Rsc2 possess shared and unique functions. Rsc1 and Rsc2 each contain two bromodomains, a bromo-adjacent homology (BAH) domain, and an AT hook. One of the bromodomains, the BAH domain, and the AT hook are each essential for Rsc1 and Rsc2 functions, although they are not required for assembly into RSC complexes. Therefore, these domains are required for RSC function. Additional genetic analysis provides further evidence that RSC function is related to transcriptional control. (+info)
Nonlinear methods in the analysis of protein sequences: a case study in rubredoxins.
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Two computational methods widely used in time series analysis were applied to protein sequences, and their ability to derive structural information not directly accessible through classical sequence comparisons methods was assessed. The primary structures of 19 rubredoxins of both mesophilic and thermophilic bacteria, coded with hydrophobicity values of amino acid residues, were considered as time series and were analyzed by 1) recurrence quantification analysis and 2) spectral analysis of the sequence major eigenfunctions. The results of the two methods agreed to a large extent and generated a classification consistent with known 3D structural characteristics of the studied proteins. This classification separated in a clearcut manner a thermophilic protein from mesophilic proteins. The classification of primary structures given by the two dynamical methods was demonstrated to be basically different from classification stemming from classical sequence homology metrics. Moreover, on a more detailed scale, the method was able to discriminate between thermophilic and mesophilic proteins from a set of chimeric sequences generated from the mixing of a mesophilic (Rubr Clopa) and a thermophilic (Rubr Pyrfu) protein. Overall, our results point to a new way of looking at protein sequence comparisons. (+info)
Structure of the human glycogen-associated protein phosphatase 1 regulatory subunit hGM: homology modeling revealed an (alpha/beta)8-barrel-like fold in the multidomain protein.
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Protein phosphatase 1 (PP1) is widely distributed among tissues and species and acts as a regulator of many important cellular processes. By targeting the catalytic part of PP1 (PP1C) toward particular loci and substrates, regulatory subunits constitute key elements conferring specificity to the holoenzyme. Here, we report the identification of an (alpha/beta)8-barrel-like structure within the N-ter stretch of the human PP1 regulatory subunit hGM, which is part of the family of diverse proteins associated with glycogen metabolism. Protein homology modeling gave rise to a three-dimensional (3D) model for the 381 N-ter residue stretch of hGM, based on sequence similarity with Streptomyces olivochromogenes xylose isomerase, identified by using FASTA. The alignment was subsequently extended by using hydrophobic cluster analysis. The homology-derived model includes the putative glycogen binding area located within the 142-230 domain of hGM as well as a structural characterization of the PP1C interacting domain (segment 51-67). Refinement of the latter by molecular dynamics afforded a topology that is in agreement with previous X-ray studies (Egloff et al., 1997). Finite difference Poisson-Boltzmann calculations performed on the interacting domains of PP1C and hGM confirm the complementarity of the local electrostatic potentials of the two partners. This work highlights the presence of a conserved fold among distant species (mammalian, Caenorhabditis elegans, yeast) and, thus, emphasizes the involvement of PP1 in crucial basic cellular functions. (+info)
Alpha1-microglobulin chromophores are located to three lysine residues semiburied in the lipocalin pocket and associated with a novel lipophilic compound.
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Alpha1-microglobulin (alpha1m) is an electrophoretically heterogeneous plasma protein. It belongs to the lipocalin superfamily, a group of proteins with a three-dimensional (3D) structure that forms an internal hydrophobic ligand-binding pocket. Alpha1m carries a covalently linked unidentified chromophore that gives the protein a characteristic brown color and extremely heterogeneous optical properties. Twenty-one different colored tryptic peptides corresponding to residues 88-94, 118-121, and 122-134 of human alpha1m were purified. In these peptides, the side chains of Lys92, Lys118, and Lys130 carried size heterogeneous, covalently attached, unidentified chromophores with molecular masses between 122 and 282 atomic mass units (amu). In addition, a previously unknown uncolored lipophilic 282 amu compound was found strongly, but noncovalently associated with the colored peptides. Uncolored tryptic peptides containing the same Lys residues were also purified. These peptides did not carry any additional mass (i.e., chromophore) suggesting that only a fraction of the Lys92, Lys118, and Lys130 are modified. The results can explain the size, charge, and optical heterogeneity of alpha1m. A 3D model of alpha1m, based on the structure of rat epididymal retinoic acid-binding protein (ERABP), suggests that Lys92, Lys118, and Lys130 are semiburied near the entrance of the lipocalin pocket. This was supported by the fluorescence spectra of alpha1m under native and denatured conditions, which indicated that the chromophores are buried, or semiburied, in the interior of the protein. In human plasma, approximately 50% of alpha1m is complex bound to IgA. Only the free alpha1m carried colored groups, whereas alpha1m linked to IgA was uncolored. (+info)
Novel prenyltransferase gene encoding farnesylgeranyl diphosphate synthase from a hyperthermophilic archaeon, Aeropyrum pernix. Molecularevolution with alteration in product specificity.
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Prenyltransferases catalyse sequential condensations of isopentenyl diphosphate with allylic diphosphates. Previously, we reported the presence of farnesylgeranyl diphosphate (FGPP) synthase activity synthesizing C25 isoprenyl diphosphate in Natronobacterium pharaonis which is a haloalkaliphilic archaeon having C20-C25 diether lipids in addition to C20-C20 diether lipids commonly occurring in archaea [Tachibana, A. (1994) FEBS Lett. 341, 291-294]. Recently, it was found that a newly isolated aerobic hyperthermophilic archaeon, Aeropyrum pernix, had only C25-C25 diether lipids, not the usual C20-containing lipids [Morii, H., Yagi, H., Akutsu, H., Nomura, N., Sako, Y. & Koga, Y. (1999) Biochim. Biophys. Acta 1436, 426-436]. In this report, we describe the isoloation from A. pernix of the novel prenyltransferase gene, fgs, encoding FGPP synthase. The protein encoded by fgs was expressed in Escherichia coli as a glutathione S-transferase fusion protein and produced FGPP as a final product. Phylogenetic analysis of fgs with other prenyltransferases revealed that the short-chain prenyltransferase family is divided into three subfamilies: bacterial subfamily I, eukaryotic subfamily II, and archaeal subfamily III. fgs is clearly contained within the archaeal geranylgeranyl diphosphate (GGPP) synthase group (subfamily III), suggesting that FGPP synthase evolved from an archaeal GGPP synthase with an alteration in product specificity. (+info)
Phylloxin, a novel peptide antibiotic of the dermaseptin family of antimicrobial/opioid peptide precursors.
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A novel family of peptide precursors that have very similar N-terminal preprosequences followed by markedly different C-terminal domains has been identified in the skin of hylid frogs belonging to the genus Phyllomedusinae. Biologically active peptides derived from the variable domains include the dermaseptins, 28-34-residue peptides that have a broad-spectrum microbicidal activity, and dermorphin and the deltorphins, D-amino acid containing heptapeptides that are very potent agonists for the micro-opioid and delta-opioid receptors, respectively. This report describes the isolation, synthesis and cloning of phylloxin, a prototypical member of a novel family of antimicrobial peptides derived from the processing of a dermaseptin/dermorphin-like precursor. The structure of phylloxin (GWMSKIASGIGTFLSGIQQ amide) shows no homology to the dermaseptins, but bears some resemblance to the levitide-precursor fragment and the xenopsin-precursor fragment, two antimicrobial peptides isolated from the skin of an evolutionarily distant frog species, Xenopus laevis. Circular dichroism spectra of phylloxin in low polarity medium, which mimics the lipophilicity of the membrane of target microorganisms, indicated 60-70% alpha-helical conformation, and predictions of secondary structure suggested that the peptide can be configured as an amphipathic helix spanning residues 1-19. Phylloxin is an addition to the structurally and functionally diverse peptide families encoded by the rapidly evolving C-terminal domains of the dermorphin/dermaseptin group of precursors. (+info)