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      Prolyl 4-Hydroxlase Activity Is Essential for Development and Cuticle Formation in the Human Infective Parasitic Nematode Brugia malayi*

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          Abstract

          Background: Collagen prolyl 4-hydroxylases (C-P4H) are involved in the formation of extracellular matrices.

          Results: The full complement of C-P4H enzymes from the human infective parasite Brugia malayi have been bioinformatically, biochemically, and functionally characterized.

          Conclusion: C-P4H enzymes are essential for development in B. malayi.

          Significance: Unique features of these essential enzymes may be exploited in future control mechanisms.

          Abstract

          Collagen prolyl 4-hydroxylases (C-P4H) are required for formation of extracellular matrices in higher eukaryotes. These enzymes convert proline residues within the repeat regions of collagen polypeptides to 4-hydroxyproline, a modification essential for the stability of the final triple helix. C-P4H are most often oligomeric complexes, with enzymatic activity contributed by the α subunits, and the β subunits formed by protein disulfide isomerase (PDI). Here, we characterize this enzyme class in the important human parasitic nematode Brugia malayi. All potential C-P4H subunits were identified by detailed bioinformatic analysis of sequence databases, function was investigated both by RNAi in the parasite and heterologous expression in Caenorhabditis elegans, whereas biochemical activity and complex formation were examined via co-expression in insect cells. Simultaneous RNAi of two B. malayi C-P4H α subunit-like genes resulted in a striking, highly penetrant body morphology phenotype in parasite larvae. This was replicated by single RNAi of a B. malayi C-P4H β subunit-like PDI. Surprisingly, however, the B. malayi proteins were not capable of rescuing a C. elegans α subunit mutant, whereas the human enzymes could. In contrast, the B. malayi PDI did functionally complement the lethal phenotype of a C. elegans β subunit mutant. Comparison of recombinant and parasite derived material indicates that enzymatic activity may be dependent on a non-reducible covalent link, present only in the parasite. We therefore demonstrate that C-P4H activity is essential for development of B. malayi and uncover a novel parasite-specific feature of these collagen biosynthetic enzymes that may be exploited in future parasite control.

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          Most cited references 39

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          Improved prediction of signal peptides: SignalP 3.0.

          We describe improvements of the currently most popular method for prediction of classically secreted proteins, SignalP. SignalP consists of two different predictors based on neural network and hidden Markov model algorithms, where both components have been updated. Motivated by the idea that the cleavage site position and the amino acid composition of the signal peptide are correlated, new features have been included as input to the neural network. This addition, combined with a thorough error-correction of a new data set, have improved the performance of the predictor significantly over SignalP version 2. In version 3, correctness of the cleavage site predictions has increased notably for all three organism groups, eukaryotes, Gram-negative and Gram-positive bacteria. The accuracy of cleavage site prediction has increased in the range 6-17% over the previous version, whereas the signal peptide discrimination improvement is mainly due to the elimination of false-positive predictions, as well as the introduction of a new discrimination score for the neural network. The new method has been benchmarked against other available methods. Predictions can be made at the publicly available web server
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            Helminth infections: the great neglected tropical diseases.

            Helminths are parasitic worms. They are the most common infectious agents of humans in developing countries and produce a global burden of disease that exceeds better-known conditions, including malaria and tuberculosis. As we discuss here, new insights into fundamental helminth biology are accumulating through newly completed genome projects and the nascent application of transgenesis and RNA interference technologies. At the same time, our understanding of the dynamics of the transmission of helminths and the mechanisms of the Th2-type immune responses that are induced by infection with these parasitic worms has increased markedly. Ultimately, these advances in molecular and medical helminth biology should one day translate into a new and robust pipeline of drugs, diagnostics, and vaccines for targeting parasitic worms that infect humans.
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              Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans.

              Genetic interference mediated by double-stranded RNA (RNAi) has been a valuable tool in the analysis of gene function in Caenorhabditis elegans. Here we report an efficient induction of RNAi using bacteria to deliver double-stranded RNA. This method makes use of bacteria that are deficient in RNaseIII, an enzyme that normally degrades a majority of dsRNAs in the bacterial cell. Bacteria deficient for RNaseIII were engineered to produce high quantities of specific dsRNA segments. When fed to C. elegans, such engineered bacteria were found to produce populations of RNAi-affected animals with phenotypes that were comparable in expressivity to the corresponding loss-of-function mutants. We found the method to be most effective in inducing RNAi for non-neuronal tissue of late larval and adult hermaphrodites, with decreased effectiveness in the nervous system, in early larval stages, and in males. Bacteria-induced RNAi phenotypes could be maintained over the course of several generations with continuous feeding, allowing for convenient assessments of the biological consequences of specific genetic interference and of continuous exposure to dsRNAs.
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                Author and article information

                Affiliations
                From the []Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Garscube Estate, Bearsden Road, Glasgow G61 1QH, Scotland, United Kingdom and
                the [§ ]Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, Oulu Center for Cell Matrix Research, University of Oulu, FIN-90014 Oulu, Finland
                Author notes
                [1 ] To whom correspondence may be addressed: Institute of Infection, Immunity, and Inflammation, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Garscube Estate, Bearsden Rd., Glasgow G61 1QH, UK. Tel,: 44-141-330-5759; Fax: 44-141-330-2271; E-mail: Alan.Winter@ 123456glasgow.ac.uk .
                [2 ] To whom correspondence may be addressed: Institute of Infection, Immunity, and Inflammation, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Garscube Estate, Bearsden Rd., Glasgow G61 1QH, UK. Tel.: 44-141-330-1997; Fax: 44-141-330-2271; E-mail: Tony.Page@ 123456glasgow.ac.uk .
                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                18 January 2013
                7 December 2012
                7 December 2012
                : 288
                : 3
                : 1750-1761
                23223450
                3548485
                M112.397604
                10.1074/jbc.M112.397604
                © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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                Glycobiology and Extracellular Matrices

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