Prolyl 4-hydroxlase activity is essential for development and cuticle formation in the human infective parasitic nematode Brugia malayi

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 triple helix. C-P4Hs 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 characterise 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, while 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, inter-subunit cross-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

Increased expression of a microRNA correlates with anthelmintic resistance in parasitic nematodes

Resistance to anthelmintic drugs is a major problem in the global fight against parasitic nematodes infecting humans and animals. While previous studies have identified mutations in drug target genes in resistant parasites, changes in the expression levels of both targets and transporters have also been reported. The mechanisms underlying these changes in gene expression are unresolved. Here, we take a novel approach to this problem by investigating the role of small regulatory RNAs in drug resistant strains of the important parasite Haemonchus contortus. microRNAs (miRNAs) are small (22 nt) non-coding RNAs that regulate gene expression by binding predominantly to the 3′ UTR of mRNAs. Changes in miRNA expression have been implicated in drug resistance in a variety of tumor cells. In this study, we focused on two geographically distinct ivermectin resistant strains of H. contortus and two lines generated by multiple rounds of backcrossing between susceptible and resistant parents, with ivermectin selection. All four resistant strains showed significantly increased expression of a single miRNA, hco-miR-9551, compared to the susceptible strain. This same miRNA is also upregulated in a multi-drug-resistant strain of the related nematode Teladorsagia circumcincta. hco-miR-9551 is enriched in female worms, is likely to be located on the X chromosome and is restricted to clade V parasitic nematodes. Genes containing predicted binding sites for hco-miR-9551 were identified computationally and refined based on differential expression in a transcriptomic dataset prepared from the same drug resistant and susceptible strains. This analysis identified three putative target mRNAs, one of which, a CHAC domain containing protein, is located in a region of the H. contortus genome introgressed from the resistant parent. hco-miR-9551 was shown to interact with the 3′ UTR of this gene by dual luciferase assay. This study is the first to suggest a role for miRNAs and the genes they regulate in drug resistant parasitic nematodes. miR-9551 also has potential as a biomarker of resistance in different nematode species

Combined extracellular matrix cross-linking activity of the peroxidase MLT-7 and the dual oxidase BLI-3 is critical for post-embryonic viability in <i>Caenorhabditis elegans</i>

The nematode cuticle is a protective collagenous extracellular matrix that is modified, cross-linked, and processed by a number of key enzymes. This Ecdysozoan-specific structure is synthesized repeatedly and allows growth and development in a linked degradative and biosynthetic process known as molting. A targeted RNA interference screen using a cuticle collagen marker has been employed to identify components of the cuticle biosynthetic pathway. We have characterized an essential peroxidase, MoLT-7 (MLT-7), that is responsible for proper cuticle molting and re-synthesis. MLT-7 is an active, inhibitable peroxidase that is expressed in the cuticle-synthesizing hypodermis coincident with each larval molt. mlt-7 mutants show a range of body morphology defects, most notably molt, dumpy, and early larval stage arrest phenotypes that can all be complemented with a wild type copy of mlt-7. The cuticles of these mutants lacks di-tyrosine cross-links, becomes permeable to dye and accessible to tyrosine iodination, and have aberrant collagen protein expression patterns. Overexpression of MLT-7 causes mutant phenotypes further supporting its proposed enzymatic role. In combination with BLI-3, an H2O2-generating NADPH dual oxidase, MLT-7 is essential for post-embryonic development. Disruption of mlt-7, and particularly bli-3, via RNA interference also causes dramatic changes to the in vivo cross-linking patterns of the cuticle collagens DPY-13 and COL-12. This points toward a functionally cooperative relationship for these two hypodermally expressed proteins that is essential for collagen cross-linking and proper extracellular matrix formation

DNA methylation-associated colonic mucosal immune and defense responses in treatment-naïve pediatric ulcerative colitis

Inflammatory bowel diseases (IBD) are emerging globally, indicating that environmental factors may be important in their pathogenesis. Colonic mucosal epigenetic changes, such as DNA methylation, can occur in response to the environment and have been implicated in IBD pathology. However, mucosal DNA methylation has not been examined in treatment-naïve patients. We studied DNA methylation in untreated, left sided colonic biopsy specimens using the Infinium HumanMethylation450 BeadChip array. We analyzed 22 control (C) patients, 15 untreated Crohn’s disease (CD) patients, and 9 untreated ulcerative colitis (UC) patients from two cohorts. Samples obtained at the time of clinical remission from two of the treatment-naïve UC patients were also included into the analysis. UC-specific gene expression was interrogated in a subset of adjacent samples (5 C and 5 UC) using the Affymetrix GeneChip PrimeView Human Gene Expression Arrays. Only treatment-naïve UC separated from control. One-hundred-and-twenty genes with significant expression change in UC (> 2-fold, P &lt; 0.05) were associated with differentially methylated regions (DMRs). Epigenetically associated gene expression changes (including gene expression changes in the IFITM1, ITGB2, S100A9, SLPI, SAA1, and STAT3 genes) were linked to colonic mucosal immune and defense responses. These findings underscore the relationship between epigenetic changes and inflammation in pediatric treatment-naïve UC and may have potential etiologic, diagnostic, and therapeutic relevance for IBD

Alien Registration- Winter, Alan John D. (Bangor, Penobscot County)

https://digitalmaine.com/alien_docs/13968/thumbnail.jp

Differences in collagen prolyl 4-hydroxylase assembly between two Caenorhabditis nematode species despite high amino acid sequence identity of the enzyme subunits

The collagen prolyl 4-hydroxylases (P4Hs) are essential for proper extracellular matrix formation in multicellular organisms. The vertebrate enzymes are &#945;2&#946;2 tetramers, in which the &#946; subunits are identical to protein disulfide isomerase (PDI). Unique P4H forms have been shown to assemble from the &lt;i&gt;Caenorhabditis&lt;/i&gt; &lt;i&gt;elegans&lt;/i&gt; catalytic &#945; subunit isoforms PHY-1 and PHY-2 and the &#946; subunit PDI-2. A mixed PHY-1/PHY-2/(PDI-2)&lt;sub&gt;2&lt;/sub&gt; tetramer is the major form, while PHY-1/PDI- 2 and PHY-2/PDI-2 dimers are also assembled but less efficiently. Cloning and characterization of the orthologous subunits from the closely related nematode &lt;i&gt;Caenorhabditis&lt;/i&gt; &lt;i&gt;briggsae&lt;/i&gt; revealed distinct differences in the assembly of active P4H forms in spite of the extremely high amino acid sequence identity (92-97%) between the &lt;i&gt;C. briggsae&lt;/i&gt; and &lt;i&gt;C. elegans&lt;/i&gt; subunits. In addition to a PHY-1/PHY-2(PDI-2)&lt;sub&gt;2&lt;/sub&gt; tetramer and a PHY-1/PDI-2 dimer, an active (PHY- 2)&lt;sub&gt;2&lt;/sub&gt;(PDI-2)&lt;sub&gt;2&lt;/sub&gt; tetramer was formed in &lt;i&gt;C. briggsae&lt;/i&gt; instead of a PHY-2/PDI-2 dimer. Site-directed mutagenesis studies and generation of inter-species hybrid polypeptides showed that the N-terminal halves of the &lt;i&gt;Caenorhabditis&lt;/i&gt; PHY-2 polypeptides determine their assembly properties. Genetic disruption of &lt;i&gt;C. briggsae phy-1&lt;/i&gt; (&lt;i&gt;Cb-dpy-18&lt;/i&gt;) via a &lt;i&gt;Mos1&lt;/i&gt; insertion resulted a small (short) phenotype that is less severe than the dumpy (short and fat) phenotype of the corresponding &lt;i&gt;C. elegans&lt;/i&gt; mutants (&lt;i&gt;Ce-dpy-18&lt;/i&gt;). &lt;i&gt;C. briggsae&lt;/i&gt; &lt;i&gt;phy-2&lt;/i&gt; RNA interference produced no visible phenotype in the wild type nematodes but produced a severe dumpy phenotype and larval arrest in &lt;i&gt;phy-1&lt;/i&gt; mutants. Genetic complementation of the &lt;i&gt;C. briggsae&lt;/i&gt; and &lt;i&gt;C. elegans&lt;/i&gt; &lt;i&gt;phy-1&lt;/i&gt; mutants was achieved by injection of a wild type &lt;i&gt;phy-1&lt;/i&gt; gene from either species

Characterisation of nematode prolyl 4-hydroxylase collagen modifying enzymes

The function of prolyl 4-hydroxylases (P4H) in the formation of the nematode cuticle was studied. The cuticle is one of the two major forms of extracellular matrix (ECM) in the nematode and performs vital roles in these animals including acting as an exoskeleton to maintain body morphology. Nematodes develop from an embryo through four larval moults to the adult stage. Each larval stage is characterised by the synthesis of a new cuticle and shedding of the existing structure. The nematode cuticle is a complex multi-layered structure formed principally from collagens that are synthesised by the underlying hypodermal tissue. Collagens are characterised by repeats of the amino acid sequence Gly-X-Y, where Gly is glycine and X and Y can be any residue but are most commonly proline and 4-hydroxyproline respectively. Three collagen monomers combine to form a triple helix, with the presence of 4- hydroxyproline residues stabilising the structure. The enzyme P4H modifies Y position proline residues in newly synthesised collagen molecules within the endoplasmic reticulum (ER) of the cell to produce 4-hydroxyproline. P4H role in cuticular ECM formation was examined primarily using the free-living species Caenorhabditis elegans due to its ease of handling and culture in the laboratory, the range of genetic and transgenic techniques available, and the complete genome sequence. Characterised collagen P4H from other species are most often multi-enzyme complexes formed from catalytically active beta subunits with the alpha subunit being the enzyme protein disulphide isomerase (PDI). The role of PDI in these complexes is not connected to its enzymatic activity but instead functions to keep the beta subunits in a catalytically active form within the ER of the cell. The described vertebrate P4Hs are alpha2beta2 tetramer complexes. Two different alpha subunits have been identified which form into separate enzyme complexes with a common PDI beta subunit. In this study the genes Ce-phy-1, Ce-phy-2 and Ce-pdi-2 were examined for their role in cuticular ECM formation in C. elegans. These genes were shown to be expressed throughout development in cuticle collagen synthesising hypodermal tissue in a manner reflecting the expression of their substrate, placing the encoded enzymes in the appropriate tissue for collagen modification, at times of maximal collagen synthesis. Disruption of Ce-phy-1 gene function produced nematodes with a mutant body shape known as dumpy (Dpy). This demonstrated that this gene is required for correct body morphology and led to the identification of the strain dpy-18 as a Ce-phy-1 mutant. This represented the first identification of a P4H mutant in any organism. Double disruption of Ce-phy-1 and Ce-phy-2 or Ce-pdi-2 singly resulted in an embryonic lethal phenotype due to the loss of P4H activity resulting in a cuticle unable to maintain nematode body shape. Disruption of these genes was demonstrated to have an affect on the localisation of specific cuticle collagens. The forms of P4H complex present were examined which revealed the presence of a unique mixed a subunit tetramer, in addition to the already described dimer form of the enzyme. Examination of three divergent putative Ce phy genes did not reveal any role for these in modification of the major ECMs in this nematode and showed that only Ce-PHY-1, Ce-PHY-2 and Ce-PDI-2 perform the essential modification of cuticle collagens. P4H was also studied in the human infective filarial nematode Brugia malayi which is one of the causative agents of lymphatic filariasis in humans, a disease that affects over 120 million people. A phy gene homologue, named Bm phy-1, was cloned and characterised from this organism. In contrast to the both human a subunit-encoding genes, which were shown to rescue the C. elegans dpy-18 P4H mutant, expression of Bm-phy-1 was not sufficient to repair the mutant form of these nematodes. Expression of this gene was demonstrated in all life cycle stages examined, with the gene promoter directing expression of a reporter gene to the hypodermal cells of C. elegans

Metal-substituted protein MRI contrast agents engineered for enhanced relaxivity and ligand sensitivity

Engineered metalloproteins constitute a flexible new class of analyte-sensitive molecular imaging agents detectable by magnetic resonance imaging (MRI), but their contrast effects are generally weaker than synthetic agents. To augment the proton relaxivity of agents derived from the heme domain of cytochrome P450 BM3 (BM3h), we formed manganese(III)-containing proteins that have higher electron spin than their native ferric iron counterparts. Metal substitution was achieved by coexpressing BM3h variants with the bacterial heme transporter ChuA in Escherichia coli and supplementing the growth medium with Mn3+-protoporphyrin IX. Manganic BM3h variants exhibited up to 2.6-fold higher T1 relaxivities relative to native BM3h at 4.7 T. Application of ChuA-mediated porphyrin substitution to a collection of thermostable chimeric P450 domains resulted in a stable, high-relaxivity BM3h derivative displaying a 63% relaxivity change upon binding of arachidonic acid, a natural ligand for the P450 enzyme and an important component of biological signaling pathways. This work demonstrates that protein-based MRI sensors with robust ligand sensitivity may be created with ease by including metal substitution among the toolkit of methods available to the protein engineer.National Institutes of Health (U.S.) (NIH Grant R01-DA28299 )National Institutes of Health (U.S.) (NIH NRSA Fellowship (Award F32-GM087102))California Institute of Technology (Caltech Jacobs Grant

Loss of secretory pathway FK506-binding proteins results in cold-sensitive lethality in caenorhabditis elegans

The FK506-binding proteins (FKBs) represent ubiquitous enzymes that catalyse the rate-limiting peptidyl prolyl cis-trans isomerization step in protein folding. The nematode &lt;i&gt;Caenorhabditis elegans&lt;/i&gt; has eight FKBs, three of which (FKB-3, -4 and -5) have dual peptidyl prolyl &lt;i&gt;cis-trans&lt;/i&gt; isomerase (PPIase) domains, signal peptides and ER-retention signals. PPIase activity has been detected for recombinant FKB-3. Both FKB-3 and -5 are expressed in the exoskeleton-synthesising hypodermis with transcript peaks that correspond to the molting and collagen synthesis cycles. FKB-4 is expressed at a low level throughout development. No phenotypes were observed in deletion mutants in each of the secretory pathway FKBs. Combined triple and &lt;i&gt;fkb-4/-5&lt;/i&gt; double deletion mutants were found to arrest at 12°C, but developed normally at 15-25°C. This cold-sensitive larval lethal effect was not maternally-derived, occurred during embryogenesis and could be rescued following the transgenic introduction of a wild type copy of either &lt;i&gt;fkb-4 or fkb-5&lt;/i&gt;. The temperature-sensitive defects also affected molting, cuticle collagen expression, hypodermal seam cell morphology and the structural integrity of the cuticular extracellular matrix. This study establishes that the secretory pathway FK506-binding PPIase enzymes are essential for normal nematode development, collagen biogenesis and the formation of an intact exoskeleton under adverse physiological conditions

A novel member of the let-7 microRNA family is associated with developmental transitions in filarial nematode parasites

Background: Filarial nematodes are important pathogens in the tropics transmitted to humans via the bite of blood sucking arthropod vectors. The molecular mechanisms underpinning survival and differentiation of these parasites following transmission are poorly understood. microRNAs are small non-coding RNA molecules that regulate target mRNAs and we set out to investigate whether they play a role in the infection event. Results: microRNAs differentially expressed during the early post-infective stages of Brugia pahangi L3 were identified by microarray analysis. One of these, bpa-miR-5364, was selected for further study as it is upregulated ~12-fold at 24 hours post-infection, is specific to clade III nematodes, and is a novel member of the let-7 family, which are known to have key developmental functions in the free-living nematode Caenorhabditis elegans. Predicted mRNA targets of bpa-miR-5364 were identified using bioinformatics and comparative genomics approaches that relied on the conservation of miR-5364 binding sites in the orthologous mRNAs of other filarial nematodes. Finally, we confirmed the interaction between bpa-miR-5364 and three of its predicted targets using a dual luciferase assay. Conclusions: These data provide new insight into the molecular mechanisms underpinning the transmission of third stage larvae of filarial nematodes from vector to mammal. This study is the first to identify parasitic nematode mRNAs that are verified targets of specific microRNAs and demonstrates that post-transcriptional control of gene expression via stage-specific expression of microRNAs may be important in the success of filarial infection