Two thioredoxin reductases, trxr-1 and trxr-2, have differential physiological roles in Caenorhabditis elegans

Thioredoxin reductase (TrxR) is a member of the pyridine nucleotide-disulfide reductase family, which mainly functions in the thioredoxin system. TrxR is found in all living organisms and exists in two major ubiquitous isoenzymes in higher eukaryotic cells; One is cytosolic and the other mitochondrial. Mitochondrial TrxR functions to protect mitochondria from oxidative stress, where reactive oxidative species are mainly generated, while cytosolic TrxR plays a role to maintain optimal oxido-reductive status in cytosol. In this study, we report differential physiological functions of these two TrxRs in C. elegans. trxr-1, the cytosolic TrxR, is highly expressed in pharynx, vulva and intestine, whereas trxr-2, the mitochondrial TrxR, is mainly expressed in pharyngeal and body wall muscles. Deficiency of the non-selenoprotein trxr-2 caused defects in longevity and delayed development under stress conditions, while deletion mutation of the selenoprotein trxr-1 resulted in interference in acidification of lysosomal compartment in intestine. Interestingly, the acidification defect of trxr-1(jh143) deletion mutant was rescued, not only by selenocystein-containing wild type TRXR-1, but also cysteine-substituted mutant TRXR-1. Both trxr-1 and trxr-2 were up-regulated when worms were challenged by environmental stress such as heat shock. These results suggest that trxr-1 and trxr-2 function differently at organismal level presumably by their differential sub-cellular localization in C. elegans.     

Structural basis of poly(3-hydroxybutyrate) hydrolysis by PhaZ7 depolymerase from Paucimonas lemoignei

The crystal structure of poly(3-hydroxybutyrate) (PHB) depolymerase PhaZ7 purified from Paucimonas lemoignei was determined at 1.90 Å resolution. The structure consists of a single domain with an α/β hydrolase fold in its core. The active site is analogous to that of serine esterases/lipases and is characterized by the presence of a catalytic triad comprising Ser136, Asp242, and His306. Comparison with other structures in the Protein Data Bank showed a high level of similarity with the Bacillus subtilis lipase LipA (RMSD, 1.55 Å). Structural comparison with Penicillium funiculosum PHB depolymerase, the only PHB depolymerase whose structure is already known, revealed significant differences, resulting in an RMSD of 2.80-3.58 Å. The two enzymes appear to utilize different types of solvent-exposed residues for biopolymer binding, with aliphatic and hydroxyl residues used in P. funiculosum PHB depolymerase and aromatic residues in PhaZ7. Moreover, the active site of P. funiculosum PHB depolymerase is accessible to the substrate in contrast to the active site of PhaZ7, which is buried. Hence, considerable conformational changes are required in PhaZ7 for the creation of a channel leading to the active site. Taken together, the structural data suggest that PhaZ7 and P. funiculosum PHB depolymerase have adopted different strategies for effective substrate binding in response to their diverse substrate specificity and the lack of a substrate-binding domain.     

Caffeine Induces High Expression of cyp-35A Family Genes and Inhibits the Early Larval Development in Caenorhabditis elegans

Intake of caffeine during pregnancy can cause retardation of fetal development. Although the significant influence of caffeine on animal development is widely recognized, much remains unknown about its mode of action because of its pleiotropic effects on living organisms. In the present study, by using Caenorhabditis elegans as a model organism, the effects of caffeine on development were examined. Brood size, embryonic lethality, and percent larval development were investigated, and caffeine was found to inhibit the development of C. elegans at most of the stages in a dosage-dependent fashion. Upon treatment with 30 mM caffeine, the majority (86.1 ± 3.4%) of the L1 larvae were irreversibly arrested without further development. In contrast, many of the late-stage larvae survived and grew to adults when exposed to the same 30 mM caffeine. These results suggest that early-stage larvae are more susceptible to caffeine than later-stage larvae. To understand the metabolic responses to caffeine treatment, the levels of expression of cytochrome P450 (cyp) genes were examined with or without caffeine treatment using comparative micro-array, and it was found that the expression of 24 cyp genes was increased by more than 2-fold (p < 0.05). Among them, induction of the cyp-35A gene family was the most prominent. Interestingly, depletion of the cyp-35A family genes one-by-one or in combination through RNA interference resulted in partial rescue from early larval developmental arrest caused by caffeine treatment, suggesting that the high-level induction of cyp-35A family genes can be fatal to the development of early-stage larvae.     

The C. elegans glycosyltransferase BUS-8 has two distinct and essential roles in epidermal morphogenesis

Ventral enclosure in Caenorhabditis elegans involves migration of epidermal cells over a neuroblast substrate and subsequent adhesion at the ventral midline. Organisation of the neuroblast layer by ephrins and their receptors is essential for this migration. We show that bus-8, which encodes a predicted glycosyltransferase, is essential for embryonic enclosure and acts in or with ephrin signalling to mediate neuroblast organisation and to permit epidermal migration. BUS-8 acts non-cell-autonomously in this process, and likely modifies an extracellular regulator of ephrin signalling and cell organisation. Weak and cold-sensitive alleles of bus-8 show that the gene has a separate and distinct post-embryonic role, being essential for epidermal integrity and production of the cuticle surface. This disorganisation of the epidermis and cuticle layers causes increased drug sensitivity, which could aid the growing use of C. elegans in drug screening and chemical genomics. The viable mutants are also resistant to infection by the pathogen Microbacterium nematophilum, due to failure of the bacterium to bind to the host surface. The two separate essential roles of BUS-8 in epidermal morphogenesis add to our growing understanding of the widespread importance of glycobiology in development.     

Nematotoxicity of Marasmius oreades agglutinin (MOA) depends on glycolipid binding and cysteine protease activity

Fruiting body lectins have been proposed to act as effector proteins in the defense of fungi against parasites and predators. The Marasmius oreades agglutinin (MOA) is a Galα1,3Gal/GalNAc-specific lectin from the fairy ring mushroom that consists of an N-terminal ricin B-type lectin domain and a C-terminal dimerization domain. The latter domain shows structural similarity to catalytically active proteins, suggesting that, in addition to its carbohydrate-binding activity, MOA has an enzymatic function. Here, we demonstrate toxicity of MOA toward the model nematode Caenorhabditis elegans. This toxicity depends on binding of MOA to glycosphingolipids of the worm via its lectin domain. We show further that MOA has cysteine protease activity and demonstrate a critical role of this catalytic function in MOA-mediated nematotoxicity. The proteolytic activity of MOA was dependent on high Ca(2+) concentrations and favored by slightly alkaline pH, suggesting that these conditions trigger activation of the toxin at the target location. Our results suggest that MOA is a fungal toxin with intriguing similarities to bacterial binary toxins and has a protective function against fungivorous soil nematodes.     

GCK-3, a newly identified Ste20 kinase, binds to and regulates the activity of a cell cycle-dependent ClC anion channel

CLH-3b is a Caenorhabditis elegans ClC anion channel that is expressed in the worm oocyte. The channel is activated during oocyte meiotic maturation and in response to cell swelling by serine/threonine dephosphorylation events mediated by the type 1 phosphatases GLC-7alpha and GLC-7beta. We have now identified a new member of the Ste20 kinase superfamily, GCK-3, that interacts with the CLH-3b COOH terminus via a specific binding motif. GCK-3 inhibits CLH-3b in a phosphorylation-dependent manner when the two proteins are coexpressed in HEK293 cells. clh-3 and gck-3 are expressed predominantly in the C. elegans oocyte and the fluid-secreting excretory cell. Knockdown of gck-3 expression constitutively activates CLH-3b in nonmaturing worm oocytes. We conclude that GCK-3 functions in cell cycle- and cell volume-regulated signaling pathways that control CLH-3b activity. GCK-3 inactivates CLH-3b by phosphorylating the channel and/or associated regulatory proteins. Our studies provide new insight into physiologically relevant signaling pathways that control ClC channel activity and suggest novel mechanisms for coupling cell volume changes to cell cycle events and for coordinately regulating ion channels and transporters that control cellular Cl- content, cell volume, and epithelial fluid secretion.     

A possible role for FRM-1, a C. elegans FERM family protein, in embryonic development

FRM-1 is a member of the FERM protein superfamily containing a FERM domain, which is a highly conserved protein-protein interaction module found in most eukaryotes. Although FRM-1 is thought to be involved in linking intracellular proteins to membrane proteins, the specific role for FRM-1 remains to be elucidated. In an effort to explore the biological function of FRM-1, we examined the phenotype of frm-1(tm4168) mutant worms. We observed that frm-1(tm4168) worms have a delayed hatching phenotype. Twelve hours after being laid, when virtually all wild-type eggs had hatched, only 64% of frm-1(tm4168) eggs had hatched. About 3% of frm-1(tm4168) eggs failed to hatch, even 3 days after they had been laid. We also found that frm-1(tm4168) mutants displayed a temperature-sensitive sterility phenotype. About 13% of frm-1(tm4168) worms were unable to produce eggs or produced nonviable eggs at 25°C. In contrast, less than 1% of wild-type animals were sterile at this temperature. At 20°C, neither the mutant nor wild type appeared to be sterile. Western blot analysis indicates that FRM-1 is expressed throughout the developmental stages with the strongest expression at the egg stage. Immunostaining experiments revealed that FRM-1 is mainly localized to the plasma membrane of most if not all cells at an early embryonic stage and to the plasma membrane of P cells during the late embryonic stages. GFP fusion experiments showed that FRM-1 can be expressed in the pharynx and intestine at the larval and adult stages. Our data suggest that FRM-1 may participate in diverse biological processes, including embryonic development.     

Caenorhabditis elegans wsp-1 Regulation of Synaptic Function at the Neuromuscular Junction

The Rho GTPase members and their effector proteins, such as the Wiskott-Aldrich syndrome protein (WASP), play critical roles in regulating actin dynamics that affect cell motility, endocytosis, cell division, and transport. It is well established that Caenorhabditis elegans wsp-1 plays an essential role in embryonic development. We were interested in the role of the C. elegans protein WSP-1 in the adult nematode. In this report, we show that a deletion mutant of wsp-1 exhibits a strong sensitivity to the neuromuscular inhibitor aldicarb. Transgenic rescue experiments demonstrated that neuronal expression of WSP-1 rescued this phenotype and that it required a functional WSP-1 Cdc42/Rac interactive binding domain. WSP-1-GFP fusion protein was found localized presynaptically, immediately adjacent to the synaptic protein RAB-3. Strong genetic interactions with wsp-1 and other genes involved in different stages of synaptic transmission were observed as the wsp-1(gm324) mutation suppresses the aldicarb resistance seen in unc-13(e51), unc-11(e47), and snt-1 (md290) mutants. These results provide genetic and pharmacological evidence that WSP-1 plays an essential role to stabilize the actin cytoskeleton at the neuronal active zone of the neuromuscular junction to restrain synaptic vesicle release.     

RKN Lethal DB: A database for the identification of Root Knot Nematode (Meloidogyne spp.) candidate lethal genes

Root Knot nematode (RKN; Meloidogyne spp.) is one of the most devastating parasites that infect the roots of hundreds of plant species. RKN cannot live independently from their hosts and are the biggest contributors to the loss of the world''s primary foods. RNAi gene silencing studies have demonstrated that there are fewer galls and galls are smaller when RNAi constructs targeted to silence certain RKN genes are expressed in plant roots. We conducted a comparative genomics analysis, comparing RKN genes of six species: Meloidogyne Arenaria, Meloidogyne Chitwoodi, Meloidogyne Hapla, Meloidogyne Incognita, Meloidogyne Javanica, and Meloidogyne Paranaensis to that of the free living nematode Caenorhabditis elegans, to identify candidate genes that will be lethal to RKN when silenced or mutated. Our analysis yielded a number of such candidate lethal genes in RKN, some of which have been tested and proven to be effective in soybean roots. A web based database was built to house and allow scientists to search the data. This database will be useful to scientists seeking to identify candidate genes as targets for gene silencing to confer resistance in plants to RKN.

Availability

The database can be accessed from http://bioinformatics.towson.edu/RKN/     

Rendering the Intractable More Tractable: Tools from Caenorhabditis elegans Ripe for Import into Parasitic Nematodes

Recent and rapid advances in genetic and molecular tools have brought spectacular tractability to Caenorhabditis elegans, a model that was initially prized because of its simple design and ease of imaging. C. elegans has long been a powerful model in biomedical research, and tools such as RNAi and the CRISPR/Cas9 system allow facile knockdown of genes and genome editing, respectively. These developments have created an additional opportunity to tackle one of the most debilitating burdens on global health and food security: parasitic nematodes. I review how development of nonparasitic nematodes as genetic models informs efforts to import tools into parasitic nematodes. Current tools in three commonly studied parasites (Strongyloides spp., Brugia malayi, and Ascaris suum) are described, as are tools from C. elegans that are ripe for adaptation and the benefits and barriers to doing so. These tools will enable dissection of a huge array of questions that have been all but completely impenetrable to date, allowing investigation into host–parasite and parasite–vector interactions, and the genetic basis of parasitism.     

Guidelines for monitoring autophagy in Caenorhabditis elegans

The cellular recycling process of autophagy has been extensively characterized with standard assays in yeast and mammalian cell lines. In multicellular organisms, numerous external and internal factors differentially affect autophagy activity in specific cell types throughout the stages of organismal ontogeny, adding complexity to the analysis of autophagy in these metazoans. Here we summarize currently available assays for monitoring the autophagic process in the nematode C. elegans. A combination of measuring levels of the lipidated Atg8 ortholog LGG-1, degradation of well-characterized autophagic substrates such as germline P granule components and the SQSTM1/p62 ortholog SQST-1, expression of autophagic genes and electron microscopy analysis of autophagic structures are presently the most informative, yet steady-state, approaches available to assess autophagy levels in C. elegans. We also review how altered autophagy activity affects a variety of biological processes in C. elegans such as L1 survival under starvation conditions, dauer formation, aging, and cell death, as well as neuronal cell specification. Taken together, C. elegans is emerging as a powerful model organism to monitor autophagy while evaluating important physiological roles for autophagy in key developmental events as well as during adulthood.     

ePAT: a simple method to tag adenylated RNA to measure poly(A)-tail length and other 3' RACE applications

The addition of a poly(A)-tail to the 3' termini of RNA molecules influences stability, nuclear export, and efficiency of translation. In the cytoplasm, dynamic changes in the length of the poly(A)-tail have long been recognized as reflective of the switch between translational silence and activation. Thus, measurement of the poly(A)-tail associated with any given mRNA at steady-state can serve as a surrogate readout of its translation-state. Here, we describe a simple new method to 3'-tag adenylated RNA in total RNA samples using the intrinsic property of Escherichia coli DNA polymerase I to extend an RNA primer using a DNA template. This tag can serve as an anchor for cDNA synthesis and subsequent gene-specific PCR to assess poly(A)-tail length. We call this method extension Poly(A) Test (ePAT). The ePAT approach is as efficient as traditional Ligation-Mediated Poly(A) Test (LM-PAT) assays, avoids problems of internal priming associated with oligo-dT-based methods, and allows for the accurate analysis of both the poly(A)-tail length and alternate 3' UTR usage in 3' RACE applications.     

VHA-8, the E subunit of V-ATPase, is essential for pH homeostasis and larval development in C. elegans

Vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump, which transports protons across the membrane. It is a multi-protein complex which is composed of at least 13 subunits. The Caenorhabditis elegans vha-8 encodes the E subunit of V-ATPase which is expressed in the hypodermis, intestine and H-shaped excretory cells. VHA-8 is necessary for proper intestinal function likely through its role in cellular acidification of intestinal cells. The null mutants of vha-8 show a larval lethal phenotype indicating that vha-8 is an essential gene for larval development in C. elegans. Interestingly, characteristics of necrotic cell death were observed in the hypodermis and intestine of the arrested larvae suggesting that pH homeostasis via the E subunit of V-ATPase is required for the cell survival in C. elegans.     

RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let-7 miRNA pathways in Caenorhabditis elegans

Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1, two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways.     

Decapping Scavenger (DcpS) enzyme: Advances in its structure,activity and roles in the cap-dependent mRNA metabolism

Decapping Scavenger (DcpS) enzyme rids eukaryotic cells of short mRNA fragments containing the 5′ mRNA cap structure, which appear in the 3′ → 5′ mRNA decay pathway, following deadenylation and exosome-mediated turnover. The unique structural properties of the cap, which consists of 7-methylguanosine attached to the first transcribed nucleoside by a triphosphate chain (m⁷GpppN), guarantee its resistance to non-specific exonucleases. DcpS enzymes are dimers belonging to the Histidine Triad (HIT) superfamily of pyrophosphatases. The specific hydrolysis of m⁷GpppN by DcpS yields m⁷GMP and NDP. By precluding inhibition of other cap-binding proteins by short m⁷GpppN-containing mRNA fragments, DcpS plays an important role in the cap-dependent mRNA metabolism. Over the past decade, lots of new structural, biochemical and biophysical data on DcpS has accumulated. We attempt to integrate these results, referring to DcpS enzymes from different species. Such a synergistic characteristic of the DcpS structure and activity might be useful for better understanding of the DcpS catalytic mechanism, its regulatory role in gene expression, as well as for designing DcpS inhibitors of potential therapeutic application, e.g. in spinal muscular atrophy.     

Differential physiological roles of ESCRT complexes in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Endosomal sorting complex required for transport (ESCRT) complexes are involved in endosomal trafficking to the lysosome, cytokinesis, and viral budding. Extensive genetic, biochemical, and structural studies on the ESCRT system have been carried out in yeast and mammalian systems. However, the question of how the ESCRT system functions at the whole organism level has not been fully explored. In C. elegans, we performed RNAi experiments to knock-down gene expression of components of the ESCRT system and profiled their effects on protein degradation and endocytosis of YP170, a yolk protein. Targeted RNAi knock-down of ESCRT-I (tsg-101 and vps-28) and ESCRT-III (vps-24, and vps-32.2) components interfered with protein degradation while knock-down of ESCRT-II (vps-25 and vps-36) and ESCRT-III (vps-20 and vps-24) components hampered endocytosis. In contrast, the knockdown of vps-37, another ESCRT-I component, showed no defect in either YP170 uptake or degradation. Depletion of at least one component from each complex — ESCRT-0 (hgrs-1), ESCRT-I (tsg-101, vps-28, and vps-37), ESCRT-II (vps-36), ESCRT-III (vps-24), and Vps4 (vps-4) — resulted in abnormal distribution of embryos in the uterus of worms, possibly due to abnormal ovulation, fertilization, and egglaying. These results suggest differential physiological roles of ESCRT-0, -I, -II, and -III complexes in the context of the whole organism, C. elegans.