Stephen Pak et al. (2001) International C. elegans Meeting "Emergence of an Ancient Intracellular Antiproteinase Defense System"

Stephen Pak, Yuko Sakaguchi, David Askew, Gary A Silverman, Chris Tsu, Cliff Luke, Sule Cataltepe, Vasantha Kumar

[International C. elegans Meeting, 2001]

Background: High molecular weight ser ine p roteinase in hibitor s (serpins) such as antithrombin III, a 2-antiplasmin and a 1-antitrypsin are critical regulators of intravascular (clotting and thrombolytic) and extravascular proteolytic cascades. We have characterized a family (n>13) of human ovalbumin–like Serpins. Unlike the fluid phase serpins, the ov-serpins lack N-terminal signal peptides and reside within the cytosol of epithelial cells. However, the function of these molecules is unknown. Objective: Determine whether the function of the ov-serpins can be elucidated using a comparative genomics approach and a simpler organism, C. elegans . Design/Methods: Database analysis and molecular cloning were used to identify serpins. Results: Screening of ACeDB revealed the presence of 9-10 functional serpin genes ( srp-1-10 ). They are tandemly arrayed along Chromosome V. By a combination of cDNA cloning and RT-PCR, cDNAs corresponding to 6 of the genes were isolated. The genes contain 5-6 exons with conserved splice sites at the 5' and 3' ends but some variation within. Like the ov-serpins, none of the worm serpins encode for typical N-terminal signal peptides. Thus, C.elegans serpins also are likely to reside intracellularly. Each serpin contains different residues in the reactive site loop (the reactive site loop is that portion of the serpin that binds to the active site of the proteinase). This variation suggests that each serpin will inhibit different types of proteinases. Indeed, kinetic analysis in vitro, using recombinant proteins expressed in E.coli , showed that srp-2 and srp-3 are inhibitors ( k ass ~1x10 4 M -1 sec -1 ) of granzyme B and chymotrypsin-like serine proteinases, respectively. Preliminary transgenic studies using a vector containing the srp-2 promotor fused to a GFP reporter gene showed that, like the ov-serpins, srp-2 is highly expressed in the hypoderm. Conclusions: Members of the ov-serpin family are functionally and topologically conserved in C.elegans . Reverse genetic studies by the generation of null mutations should reveal the functions of this intracellular anti-proteinase defense system that is highly conserved through out metazoan evolution.

Ranganathan, Supraja et al. (2021) International Worm Meeting "Regulation of antiviral responses in C. elegans embryos"

Ranganathan, Supraja, Bass, Brenda

[International Worm Meeting, 2021]

C. elegans embryos utilize various endogenous RNA interference (RNAi) pathways to modulate signaling and ensure normal development. One such pathway is the 26G endogenous RNAi pathway, where certain transcripts are processed by a complex which includes Dicer (DCR-1), RDE-4, and the RNA-dependent RNA polymerase, RRF-3. Using the transcript as a template, RRF-3 synthesizes an antisense strand, and DCR-1 cleaves the resulting dsRNA into small-interfering RNAs (siRNAs) that are 26 nucleotides long with a bias for guanosine at the 5′ end (26G siRNAs). Deleting rrf-3 disrupts the 26G pathway and makes the worms hypersensitive to extracellular dsRNA. Recently, the Bass lab discovered that genes upregulated in rrf-3(-/-) embryos overlap with a set of genes known as the Orsay virus-induced genes (OVIGs). OVIGs are genes that are upregulated during infection with Orsay virus (OV), a naturally infecting C. elegans pathogen. Interestingly, worms that lack rrf3 evade the virus better than wildtype and have significantly less viral load relative to wildtype. Upon OV infection and OV genomic RNA replication, the viral replication intermediates are recognized by DRH-1, a Dicer-related helicase, which leads to the upregulation of OVIGs. We observed that two-thirds of the OVIGs upregulated in rrf-3(-/-) embryos are only DRH-1-dependent during OV infection. We show that DRH-1 is not necessary for induction of OVIGs in rrf-3(-/-) embryos. These results suggest that other pathways are involved in the regulation of OVIG expression. We propose that the resulting accumulation of 26G target transcripts and proteins upon disrupting the 26G RNAi pathway leads to the induction of an OVIG response. We are developing a fluorescent reporter to identify the triggers that induce an antiviral response in rrf-3(-/-) embryos. Keywords: 26G RNAi pathway, rrf-3, OVIGs, DRH-1

Stephen C Pak et al. (2002) East Coast Worm Meeting "SERPIN FUNCTION IN C. ELEGANS: A GENETIC AND BIOCHEMICAL ANALYSIS"

Cliff J Luke, Gary A Silverman, David J Askew, Yuko S Askew, David R Mills, Stephen C Pak, Sule Cataltepe, Christopher Tsu, Vasantha Kumar

[East Coast Worm Meeting, 2002]

To better understand the biology of ov-serpins, we undertook a comparative genomics study using C. elegans as a model organism. A screen of the C. elegans database (ACeDB ) using the human serpin, SCCA2, amino acid sequence identified the presence of 9-10 serpin genes (srp-1-10). These genes are arranged in tandem and are distributed throughout the length of chromosome V. By a combination of cDNA cloning and RT-PCR, cDNAs corresponding to 6 of these genes were isolated. Like the human ov-serpins, no typical N-terminal signal peptides were found suggesting that the worm serpins may also reside intracellularly. Each serpin contains different residues in the reactive site loop suggesting that they have distinct proteinase targets. Interestingly and reminiscent of the Manduca sexta serpin gene-1, srp-7 was found to have 3 distinct reactive site loops resulting from alternative splicing of exon 5. In vitro kinetic analysis, using recombinant proteins expressed in E. coli, showed that srp-2, srp-3 and srp-6 are inhibitors (kass _1x104 M-1 sec-1 ) of granzyme B, chymotrypsin-like serine proteinases, and papain-like cysteine proteinases, respectively. Expression studies using promoter::GFP-fusions indicate that serpins are present in the hypoderm, intestine, neurons and excretory glands. Null deletion mutants of srp-1, srp-2, srp-6, srp-7(a-c) and srp-8 have been isolated and initial characterizations suggest that serpins play important roles in osmoregulation and host defense against bacterial infection.

Andreas Kampkoetter et al. (2001) International C. elegans Meeting "The Onchocerca volvulus glutathione S-transferase 3 (Ov-GST-3) confers an increased resistance to oxidative stress in transgenic Caenorhabditis elegans"

Andreas Kampkoetter, Britta Leiers, Sarah Hegi de Castro, Lars-Oliver Klotz, Kimberly Henkle-Duehrsen, Chris Link, Helmut Sies

[International C. elegans Meeting, 2001]

Onchocerca volvulus is a parasitic nematode which is infectious for humans, resulting in the disease called River Blindness or Onchocerciasis. Onchocerciasis is the worlds second cause of blindness, due to an infection. A total of 18 million people are infected with this parasite, 120 million live at risk, and roughly half a million are completely blind due to this disease. The adult parasites live for many years in nodules under the skin of the human host and produce large numbers of offspring called microfilariae. These microfilariae migrate in the subcutaneous tissue, can be swallowed by a blood sucking black fly and mature to juveniles. During another blood meal of the mosquito, the larval parasites can enter the human host to continue the life cycle. Like all aerobic organisms O. volvulus must defend itself against cellular oxidative stress generated by metabolism and incomplete respiration. Therefore, protective enzymatic and non-enzymatic antioxidants exist that prevent, repair or eliminate oxidative damage. Detoxification enzymes, like the glutathione S-transferases (GSTs), minimize the concentration of oxidatively damaged cellular components, particularly oxidized DNA, proteins and lipids. Nevertheless, the parasite is able to persist for a long time in the human host even though it is heavily attacked by the immune system of the human host. This attack includes the release of a variety of reactive oxygen species from stimulated host phagocytic cells which cause an external oxidative stress. This means that O. volvulus must have evolved additional mechanisms to protect itself against the immune response of the human host to ensure its own survival. A glutathione S-transferase of O. volvulus ( Ov -GST-3), shown to be upregulated in response to oxidative stress, is suspected to be involved in the defense against the host's immune system. To have the chance to investigate the function of this molecule in more detail, transgenic C. elegans (AK1) containing the Ov -GST-3 gene under the control of a C. elegans promotor (let-858) were generated. The comparison of AK1 worms and control worms on plates with artificially produced oxidative stress revealed significant higher survival rates of the transgenic line. The extent of lipid peroxidation, one type of damage caused by oxidative stress, is also reduced in the transgenic worms as determined by the measurement of the level of malondialdehyde (MDA). These findings clearly demonstrate that the overexpression of Ov -GST-3 confers an increased resistance to oxidative stress in the transgenic AK1 worms.

Sarwar Hashmi et al. (2002) East Coast Worm Meeting "Cathepsin Z is expressed in major hypodermal cells of C elegans and has a role in worm's molting/development"

Sara Lustigman, Sarwar Hashmi, Jun Zhang

[East Coast Worm Meeting, 2002]

We identified two C elegans homologues Ce-cpz-1 and Ce-cpz-2 of Onchocerca volvulus cathepsin Z-like enzyme. O. volvulus is the causal agent of river blindness in human. In O. volvulus it was postulated that Ov-CPZ (LOVCP) has a role in molting of L3 to L4 larvae. We used C. elegans as a model to study the functional roles of CPZ-1 (82% identity to Ov-CPZ) in C. elegans and thus to elaborate on its potential function of this gene family in nematodes. We generated several lines of transgenic worms by injecting either gfp or LacZ reporter constructs containing a 2.1 kb encoding sequences plus 1.1 or 2.3 kb upstream sequences of the Ce-cpz-1 gene (Ce-cpz-1:gfp or lacZ) into wild type C. elegans and studied the expression pattern of the gene during worm's development. The expression of cpz-1:gfp or lacZ fusion gene was in the hypodermal and pharyngeal muscle cells the same regions where the native protein was identified; few cells in early embryo. At late-stage embryos and other larval stages CPZ-1 localized in the hypodermal region as well as in the pharyngeal lining of the worms. In thin sections of C elegans, the native CPZ-1 was present in the cuticular region of the worm and in molting larvae the protein was localized between the old and the new cuticle similar to what was observed in O volvulus. To test whether Ce-cpz-1 is essential for worm's development, we injected dsRNA prepared from relevant cDNA of Ce-cpz-1 gene into the gonad of young adult hermaphrodite. We found that 100% of injected hermaphrodites gradually became paralyzed over three day period after injection, produced several embryos and later died. Embryonic defects and lethality was seen also in laid embryos. 60% of L4 soaked animals had phenotypic defects very similar to injected animals. Although embryonic defects and lethality were observed in some laid embryos, majority of the embryos escaped lethality, developed to L2, but had severe defect in the L2/L3 molt. To determine more precisely whether Ce-cpz-1 has a role in larval molts, L1 larvae were soaked in dsRNA. A number of phenotypes were observed 8-24 hours after transfer of dsRNA treated larvae onto agar plates. Majority of the soaked L1 had siginificant but transient reduced growth, and some of the affected larvae did not go through L1/L2 molt. We suggest that CPZ-1 functions in molting either by taking part in the process of ecdysis or in the synthesis of new cuticle, similar to what was observed in O. volvulus. Its role however, during embryonic development is still under investigation.