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.

David R. Bell et al. (2006) European Worm Meeting "The Function of Latrophilin in C. elegans"

S. van der Poel, P.N.R. Usherwood, D. Kendall, J. Davy, A. Ademola, I.R. Duce, D. de Pomerai, David R. Bell, I. R. Mellor

[European Worm Meeting, 2006]

David R. Bell, A. Ademola, J. Davy, S. van der Poel, D. Kendall, P.N.R. Usherwood, I.R. Duce, I. R. Mellor and D. de Pomerai. Black Widow spider venom (BWSV) contains latrotoxins that induce catastrophic neurotransmitter release in mammals, and is known to bind with high affinity to three neural proteins in mammals, including latrophilin. We have investigated C. elegans as a model system for studying the function of these proteins, and their role in regulating neurotransmitter release by latrotoxins.. We have shown that latrophilin is required for the lethality of BWSV in C. elegans by RNAi experiments. We now present data on the characterisation of null mutants of the C. elegans latrophilin, lat-1, illustrating the function of this gene in C. elegans, and show that lat-1 nulls are resistant to emodepside. The expression of latrophilin-GFP constructs is examined, and compared with the phenotypes seen in null mutants. Genetic interactions between endogenous signaling pathways and the lat-1 nulls have been examined by the construction of double mutant worms, revealing novel facets of lat-1 function.

Stephen C Pak et al. (2004) East Coast Worm Meeting "SRP-2 is an intracellular serpin that participates in postembryonic development"

Anthony C Clark, David R Mills, David J Askew, Christopher Tsu, Stephen C Pak, Vasantha Kumar, Gary Silverman, Yuko S Askew, Cliff J Luke

[East Coast Worm Meeting, 2004]

Serpins are high molecular weight serine proteinase inhibitors that inactivate target proteinases by a suicide substrate-like mechanism. Serpins can be categorized into two broad groups, intracellular and extracellular. Most extracellular serpins function in the circulation and regulate proteinases involved in blood coagulation, fibrinolysis, complement activation, inflammation, and extracellular matrix remodeling. In contrast, serpins belonging to the intracellular group have been implicated in regulating apoptosis, tumor progression, and metastasis. However, their functions in terms of an intact organism have not been well defined. Database analysis of the C. elegans genome reveals the presence of several intracellular serpins. To determine whether nematode serpins function as proteinase inhibitors, one family member, srp-2 , was chosen for further characterization. Biochemical analysis of recombinant SRP-2 protein revealed SRP-2 to be a potent inhibitor of the lysosomal cysteine proteinases, cathepsins K, L, S, and V. Analysis of temporal and spatial expression indicated that SRP-2 was present during early embryonic development and highly expressed in the hypoderm of larval and adult worms. To determine whether SRP-2 plays a role in C. elegans development, null mutants and transgenic animals overexpressing SRP-2 were generated. Whereas null mutants showed no overt developmental phenotype, of the animals overexpressing SRP-2 ~30% displayed developmental abnormalities characterized by early (L1/L2) larval arrest/death, slow growth and molting defects at 25 degC. However, at 27 degC the phenotype was considerably more penetrant with ~95% of the animals being abnormal. Although the mode of SRP-2 action is currently unknown, we hypothesize that SRP-2 plays a role in regulating proteinase activity during development and that an imbalance in the serpin/proteinase equilibrium has deleterious consequences during C. elegans development.

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Kohn RE, Rand JB, McManus JR, Moulder GL, Duke A, Barstead RJ, Duerr JS

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.

John Peloquin et al. (2007) International Worm Meeting "Worms and Filthy Lucre: Applying C. elegans to natural product development in industry."

Stuart Reeves, John Peloquin

[International Worm Meeting, 2007]

Diamond V Mills is a medium sized Agricultural Biotechnology company with more than 63 years of success in producing dietary supplements and ingredients. Recently the company has moved into human nutrition through its formation of Embria Health Sciences. Thus, the product repertoire has increased and is increasing. Our products arise naturally as the result of subtle alterations of fermentative processes, which are still not completely understood. Furthermore, their effects on animal biology are pleotropic. This presents some serious economic challenges in that far more product prototypes can be produced than economically tested in vertebrate models to demonstrate efficacy. Because many biochemical and signal transduction pathways are conserved between C. elegans and other animals, we have begun to develop C. elegans-based medium through-put screens to identify new product prototypes. We present preliminary data on anti-aging effects of an Embria product as an example of our future goals. (this work was initiated with technical assistance from Mark A. Wilson and Cathy Wolkow of the NIA).

Schein JE et al. (1997) International C. elegans Meeting "POSITIONING OF ESSENTIAL GENES IN THE lin-3 - let-60 INTERVAL"

Franz NW, Rose AM, Janke DL, Bilkhu N, Baillie DL, Schein JE

[International C. elegans Meeting, 1997]

The lin-3 - let-60 interval of the right arm of chromosome IV contains thirteen essential gene mutations and 22 overlapping sequenced cosmids. The Sanger Center has nearly finished sequencing the entire interval. Analysis of cosmid sequences shows over one hundred potential coding regions. Of the coding regions with proposed protein function, more than 50% have strong homology to human genes. Most notably, the putative genes include three phosphatases, four protein kinases, a kinesin, and a cluster of histones. C. elegans strains carrying sequenced cosmids (in extrachromosomal arrays) have been constructed (see abstract for D. Janke et al., this meeting) and will be used in rescue experiments involving the essential genes in this interval. lin-3 and let-60(ras), the two genes bracketing this interval, were previously placed on the physical map by others. One of the remaining eleven essential genes has been already positioned to a cosmid: let-70(ubc2) (in collaboration with Mei Zhen and Peter Candido). By the time of the meeting, we anticipate that several of the remaining mutations will have been positioned to cosmids. This work is being funded by a grant from the Medical Research Council to Ann Rose and David Baillie and by a grant from the Natural Sciences & Engineering Research Council to David Baillie.

HA Tissenbaum et al. (1999) International C. elegans Meeting "Using model organisms to identify genes that affect life span"

L Guarente, HA Tissenbaum

[International C. elegans Meeting, 1999]

Previous work in C. elegans has identified a number of mutants that affect life span. We are trying to identify new components of the aging mechanism by isolating mutations in genes that are conserved in C. elegans and have previously been shown be important in the aging process of other eukaryotic organisms. 1) Werner Syndrome : People with Werner Syndrome (WRN) show many signs of premature aging (reviewed in Yu et al. 1997). The WRN protein is a member of the RecQ family of helicases. In yeast, mutations in the WRN homolog, SGS1 , cause yeast to age prematurely (Sinclair et al. 1997). Four members of the RecQ family have been identified in the C. elegans database. We are currently isolating knockout mutations of all four of the family members. As well, we are generating GFP fusions and doing RNAi to help elucidate the different functions of these four genes. These studies will help to determine if any of the four genes is the true WRN homolog in C. elegans and whether they function to control aging. 2) SIR2 homologs: Studies from yeast ( S. cerevisiae ) have identified the SIR proteins (SIR2, 3, 4) as important components for yeast aging (reviewed in Sinclair et al. 1998). SIR2, however, is unique among the SIR proteins in several respects including that it is highly conserved from yeast to man. There are two SIR2 homologs identified in the C. elegans database. To understand the role of C. elegans SIR2 in aging, we are generating knockout mutations and GFP fusions as well as performing RNAi. We are also using the candidate gene approach to identify mutants in the SIR2 homologs. References: Sinclair D.A., Mills K., and Guarente L. (1997). Science 277 :1313-6 Sinclair D.A., Mills K., and Guarente L. (1998). Annu Rev Microbiol 52 :533-60 Yu C.E., Oshima J., Wijsman E.M., et al. (1997) Am J Hum Genet 60 :330-41

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

A V Bicknell et al. (2002) European Worm Meeting "The C. elegans F47F2.1 gene encodes a cyclic AMP-dependent protein kinase (PK-A) catalytic subunit"

A V Bicknell, M J Fisher, M Tabish, R A Clegg, H H Rees

[European Worm Meeting, 2002]

PK-A mediates all known effects of cyclic AMP on cellular activity in eukaryotes. The holoenzyme is an inactive tetramer of two regulatory (R) subunits and two catalytic (C) subunits. Following binding of cyclic AMP to the R subunits, dissociation of active C-subunits occurs. In mammals, , and isoforms of C-subunit, encoded by different genes, have been identified.

Angelo RG et al. (1997) International C. elegans Meeting "DISCOVERY OF A POTENTIAL ANCHOR/SCAFFOLD PROTEIN FOR C. ELEGANS PROTEIN KINASE A (PKA)"

Angelo RG, Rubin CS

[International C. elegans Meeting, 1997]

C. elegans PKA is composed exclusively of catalytic and regulatory (R) subunits encoded by the kin-1 and kin-2 genes. Since C. elegans lacks other PKA isoforms, this enzyme must disseminate signals carried by cAMP to all cell compartments. Approximately 60% of total R (PKA) is in the particulate fraction of disrupted C. elegans, indicating that protein/protein interactions may diversify PKA signaling by anchoring the kinase at specific intracellular locations. Co-localization of PKA with upstream activators and/or downstream effectors can create target sites for cAMP action. To understand the mechanism of PKA localization in C. elegans, a cDNA expression library was screened with recombinant radiolabeled-R (0.5 nM) to obtain high-affinity binding proteins. A cDNA encoding a novel, 143 kDa A kinase anchor protein (AKAP1) was retrieved and sequenced. The corresponding gene (rap-1) is located in LGII and contains 17 exons. AKAP1 is an acidic protein (pI=4.4) that is unrelated to previously characterized proteins. C. elegans R is avidly bound by both soluble and immobilized fragments of AKAP1. Competition binding studies indicate that C. elegans R is a preferred ligand, whereas mammalian RII and RI isoforms are only weakly sequestered. Scatchard analysis yielded a Kd value of ~10 nM for the R/AKAP1 complex. Deletion mutagenesis, coupled with protein expression in E. coli and in vitro assays, demonstrated that residues 235 to 255 govern high-affinity binding of R by AKAP1. A hydrophobic surface generated by amino acids with branched aliphatic side chains may be a key determinant of R binding activity. Site directed mutagenesis will pinpoint essential residues involved in PKA binding. Studies aimed at identifying the AKAP1 binding site on R are also in progress. High-affinity anti-AKAP1 IgGs are being used to determine (a) whether AKAP1 expression is developmentally-regulated and cell- specific and (b) the relationship between R (PKA) and AKAP1 in vivo.