Pispa, Johanna et al. (2021) International Worm Meeting "Identification of a potential regulator of proteasome nuclear localisation"

Pispa, Johanna, Mikkonen, Elisa, Arpalahti, Leena, Holmberg, Carina I

[International Worm Meeting, 2021]

The proteasome is the major enzymatic machinery of the ubiquitin-proteasome system (UPS), which is required for degradation of misfolded, aged and unnecessary proteins. It is a multisubunit complex, consisting of a core particle, 20S, capped by regulatory particles, 19S. The barrel-shaped 20S particle is responsible for the proteolytic activity, whereas the 19S particles bind polypeptide substrates via polyubiquitin chains, remove the ubiquitins for recycling, and unfold the polypeptides prior to transferring them into the lumen of the core particle. Recent studies by us and others have shown that the proteasome's expression and activity are differentially regulated in specific spatial, temporal and physiological contexts in vivo to facilitate its essential function in maintenance of cellular proteostasis. Subcellularly, the proteasome is localised both to the cytoplasm and in the nucleus. The nuclear role of the proteasome is not fully understood, but it may involve also functions not related to its proteolytic activity. In order to discover new regulators of the proteasome, we have performed a genome-wide C. elegans RNAi screen using a transgenic intestinal polyubiquitin-binding reporter strain previously published by our laboratory. This fluorescent reporter has been shown to respond to changes in the endogenous amount of polyubiquitinated proteins, and can be used as a tool for identifying disturbances in protein degradation mechanisms in live animals. After exclusion of hits affecting protein synthesis or having a developmental phenotype, we obtained 33 potential candidate genes. Of these, downregulation of 19 hits increased the fluorescence level of the polyubiquitin-binding reporter animals whereas 14 hits decreased the fluorescence. Particularly, one RNAi clone stood out as it not only resulted in increased reporter fluorescence but also changed the reporter's subcellular localisation from being expressed throughout the intestinal cell into a predominantly nuclear location. Immunostaining for proteasome 20S subunits showed that the intestinal nuclei have reduced amount of the proteasome, which could cause the accumulation of polyubiquitinated substrates. Further characterization of the potential regulator will be presented.

Masler EP et al. (1998) East Coast Worm Meeting "FMRFamide-Like Immunoreactivity in Heterodera glycines: Changes During Development and Comparison with Caenorhabditis elegans and Panagrellus redivivus."

Sardanelli S, Kovaleva ES, Masler EP

[East Coast Worm Meeting, 1998]

A peptide family with a characteristic arginine-phenylalanine-amide C-terminal motif ("FMRFamide") and myoactive properties has been described for both free-living and animal parasitic nematodes. Primary structures reported to date probably represent only a fraction of those present in these animals, and their neuromodulatory nature suggests a potentially extensive involvement of this peptide family in the behavior, development and reproduction of nematodes. Immunocytochemical studies (Atkinson et al. 1988) detected the presence of FMRFamide-like peptides in the nervous system of the plant parasitic nematode Heterodera glycines, but no analyses of these peptides from plant parasitic nematodes have been available. We have developed methods for the collection and extraction of H. glycines and the detection of FMRFamide-like peptides using a competitive ELISA. HPLC-ELISA procedures reveal species-specific differences in the quality and quantity of FMRFamide-like peptides from H. glycines and the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus. In addition, changes in FMRFamide-like peptide immunoreactivity were observed during H. glycines development and maturation. The significance of these developmental changes and species differences is discussed, and arguments for the involvement of FMRFamides in parasitic nematode growth and development are presented.

Chi-Hao Luan et al. (2003) International Worm Meeting "High Throughput Recombinant Protein Expression for C. elegans"

Ming Luo, Rita J Gray, Lawrence J DeLucas, Wenying Huang, Chi-Hao Luan, James B Finley, Shihong Qiu

[International Worm Meeting, 2003]

C. elegans is one of the most investigated multicellular organisms. The complete sequencing of the worm's genome and the cloning of its protein encoding open reading frames (ORFs) using the Gateway system (Invitrogen) greatly facilitates the genomic and proteomic study of the model Eukaryotic system. Instead of traditional approach whereby genes and gene products are studied a few at a time, the entirety of 19,099 predicted C. elegans genes are selected by Southeast Collaboratory for Structural Genomics as targets in a genomic-scale structural biology initiative. To support this effort, we are establishing a state-of-the-art facility for high throughput recombinant protein expression and refolding. We developed an automated recombinant protein expression system using the Gateway cloning and expression technology. The system, based on a strategy of step-wise automation on an integrated robotic platform with in-house technology development, successfully automated nearly all aspects of recombinant protein expression. To achieve total automation, we investigated and optimized conditions for miniaturization of bacterial growth, DNA plasmid propagation, protein expression and purification in 96-well plate of 0.6 ml. We developed a novel transformation platform including a robotic device for heat-shock in 96-well plate, as well as an ElectroTip for automated electroporation. We developed fully automated dynamic ELISA for profiling protein expression level and solubility, which increased the dynamic range of 96-well plate ELISA up to two orders of magnitude. We also introduced sigma, a new parameter defined as the ratio of ELISA signal in soluble fraction to that of insoluble fraction, to predict a proteins solubility and how to improve it in scale up production, and to predict the easiness of refolding if the protein is not readily soluble. The solubility profiling is almost 100% accurate as confirmed by 1 L scale up expressions. Currently, 384 unique C. elegans ORFs in Gateway entry vector are processed in 3-weeks, processing four 96-well plates in parallel. This throughput can be further increased in an assembly line fashion by staggering new plates in the process each day as demand dictates. To increase the number of soluble proteins, we developed new Gateway compatible destination vectors, utilized multiple E. coli strains, and are exploring alternative expression systems, for instance, the Baculovirus system. The automation developed using the E. coli expression system can be readily adapted to other expression systems.

Sithigorngul P et al. (1995) International C. elegans Meeting "AF1-like and other -RFamide like immunoreactive substances in Caenorhabditis elegans."

Sithigorngul P, Stretton AOW

[International C. elegans Meeting, 1995]

A high sensitivity dot-ELISA technique has been used to detect -RFamide like peptides in an extract of C. elegans fractionated by HPLC. Monoclonal antibodies AF1-243 (highly specific to AF1), AF2-003 (detects AF2), AF3-442 (recognizes the -VLRFamide family of peptides) and AF1-62 (recognizes -RFamide peptides) were used to monitor the fractionation. AF1-like immunoreactivity was detected in the fractions with an elution time similar to AF1 in Ascaris. Neither AF2 nor -VLRFamide like immunoreactive substances were found, however numerous -RFamide like immunoreactive peptides were revealed with antibody AF1-62. This indicates that additional of-RFamide like peptides, besides those encoded by the flp-1 gene (Rosoff et al., 1992) are present in C. elegans. Purification of -RFamide like peptides from C. elegans is in progress.

Link CD (1996) West Coast Worm Meeting "MAKING AMYLOID WORMS MORE SENILE?"

Link CD

[West Coast Worm Meeting, 1996]

Transgenic worms containing a signal peptide/ beta peptide 1-42 minigene (derived from the human Amyloid Precursor Protein, APP) driven by the unc-54 promoter/enhancer produce intracellular immunoreactive beta peptide deposits that react with the amyloid specific dye thioflavin S. Animals containing an identical construct expressing the 1-40 version of the beta peptide (lacking two C-terminal amino acids) also produce immunoreactive beta peptide deposits, but these deposits do not react with thioflavin S. Recent experiments have been directed towards: 1) determining if the difference in thioflavin reactivity between transgenic lines expressing the 1-42 and 1-40 is due to quantitative or qualitative differences in the expressed peptide, 2) determining what the engineered signal peptide is (or isn't) doing in these constructs, and 3) investigating whether formation of thioflavin-reactive (amyloid) deposits can be enhanced by co-expression of human apolipoprotein E 4, which is strongly associated with increased Alzheimer's Disease susceptibility and has been postulated to directly interact with beta peptide to form amyloid. Quantitative ELISA assays done in collaboration with the lab of S. Younkin (Mayo Clinic) indicate that the failure of beta 1-40 peptide to make amyloid deposits is due to qualitative differences in the expressed peptide (not due to lower overall levels of peptide expression). However, both peptide versions also show evidence of N-terminal modification, raising questions about the role of the signal peptide in beta peptide expression, and complicating interpretation of the ELISA data. I have therefore generated a new series of transgenic constructs with the signal peptide replaced with an initiator methionine, employing unc-119, mec-7, and unc-54 promoters. For currently unknown reasons, none of these lines express immunohistochemically detectable beta peptide. Extrachromosomal transgenic lines have also been constructed that coexpress an unc-54/Apo E4 construct and either of the original unc-54/signal peptide/beta peptide constructs. Although immunohistochemistry clearly demonstrates that these lines coexpress both proteins in muscle cells, significant enhancement of thioflavin S-reactive deposits has not been observed in the extrachromosomal lines examined so far. Integrated lines are currently being generated to more carefully examine the interaction of Apo E and beta peptide in this model system.

Singh, Akanksha (2021) International Worm Meeting "CRISPR- Nanobodies from C. elegans as an therapeutic approach for Erythroblastosis Fetalis"

Singh, Akanksha

[International Worm Meeting, 2021]

Erythroblastosis fetalis is a consequence of incompatibility amongst Rh-negative antigen from mother and Rh-positive antigen of foetus thus resulting in hemolysis. The hemolysis usually results in respiratory problems, neurological disorders or even heart failure. C.elegans is known to be homologous to some extent with human genome and produces monoclonal antibodies. The nanobodies from C. elegans are engineered with CRISPR Cas 9 technology where Cas 9 protein has been incorporated into nanobodies that will accelerate its efficiency. The engineered nanobodies will target genes like RHD and RhCE that are prominent in RBC membranous environment. The nanobodies with Cas-9 might block the interaction of Rh-negative antibodies with Rh-positive antibodies of foetus as by refusing the recruitment of Rh-negative antibodies in placenta. Antibodies from the C. elegans are extracted and engineered to produce nanobodies with encoded Cas 9 protein. The haemoglobin obtained from foetus are mixed with haemoglobin from mother with Rh-antigens in vitro. After a period of 24-48 hours, when engineered nanobodies are treated with blood by ELISA technique indicated reduced count of Rh-positive antibodies thus blocking RHD gene not allowing the interaction with Rh-negative antibodies. The aim of the study was to study molecular interaction occurring after exposure of nanobodies to Rh- antigens present in foetal blood. Keywords: Erythroblastosis fetalis, Rh-negative, hemolysis, C. elegans, CRISPR Cas 9, RHD , RHCE, haemoglobin, nanobodies.

Brad D Barrows et al. (2005) International Worm Meeting "Analysis of a gene associated with resistance to a specific crystal protein produced by Bacillus thuringiensis"

Brad D Barrows, Raffi V Aroian

[International Worm Meeting, 2005]

The crystal proteins made by Bacillus thuringiensis (Bt toxins) are important, naturally occurring agents for the control of insects that eat crops and carry disease. Mutant animals (C. elegans) resistant to the nematode-active crystal toxin (Cry 5B) can be isolated, providing the first genetic system for the study of these important toxins. Resistant animals are called bre mutants. All of the four bre mutants cloned to date (bre-2 bre-5) encode putative glycosyltransferases. The bre-1 gene has been found to be an exception to the pattern established by other bre genes. Instead of functioning as a glycosyltransferase involved in a carbohydrate biosynthetic pathway, bre-1 appears to be involved in a monosaccharide biosynthetic pathway. Collected data suggests that bre-1, like the other bre genes, is involved in the biosynthesis of a glycolipid receptor, but unlike the other bre genes, the involvement of bre-1 is less direct. bre-1 is also intriguing in other aspects. Unlike the other bre mutants, bre-1 has an overall reduced brood size that is more severe than that of other resistance mutants in addition to also having a weak resistance phenotype. bre-1 mutant animals have been characterized by examining pharyngeal pumping in response to toxin, temperature sensitivity, intestinal injection of specific sugars, glycolipid analysis by thin layer chromatography, and ELISA-based receptor binding studies. These results will be presented.

Wong WL et al. (1997) International C. elegans Meeting "PATTERN OF MAB-21 GENE EXPRESSION IN CAENORHABDITIS ELEGANS IS REGULATED DEVELOPMENTALLY."

Lee EF, Wong WL, Chow K-L

[International C. elegans Meeting, 1997]

The differentiation of the male specific peripheral sensory organs in C. elegans is a complex process. A gene mab-21, which encodes a protein of 386 amino acids, is required for the choice of alternate cell fates of several cells in the male tail through its interaction with homeobox containing genes. Tagged MAB-21 protein has been shown to be localized in the nucleus with residual expression in the cytoplasm in nematode by both galactosidase assay and anti-b-galactosidase antibody staining against the fusion protein in transgenic worms. Such observation has also been confirmed by immnostaining in yeast and subcellular fractionation analysis. Using antibody against b-galactosidase protein, we have analyzed the expression pattern at different stages in transgenic lines. mab-21 promoter is active in distinct cells in the body wall muscle around the pharynx, lateral hypodermis, and male tail. The observation, which is at a higher resolution, is consistent with our previously reported galactosidase staining result. To confirm the expression pattern at the protein level, a maltose binding protein (MBP)-MAB-21 fusion protein was expressed and purified from bacteria for raising antisera from mice, rats, and rabbits. ELISA and western blotting analysis showed the antisera could detect both the fusion protein expressed in bacteria and affinity purified fusion protein. However, only the rabbit serum could recognize the MAB-21 protein in worm. This antiserum is currently used to determine the expression pattern of MAB-21 protein in wildtype and mutant nematode by immunostaining. We intend to perform co-immunoprecipitation to identify additional cellular components that interact with the mab-21 products.

Nicolai, M.M. et al. (2019) International Worm Meeting "Investigations on manganese-induced oxidative stress and its impact on DNA repair in C. elegans."

Nicolai, M.M., Bornhorst, J., Schwerdtle, T., Baesler, J., Aschner, M.

[International Worm Meeting, 2019]

The association between manganese (Mn)-induced oxidative stress and neuropathological diseases due to chronic Mn overexposure has been discussed extensively. Nevertheless, the underlying mechanisms have yet to be explained. Since an excessive production of reactive oxygen and nitrogen species (RONS) can lead to interactions with macromolecules, such as DNA, it has been proposed that DNA damage and defective DNA repair may contribute to neurological dysfunction. Due to the widely recognized difficulty in quantifying oxidative stress/ damage markers, multiple endpoints were investigated in C. elegans. Cellular DNA damage response poly(ADP-ribosyl)ation and the GSH/GSSG ratio were quantified by established and validated LC-MS/MS methods and indicate Mn-induced oxidative stress, while Mn seems not to affect DNA damage response as assessed by PARylation. Gene expression studies further revealed that Mn exposure results in increased mRNA levels of genes for initial recognition of lesions in base excision repair (BER). Using transgenic BER mutants points out that loss of nth-1, normally initiating the removal of oxidized purines and pyrimidines, results in hypersensitivity with respect to Mn-induced lethality, as well as Mn-induced oxidative stress compared to wildtype (WT) worms. To further investigate the hypersensitivity of this particular BER mutant, multiple methods for qualification and quantification of oxidative DNA base modification 8-oxodG were established. While 8-oxodG levels in the nth-1 mutants showed no difference to WT worms using the semi-quantitative ELISA, immunofluorescence staining indicated higher levels compared to WT worms. Further mechanistic studies and improvement of existing methods need to be established to mechanistically clarify the development of manganism and the role of aberrant DNA repair mechanisms in such etiologies.

Valenzuela MRL et al. (1991) International C. elegans Meeting "STRUCTURES AND FUNCTIONS OF THE REPEATING MOTIFS IN TWITCHIN."

Valenzuela MRL, Benian GM

[International C. elegans Meeting, 1991]

Twitchin, the 753,570 Da polypeptide encoded by the unc-22 gene, was the first member of a growing family of intracellular and mostly muscle proteins, found in diverse animals, composed of multiple copies of motif I (fibronectin type III domain-like) and motif II ( immunoglobulin C2 domain-like). To date, this family includes, smooth muscle and non-muscle myosin light chain kinases, titin, C-protein, 86 kDa protein, skelemin and probably insect projectin. The similarities to motifs found in extracellular and cell surface proteins engaged in recognition or adhesion suggests that motifs I and II of muscle proteins are also involved in binding--probably to myosin, other thick filament components, and also binding of these proteins to themselves and other family members. We hope to be able to demonstrate binding of small numbers of these motifs to thick filament components both in vitro and in vivo. Crystallographic structure determination will be done in collaboration with Pamela Bjorkman (Cal Tech). Using the pGEX- 2T vector, we have been able to express and purify large quantities of motif I, motif II and the predominant triplet I,I,II as glutathione-S- transferase (GST) fusion proteins in E. coli. We have succeeded with the thrombin cleavage of the motifs from GST and are trying ELISA-type binding assays with nematode and rabbit myosin. For in vivo 'binding' studies, we have cloned motif I, motif II, the trio I,I,II and the C- terminal 5 motif IIs into Andy Fire's pPD30.38 vector which directs body wall muscle expression. These constructs will be microinjected into unc-22 nulls and transgenics examined by immunofluorescence for co-localization to myosin.