Garcia-Sanchez, Juan A. et al. (2021) International Worm Meeting "Ubiquitin-related modifying enzymes in the regulation of HLH-30 signaling during S. aureus infection"

Harding, Benjamin, Ewbank, Jonathan J., Belougne, Jerome, Garcia-Sanchez, Juan A., Visvikis, Orane, Loubatier, Celine, Boyer, Laurent

[International Worm Meeting, 2021]

Ubiquitination and ubiquitination-like modifications are conserved post-translational modifications that influence virtually all physiological processes, including innate immunity. They are controlled by hundreds of ubiquitin (Ub) and Ub-like modifying enzymes that regulate their targets by modulating their stability, localization or activity. In recent years, the importance of these enzymes in C. elegans' innate immunity has been revealed, with the identification of key Ub-related regulators of innate immune pathways, as well as Ub-related effectors in the host response (Garcia-Sanchez et al, 2021). Little is known, however, about the role of these processes in the immune response to the intestinal bacterium Staphylococcus aureus, which is coordinated by the transcription factor TFEB/HLH-30 (Visvikis et al, 2014; Najibi et al, 2016). Our research focuses on deciphering the importance of Ub-modifying enzymes in regulating this immune pathway, as well as their role as effectors in the host response. Firstly, we undertook an RNAi-based screen to identify new immune regulatory enzymes. We generated a fluorescent reporter strain which allowed us to screen the effect of 216 enzymes on the activity of HLH-30 during S. aureus infection. We identified 22 genes whose downregulation reduced reporter fluorescence. To distinguish between specific and more general regulators, we performed a parallel screen using the model of epidermal infection by the fungus Drechmeria coniospora, which triggers an immune response mainly controlled by the p38 MAPK/PMK-1 pathway (Zugasti et al, 2014; 2016). We found 6 enzymes that are common to both screens; the remaining 16 enzymes are potentially specific regulators of the HLH-30-dependent immune response to S. aureus. Secondly, to identify Ub-related immune effectors important for the response to S. aureus infection, we analyzed previously published RNA-seq data (Visvikis et al, 2014) and determined that 16 out 19 Ub-related enzymes induced during infection are controlled by HLH-30. Focusing on 3 conserved E3 Ub-ligases, we confirmed their HLH-30 dependent induction by qPCR. Interestingly, using survival assays, we could demonstrate their importance in C. elegans' host defense against S. aureus infection suggesting they play a specific effector role in infection. Altogether, these results demonstrate the importance of Ub-related enzymes in HLH-30 dependent host response to S. aureus infection and have revealed specific and general Ub-related mechanisms in the regulation of immune pathways.

Shingo Tazawa et al. (2004) East Asia Worm Meeting "Functional analysis of C.elegans DUO-1, a ubiquitin specific protease"

Hirofumi Tanaka, Hideshi Inoue, Kenji Takahashi, Mikiko Kusano, Shingo Tazawa

[East Asia Worm Meeting, 2004]

Ubiquitin specific proteases (USPs) of family C19 is important for the regulation of the processes in which ubiquitin (Ub) is involved. C. elegans has 26 genes that code for the proteins of this family. We performed RNAi on 21 of them. The most severe effect was observed by RNAi of F09D1.1 (100% embryonic lethal). The gene product of F09D1.1, however, is predicted to be U4/U6.U5 tri-snRNP-associated protein with no protease activity because the catalytic residue is not conserved. Some mildly abnormal phenotypes were observed by RNAi of several other genes. Feeding RNAi of duo-1 caused no abnormality in the P0 and F1 animals but 80% lethality on the F2 embryo. The knockout mutant duo-1 ( tm934 ) showed 80% larval lethality. Thus, duo-1 is thought to be important for viability of C. elegans . The duo-1 gene codes for a protein of 822 amino acid residues, which is predicted to have a unique domain structure; it has an insertion of an OTU-like cysteine protease domain in the USP domain. Since OTU-like cysteine proteases have been reported to be a novel type of deubiquitinating enzyme, DUO-1 is presumed to have dual catalytic sites for ubiquitinated proteins. To confirm this, the OTU-like cysteine protease domain was expressed as a GST-fusion protein in E. coli and purified. The recombinant OTU domain showed C-terminal hydrolase activity toward recombinant Ub fused with HSV and His-tags at the C-terminus, but not toward SUMO-1- and NEDD8-fused substrates. To evaluate the ability of each catalytic site to cleave branched poly-Ub chains, we performed in vivo isopeptidase assay by co-expression of HA-tagged Ub and DUO-1 in the COS7 cells. Upon expression of wild-type DUO-1, Ub-conjugate level in the transfected COS7 cytosol was found to decrease. When Cys223 and Cys607, the predicted catalytic residues in the USP and OTU domains, respectively, were individually replaced with Ala, a low level of Ub-conjugates was observed on each case, while the double mutation of C223A and C607A showed a high level of Ub-conjugates. These results suggest that each of the predicted catalytic sites of DUO-1 has isopeptidase activity toward polyubiquitylated proteins.

Jolanta Polanowska et al. (2006) European Worm Meeting "Regulation of BRC-1 - Dependent Ubiquitylation at DNA Damage Sites"

Jolanta Polanowska, Simon Boulton, Tatiana Garcia-Muse, Julie Martin

[European Worm Meeting, 2006]

Jolanta Polanowska1,2, Julie Martin1, Tatiana Garcia-Muse1 and Simon Boulton1. The BRCA1 tumour suppressor and its heterodimeric partner BARD1 constitute an E3-ubiquitin (Ub) ligase and function in DNA repair by unknown mechanisms. We have previously described C. elegans BRCA1 and BARD1 orthologues (brc-1 and brd-1, respectively) that possess many of the functional domains present in their human counterparts, including RING, ankyrin, and BRCT domains (Boulton et al., 2004). Consistent with conserved roles in DNA repair, BRC-1 and BRD-1 interact to form a heterodimer via their respective RING domains. To explore the mechanistic role of BRC-1 and BRD-1 in DNA repair processes we have characterized a C. elegans BRC-1/BRD-1 complex (CeBCD) purified by tandem immunoaffinity before and at different time points after IR-treatment. This approach is a first for C. elegans and demonstrates that protein complexes purified in this manner are amenable to biochemical analysis and can be used in combination with genetics and cell biology to accelerate functional discoveries. We present evidence that the CeBCD complex possesses an E3-Ub ligase that is activated on chromatin in response to IR-treatment and further demonstrate that the DNA damage checkpoint promotes association of the CeBCD complex with E2-Ub conjugating enzyme, Ubc5(LET-70), to form an active E3-Ub ligase in response to DNA damage. We also show that ubiquitylation events at DNA damage sites require brc-1, brd-1, ubc5(let-70), mre-11 and atl-1, thus providing in vivo evidence to support our biochemical analysis.. Boulton, S.J., Martin, J.S., Polanowska, J., Hill, D.E., Gartner, A. and Vidal, M. (2004) Curr Biol, 14, 33-39.

Chiaki Fujitake et al. (2005) International Worm Meeting "Enzymological and Functional Analysis of Ubiquitin C-terminal Hydrolases"

Kenji Takahashi, Yoshie Watanabe, Hideshi Inoue, Chiaki Fujitake

[International Worm Meeting, 2005]

Ubiquitin C-terminal hydrolases (UCHs) contribute to the ubiquitin (Ub) system by processing Ub precursors or by removing Ub from ubiquitinated peptides or small C-terminal Ub adducts, and are thought to play a central role for a number of cellular processes including cytoplasmic protein degradation, endocytosis, etc. However, the functions of UCHs are still uncertain. C. elegans has four genes coding for UCH; ubh-1~4. The ubh-1, ubh-2, and ubh-3 genes are predicted to be organized into an operon, and the encoded proteins are 32-38% and 34-38% identical to human UCH-L1 and UCH-L3, respectively, while UBH-4 is 47% identical to human UCH-L5 that is associated with 26S proteasome. Enzymological characterization: All the UBH proteins were expressed in E. coli as fusion proteins with a His-tag and purified. UBH-1, UBH-2, and UBH-3 showed C-terminal hydrolase activity toward C. elegans Ub and NEDD8 fused with HSV- and His-tags at the C-termini but not toward SUMO-1 fused with HSV- and His-tags. This substrate specificity is consistent with that of UCH-L3. UBH-4 showed significant but far less activity toward Ub and NEDD8. Expression patterns: We made ubh-1::GFP and ubh-4::GFP translational fusion constructs. In the case of ubh-1, GFP-reporter expression was observed in some neuronal cells including the HSN and DVB neurons. The expression pattern observed by immunostaining with anti-UBH-1 antibody was similar to that by the GFP-reporter analysis. The neuron-specific expression of UBH-1 is analogous to UCH-L1. On the other hand, ubh-4::GFP was observed to be expressed almost ubiquitously. Phenotype of ubh-1-deletion mutant: The mutant allele tm526 has a deletion in the 3 half of the ubh-1 gene so that the gene product is considered to have lost its function. By RT-PCR analysis, ubh-2 and ubh-3 are confirmed to be expressed in the deletion mutant ubh-1(tm526). The mutant animals have a slower rate of larval growth and have a smaller brood size (63%). Additionally, the defecation cycle of the knockout animals was longer (60 sec) than that of the wild-type animals (45 sec). Transgenic expression of ubh-1::GFP rescued the extended defecation cycle of the ubh-1(tm526) mutant animals. Conclusions: UBH-1, UBH-2, and UBH-3 are structurally and enzymatically related to human UCH-L1 and UCH-L3. Analogous to UCH-L1, mutations of which are linked to certain familial forms of Parkinsons disease, UBH-1 is important for neuronal functions.

Lee JH et al. (2000) East Coast Worm Meeting "Identification and characterization of a Ubiquitin C-terminal hydrolase in C. elegans"

Ahnn JH, Lee JH, Lee MH, Chung CH

[East Coast Worm Meeting, 2000]

Ubiquitin (ub), a protein of 76 amino acid residues, is extremely well conserved in all eukaryotic cells. It plays a key role in a variety of cellular functions, including the regulation of intracellular protein breakdown, cell cycle regulation, and stress response. Ubiquitins, either by themselves or conjugated to protein, can also be ligated to additional ub molecules to form branched poly-ub molecules. Ubiquitin C-terminal hydrolase (UCH) is a thiol protease that recognizes and hydrolyzes the peptide bond at the C- terminal glycine of ubiquitin. Since we are interested in identifying and characterizing UCH in C. elegans, we searched the worm genome database and located a putative homologue of UCH in the cosmid, ZK328 (LGIII). C. elegans UCH (ZK328.1) consists of 12 exons encoding 1041 amino acids and contains two functional domains that are well conserved in other organisms. As a first step to characterize the worm UCH, expression pattern was investigated using the GFP reporter system. ZK328.1 is shown to express in neuronal cells, muscle cells, and seam cells throughout the body. Northern blot experiments with total RNAs from wild type mixed-staged animals reveal two mRNA transcripts, the dominant one being 3.6 kb in size. To better understand the function of UCH in C. elegans, RNA-mediated interference (RNAi) technology is being carried out currently.

Eri Ikeda et al. (2007) International Worm Meeting "Ubiquitin C-terminal hydrolases is required for normal defecation."

Hideshi Inoue, Chiaki Fujitake, Eri Ikeda, Maiko Takahashi

[International Worm Meeting, 2007]

Rhythmic behaviors are fundamental biological phenomena of all organisms. Of these rhythmic behaviors, the rhythms less than a day such as heartbeat are referred to as an ultradian rhythm. Some of the ultradian rhythmic behaviors are regulated by changes in intracellular Ca2+ concentration, while the molecular mechanisms underlying the generations and regulations of them are not well understood. Although the ubiquitin (Ub)-proteasome system is known to mediate circadian rhythms of roughly 24 hours, it has not been reported to be involved in ultradian rhythm. Here, we show that ubiquitin C-terminal hydrolase (UCH) has an important role in defecation, one of the ultradian rhythmic behaviors of C. elegans. UCHs contribute to the Ub system by processing Ub precursors or by removing Ub from ubiquitylated peptides or small C-terminal adducts, and are thought to play a central role in a number of cellular processes including cytoplasmic protein degradation, endocytosis, etc. However, the functions of UCHs are still uncertain. C. elegans has four genes that code for UCH-like proteins. Among them, the ubh-1, ubh-2, and ubh-3 genes are predicted to be organized in an operon, and were shown to be expressed in some neuronal cells including HSN and DVB, which are responsible for egg-laying behavior and defecation respectively, and in intestinal cells by analyses using transcriptional GFP reporter and anti-UBH antibodies. The proteins encoded by these three genes are 32-38% identical to human UCH-L1 and UCH-L3. Enzymological assay using recombinant proteins demonstrated that all of them exhibit UCH activity equivalent with UCH-L3 rather than UCH-L1. The null mutant animals ubh-1(tm526) showed slower growth rate, smaller brood size (63%), and a longer defecation cycle than the wild-type animals. While the defecation cycle of the wild-type animals was approximately 45 sec at 25C, that of the ubh-1(tm526) mutants was about 60 sec. Transgenic expression of ubh-1::GFP in the mutant animals restored the defecation cycle, suggesting that the ubh-1gene is important for the defecation program. The defecation cycle was normalized also when UBH-1::GFP was expressed under the control of the promoter of the flr-4 gene, which is expressed in intestine. However, the promoter of the unc-47 gene, which is expressed in some neuronal cells including DVB, could not be substituted for the ubh-1 promoter in restoring the defecation cycle. These results suggest that the Ub system in the intestine is involved in control of the defecation cycle.

L Wille et al. (1999) International C. elegans Meeting "The Anaphase-Promoting Complex is Required for Meiosis I Anaphase"

M Abdolrasulnia, L Wille, D Shakes

[International C. elegans Meeting, 1999]

The mitotic metaphase to anaphase transition in both yeast and vertebrates is driven by a multimeric E3 ubiquitin (Ub) ligase known as the anaphase promoting complex, APC. APC drives mitotic progression by sequentially targeting specific substrates for destruction, but its meiotic role is poorly characterized. To analyze APC's role during C. elegans meiosis, we are depleting maternal stores of APC components by RNA-mediated interference. Our goal is to analyze all APC genes in C. elegans , with initial studies focusing on APC1(BIME) and APC2. APC1 is postulated to be a negative regulator of mitosis since bimE mutants enter mitosis prematurely and can bypass a G2 or S phase arrest (Osmani et al., 1988), subsequently arresting in mitotic metaphase. RNAi of the APC1 C.elegans homolog (W10C6.1) resulted in fertilized oocytes which arrest in meiosis metaphase I (Chase et al., 1998). Later studies using a different RNA preparation, resulted in "weaker" phenotypes, including embryos with cytokinesis defects and hatching larvae which develop into sterile adults. Such adults are often severely GLP, but others have abnormal vulvals and small, highly disorganized gonads filled with differentiated gametes. We are currently testing whether the observed variability is dosage dependent. APC2 shares a conserved cullin domain with the G1-S Ub ligase component cdc53 which suggests that, like cdc53 , APC2 may interact directly with Ub conjugating enzymes (Yu et al., 1998). Consistent with its role as a core APC component, RNA-mediated interference of APC2 expression results in a strong 1-cell arrest phenotype in which maternal chromosomes lock in a prolonged meiosis metaphase I configuration. We have also observed weaker phenotypes, suggesting that APC2 RNAi effects are also dosage dependent. Our results suggest that APC function is not required for oocyte maturation, oocyte ovulation, fertilization, or the initial post-fertilization congression of the oocyte chromosomes. However APC function is critical during the meiosis I metaphase to anaphase transition, embryonic mitosis, and germline proliferation. Chase et al. (1998) WBG 15(4):45 Yu et al. (1998) Science 279: 1219-22 Osmani et al. (1988) Cell 52: 241-251

Chiaki Fujitake et al. (2004) East Asia Worm Meeting "CHARACTERIZATION OF C.elegans UBIQUITIN C-TERMINAL HYDROLASES"

Kenji Takahashi, Hideshi Inoue, Maiko Takahashi, Chiaki Fujitake

[East Asia Worm Meeting, 2004]

Ubiquitin C-terminal hydrolases (UCHs) are presumed to contribute greatly to the ubiquitin system by removing ubiquitin (Ub) from ubiquitinated peptides or amides. This activity is thought to be essential for cytoplasmic protein degradation. C.elegans has four genes coding for UCH; ubh-1, ubh-2, ubh-3, and ubh-4 . Expression of all the four genes was confirmed by RT-PCR. The ubh-1, ubh-2 , and ubh-3 genes are predicted to be organized into an operon, and the encoded protein UBH-1, UBH-2, and UBH-3 are 37%, 32%, and 34% identical to human UCH-L1 and 40%, 34%, and 35% identical to human UCH-L3, respectively, while UBH-4 is 47% identical to human UCH-L5. UCH-L1 is an abundant neuronal enzyme, whose deficiency has been reported to link with neurodegenerative disorders such as Parkinson disease. UBH-1 and UBH-3 were expressed in E.coli as fusion proteins with a His-tag and purified. We assayed these enzymes using C.elegans UB, NEDD8, and SUMO-1 fused with HSV- and His-tags at the C-termini as substrates. Both UBH-1 and UBH-3 showed C-terminal hydrolase activity toward Ub and NEDD8, but not toward SUMO-1. In order to examine the expression pattern of ubh-1 , we performed GFP expression analysis using ubh-1::gfp . GFP fluorescence was observed in some neuronal cells, two of which were identified as HSN neurons that are responsible for egg laying. To reveal the function of ubh-1 , we analyzed the phenotypes of knockout mutant ubh-1 ( tm526 ) and found egg laying defect; the total number of eggs laid by ubh-1 ( tm526 ) was 63% of that by the wild-type animals. Additionally, the defecation cycle of the ubh-1 ( tm526 ) was found to be longer (av. 60 sec) than that of the wild type animals (av. 45 sec). Thus, ubh-1 is thought to be important for neuronal functions. In the genetic background of rrf-3 ( pk1246 ), feeding RNAi of ubh-4 caused burst of gonad arms (60%) with egg laying defect, and abnormal DTC migration. This result suggests the possibility that ubh-4 may be required for gonadogenesis.

Glotzer M et al. (2000) European Worm Meeting "The cyk-3 ubiquitin C-terminal hydrolase regulates osmotic responsiveness in the early C. elegans embryo"

Gonczy P, Glotzer M, Hyman AA, Kaitna S

[European Worm Meeting, 2000]

Cytokinesis divides one cell into two daughter cells. This process is not well understood at the molecular level. In our lab we are interested in identifying proteins required for cytokinesis and to understand how they contribute to the assembly and function of the contractile ring. We are currently using forward genetics in Caenorhabditis elegans to help identify additional key players. cyk-3(t1525) is a mutant originally identified in a screen for maternal effect embryonic lethal (MEL) mutants on linkage group III (LGIII). cyk-3 homozygous hermaphrodites are viable, but produce only dead embryos. cyk-3 mutant embryos successfully complete karyokinesis, but fail to form a cleavage furrow that separates the daughter cells. Time-lapse analysis of early embryos reveals that neither pseudocleavage nor any furrowing reminiscent of cytokinesis occurs. Since the contractile ring is actomyosin based, we examined the distribution of actin throughout the first cell cycle. cyk-3 mutant embryos fail to reorganize actin into an "anterior cap" during pronuclear migration. Surprisingly, an actin ring assembles at the correct time and place. cyk-3 mutant embryos are also osmotically sensitive. We have analyzed the response of wild-type blastomeres and cyk-3 mutant blastomeres to varying osmotic conditions and have found that wild-type blastomeres readily shrink in hypertonic medium whereas cyk-3 mutant embryos do not. Moreover we have found that we can partially rescue the cyk-3 cytokinesis defect by providing osmotic support. Thus we conclude that the primary defect in cyk-3 mutant embryos is a failure to regulate the osmotic balance of the cell and that the cytokinesis defect is a secondary phenotype. cyk-3 maps to position -1.4cM on LGIII. We have cloned the cyk-3 gene by functional rescue and found that it encodes a ubiquitin C-terminal hydrolase, an enzyme that is predicted to remove ubiquitin moieties from Ub-conjugates. Biochemical analysis shows that Cyk-3 is indeed a functional ubiquitin C-terminal hydrolase, moreover it is specific for ubiquitin, it does not remove Ub-like conjugates from a model substrate. We speculate that the Cyk-3 Ub C-terminal hydrolase may regulate osmotic responsiveness by preventing the degradation or endocytosis of a protein that regulates osmotic balance, such as a ion channel. Interestingly, pod-1 mutant embryos have a similar range of phenotypes as cyk-3 mutant embryos. Since Pod-1 is a coronin-like protein involved in endocytosis, these two proteins may effect a common pathway.

Khanikar, Jayshree et al. (2013) International Worm Meeting "Characterization of non-SMC elements of the SMC-5/6 complex in C. elegans."

Bickel, Jeremy, Chan, Raymond, Fingerhut, Jaclyn, Khanikar, Jayshree

[International Worm Meeting, 2013]

Meiosis is a specialized cell cycle that produces haploid gametes from diploid germ cells. In preparation for meiotic chromosome segregation, DNA double-strand breaks (DSB) are produced and repaired via homologous recombination to generate inter-homolog crossovers, which in turn promote accurate chromosome segregation and increased genetic diversity in the offspring. Given the importance of inter-homolog crossovers, an excess of DSB are produced in meiotic prophase to ensure that the obligate crossovers are generated. Repair of the excess DSB that do not result in inter-homolog crossovers is nonetheless important for successful meiosis, but our understanding of this type of repair is poor compared to inter-homolog repair. We showed previously that the Structural Maintenance of Chromosomes (SMC) 5 and 6 proteins are important for successful repair of these excess DSB by homolog-independent mechanism(s). The SMC-5/6 proteins are known to associate with multiple subunits, four of which are conserved in human and yeast cells, termed the non-SMC elements (NSE). Three of the NSE subunits promote post-translational modification by the ligation of ubiquitin (Ub) and SUMO to targeted proteins. The mechanism by which SMC-5/6 protein complexes regulated DSB repair has not been defined, however the Ub and SUMO ligase activities are postulated to be involved in this process. Our goal is to identify and characterize the NSE subunits for the C. elegans SMC-5/6 protein complex in order to test and understand their relevance in meiotic DSB repair. We have identified putative homologs to NSE-1, -2, -3 and -4 based on protein sequence homology. Using RNAi and genetic mutants, we have observed aberrant localization of SMC-5/6 complex subunits. We will present the on-going characterization of these putative homologs in terms of their subcellular location, protein interactions and functional impact on SMC-5/6-related roles in meiosis.