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.

Yuki Yanagi et al. (2010) East Asia Worm Meeting "C.elegans DUO-1 is Important for Osmotic Regulation by Excretory System"

Mikiko Kusano, Shingo Tazawa, Aiko Baba, Yosuke Kaku, Hideshi Inoue, Yuki Yanagi, Hirofumi Tanaka

[East Asia Worm Meeting, 2010]

Deubiquitinating enzymes (DUB) are classified into five subclasses; USP, UCH, OTU, MJD, and JAMM. The C. elegans duo-1 gene encodes a unique protein with two distinct DUB domains, USP and OTU. The DUO-1 protein was detected mainly at the L1 and L2 stages by Western blotting. To reveal physiological roles of duo-1, phenotype of a deletion mutant duo-1(tm934) was analyzed. The mutant animals showed rod-like-larval lethality: they had abnormal vacuoles in the hypodermis and 85% of them died at the larval stages. The rod-like larval lethal phenotype is relevant to defects in osmotic regulation. The duo-1(tm934) mutant animals are hypersensitive to osmotic stress; they lost mobility in a 0.5 M NaCl solution in a shorter time than the wild-type animals. In general, excretory system is important for protection against osmotic stress. The mutants of let-60/Ras signaling pathway genes which exhibit rod-like larval lethality have been reported to have defects in excretory duct development. Also the duo-1(tm934) mutant has defects in the excretory duct cell, because no expression of transgenic Plin-48::GFP, an expression marker for the excretory duct cell, was observed in the duo-1(tm934) genetic background. In order to examine whether duo-1 is a positive regulator of the Ras signaling pathway, sli-1 and gap-2, negative regulators of the Ras signal, were silenced by RNAi in the duo-1(tm934) genetic background. Down regulation of these genes partly suppressed the rod-like larval lethality of duo-1(tm934). In addition, RNAi of duo-1 in the genetic background of let-60(n1046) gain-of-function mutant suppressed the exploded-through-vulva phenotype observed under the negative control conditions. Thus, DUO-1 is suggested to be important for activation of the let-60/Ras signaling pathway. EOR-1 is a transcription factor downstream of the pathway. Besides rod-like larval lethality, the mutant of eor-1 shows defects in DiO uptake of the amphid and phasmid neuronal cells. Also the duo-1(tm934) animals showed dye-filling defective of the phasmid neuronal cells. In conclusion, DUO-1 is important for osmotic regulation controlled by excretory system probably via positively regulating the let-60/Ras signaling pathway or EOR-1.

Galvin BD et al. (2000) East Coast Worm Meeting "Two screens for new genes involved in programmed cell death"

Galvin BD, Bob Horvitz, Cameron S

[East Coast Worm Meeting, 2000]

Many genes involved in programmed cell death in C. elegans have been identified by screening for mutations that allow survival of cells that are normally destined to die. The opposite approach, screening for mutations that result in the deaths of cells, has been comparatively unexplored, in part because of a lack of efficient methods to identify such mutations. A lin-11::gfp reporter (see abstract by Cameron and Horvitz) expresses in Pn.aap cells. In wild-type animals, only the six Pn.aap cells of the P3-P8 lineages survive and express GFP, while in mutants defective for cell killing all 12 Pn.aap cells survive and express GFP. A strain carrying this reporter allows one to monitor the survival of cells using a dissecting microscope rather than Nomarski optics, greatly facilitating screening. In this way, I am performing a ced-4 suppression screen by looking for a reduction in the number GFP-positive cells and thus an increase in cell death in the ventral cords of partial loss-of-function ced-4 mutants. To identify substrates of the caspase CED-3, which is required for programmed cell death, I am using a modified two-hybrid screen. An active caspase is composed of two subunits proteolytically derived from an inactive procaspase. The active site of the caspase contains a cysteine, which when mutated prevents the cleavage activity of the enzyme. In this screen the two active subunits of the CED-3 caspase are expressed separately, with the active-site cysteine changed to a serine. This amino acid substitution should permit an interaction with substrates but prevent their cleavage, allowing for the identification of interacting proteins. Other groups have identified substrates of mammalian caspases using this approach (1, 2). Candidates from the screen will be analyzed by testing in vitro cleavage by CED-3 and assayed by RNAi to examine their roles in programmed cell death. 1. Van Criekinge, W., van Gurp, M., Decoster, E., Schotte, P., Van de Craen, M., Fiers, W., Vandenabeele, P., and Beyaert, R. Analytical Biochemistry. 263, 62-66 (1998). 2. Kamada, S, Kusano, H., Fujita, H., Ohtsu, M., Koya, R. C., Kuzumaki, N., and Tsujimoto, Y. Proc. Natl. Acad. Sci. USA. 95, 8532-8537 (1998).