Functional analysis of the C. elegans cyld-1 gene reveals extensive similarity with its human homolog

The human cylindromatosis tumor suppressor (HsCyld) has attracted extensive attention due to its association with the development of multiple types of cancer. HsCyld encodes a deubiquitinating enzyme (HsCYLD) with a broad range of functions that include the regulation of several cell growth, differentiation and death pathways. HsCyld is an evolutionarily conserved gene. Homologs of HsCyld have been identified in simple model organisms such as Drosophila melanogaster and Caenorhabditis elegans (C. elegans) which offer extensive possibilities for functional analyses. In the present report we have investigated and compared the functional properties of HsCYLD and its C. elegans homolog (CeCYLD). As expected from the mammalian CYLD expression pattern, the CeCyld promoter is active in multiple tissues with certain gastrointestinal epithelia and neuronal cells showing the most prominent activity. CeCYLD is a functional deubiquitinating enzyme with similar specificity to HsCYLD towards K63- and M1-linked polyubiquiting chains. CeCYLD was capable of suppressing the TRAF2-mediated activation of NF-kappaB and AP1 similarly to HsCYLD. Finally, CeCYLD could suppress the induction of TNF-dependent gene expression in mammalian cells similarly to HsCYLD. Our results demonstrate extensively overlapping functions between the HsCYLD and CeCYLD, which establish the C. elegans protein as a valuable model for the elucidation of the complex activity of the human tumor suppressor protein.


Introduction
Inactivating mutations in the human Cyld gene (HsCyld) predispose individuals to the development of skin tumors that include cylindromas, spiradenomas and trichoepitheliomas (reviewed in [1]). A tumor suppressing activity of HsCyld has been associated with several other types of human malignancies including multiple myeloma, melanoma, breast colon and hepatocellular carcinoma [2][3][4][5][6]. These findings have fueled an intense effort to understand the molecular mechanisms that underlie the homeostatic functions of HsCyld. HsCyld encodes a 956 amino acid polypeptide (HsCYLD) which has a carboxyl-terminal deubiquitinating domain and three amino-terminal CAP-Gly domains, two of which mediate the interaction of HsCYLD with microtubules ( Fig 1A, [7]). Shorter variants of HsCYLD are predicted from multiple alternatively spliced mRNA species. HsCYLD preferentially hydrolyzes K63-and M1-linked polyubiquitin chains [8,9]. The deubiquitinating activity of HsCYLD has been associated with its ability to regulate several growth and survival pathways which include the NF-kappaB, JNK, p38, TGFbeta, Wnt and Notch pathways. The mechanism of HsCYLD-mediated inhibition of NF-kappaB and JNK activation by members of the TNFand Toll/IL-1-receptor families has been studied more extensively than other signaling pathways. These studies have indicated that the inhibitory action of HsCYLD is mediated by its ability to hydrolyze polyubiquitin chains that may be free or conjugated onto specific proteins such as RIPK1, TAK1, TRAF-family members and Bcl3. The polyubiquitin chains that are targeted by HsCYLD mediate the assembly of multiprotein complexes that lead to proximityinduced activation of protein kinases and the propagation of signaling. Therefore, the hydrolysis of these polyubiquitin chains by HsCYLD disrupts the signaling process. By inhibiting NF-kappaB activation, HsCYLD can promote apoptosis which is inhibited by NF-kappaB. HsCYLD can promote also necroptosis, which is an alternative type of programmed cell death. The promotion of necroptosis by HsCYLD is based on its ability to deubiquitinate RIPK1 and facilitate its interaction with RIPK3 and the subsequent activation of MLKL. The ability of HsCYLD to facilitate various types of programmed cell death and inhibit growth and differentiation pathways is consistent with its broad tissues range of homeostatic functions.
Putative HsCyld homologues have been identified in multiple species including simple experimental model organisms such as Drosophila melanogaster (DmCyld) and C. elegans (cyld-1, called CeCyld hereafter) as well as evolutionarily more distant organisms belonging to phyla Cnidaria (Acropora digitifera, Hydra Vulgaris and Nematostlella Vectensis) and Porifera (Amphimedon queenslandica). The functional characterization of HsCyld homologues from model organisms will permit the use of genetically tractable organisms for the elucidation of the complex functional role of CYLD in animal pathophysiology. Fig 1 shows a schematic representation of HsCyld and its putative homologues from four representative species along with an amino acid alignment of their deubiquitinating domains and the results of pairwise comparisons of their amino acid sequences. These proteins have a similar domain organization with a highly conserved carboxyl-terminal deubiquitinating domain and amino terminal CAP-Gly domains. Previous work has demonstrated the ability of the Drosophila HsCYLD homologue to act as a deubiquitinating enzyme and regulate the IMD and JNK pathways in Drosophila, indicating the functional conservation between the human and fruit fly genes [10,11]. Furthermore, a recent evaluation of CeCyld-deficient C. elegans demonstrated an important role of CeCYLD in mediating p53-induced apoptosis similarly to the role of HsCYLD in preventing DNA-damage induced apoptosis [12]. However, a comparative biochemical and signaling characterization of CeCYLD and HsCYLD has not been performed. In the present report we have analyzed and compared the deubiquitinating and signaling properties of CeCYLD and HsCYLD and demonstrated an extensively overlapping functional pattern. Our findings support the use of C. elegans as a valid animal model for the exploration of the functional repertoire of HsCYLD.

Plasmids
Expression vectors for HsCYLD and TRAF2 were previously described [14,15]. The C. elegans cyld-1 cDNA was prepared from total RNA obtained from mixed developmental stages of wild-type worms. In brief, total RNA was extracted using Trizol reagent (Thermo Fisher Scientific) and the first strand cDNA synthesis was performed using SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. The 3.7 Kb cyld-1 cDNA was subsequently synthesized via PCR with Phusion High-Fidelity DNA polymerase (New England Biolabs) using cyld-1-specific primers: Forw: 5'-atgcccggcgaat ttgcaaagtttc-3' and Rev: 5'-caactagttgaattacattcaccattctaag-3'. An amplicon, that corresponded to the cyld-1 isoform a (http://www.wormbase.org) was cloned into pJet1.2 to create pMGN32 and verified by sequencing. The latter was used as a template for PCR-based site directed mutagenesis. In order to introduce a mutation in the cysteine residue 774 of the CeCyld and replace it with serine, the BsiWI-ClaI fragment was mutagenized and used to replace the corresponding wild type fragment. The sequences of the mutagenic primers that were used are: For Mut 5'-TGTAACTCGTCTTATCTGGATGC-3' and Rev Mut 5'-CCAGATAAGACGAGTTACAGTAACCC-3', whereas the external primer pair used is For WT 5'-CAATGTGAATGGAAAGCAAGC-3' and Rev WT 5'-TGACATGATCCGCTCGC ACA-3'. The presence of the point mutation was verified by sequencing.

Luciferase and β-galactosidase assays
HEK293T cells were cotransfected with a β-galactosidase expressing plasmid to test for transfection efficiency (pGKβ-gal [16]), and a plasmid containing the firefly luciferase gene under the control of three NF-kappaB binding sites (3xKBL [17]) or a plasmid containing the firefly luciferase gene under the control of seven AP1 binding sites (7xAP1, Stratagene) in the presence or absence of plasmids expressing FLAG-tagged TRAF2, HsCYLD, CeCYLD or CeCYLDC774S. The luciferase and β-galactosidase activities were determined by the Luciferase Assay System (Promega, E1501) and the Galacto-light Plus Reporter Gene Assay System (Applied biosystems, T1011) respectively. The cells were collected approximately 18 h after transfection and lysed in passive lysis buffer provided in the Tropix kit supplemented with 1mM DTT. The activities of the lysates were measured using a TD20/20 luminometer (Turner Designs). Relative luciferase activities were determined by dividing the values of luciferase activities by the corresponding values of β-galactosidase activities. The mean and the standard error were calculated using Microsoft Office Excel 2013 software.

CeCYLD is a functional deubiqutinating enzyme with a broad pattern of expression
The expression pattern of CeCyld was evaluated to determine whether CeCyld has a broad or tissue specific function. Towards this goal transgenic worms that express DsRed under the control of the CeCyld promoter were generated (Fig 2). The analysis of the transgenic animal showed a rather broad pattern of reporter gene expression with prominent signals in the pharynx (Fig 2A, 2B and 2E), the presumptive AVB/AVD interneurons in the nerve ring (Fig 2B  and 2C) and the intestine (Fig 2D). These findings are consistent with a functional role for CeCyld in multiple tissues similarly to the human gene.
The extensive homology between the deubiquitinating domain of HsCYLD and the corresponding region of CeCYLD prompted an investigation into the ability of CeCYLD to act a deubiquitinating enzyme [18]. Towards this goal FLAG-tagged CeCYLD or HsCYLD were Comparative analysis of human and C. elegans CYLD proteins expressed in HEK293T cells and isolated by immunoprecipitation using an anti-FLAG antibody. The immunoprecipitated proteins were incubated with K63-or M1-linked polyubiquitin substrates and the products of the reaction were analyzed by electrophoresis. As shown in Fig  3 CeCYLD was capable of hydrolyzing both K63-and M1-linked chains with a similar efficiency to the human protein. The deubiquitinating activity of HsCYLD depends on several amino acid residues of the active site including cysteine 601 (C601). C601 of HsCYLD corresponds to C774 of CeCYLD (Fig 1B). In order to determine whether the catalytic activity of CeCYLD depends on C774 a mutated form of CeCYLD (CeCYLDC774S) containing a serine residue in place of C774 was generated and tested for its ability to hydrolyze K63-and Comparative analysis of human and C. elegans CYLD proteins M1-linked polyubiquitin chains. As shown in Fig 3 the deubiquitinating activity of CeCYLD towards both substrates was practically abolished by the replacement of C774 with serine. This result highlights an additional similarity between HsCYLD and CeCYLD and practically excludes the possibility of attributing the deubiquitinating activity of immunoprecipitated CeCYLD to an associated enzyme. Taken together these results demonstrate a comparable deubiquitinating activity and specificity between the human and C. elegans proteins, which strengthens further the evolutionary relationship of these proteins.

CeCYLD inhibits the NF-kappaB and JNK activation pathways in human cells
HsCYLD has a well-established role as an inhibitor of NF-kappaB and JNK activation pathways by members of the TNF and Toll/IL-1 receptor families. In order to explore further the functional relationship between CeCYLD and HsCYLD, the ability of CeCYLD to inhibit the NF-kappaB and JNK activation pathways was evaluated. The activation of these pathways was simulated in HEK293T cells by overexpression of TRAF2. The activity of NF-kappaB was monitored by a co-transfected NF-kappaB-dependent luciferase reporter plasmid (3xKBL). As shown in Fig 4 TRAF2 induced a significant activation of NF-kappaB-dependent luciferase activity, which was inhibited by the coexpression of HsCYLD as expected (Fig 4, S1 File). Coexpression of CeCYLD with TRAF2 also inhibited the activation of NF-kappaB similarly to HsCYLD despite the fact that CeCYLD was expressed at lower levels than HsCYLD. The inhibitory activity of CeCYLD on TRAF2-mediated NF-kappaB activation was dependent on its deubiquitinating activity since CeCYLDC774S was severely compromised in its ability to inhibit TRAF2-mediated activation of NF-kappaB. Immunoblotting demonstrated comparable expression of TRAF2 in these experiments. The JNK activation pathway was monitored by a cotransfected AP1-dependent luciferase reporter plasmid (7xAP1), since JNK activation induces the activity of AP1 transcription factor complex. As shown in Fig 5 TRAF2 induced a significant activation of AP1-dependent luciferase activity which was inhibited by the coexpression of HsCYLD as expected (Fig 5, S2 File). Coexpression of CeCYLD with TRAF2 also inhibited the activation of AP1 similarly to HsCYLD. The inhibitory activity of CeCYLD on TRAF2-mediated AP1 activation was dependent on its deubiquitinating activity since CeCYLDC774S was compromised in its ability to inhibit TRAF2-mediated activation of AP1. Immunoblotting demonstrated comparable expression of TRAF2 in these experiments. Collectively, these results expand the repertoire of similar functional properties between CeCYLD and HsCYLD.

CeCYLD can inhibit TNF-dependent IL8 expression
Our previous experiments indicated the ability of CeCYLD to interfere with the activation of the NF-kappaB and JNK signaling pathways similarly to HsCYLD. In order to determine whether CeCYLD could affect NF-kappaB-dependent endogenous gene expression, its ability Comparative analysis of human and C. elegans CYLD proteins to inhibit the induction of IL8 by TNF was evaluated in HeLa cells. For this purpose, HeLa cells were transfected with empty vector or vectors expressing HsCYLD or CeCYLD and stimulated with TNF. The relative expression of IL8 mRNA was determined by real time PCR. As shown in Fig 6, TNF stimulation caused a dramatic increase in the levels of IL8 mRNA (Fig 6,  S3 File). As expected the exogenous expression of HsCYLD reduced the levels of TNF-induced IL8 mRNA. CeCYLD expression caused also a significant reduction to the levels of TNFinduced IL8 mRNA. On the other hand, CeCYLDC774S overexpression did not affect the induction levels of IL8 by TNFα (Fig 6). These results extend the functional similarity of HsCYLD and CeCYLD to include their common effect on NF-kappaB-driven endogenous gene expression.

Discussion
The present report was undertaken to examine the extent of functional similarity between the human tumor suppressor protein HsCYLD and its C. elegans homologue CeCYLD. The two proteins have a relatively low overall amino acid sequence homology but a similar domain organization which includes amino terminal sequences with homology to CAP-Gly domains and a highly similar carboxyl terminal region with homology to deubiquitinating domains. Our experiments demonstrated that CeCYLD has a functional deubiquitinating domain with a similar substrate specificity to HsCYLD. In addition, CeCYLD was capable of inhibiting NF- Comparative analysis of human and C. elegans CYLD proteins kappaB and JNK pathway activation and TNF-induced gene expression in a manner that is similar to HsCYLD. These findings demonstrate an extensively overlapping range of functional properties between the worm and human proteins that support strongly an evolutionary relationship between these molecules.
KGB-1 is the JNK homologue in C. elegans [19]. It is primarily expressed in the nervous system where it controls coordinated movement. Furthermore C. elegans JNK plays important roles in stress responses to heavy metals, unfolded protein generation and bacterial pore forming toxins. Our findings on the expression pattern and activity of CeCYLD suggest that at least some of the functions associated with C. elegans JNK might be regulated by CeCYLD. Interestingly, a recent report demonstrated the importance of CeCYLD in the response of worms to DNA-damage induced apoptosis through the stabilization of the C. elegans homologue of p53 [12].
The activation of NF-kappaB by TNF-and Toll/IL-1-receptor families is another pathway that can be inhibited by HsCYLD and CeCYLD. Intriguingly, C. elegans has no known NF-kappaB protein homologues and therefore the biological significance of NF-kappaB activation inhibition by CeCYLD is unclear. However, it should be noted that the negative effects of HsCYLD on NF-kappaB activation are mediated by targeting signaling molecules functioning upstream from the NF-kappaB transcription factors (see introduction). Therefore, it is conceivable that CeCYLD may affect the activity of similar molecules that serve other functions in C. elegans and were later adopted to facilitate the activation of NF-kappaB in mammals. Possible targets of CeCYLD would include molecules that are implicated in the signaling of the C. elegans Toll-like receptor TOL-1 which is important for innate immune responses in the worm [20,21]. Therefore, it would be interesting to investigate whether CeCYLD might affect TOL-1 associated functions possibly in a ubiquitination-dependent manner. Likewise, HsCYLD homologues in Cnidaria and Porifera may regulate the signaling activity of Toll-like receptor, TRAF and NF-kappaB homologues that have been identified in these organisms [22].
Our experiments demonstrated a similar substrate specificity between CeCYLD and HsCYLD. These proteins can hydrolyze with similar efficiency K63-linked or M1-linked polyubiquitin chains. An examination of the amino acid sequence of the CeCYLD deubiquitinating domain is consistent with our findings and previously reported crystal structures. The catalytic triad of amino acid residues C-H-D which is conserved in all deubiquitinating enzymes of the USP class is conserved in CeCYLD and corresponds to amino acids C774, H1034 and D1050 (Fig 1, [8]). In accordance with this notion, our experiments demonstrated the functional significance of C774 for the deubiquitinating activity of CeCYLD. In addition, several structural features of HsCYLD and the zebrafish CYLD homologue, which are critical for the specific recognition of K63-linked and M1-linked polyubiquitin chains are conserved in CeCYLD. For example, the sequences of two short β-strands which are important for the specificity of zebrafish CYLD homologue towards K63-linked and M1-linked polyubiquitin chains are highly conserved in CeCYLD and correspond to amino acids 1053-1056 and 1062-1064 respectively [9]. The amino-terminal β-strand which spans amino acids 1053-1056 includes R1055 which was shown to be critical for the specific hydrolysis of K63-linked and M1-linked polyubiquitin chains by the zebrafish CYLD homologue. Furthermore, the shorter length of a loop between β-strands β6 and β7 in the zebrafish CYLD homologue is critical for the specificity of the enzyme towards K63-linked and M1-linked polyubiquitin chains [9]. The length and amino acid sequence of this loop is highly conserved in CeCYLD and spans amino acids 1030-1035.
Our study demonstrated for the first time the ability of CeCYLD to act as a deubiquitinating enzyme with similar activity and specificity to HsCYLD. In addition, a broadly overlapping spectrum of functions was demonstrated for the C. elegans and human proteins. These findings establish CeCyld as an orthologue of HsCyld and support the use of C. elegans as a valid model organism for the identification and elucidation of the functional properties of the human tumor suppressor gene by high throughput functional genomic analyses.
Supporting information S1 File. Supplemental data for Fig 4A. The numerical values from six independent experiments (A-F) along with the corresponding average and standard error (std err) values and the t-test p-values that were used to generate the plot shown in Fig 4A are