The WD40-Repeat Protein WDR-48 Promotes the Stability of the Deubiquitinating Enzyme USP-46 by Inhibiting Its Ubiquitination and Degradation

bioRxiv preprint doi: https://doi.org/10.1101/679415; this version posted June 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The WD40-repeat protein WDR-48 promotes the stability of the deubiquitinating enzyme USP-46 by inhibiting its ubiquitination and degradation

Molly Hodul1,2, Rakesh Ganji 2, Caroline L Dahlberg3, Malavika Raman1,2, and Peter Juo1,2*

From the 1Graduate Program in Neuroscience, and 2Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111; 3Department of Biology, Western Washington University, Bellingham, WA 98225

Running Title: WDR-48 promotes USP-46 stability

*To whom correspondence should be addressed: Peter Juo, Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111, USA; [email protected]; Tel: (617) 636-3950; Fax: (617) 636-0445;

Keywords: ubiquitin, deubiquitinating enzyme, DUB, USP46, WD40-repeat, WDR, regulation, C. elegans ______

ABSTRACT unable to promote USP-46 abundance in vivo. Ubiquitination is a reversible post-translational Together, these data support a model in which modification that has emerged as a critical WDR-48 binds and stabilizes USP-46 protein regulator of synapse development and function. levels by preventing the ubiquitination and However, mechanisms that regulate the degradation of USP-46 in the proteasome. Given deubiquitinating enzymes (DUBs) that are that a large number of USPs interact with WDR responsible for the removal of ubiquitin from proteins, we propose that stabilization of DUBs target proteins are poorly understood. We by their interacting WDR proteins may be a previously showed that the DUB USP-46 conserved and widely used mechanism to control removes ubiquitin from the glutamate receptor DUB availability and function. GLR-1 and regulates it trafficking and ______degradation in C. elegans. We found that WD40- repeat proteins WDR-20 and WDR-48 bind and Ubiquitination is a widely used post- stimulate the catalytic activity of USP-46. Here, translational modification that regulates a large we identify another mechanism by which WDR- variety of neuronal processes including synapse 48 regulates USP-46. We found that increased development and function (1-3). The covalent expression of WDR-48, but not WDR-20, attachment of ubiquitin to lysine residues on promotes USP-46 abundance in mammalian cells substrates by ubiquitin E3 ligases has many in culture and in C. elegans neurons in vivo. consequences for target proteins including Inhibition of the proteasome promotes the degradation in the proteasome, changes in protein abundance of USP-46, and this effect is non- trafficking, or altered function. Ubiquitination is additive with increased expression of WDR-48. a highly regulated and reversible process, where We found that USP-46 is ubiquitinated, and the removal of ubiquitin is achieved by a family expression of WDR-48 reduces the levels of of proteases called deubiquitinating enzymes ubiquitin-USP-46 conjugates and increases the (DUBs). Although there are about 600 ubiquitin half-life of USP-46. A point mutant version of ligases encoded by the human WDR-48 that disrupts binding to USP-46 is

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WDR-48 promotes USP-46 stability

genome, there are only about 95 DUBs, translesion synthesis pathways (22,23). The suggesting that DUB function is tightly regulated critical roles of these USPs in nervous system (4,5). Indeed, increasing evidence shows that function, cell proliferation and cancer DUBs can be highly selective for substrates and progression underscore the need to understand appear to have very specific cellular functions (5- how these DUBs are regulated. 7). Additionally, because DUBs are intracellular Recent work shows that DUBs can be proteases and the ubiquitination of proteins regulated by a variety of mechanisms including affects their function with profound cellular transcription, post-translational modifications, consequences, tight regulation of DUBs is needed and by interaction with other proteins (8,9). to prevent indiscriminate proteolytic cleavage of Large-scale proteomic studies revealed that the ubiquitin conjugates (8). vast majority of DUBs interact with multiple The ubiquitin-specific protease (USP) proteins (24,25) that can regulate their subcellular family represents the largest sub-family of DUBs localization, substrate recognition, and catalytic comprised of 56 members that regulate diverse activity (8,9). Interestingly, 35% of USP enzymes cellular functions (4,7,9). Three related DUBs, interact with one class of protein, WD40-repeat USP-46, USP-12, and USP-1, have received (WDR) proteins, and 45% of those interact with particular attention due to their roles in regulating multiple WDR proteins (10,24). The WDR neuronal function, cell growth and division, and domain forms a rigid β-propeller structure that DNA damage (10,11). We previously showed provides a stable binding surface for protein- that USP-46 regulates glutamate receptor levels protein interactions (26,27). in neurons to control glutamatergic behavior in C. In this study, we investigate the elegans (12). USP-46 deubiquitinates the regulation of USP-46 protein levels by the WDR glutamate receptor GLR-1 and protects it from proteins WDR-48 (also known as USP-1- degradation in the lysosome. Similarly, in associated factor 1, UAF1) and WDR-20. USP- mammalian neurons, USP-46 can promote 46 and its homolog USP-12 have low intrinsic glutamate receptor subunit stability by catalytic activity (28-30). Biochemical studies deubiquitinating the glutamate receptor subunits showed that WDR-48 interacts with USP-46, GluA1 and GluA2 (13), indicating that this USP-12 and USP-1 and stimulates their catalytic mechanism is conserved. Characterization of activity (24,28,30-32). WDR-20 forms a ternary Usp46 mutant mice suggests that the DUB can complex with WDR-48 and USP-46/USP-12, but also regulate GABA signaling and depression- not USP-1 (24,29), and further enhances their like behaviors (14,15). In non-neuronal cells, catalytic activity in vitro (29,30). The USP-46 and USP-12 regulate a variety of cellular mechanisms underlying the ability of the WDR processes including cell proliferation and proteins to activate USP-12 and USP-46 were tumorigenesis (16-18). For example, USP-12 and recently revealed by their crystal structures. The USP-46 promote the stability of the Akt crystal structures of both DUBs were solved in phosphatase PHLPP1 resulting in decreased cell complex with WDR-48 (33-35), and USP-12 was proliferation and tumorigenesis in colon cancer additionally solved in a ternary complex with cells (17,18). PHLPP1 is a tumor suppressor that WDR-48 and WDR-20 (34). Together, these has been implicated in several cancers including structural studies revealed that the WDR proteins glioblastoma, colon and breast cancer (19-21). In interact at a site distal to the active site, contrast, overexpression of USP-12 promotes the suggesting an allosteric mechanism underlies the stability and function of androgen receptors ability of the WDR proteins to stimulate catalytic resulting in increased proliferation and survival activity of the DUBs. of prostate cancer cells (16). Lastly, USP-1 is a Here we identify another mechanism by critical mediator of two major DNA damage which WDR proteins regulate DUBs. We found response pathways, the Fanconi anemia and DNA that USP-46 is ubiquitinated and degraded in the bioRxiv preprint doi: https://doi.org/10.1101/679415; this version posted June 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

proteasome. We show that WDR-48 promotes these neurons by measuring the average USP-46 protein abundance by binding to the fluorescence of USP-46::GFP in a defined DUB and inhibiting its ubiquitination. We anterior region of the VNC or in the cell body of propose that binding of WDR proteins to DUBs the VNC neuron PVC (see Experimental may provide a general mechanism to regulate the Procedures). We found that overexpression of stability and thus availability of DUBs to carry WDR-48 and WDR-20 in these interneurons out their various cellular functions. increases USP-46::GFP abundance by 2.6-fold (Fig. 1, C and D). This effect can be largely Results attributed to WDR-48, as overexpression of WDR-48 promotes USP-46 protein abundance WDR-48 alone results in a 2.2-fold increase in in vivo USP-46::GFP levels, whereas overexpression of We previously showed that the C. WDR-20 alone results in a smaller increase of a elegans WD40-repeat proteins WDR-48 and 1.4-fold in USP-46::GFP levels (Fig. 1, C and D). WDR-20 bind to the deubiquitinating enzyme We observed similar effects in the soma of VNC USP-46 and increase its catalytic activity (36). neurons (Fig. 3B), suggesting that the WDR We noticed that expression of WDR-48 but not proteins increase USP-46::GFP levels WDR-20 increases USP-46 protein levels in throughout the neuron. HEK293T cells. To test whether this effect of Because our USP-46::GFP fluorescence WDR-48 occurs in neurons in vivo, we generated reporter is expressed under control of the nmr-1 transgenic worms (pzIs40) expressing GFP- promoter, the effects of the WDR proteins on tagged USP-46 (USP-46::GFP) under control of USP-46 protein levels could be indirectly due to the nmr-1 promoter (37) in ventral cord increased nmr-1 promoter activity. We performed interneurons where the WDR proteins and USP- two experiments to test this possibility. First, we 46 are known to act (12,36). GFP-tagged USP-46 analyzed the effects of the WDR proteins on a is functional because this transgene rescues second USP-46::GFP reporter transgene (pzIs37) specific glutamatergic behavioral defects under control of an independent promoter, the observed in usp-46 null mutants (Supplemental glr-1 promoter, which is also expressed in the Fig. 1, see Experimental Procedures). We first ventral cord interneurons (38-40). We found that tested whether expression of the WDR proteins in expression of WDR-20 and WDR-48 (wdr- ventral cord interneurons altered total USP- 48(xs);wdr-20(xs)) resulted in a similar 3-fold 46::GFP levels by immunoblotting total worm increase in USP-46::GFP fluorescence in the lysates with anti-GFP antibodies. Consistent with VNC using this independent transgene (Fig. 1E). our previous findings in HEK293T cells (36), we Second, we explicitly measured the activity of the found that overexpression of WDR-48 alone nmr-1 promoter using a transcriptional reporter (wdr-48(xs)) or WDR-48 and WDR-20 together (Pnmr-1::GFP). We found that co-expression of (wdr-48(xs);wdr-20(xs)) in GLR-1-expressing WDR-48 and WDR-20 had no effect on GFP interneurons increased USP-46::GFP protein fluorescence (Fig. 1F). These data, together with levels. In contrast, overexpression of WDR-20 the fact that we previously observed similar alone (wdr-20(xs)) had no effect on USP-46::GFP effects of the WDR proteins on USP-46 protein protein levels (Fig. 1, A and B). Next, we directly levels in HEK293T cells (where USP-46 was measured the levels of USP-46::GFP expressed using the mammalian expression fluorescence in glr-1-expressing neurons in the vector pMT3)(36), suggest that the WDR ventral nerve cord (VNC) of C. elegans. In wild- proteins increase the abundance of USP-46 type animals, USP-46::GFP is localized in a protein. diffuse pattern throughout the cell bodies and ventral nerve cord processes (Fig. 1C). We USP-46 is regulated by the proteasome estimated the levels of USP-46::GFP protein in

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We next sought to determine the point (compare lanes 5, 9, 17 and 21). In contrast, mechanism by which WDR-20 and WDR-48 expression of HA-WDR-20 alone (lanes 10-13) regulate USP-46 protein abundance by testing the did not stabilize FLAG-USP-46 resulting in a hypothesis that the WDR proteins promote the similar decline in USP-46 levels as observed with stability of the DUB. We first measured the half- CHX treatment alone. Because USP-46 is life of transiently transfected FLAG-tagged C. degraded in the proteasome, we wanted to elegans USP-46 (FLAG-USP-46) in HEK293T determine if the promotion of USP-46 abundance cells. We blocked protein synthesis with the via WDR-48 is acting through the same translational inhibitor cycloheximide (CHX) and mechanism in vivo. As described above, measured the levels of FLAG-tagged USP-46 treatment of transgenic animals expressing USP- over time by western blotting with an anti-FLAG 46::GFP in VNC interneurons with BTZ results antibody. We found that USP-46 is relatively in a 3-fold increase in USP-46::GFP fluorescence unstable and is degraded over time with a half- (Fig. 3B). Co-expression of WDR-48 alone (wdr- life of about 3-4 hours (Fig. 2, A and B, Fig. 3A). 48(xs)), or WDR-48 and WDR-20 together (wdr- We next tested if USP-46 is degraded by the 48(xs);wdr-20(xs)) result in similar increases in proteasome by co-incubating CHX-treated cells USP-46::GFP fluorescence levels, and in the with the proteasome inhibitor bortezomib (BTZ). presence of the proteasome inhibitor BTZ, Compared to cells treated with CHX alone, we expression of WDR-48 does not confer any found that FLAG-tagged USP-46 was degraded additional stability (Fig. 3B). Together, these data more slowly over time and had an increased half- suggest that WDR-48 acts to reduce the life (>8 hours) in the presence of CHX and BTZ proteasomal degradation of USP-46. (Fig. 2, A and B). To test whether USP-46 is also regulated by the proteasome in neurons in vivo, WDR-48 inhibits ubiquitination of USP-46 we treated USP-46::GFP expressing worms with If USP-46 is degraded by the BTZ for 6 hours and measured USP-46::GFP proteasome, we would expect the DUB to be fluorescence in the soma of the VNC neuron directly regulated by ubiquitin (Ub). We tested PVC. We found that USP-46::GFP levels this idea using a HEK293T cell line stably increase about 3-fold in the presence of BTZ (Fig. expressing FLAG-tagged human USP-46. We 2C). Together, these data suggest that USP-46 treated these cells with BTZ for 6 hours to block protein stability is regulated by the proteasome. degradation by the proteasome, immunoprecipitated FLAG-USP-46 under WDR-48 increases USP-46 protein stability denaturing conditions, and used anti-Ubiquitin We next tested whether the WDR antibodies to probe for Ubiquitin-USP-46 protein-mediated increase in USP-46 levels we conjugates. We found a low level of Ubiquitin- observed (Fig. 1) is due to increased USP-46 USP-46 conjugates in the absence of BTZ (Fig. stability. We measured levels of transiently 4A, lane 1), however treatment with BTZ results transfected FLAG-tagged USP-46 over time in in readily detectable high molecular weight Ub- HEK293T cells treated with CHX in the presence USP-46 conjugates (lane 2) consistent with or absence of HA-WDR-20 alone, Myc-WDR-48 polyubiquitination of USP-46. Interestingly, alone, or HA-WDR-20 and Myc-WDR-48 expression of Myc-WDR-48 alone (lane 4) or together. Similar to the effect of BTZ on USP-46 Myc-WDR-48 together with HA-WDR-20 (lane protein levels (Fig. 3A, lanes 6-9), we found that 5) in the presence of BTZ results in decreased expression of Myc-WDR-48 alone (lanes 14-17), levels of Ubiquitin-USP-46 conjugates compared or HA-WDR-20 together with Myc-WDR-48 to cells treated with BTZ alone (lane 2). In (lanes 18-21), increased the stability of FLAG- contrast, expression of HA-WDR-20 by itself USP-46 compared to CHX treatment alone (lanes (lane 3) did not reduce the levels of Ubiquitin- 2-5). This effect is most evident at the 8 hour time USP-46 conjugates, suggesting that WDR-48

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specifically blocks ubiquitination of USP-46. identical or similar amino acids (Fig. 5A). We Together, our data are consistent with the idea mutated these three residues in C. elegans WDR- that USP-46 is ubiquitinated and degraded in the 48 (R224E, W266A, K282D, hereafter referred to proteasome, and that WDR-48 inhibits USP-46 as WDR-48(3Xmut)) to test if they would disrupt ubiquitination to promote its availability in the binding between WDR-48 and USP-46, as cell. predicted by the high homology of these residues. We, and others, previously showed that We co-transfected HEK293T cells with FLAG- WDR-48 and WDR-20 bind to USP-46 and USP-46 and either wild-type Myc-WDR-48 or increase its catalytic activity (28-30,36). Several Myc-WDR-48(3Xmut). As we showed USPs, such as USP4 and USP37, have been previously (36), FLAG-USP-46 co- shown to auto-deubiquitinate in trans (41,42). immunoprecipitates with wild-type Myc-WDR- Thus, we tested whether the ability of WDR-48 48 (Fig. 5B, lane 1). In contrast, a much lower and WDR-20 to increase USP-46::GFP levels in amount of Myc-WDR-48(3Xmut) is pulled down vivo was due to increased auto-deubiquitination with FLAG-USP-46 (Fig. 5B, lane 2), suggesting of USP-46. We mutated the active site cysteine of that the triple point mutant has a diminished USP-46, which is known to eliminate the ability to interact with USP-46. We next tested catalytic activity of the DUB (36), and expressed whether the ability of WDR-48 to promote USP- catalytically-inactive USP-46(C>A)::GFP in an 46 protein levels required a direct interaction usp-46 null mutant background. We found that between the two proteins. We generated co-expression of WDR-48 and WDR-20 (wdr- transgenic animals expressing wild-type Myc- 48(xs);wdr-20(xs)) was still able to increase the WDR-48 or Myc-WDR-48(3Xmut), under abundance of USP-46(C>A)::GFP in the VNC by control of the glr-1 promoter in the VNC neurons. 3-fold (Fig. 4B), which is similar in magnitude to Consistent with our previous data, we found that the effect we observed on wild-type USP- overexpression of wild-type Myc-WDR-48 46::GFP (Fig. 1D). These results suggest that the results in increased levels of USP-46::GFP as WDR proteins increase USP-46 levels determined by western blotting of whole worm independent of its own catalytic activity. lysates (Fig. 5C) and by measuring USP-46::GFP fluorescence in the VNC (Fig. 5D). In contrast, WDR-48 binding to USP-46 is required to overexpression of Myc-WDR-48(3Xmut) was stabilize the DUB unable to increase levels of USP-46::GFP (Fig. 5, WDR-48, WDR-20 and USP-46 form a C and D). The inability of Myc-WDR-48(3Xmut) complex in vitro (24,28,29,36), and recently, the to increase USP-46::GFP levels in vivo is not due crystal structures of mammalian WDR-48/USP- to differences in expression levels because the 46, WDR-48/USP-12 and WDR-48/WDR- wild-type Myc-WDR-48 and Myc-WDR- 20/USP-12 were recently solved (33-35). C. 48(3Xmut) transgenes were expressed at elegans USP-46 is the sole homolog of both comparable levels as assessed by western blotting mammalian USP-46 and USP-12, which share whole worm lysates (Fig. 5E). These data suggest 90% sequence similarity with each other (12). that direct binding of WDR-48 to USP-46 is The crystal structures defined the critical required for the ability of WDR-48 to promote interaction interface between WDR-48 and USP- USP-46 protein levels in neurons in vivo. 46/USP-12 revealing several key amino acid residues that are required for the interaction. Yin Discussion et al. showed that mutation of 3 key residues in Ubiquitin can be added and removed WDR-48 (K214E, W256A, R272D) at the from target proteins, however less is known about interface with USP-46 disrupted binding between the regulatory mechanisms that control the two proteins (35). These three residues are deubiquitinating enzymes. We previously conserved in C. elegans WDR-48 as either showed that USP-46 regulates glutamate receptor

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levels in the VNC of C. elegans and that two and Lys235) at the interface of USP-46 and WDR proteins bind and stimulate the catalytic WDR-48 (35). It will be interesting in the future activity of USP-46 (12,36), consistent with other to test if WDR-48 inhibits ubiquitination of USP- studies (24,28,29). Here, we identify another 46 by blocking access of an E3 ligase to these mechanism by which the WDR proteins regulate surface lysines. Interestingly, one or both of these DUBs. We find that expression of WDR-48, but lysine residues are conserved in USP-1 (Lys503) not WDR-20, increases the abundance of USP-46 and USP-12 (Lys230 and Lys239), suggesting in cultured mammalian cells and C. elegans that WDR-48 may also stabilize these closely neurons in vivo. We show that USP-46 is related USPs via the same mechanism. The WDR ubiquitinated and degraded in the proteasome. proteins and USP-46/USP-12 are conserved Overexpression of WDR-48 blocks across phylogeny from yeast to humans, where ubiquitination of USP-46, and binding of WDR- they regulate several important processes 48 to USP-46 is required to promote DUB protein including endocytosis, cell polarity, signal levels. These data show that WDR-48 stabilizes transduction and mitochondrial biogenesis USP-46 and suggest that controlling WDR-48 (11,25,43,44). These WDR protein homologs expression may provide a novel mechanism to may also regulate the stability of USP-46 control DUB availability. homologs in non-neuronal cells in other species. What is the mechanism by which WDR- For example, overexpression of WDR-48, but not 48 decreases ubiquitination of USP-46? Our data WDR-20, can increase protein levels of human show that expression of WDR-48 decreases USP-12 resulting in stabilization of androgen levels of ubiquitin-USP-46 conjugates (Fig. 4A) receptors and proliferation of prostate cancer and that WDR-48 binding to USP-46 is required cells (16). In the filamentous fungi Aspergillus for its ability to stabilize the DUB (Fig. 5). nidulans, protein levels of the USP-46 homolog Because WDR proteins can stimulate the CreB, were dramatically increased after catalytic activity of USP-1, USP-12 and USP-46 expression of the WDR protein CreC (45). (28,30,31,36) and several DUBs such as USP4 Although the precise mechanism controlling and USP37 have been shown to deubiquitinate DUB levels was not investigated in these studies, themselves in trans (41,42), we tested if USP-46 our work suggests that these WDR proteins may catalytic activity was required for the ability of increase protein levels of their interacting DUB WDR-48 to regulate USP-46 levels. We found partners by preventing their ubiquitination and that expression of WDR-48 increased the levels degradation in the proteasome. We propose that of catalytically-inactive USP-46(C>A)::GFP by this mechanism of stabilizing USP-46 and its about 3 fold (Fig. 4B), which is similar to the homologs is likely conserved across phylogeny magnitude of its effect on wild-type USP- and may be a widely used mechanism to stabilize 46::GFP (Fig. 1D). These data are consistent with DUBs given that over 35% of all USPs interact the idea that WDR-48 decreases ubiquitination of with WDR proteins (24). USP-46 and promotes USP-46 protein levels WDR-48 regulates USP-46 related independent of its own catalytic activity. DUBs via multiple mechanisms and is thus Although we do not know the precise mechanism emerging as a critical DUB regulatory protein. by which WDR-48 prevents USP-46 First, WDR-48, either alone or together with ubiquitination, we propose that WDR-48 binding WDR-20, can bind and stimulate the catalytic to USP-46 either prevents the recruitment of an activity of USP-1, USP-46 and USP-12 (28- E3 ligase or blocks the ability of an E3 ligase to 31,36). Second, WDR-48 can function as a ubiquitinate key lysine residues on the surface of substrate adaptor. For example, WDR-48 can USP-46. Intriguingly, the crystal structure of bind substrates such as FANCD2/FANCI WDR-48 bound to USP-46 reveals the presence heteromers via its SUMO-like domain (SLD) and of two USP-46 surface lysine residues (Lys226 recruit them to USP-1 (46). Similarly, the C-

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terminal region of WDR-48 which includes the in response to changes in synaptic activity. SLD is sufficient to bind the USP-12 substrate Interestingly, another DUB that can also regulate PHLPP1 (17). Third, WDR-48 can regulate the mammalian AMPA-type glutamate receptors, subcellular localization of its interacting USP. USP8, has been shown to be regulated by Bun107 and Bun62, the yeast homologs of WDR- synaptic activity. In this case, activation of 48 and WDR-20, respectively, can recruit the NMDARs leads to rapid dephosphorylation and USP-46 homolog Ubp9, to specific locations in activation of the DUB (48). Further studies will the cytoplasm (25). The ability of WDR-48 to be necessary to identify the upstream signals that relocalize USPs is underscored by a study control expression of both WDR-48 and WDR- showing that human papilloma viruses (HPVs) 20 in neurons to regulate USP-46 function. have evolved to manipulate WDR-48 in order to USP-46 and USP-12 have been control the subcellular localization of their implicated in several cancers (including colon interacting USPs. Specifically, HPV protein E1 cancer, prostate cancer and glioblastoma) and in binds to WDR-48 and recruits USP-1, USP-12 regulating both glutamatergic and GABAergic and USP-46 to viral origins of replication in the signaling in the nervous system. The fact that nucleus (47). These studies, together with our DUBs are proteases makes them an attractive data showing that WDR-48 promotes USP-46 drug target; however, blocking the catalytic stability, reveal that WDR-48 is a versatile cysteine residue present in the active sites of most regulator of this group of USPs. DUBs and many other proteases, could lead to Our data suggest that USP-46 is non-selective effects. Our study shows that continuously degraded and that increased WDR-48 stabilizes USP-46 and that interaction expression of WDR-48 promotes the stability of of WDR-48 with USP-46 is required for this the DUB. We suspect that WDR-48 levels are effect. Designing drugs that disrupt the kept at a relatively low level resulting in low interaction of WDR-48 with USP-46 could be an levels of USP-46. We propose that increased effective and more specific strategy to inhibit expression of WDR-48 stabilizes USP-46 as a USP-46 function. We propose that targeting the mechanism to control DUB availability. We interaction interface between WDR proteins and previously showed that overexpression of USP- their DUB partners may be a promising and more 46 alone does not increase its ability to regulate specific approach to destabilize DUBs and thus GLR-1 (36), suggesting that in vivo there are inhibit their function. other limiting factors required to regulate USP- 46. Although our data show that expression of Experimental Procedures WDR-48 alone is sufficient to increase the Strains—The following strains were used for stability of USP-46, co-expression of WDR-48 experiments described in this manuscript: and WDR-20 are required to fully stimulate the N2 (Bristol) wild type, pzIs40 (Pnmr-1::USP- catalytic activity of the DUB and modulate 46::GFP) II, ljIs114 (Pgpa-13::FLPase; Psra- glutamatergic behavior (36). Together, these data 6::FTF::ChR2::YFP) X (gift from William suggest that increasing the expression of WDR- Schafer), pzIs37 (Pglr-1::USP-46::GFP) III, 48 alone is not sufficient to promote USP-46 pzEx386 (Pnmr-1::GFP), usp-46 (ok2232) III, function. A signal that increases expression of pzIs25 (Pglr-1::WDR-20; Pglr-1::WDR-48) I, WDR-48 together with another signal that pzEx230 (Pglr-1::WDR-20), pzEx231 (Pglr- controls WDR-20 expression could function as an 1::WDR-48), pzEx378 (Pglr-1::USP- “AND” gate to precisely control USP-46 46(C38A)::GFP), pzEx456 (Pglr-1::Myc-WDR- function. Although the relevant signals that 48), pzEx457 (Pglr-1::Myc-WDR-48(W266A, regulate WDR-48 expression in vivo are not yet R224E, K282D)) (also referred to as 3Xmut). All known, we speculate that WDR-48 expression in strains were maintained at 20 °C as described neurons may be regulated during development or previously (49).

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Constructs, Transgenes and Germ-line Imaging—Fluorescence imaging of USP- Transformation—pzIs25, pzEx230, pzEx231 and 46::GFP was performed as follows. Briefly, L4 pzEx378 were described previously (36). Pnmr- larval-stage animals were immobilized using 30 1::USP-46::GFP (FJ#129) was generated by mg/ml 2,3-butanedione monoxamine (Sigma- replacing the Pglr-1 promoter in Pglr-1::USP- Aldrich), and the ventral nerve cord (VNC) was 46::GFP (FJ#109) with Pnmr-1 (~1 kb) from imaged in the anterior region of the animals just pBM16 (50) using SphI and BamHI sites. pzIs40 posterior to the RIG neuronal cell bodies or in the was created by injecting FJ#129 (50 ng/µl) with cell body of the VNC neuron PVC. 1 µm (total the coinjection marker Pttx-3::GFP (50 ng/µl) depth) Z-series stacks were collected using a Carl followed by integration using a UV Stratalinker. Zeiss Axioscope M1 microscope with a 100X pzIs40 was backcrossed 3 times prior to imaging. Plan Apochromat (1.4 numerical aperture) pzIs37 was created by injecting FJ#109 (50 ng/ul) objective equipped with GFP and Cy3.5 filters. with the coinjection marker Pttx-3::GFP (50 Images were collected with an Orca-ER charge ng/µl) followed by integration using a UV coupled device (CCD) camera (Hamamatsu) and Stratalinker. pzIs37 was backcrossed 3 times MetaMorph (version 7.1) software (Molecular prior to imaging. pzEx378 was created by Devices). Maximum intensity projections of Z- injecting FJ#109 (50 ng/µl) with the coinjection series stacks were used for quantitative analyses marker Pttx-3::dsRed (50 ng/µl). pzEx386 was of fluorescence. Exposure settings and gain were created by injecting pBM16 (50 ng/µl) with the adjusted to fill the 12-bit dynamic range without coinjection marker Pttx3::GFP (50ng/µl). saturation and were identical for all images. The pMT3-FLAG-USP-46 (FJ#66), pMT3-HA- intensity for each worm was determined by WDR-20 (FJ#94), pMT3-Myc-WDR-48 (FJ#96), averaging the maximum intensity from three have been previously described (36). The separate ROIs. For proteasome inhibitor mammalian expression vector pMT3 was kindly pretreatment, animals were placed on NGM provided by Dr. Larry Feig. Myc-WDR-48 (~2 plates containing 50 µM Bortezomib for 6 hours kb) was subcloned into the C. elegans expression prior to imaging (51). vector pV6 for expression under the glr-1 promoter to create Pglr-1::Myc-WDR-48 Cell culture and Transfections—HEK293T cells (FJ#130) by first cloning into pBlueScript using were cultured in Dulbecco's modified Eagle's HindIII and KpnI sites and then into FJ#109 using medium (DMEM) supplemented with 10% fetal BamHI and KpnI sites. pzEx456 was created by bovine serum (FBS) and 100 U/ml penicillin. injecting FJ#130 (50 ng/µl) with the coinjection Cells were maintained in a humidified, 5% CO2 marker Pmyo-2::NLS-mCherry (50 ng/µl). atmosphere at 37°C. pMT3-Myc-WDR-48(R224E,W266A, K282R) For protein synthesis inhibition also referred to as Myc-WDR-48(3Xmut) experiments, HEK293T cells were seeded into a (FJ#131) was generated by using Quikchange 100 mm dish and transfected with 6 µg of FLAG- (Invitrogen) to generate point mutations in the USP-46 using Polyethylenimine (PEI, parent plasmid pMT3-Myc-WDR-48 PolySciences) for 16-18 hours. Cells were (FJ#96)(36). Myc-WDR-48(R224E,W266A, trypsinized and plated into a 6 well plate and K282R) (~2 kb) was subcloned into the C. reverse transfected with Myc-WDR-48 (3 µg elegans expression vector pV6 for expression DNA), and/or HA-WDR-20 (2 µg DNA) using under the glr-1 promoter to create Pglr-1::Myc- Lipofectamine 2000 (Invitrogen). 24 hours post- WDR-48 (R224E,W266A, K282R)(FJ#132) as transfection the cells were again split into 12-well described for FJ#130. pzEx457 was created by plates at 100,000 cells per well for cycloheximide injecting FJ#132 (50 ng/µl) with the co-injection time course studies. Cells were lysed in marker Pmyo-2::NLS-mCherry (50 ng/µl). mammalian cell lysis buffer (MCLB, 50 mM Tris

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(pH 7.5), 150 mM NaCl, 0.5% Nonidet P-40, to acquire a final SDS concentration of 0.1%. HALT Inhibitors (Pierce)). Cells were incubated Lysates were incubated with anti-FLAG at 4°C for 10 min and then centrifuged at 14,000 antibody–coupled magnetic beads overnight at 4 rpm for 15 min at 4°C. The supernatant was ºC and then washed 3 times with MCLB. Beads removed, and the protein concentration was were suspended in 2X SDS sample buffer, estimated using the Bradford method (Bio-Rad). resolved by SDS-PAGE and transferred to For immunoprecipitation studies, nitrocellulose membranes. Membranes were HEK293T cells in 6-well plates were transfected blocked in Tris-buffered saline with Tween with FLAG-USP-46, Myc-WDR-48, Myc-WDR- (TBS-T) and 5% milk prior to incubation with 48(R224E, W266A, K282D) or HA-WDR-20, various primary antibodies. with a total of 2 µg of DNA per well, using Whole worm lysates were obtained by Lipofectamine 3000 and harvested and lysed at placing 100 animals in 2X SDS sample buffer and 24-36 hours post-transfection for boiling at 95˚C for 5 min, vortexing for 1 min and immunoprecipitations. HEK293T cells stably boiling again at 95˚C for 5 min. Samples were expressing human FLAG/HA-USP46 was subjected to SDS-PAGE and immunoblotting. created by lentiviral transduction as described The following antibodies were used for elsewhere (52). Stable clones were selected using immunoblotting: mouse-anti-GFP (Covance), 1 µg/ml puromycin. rabbit anti-tubulin (ab4074, Abcam), rabbit anti- FLAG (M2, Sigma), mouse anti-Myc (clone Immunoprecipitation and Immunoblotting— 9E10, Santa Cruz), rat anti-HA (Covance), mouse For immunoprecipitation experiments, anti-PCNA (clone PC10, Santa Cruz), and mouse HEK239T cells were washed once with PBS and anti-GAPDH (clone 0411, Santa Cruz), and lysed after 24 h with MCLB. Lysed cells were mouse anti-ubiquitin (clone P4D1, Santa Cruz). centrifuged at 14,000 rpm for 10 min at 4°C. Cleared lysate was incubated for 4–12 hours with Behavioral assays—All behavioral assays were anti-FLAG antibody-coupled magnetic beads performed using at least 10 young adult (Sigma) in the presence of 10 µg/ml HALT hermaphrodites over at least 3 independent protease inhibitors and phosphatase inhibitors experiments and by an experimenter who was (sodium fluoride 50 µg/ml, sodium blinded to the genotypes of the animals being orthovanadate 5 µg/ml). Immunoprecipitated tested. Optogenetic activation of the ASH- complexes were washed four times with MCLB dependent nose touch response was performed and resuspended in 2X SDS-PAGE sample similarly to what has been described previously buffer. Samples were subjected to SDS-PAGE on (53). Gentle touch to the nose of the worm (nose- 10% acrylamide gels and subsequently touch) (39,40,54) or photostimulation of ASH transferred to a nitrocellulose membrane. expressing channelrhodopsin2 (ChR2)(53,55) Membranes were blocked in Tris-buffered saline result in locomotion reversals away from the with Tween (TBS-T) and 5% milk prior to stimulus. This nose-touch response is dependent incubation with various primary antibodies. on presynaptic glutamate and GLR-1 in For denaturing immunoprecipitation postsynaptic interneurons (39,40,54). We studies, FLAG/HA USP46 293T cells were lysed previously showed that mutants with decreased in MCLB containing 1% SDS, and vortexed at levels of GLR-1 in the VNC exhibit defects in the room temperature for 10 min. Insoluble material nose-touch response (12). Animals expressing was removed by centrifugation (14,000rpm, 5 ChR2 specifically in ASH sensory neurons min). Protein concentration in the supernatants (ljIs114, Pgpa-13::FLPase; was determined by BCA assay (Pierce). Equal Psra6::FTF::ChR2::YFP)(56) were grown for amounts of protein from each experimental one generation in the dark on NGM agar plates sample were taken and diluted 10 fold in MCLB spotted with OP50 and the ChR2 co-factor all-

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trans-retinal (ATR, 100 µM). Animals were locomotor reversal was scored as a positive subsequently transferred to an NGM agar plate response if the backward movement was greater spotted with OP50 (without ATR) and than the distance from the nose to the terminal illuminated with 1 s pulses of blue light (0.47 bulb of the pharynx observed during or mW/mm2) from a mercury bulb filtered through a immediately after blue light illumination. GFP excitation filter (480 nm) under 32x total magnification on a Leica MZ16F microscope. A

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WDR-48 promotes USP-46 stability

Acknowledgements: We would like to thank William Schafer and the Caenorhabditis Genetics Center for strains, and Larry Feig for reagents. We thank Colleen Kreiser for help with preliminary experiments, and Eric Luth, Bethany Rennich and Betty Ortiz for advice and critical comments on this manuscript.

Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this article.

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25. Kouranti, I., et al. (2010) A global census of fission yeast deubiquitinating enzyme localization and interaction networks reveals distinct compartmentalization profiles and overlapping functions in endocytosis and polarity. PLoS Biol 8, e1000471 26. Pashkova, N., et al. (2010) WD40 repeat propellers define a ubiquitin-binding domain that regulates turnover of F box proteins. Mol Cell 40, 433-443 27. Stirnimann, C. U., et al. (2010) WD40 proteins propel cellular networks. Trends Biochem Sci 35, 565-574 28. Cohn, M. A., et al. (2009) UAF1 is a subunit of multiple deubiquitinating enzyme complexes. J Biol Chem 284, 5343-5351 29. Kee, Y., et al. (2010) WDR20 regulates activity of the USP12 x UAF1 deubiquitinating enzyme complex. J Biol Chem 285, 11252-11257 30. Faesen, A. C., et al. (2011) The differential modulation of USP activity by internal regulatory domains, interactors and eight ubiquitin chain types. Chem Biol 18, 1550-1561 31. Cohn, M. A., et al. (2007) A UAF1-containing multisubunit protein complex regulates the Fanconi anemia pathway. Mol Cell 28, 786-797 32. Villamil, M. A., et al. (2012) Serine phosphorylation is critical for the activation of ubiquitin- specific protease 1 and its interaction with WD40-repeat protein UAF1. Biochemistry 51, 9112- 9123 33. Dharadhar, S., et al. (2016) A conserved two-step binding for the UAF1 regulator to the USP12 deubiquitinating enzyme. J Struct Biol 196, 437-447 34. Li, H., et al. (2016) Allosteric Activation of Ubiquitin-Specific Proteases by beta-Propeller Proteins UAF1 and WDR20. Mol Cell 63, 249-260 35. Yin, J., et al. (2015) Structural Insights into WD-Repeat 48 Activation of Ubiquitin-Specific Protease 46. Structure 23, 2043-2054 36. Dahlberg, C. L., and Juo, P. (2014) The WD40-repeat proteins WDR-20 and WDR-48 bind and activate the deubiquitinating enzyme USP-46 to promote the abundance of the glutamate receptor GLR-1 in the ventral nerve cord of Caenorhabditis elegans. J Biol Chem 289, 3444-3456 37. Brockie, P. J., et al. (2001) The C. elegans glutamate receptor subunit NMR-1 is required for slow NMDA-activated currents that regulate reversal frequency during locomotion. Neuron 31, 617- 630 38. Brockie, P. J., et al. (2001) Differential expression of glutamate receptor subunits in the nervous system of Caenorhabditis elegans and their regulation by the homeodomain protein UNC-42. J Neurosci 21, 1510-1522 39. Hart, A. C., et al. (1995) Synaptic code for sensory modalities revealed by C. elegans GLR-1 glutamate receptor. Nature 378, 82-85 40. Maricq, A. V., et al. (1995) Mechanosensory signalling in C. elegans mediated by the GLR-1 glutamate receptor. Nature 378, 78-81 41. Huang, X., et al. (2011) Deubiquitinase USP37 is activated by CDK2 to antagonize APC(CDH1) and promote S phase entry. Mol Cell 42, 511-523 42. Zhang, L., et al. (2012) USP4 is regulated by AKT phosphorylation and directly deubiquitylates TGF-beta type I receptor. Nat Cell Biol 14, 717-726 43. Kanga, S., et al. (2012) A deubiquitylating complex required for neosynthesis of a yeast mitochondrial ATP synthase subunit. PLoS One 7, e38071 44. Moretti, J., et al. (2012) The Ubiquitin-specific Protease 12 (USP12) Is a Negative Regulator of Notch Signaling Acting on Notch Receptor Trafficking toward Degradation. J Biol Chem 287, 29429-29441 45. Lockington, R. A., and Kelly, J. M. (2002) The WD40-repeat protein CreC interacts with and stabilizes the deubiquitinating enzyme CreB in vivo in Aspergillus nidulans. Mol Microbiol 43, 1173-1182 46. Yang, K., et al. (2011) Regulation of the Fanconi anemia pathway by a SUMO-like delivery network. Genes Dev 25, 1847-1858

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47. Lehoux, M., et al. (2014) E1-mediated recruitment of a UAF1-USP deubiquitinase complex facilitates human papillomavirus DNA replication. J Virol 88, 8545-8555 48. Scudder, S. L., et al. (2014) Synaptic strength is bidirectionally controlled by opposing activity- dependent regulation of Nedd4-1 and USP8. J Neurosci 34, 16637-16649 49. Brenner, S. (1974) The genetics of Caenorhabditis elegans. Genetics 77, 71-94 50. Moss, B. J., et al. (2016) The CaM Kinase CMK-1 Mediates a Negative Feedback Mechanism Coupling the C. elegans Glutamate Receptor GLR-1 with Its Own Transcription. PLoS Genet 12, e1006180 51. Chen, Y., et al. (2016) Caenorhabditis elegans paraoxonase-like proteins control the functional expression of DEG/ENaC mechanosensory proteins. Mol Biol Cell 27, 1272-1285 52. Raman, M., et al. (2015) Systematic proteomics of the VCP-UBXD adaptor network identifies a role for UBXN10 in regulating ciliogenesis. Nat Cell Biol 17, 1356-1369 53. Schmitt, C., et al. (2012) Specific expression of channelrhodopsin-2 in single neurons of Caenorhabditis elegans. PLoS One 7, e43164 54. Kaplan, J. M., and Horvitz, H. R. (1993) A dual mechanosensory and chemosensory neuron in Caenorhabditis elegans. Proc Natl Acad Sci U S A 90, 2227-2231 55. Kindt, K. S., et al. (2007) Caenorhabditis elegans TRPA-1 functions in mechanosensation. Nat Neurosci 10, 568-577 56. Ezcurra, M., et al. (2011) Food sensitizes C. elegans avoidance behaviours through acute dopamine signalling. EMBO J 30, 1110-1122

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FOOTNOTES Funding for this work was supported in part by a National Science Foundation Grant (IOS1353862 to P.J.), National Institutes of Health Grants (R56NS059953 to P.J., and R01GM127557 to M.R.) and the Synapse Neurobiology Training Grant (T32 NS061764 to M.H.), the Tufts Center for Neuroscience Research Grant (P30NS047243) and the NIH Office of Research Infrastructure Programs Grant (P40OD010440) to the Caenorhabditis Genetics Center.

The abbreviations used are: DUB, deubiquitinating enzyme; WDR, WD40-repeat; VNC, ventral nerve cord; IP, immunoprecipitation; USP, ubiquitin-specific protein; CHX, cycloheximide; BTZ, bortezomib; UAF1, USP1-associated factor-1

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WDR-48 promotes USP-46 stability

A. B. USP-46::GFP in Whole Worm Lysates USP-46::GFP 2 in Whole Worm Lysates # #

75 kDa USP-46::GFP n.s. 1 Tubulin 50 kDa (Normalized to WT)

Wild Type USP-46::GFP Band Intensity wdr-48(xs); wdr-48(xs) wdr-20(xs) 0 wdr-20(xs)

Wild Typewdr-48(xs); wdr-48(xswdr-20) (xs) wdr-20(xs) C. USP-46::GFP in D. USP-46::GFP in ventral cord neurons ventral cord neurons 3 ** Wild Type ** wdr-48(xs); 2 wdr-20(xs) ** wdr-48(xs) 1 GFP Fluorescence wdr-20(xs) (Normalized to WT ) 10µm 0

Wild Typewdr-48(xs); wdr-48(xswdr-20) (xs) wdr-20(xs) E. Pglr-1::USP-46::GFP F. Pnmr-1::GFP in ventral cord neurons in ventral cord neurons 4 2 ** 3

n.s. 2 1

1 GFP Fluorescence GFP Fluorescence (Normalized to WT ) (Normalized to WT )

0 0

Wild Typewdr-48(xs); Wild Typewdr-48(xs); wdr-20(xs) wdr-20(xs)

Figure 1. WDR-48 promotes USP-46 protein abundance. A, Representative immunoblots showing USP-46::GFP expression levels (top), as detected with anti-GFP antibodies, in total worm lysates isolated from L4 stage larval animals harboring a USP-46::GFP transgene expressed under control of the nmr-1 promoter (pzIs40) in either a wild type background, or in animals overexpressing wdr-48 and wdr-20 together (wdr-48(xs);wdr-20(xs)), wdr-48 alone (wdr-48(xs)), or wdr-20 (wdr-20(xs)) alone. Tubulin was also detected in these lysates (bottom) as a loading control. B, Quantification of the USP-46::GFP bands from three independent western blots from the strains described in A. C, Representative images of USP-46::GFP in the VNCs of L4 stage larval pzIs40 animals for wild-type (n=57), wdr-48(xs);wdr-20(xs) (n=49), wdr-48(xs) (n=16), and wdr-20(xs) (n=27) animals. D, Quantification of USP-46::GFP fluorescence intensities (normalized) for the strains described in C. E, Quantification of the USP-46::GFP fluorescence intensities in the VNCs of L4 larval animals harboring a USP-46::GFP transgene expressed under the control of the glr-1 promoter (pzIs37). Shown are the GFP fluorescence intensities (normalized) for

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WDR-48 promotes USP-46 stability

wild-type (n=20) and wdr-48(xs);wdr-20(xs) animals (n=20). F, Quantification of VNCs of L4 larval animals harboring a GFP transgene expressed under the control of the nmr-1 promoter (pzEx386). Shown are the GFP fluorescence intensities (normalized) for wild-type (n=47) and wdr-48(xs);wdr-20(xs) animals (n=28). For all graphs, mean intensity ± S.E.M. are shown. Values that differ significantly from the wild type (Student’s t-test or ANOVA followed by Dunnett’s multiple comparison tests) are indicated as follows: #, p<0.05; **, p ≤ 0.001; n.s., p>0.05.

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WDR-48 promotes USP-46 stability

A. CHX BTZ (1µM) C. USP-46::GFP (100 µg/mL) + CHX (100 µg/mL) in ventral cord neuron PVC Time (hr) UT 0 2 4 6 8 0 2 4 6 8 4 FLAG-USP-46 50 kDa ** 37 kDa PCNA 3

Lane: 1 2 3 4 5 6 7 8 9 10 11 B. 2 CHX 100 * CHX+BTZ GFP Fluorescence ** (Normalized to WT ) 1 80 ** ** 60 0

40 BTZ Remaining DMSO 20 Percent FLAG-USP-46 0 0 2 4 6 8 Time (hr) Figure 2. USP-46 is regulated by the proteasome. A, Representative immunoblot analysis of HEK293T cells transiently transfected with FLAG-USP-46 and either left untreated (UT, lane 1), or treated for the indicated times with cycloheximide (CHX, 100 µg/mL) alone (lanes 2-6) or CHX together with bortezomib (BTZ, 1 µM) (lanes 7-11). Cell lysates were immunoblotted for FLAG-USP-46 or PCNA (as a loading control). B, Quantification of the levels of FLAG-USP-46 from three independent experiments as described in A (normalized) are shown (means ± S.D.). C, Quantification of USP-46::GFP fluorescence intensities (normalized) in the soma of PVC ventral cord neurons of L4 larval animals harboring a USP-46::GFP transgene expressed under the control of the nmr-1 promoter (pzIs40) treated with vehicle alone (DMSO) (n=12) or 50 µM BTZ (n=26, means ± S.E.M.) for 6 hours. Values that differ significantly from the control (Student’s t-test) are indicated as follows: *, p ≤ 0.01; **, p ≤ 0.001.

WDR-48 promotes USP-46 stability

A. FLAG-USP-46 in HEK283T Cells CHX (100 µg/mL)

HA-WDR-20 + BTZ (1µM) HA-WDR-20 Myc-WDR-48 Myc-WDR-48 UT 0 2 4 8 0 2 4 8 0 2 4 8 0 2 4 8 0 2 4 8 :Time (hr)

50 kDa FLAG-USP-46

1 0.8 0.5 0.2 1 0.9 0.7 0.4 1 0.7 0.4 0.2 1 0.8 0.6 0.4 1 0.8 0.6 0.5 :Fold Change 100 kDa HA-WDR-20

100 kDa HA-WDR-20 75 kDa Myc-WDR-48

37 kDa GAPDH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 :Lane

B. USP-46::GFP in ventral cord neuron PVC DMSO 4 BTZ n.s. 3 ** n.s.

2 GFP Fluorescence (Normalized to WT ) 1

Wild Type wdr-48(xs)

wdr-48(xs); wdr-20(xs) Figure 3. WDR-48 increases USP-46 protein stability. A, Representative immunoblot analysis of HEK- 293T cells transiently transfected with FLAG-USP-46 and either left untreated (UT, lane 1) or treated with cycloheximide (CHX, 100 µg/mL) for the indicated times (lanes 2-21). In addition, cells were either treated with bortezomib (BTZ, 1 µM) (lanes 6-9) or transiently co-transfected with HA-WDR-20 (lanes 10-13), Myc-WDR-48 (lanes 14-17), or both HA-WDR-20 and Myc-WDR-48 together (lanes 18-21). GAPDH was used as a loading control. Whole cell lysates were immunoblotted for the various epitope-tagged proteins or GAPDH, as indicated. B, Quantification of USP-46::GFP fluorescence intensities (normalized) in the soma of PVC ventral cord neurons of L4 larval animals harboring a USP-46::GFP transgene expressed under the control of the nmr-1 promoter (pzIs40) with or without 6 hour exposure to BTZ. Shown are the USP-46::GFP fluorescence intensities for wild-type animals treated with either DMSO vehicle control (n=21) or BTZ (n=10), wdr-48(xs);wdr-20(xs) animals in DMSO (n=10), wdr-48(xs);wdr-20(xs) animals in BTZ (n=8), wdr-48(xs) animals in DMSO (n=10), and wdr-48(xs) animals in BTZ (n=8). Both wdr-48 and wdr-20 were expressed under control of the glr-1 promoter. BTZ treatment values that differ significantly from DMSO control within genotypes (ANOVA followed by Dunnett’s multiple comparison test) are indicated as follows: **, p ≤ 0.001; n.s., p>0.05.

18 bioRxiv preprint that differ significantlyfromthecontrol(Student’s t-test)areindicatedasfollows: **,p≤0.001. either without(n=19)orwith co-expressionofwdr-48 andwdr-20 (wdr-48(xs);wdr-20 (xs))(n=20). harboring aUSP-46(C38A)::GFP transgene expressedunderthecontrolofglr-1 promoter(pzEx378) GFP fluorescence intensity (means ±S.E.M.)inthe VNCs ofL4larvalusp-46(ok2232)nullmutants as indicated. Similar resultswereobtainedinthreeindependentexperiments.B, GAPDH, as indicated. Whole cell lysates (WCL)wereimmunoblotted forthevarious epitope-taggedproteinsand conditions followedbyimmunoblot analysis withanti-ubiquitin antibodiesoranti-HA/FLAGantibodies, (lane 5). Cell lysates were subjected to immunoprecipitation withanti-FLAGantibodyunderdenaturing HA-WDR-20 (lane 3) alone, Myc-WDR-48 (lane 4) alone,or Myc-WDR-48 andHA-WDR-20together Bortezomib (BTZ, 1µM)for6hours(lanes2-5).Somecellswerealsotransientlytransfectedwitheither wereeither leftuntreated(lane1)ortreatedwith (HA/FLAG-USP46) ing HA/FLAG-taggedhumanUSP46 ubiquitin-USP-46 conjugatesinHEK293T cellsundervariousconditions.HEK293T cellsstablyexpress- Figure 4. WDR-48 blocks ubiquitination of USP-46. Representative immunoblot comparing the levels of B. A. HA/FLAG-USP46 BTZ (1µM,6hr) Myc-WDR-48 FLAG-USP46 Myc-WDR-48 HA-WDR-20 HA-WDR-20 GAPDH Lanes: Ub Ub GFP Fluorescence doi: certified bypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission.

wdr-48(xs); wdr-20(xs) (Normalized to usp-46(lf)) Stable HEK293T Cell Line 12345

in ventral cord neurons https://doi.org/10.1101/679415

usp-46 (lf) 0 1 2 3 4 usp-46( USP-46(C>A)::GFP - -

FLAG-USP46 -+++ + + +- lf) - - + - ** + - + + 25 kDa 75 kDa 37 kDa 25 kDa 75 kDa 50 kDa 75 kDa 75 kDa 50 kDa 50 kDa

WCL IP: FLAG ; this versionpostedJune21,2019. 19 The copyrightholderforthispreprint(whichwasnot WDR-48 promotes USP-46stability Quantification of Values bioRxiv preprint Shown areUSP-46::GFP fluorescence intensities (normalized) forwild type(n=15),wdr-48(xs, n=16) animals harboring a USP-46::GFP transgene expressed underthecontrolofnmr-1 promoter(pzIs40). (bottom) asaloadingcontrol. D,Quantification of USP-46::GFP fluorescenceinthe VNCs ofL4 larval wdr-48 promoter (pzIs40,top)fromwild-type animals, or animalsexpressingeitherwild typewdr-48 (xs)or lysates ofL4larvalanimalsharboringaUSP-46::GFP transgeneexpressedunderthecontrolofnmr-1 Representative immunoblot for total USP-46::GFP, asdetectedwithanti-GFP antibodies,inwholeworm epitope-tagged proteins, asindicated. Similar resultswereobtainedinthreeindependentexperiments. C, noblotting withanti-Mycantibodies. Whole celllysates(WCL)wereimmunoblotted forthevarious lane 2).Celllysatesweresubjectedtoimmunoprecipitation withanti-FLAGantibodiesfollowedbyimmu- fected together with FLAG-USP-46 with either wild typeMyc-WDR-48(lane1)orMyc-WDR-48(3Xmut, that was co-immunoprecipitated from HEK293Tby FLAG-USP-46 cells.Cells weretransientlyco-trans- asterisks. Myc-WDR-48(R224E, W266A, andK282D),alsoreferredtoasMyc-WDR-48(3Xmut),aremarkedby arrows. The positions of the corresponding amino acids inC.elegans WDR-48 thatweremutatedin tions in H.sapiens WDR48 (K214E, binding totheDUBaremarkedby W256A, andR272D)thatdisrupt between sequence alignment illustrating the similarity (grey shading)oridentity(blackofaminoacids Figure 5.Directbindingof WDR-48 toUSP-46isrequired tostabilizetheDUB. D. B. A. Myc-WDR-48(3xmut)

GFP Fluorescence FLAG-USP-46 FLAG-USP-46 C. elegans H. sapiens C. elegans H. sapiens Myc-WDR-48 Myc-WDR-48 FLAG-USP-46 (Normalized to WT) Myc-WDR-48 (3xmut, xs)expressedunder control of theglr-1 promoter. Tubulin wasalsodetectedintheselysates 0 1 2 3 H. sapiensand B, Representative the amountofMyc-WDR-48 and Myc-WDR-48(3Xmut) immunoblot showing doi: Wild Type Lane:

in ventral cord neurons certified bypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. IgG 148 QQRCIAT 138 QQRCIAT 112 KLRGHTDNVRALVVN 102 KLKGHTDNVKALLLN T https://doi.org/10.1101/679415 12 wdr-48(xs) USP-46::GFP wdr-48(3xmut,xs) + + - ** + + - n.s. C. elegans WDR48 in the region thatinteractswithUSP46(34). Three pointmuta- 50 kDa 50 kDa 50 kDa 50 kDa 50 kDa CIAHEEGVW YRVHDEGVW E * WCL IP: α-FLAG Myc-WDR-48 E. C. USP-46::GFP DDGTRALSAGSDTIRLWIG RDGTQCLSGSSDGTIRLWSLG A * ; Tubulin TLQVDSSFTVYSAGKDKMV ALQVNDAFTHVYSGGRDRKI Tubulin this versionpostedJune21,2019.

Wild Type Wild Type

in Whole Worm Lysates

Myc-WDR-48 USP-46::GFP wdr-48(xs) wdr-48(xs) 20 wdr-48(3xmut,xs) wdr-48(3xmut,xs) D * 50 kDa 75 kDa 50 kDa 50 kDa The copyrightholderforthispreprint(whichwasnot WDR-48 promotes USP-46stability A, Partialprotein bioRxiv preprint doi: https://doi.org/10.1101/679415; this version posted June 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

WDR-48 promotes USP-46 stability

and wdr-48(3xmut, xs, n=12) (means ± S.E.M). E, Representative immunoblots for Myc-WDR-48 and Myc-WDR-48(3Xmut) expression, as detected with anti-Myc antibodies, of lysates from L4 larval animals of wild-type, wdr-48(xs), and wdr-48(3xmut,xs) animals (top). Tubulin was also detected in these lysates (bottom) as a loading control. Results from three independent experiments show that the relative abundance of Myc-WDR-48 is similar to that of Myc-WDR-48(3xmut, means ± S.E.M.). Values that differ significantly from the wild type (ANOVA followed by Dunnett’s multiple comparison test) are indicated as follows: **, p ≤ 0.001; n.s., p>0.05.

21 bioRxiv preprint upeetl iue 1. Figure Supplemental multiple comparisontests)areindicatedasfollows:**, (pzIs40);usp-46ok2232, (n=10), type wild mental procedures). The average number of reversals per 10 stimuli are shown for the following genotypes: Experi- (see measured was stimuli reversals/10 of number the and light blue with stimulated were worms F::ChR2::YFP) were fed with OP50 along with the ChR2 co-factor all- ing ASH-dependent nose touch response was performed, as described previously (53). Animals stably express- Reversals (per 10 stimuli) hneroosn (h2 i AH esr nuos (ljIs114, Pgpa-13::FLPase; neurons sensory ASH in (ChR2) channelrhodopsin2 10

Wild Type 0 2 4 6 8

usp-46 Optogenetic Nose doi:

Touch Response **

usp-46 certified bypeerreview)istheauthor/funder.Allrightsreserved.Noreuseallowedwithoutpermission. (lf n.s. ); USP-46::GFP https://doi.org/10.1101/679415

(lf) ** ** n.s. usp-46(ok2232, =0.Vle ta dfe sgiiaty rm h wl-ye (ANOVA, Dunnett’s wild-type the from significantly differ that Valuesn=10). GFP-tagged USP-46isfunctionalinvivo =0, n UP4:GP xrse udr the under expressed USP-46::GFP and n=10), ; this versionpostedJune21,2019. p ≤0.001.n.s.p>0.05. The copyrightholderforthispreprint(whichwasnot trans-retinal (ATR, 100 μM). Single . Otgntc ciain f the of activation Optogenetic A, nmr-1 Psra-6::FT- promoter