TRABID antibody production
Owing to the poor quality of commercially available TRABID antibodies, we intended to generate recombinant TRABID protein for antibody production. To this end, we cloned two fragments of TRABID, the 3 NZF domain and Ankyrin-repeat domain, into pET32 vector, which carries a 6xHis tag, and transformed them into Rosetta competent cells. After inoculation and IPTG induction, recombinant proteins of 3NZF and ankyrin repeats were purified with Ni-sepharose under denaturing and native conditions, respectively, and quantified by SDS-PAGE (Figure 1A). The result indicated the recovery of recombinant proteins with high purity and yield. Purified antigens were provided to a biotech company for polyclonal antibody production. To test the resulting antisera, cell lysates obtained from TRABID knockdown clones, transiently expressing Flag-TRABID cells and several nontransfected cell lines were used. Although the antisera showed higher sensitivity to ectopically expressed Flag-TRABID compared with commercial Flag and TRABID antibodies, endogenous level of TRABID still could not be detected (Figure 1B). Hence, we tried to purify antibodies by affinity chromatography using the soluble antigens. Western blot analysis revealed that purified antibodies were able to recognize both overexpressed and endogenous TRABID proteins. Furthermore, the reduced Western blot signal in TRABID knockdown cells indicated the specificity of the antibodies (Figure 1C). These antibodies were then used in the following studies presented in this thesis.
TRABID promotes autophagy induction under both basal and starved conditions
To further study the role of TRABID in autophagy regulation, we established a TRABID overexpression HeLa cell line and assayed for the abundance of autophagosomes in fed and starved cells. As shown in Fig. 2, the LC3 puncta (a marker of autophagosome) were increased upon TRABID overexpression in both fed and starved cells. However, increased autophagosome levels could result from either an increased onset of autophagy or a decreased autophagic flux. To discriminate between the two possibilities, we treated cells with Bafilomycin A1, which blocks autophagic flux by preventing the fusion of autophagosome with lysosome. Under this circumstance, TRABID overexpression significantly enhanced LC3 lipidation (LC3-II), at both basal state and upon starvation (Figure 3). Together, our data are consistent with the results of previous study in our laboratory using TRABID knockdown strategy and confirm a positive regulatory role of TRABID in autophagy.
TRABID interacts with several components within VPS34 complex
Studies in our laboratory revealed that TRABID knockdown attenuated ATG16 puncta and DFCP-1 puncta (markers of forming autophagosome) without affecting mTOR activity (Yu-Hsuan Chen, unpublished data). These findings suggest a functional place of TRABID at the autophagosome nucleation stage. We therefore tested whether VPS34 complex serves as a target of TRABID. To this end, interaction between transiently expressed Flag-WT-TRABID and endogenous VPS34 complex subunits under fed and starved conditions was examined by co-immunoprecipitation, in which Flag-TRABID was pulled down with M2 beads. Owing to the availability of antibodies, only VPS34, Beclin1, Ambra1 and Rubicon were analyzed by Western blot. The result showed that VPS34, Beclin1, Ambra1, but not Rubicon, the negative regulator of
autophagy, were co-immunoprecipitated with TRABID (Figure 4). Furthermore, the association between TRABID and these proteins was not altered upon starvation, indicating a constitutive interaction.
TRABID promotes VPS34 deubiquitination
Next, we explored whether TRABID can influence on the ubiquitination status of VPS34 complex components. To this end, 293T cells were transfected with His-WT-ubiquitin, Flag-VPS34 together with empty vector, V5-WT-TRABID or the catalytically dead mutant V5-C443S-TRABID. Ubiquitinated proteins were pulled down with Ni sepharose under denaturing condition and followed by Western blot analysis.
Remarkably, WT-TRABID significantly reduced ubiquitination level of VPS34 while the catalytic dead mutant failed to remove ubiquitin from VPS34 (Appendix 2).
Subsequently, direct deubiquitination of VPS34 by TRABID was tested by in vitro deubiquitination assay, in which ubiquitinated Flag-VPS34 and V5-TRABID were separately purified from 293T cells and then were incubated together. Similarly, WT-TRABID, but not C443S TRABID mutant, was capable of deubiquitinating VPS34 (Figure 5). These results indicate that VPS34 as a substrate of TRABID.
Notably, most of the DUBs in the OTU family display intrinsic linkage specificity. TRABID was also reported to preferentially cleave Lys29/33-linked di-ubiquitin in vitro and Lys11/29-linked or Lys63-linked polydi-ubiquitin chains in vivo (Afonina and Beyaert, 2016; Fernando et al., 2014; Licchesi et al., 2012; Tran et al., 2008). To examine the types of VPS34 polyubiquitin chains affected by TRABID, we conducted in vivo deubiquitination assay by introducing 3xFlag-VPS34, V5-WT-TRABID together with a set of ubiquitin mutants with individual Lys residues are
mutated to Arg. Ubiquitinated VPS34 was pulled down with Ni sepharose under denaturing condition and subjected to Western blot analysis. Overexpression of WT-TRABID was less able to remove ubiquitin molecules lacking Lys29 and Lys48, suggesting that TRABID tends to hydrolyze Lys29 and Lys48-linked polyubiquitin chains on VPS34 (Appendix 3). Although TRABID’s ability to cleave Lys48-linked polyubiquitin chain in vivo has never been demonstrated, its first NZF domain was reported to capture Lys29-linked heterotypic polyubiquitin chains containing Lys48 linkages from cell extracts (Kristariyanto et al., 2015), which is consistent with our finding. Collectively, these data indicate that catalytic activity of TRABID is essential for VPS34 deubiquitination through Lys29/48 linkages.
TRABID does not facilitate complex formation but regulates stability of VPS34
After identifying VPS34 as TRABID’s substrate, we next sought to elucidate how TRABID promotes autophagy by interacting and deubiquitinating VPS34. Here, two possible downstream events were proposed: deubiquitination of VPS34 may lead to impacts on either its interaction with other components in the complex or its lipid kinase activity. We first tested whether TRABID affects the formation of VPS34 complex by conducting endogenous co-immunoprecipitation. Cell lysates of control or stably expressing Flag-TRABID HeLa cells under fed or starved conditions were subjected to IgG or anti-VPS34 antibody. Western blot analysis revealed that overexpressing TRABID did not alter the amount of endogenous VPS15 and Beclin1 that co-precipitated with endogenous VPS34 regardless of serum starvation (Figure 6), implying that the complex assembly was not influenced by TRABID.
Although overexpression of TRABID did not modulate the formation of VPS34 complex, we noticed a remarkably elevated protein abundance of endogenous VPS34, VPS15 and Beclin1 in cell lysates, pointing to a potential role of TRABID on stabilizing VPS34. To assess whether TRABID regulates the stability of VPS34, we examined the half-life of endogenous VPS34 by cycloheximide (CHX) treatment, which blocks de novo protein synthesis. Endogenous VPS34 protein displayed a prolonged half-life in
stably expressing Flag-TRABID HeLa cells compared with control HeLa cells (Figure 7A), whereas the accelerated turnover rate was observed in two TRABID knockdown clones (Figure 7B). Considering that the stability of components within the VPS34 complex is interdependent on each other (Liu et al., 2011; Platta et al., 2012; Thoresen et al., 2010), we therefore deduced that increased level of VPS15 and Beclin1 might attribute to the stabilization of VPS34, which possibly increases the population of VPS34-VPS15-Beclin1 complex in cells.
TRABID interacts with both ATG14L and UVRAG-containing VPS34 complexes and increases intracellular PI3P production
ATG14L and UVRAG are two additional proteins that exist in distinct VPS34 complex in a mutually exclusive manner. While ATG14L is reported to facilitate autophagy process, UVRAG participates in both endocytic and autophagic pathways.
Since our previous data suggested that TRABID regulates VPS34, we were also interested in investigating the pool of VPS34 complexes that TRABID interacts with.
First, interaction between ectopically expressed V5-WT-TRABID and EGFP-ATG14L or EGFP-UVRAG in 293T cells was examined by co-immunoprecipitation, in which V5-TRABID was pulled down with V5 beads and followed by Western blot analysis.
Both EGFP-fused ATG14L and UVRAG could be detected in the immunoprecipitants (Figure 8A). The association between transiently expressed ATG14L or EGFP-UVRAG and endogenous TRABID were also determined by using GFP-Trap_A.
Consistently, endogenous TRABID could associate with endogenous VPS34, VPS15, Beclin1 and both EGFP-fused ATG14L and UVRAG (Figure 8B), indicating that TRABID is a binding partner of both ATG14L and UVRAG-containing VPS34 complexes.
Localized production of PI3P after membrane targeting of VPS34 complex is believed to be essential to both endocytic and autophagic pathways for passing down the signals via recruiting effector proteins, which contain PI3P-binding domains. Our aforementioned data indicate that TRABID could deubiquitinate and stabilize VPS34, which also led to stabilization of VPS15 and Beclin1 and possible increase of VPS34-VPS15-Beclin1 complexes. Therefore, we postulated that TRABID may also have influence on PI3P production. Intracellular PI3P of control and stably expressing Flag-TRABID HeLa cells were extracted and subjected to quantitative mass PI3P ELISA analysis. As expected, overexpression of TRABID elevated intracellular PI3P level under nutrient rich condition (Figure 9), supporting our hypothesis that TRABID increases the population of functional VPS34 complexes through deubiquitinating and stabilizing VPS34.
TRABID also regulates endocytic pathway
Given that TRABID interacts with UVRAG-containing VPS34 complex, we wonder if TRABID also modulates endocytic pathway via regulating VPS34. PI3P signaling, occurred on endosomal membrane, controls sorting of internalized receptor
molecules, including recycling of transferrin receptor (Tfr) to plasma membrane or targeting epidermal growth factor receptor (EGFR) to multivesicular bodies (MVB) for later degradation (Jaber et al., 2012; Raiborg et al., 2003). Therefore, we carried out EGFR degradation assay to monitor TRABID’s effect on endocytic trafficking. After serum starvation for overnight, EGF was employed for indicated time to stimulate EGFR activation and subsequent degradation. In stably expressing Flag-TRABID HeLa cells, EGFR degradation rate was accelerated compared with that in control cells (Figure 10A). In contrast, EGFR degradation was drastically attenuated in TRABID knockdown clones (Figure 10B) in accordance with previous results described in VPS34-null MEFs (Jaber et al., 2012). Thus, our findings implied that regulation of VPS34 by TRABID also has an impact on endocytosis in addition to autophagy.