In this study, we first confirmed the contribution of a deubiquitinase - TRABID in basal and starvation-induced autophagy. We further identified VPS34 as a novel substrate of TRABID. Through modifying the ubiquitin chains thereby affecting the turnover of VPS34 protein, TRABID enhanced the complex stability of VPS34-VPS15-Beciln1, leading to an increase in the overall abundance of functional VPS34 complexes. Importantly, TRABID’s impact on VPS34 complexes is not restricted to autophagic-specific ATG14L-containing complex but also extended to UVRAG-containing complex, thereby affecting endocytosis. All together, we demonstrated that TRABID positively regulates endocytic and autophagic pathways via modulating the abundance of VPS34 complexes.
We discovered that TRABID could remove Lys29/Lys48-linked heterotypic ubiquitin chains on VPS34, thereby stabilizing it. As mentioned, linkage specificity of TRABID in vitro was reported to be Lys29/33-linked ubiquitin chains, which is slightly different from our finding and other in vivo studies (Fernando et al., 2014; Jin et al., 2016; Licchesi et al., 2012; Tran et al., 2008; Virdee et al., 2010). In fact, some of the members in OTU family such as A20 and OTUD5 exhibit the same conundrum of unmatched linkage preference in vivo and in vitro. One plausible explanation is that post-translational modifications like phosphorylation or interactions unique in mammalian cells could account for DUBs’ activities toward a specific subset of chain type in vivo (Huang et al., 2012; Wertz et al., 2015). Numerous phosphorylation sites on TRABID were reported according to PhosphoSitePlus®, supporting the above-mentioned theory (Hornbeck et al., 2004). Besides, DUBs often contain additional protein interacting domains that selectively recognize their substrates (Komander,
2010). Hence using di-ubiquitins as substrates while performing in vitro DUB activity analysis could not completely represent the actual situation in cells.
Since the deubiquitination of VPS34 leads to its stabilization, Lys29/Lys48-linked heterotypic ubiquitin chains may dictate VPS34 to proteasomal or lysosomal degradation. Previous studies implicated that apart from Lys48-linked homotypic chains, heterotypic chains might be involved in proteasomal degradation as well (Grice et al., 2015; Meyer and Rape, 2014; Xu et al., 2009). Branched ubiquitin chains were proposed to possess higher signal capacity than longer chains as they provide multivalent recognition sites for effectors with several ubiquitin binding sites (Meyer and Rape, 2014; Thrower et al., 2000). For instance, Lys11/Lys48-linked chains were reported to adopt a conformation which allows its binding with 19S subunit of proteasome and facilitate degradation preferentially compared to homotypic Lys11 or Lys48-linked chains of same molecular weight (Grice et al., 2015; Meyer and Rape, 2014). Though proteasome-targeting Lys29/Lys48-linked chains in vivo has not been elaborately demonstrated so far, multiple lines of evidence suggest the importance of Lys29 linkages in proteasomal degradation. Quantitative mass spectrometry analysis showed increased abundance of Lys29 linkages in MG132-treated yeast or 26S proteasome knockout mouse neurons (Bedford et al., 2011; Xu et al., 2009). In addition, hydrolysis of Lys29/Lys11-linked chains by TRABID was discovered to protect Jmjd2d from proteasomal degradation (Jin et al., 2016). In the aspect of lysosomal degradation, heterotypic chains are less-addressed. Itch/AIP4, a HECT-type E3 ligases, was revealed to catalyze the formation of Lys29/Lys48-linked chains on DTX, expediting its destruction through lysosomal degradation (Chastagner et al., 2006, 2008).
Our previous data revealed that TRABID promotes autophagy under both basal and serum-starved conditions. Consistently, the interaction between VPS34 and TRABID was not enhanced in response to nutrient starvation, indicating a constitutive binding. To understand the potential upstream regulating mechanism of TRABID-induced VPS34 deubiquitination, several autophagy-promoting signals including serum or glucose starvation, H2O2-induced oxidative stress and ER stress were tested.
However, none of these stimuli were able to induce expression of TRABID proteins (Yu-Hsuan Chen, data not shown). In addition, results of in vivo deubiquitination assay implies that VPS34 deubiquitination by TRABID remains unaltered under these conditions (Yu-Hsuan Chen, data not shown). While we have some other autophagy-inducing agents such as mitophagy inducer and hypoxia still in line of testing, we have not excluded the possibility that TRABID is a constitutive component of VPS34 complexes regardless of autophagy-initiating signals. In that scenario, there are probably other proteins that are associated with TRABID-interacting VPS34 complexes and could be regulated by autophagy. As many E3 ligases act as a pair with DUBs and fine-tune the fate of their substrates, there might be an autophagy-regulating E3 ligase capable of counteracting the function of TRABID on VPS34. Since HECT-type E3s are assumed to possess enzymatic activity and display linkage specificity while assembling ubiquitin chains (Kim and Huibregtse, 2009; Wang and Pickart, 2005), it is possible that one of them potentiates the heterotypic chains on VPS34. Among them, Itch/AIP4 and UBE3C/KIAA10 are two HECT-E3s that were capable of catalyzing the formation Lys29/Lys48-linked heterotypic chains (Chastagner et al., 2006; You and Pickart, 2001).
Therefore, whether they are involved in the regulation of VPS34 could be further studied.
We also addressed that TRABID has an impact on endocytic trafficking presumably via regulating UVRAG-containing VPS34 complex. Considering that UVRAG-containing complex also contributes to autophagosome maturation, whether TRABID elicits effects on later stages of autophagy needs to be elucidated through utilizing tandem RFP-GFP-LC3 expressing cells (Klionsky et al., 2016). While the GFP, which is modified into an acid-sensitive manner, is quenched once entering the autolysosome, RFP remains stable under the acidic environment. Thus, the progression of autophagosome to autolysosome could be monitored by calculating the ratio of RFP-positive puncta to GFP and RFP double RFP-positive puncta. If the hypothesis stands, it might define a broader range of TRABID’s function in autophagy.
After identifying the role of TRABID in autophagy and endocytosis in the cell-based experiments, we also wonder what its physiological role is. In our findings, depletion of TRABID impaired autophagic process even under nutrient-rich condition.
Given its significant impact on basal state of autophagy, TRABID might participate in homeostasis maintenance or organelle quality control in cells. Notably, autophagic process is known to be crucial for homeostasis and development of the immune system (Levine et al., 2011; Mizushima and Levine, 2010). For instance, clearance of mitochondria is an intricately regulated developmental transition from thymocyte to mature circulating T cells and contributes to T cells’ survival (Mizushima and Levine, 2010). Increased accumulation of mitochondria and enhanced apoptosis were observed in T cell-specific Atg5,Atg7 and Vps34 knockout mice, leading to reduction of peripheral T cells (Pua et al., 2009; Stephenson et al., 2009; Willinger and Flavell, 2012). Besides, autophagy has been implicated in various facets of MHC-mediated antigen presentation including delivery of antigens to MHC class I and class II for
presentation and cross-presentation of phagocytosed antigens in dendritic cells (Crotzer and Blum, 2009; Mintern et al., 2015). Of note, numerous OTU family DUBs including A20, OTUD5, Cezanne, otubain-1 are reported to participate in the regulation of immune response (Sun, 2008). TRABID’s role in immune system was elicited in a recent study, in which it contributes to proinflammatory cytokine production in both dendritic cells and macrophages and mediates inflammatory T cells responses (Jin et al., 2016). Whether TRABID, with the newly discovered regulatory impact on autophagy, possesses other functional role in immune system could be further investigated.
On the other hand, higher expression levels of TRABID were detected in brain, endocrine tissues and reproductive system based on information integrated by Human Protein Atlas (HPA) Program and other literatures (Clague et al., 2013). Dysregulation of autophagy has been widely reported in several neurodegenerative diseases (Choi et al., 2013). Emerging evidence suggests that except for maintaining basal level of autophagy as other cells, highly specified neurons also have adapted autophagy to a more sophisticated manner in order to suit their specific needs such as turnover of receptors, synaptic vesicles and proteins or clearance of aggregates (Yamamoto and Yue, 2014). Accumulation of protein aggregates and mitochondria dysfunction are two common features in the pathogenesis of neurodegenerative disorders including Alzheimer’s disease, Huntington’s disease and Parkinson’s diseases, pointing to potential disruption of autophagy in these diseases (Choi et al., 2013; Yamamoto and Yue, 2014). Interestingly, similar features such as increased protein aggregates and disorganized mitochondria were observed in VPS34-null cells and mouse organs (Jaber et al., 2012). Thus, whether deficiency or functional impairment of TRABID is involved in pathogenesis or progression of above-mentioned diseases could be further examined.
If the physiopathological role of TRABID holds true, TRABID-mediated autophagy induction that potentially alleviates described symptoms might offer novel therapeutic strategies for medical applications.
Our study aims to address the significance of DUBs in autophagy. A promising DUB - TRABID was identified to hydrolyze less-addressed Lys29/Lys48-linked heterotypic chains on VPS34, which stands at the hub of both autophagic and endocytic pathways. Through regulating the turnover of VPS34 protein, TRABID modulates the stability of various VPS34 complexes and potentiates the maintenance of basal and stress-induced autophagy. Hence, the essential engagement of TRABID in autophagy implies its potential role in diseases associated with autophagy dysregulation.