Deubiquitinating enzymes (DUBs) regulate almost every aspect of cell metabolism, by counteracting the E1-E2-E3 enzymatic cascade reaction of substrate ubiquitination, or ubiquitin chain formation (Wilkinson, 2009; Komander and Rape, 2012). We now know that ubiquitin chains come in different flavors, depending on the lysine residue or N-terminal methionine via which they are linked. This results in different chains having different topologies that are subsequently recognized by different ubiquitin binding domains, hence resulting in a different cellular outcome (Komander, Clague and Urbé, 2009). It is well established that lysine-48 (K48) linked ubiquitin chains on substrates serve as a signal for a targeted degradation by the proteasome (Glickman and Ciechanover, 2002; Jacobson et al., 2009), and are counteracted by three deubiquitinating enzymes that are a part of the 19S regulatory particles. Those are USP14, UCHL5, and RPN11, two cysteine proteases, and a Zn2+-dependent metalloprotease, respectively (Lam et al., 1997; Borodovsky et al., 2001; Leggett et al., 2002; Yao and Cohen, 2002). Given that DUBs are becoming more and more attractive as drug targets (Bedford et al., 2011), and both proteasome activating and inhibiting strategies are desirable, understanding the molecular mechanism of action, and how they achieve substrate selectivity is crucial for rational drug design.
In this well structured and systematic study, the authors set out to elucidate the link between accumulation of aggregation-prone neurodegenerative proteins and USP14 levels or activity. By blocking the activity of the DUB with inhibitors (IU1 and b-AP15), and complementary, using catalytically inactive enzymatic counterpart, they show it leads to accumulation of ubiquitinated TDP‐43, TDP‐43 ALS causing mutant protein, tau and α‐synuclein (endogenousand/or overexpressed) in Hek293 cells. Surprisingly, this does not affect their protein levels. Hypothesizing the enriched ubiquitin species could be K48-, or K63-linked, they show indeed it corresponds to K48-linked ubiquitin chains. Furthermore, through mass spectrometry analysis of immunoprecipitated K48-linked ubiquitinated proteins from cells overexpressing active or inactive USP14, they successfully identified substrates specifically enriched in lysates containing the inactive DUB. Among those are proteins known to undergo proteasomal degradation. To validate the screen, they chose β-catenin, a very important signaling molecule in the Wnt pathway, potentially due to previously demonstrated link between USP14 and β-catenin levels (Wu et al., 2013; Xu et al., 2017). In subsequent experiments, they aimed to establish if ubiquitinatedβ-catenin accumulation could also be achieved by overexpression of catalytically inactive UCHL5, another proteasome-associated DUB. Again, using mass spectrometry approach, they show that catalytically inactive UCHL5 leads to accumulation of ubiquitinated proteins, but interestingly β-catenin was not among them, suggesting it is a USP14 specific substrate. Combining both data sets, they identify overlapping hits, but also others specific for either USP14, or UCHL5. In addition, they show proteasomal subunits themselves are on both of the accumulated protein lists, indicating their ubiquitination status is regulated by USP14 and UCHL5, too.
What I like about this preprint
I chose to highlight this preprint because of the scientific dissemination effect I believe it has. Systematic mass spectrometry screens allow unbiased and global data analysis, and could bring us one step closer to understanding the underlying molecular mechanism of USP14 and UCHL5 substrate specificity, especially as USP14 is a promising target in neurodegenerative diseases. Quite a lot of structural and biochemical work has been done to elucidate this question (Hu et al., 2005; Chen et al., 2009, Lee et al., 2016), and this approach nicely complements previous efforts.
Evolutionary, it is quite exciting that USP14 has a yeast counterpart, but UCHL5 does not, and brings forward the question of the evolutionary advantage in having two, instead of one DUB (in addition to both yeast and humans having essential RPN11). How the substrate load is distributed among DUBs in human cells could explain the gain of having two enzymes at work.
Moreover, the link between proteasomal degradation and autophagy, a second major (protein) turnover mechanism is well established and recently Kim et al. show that USP14 inhibition leads to increase in proteasomal activity, and through negative feedback loop inhibits autophagy induction via regulation of UVRAG levels. This raises questions about other proteins that enable the crosstalk between the pathways, and how proteins like tau utilize both degradation pathways. This particular preprint makes a step forward to understanding this complex question, and the answers may be among these substrates.
Which identified ubiquitinated proteins are actually direct substrates of the DUBs investigated? Are they indeed targets for the proteasomal degradation? Why does inactivation of USP14 lead to TDP-43, tau, and α‐synuclein ubiquitination, but does not change the levels of these proteins?
How does the substrate profile of USP14 and UCHL5 change upon different stimuli, e.g. autophagy induction compared to physiological conditions? How conserved are they across different cell lines?
In case of loss (knockdown or knockout) or inhibition of either USP14 or UCHL5, does the other DUB eventually compensate and facilitate degradation of substrates that otherwise it would not? Does this also apply to the ubiquitination status of proteasomal subunits themselves identified in this study? Furthermore, what is the function of here reported proteasomal subunit ubiquitination?
- WilkinsonKD (2009) DUBs at a glance. J Cell Sci 122: 2325-2329
- Komander D and Rape M (2012) The ubiquitin code. Annual review of biochemistry 81:203‐
- Komander D, Clague MJ, Urbé S (2009) Breaking the chains: structure and function of the deubiquitinases. Nat Rev Mol Cell Biol10: 550–563
- Glickman MH and Ciechanover A (2002) The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82:373‐
- Jacobson AD, Zhang NY, Xu P, Han KJ, Noone S, Peng J and Liu CW (2009) The lysine 48 and lysine 63 ubiquitin conjugates are processed differently by the 26 s proteasome. J Biol Chem284:35485‐
- Lam YA, Xu W, DeMartino GN and Cohen RE (1997) Editing of ubiquitin conjugates by an isopeptidase in the 26S proteasome. Nature385:737‐
- Borodovsky A, Kessler BM, Casagrande R, Overkleeft HS, Wilkinson KD and Ploegh HL (2001) A novel active site‐directed probespecific for deubiquitylating enzymes reveals proteasome association of EMBO J 20:5187‐5196.
- Leggett DS, Hanna J, Borodovsky A, Crosas B, Schmidt M, Baker RT, Walz T, Ploegh H and Finley D (2002) Multiple associated proteins regulate proteasome structure and function. Mol Cell10:495‐
- Yao T and Cohen RE (2002) A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 419:403‐
- Bedford L, Lowe J, Dick LR, Mayer RJ, & Brownell JE (2011). Ubiquitin-like protein conjugation and the ubiquitin-proteasome system as drug targets. Nat Rev Drug Discov10: 29–46.
- Wu N, Liu C, Bai C, H YP, Cho WCS, Li Q (2013) Over-Expression of Deubiquitinating Enzyme USP14 in Lung Adenocarcinoma Promotes Proliferation through the Accumulation ofβ-Catenin J. Mol. Sci., 14(6), 10749-10760
- Xu X, Liu J, Shen C, Ding L, Zhong F, Ouyang Y, Wang Y, He S (2017) The role of ubiquitin-specific protease 14 (USP14) in cell adhesion-mediated drug resistance (CAM-DR) of multiple myeloma cells. J. Haematol.98, 4–12
- Hu M, Li, P, Song L, Jeffrey PD, Chenova TA, Wilkinson KD, et al. (2005). Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14. The EMBO Journal, 24(21), 3747–3756.
- Chen P.C, Qin, LN, Li XM, Walters BJ, Wilson JA, Mei L, & Wilson SM (2009). The proteasome-associated deubiquitinating enzyme Usp14 is essential for the maintenance of synaptic ubiquitin levels and the development of neuromuscular junctions. The Journal of Neuroscience : the Official Journal of the Society for Neuroscience, 29(35), 10909–10919.
- Lee B.-H, Lu Y, Prado MA, Shi Y, Tian G, Sun S, et al. (2016). USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites. Nature, 532(7599), 398–401
- Kim E, Park S, Lee JH, Mun JY, Choi WH, Yun Y, et al. (2018). Dual Function of USP14 Deubiquitinase in Cellular Proteasomal Activity and Autophagic Flux. Cell Reports, 24(3), 732–743.
Posted on: 31st January 2019