Menu

Close

Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells

Jayashree Chadchankar, Victoria Korboukh, Peter Doig, Steve Jacobsen, Nicholas Brandon, Stephen Moss, Qi Wang

Preprint posted on November 26, 2018 https://www.biorxiv.org/content/early/2018/11/26/479758.article-info

Together at the proteasome, but substrates apart...? Shedding light on USP14 and UCHL5 substrate specificity.

Selected by Mila Basic

 

Background

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.

 

Key Findings

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.

 

Open questions

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?

 

References

  • 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

Read preprint (2 votes)




  • Have your say

    Your email address will not be published. Required fields are marked *

    This site uses Akismet to reduce spam. Learn how your comment data is processed.

    Sign up to customise the site to your preferences and to receive alerts

    Register here

    Also in the biochemistry category:

    The autophagic membrane tether ATG2A transfers lipids between membranes

    Shintaro Maeda, Chinatsu Otomo, Takanori Otomo



    Selected by Sandra Malmgren Hill

    LTK is an ER-resident receptor tyrosine kinase that regulates secretion

    Federica G. Centonze, Veronika Reiterer, Karsten Nalbach, et al.



    Selected by Nicola Stevenson

    1

    Plant photoreceptors and their signaling components compete for binding to the ubiquitin ligase COP1 using their VP-peptide motifs

    Kelvin Lau, Roman Podolec, Richard Chappuis, et al.



    Selected by Martin Balcerowicz

    HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly

    C. Favard, J. Chojnacki, P. Merida, et al.



    Selected by Amberley Stephens

    Aqueous synthesis of a small-molecule lanthanide chelator amenable to copper-free click chemistry

    Stephanie Cara Bishop, Robert Winefield, Asokan Anbanandam, et al.



    Selected by Zhang-He Goh

    Hepatocyte-specific deletion of Pparα promotes NASH in the context of obesity

    Marion Regnier, Arnaud Polizzi, Sarra Smati, et al.



    Selected by Pablo Ranea Robles

    Microfluidic protein isolation and sample preparation for high resolution cryo-EM

    Claudio Schmidli, Stefan Albiez, Luca Rima, et al.



    Selected by David Wright

    ENDOSOMAL MEMBRANE TENSION CONTROLS ESCRT-III-DEPENDENT INTRA-LUMENAL VESICLE FORMATION

    Vincent Mercier, Jorge Larios, Guillaume Molinard, et al.



    Selected by Nicola Stevenson

    1

    Dynamic Aha1 Co-Chaperone Binding to Human Hsp90

    Javier Oroz, Laura J Blair, Markus Zweckstetter



    Selected by Reid Alderson

    1

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    1

    Structures of the Otopetrin Proton Channels Otop1 and Otop3

    Kei Saotome, Bochuan Teng, Che Chun (Alex) Tsui, et al.



    Selected by David Wright

    S-acylated Golga7b stabilises DHHC5 at the plasma membrane to regulate desmosome assembly and cell adhesion.

    Keith T Woodley, Mark O Collins



    Selected by Abagael Lasseigne

    3

    A complex containing lysine-acetylated actin inhibits the formin INF2

    Mu A, Tak Shun Fung, Arminja N. Kettenbach, et al.



    Selected by Laura McCormick

    1

    Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

    Quynh Nguyen, Zhen Liu, Rashmi Nanjundappa, et al.



    Selected by Robert Mahen

    Atlas of Subcellular RNA Localization Revealed by APEX-seq

    Furqan M Fazal, Shuo Han, Pornchai Kaewsapsak, et al.

    AND

    Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

    Alejandro Padron, Shintaro Iwasaki, Nicholas Ingolia



    Selected by Christian Bates

    Applications, Promises, and Pitfalls of Deep Learning for Fluorescence Image Reconstruction

    Chinmay Belthangady , Loic A. Royer



    Selected by Romain F. Laine

    Also in the cancer biology category:

    Oncogenic hijacking of a developmental transcription factor evokes therapeutic vulnerability for ROS-induction in Ewing sarcoma

    Aruna Marchetto, Shunya Ohmura, Martin F. Orth, et al.



    Selected by Hannah Brunsdon

    Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

    Adam K. Glaser, Nicholas P. Reder, Ye Chen, et al.



    Selected by Tim Fessenden

    1

    Myc instructs and maintains pancreatic adenocarcinoma phenotype

    Nicole M Sodir, Roderik M Kortlever, Valentin JA Barthet, et al.



    Selected by William Hill

    Quantification of microenvironmental metabolites in murine cancer models reveals determinants of tumor nutrient availability

    Mark R Sullivan, Laura V Danai, Caroline A Lewis, et al.



    Selected by Maria Rafaeva

    1

    Mechanical Stretch Kills Transformed Cancer Cells

    Ajay Tijore, Mingxi Yao, Yu-Hsiu Wang, et al.



    Selected by Joseph Jose Thottacherry

    Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells

    Jayashree Chadchankar, Victoria Korboukh, Peter Doig, et al.



    Selected by Mila Basic

    A conserved MFS orchestrates a subset of O-glycosylation to facilitate macrophage dissemination and tissue invasion

    Katarina Valoskova, Julia Biebl, Marko Roblek, et al.



    Selected by Giuliana Clemente

    1

    MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

    Emily Yun-Chia Chang, James P Wells, Shu-Huei Tsai, et al.



    Selected by Katie Weiner

    1

    Basal extrusion drives cell invasion and mechanical stripping of E-cadherin

    John Fadul, Gloria M Slattum, Nadja M Redd, et al.



    Selected by William Hill

    Molecularly distinct models of zebrafish Myc-induced B cell leukemia

    Chiara Borga, Clay Foster, Sowmya Iyer, et al.



    Selected by Hannah Brunsdon

    1

    The landscape of antigen-specific T cells in human cancers

    Bo Li, Longchao Liu, Jian Zhang, et al.



    Selected by Rob Hynds

    1

    Unjamming overcomes kinetic and proliferation arrest in terminally differentiated cells and promotes collective motility of carcinoma.

    Andrea Palamidessi, Chiara Malinverno, Emanuela FRITTOLI, et al.



    Selected by Tim Fessenden

    1

    STAT3 promotes melanoma metastasis by CEBP-induced repression of the MITF pigmentation pathway

    Alexander Swoboda, Robert Soukup, Katharina Kinslechner, et al.



    Selected by Hannah Brunsdon

    Memory sequencing reveals heritable single cell gene expression programs associated with distinct cellular behaviors

    Sydney M Shaffer, Benjamin L Emert, Ann E. Sizemore, et al.



    Selected by Leighton Daigh

    2

    Anti-angiogenic effects of VEGF stimulation on endothelium deficient in phosphoinositide recycling

    Amber N Stratman, Olivia M Farrelly, Constantinos M Mikelis, et al.



    Selected by Coert Margadant

    Profiling the surface proteome identifies actionable biology for TSC1 mutant cells beyond mTORC1 signaling

    Junnian Wei, Kevin K. Leung, Charles Truillet, et al.



    Selected by Rob Hynds

    1

    Also in the cell biology category:

    The autophagic membrane tether ATG2A transfers lipids between membranes

    Shintaro Maeda, Chinatsu Otomo, Takanori Otomo



    Selected by Sandra Malmgren Hill

    LTK is an ER-resident receptor tyrosine kinase that regulates secretion

    Federica G. Centonze, Veronika Reiterer, Karsten Nalbach, et al.



    Selected by Nicola Stevenson

    1

    Distinct RhoGEFs activate apical and junctional actomyosin contractility under control of G proteins during epithelial morphogenesis

    Alain Garcia De Las Bayonas, Jean-Marc Philippe, Annemarie C. Lellouch, et al.



    Selected by Ivana Viktorinová

    1

    In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor

    Jacob P. Keller, Jonathan S. Marvin, Haluk Lacin, et al.



    Selected by Stephan Daetwyler

    1

    The spindle assembly checkpoint functions during early development in non-chordate embryos

    Janet Chenevert, Marianne Roca, Lydia Besnardeau, et al.



    Selected by Maiko Kitaoka

    Blue light induces neuronal-activity-regulated gene expression in the absence of optogenetic proteins

    Kelsey M. Tyssowski, Jesse M. Gray



    Selected by Zheng-Shan Chong

    Mutations in the Insulator Protein Suppressor of Hairy Wing Induce Genome Instability

    Shih-Jui Hsu, Emily C. Stow, James R. Simmons, et al.



    Selected by Maiko Kitaoka

    1

    Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

    Adam K. Glaser, Nicholas P. Reder, Ye Chen, et al.



    Selected by Tim Fessenden

    1

    ATAT1-enriched vesicles promote microtubule acetylation via axonal transport

    Aviel Even, Giovanni Morelli, Chiara Scaramuzzino, et al.



    Selected by Stephen Royle

    1

    HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly

    C. Favard, J. Chojnacki, P. Merida, et al.



    Selected by Amberley Stephens

    Hepatocyte-specific deletion of Pparα promotes NASH in the context of obesity

    Marion Regnier, Arnaud Polizzi, Sarra Smati, et al.



    Selected by Pablo Ranea Robles

    Mitochondrial biogenesis is transcriptionally repressed in lysosomal lipid storage diseases

    King Faisal Yambire, Lorena Fernandez-Mosquera, Robert Steinfeld, et al.



    Selected by Sandra Franco Iborra

    1

    Thyroid hormone regulates distinct paths to maturation in pigment cell lineages

    Lauren Saunders, Abhishek Mishra, Andrew J Aman, et al.



    Selected by Hannah Brunsdon

    1

    Kinesin-6 Klp9 plays motor-dependent and -independent roles in collaboration with Kinesin-5 Cut7 and the microtubule crosslinker Ase1 in fission yeast

    Masashi Yukawa, Masaki Okazaki, Yasuhiro Teratani, et al.



    Selected by I. Bouhlel

    A pair of E3 ubiquitin ligases compete to regulate filopodial dynamics and axon guidance

    Nicholas P Boyer, Laura E McCormick, Fabio L Urbina, et al.



    Selected by Angika Basant

    1

    SorCS1-mediated Sorting of Neurexin in Dendrites Maintains Presynaptic Function

    Luis Filipe Ribeiro, Ben Verpoort, Julie Nys, et al.



    Selected by Carmen Adriaens

    1

    Also in the molecular biology category:

    The autophagic membrane tether ATG2A transfers lipids between membranes

    Shintaro Maeda, Chinatsu Otomo, Takanori Otomo



    Selected by Sandra Malmgren Hill

    LTK is an ER-resident receptor tyrosine kinase that regulates secretion

    Federica G. Centonze, Veronika Reiterer, Karsten Nalbach, et al.



    Selected by Nicola Stevenson

    1

    Accurate detection of m6A RNA modifications in native RNA sequences

    Huanle Liu, Oguzhan Begik, Morghan C Lucas, et al.



    Selected by Christian Bates

    1

    Blue light induces neuronal-activity-regulated gene expression in the absence of optogenetic proteins

    Kelsey M. Tyssowski, Jesse M. Gray



    Selected by Zheng-Shan Chong

    Slide-seq: A Scalable Technology for Measuring Genome-Wide Expression at High Spatial Resolution

    Samuel G Rodriques, Robert R Stickels, Aleksandrina Goeva, et al.

    AND

    High-density spatial transcriptomics arrays for in situ tissue profiling

    Sanja Vickovic, Goekcen Eraslan, Johanna Klughammer, et al.



    Selected by Carmen Adriaens

    Optical determination of absolute membrane potential

    Julia R. Lazzari-Dean, Anneliese M.M. Gest, Evan Miller



    Selected by James Marchant

    MicroRNA-mediated control of developmental lymphangiogenesis

    Hyun Min Jung, Ciara Hu, Alexandra M Fister, et al.



    Selected by Rudra Nayan Das

    Microfluidic protein isolation and sample preparation for high resolution cryo-EM

    Claudio Schmidli, Stefan Albiez, Luca Rima, et al.



    Selected by David Wright

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    1

    Structures of the Otopetrin Proton Channels Otop1 and Otop3

    Kei Saotome, Bochuan Teng, Che Chun (Alex) Tsui, et al.



    Selected by David Wright

    Central spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

    Che-Hang Yu, Stefanie Redemann, Hai-Yin Wu, et al.



    Selected by Federico Pelisch

    1

    Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division

    Evgeny Zatulovskiy, Daniel F. Berenson, Benjamin R. Topacio, et al.



    Selected by Zaki Ahmad

    1

    Distinct ROPGEFs successively drive polarization and outgrowth of root hairs

    Philipp Denninger, Anna Reichelt, Vanessa Aphaia Fiona Schmidt, et al.



    Selected by Marc Somssich

    Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells

    Jayashree Chadchankar, Victoria Korboukh, Peter Doig, et al.



    Selected by Mila Basic

    Bacteriophage resistance alters antibiotic mediated intestinal expansion of enterococci

    Anushila Chatterjee, Cydney N Johnson, Phat Luong, et al.



    Selected by Yasmin Lau

    On-site ribosome remodeling by locally synthesized ribosomal proteins in axons

    Toshiaki Shigeoka, Max Koppers, Hovy Ho-Wai Wong, et al.



    Selected by Srivats Venkataramanan
    Close