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In Vivo Strategies to Isolate and Characterize the Neuronal Ubiquitinated Proteome

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Current Proteomic Approaches Applied to Brain Function

Part of the book series: Neuromethods ((NM,volume 127))

Abstract

Protein ubiquitination is essential for the development of neurons and their proper functioning. Indeed, its failure is associated with a number of neurological disorders. The identification of the proteins that are ubiquitinated in vivo in neurons can greatly contribute to our understanding of the roles that this modification plays in the brain. However, the low stoichiometry at which ubiquitin-modified proteins are found within the cells makes the study of this modification quite challenging.

Here we describe two methodologies that have proven to be suitable approaches for the in vivo analysis of neuronal ubiquitinated proteins. The first approach is based on the in vivo biotinylation of ubiquitin and allows the isolation and enrichment of hundreds of ubiquitin conjugates. The second approach is designed to selectively isolate particular proteins in order to characterize their ubiquitinated fraction.

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References

  1. Komander D, Rape M (2012) The ubiquitin code. Annu Rev Biochem 81:203–229. doi:10.1146/annurev-biochem-060310-170328

    Article  CAS  PubMed  Google Scholar 

  2. Ciechanover A (2013) Intracellular protein degradation: from a vague idea through the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting. Bioorg Med Chem 21:3400–3410. doi:10.1016/j.bmc.2013.01.056

    Article  CAS  PubMed  Google Scholar 

  3. Hershko A, Ciechanover A, Heller H et al (1980) Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis. Proc Natl Acad Sci U S A 77:1783–1786

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Swatek KN, Komander D (2016) Ubiquitin modifications. Cell Res 26:399–422. doi:10.1038/cr.2016.39

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hoeck JD, Jandke A, Blake SM et al (2010) Fbw7 controls neural stem cell differentiation and progenitor apoptosis via Notch and c-Jun. Nat Neurosci 13:1365–1372. doi:10.1038/nn.2644

    Article  CAS  PubMed  Google Scholar 

  6. Hamilton AM, Oh WC, Vega-Ramirez H et al (2012) Activity-dependent growth of new dendritic spines is regulated by the proteasome. Neuron 74:1023–1030. doi:10.1016/j.neuron.2012.04.031

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Rinetti GV, Schweizer FE (2010) Ubiquitination acutely regulates presynaptic neurotransmitter release in mammalian neurons. J Neurosci 30:3157–3166. doi:10.1523/JNEUROSCI.3712-09.2010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ramser J, Ahearn ME, Lenski C et al (2008) Rare missense and synonymous variants in UBE1 are associated with X-linked infantile spinal muscular atrophy. Am J Hum Genet 82:188–193. doi:10.1016/j.ajhg.2007.09.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Shimura H, Hattori N, Si K et al (2000) Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase. Nat Genet 25:302–305. doi:10.1038/77060

    Article  CAS  PubMed  Google Scholar 

  10. Kishino T, Lalande M, Wagstaff J (1997) UBE3A/E6-AP mutations cause Angelman syndrome. Nat Genet 15:70–73. doi:10.1038/ng0197-70

    Article  CAS  PubMed  Google Scholar 

  11. Choi J, Levey AI, Weintraub ST et al (2004) Oxidative modifications and down-regulation of ubiquitin carboxyl-terminal hydrolase L1 associated with idiopathic Parkinson’s and Alzheimer’s diseases. J Biol Chem 279:13256–13264. doi:10.1074/jbc.M314124200

    Article  CAS  PubMed  Google Scholar 

  12. Beaudette P, Popp O, Dittmar G (2016) Proteomic techniques to probe the ubiquitin landscape. Proteomics 16:273–287. doi:10.1002/pmic.201500290

    Article  CAS  PubMed  Google Scholar 

  13. Mayor U, Peng J (2012) Deciphering tissue-specific ubiquitylation by mass spectrometry. Methods Mol Biol 832:65–80. doi:10.1007/978-1-61779-474-2_3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Peng J, Schwartz D, Elias JE et al (2003) A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 21:921–926. doi:10.1038/nbt849

    Article  CAS  PubMed  Google Scholar 

  15. Matsumoto M, Hatakeyama S, Oyamada K et al (2005) Large-scale analysis of the human ubiquitin-related proteome. Proteomics 5:4145–4151. doi:10.1002/pmic.200401280

    Article  CAS  PubMed  Google Scholar 

  16. Vasilescu J, Smith JC, Ethier M, Figeys D (2005) Proteomic analysis of ubiquitinated proteins from human MCF-7 breast cancer cells by immunoaffinity purification and mass spectrometry. J Proteome Res 4:2192–2200. doi:10.1021/pr050265i

    Article  CAS  PubMed  Google Scholar 

  17. Bennett EJ, Shaler TA, Woodman B et al (2007) Global changes to the ubiquitin system in Huntington’s disease. Nature 448:704–708. doi:10.1038/nature06022

    Article  CAS  PubMed  Google Scholar 

  18. Greer PL, Hanayama R, Bloodgood BL et al (2010) The Angelman Syndrome protein Ube3A regulates synapse development by ubiquitinating arc. Cell 140:704–716. doi:10.1016/j.cell.2010.01.026

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Xu G, Paige JS, Jaffrey SR (2010) Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat Biotechnol 28:868–873. doi:10.1038/nbt.1654

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lopitz-Otsoa F, Rodriguez-Suarez E, Aillet F et al (2012) Integrative analysis of the ubiquitin proteome isolated using Tandem Ubiquitin Binding Entities (TUBEs). J Proteomics 75:2998–3014. doi:10.1016/j.jprot.2011.12.001

    Article  CAS  PubMed  Google Scholar 

  21. Kim W, Bennett EJ, Huttlin EL et al (2011) Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol Cell 44:325–340. doi:10.1016/j.molcel.2011.08.025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wagner SA, Beli P, Weinert BT et al (2011) A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol Cell Proteomics 10:M111.013284. doi:10.1074/mcp.M111.013284

    Article  PubMed  PubMed Central  Google Scholar 

  23. Na CH, Jones DR, Yang Y et al (2012) Synaptic protein ubiquitination in rat brain revealed by antibody-based ubiquitome analysis. J Proteome Res 11:4722–4732. doi:10.1021/pr300536k

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Wagner SA, Beli P, Weinert BT et al (2012) Proteomic analyses reveal divergent ubiquitylation site patterns in murine tissues. Mol Cell Proteomics 11:1578–1585. doi:10.1074/mcp.M112.017905

    Article  PubMed  PubMed Central  Google Scholar 

  25. Sarraf SA, Raman M, Guarani-Pereira V et al (2013) Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. Nature 496:372–376. doi:10.1038/nature12043

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Franco M, Seyfried NT, Brand AH et al (2011) A novel strategy to isolate ubiquitin conjugates reveals wide role for ubiquitination during neural development. Mol Cell Proteomics 10:M110.002188. doi:10.1074/mcp.M110.002188

    Article  PubMed  Google Scholar 

  27. Lectez B, Migotti R, Lee SY et al (2014) Ubiquitin profiling in liver using a transgenic mouse with biotinylated ubiquitin. J Proteome Res 13:3016–3026. doi:10.1021/pr5001913

    Article  CAS  PubMed  Google Scholar 

  28. Lee SY, Ramirez J, Franco M et al (2014) Ube3a, the E3 ubiquitin ligase causing Angelman syndrome and linked to autism, regulates protein homeostasis through the proteasomal shuttle Rpn10. Cell Mol Life Sci 71:2747–2758. doi:10.1007/s00018-013-1526-7

    Article  CAS  PubMed  Google Scholar 

  29. Ramirez J, Martinez A, Lectez B et al (2015) Proteomic analysis of the ubiquitin landscape in the Drosophila embryonic nervous system and the adult photoreceptor cells. PLoS One 10:e0139083. doi:10.1371/journal.pone.0139083

    Article  PubMed  PubMed Central  Google Scholar 

  30. Min M, Mayor U, Lindon C (2013) Ubiquitination site preferences in anaphase promoting complex/cyclosome (APC/C) substrates. Open Biol 3:130097. doi:10.1098/rsob.130097

    Article  PubMed  PubMed Central  Google Scholar 

  31. Min M, Mevissen T, Luca MD et al (2015) Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages. Mol Biol Cell 26(24):4325–4332. doi:10.1101/016139

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kwon K, Beckett D (2000) Function of a conserved sequence motif in biotin holoenzyme synthetases. Protein Sci 9:1530–1539

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Chandler CS, Ballard FJ (1985) Distribution and degradation of biotin-containing carboxylases in human cell lines. Biochem J 232:385–393

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Beckett D, Kovaleva E, Schatz PJ (1999) A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci 8:921–929. doi:10.1110/ps.8.4.921

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kimura Y, Tanaka K (2010) Regulatory mechanisms involved in the control of ubiquitin homeostasis. J Biochem 147:793–798. doi:10.1093/jb/mvq044

    Article  CAS  PubMed  Google Scholar 

  36. Marttila AT, Laitinen OH, Airenne KJ et al (2000) Recombinant NeutraLite Avidin: a non-glycosylated, acidic mutant of chicken avidin that exhibits high affinity for biotin and low non-specific binding properties. FEBS Lett 467:31–36. doi:10.1016/S0014-5793(00)01119-4

    Article  CAS  PubMed  Google Scholar 

  37. Min M, Mayor U, Dittmar G, Lindon C (2014) Using in vivo biotinylated ubiquitin to describe a mitotic exit ubiquitome from human cells. Mol Cell Proteomics 13:2411–2425. doi:10.1074/mcp.M113.033498

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We would like to acknowledge Maribel Franco and So Young Lee who also contributed to the optimization of these two approaches. We would also like to thank James Sutherland, Rosa Barrio, Michael Clague, Sylvie Urbe, Catherine Lindon, and Jesus Mari Arizmendi for all their advice and support.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Barrio Sarriena s/n, 48940, Leioa, Bizkaia, Spain

    Juanma Ramirez, Nagore Elu, Benoit Lectez & Ugo Mayor

  2. Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Crown Street, Liverpool, Merseyside, L69 3BX – SBM1, UK

    Aitor Martinez

  3. Ikerbasque, Basque Foundation for Science, Bilbao, Bizkaia, Spain

    Ugo Mayor

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  1. Juanma Ramirez
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  2. Nagore Elu
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  3. Aitor Martinez
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  4. Benoit Lectez
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  5. Ugo Mayor
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Corresponding author

Correspondence to Ugo Mayor .

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Editors and Affiliations

  1. Clinical Neuroproteomics Unit, Navarrabiomed, Navarra Health Department, Public University of Navarra, Proteored-Institute of Health Carlos III (ISCIII), Navarra Institute for Health Research (IdiSNA), Pamplona, Spain

    Enrique Santamaría

  2. Clinical Neuroproteomics Unit, Navarrabiomed, Navarra Health Department, Public University of Navarra, Proteored-Institute of Health Carlos III (ISCIII), Navarra Institute for Health Research (IdiSNA), Pamplona, Spain

    Joaquín Fernández-Irigoyen

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Ramirez, J., Elu, N., Martinez, A., Lectez, B., Mayor, U. (2017). In Vivo Strategies to Isolate and Characterize the Neuronal Ubiquitinated Proteome. In: Santamaría, E., Fernández-Irigoyen, J. (eds) Current Proteomic Approaches Applied to Brain Function. Neuromethods, vol 127. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7119-0_11

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  • DOI: https://doi.org/10.1007/978-1-4939-7119-0_11

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7118-3

  • Online ISBN: 978-1-4939-7119-0

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