Abstract
Ubiquitination pathways are widely used within eukaryotic cells. The complexity of ubiquitin signaling gives rise to a number of problems in the study of specific pathways. One problem is that not all processes regulated by ubiquitin are shared among the different cells of an organism (e.g., neurotransmitter release is only carried out in neuronal cells). Moreover, these processes are often highly temporally dynamic. It is essential therefore to use the right system for each biological question, so that we can characterize pathways specifically in the tissue or cells of interest. However, low stoichiometry, and the unstable nature of many ubiquitin conjugates, presents a technical barrier to studying this modification in vivo. Here, we describe two approaches to isolate ubiquitinated proteins to high purity. The first one favors isolation of the whole mixture of ubiquitinated material from a given tissue or cell type, generating a survey of the ubiquitome landscape for a specific condition. The second one favors the isolation of just one specific protein, in order to facilitate the characterization of its ubiquitinated fraction. In both cases, highly stringent denaturing buffers are used to minimize the presence of contaminating material in the sample.
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References
Komander D, Rape M (2012) The ubiquitin code. Annu Rev Biochem 81:203–229
Lopitz-Otsoa F, Rodriguez-Suarez E, Aillet F, Casado-Vela J, Lang V, Matthiesen R et al (2012) Integrative analysis of the ubiquitin proteome isolated using tandem ubiquitin binding entities (TUBEs). J Proteomics 75:2998–3014
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
Xu G, Paige JS, Jaffrey SR (2010) Global analysis of lysine ubiquitination by ubiquitin remnant immunoaffinity profiling. Nat Biotechnol 28:868–873
Peng J, Schwartz D, Elias JE, Thoreen CC, Cheng D, Marsischky G et al (2003) A proteomics approach to understanding protein ubiquitination. Nat Biotechnol 21:921–926
Greer PL, Hanayama R, Bloodgood BL, Mardinly AR, Lipton DM, Flavell SW et al (2010) The Angelman syndrome protein Ube3A regulates synapse development by ubiquitinating arc. Cell 140:704–716
Franco M, Seyfried NT, Brand AH, Peng J, Mayor U (2011) A novel strategy to isolate ubiquitin conjugates reveals wide role for ubiquitination during neural development. Mol Cell Proteomics 10, M110.002188
Lectez B, Migotti R, Lee SY, Ramirez J, Beraza N, Mansfield B et al (2014) Ubiquitin profiling in liver using a transgenic mouse with biotinylated ubiquitin. J Proteome Res 13:3016–3026
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
Min M, Mayor U, Lindon C (2013) Ubiquitination site preferences in anaphase promoting complex/cyclosome (APC/C) substrates. Open Biol 3:130097
Min M, Mevissen T, Luca MD, Komander D, Lindon C (2015) Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages. Mol Biol Cell. 26:4325–32
Lee SY, Ramirez J, Franco M, Lectez B, Gonzalez M, Barrio R 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
Tsirigotis M, Thurig S, Dubé M, Vanderhyden BC, Zhang M, Gray DA (2001) Analysis of ubiquitination in vivo using a transgenic mouse model. Biotechniques 31:120–126, 128, 130
Tirard M, Hsiao H-H, Nikolov M, Urlaub H, Melchior F, Brose N (2012) In vivo localization and identification of SUMOylated proteins in the brain of His6-HA-SUMO1 knock-in mice. Proc Natl Acad Sci U S A 109:21122–21127
Kim W, Bennett EJ, Huttlin EL, Guo A, Li J, Possemato A et al (2011) Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol Cell 44:325–340
Wagner SA, Beli P, Weinert BT, Nielsen ML, Cox J, Mann M et al (2011) A proteome-wide, quantitative survey of in vivo ubiquitylation sites reveals widespread regulatory roles. Mol Cell Proteomics 10, M111.013284
Sarraf SA, Raman M, Guarani-Pereira V, Sowa ME, Huttlin EL, Gygi SP et al (2013) Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. Nature 496:372–376
Wagner SA, Beli P, Weinert BT, Schölz C, Kelstrup CD, Young C et al (2012) Proteomic analyses reveal divergent ubiquitylation site patterns in murine tissues. Mol Cell Proteomics 11:1578–1585
Na CH, Jones DR, Yang Y, Wang X, Xu Y, Peng J (2012) Synaptic protein ubiquitination in rat brain revealed by antibody-based ubiquitome analysis. J Proteome Res 11:4722–4732
Leidecker O, Matic I, Mahata B, Pion E, Xirodimas DP (2012) The ubiquitin E1 enzyme Ube1 mediates NEDD8 activation under diverse stress conditions. Cell Cycle Georget Tex 11:1142–1150
Shi Y, Xu P, Qin J (2010) Ubiquitinated proteome: ready for global? Mol Cell Proteomics 10, R110.006882
Williams C, van den Berg M, Sprenger RR, Distel B (2007) A conserved cysteine is essential for Pex4p-dependent ubiquitination of the peroxisomal import receptor Pex5p. J Biol Chem 282:22534–22543
Hensel A, Beck S, Magraoui FE, Platta HW, Girzalsky W, Erdmann R (2011) Cysteine-dependent ubiquitination of Pex18p Is linked to cargo translocation across the peroxisomal membrane. J Biol Chem 286:43495–43505
Wang X, Herr RA, Hansen TH (2012) Ubiquitination of substrates by esterification. Traffic Cph Den 13:19–24
Talamillo A, Herboso L, Pirone L, Pérez C, González M, Sánchez J et al (2013) Scavenger receptors mediate the role of SUMO and Ftz-f1 in Drosophila steroidogenesis. PLoS Genet 9:e1003473
Beckett D, Kovaleva E, Schatz PJ (1999) A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci 8:921–929
Acknowledgments
While this chapter was written by the authors listed above, we would like to acknowledge other lab members who contributed to optimization of these techniques as described: Our thanks therefore to Maribel Franco, James Sutherland, Aitor Martinez, Benoit Lectez, and So Young Lee. We would also like to thank Junmin Peng and Gunnar Dittmar, without whose excellent MS support we would never have confirmed how well our bioUb pulldown strategy was performing. The authors would like to acknowledge networking support by the Proteostasis COST Action (BM1307).
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Ramirez, J., Min, M., Barrio, R., Lindon, C., Mayor, U. (2016). Isolation of Ubiquitinated Proteins to High Purity from In Vivo Samples. In: Matthiesen, R. (eds) Proteostasis. Methods in Molecular Biology, vol 1449. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3756-1_10
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DOI: https://doi.org/10.1007/978-1-4939-3756-1_10
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